• 专利附录
  • 专利说明
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    • 专利摘要:
    The invention relates to a cell microcompartment comprising successively, organized around a light, at least one layer of pluripotent cells, an extracellular matrix layer and an outer layer of hydrogel. The invention also relates to methods for preparing such microcompartmental cells.
    公开号:FR3059009A1
    申请号:FR1661377
    申请日:2016-11-23
    公开日:2018-05-25
    发明作者:Maxime Feyeux；Kevin Alessandri；Pierre Nassoy；Laurent Cognet；Gaelle Recher；Erwan Bezard
    申请人:Centre National de la Recherche Scientifique CNRS；Institut National de la Sante et de la Recherche Medicale INSERM；Universite de Bordeaux；Institut dOptique Graduate School；
    IPC主号:
    专利说明:

    Holder (s): UNIVERSITY OF BORDEAUX Public establishment, NATIONAL CENTER OF SCIENTIFIC RESEARCH, NATIONAL INSTITUTE OF HEALTH AND MEDICAL RESEARCH - INSERM, GRADUATE SCHOOL DOPTICAL INSTITUTE.
    Agent (s): CABINET BECKER ET ASSOCIES.
    164 / CELL MICROCOMPARTMENT AND PREPARATION METHODS.
    16 /) The invention relates to a cellular microcompartment successively comprising, organized around a lumen, at least one layer of pluripotent cells, a layer of extracellular matrix and an outer layer of hydrogel. The invention also relates to methods of preparing such cellular microcompartment.
    FR 3 059 009 - A1
    i
    Cellular micro-compartment and methods of preparation
    The invention relates to a cellular micro-compartment enabling the pluripotent character of human cells to be preserved. The invention also relates to a preparation process for obtaining such three-dimensional (3D) cell culture compartments.
    Pluripotent cells are considered an important human cell resource, and their culture is of growing interest, especially in the medical and pharmaceutical fields. Thus, obtaining pluripotent cells in large quantities would make it possible to meet the new needs formulated by the pharmaceutical industry which tends to reduce the use of animal models and to use cellular models more relevant than the numerous cell lines used at the time. actual hour. High-throughput tests developed by pharmaceutical groups already use large quantities of human pluripotent cells. Likewise, tissue engineering and cell therapy in humans are conditioned by the availability of industrial quantities of human pluripotent cells.
    Currently, the culture of human pluripotent cells is done mainly in a two-dimensional (2D) environment, such as on petri dishes, very far from the 3D environment in which cells evolve normally. The manipulation of these cells cultured in 2D is often delicate, and in particular requires purification steps, enzymatic detachment, etc. In addition, these cells are difficult to maintain and have a very low freezing survival rate. However, the possibility of sending by conventional transporter cultures of pluripotent cells frozen in massive quantities, and compatible with liquid culture, represents a major challenge both for research laboratories and for the pharmaceutical industries.
    Faced with this fact, three-dimensional culture systems have been developed, which seek in particular to increase the throughput, the efficiency and the quality of the culture systems of human pluripotent stem cells.
    However, the existing 3D culture systems are not entirely satisfactory. Uncontrolled fusion phenomena are often observed, giving rise to cellular aggregates whose size (> 200 μm in diameter) makes the diffusion of the culture medium insufficient. Thus, within such 3D culture systems, cell differentiation is difficult to control and / or the rate of cell death is very high. In general, the lack of homogeneity of the products resulting from the culture of 3D cells, as well as the cost of such techniques make this technology uncompetitive compared to 2D culture which however is not satisfactory.
    There is therefore a need for a 3D cell culture system which makes it possible to supply large quantities of pluripotent cells whose phenotype is controlled, and which can be used easily both for basic research and at industrial level.
    Summary of the invention
    By working on the development of cellular microcompartments for cell culture in 3D, the inventors have developed a system allowing mass culture in liquid suspension of human pluripotent cells while retaining their phenotype. The micro-compartments developed allow cells to be cultured in a liquid medium, using the media conventionally used in 2D culture, while protecting the cells and controlling their phenotype to avoid wild differentiations and maintain pluripotency. More specifically, the micro-compartments, or capsules, developed by the inventors successively comprise, organized in a substantially homocentric manner, a hydrogel shell, a layer of extracellular matrix and one or more layers of human pluripotent cells surrounding a central lumen. The hydrogel shell of the capsules according to the invention, unlike existing culture systems, protects the cells from mechanical stresses linked to collisions or fusions during culture in liquid suspension. In a particularly advantageous manner, the organization into “cysts” of the micro-compartments according to the invention allows them to be frozen with a high cell survival rate. The cells can also be differentiated before use, directly within the micro-compartment, or be used at the pluripotent stage, in 3D culture as in 2D culture. The inventors have also developed methods for the preparation of such cellular micro-compartments, ensuring that the cyst form is obtained and maintained, which is suitable both for freezing and for controlling the phenotype of the cells which they contain.
