In vitro preservation of therapeutic cells

专利摘要:
The invention relates to the in vitro preservation of living animal cells. In particular, the invention provides a method of in vitro preservation of cells, comprising maintaining adherent mesenchymal stem cells (MSC) or adherent cells derived from MSC in suspension in a composition comprising at least 20% v / v of human plasma. or human serum or a mixture thereof.
公开号:BE1023315B1
申请号:E2016/5278
申请日:2016-04-22
公开日:2017-02-01
发明作者:Enrico Bastianelli；Carmen Brenner；Elodie Deltour
申请人:Bone Therapeutics Sa；
IPC主号:

专利说明:

IN VITRO PRESERVATION OF THERAPEUTIC CELLS
FIELD The invention is generally in the medical field, more specifically in the field of cell-based products useful for cell therapy. In particular, the invention relates to the in vitro preservation of living animal cells.
BACKGROUND
Animal cells, such as mesenchymal stem cells (MSCs) or MSC-derived cells, which are intended for cell therapy, should be conveniently stored prior to their use for medical or medical research purposes, so that remain viable and maintain desired properties.
Such cells can be stored virtually indefinitely at the temperature of liquid nitrogen at atmospheric pressure (-196 ° C), where cryo-preservatives are usually added to the cell suspension prior to freezing to prevent damage to the cells. cells caused by the freezing process. However, because of the need to constantly maintain very low temperatures, the transport of cryo-preserved cells from the place of their production (for example, a cell therapy company) to the place of their use (for example, a hospital) is logistically, and the continuous storage of cells instead of their use requires specialized equipment (eg, liquid nitrogen tanks). In addition, prior to use, such as prior to administration to a patient, the cells should be thawed, washed to remove any cryo-preservative, and resuspended in a suitable excipient composition. Such additional sample handling steps reduce, among other things, cell viability, increase the risk of cell contamination, and increase variability between samples.
Non-cryogenic methods of cell preservation are also known. In many of such methods, the cells may generally be stored as a cell suspension in a suitable aqueous liquid phase at temperatures typically ranging from about 0 ° C to about 8 ° C. Such aqueous storage liquids are usually based on well-known tissue cell culture growth media, such as, for example, Eagle Minimal Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), or the Middle of the Roswell Park Memorial Institute (RPMI). Alternatively, such aqueous storage liquids may be based on physiological electrolyte solutions, such as Plasma-Lyte® A (e.g., Plasma-Lyte® Injection pH 7.4 (Multiple Electrolyte Injection, Type 1, USP) from Baxter, Deerfield, IL, USA), Ringer Lactate solution (e.g., Ringer Lactate USP injection from Baxter, Deerfield, IL, USA) or a Hartmann solution, optionally supplemented with Human Albumin Serum (eg, 5% w / v).
Historically, plasma or plasma-containing solutions have been used for the extended non-cryogenic storage of cellular blood components, particularly platelets or erythrocytes, in suspension. However, to the knowledge of the Applicant, serum or plasma are not usually employed as excipients in cell-suspension products of cell types other than the cellular components of blood, in particular suspended cell products intended for storage. not cryogenic of such cellular products.
Sometimes low concentrations, such as 10% v / v or 5% v / v or less, of autologous serum, autologous plasma, or platelet-rich plasma are included in solutions used to suspend therapeutic cells prior to their use. injection or infusion to patients. In other cases, therapeutic cells may be suspended in solutions containing predominantly or exclusively autologous serum, autologous plasma, or platelet-rich plasma prior to injection or infusion into patients. However, cells are typically suspended in such serum or plasma containing solutions just prior to administration to patients, and non-cryogenic storage of cell suspensions has not been contemplated.
Several studies, including US 2014/242181, have explored methods of repair or bone regeneration involving implantation of scaffolds consisting of autologous plasma or platelet-rich plasma containing therapeutic cells included. These studies do not include suspended cellular products or prolonged non-cryogenic storage of these products.
Garvican et al. (Stem Cell Res Ther, 2014, vol.5, 94) compared the effects of various allogeneic media on the viability and proliferative properties of horse MSCs suspended in the medium up to 72 hours to 4 hours. at 8 ° C. Cell viability was clearly retained for longer durations in isotonic saline or DMEM supplemented with 10% v / v fetal calf serum relative to allogeneic horse plasma or allogeneic horse serum. According to Garvican et al., The viability of horse MSCs stored in plasma, serum or equine platelet rich plasma (PRP) for 72 hours was considerably less than 70%, which constitutes a cell viability threshold during storage usually required by regulatory authorities for therapeutic cellular products. Garvican et al. advocate instead maintaining horse MSCs in physiological saline for up to 24 hours, and suggest cryopreservation for longer storage periods.
The CellSeal® system from Cook Medical (Bloomington, IN, USA) is a closed system container for the protection of specimens during shipment of unfrozen cell therapy products for direct clinical use (http: // cellseal. cookmedical.com/wp-content/uploads/2014/04/PI-BP-SCIPOSJ-EN-201404.pdf). Horse MSCs suspended in autologous plasma platelet lysate were packaged in CellSeal® vials and transported overnight at 4-8 ° C.
WO 2014/049063 discloses cell delivery formulations comprising solvent / detergent treated plasma (S / D plasma) and mesenchymal stem cells configured for intraosseous administration to subjects. It is contemplated that the formulations exhibit or achieve gel consistency upon administration.
An object of the present invention is to provide additional and improved methods of in vitro preservation of living animal cells, such as therapeutic cells useful for research or medical purposes.
ABSTRACT
As corroborated by the experimental section, which illustrates some representative embodiments of the invention, the inventors have realized that mesenchymal stem cells (MSCs) or MSC-derived cells subjected to non-cryogenic suspension storage in Liquid storage based on human plasma or human serum has exhibited advantageous viability and stability of cell product, even after storage for extended periods of time. The present findings are unexpected, inter alia, because equine plasma and serum have been reported to be less satisfactory than a number of other liquid media, particularly physiological saline, for suspended MSC suspension storage. for 24 hours, and because cryopreservation has certainly never been recommended for longer storage periods of horse MSC (Garvican et al).
Accordingly, in one aspect the invention provides a method for in vitro storage of cells comprising maintaining adherent mesenchymal stem cells (MSC) or adherent cells derived from MSC in suspension in a composition comprising at least 20% v / v of human plasma or human serum or a mixture thereof. Preferably, the invention provides a method for non-cryogenic in vitro preservation of cells comprising maintaining adherent MSCs or MSC-derived adherent cells in suspension in a composition comprising at least 20% v / v of human plasma or human serum or a mixture of these.
The above and other aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject of the appended claims is thus specifically incorporated in the present specification.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the proportion of living cells in a population of osteoblastic cells derived from human MSC after suspension storage at 2-8 ° C in the indicated storage medium for 24, 48, 72, 96, 144 or 192 hours. NA (in this figure and following): not available. "Standard Excipient" (in this figure and the following): Plasma-Lyte® supplemented with 5% w / v of HSA; "Plasma" (in this figure and the following): 100% Octaplas®; "Serum" (in this figure and the following): 100%, auto-serum; Serum enriched in HSA: Auto-serum supplemented with 50% w / v of human serum albumin (HSA).
Fig. Figure 2 illustrates optical microscopy images of osteoblastic cells derived from human MSCs after suspension storage at 2-8 ° C for 72 hours in the indicated storage medium, reseeding, and culture for 24 hours.
Fig. Figure 3 illustrates flow cytometric analyzes of marker expression by osteoblastic cells derived from human MSCs after storage in suspension at 2-8 ° C in the medium indicated for the indicated periods. The results show the proportion (%, mean ± standard deviation) of the cells considered positive for the respective markers.
Fig. 4 illustrates bone formation in the cranial vault of mice, 14 days after injection of cell-free standard excipient (top row), or osteoblastic cells derived from human MSCs previously stored in suspension at 2-8 ° C in an excipient standard (middle row) or human plasma (lower row), for the periods indicated. The mineralized bone prior to cell injection appeared to be comparatively darker, and the mineralized bone after the cell injection appeared comparatively clearer, due to the incorporation of fluorochromes, particularly green calcein and yellow tetracycline.
Fig. 5 illustrates (A) mouse cranial vault bone density, 14 days after injection of MSC derived osteoblastic cells previously stored in suspension at 2-8 ° C in the medium indicated for the indicated periods. The Y axis represents the number of pixels measured on x-ray images. (B) Bone formation within the mouse cranial vault, 14 days after injection of MSC-derived osteoblastic cells previously stored in suspension at 2-8 ° C in the medium indicated for the periods indicated. The Y axis represents the average value of bone formation (expressed in%). ° p <0.1, * p <0.05, ** p <0.01, *** p <0.001.
DESCRIPTION OF THE EMBODIMENTS
As used herein, the singular forms "one", "one", and "the" include singular and plural referents unless otherwise indicated and clear from context.
The terms "comprising", "includes" and "constituted by" as used herein are synonymous with "comprising", "includes" or "containing", "contains", and are with or without terms and do not exclude additional, unlisted members, elements, or method steps. The terms also include "constituted by" and "essentially constituted by", which have well-established meanings in the terminology of patents. The enumeration of the digital ranges by the end points includes all digits and subsumed fractions within the respective ranges, as well as the endpoints enumerated.
By the terms "about" or "approximately", as used herein when referring to a measurable value such as a parameter, a quantity, a time duration, and the like, is meant to encompass the variations of and from of the specified value, such as variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and still more preferably +/- 0.1 % or less and from the specified value, as long as such variations are appropriate to execute in the disclosed invention. It should be understood that the value to which the "about" modifier refers is itself also specifically, and preferably, disclosed.
Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, are self-explanatory, by way of additional exemplification, the term includes, inter alia, a reference to any one of said members, or to any two or more of said members, such as, for example,> 3,> 4,> 5,> 6 or> 7 etc. any of said members, and up to all of said members. In another example, "one or more" or "at least one" may denote 1, 2, 3, 4, 5, 6, 7 or more.
The discussion of the background of the invention herein is included to explain the context of the invention. It shall not be construed as an admission that any of the materials cited have been published, known, or were part of general common knowledge in any country from the priority date of any of the claims .
Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identification citation. All documents cited in this specification are thus incorporated by reference in their entirety. In particular, the teachings or sections of such documents specifically cited herein are incorporated by reference.
Unless otherwise defined, all terms used in the disclosure of the invention, including technical and scientific terms, have the meaning as commonly understood by those skilled in the art to which the present invention belongs. As additional guidance, definitions of terms are included to better appreciate the teaching of the invention. When specific terms are defined in relation to a particular aspect of the invention or a particular embodiment of the invention, such connotation is intended to apply throughout the present specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined.
In the following passages, various aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with one or more other aspects or embodiments, unless otherwise indicated and clearly indicated. In particular, any feature indicated as being preferred or advantageous may be combined with one or more of any other features indicated as being preferred or advantageous.
Reference throughout the present specification to "an embodiment" means that a particular feature, structure or feature described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the presences of the terms "in one embodiment" at various points throughout this specification do not necessarily all refer to the same embodiment, but may do so. In addition, particular features, structures, or features may be combined in any suitable manner, as will be apparent to those skilled in the art from the present disclosure, in one or more embodiments. Further, while some embodiments described herein include some, but not others, features included in other embodiments, combinations of features of different embodiments are expected to lie within the scope of the invention. invention, and form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.
As noted above, the inventors have realized that mesenchymal stem cells (MSC) or MSC-derived cells subjected to non-cryogenic suspension storage in a liquid storage medium comprising a given minimum amount of human plasma or serum Humans exhibited advantageous cell product stability even after storage for extended periods of time.
Accordingly, one aspect of the invention provides a method for in vitro preservation of cells comprising maintaining adherent mesenchymal stem cells (MSC) or adherent cells derived from MSC in suspension in a composition comprising at least 20% v / v ( relative to the volume of the composition) of human plasma or human serum or a mixture thereof. Preferably, the invention provides a method for non-cryogenic in vitro preservation of cells comprising maintaining adherent MSCs or MSC-derived adherent cells in suspension in a composition comprising at least 20% v / v of human plasma or human serum or a mixture of these.
The present methods allow non-cryogenic maintenance of the cells, so that the compositions remain in the unfrozen state (i.e., in the liquid state, typically having a temperature above the freezing point of the compositions). during storage.
The terms "composition", "formulation", or "preparation" may be used interchangeably herein.
By the term "mesenchymal stem cell" or "MSC" as used herein is meant an adult stem cell derived from the mesoderm that is capable of generating cells of mesenchymal lineages, typically two or more mesenchymal lineages, by For example, the osteoblastic (bone), chondroblastic (cartilage), myocytic (muscle), tendinocytic (tendon), fibroblastic (connective tissue), adipocyte (fat) and stromal (bone marrow stroma) lineage. MSCs can be isolated from, for example, bone marrow, trabecular bone, blood, umbilical cord, placenta, fetal yolk sac, skin (dermis), specifically fetal skin, and adolescent, periosteum and adipose tissue. Human MSCs, their isolation, in vitro expansion, and differentiation have been described, for example, in U.S. Patent No. 5,486,359; U.S. Patent No. 5,811,094; U.S. Patent No. 5,736,396; U.S. Patent No. 5,837,539; or U.S. Patent No. 5,827,740. Any MSC described in the state of the art and isolated by any method described in the state of the art may be suitable in the present method.