    The subject of the invention is therefore a cellular microcompartment successively comprising, organized around a light:
    - at least one layer of human pluripotent cells;
    - a layer of extracellular matrix;
    - an outer hydrogel layer.
    Advantageously, culture medium fills the spaces left between the layers.
    The subject of the invention is also a method for preparing a cellular microcompartment according to the invention, comprising the steps consisting in (a) incubating human pluripotent stem cells in a culture medium containing an inhibitor of the RHO / ROCK pathways,;
    (b) mixing the pluripotent stem cells from step (a) with an extracellular matrix;
    (c) encapsulating the mixture of step (b) in a layer of hydrogel;
    (d) cultivating the capsules obtained in step (c) in a culture medium containing an inhibitor of the RHO / ROCK pathways;
    (e) rinse the capsules from step (d), so as to eliminate the pathway inhibitor
    RHO / ROCK;
    (f) cultivate the capsules resulting from step (e) for 3 to 20 days, preferably 5 to 10 days, in a culture medium devoid of inhibitor of the RHO / ROCK pathways, and optionally recover the cellular microcompartments obtained.
    The subject of the invention is also a method for preparing a cellular microcompartment according to the invention, comprising the steps consisting in (a) mixing differentiated human cells with an extracellular matrix and cell reprogramming agents;
    (b) encapsulating the mixture of step (a) in a layer of hydrogel;
    (c) cultivate the capsules resulting from step (b) for 10 to 40 days, and optionally recover the cellular microcompartment obtained.
    Brief description of the figures
    Ligure 1: Photo (A) and schematic representation (B) of a cellular microcompartment forming a cyst according to the invention (1: layer of hydrogel; 2: layer of extracellular matrix; 3: layers of pluripotent cells; 4: light ).
    detailed description
    The subject of the invention is a cellular microcompartment, in 3D, comprising human pluripotent cells, in which the pluripotent character of the cells is preserved.
    Cellular micro-compartment
    The cellular microcompartment according to the invention forms a cyst, the hollow center, or lumen, is preferably aqueous. In the context of the invention, a “cyst” means a closed hollow structure, containing substantially homocentric layers, in the sense that they are successively organized around the same point, the outer layer enveloping the layer of matrix that envelops the layer of cells that surrounds the light. In general, the pluripotent cells constituting the cyst are polarized The polarity of these cells inside the cyst can be demonstrated by the proteins TJP-1 or ZO-1, both located on the internal / apical side of the layer of pluripotent cells, which adjoins the light.
    Light is generated, when the cyst forms, by the cells that multiply and develop on the layer of extracellular matrix. Advantageously, the light contains a liquid and more particularly culture medium.
    In the context of the invention, the “hydrogel layer” designates a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, and preferably water. Advantageously, the hydrogel used is biocompatible, in the sense that it is not toxic to the cells. In addition, the hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the micro-compartment and allow their survival. For example, the outer hydrogel layer contains alginate. Preferably, the outer layer only comprises alginate. In the context of the invention, the term "alginate" means linear polysaccharides formed from β-D-mannuronate (M) and cc-L-guluronate (G), salts and derivatives thereof. Advantageously, the alginate is a sodium alginate, composed of more than 80% of G and less than 20% of M, with an average molecular weight of 100 to 400 KDa (for example: PRONOVA® SLG100) and a total concentration between 0.5% and 5% by mass. According to the invention, the hydrogel layer is devoid of cells. In one embodiment of the cellular microcompartment according to the invention, the outer layer comprises alginate.
    The extracellular matrix layer may in turn comprise a few cells. In fact, when the cyst forms, the cells widen their space in the matrix and multiply, filling the micro-compartment. The delimitation between the layer of extracellular matrix and the layer of pluripotent cells may therefore not be perfectly clear. At the level of the surface in contact with the cell layer, the extracellular matrix can thus contain a few pluripotent cells. Conversely, the surface of the extracellular matrix layer in contact with the hydrogel layer is free of cells.
    The extracellular matrix layer is necessary for the survival of pluripotent cells in the micro-compartment and for the creation of the cyst.
    Preferably, the layer of extracellular matrix forms a gel on the internal face of the hydrogel layer, that is to say the face directed towards the lumen of the micro-compartment. The extracellular matrix layer comprises a mixture of proteins and extracellular compounds necessary for cell culture, and more particularly pluripotent cells. Preferably, the extracellular matrix comprises structural proteins, such as laminins containing the subunits a1, a4 or a5, the subunits βΐ or β2, and the subunits γΐ or γ3, of entactin, of vitronectin , laminins, collagen, as well as growth factors, such as TGF-beta and / or EGL. In one embodiment, the extracellular matrix layer consists of, or contains Matrigel® and / or Geltrex®.
    According to the invention, the cellular micro-compartment contains one or more layers of human pluripotent stem cells. A pluripotent stem cell, or pluripotent cell, means a cell that has the capacity to form all the tissues present in the organism of whole origin, without being able to form an entire organism as such.
    In particular, the cellular microcompartment according to the invention may contain pluripotency-induced stem cells (IPS), or MUSE (“Multilineage-differentiating Stress Enduring”) cells which are found in the skin and bone marrow of adult mammals. . More generally, in the context of the invention, the stem cells used for the micro-compartments according to the invention are not derived from a human embryo. In one embodiment, the human pluripotent stem cells used for the micro-compartments according to the invention are induced to pluripotency from somatic cells.
    Advantageously, the layer of cells contains at least 95% by volume, preferably at least 96%, 97%, 98%, 99% of cells and of matrix produced by said cells. Cells are essentially pluripotent cells. By "essentially" is meant that at least 90% of the cells contained in the cell layer are pluripotent cells, preferably at least 95%, 96%, 97%, 98%, 99%, 100%, are cells pluripotent.
    Advantageously, the lumen of the cyst contains culture medium. In particular, any culture medium allowing the suspension culture of pluripotent cells can be used, and in particular any culture medium conventionally used in 2D culture.
    Preferably, the cellular microcompartment is closed. It is the outer hydrogel layer that gives its size and shape to the cellular micro-compartment. The micro-compartment can have any shape compatible with cell encapsulation.
    Advantageously, the dimensions of the cellular micro-compartment are controlled. In one embodiment, the cellular micro-compartment according to the invention has a spherical shape. Preferably, the diameter of such a micro-compartment is between 10 μm and 1 mm, more preferably between 50 μ m and 500 μ m, even more preferably less than 500 μm, preferably less than 400 μm.
    In another embodiment, the cellular microcompartment according to the invention has an elongated shape. In particular, the micro-compartment may have an ovoid or tubular shape. Advantageously, the smallest dimension of such an ovoid or tubular micro-compartment is between 10 μm and 1 mm, more preferably between 50 μm and 500 μm, even more preferably less than 500 μm, preferably less than 400 μm. By “smallest dimension” is meant double the minimum distance between a point located on the external surface of the hydrogel layer and the center of the micro-compartment.
    In a particular embodiment, the thickness of the outer hydrogel layer represents 5 to 40% of the radius of the micro-compartment. The thickness of the layer of extracellular matrix represents 5 to 80% of the radius of the micro-compartment and is advantageously attached to the internal face of the hydrogel shell. The thickness of the pluripotent cell layer represents approximately 10% of the radius of the micro-compartment. The pluripotent cell layer is in contact at least at one point with the extra cellular matrix layer, a space filled with culture medium may be present between the matrix layer and the cyst. Light then represents from 5 to 30% of the radius of the micro-compartment. In the context of the invention, the "thickness" of a layer is the dimension of said layer extending radially relative to the center of the micro-compartment.
    In a particular example, the cellular microcompartment has a spherical shape with a radius equal to lOOpm. The hydrogel layer has a thickness of 5pm to 40pm. The extracellular matrix layer has a thickness of 5pm to about 80pm. The pluripotent cell layer has a thickness of 10 to 30 µm, the lumen having a radius of about 5 to 30 µm.