The term "MSC" also includes MSC offspring, for example, progeny obtained by in vitro or ex vivo (propagation) proliferation of MSCs obtained from a biological sample of an animal or human subject.
Preferable MSCs can potentially generate cells of at least the osteoblastic lineage (bone), such as, for example, osteoprogenitor and / or pre-osteoblasts and / or osteoblasts and / or osteocytes, etc. or at least the chondroblastic line (cartilage), such as, for example, chondrogenic cells and / or chondroblasts and / or chondrocytes, the fibroblastic line (connective tissue), such as, for example, fibroblasts, fibrocytes or at least synoviocytes (synovial fluid) or tenocytes etc.
The term "stem cell" is generally understood to mean a non-specialized or relatively less specialized and proliferating cell which is capable of self-renewal, that is, capable of proliferation without differentiation, and which, or the whose offspring, can lead to a relatively more specialized cell type. The term encompasses stem cells capable of substantially unlimited self-renewal, i.e. where the offspring of a stem cell or at least a portion thereof retain substantially the non-specialized or relatively less specialized phenotype. , the potential for differentiation, and the proliferative capacity of the parent stem cell, as well as the stem cells that exhibit limited self-renewal, that is, the capacity of the offspring of a part of This for further proliferation and / or differentiation is demonstrably reduced relative to the parent cell. By way of example and without limitation, a stem cell can lead to differentiable offspring in one or more lines to produce more and more specialized cells, where such offspring and / or such More and more relatively more specialized cells can themselves be stem cells as defined herein, or even produce finally differentiated cells, i.e., totally specialized cells, which may be post-mitotic.
By the term "adult stem cell" as used herein is meant a stem cell present in or obtained from (or isolated from) an organism at the fetal stage or preferably after birth (e.g. in particular but without limitation for a human organism, an age of at least one month after birth, for example, at least 2 months, at least 3 months, for example, at least 4 months, at least 5 months, for example an age of at least 6 months after birth, such as, for example, an age of 1 year or over, 5 years or more, at least 10 years or more, 15 years or older, 20 years or older, or 25 years or more after birth), for example after reaching adulthood. By way of example, adult stem cells can be obtained from human subjects that would otherwise be described in the conventional terms "infant", "child", "young", "adolescent" or "adult".
By the term "mesenchymal stem cell-derived cells" or "MSC-derived cells" as used herein is meant mesenchymal lineage cells (e.g., osteoblastic (bone), chondroblastic (cartilage), myocytic (muscle) ), tendinocytic (tendon), fibroblastic (connective tissue), adipocyte (fat) or stromal (bone marrow stroma)) obtained by the differentiation of MSC, in particular obtained by the in vitro differentiation (including ex vivo) of MSC .
Differentiation of MSC may involve culture of MSCs under conditions capable of inducing differentiation of MSC to the desired cell type, more typically culturing of MSCs in a medium comprising one or more factors (e.g., growth factors ) capable of inducing the differentiation of MSC to the desired cell type. The MSC differentiation protocols are known per se (see, inter alia, WO 2007/093431, and in addition REGER, RL et al., "Differentiation and Characterization of Human MSCs" in: "Mesenchymal Stem Cells: Methods and Protocols". Methods in Molecular Biology), Edited by DJ Prockop et al Humana Press, 2008, Vol 449, 93-107, VERMURI, MC et al (Eds.), "Mesenchymal Stem Cell Assays and Applications" (Methods in Molecular Biology). Molecular Biology), Humana Press, 2011, Vol 698, especially pages 201 to 352).
By the term "in vitro" is generally meant outside, or outside, of the animal or human organism. The term "ex vivo" typically refers to tissues or cells taken from an animal or human body organism and maintained or propagated out of the body, for example, into a culture vessel. The term "in vitro" as used herein should be understood to include "ex vivo". By the term "in vivo" is generally meant within, or within, the animal or human organism.
In some embodiments, MSC-derived cells may include osteoblastic (bone), chondroblastic (cartilage) line, myocytic (muscle), tendinocytic (tendon), fibroblast (connective tissue) lineage cells. , adipocyte lineage (fat) or stromal line (stroma of the bone marrow).
In some embodiments, cells derived from MSC may include cells of osteoblastic (bone) or chondroblastic (cartilage) lineage.
In some embodiments of the methods as taught herein, MSC-derived cells may include osteoprogenitor, osteoblast cells, osteocytes, chondroblast cells, chondrocytes, fibroblast cells, fibroblasts, fibrocytes, tenoblasts, tenocytes, or synoviocytes, for example, cells derived from MSC can be chosen from the group comprising or consisting of osteoprogenitor, osteoblast cells, osteocytes, chondroblastic cells, chondrocytes, fibroblast cells, fibroblasts, fibrocytes, tenoblasts, tenocytes, synoviocytes, and optionally mixtures thereof. In some preferred embodiments of the methods as taught herein, MSC-derived cells may include osteoprogenitor, osteoblast cells, or osteocytes, for example, MSC derived cells may be selected from the group consisting of or consisting of osteoprogenitor, osteoblast cells, osteocytes, and possibly mixtures thereof. In some more preferred embodiments of the methods as taught herein, MSC derived cells may include osteoprogenitor or osteoblast cells, for example, MSC derived cells may be selected from the group consisting of or consisting of osteoprogenitor, osteoblastic cells, and optionally mixtures thereof. In some more preferred embodiments of the methods as taught herein, MSC-derived cells may be osteoblastic cells.
As known in the state of the art, osteoblastic (bone) lineage cells typically encompass cell types having an osteogenic phenotype, and which may contribute to, or are capable of developing into, cells that may contribute to the formation of bone material or bone matrix. As used herein, "osteoprogenitor" can include, in particular, early and late osteoprogenitor therapy. "Osteoblastic cells" may include in particular pre-osteoblasts and osteoblasts. All these terms are well known per se. As additional and unrestricted guidance, osteoprogenitor and osteoblastic cells, as well as cell populations comprising osteoprogenitor and / or osteoblast cells, may have the following characteristics: a) the cells comprise the expression of Runx2, a multifunctional transcription factor that regulates the differentiation of osteoblasts and the expression of many extracellular matrix protein genes during osteoblast differentiation; b) the cells comprise the expression of at least one of the following: alkaline phosphatase (ALP), more specifically bone-liver-kidney ALP; and more preferably also include the expression of one or more additional bone markers such as osteocalcin (OCN), the amino-terminal propeptide of procollagen type 1 (P1NP), osteonectin (ON), osteopontin ( OP) and / or bone sialoprotein (BSP), and / or one or more other bone matrix proteins such as decorin and / or osteoprotegerin (OPG); c) the cells do not substantially express CD45 (e.g., less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45); d) the cells show signs of ability to mineralize the external environment, or to synthesize an extracellular matrix containing calcium (for example, when exposed to an osteogenic medium, see Jaiswal et al., J Cell Biochem, 1997). 64, 295-312). The accumulation of calcium inside the cells and the deposition in matrix proteins can be measured conventionally for example by culturing in 45Ca2 +, washing and re-culturing, and then determining any radioactivity present inside. of the cell or deposited in the extracellular matrix (US 5,972,703), or using an Alizarin red mineralization assay (see, for example, Gregory et al., Analytical Biochemistry, 2004, vol 329, 77 -84); e) the cells do not substantially differentiate towards adipocyte lineage cells (for example, adipocytes) or chondroblastic lineage (for example, chondroblasts, chondrocytes). The lack of differentiation to such cell lines can be tested using standard differentiation induction conditions established in the state of the art (e.g., see Pittenger et al., Science, 1999, vol 284, 143- 7), and assay methods (for example, when they are induced, the adipocytes typically stain with the red oil O showing lipid accumulation, the chondrocytes typically stain with alcian blue or safranin O). A substantially absent trend towards adipogenic and / or chondrogenic differentiation can typically mean that less than 20%, or less than 10%, or less than 5%, or less than 1% of the cells tested would show signs of adipogenic or chondrogenic differentiation when applying the respective test.
As known in the state of the art, chondroblastic (cartilage) line cells typically include cell types having a chondrogenic phenotype, and which may contribute to, or are capable of developing into, cells that may contribute to the formation of cartilage or cartilaginous matrix. As used herein, "chondro-progenitors" may in particular include early and late chondro-progenitors. "Chondroblastic cells" can include, in particular, pre-chondroblasts and chondroblasts. All these terms are well known per se. As additional and unrestricted guidance, chondro-progenitors and chondroblastic cells, as well as cell populations comprising chondro-progenitors and / or chondroblastic cells may exhibit the following characteristics: a) the cells comprise the expression of SOX9, a transcription factor that plays a central role in the differentiation of chondroblasts and cartilage formation; b) the cells comprise the expression of at least one of the following: aggrecan (AGG), collagen type II, or CD90; c) the cells do not substantially express CD45 (e.g., less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45); d) the cells show evidence of an ability to produce high levels of type II, IX, and XI collagen and proteoglycans, the main components of the extracellular hyaline matrix (ECM) in situ. Cartilage formation can be measured conventionally, for example, using a solid safranin-O / solid assay to stain glysaminoglycans and non-collagenous proteins, respectively (see, for example, Lee et al. Tissue Engineering, 2011, vol 18, 484-98); e) human articular chondrocytes may exhibit cell expression characteristics as summarized in Diaz-Romero et al. 2005 (J Cell Physiol, vol 202 (3), 731-42), for example, they can express integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51 / 61, CD54 , CD106, CD166, CD58, CD44), tetraspanines (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ecto-enzymes (CD10, CD26), and other surface molecules (CD90, CD99). During monolayer culture, chondrocytes may up-regulate certain markers thought to be distinctive of mesenchymal stem cells (CD10, CD90, CD105, CD166). Such markers can therefore also be expressed by less mature chondroblastic cells. f) the cells do not substantially differentiate to adipocyte lineage (e.g., adipocytes) or osteoblast lineage cells (e.g., osteoblasts, osteocytes). The lack of differentiation to such cell lines can be tested using standard differentiation induction conditions established in the state of the art (e.g., see Pittenger et al., Science, 1999, vol 284, 143- 7), and assay methods (for example, when they are induced, the adipocytes typically stain with the red oil O showing lipid accumulation, osteoblastic cells typically stain for ALP). A substantially absent trend toward adipogenic and / or osteoblastic differentiation may typically mean that less than 20%, or less than 10%, or less than 5%, or less than 1% of the cells tested would exhibit signs of adipogenic or osteoblastic differentiation when applying the respective test.
As known in the state of the art, fibroblastic lineage cells can contribute to, or are able to grow into, cells that can contribute to connective tissue formation. As additional and unrestricted guidance, fibroblast cells may have the following characteristics: a) the cells comprise the expression of FSP1 (fibroblast-specific protein 1); b) the cells comprise the expression of at least one of the following: collagen, vimentin, desmin or CD90; c) the cells do not substantially express CD45 (e.g., less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45); d) the cells show signs of ability to produce collagen, glycosaminoglycans, reticular and elastic fibers, glycoproteins to form the extracellular matrix of connective tissues. Fibroblasts contribute to the structural integrity of ligaments and tendons and have a tissue repair function. Collagen deposition can be visualized by Trichrome staining (Li et al., World J Gastroenterol, 2014, Vol 20 (16), 4648-61). Type I collagen (Chondres, Redmond, WA) and tenascin-C (Tn-C, IBL-America, Minneapolis, MN) are two markers for ligament fibroblasts, and can be assayed by ELISA (Brissett et al. Arthritis Rheum, 2012, 64 (1), 272-80).
As known in the state of the art, tendinocytic lineage cells may contribute to the formation of tendon or tendon matrix material. The tendon consists of large bundles of fibers that include a network of collagen fibrils and different types of cells, including synovial cells, endothelial cells, tenoblasts, and tenocytes lying longitudinally in rows in collagen molecules. Tenoblasts are immature forms of tendon cells that differentiate into tenocytes as they age with reduced metabolic activity. As additional and unrestricted guidance, tenocytes may have the following characteristics: a) the cells include the expression of "scleraxis" (SCX), a member of the basic helix-loop-helix family of transcription factors involved in cell differentiation and organization of the extracellular matrix in the tendons; b) the cells comprise the expression of at least one of the following: tnnodulin (TNMD) and Tenascin-C (TNC); c) the cells express substantially CD44, CD73, CD90 and CD105 but do not express CD34, CD45, CD146, or stro-1; d) the cells exhibit evidence of an ability to produce an extracellular tendon component which consists of type I, III and V collagens, proteoglycans, fibronectin, and elastic fibrils for regeneration of tendon tissue (Güngormüs and Connect Tissue Res, 2008, 53 (6), 485-91); e) the cells do not substantially differentiate towards adipocyte lineage (e.g., adipocytes), or chondroblastic lineage (e.g., chondroblasts, chondrocytes), or osteoblast lineage (e.g., osteoblasts, osteocytes).
As known in the state of the art, cells of the synoviocyte lineage (synovial fluid) typically include synovial type A or macrophage-like cells, and type B or fibroblast-like synoviocytes (FLCs). , and may contribute to the formation of synovial membranes and synovial fluid. All these terms are well known per se. By the term "synoviocyte" as used herein is meant any, as well as collectively all, of such cell types. As additional and unrestricted guidance, the synoviocytes may have the following characteristics: a) the cells exhibit evidence of ability to secrete proteoglycan 4 (PRG4) and are the primary source of surfactant phospholipids (SAPL) as well as hyaluronan (HA) present in the synovial fluid (Tamer et al., Toxicol Disc, 2013, 6 (1), 111-125); b) Synovial type A or macrophage-like cells include expression of markers of hematopoietic origin including CD11b, CD86, CD14, CD163, DR antigen and Fc receptor. Type B or fibroblast-like synoviocytes are mesenchymal cells that exhibit numerous fibroblast characteristics, including expression of type IV and V collagens, vimentin, and CD90. In addition, type B cells have some unique in situ properties that distinguish them from many other fibroblast lineages, including subintima resident fibroblasts. For example, cadherin-11 (a specific adhesion molecule that plays a key role in FLS homotypic aggregation), CD55 (degradation acceleration factor), VCAM-1 (vascular adhesion molecule 1), and ICAM-1 (Intercellular Adhesion Molecule 1) (Bartok et al., Immunol Rev, 2011, 233 (1), 233-255); c) the cells do not substantially express CD45 (e.g., less than about 10%, preferably less than about 5%, more preferably less than about 2% of the cells can express CD45); d) the cells do not substantially differentiate to adipocyte lineage (e.g., adipocytes), or chondroblastic lineage (e.g., chondroblasts, chondrocytes), or osteoblast lineage (e.g., osteoblasts, osteocytes).
When a cell is said to be positive for (or to express or understand the expression of) a particular marker, it is meant that those skilled in the art will conclude the presence or indications of a distinct signal, for example, detectable by antibody or reverse transcription detection followed by a polymerase chain reaction, for this marker when carrying out the appropriate measurement, with respect to suitable controls. When the method allows the quantitative evaluation of the marker, the positive cells can generate on average a signal which is significantly different from the control, for example, but without limitation, at least 1.5 times greater than such a signal generated by control cells for example, at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more. The expression of the above cell-specific markers can be detected using any suitable immunological technique known in the art, such as immunocytochemistry or affinity adsorption, Western blot analysis, flow cytometry, ELISA, etc., or by any suitable biochemical assay for enzymatic activity (eg, for ALP), or any suitable technique for measuring the amount of marker mRNA, for example, a Northern blotting, semi-quantitative or quantitative RT-PCR, etc. The sequence data for the markers enumerated in this disclosure are known and can be obtained from public data store such as GenBank (http://www.ncbi.nlm.nih.gov/).
When a detection method allows the evaluation of marker expression at the single cell level (e.g., flow cytometry), and when said cells understand expression of the marker a large enough fraction of the cells tested may be positive for the label, for example, at least about 20%, at least about 40%, preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, still more preferably at least about 80%, still more preferably at least about 90%, and still more preferably at least about 95% and up to 100% of the cells tested may be positive for the marker.
In some embodiments of the methods as taught herein, the MSCs or MSC-derived cells in the composition may be animal cells, preferably warm-blooded animal cells, more preferably mammalian cells, such as human cells or non-human mammalian cells, and most preferably human cells.
MSCs or MSC-derived cells as provided herein are adherent, i.e. they require a surface for growth, and typically grow as an adherent monolayer on said surface (i.e. ie cultured adherent cells), rather than in the form of cells floating freely in a culture medium (suspension culture). Adhesion of cells to a surface, such as the surface of a plastic cell culture vessel, can be easily examined by visual inspection using an inverted microscope. Cultured cultured cells require periodic reseeding, where cells can be enzymatically removed from the surface (e.g., with trypsin), suspended in culture medium, and reseeded in one or more new culture vessels. In general, a surface or substrate that allows cell adhesion thereto may be any substantially hydrophilic substrate. As known in the state of the art, cell culture vessels, for example, culture flasks, microtiter plates, cans, or the like, can usually be made with a wide variety of polymeric materials. , suitably surface-treated or post-molded to provide hydrophilic substrate surfaces.
The methods as taught herein generally relate to the in vitro conservation of MSCs or cells derived from living (viable) MSCs. Preferably, the MSCs or MSC-derived cells in the composition are functional cells.
By the terms "living cells" or "viable cells" as used herein is meant cells that can be described as viable by tests known per se, and more particularly MSCs or MSC-derived cells that are capable of being viable. to divide and proliferate. When MSC or MSC-derived cells are said to be alive or viable, a fairly large fraction of the cells tested may be viable, for example, at least about 20%, at least about 40%, preferably at least about 50%. more preferably at least about 60%, still more preferably at least about 70%, still more preferably at least about 80%, still more preferably at least about 90%, and still more preferably at least about 95% and up to about 100%. % of the cells tested may prove viable.
The viability of cells can be measured using techniques known in the state of the art. Techniques for determining viability or survival of cells are usually referred to as viability assays. For example, cell viability can be measured by conventional dye exclusion assays, such as trypan blue exclusion assay or propidium iodide exclusion assay. In such assays, the viable cells exclude the dye and thus remain unstained, while the nonviable cells absorb the dye and are stained. The cells and their dye uptake can be visualized and revealed by a suitable technique (eg, conventional light microscopy, fluorescence microscopy, flow cytometry), and viable (non-stained) and nonviable (stained) cells in the sample tested can be counted. Cell survival can be conveniently expressed as the absolute number of living cells, or as cell viability (i.e., the ratio or proportion (%) of viable cells to total cells (this is ie sum of viable and non-viable cells)).
By the term &quot; functional cells &quot; as used herein is meant MSCs or MSC-derived cells that retain their natural cellular properties and are capable of performing their natural cellular functions, such as cellular recovery (e.g. ability of cells to adhere to a surface), cell identity (e.g., expression of key cell surface markers), and cell activity (e.g., bone formation properties). When said MSC or MSC-derived cells are functional, a fairly large fraction of the cells tested may have retained its natural cellular properties, for example, at least about 20%, at least about 40%, preferably at least about 50%. %, more preferably at least about 60%, still more preferably at least about 70%, still more preferably at least about 80%, still more preferably at least about 90%, and still more preferably at least about 95% and up to about 100% of the cells tested.
The functionality of the cells can be measured using techniques known in the state of the art. Techniques for determining cell functionality include assays for measuring known cellular functions, such as cell recovery, cell identity, and cell activity.
For example, cellular recovery can be measured by determining the number or fraction (%) of MSCs or MSC-derived cells that attach to a tissue culture plastic surface after a predetermined time (e.g., 24 hours) of culture. Adherent cells can be visualized by conventional light microscopy. Suitable staining methods, for example, Romanowsky dyes, can be used to improve cell visualization.
For example, cell identity can be determined by detecting the expression of key cell surface markers characterizing MSCs or MSC-derived cells. Ideally, expression of markers in MSCs or MSC-derived cells subjected to an in vitro preservation method as taught herein does not significantly differ from marker expression in MSCs or MSC derived cells. not subject to in vitro storage method (ie, controls). By way of example but without limitation, suitable cell surface markers for assessing the cellular identity of MSC may include CD105, CD90, CD73, and CD45. These cell surface markers may for example be detected by commercially available monoclonal antibodies, such as fluorochrome-labeled monoclonal antibodies for detection of cells by flow cytometry. In particular, CD105, CD90, and CD73 are mesenchymal markers, and are typically highly expressed by MSCs; CD45 is a hematopoietic marker, and is typically substantially absent from MSCs. By way of example but without limitation, suitable cell surface markers for assessing the cellular identity of MSC-derived osteoblastic cells may include CD105, CD90, CD73, CD45 and alkaline phosphatase (ALP), particularly ALP. bone-liver-kidney type. These cell surface markers may be detected for example by commercially available monoclonal antibodies, such as fluorochrome-labeled monoclonal antibodies for detection of cells by flow cytometry. In particular, CD105, CD90, and CD73 are mesenchymal markers, and are typically highly expressed by osteoblastic cells derived from MSC; CD45 is a hematopoietic marker, and is typically substantially absent from MSC derived osteoblastic cells; ALP is a marker of osteoblastic cells, and is typically expressed by a substantial fraction of osteoblastic cells derived from MSC.
For example, the cellular activity of MSC-derived osteoblastic cells can be determined by measuring the bone formation properties of such cells. The ability of osteoblastic cells derived from MSC to induce bone formation can be measured in vivo for example by evaluating the newly mineralized bone thickness after administration of cells to mice by subcutaneous injection into the cranial vault.
In some embodiments of the methods as taught herein, the concentration of MSCs or MSC-derived cells (preferably MSCs or cells derived from living MSCs, or preferably MSCs or cells derived from living and functional MSCs) in the composition. may be from about 1 × 10 4 to about 1 × 10 11, or from about 1 × 10 5 to about 1 × 10 10, or from about 1 × 10 6 to about 1 × 10 4 cells per ml of the composition. In some embodiments, the concentration of MSC or MSC-derived cells in the composition may range from about 1 × 10 7 to about 1 × 10 8 cells per ml of the composition. In some embodiments, the concentration of MSC or MSC-derived cells in the composition may range from about 2 × 10 7 to about 5 × 10 7 cells per ml of the composition. In some embodiments, the concentration of MSC or MSC-derived cells in the composition may range from about 2.5 to 107 cells per mL of the composition.
The term "plasma" is as defined conventionally. In some embodiments, the human plasma may be, or the source of human plasma may be, fresh plasma, lyophilized plasma, solvent / detergent treated plasma, fresh frozen plasma, thawed plasma, or a cryogenic precipitate cryogenic supernatant or a plasma concentrate as concentrated from frozen plasma, or a mixture of any two or more of these. Plasma is usually obtained from a sample obtained by apheresis or from a whole blood sample supplied or contacted with an anticoagulant (eg, heparin, citrate, oxalate or EDTA). Then, the cellular components of the blood sample are separated from the liquid component (plasma) by an appropriate technique, typically by centrifugation. As a specific but non-limiting example, in order to obtain plasma suitable for use in the present invention, a blood sample can be taken from a Vacutainer tube containing anticoagulant EDTA (ethylenediaminetetraacetic acid) (e.g. plastic BD Vacutainer EDTA, 10 ml, 1.8 mg / ml). The sample is gently stirred and then centrifuged for 10 min at room temperature at 1000-2000 g in order to separate the plasma from the erythrocytes. The supernatant (plasma) is recovered, possibly pooled (if a plurality of blood samples are used), and aliquoted into cryogenic tubes, which are stored at -80 ° C until use. The term "plasma" therefore refers to a composition devoid of the cellular components of the blood sample, which is not part of a human or animal organism. Therefore, the plasma as provided herein is a cell-free plasma, for example, the plasma comprises less than about 1.0% w / w, preferably less than about 0.5% w / w or less than 0, 1% w / w of whole cell material, or substantially no whole cell material.
The plasma may preferably be untreated plasma, i.e. plasma derived by separation from whole blood and not subject to downstream processing steps that alter its chemical, biochemical or cellular composition, other than optional thermal inactivation, storage (cryogenic or non-cryogenic), sterilization, filtration, lyophilization and / or solvent / detergent treatment.
The term "plasma" may in certain embodiments specifically exclude processed plasma, i.e., plasma subjected after separation from whole blood to one or more processing steps that alter its composition, specifically its chemical composition, biochemical or cellular. Preferably, the term "plasma" as provided herein specifically excludes platelet rich plasma (PRP), i.e., plasma having been enriched with platelets. Typically, the PRP may contain about 1.0 × 10 6 platelets, while the platelet concentration in whole blood may range from about 1.5 × 10 5 to 3.5 × 10 5 L. Therefore, the plasma as provided herein may contain less than about 8.0 * 105, preferably less than about 7.0 * 10 5, more preferably less than about 6.0 * 10 5, still more preferably less than about about 5.0 * 105, such as less than about 4.0 * 105 platelets.
Plasma as provided herein is human plasma, i.e. obtained from a single human subject or from a plurality of human subjects (e.g., mixed plasma pool).
Plasma can be used directly in the present methods. The plasma can also be stored appropriately for later use (for example, for shorter periods of time, for example, up to about 1-2 weeks, at a temperature above the plasma freezing points, but below at room temperature, this temperature usually ranging from about 4 ° C to 5 ° C, or for longer times by storage in the frozen state, usually between about -70 ° C and about -80 ° C).
The methods of the present invention may employ plasma that is autologous to the subject to be treated, and thus cells to be retained in the composition comprising the plasma. The term "autologous" with reference to plasma refers to the fact that the plasma is obtained from the same subject to be contacted or treated by the plasma. The methods of the present invention can employ plasma which is "homologous" or "allogeneic" with respect to the subject to be treated and therefore cells to be stored in the composition comprising the plasma, that is to say obtained at from one or more (combined) subjects other than the subject to be contacted or treated by the plasma. The methods of the present invention may also employ a mixture of autologous and homologous (allogeneic) plasma as defined above. Preferably, the methods of the present invention may employ a plasma that is "allogenic" to the subject to be treated. Advantageously, the allogeneic plasma is commercially available and therefore constitutes an unrestricted source of plasma. In particular, the present invention is directed to human plasma, more preferably allogeneic human plasma for in vitro preservation of MSC or cells derived from human MSCs.
The plasma can be thermally inactivated as known in the state of the art, in particular in order to eliminate the complement. When the present methods employ autologous plasma vis-à-vis the subject to be treated, it may not be necessary to thermally inactivate the plasma. When the plasma is at least partially allogeneic with respect to the subject to be treated, it may be advantageous to thermally inactivate the plasma.
In some embodiments, the plasma may be a solvent / detergent treated plasma. The terms "solvent / detergent treated plasma", "S / D treated plasma", or "S / D plasma" generally refer to a decellularized plasma (i.e. plasma without cellular blood components, acellular plasma ) obtained or obtainable by a method comprising the steps of (a) treating the plasma with a solvent and detergent and (b) filtering the solvent / detergent-treated plasma.
The plasma to be treated in step (a) may be any plasma as conventionally defined, such as fresh plasma, reconstituted freeze-dried plasma, fresh frozen plasma, thaw frozen plasma, or cryogenic precipitate, a cryogenic supernatant or concentrate from frozen plasma, as well as the dilution products thereof.
Solvents such as di- or trialkyl phosphates and detergents are described in US 4,764,369. The solvent used for the preparation of S / D plasma is preferably a dialkyl phosphate or a trialkyl phosphate, both having alkyl groups containing from 1 to 10 carbon atoms, especially from 2 to 10 carbon atoms. Illustrative examples of solvents may include tri- (n-butyl) phosphate, tri (t-butyl) phosphate, tri (n-hexyl) phosphate, tri (2-ethylhexyl) phosphate. , or tri- (n-decyl) phosphate. A preferred solvent is tri- (n-butyl) phosphate. Mixtures of different trialkyl phosphates may also be employed, as well as phosphates having alkyl groups of different alkyl chains, for example ethyl phosphate, di (n-butyl). Similarly, the respective dialkyl phosphates can be employed, including those of mixtures of different alkyl groups of dialkyl phosphate. In addition, mixtures of di- and trialkyl phosphates may be employed.
The solvent such as di- or trialkyl phosphate for use in the treatment step (a) is preferably employed in an amount of from about 0.01 mg / ml to about 100 mg / ml, and preferably from about 0.1 mg / ml to about 10 mg / ml. When stated differently, the di- or trialkyl phosphates for use in the treatment step (a) are preferably employed in an amount of from about 0.001% w / v to about 10% w / v, and preferably from about about 0.01% w / v to about 1% w / v.
The detergent used for S / D plasma preparation is preferably a non-toxic detergent. Nonionic detergents envisaged include those which disperse at the prevailing temperature at least 0.1% by weight of the fat in an aqueous solution containing it when 1 gram of detergent per 100 ml of solution is introduced therein. Illustrative examples of detergents may include polyoxyethylenated fatty acid derivatives, sorbitol anhydride partial esters, for example, products known commercially as "Tween 80" or "polysorbate 80" (polyoxyethylenated sorbitan monooleate). )), "Tween 20" or "polysorbate 20" (polyoxyethylenated (20) sorbitan monolaurate) and nonionic, water-soluble, aqueous detergents such as those sold under the tradename "Triton X-100" (oxyethylated alkylphenol, octylphenol ethoxylate) ). Sodium deoxycholate as well as "zwittergents", which are synthetic zwitterionic detergents known as "sulfobetaines" such as N-dodecyl-N, N-methyl-2-ammonio-1-ethane sulfonate and its congeners, are also contemplated. nonionic detergents such as octyl-beta-D-glucopyranoside.
The amount of detergent can range from about 0.001% v / v to about 10% v / v, preferably from about 0.01% v / v to 1.5% v / v.
The treatment with solvent and detergent is preferably carried out at a temperature of from -5 ° C to 70 ° C, preferably from 0 ° C to 60 ° C. The time for such treatment (contact) is at least 1 minute, preferably at least 1 hour, and generally ranges from 4 to 24 hours. The treatment is normally effective at atmospheric pressure, although subatmospheric and super-atmospheric pressures may also be employed.
Usually, after the treatment, the solvent such as trialkyl phosphate and detergent are removed. The solvent and the detergent can be removed by any technique suitable for separating the solvent and detergent from the plasma. When using a nonionic detergent with the solvent such as trialkyl phosphate, they can be removed by: (1) diafiltration using microporous membranes such as TEFLON which retain the plasma proteins; (2) absorption of desired plasma components on chromatographic or affinity chromatography supports; (3) precipitation, for example, by salting out the plasma proteins; (4) lyophilization, etc.
Solvents such as dialkyl phosphate or trialkyl phosphate can be removed as follows: (a) elimination of anti-hemophilic factor (AHF) can be accomplished by precipitation of AHF with 2.2M glycine and 2.0M Sodium Chloride (b) The removal of fibronectin can be performed by fixing the fibronectin on a gelatin column made insoluble and removing the fibronectin from the reagent by washing. The filtration step (b) is usually performed with a 1 μm filter to remove cells and debris, followed by sterile filtration using a 0.2 μm filter. As a preferred example, as described by Horowitz et al., 1992 (Blood, 3, 826-831), S / D plasma can be prepared as follows: fresh frozen plasma can be quickly thawed and can be processed with stirring for 4 hours with 1% (v / v) of tri- (N-butyl) (TNBP) phosphate and 1% (v / v) of polyoxyethylene-p-octylphenol (Triton X-100) at 30 ° C . After treatment, edible oil such as soybean oil (5% v / v) or castor oil can be added, gently mixed for 30 minutes, and can be removed by centrifugation at 10,000 g. for 20 minutes. The clarified plasma can be applied to a Waters Prep C18 resin column so that the ratio of plasma to column volume is 6 and the contact time can be 3 minutes. The column eluate can be filtered on a 0.2 μm filter.
The term "S / D plasma" encompasses plasma comprising a reduced concentration or activity of Plasmine inhibitor, such as a Plasmin inhibitor level equal to or less than 0.60 IU / ml or equal to or less than 0, 50 IU / ml, for example a level of Plasmine inhibitor of between 0.20 and 0.30 IU / ml, more specifically between 0.22 and 0.25 IU / ml.
In comparison with fresh frozen plasma, the S / D plasma may comprise a reduced amount and / or activity of one or more of a plasmin inhibitor, protein S, Factor XI, Factor V, Factor VIII, Factor X, α 2 anti-plasmin, antitrypsin, von Willebrand factor (vWF), and von Willebrand factor cleavage protease (VWFCP) also known as disintegrin and a metalloproteinase having a thrombospondin type 1 motif , member 13 (ADAMTS-13), tumor necrosis factor alpha (TNFα), interleukin-8 (IL-8), interleukin-10 (IL-10) (Benjamin and McLaughlin, 2012, Svae et al. 2007, Beeck and Hellstern 1998, Doyle et al., 2003, Mast et al., 1999, Theusinger et al., 2011) and / or may include an increased amount and / or activity of Factor VII (Doyle et al. ., 2003). Overall, treatment with S / D may induce a reduction in pro-coagulant factors, although their rates remain within the normal range.
For example, S / D plasma is commercially available as Octaplas® (Octapharma AG, Lachen, Switzerland).
The term "serum" is as conventionally defined and includes fresh serum, thawed frozen serum or serum prepared from plasma, or a mixture of any two or more of these. The serum can usually be obtained from a whole blood sample by first allowing coagulation to be produced in the sample and then separating the clot thus formed and the cellular components of the blood sample from the component liquid (serum) by an appropriate technique, typically by centrifugation. Coagulation can be facilitated by an inert catalyst, for example, beads or glass powder. Alternatively, the serum can be obtained from plasma by removing anticoagulant and fibrin. As a specific but non-limiting example, in order to obtain serum suitable for use in the present invention, a blood sample may be taken from a Vacutainer tube containing no anticoagulant (eg BD Vacutainer serum plastic tube). Plus, 10 ml) and incubated for 30-45 min at room temperature to allow coagulation. The tube is then centrifuged for 15 min at room temperature at 1000-2000 g to separate the serum from the erythrocytes. The supernatant (serum) is recovered, possibly pooled (if a plurality of blood samples are used), and aliquoted into cryogenic tubes, which are stored at -80 ° C until use. The term "serum" therefore refers to an acellular composition that is not part of a human or animal organism.
The serum as provided herein is human serum, i.e. obtained from a single human subject or from a plurality of human subjects (e.g., mixed serum pool).
The serum may be preferably untreated serum, i.e., serum derived by separation from whole blood and not subjected to downstream processing steps that alter its chemical, biochemical or cellular composition, other than optional thermal inactivation, storage (cryogenic or non-cryogenic), sterilization, filtration, lyophilization and / or filtration.
The term "serum" may in certain embodiments specifically exclude treated serum, i.e. serum subjected after separation from whole blood to one or more treatment steps that alter its composition, specifically its chemical composition, biochemical or cellular.
The serum can be used directly in the methods as taught here. The serum can also be stored appropriately for later use (eg, for shorter periods of time, for example, up to about 1-2 weeks, at a temperature above the freezing point of the serum, but below at room temperature, this temperature usually ranging from about 4 ° C to 5 ° C, or for longer times by storage in the frozen state, usually between about -16 ° C and about -24 ° C, or between about -70 ° C and about -80 ° C). Optionally, the serum may also be sterilized prior to storage or use, using conventional microbiological filters, preferably having a pore size of 0.2 μm or less.
In some embodiments, the methods may employ serum that is autologous to the subject to be treated, and thus cells to be retained in the composition comprising the serum. The term "autologous" with reference to serum means that the serum is obtained from the same subject as that to be brought into contact with the serum. In some embodiments, the methods may employ serum that is "homologous" or "allogeneic" to the subject to be treated, and thus cells to be retained in the composition comprising the serum, i.e. say obtained from one or more (combined) subjects other than the subject to be put in contact with the serum. In some embodiments, the methods may employ a mixture of autologous and homologous (allogeneic) sera as defined above. Specifically, human serum, more preferably allogenic human serum for the in vitro preservation of MSC or cells derived from human MSCs is particularly contemplated.
The serum may be thermally inactivated as known in the state of the art, in particular for eliminating complement. When the present methods employ autologous serum to cells grown in the presence thereof, it may not be necessary to thermally inactivate the serum. When the serum is at least partially allogeneic to cultured cells, it may be advantageous to thermally inactivate the serum.
In some embodiments, the serum can be obtained from solvent / detergent treated plasma. S / D plasma can be conveniently treated to counteract the action of the anticoagulant, such as to allow conversion of fibrinogen to fibrin and clot formation. For example, when the anticoagulant sequesters calcium ions (Ca 2+), such as EDTA, citrate or oxalate, the addition of a sufficient amount of a source of divalent calcium ions (Ca 2+) may be necessary. Suitable sources of divalent calcium ions (Ca 2+) include the pharmaceutically acceptable calcium salt (s), preferably the soluble calcium salt (s). Such Ca2 + salts can be formed with inorganic or organic acids. Examples of such salts include calcium chloride (CaCl 2), calcium glycerophosphate, calcium phosphate, calcium hydrogencarbonate, calcium citrate, calcium sulfate, calcium lactate, calcium gluconate, calcium ascorbate, and mixtures thereof. CaCl 2, which advantageously has good solubility and is well tolerated in injectable solutions, may be particularly preferred.
The S / D plasma thus treated can be incubated under conditions leading to clot formation, for example but without limitation, incubated for about 1 hour at 37 ° C, then for a minimum of 12 hours at -20 ° C, followed by defrosting, for example, at 37 ° C. Then the serum is separated from the clot in a convenient manner, for example, taken with the aid of a syringe. Optionally, prior to treatment of the S / D plasma to obtain the serum as taught above, an additional amount (e.g., between about 5% v / v and about 20% v / v, preferably about 10% v / v). v) Serum, preferably human serum, more preferably autologous or allogeneic serum, may be mixed with S / D plasma to facilitate coagulation.
In some particularly preferred embodiments of the methods or uses as taught herein, the composition may be further supplemented (enriched) with one or more fractionated plasma or serum components. In particular, an additional amount (surplus, extra) of one or more fractionated plasma or serum components may be included in the composition. A particularly preferred example of such a fractionated plasma or serum component for inclusion in the composition is human serum albumin (HSA). By way of example but without limitation, the composition may be supplemented (enriched) by at least about 1% w / v (based on composition volume) of HSA (i.e. minus about 1% w / v of HSA in addition to any HSA present in plasma or serum), such as at least about 5% w / v, or at least about 10% w / v, at least about 15% p / v, at least about 20% w / v, at least about 30% w / v, at least about 40% w / v, or at least about 50% w / v HSA. Native versions (isolated from human blood) as well as recombinant HSA with purity of up to 95% or 99% or more are commercially available.
The concentration of plasma or serum or of the mixture thereof in the composition (i.e., based on the volume of the composition) is at least 20% v / v, such as at least 25% v / v, at least 30% v / v, at least 35% v / v, at least 40% v / v, or at least 45% v / v. In some embodiments of the methods as taught herein, the concentration of plasma or serum or mixture thereof in the composition may be at least 50% v / v, such as at least 55% by weight. % v / v. In some embodiments, the concentration of plasma or serum or mixture thereof in the composition may be at least 60% v / v, such as at least 65% v / v. In some embodiments, the concentration of plasma or serum or mixture thereof in the composition may be at least 70% v / v, such as at least 75% v / v. In some embodiments, the concentration of plasma or serum or mixture thereof in the composition may be at least 80% v / v, such as at least 85% v / v. In some embodiments, the concentration of plasma or serum or mixture thereof in the composition may be at least 90% v / v. In some embodiments, the concentration of plasma or serum or mixture thereof in the composition may be at least 99-100% v / v.
In some embodiments, an undiluted and unconcentrated plasma product (e.g., fresh plasma, reconstituted lyophilized plasma, solvent / detergent treated plasma, fresh frozen plasma, and / or thawed plasma, or mixture thereof) may be included in the composition in a concentration of at least 20% v / v plasma, or an undiluted and unconcentrated serum product (e.g., fresh serum, thawed frozen serum, or serum prepared from plasma, or a mixture thereof) may be included in the composition at a concentration of at least 20% v / v of the serum, or a mixture of the undiluted and unconcentrated plasma product and undiluted and unconcentrated serum product may be included in the composition in a concentration of at least 20% v / v of the plasma and serum mixture.
In other embodiments, diluted plasma and / or serum products may instead be employed. Such diluted plasma and / or serum products are included in the composition at a concentration higher than the desired concentration of plasma and / or serum, so as to compensate (counterbalance, compensate for) the dilution factor, leading to thus to a composition equivalent to that obtained by inclusion of at least 20% v / v of plasma product and / or undiluted serum.
In still other embodiments, concentrated plasma and / or serum products (e.g., plasma concentrates such as frozen plasma concentrates) may be employed. Such concentrated plasma and / or serum products may be included in the composition at a concentration lower than the concentration of plasma and / or serum desired, so as to compensate (counterbalance, compensate for) the concentration factor, thus leading to a composition equivalent to that obtained by inclusion of at least 20% v / v of plasma product and / or unconcentrated serum. Alternatively, such concentrated plasma and / or serum products can be included in the composition in a concentration of at least 20% v / v, thus resulting in a composition equivalent to that obtained by inclusion of more than 20% v / v (depending on the concentration factor) plasma product and / or unconcentrated serum.
When the composition comprises a mixture of plasma and serum, examples of concentrations of plasma and serum in the mixture (i.e., plasma and serum constituting 100% v / v of the mixture, and the mixture constituting at least 20% v / v of the composition) may be, for example, 1-10% v / v plasma and 90-99% v / v serum (for example, 1% v / v plasma and 99% v / v serum, or 10% v / v plasma and 90% v / v serum), or 11-20% v / v plasma and 80-89% v / v serum (e.g. 11% v / v plasma and 89% v / v serum, or 20% v / v plasma and 80% v / v serum), or 21-30% v / v plasma and 70-79% v serum (e.g., 21% v / v plasma and 79% v / v serum, or 30% v / v plasma and 70% v / v serum), or 31-40% v / v plasma and 60-69% v / v serum (for example, 31% v / v plasma and 69% v / v serum, or 40% v / v plasma and 60% v / v serum), or 41-50% v / v plasma and 50-59% v / v serum (for example, 41% v / v plasma and 59% v / v serum, or 50% v / v v plasma and 50% v / v serum), or 51-60% v / v plasma and 40-49% v / v serum (eg 51% v / v plasma and 49% v / v serum, or 60% v / v plasma and 40% v / v serum), or 61-70% v / v plasma and 30-39% v / v serum (eg, 61% v / v plasma and 39% v / v serum, or 70% v / v plasma and 30% v / v serum), or 71-80% v / v plasma and 20-29% v / v serum ( for example, 71% v / v plasma and 29% v / v serum, or 80% v / v plasma and 20% v / v serum), or 81-90% v / v plasma and 10- 19% v / v serum (eg, 81% v / v plasma and 19% v / v serum, or 90% v / v plasma and 10% v / v serum), or 91-99% v / v plasma and 1-9% v / v serum (e.g. 91% v / v plasma and 9% v / v serum, or 99% v / v plasma and 1% v / v serum).
In some embodiments of the methods, as taught herein, the concentration of MSC or MSC-derived cells in the composition, and the concentration of plasma or serum or mixture thereof in the composition, may be such that as illustrated in Table 1 (each row of Table 1 providing an example of such an embodiment.
Table 1. Examples of embodiments of the compositions used in the present methods
In some embodiments of the methods, as taught herein, the composition may consist essentially or may consist of i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture of those -this.
Preferably, the invention provides a method for non-cryogenic in vitro storage of cells. Therefore, in some embodiments, the composition may be maintained (at a temperature) above the freezing point of the composition. In some embodiments of the methods as taught herein, the composition may be maintained at or above about 0 ° C, or at or above about 0.1 ° C (e.g. 0.10 ° C), or at or above about 1 ° C. In some embodiments, the composition may be maintained at or above about 2 ° C, for example, at or above about 3 ° C, or at or above about 4 ° C , or at or above about 5 ° C, or at or above about 6 ° C, or at or above about 7 ° C. In some embodiments, the composition may be maintained at or above about 8 ° C, for example, at or above about 9 ° C. In some embodiments, the composition may be maintained at or above about 10 ° C, for example, at or above about 11 ° C, or at or above about 12 ° C , or at or above about 13 ° C, or at or above about 14 ° C. In some embodiments, the composition may be maintained at or above about 15 ° C, for example, at or above about 16 ° C, or at or above about 17 ° C , or at or above about 18 ° C, or at or above about 19 ° C. In some embodiments, the composition may be maintained at or above about 20 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained at or below room temperature (i.e., "room temperature"). In some embodiments of the methods as taught herein, the composition may be maintained at or below about 25 ° C, for example, at or below about 24 ° C, or at or below at about 23 ° C, or at or below about 22 ° C, or at or below about 21 ° C. In some embodiments, the composition may be maintained at or below about 20 ° C, for example, at or below about 19 ° C. In some embodiments, the composition may be maintained at or below about 18 ° C, for example, at or below about 17 ° C, or at or below about 16 ° C. In some embodiments, the composition may be maintained at or below about 15 ° C, for example, at or below about 14 ° C, or at or below about 13 ° C, or at about or below about 12 ° C, or at or below about 11 ° C. In some embodiments, the composition may be maintained at or below about 10 ° C, for example, at or below about 9 ° C. In some embodiments, the composition may be maintained at or below about 8 ° C, for example, at or below about 7 ° C, or at or below about 6 ° C, or at about or below about 5 ° C, or at or below about 4 ° C, or at or below about 3 ° C, or at or below about 2 ° C, or at or below below about 1 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained at about 0 ° C to room temperature. In some embodiments of the methods as taught herein, the composition may be maintained at about 0 ° C to about 25 ° C, or at about 0 ° C to about 18 ° C, or at about 0 ° C to about 0 ° C. 15 ° C. In some embodiments, the composition may be maintained at about 0 ° C to about 10 ° C. In some embodiments, the composition may be maintained at about 0 ° C to about 8 ° C. In some embodiments, the composition may be maintained at about 1 ° C to about 8 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to room temperature. In some embodiments of the methods as taught herein, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C, or about 0.1 ° C C (e.g., about 0.10 ° C) and about 18 ° C, or about 0.1 ° C (e.g., about 0.10 ° C) to about 15 ° C. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 10 ° C. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 8 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained above the freezing point of the composition and at or below room temperature (i.e. the room "). In some embodiments of the methods as taught herein, the composition may be maintained above the freezing point of the composition and at or below about 25 ° C, or above the freezing point of the composition. the composition and at or below about 20 ° C, or above the freezing point of the composition and at or below about 18 ° C, or above the freezing point of the composition and at or below about 15 ° C. In some embodiments, the composition may be maintained above the freezing point of the composition and at or below about 10 ° C. In some embodiments, the composition may be maintained above the freezing point of the composition and at or below about 8 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained at about 0 ° C to room temperature. In some embodiments of the methods, as taught herein, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature, or between about 1 ° C and about room temperature, or between about 2 ° C and room temperature. In some embodiments, the composition may be maintained at about 8 ° C to room temperature. In some embodiments, the composition may be maintained at about 15 ° C to room temperature.
In some embodiments of the methods as taught herein, the composition may be maintained at about 2 ° C to about 8 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained at about 8 ° C to about 15 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained at about 15 ° C to about 25 ° C.
In some embodiments of the methods as taught herein, the composition may be maintained for at least 8 hours, preferably at least 16 hours, more preferably at least 24 hours. In some embodiments, the composition may be maintained for at least 30 hours. In some embodiments, the composition may be maintained for at least 36 hours. In some embodiments of the methods as taught herein, the composition may be maintained for at least 42 hours.
In some embodiments of the methods as taught herein, the composition may be maintained for at least 48 hours. In some embodiments of the methods as taught herein, the composition may be maintained for at least 72 hours. In some embodiments, the composition may be maintained for at least 96 hours. In some embodiments, the composition may be maintained for at least 120 hours. In some embodiments, the composition may be maintained for at least 144 hours. In some embodiments, the composition may be maintained for at least 192 hours.
In some embodiments of the methods as taught herein, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 48 hours. In some embodiments of the methods as taught herein, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 48 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to room temperature for at least 72 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 72 hours. In some embodiments, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 96 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 96 hours. In some embodiments, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 120 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 120 hours. In some embodiments, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 144 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 144 hours. In some embodiments, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 168 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 168 hours. In some embodiments, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 192 hours. In some embodiments, the composition may be maintained at about 0.1 ° C (eg, about 0.10 ° C) to about 25 ° C for at least 192 hours.
In some embodiments of the methods as taught herein, the composition may be maintained at or below ambient temperature for at least 48 hours, or for at least 72 hours, or for at least 96 hours, or at least minus 120 hours. In some embodiments, the composition may be maintained at or below room temperature for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments of the methods, as taught herein, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 48 hours, or at least 72 hours, or for at least 96 hours, or for at least 120 hours. In some embodiments, the composition may be maintained between about 0.1 ° C (eg, about 0.10 ° C) and room temperature for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments of the methods as taught herein, the composition may be maintained at about 2 ° C to about 8 ° C for at least 48 hours. In some embodiments, the composition may be maintained at about 2 ° C to about 8 ° C for at least 72 hours. In some embodiments, the composition may be maintained at about 2 ° C to about 8 ° C for at least 96 hours. In some embodiments, the composition may be maintained at about 2 ° C to about 8 ° C for at least 120 hours. In some embodiments, the composition may be maintained at about 2 ° C to about 8 ° C for at least 144 hours. In some embodiments, the composition may be maintained at about 2 ° C to about 8 ° C for at least 168 hours. In some embodiments, the composition may be maintained at about 2 ° C to about 8 ° C for at least 192 hours.
In some embodiments of the methods as taught herein, the composition may be maintained at about 8 ° C to about 15 ° C for at least 48 hours. In some embodiments, the composition may be maintained at about 8 ° C to about 15 ° C for at least 72 hours. In some embodiments, the composition may be maintained at about 8 ° C to about 15 ° C for at least 96 hours. In some embodiments, the composition may be maintained at about 8 ° C to about 15 ° C for at least 120 hours. In some embodiments, the composition may be maintained at about 8 ° C to about 15 ° C for at least 144 hours. In some embodiments, the composition may be maintained at about 8 ° C to about 15 ° C for at least 168 hours. In some embodiments, the composition may be maintained at about 8 ° C to about 15 ° C for at least 192 hours.
In some embodiments of the methods as taught herein, the composition may be maintained at about 15 ° C to room temperature for at least 48 hours. In some embodiments of the methods as taught herein, the composition may be maintained at about 15 ° C to about 25 ° C for at least 48 hours. In some embodiments, the composition may be maintained at about 15 ° C to room temperature for at least 72 hours. In some embodiments, the composition may be maintained at about 15 ° C to about 25 ° C for at least 72 hours. In some embodiments, the composition may be maintained at about 15 ° C to room temperature for at least 96 hours. In some embodiments, the composition may be maintained at about 15 ° C to about 25 ° C for at least 96 hours. In some embodiments, the composition may be maintained between about 15 ° C and room temperature for at least 120 hours. In some embodiments, the composition may be maintained at about 15 ° C to about 25 ° C for at least 120 hours. In some embodiments, the composition may be maintained at about 15 ° C to room temperature for at least 144 hours. In some embodiments, the composition may be maintained at about 15 ° C to about 25 ° C for at least 144 hours. In some embodiments, the composition may be maintained at about 15 ° C to room temperature for at least 168 hours. In some embodiments, the composition may be maintained at about 15 ° C to about 25 ° C for at least 168 hours. In some embodiments, the composition may be maintained at about 15 ° C to room temperature for at least 192 hours. In some embodiments, the composition may be maintained at about 15 ° C to about 25 ° C for at least 192 hours.
The temperature as used in the context of the present invention is expressed in degrees Celsius (° C). It should be noted, however, that the temperature can also be expressed in any other suitable unit such as Kelvin (K).
Note that the temperature is rounded to the nearest unit. For example, 8.1 ° C, 8.2 ° C, 8.3 ° C, and 8.4 ° C is rounded down to 8 ° C, while 8.5 ° C, 8.6 ° C , 8.7 ° C, 8.8 ° C, and 8.9 ° C is rounded up to 9 ° C. A reference to a temperature value not followed by any decimal also also includes in particular the same value followed by 0 to the first decimal place, for example, a reference at 8 ° C in particular encompasses 8.0 ° C.
The time, as used in the context of the present invention, is expressed in hours or days. However, it should be noted that the time can also be expressed in any other unit such as seconds or minutes.
We note that the time in hours is rounded to the nearest unit. For example, 24.1 hours (24 hours 6 minutes), 24.2 hours (24 hours 12 minutes), 24.3 hours (24 hours 18 minutes), 24.4 hours (24 hours 24 minutes) is rounded to the Low to 24 hours, while 24.5 hours (24 hours 30 minutes), 24.6 hours (24 hours 36 minutes), 24.7 hours (24 hours 42 minutes), 24.8 hours (24 hours 48 minutes) , and 24.9 hours (24 hours 54 minutes) is rounded up to 25 hours. A reference to a period of time not followed by any decimal also includes in particular the same value followed by 0 to the first decimal place, for example, a reference to 24 hours includes in particular 24.0 hours.
The terms "time", "period of time" and "duration of time" can be used interchangeably.
The terms "24 hours" and "1 day" can be used interchangeably. The terms "48 hours" and "2 days" may be used interchangeably. The terms "72 hours" and "3 days" can be used interchangeably. The terms "96 hours" and "4 days" can be used interchangeably. The terms "120 hours" and "5 days" can be used interchangeably. The terms "144 hours" and "6 days" can be used interchangeably. The terms "168 hours" and "7 days" can be used interchangeably. The terms "192 hours" and "8 days" can be used interchangeably.
In some embodiments of the methods, as taught herein, the composition may be maintained at a temperature as described in Table 2 and for a period of time as described in Table 2. In some embodiments, the The composition can be maintained at a temperature (Tx) as described in Table 2 and for a period of time (tx) as described in Table 2. In some embodiments of the methods, as taught herein, the composition may be maintained at any of the temperatures T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, T13, T14, T15, T16, T17, T18, T12 T20 , T21 or T22 as described in Table 2 and during any of the time periods t1, t2, t3, t4, t5, t6, t7, t8, t9, t10, t11, or t12 as described in For example, the composition can be maintained at a temperature T1 as described in FIG. in Table 2 and for a period of time t1 as described in Table 2.
By the expression "at a temperature Tx as described in Table 2 and for a period of time tx as described in Table 2" is meant any one or more combinations, such as all combinations, of temperatures and periods of time as described in Table 2, and in particular the combinations specified in each field of Table 2 by the expression "Tx + tx".
Table 2: Combinations of a temperature and a period of time for maintaining the compositions as taught here
As mentioned above, the present invention relates to a method of in vitro preservation of cells comprising the maintenance of adherent MSCs or adherent cells derived from MSC in suspension in a composition comprising at least 20% v / v of human plasma or human serum or a mixture of these. In particular, the composition can thus be maintained at a temperature and for a time as discussed above.
By the term "maintain a composition between a given temperature (such as about 0 ° C) and another given temperature (such as about 25 ° C) for at least a given period of time (such as 24 hours) As used herein, it is intended to ensure that the temperature of the composition remains (i.e., is maintained, kept or controlled) between said temperatures for at least the period of time mentioned.
In order to control the temperature of the composition, the composition may be conveniently exposed to an environment having the desired temperature, for example, a temperature between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and the ambient temperature or a temperature between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and about 25 ° C, for a period of time desired.
In some embodiments, the composition may be exposed to an environment having a temperature between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) to about 18 ° C, between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and about 15 ° C, about 0 ° C (for example, about 0.1 ° C, for example, about 0.10 ° C) and about 12 ° C, between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) to about 10 ° C, about 0 ° C (e.g., about 0.1 ° C, for example, about 0.10 ° C) and about 8 ° C, temperature between about 1 ° C and about 18 ° C, between about 1 ° C and about 15 ° C, between about 1 ° C and about 12 ° C, between about 1 ° C and about 10 ° C, between about 1 ° C and about 8 ° C, temperature between about 2 ° C and about 18 ° C, between about 2 ° C and about 15 ° C, between about 2 ° C and about 12 ° C, between about 2 ° C and about 10 ° C, or between about 2 ° C and about 8 ° C, or e at about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and about 25 ° C, or about 15 ° C to about 25 ° C, or about 8 ° C and about 15 ° C, or at or below room temperature, or above the freezing point of the composition and at or below ambient temperature, or between about 0 ° C (eg, about 0, 1 ° C, for example, about 0.10 ° C) and ambient temperature, or between about 15 ° C and room temperature, for a period of time effective to maintain the Tx composition as described in Table 2 and during tx as described in Table 2.
In some embodiments, the composition may be exposed to an environment having a temperature between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and ambient temperature, for a period of time. an effective time period for maintaining the composition between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and room temperature for at least 24 hours, or for at least 30 hours , or for not less than 36 hours, or for not less than 42 hours, or for not less than 48 hours, or for not less than 72 hours, or for not less than 84 hours, or for not less than 96 hours, or for not less than 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 2 ° C and about 8 ° C for a period of time effective to maintain the composition between about 2 ° C and about 8 ° C for a period of time. at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 15 ° C and room temperature for a period of time effective to maintain the composition between about 15 ° C and room temperature for at least 24 hours. hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 15 ° C and about 25 ° C for a period of time effective to maintain the composition between about 15 ° C and about 25 ° C for a period of time. at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 8 ° C and about 15 ° C for a period of time effective to maintain the composition between about 8 ° C and about 15 ° C for a period of time. at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and ambient temperature, for a period of time. effective time period to maintain the composition between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and room temperature for at least 24 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 2 ° C and about 8 ° C for a period of time effective to maintain the composition between about 2 ° C and about 8 ° C for a period of time. less 24 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 15 ° C and room temperature for a period of time effective to maintain the composition between about 15 ° C and room temperature for at least 24 hours. hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 15 ° C and about 25 ° C for a period of time effective to maintain the composition between about 15 ° C and about 25 ° C for a period of time. less 24 hours.
In some embodiments, the composition may be exposed to an environment having a temperature between about 8 ° C and about 15 ° C for a period of time effective to maintain the composition between about 8 ° C and about 15 ° C for a period of time. less 24 hours. The environment having a temperature as mentioned above may be conveniently created or generated in any way, for example, any conventional means of controlling temperature. For example, temperatures in the range of about 20 ° C to about 25 ° C correspond to ambient temperature conditions (i.e. "room temperature"), so that no means Dedicated temperature control is only required in order to maintain the composition at such temperatures. In order to maintain the composition at temperatures below room temperature, any conventional means of refrigeration may be employed, such as, for example, ordinary household refrigerators, industrial refrigerators, temperature controlled refrigerators, transportable cooling systems ( for example, cooler), etc.
It should be understood that during a desired period of time during which the temperature of the composition is maintained within a desired range, the composition may be transiently exposed to an environment having a temperature outside said range. For example, the composition may be transiently removed from a refrigerated environment for inspection, handling, packaging, labeling, etc. ; or the composition may be briefly removed from a refrigerated environment and placed in another refrigerated environment. The transient nature of such exposure, however, means that the temperature of the composition remains within the desired range.
Therefore, in some embodiments, provided that the temperature of the composition is maintained between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and the ambient temperature for at least 24 hours, the composition may be exposed to an environment having a temperature of less than about 0 ° C or greater than ambient temperature for one or more periods of time during said at least 24 hours.
In some embodiments, provided that the temperature of the composition is maintained between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) to about 25 ° C for at least 24 hours, the composition may be exposed to an environment having a temperature of less than about 0 ° C or greater than about 25 ° C for one or more periods of time during said at least 24 hours.
In some embodiments, provided that the temperature of the composition is maintained at the temperature Tx as described in Table 2 and during the time period tx as described in Table 2, the composition may be exposed to an environment. having a temperature of less than or greater than Tx (eg ambient temperature) for one or more periods of time during said time period tx.
In some embodiments, provided that the temperature of the composition is maintained between about 2 ° C and about 8 ° C for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or hours, or for at least 192 hours, the composition may be exposed to an environment having a temperature of less than about 2 ° C or greater than about 8 ° C (eg, room temperature) for one or more periods of time during said at least 24, 30, 36, 42, 48, 72, 84, 96, 120, 144, 168, or 192 hours.
In some embodiments, provided that the temperature of the composition is maintained between about 2 ° C and about 8 ° C for at least 24 hours, the composition may be exposed to an environment having a temperature below about 2 ° C and or greater than about 8 ° C (eg, room temperature) for one or more periods of time during said at least 24 hours.
The methods as taught herein are particularly suitable for in vitro preservation of MSCs or MSC-derived cells, such as during storage of the composition and / or transport of the composition, for example from the place of production of the composition ( for example, a cell therapy company) instead of using the composition (for example, a hospital). Preferably, when the MSCs or MSC-derived cells are maintained as taught herein, less than 20%, more preferably less than 15%, still more preferably less than 10% of the cells may lose their viability relative to the composition. beginning of the method.
The present methods for in vitro preservation of MSC or MSC-derived cells thus make it possible to adequately maintain the viability of MSCs or MSC-derived cells.
In some embodiments, the method may comprise: a) providing the composition comprising (i) MSCs or MSC-derived cells and (ii) human plasma or human serum or a mixture thereof, optionally wherein said providing comprises a1) culturing MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition so as to effect cell preservation, for example, maintaining the composition at about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C). C) and the ambient temperature for at least 24 hours or maintaining the composition between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and about 25 ° C for less 24 hours.
In some embodiments, the method may comprise: a) providing the composition comprising (i) MSCs or MSC derived cells and (ii) human plasma or human serum or a mixture thereof, wherein said supply comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition so as to effect cell preservation, for example, maintaining the composition at about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C). C) and the ambient temperature for at least 24 hours or maintaining the composition between about 0 ° C (eg, about 0.1 ° C, for example, about 0.10 ° C) and about 25 ° C for less 24 hours.
In some embodiments, the methods as taught herein may relate to culture (eg, maintenance, propagation and / or differentiation) of MSC or MSC-derived cells in the presence of cell culture medium or tissue as known per se, such as for example using a liquid culture medium of cells or tissues. Such culture medium may desirably support maintenance (eg, survival, genotypic, phenotypic, and / or functional stability) and propagation of MSCs or MSC-derived cells.
General techniques of cell culture and uses of media are set forth inter alia in "Large Scale Mammalian Cell Culture" (Hu et al., 1997, Curr Opin Biotechnol, 8, 148); "Serum-free Media" (K. Kitano, 1991, Biotechnology, 17, 73); or "Large Scale Mammalian Cell Culture" (Curr Opin Biotechnol, 1991, 2, 375).
In some embodiments, the method may comprise: a) providing the composition comprising (i) MSCs or MSC-derived cells and (ii) human plasma or human serum or a mixture thereof, optionally wherein said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the Tx composition as described in Table 2 and during tx as described in Table 2.
In some embodiments, the method may comprise: a) providing the composition comprising i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture thereof, optionally where said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition between about 2 ° C and about 8 ° C for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours; hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours .
In some embodiments, the method may comprise: a) providing the composition comprising i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture thereof, optionally where said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition between about 15 ° C and room temperature for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
In some embodiments, the method may comprise: a) providing the composition comprising i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture thereof, optionally where said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition between about 15 ° C and about 25 ° C for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours; hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours .
In some embodiments, the method may comprise: a) providing the composition comprising i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture thereof, optionally where said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition at about 2 ° C to about 8 ° C for at least 24 hours.
In some embodiments, the method may comprise: a) providing the composition comprising i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture thereof, optionally where said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition between about 15 ° C and room temperature for at least 24 hours.
In some embodiments, the method may comprise: a) providing the composition comprising i) MSCs or MSC-derived cells and ii) human plasma or human serum or a mixture thereof, optionally where said providing comprises a1) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition at about 15 ° C to about 25 ° C for at least 24 hours.
MSCs or cells derived from MSC are suspended in the composition.
The terms "suspension" and "cell suspension" generally refer to a heterogeneous mixture containing MSCs or cells derived from MSCs dispersed in a liquid phase. Since the composition is liquid, the MSCs or MSC-derived cells can in principle be capable of, but do not need to, deposit or sediment from the composition, even when the composition is maintained at temperatures and time periods. discussed above.
In some embodiments of the methods, as taught herein, the composition may preferably remain liquid (i.e., in the liquid state) throughout the said maintenance of the composition.
The term "liquid" is well appreciated in the state of the art as referring to a state of matter having a defined volume but a non-fixed form. In particular, in such embodiments, the composition does not become solid, semi-solid, or gel-like, i.e. does not gel / gellify.
In some embodiments, the composition may remain liquid (i.e., in a liquid state as recited above) throughout said maintenance of the composition at about 0 ° C (e.g., about 0 ° C). , 1 ° C, for example, about 0.10 ° C) and room temperature for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours hours. In some embodiments, the composition may remain liquid (i.e., in a liquid state) throughout said maintenance of the composition at about 0 ° C (eg, about 0.1 ° C, for example). for example, about 0.10 ° C) and room temperature for at least 24 hours.
In some embodiments, the composition may remain liquid (i.e., in a liquid state as recited above) throughout said maintenance of the composition at about 0 ° C (e.g., about 0 ° C). , 1 ° C, for example, about 0.10 ° C) and about 25 ° C for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours. In some embodiments, the composition may remain liquid (i.e., in a liquid state) throughout said maintenance of the composition at about 0 ° C (eg, about 0.1 ° C, for example). for example, about 0.10 ° C) and about 25 ° C for at least 24 hours.
In some embodiments, the composition may remain liquid (i.e., in a liquid state as recited above) throughout said maintenance of the Tx composition as described in Table 2 and during tx as described in Table 2.
In some embodiments, the composition may remain liquid (i.e., in a liquid state as recited above) throughout said maintenance of the composition at about 2 ° C to about 8 ° C at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours. In some embodiments, the composition may remain liquid (i.e., in a liquid state) throughout said maintenance of the composition at about 2 ° C to about 8 ° C for at least 24 hours.
In some embodiments, the composition may remain liquid (i.e., in a liquid state as recited above) throughout said maintenance of the composition at about 15 ° C and at room temperature for a period of time. at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours. In some embodiments, the composition may remain liquid (i.e., in a liquid state) throughout said maintenance of the composition between about 15 ° C and room temperature for at least 24 hours.
In some embodiments, the composition may remain liquid (i.e., in a liquid state as recited above) throughout said maintenance of the composition at about 15 ° C to about 25 ° C. at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least 48 hours, or for at least 72 hours, or for at least 84 hours, or for at least at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours. In some embodiments, the composition may remain liquid (i.e., in a liquid state) throughout said maintenance of the composition at about 15 ° C to about 25 ° C for at least 24 hours.
As illustrated in the examples, the inventors have realized that in vitro preservation of MSC or MSC-derived cells in a human plasma or human serum-based composition advantageously retains cell viability, even after storage for extended periods of time.
Accordingly, in another aspect, the present invention provides the use of a composition comprising, consisting essentially of, or consisting of human plasma or human serum or a mixture thereof as an excipient or preservative (which allows ) suspension storage of MSC or cells derived from MSC.
In some embodiments of the methods or uses, as taught herein, the composition may further comprise additional components or components compatible with or favorable for the in vitro storage of cells in suspension, including, for example, aqueous storage.
Such aqueous storage liquids are well known in the state of the art. Non-limiting examples of suitable aqueous storage liquids include the PRIME-XV® Hypothermic Storage Solution (Irvine Scientific, CA, USA); aqueous storage liquids based on cell and tissue culture growth media, such as, for example, EMEM, DMEM, or RPMI media; and aqueous storage liquids based on physiological electrolyte solutions, such as Plasma-Lyte® A (eg, Plasma-Lyte® Injection pH 7.4 (Multiple Electrolyte Injection, Type 1, USP) from Baxter, Deerfield, IL, USA), Ringer Lactate solution (e.g., Ringer Lactate USP injection from Baxter, Deerfield, IL, USA), or Hartmann's solution.
While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art from the foregoing description. Therefore, it is intended to encompass all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.
Aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples.
EXAMPLES
Example 1 In Vitro Storage of Osteoblastic Cells Derived from Human MSCs
The present example illustrates non-cryogenic storage methods, in particular hypothermic (cold) storage, of living cells derived from suspended MSC which implements the principles of the invention. In particular, osteoblastic cells derived from human MSCs were subjected to suspension storage at 2 ° C. to 8 ° C. for at least 24 hours and up to 192 hours (8 days) in a liquid storage medium based on human plasma or human serum according to certain embodiments of the invention, with respect to a liquid storage medium previously used in cell therapy products, based on a physiological solution of electrolytes supplemented with human serum albumin . The stability of the MSC-derived osteoblastic cell product after storage was assessed by determining cell survival, cell recovery (ie, the ability of cells to adhere to a surface), cell identity (i.e. expression of key cell surface markers), and cell activity (i.e., bone formation properties). The results set out below confirmed the superiority of the liquid storage medium based on human plasma and human serum.
Generation of osteoblastic cells derived from MSC
Osteoblastic cells derived from MSC were obtained essentially as described in WO 2009/087213. Briefly, 20 to 60 ml of heparinized bone marrow (BM) were obtained from the iliac crest of 8 healthy human volunteers. In parallel, serum from healthy human donors was obtained after centrifugation of 160 ml of blood taken from dry tubes. The bone marrow was seeded in culture flasks at a fixed leukocyte density (50,000 cells / cm 2) and cultured in a conventional culture medium supplemented with serum, a basic fibroblast growth factor (FGF-2), a transforming growth factor beta 1 (TGFb-1) and growth factors, in a humidified atmosphere at 37 ° C containing 5% CO2. The mesenchymal stem cells (MSC) were allowed to attach before an initial medium change. The medium has been changed regularly. At the end of the primary culture, the cells were detached with a trypsin / EDTA solution for 1-5 min at 37 ° C, counted and resuspended at 1000 cells / cm 2 for secondary culture in vials. of culture in the same medium. At the end of the secondary culture, osteoblastic cells derived from MSC were harvested and washed with phosphate buffered saline.
Cell suspension storage
After harvest, MSC-derived osteoblastic cells were suspended at a fixed cell density (sample volume ranging from 1 ml to 4 ml) in the following liquid storage medium at room temperature: Plasma-Lyte® ( Baxter, cat # AE0323) supplemented with 5% w / v human serum albumin (HSA) (diluted from a 20% w / v solution of HSA, Sanquin Blood Supply, Amsterdam, The Netherlands) ( also referred to as "standard excipient" in the following and in the drawings) - human plasma (100% Octaplas®, Octapharma AG, Lachen, Switzerland) - human serum (100% auto-serum) - human serum enriched with human serum albumin (auto-serum supplemented with 50% w / v HSA (CAF-DCF, Cat.No CR-RK-450, form: aqueous solution with 20% HSA, purity> 95%) )
Samples were stored at 2-8 ° C in a controlled temperature controlled refrigerator for 24, 48, 72, 96, 144 or 192 hours.
Human Plasma (Octaplas®) and Human Serum
According to the manufacturer, Octaplas® is made from pooled plasma (the plasma included in the pool may be from a single AB0 group, or multiple AB0 groups). Frozen plasma units are thawed and pooled. Sodium dihydrogenphosphate dihydrate is added as a buffer to counteract an increase in pH due to the loss of CO2. After filtration on a 1 μm pore size membrane, the plasma pool is treated with solvent / detergent (S / D) reagents (1% v / v tri (n-butyl) phosphate (TNBP) and 1% v / v octoxynol for 1-1.5 hours at 30 ° C). S / D reagents are removed by sequential oily and solid phase extraction procedures. Glycine is added to adjust the osmolality. Optionally, the plasma with glycine is applied to a column loaded with an affinity ligand resin provided for selective binding of prion protein (PrPSc). Finally, the product is sterilized by filtration and typically loaded into sterile polyvinylchloride blood bags, labeled, frozen and stored at -18 ° C or lower.
Survival of the cells
After storage in suspension at 2-8 ° C for 24, 48, 72, 96, 120, 144 or 192 hours, samples of cell suspensions of osteoblastic cells derived from MSC in the various liquid storage media (50 μL) were diluted 1: 1 v / v with a 0.4% w / v solution of Trypan blue (fresh and filtered). About 50 μL of the mixture was deposited on a slide in a counting chamber. Cells (including unstained viable cells, and non-viable cells stained blue) were counted manually using a microscope. Cell survival for each sample was expressed as cell viability (i.e., proportion of viable cells versus total cells counted) (Figure 1).
Storage of osteoblastic cells derived from MSC in human plasma or serum allowed better survival of cells over time, compared to the standard excipient (Figure 1). Cell recovery
After storage in suspension at 2-8 ° C for 72 hours, a fixed number of osteoblastic cells derived from MSC (dead as well as living cells, as assessed by Trypan blue staining), were resown in plates. plastic culture at a density of 55,000 cells / cm 2. The cells were cultured in a conventional medium as described above supplemented with 5% v / v serum for 24 hours in a humidified atmosphere at 37 ° C containing 5% CO 2. After 24 hours, the culture plates were analyzed to evaluate the ability of the cells to adhere back to a substrate after storage. In particular, the cells were visualized using a Romanowsky dye (Diff-Quik® stain, Medion Diagnostics AG, Düdingen, Switzerland). Briefly, the cells were fixed in a methanol solution, and stained with sodium azide / eosin for the cytoplasm (pink) and with a thiazine dye for the nucleus (blue).
As shown in Figure 2, only a few cells adhered to the substrate after 72 hours of storage in the standard excipient. Conversely, in the case of human plasma or human serum, the cells readily adhered to the plastic substrate with a spindle-like shape, reaching confluency.
The number of cells re-attached to the substrate qualitatively reflects the percentage of cell viability in the corresponding population before reseeding (see Figure 1). Thus, cells that are alive after 72 hours of suspension in human plasma or human serum have retained the ability to adhere back to plastic. In the case of the standard excipient, the number of live cells before reseeding was already quite low (Figure 1), which made it difficult to draw conclusions about their ability to refix based on the results. illustrated in Figure 2.
Identity of the cells
After storage in suspension at 2-8 ° C for 0-2, 24, 48, 72, 96, 120, 144 or 192 hours, cell surface markers were analyzed by flow cytometry in osteoblastic cells derived from MSC. . In order to detect the cell surface markers, the cells were incubated with the following conjugated monoclonal antibodies: anti-CD45 (CD45 is a hematopoietic marker, and must be substantially absent from osteoblastic cells derived from MSC), anti-CD73 (CD73 is a mesenchymal marker, and must be highly expressed by osteoblastic cells derived from MSC) and anti-ALP for 15 min at room temperature, then washed with phosphate buffered saline (PBS) before centrifugation and resuspended in 0.3 mL of PBS. The results are summarized in the table in Figure 3.
As shown in Figure 3, marker expression by the cells was maintained during storage times, regardless of the medium used. Thus, the use of human plasma or human serum, instead of the standard excipient, did not have a detrimental effect on maintaining the cellular identity of MSC-derived osteoblastic cells during storage.
Cell activity (in vivo)
The ability of osteoblastic cells derived from MSC to induce bone formation in mice after storage in the various media was also evaluated.
One hundred and eight (108) NMRI female nude mice were divided into three groups of equivalent size: 1) A control group, which was injected with the standard excipient (Plasma-Lyte® supplemented with 5% w / v HSA) without no cell; 2) A group injected with MSC-derived osteoblastic cells suspended in the standard excipient; and 3) A group injected with MSC-derived osteoblastic cells suspended in human plasma (100% Octaplas LG®, Octapharma AG, Lachen, Switzerland).
Each group was subdivided into 6 subgroups of 6 mice each, corresponding to 6 different storage times of osteoblastic cells derived from MSC (0-2, 48, 72, 96, 120 or 144 hours).
On day 0 (D0), the mice were injected with the cell product - at a single cell dose of 2.5 χ 106/100 μL - or the standard cell free excipient by subcutaneous injection under the cranial vault (100 μL). .
In this experiment, alizarin red (red) was used to stain the basal line, ie the mineralization front at the time of cell implantation, calcein (green) was used. to color the early-stage mineralization front, roughly corresponding to newly formed bone in the first week following cell implantation, and tetracycline (yellow) was used to stain the mineralization front at a stage subsequent, corresponding roughly to the newly formed bone in the second week following cell implantation. The mineralized bone appeared in blue with the microscope. At day 14 (that is, 14 days after administration of cells or control), the mice were sacrificed. The bones of the cranial vault were removed and subjected to X-ray imaging (Faxitron®) to assess bone density: the signal intensity (ie the average of the pixel values) ), associated with the mineralized content, measured for each cranial vault from X-ray images, and analyzed using the Adobe Photoshop® histogram tool.
In addition, the bones of the cranial vault were embedded in plastic and cut transversely (5 μm sections) using a microtome. The thickness of the mineralized bone before and after the cell injection was measured by fluorochrome incorporation. See Figure 4 for illustration, in which the mineralized bone prior to cellular injection appears to be comparatively darker, and the mineralized bone after cellular injection appears comparatively clearer, due to the incorporation of fluorochromes, particularly green calcein and yellow tetracycline. Percentage of bone formation was calculated as follows: thickness of mineralized bone 14 days after cell injection χ 100 thickness of mineralized bone before cell injection that was measured, and expressed in%, for each cranial vault. The initial and final thickness values are the average values of named measurements on four different areas of the cranial vault.
As shown in Figures 5A and 5B, freshly prepared MSC-derived osteoblastic cells stored for a very short time (0-2 hours) in the standard excipient or human plasma have similar activity for the formation of new bone.
In contrast, when the cells have been previously stored for at least 48 hours, their activity for new bone formation is highly dependent on the storage medium. Indeed, for virtually all the storage times tested, the number of pixels measured on X-ray images (reflecting the bone density) and the percentage of bone formation were higher in the cranial vault having received an injection of cells stored in human plasma, relative to the standard excipient. In fact, MSC-derived osteoblastic cells that have been stored in the standard excipient for 72 hours or more have similar activity for cell-free control vehicle bone formation. Therefore, the activity of osteoblastic cells to promote new bone formation is maintained much longer when stored in human plasma, ie, in this experiment, at least 120 hours compared to less than 48 hours. hours in the standard excipient.

权利要求:
Claims (16)
[1]
MODIFIED CLAIMS (accepted modifications version)
A method of non-cryogenic in vitro storage of cells, comprising maintaining adherent mesenchymal stem cells (MSC) or adherent cells derived from MSC in suspension in a composition comprising at least 20% v / v of human plasma or human serum or a mixture thereof, wherein the composition is maintained at or below room temperature.
[2]
The method of claim 1 comprising: a) providing the composition comprising (i) MSCs or MSC derived cells and (ii) human plasma or human serum or mixture thereof, optionally where said providing comprises al) recovering MSCs or MSC-derived cells and a2) suspending MSCs or MSC-derived cells with at least human plasma or human serum or mixture thereof; and b) maintaining the composition, so as to effect cell preservation.
[3]
3. A method according to any one of claims 1 or 2, wherein the composition is maintained between about 0.1 ° C and room temperature.
[4]
The method of any one of claims 1 or 2, wherein the composition is maintained at about 0.1 ° C to about 25 ° C.
[5]
The method of any of claims 1 or 2, wherein the composition is maintained at about 2 ° C to about 8 ° C.
[6]
The method according to any one of claims 1 to 5, wherein the composition is maintained for at least 24 hours, or for at least 30 hours, or for at least 36 hours, or for at least 42 hours, or for at least at least 48 hours, or for at least 72 hours, or for at least 96 hours, or for at least 120 hours, or for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
[7]
The method of claim 6, wherein the composition is maintained for at least 48 hours, preferably for at least 72 hours, more preferably for at least 96 hours, even more preferably for at least 120 hours, such as for at least 144 hours. hours, or for at least 168 hours, or for at least 192 hours.
[8]
The method according to any one of claims 1 to 7, wherein the composition is maintained at about 2 ° C to about 8 ° C for at least 48 hours, preferably at least 72 hours, more preferably at least 96 hours. still more preferably for at least 120 hours, such as for at least 144 hours, or for at least 168 hours, or for at least 192 hours.
[9]
The method according to any one of claims 1 to 8, wherein the source of human plasma is fresh plasma, lyophilized plasma, solvent / detergent treated plasma, fresh frozen plasma, thawed plasma, or a precipitate cryogenic, a cryogenic supernatant or a plasma concentrate such as concentrate from frozen plasma, or a mixture of any two or more of these, or wherein the human serum is fresh serum, thawed frozen serum , or serum prepared from plasma, or a mixture of any two or more of these.
[10]
The method according to any one of claims 1 to 9, wherein the composition remains liquid throughout said maintenance of the composition.
[11]
The method according to any one of claims 1 to 10, wherein the concentration of human plasma or human serum or a mixture thereof in the composition is at least 50% v / v, preferably from minus 60% v / v, more preferably at least 70% v / v, still more preferably at least 80% v / v, still more preferably at least 90% v / v, still more preferably at least at least 99-100% v / v.
[12]
The method according to any one of claims 1 to 11, wherein the concentration of MSC or MSC-derived cells in the composition is between about 1 χ 104 and about 1 χ 1011, or between about 1 χ 105 and about 1 χ 1010, or between about 1 χ 106 and about 1 9 7 8 7 7 χ 10, or between about 1 χ 10 and about 1 χ 10, or between about 2 χ 10 and about 5 χ 10, such as about 2.5 - 107 cells per ml of the composition.
[13]
The method of any one of claims 1 to 12, wherein the composition consists essentially or consists of i) MSCs or MSC-derived cells and ii) human plasma or human serum or mixture thereof. this.
[14]
The method according to any one of claims 1 to 12, wherein the composition is further complemented by one or more fractionated plasma or serum components, preferably wherein the composition is supplemented with human serum albumin (HSA).
[15]
The method according to any one of claims 1 to 14, wherein the MSC derived cells comprise osteoprogenitor, osteoblast cells, osteocytes, chondroblast cells, chondrocytes, fibroblast cells, fibroblasts, fibrocytes, tenoblasts, tenocytes, or synoviocytes.
[16]
The method of any one of claims 1 to 15, wherein the MSCs or MSC-derived cells are MSCs or cells derived from human MSCs.

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同族专利:

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公开号 | 公开日

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KR20170138448A|2017-12-15|

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JP2018513208A|2018-05-24|

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BE1023315A1|2017-02-01|

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JP6450478B2|2019-01-09|

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CN107592789A|2018-01-16|

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AU2016251313A1|2017-10-05|

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CA2983373A1|2016-10-27|

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WO2016170112A1|2016-10-27|

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AU2016251313B2|2018-05-17|

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EP3285575A1|2018-02-28|

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IL254732D0|2017-11-30|

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EP3285575B1|2019-08-14|

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KR101938182B1|2019-01-14|

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SG11201708434PA|2017-11-29|

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US20180110217A1|2018-04-26|

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CA2983373C|2018-12-11|

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BR112017022656A2|2018-07-17|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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EP15164903|2015-04-23|

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EP15164903.5|2015-04-23|

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