    In general, the presence of the outer hydrogel layer imposes a maximum size on the cell layer and makes it possible, by confinement, to limit the uncontrolled proliferation of cells, which could lead to the death by anoxia of the cells and / or uncontrolled differentiations of cells at the level of the deepest layers, that is to say those closest to the lumen of the cyst. In 2D, on a Petri dish, the colonies are discs, the cells at the heart of the disc tend to die (each new cell resulting from a division is excluded from the colony by the lack of space) or to differentiate under the constraints of the cells surrounding them, the edge cells tend to differentiate and only a band at the right distance presents the optimal phenotype. The topology of the microcompartment presented here, the internal surface of the sphere formed by the capsule, makes it possible to generate a “colony” of stem cells (the pluripotent layer of cells) “without edges” where all the cells are positioned in an optimal and equivalent manner. both for the diffusion of small molecules and in terms of mechanical stresses. Advantageously, the cell density in the micro-compartment is between 1 and several thousand cells per micro-compartment, preferably between 50 and 1000 cells per micro-compartment with a radius of 100 μm.
    Methods for preparing cellular microcompartment
    The invention also relates to processes for the preparation of cellular micro-compartments making it possible to obtain the cellular micro-compartment according to the invention. More particularly, the invention proposes to produce cellular micro-compartments containing pluripotent stem cells organized into cysts directly from pluripotent stem cells, or from differentiated cells which will be reprogrammed into pluripotent cells inside the hydrogel capsule. during the formation of micro-compartments.
    Any method of production of cellular microcompartments containing inside a hydrogel capsule of the extracellular matrix and pluripotent stem cells can be used for the implementation of the preparation process according to the invention. In particular, it is possible to prepare micro-compartments by adapting the method and the microfluidic device described in Alessandri et al., 2016 (“A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human
    Neuronal Stem Cells (hNSC) ”, Lab on a Chip, 2016, vol. 16, no. 9, p. 1593-1604), in accordance with the steps described below.
    In a first embodiment, the preparation method according to the invention comprises the steps consisting in (a) incubating human pluripotent stem cells in a culture medium containing an inhibitor of the RHO / ROCK pathways;
    (b) mixing the pluripotent stem cells from step (a) with an extracellular matrix;
    (c) encapsulating the mixture of step (b) in a layer of hydrogel;
    (d) cultivating the capsules obtained in step (c) in a culture medium containing an inhibitor of the RHO / ROCK pathways;
    (e) rinse the capsules from step (d), so as to eliminate the inhibitor of the RHO / ROCK pathways;
    (f) cultivate the capsules resulting from step (e) for 3 to 20 days, preferably 5 to 10 days, until a cyst is obtained, and optionally recover the cellular microcompartment obtained.
    Stage (a) of incubation and stage (d) of culture in a medium containing one or more inhibitors of the RHO / ROCK pathways (“Rho-associated protein kinase”), such as thiazovivin (C15H13N5OS) and / or Y-27632 (C14H21N3O) make it possible to promote the survival of pluripotent stem cells, and the adhesion of the cells to the extracellular matrix at the time of the formation of the outer layer of hydrogel around said extracellular matrix. However, it is desirable that these steps be limited in time, so that inhibitors of the RHO / ROCK pathways do not prevent the formation of cysts.
    Thus, preferably, the incubation of step (a) is carried out for a time between a few minutes and a few hours, preferably between 2 minutes and 2 hours, more preferably between 10 minutes and 1 hour.
    Likewise, preferably, the culture step (d) is carried out for a time between 2 and 48 hours, preferably for a time between 6 and 24 hours, more preferably for a time between 12 and 18 hours.
    Step (e) is necessary to guarantee the elimination of all traces of inhibitors of the RHO / ROCK pathways. Step (e) is for example carried out by rinsing, and preferably by several rinses, in successive culture media free of inhibitors of the RHO / ROCK pathways.
    Advantageously, step (f) is carried out for a time sufficient to obtain a cellular micro-compartment in which the layer of pluripotent cells and the light have a cumulative thickness equal to 10 to 95% of the radius of the micro-compartment, ie for a sufficient time for allow to go from two cells to about a thousand cells. Any culture medium suitable for the cultivation of pluripotent stem cells can be used, and in particular saline phosphate buffer such as the “Roswell Park Memorial Institute medium” medium.
    In one embodiment, the method according to the invention comprises an intermediate step (a ') consisting in dissociating the pluripotent stem cells originating from step (a) before step (b), preferably by means of an enzyme-free reagent. Advantageously, said reagent is inhibited or rinsed before the encapsulation step, in particular by successive rinses in a specific medium for pluripotent cells. For example, the reagent used is an isoosmotic buffer containing EDTA or EGTA such as ReLeSR®. Of course, it is also possible to use trypsin or a reagent containing an enzyme, but the survival rate of pluripotent cells at the end of this step may then be less compared to the use of a reagent free of 'enzyme. In all cases, the rinsing step is necessary to remove all traces of the reagent used for dissociating the cells.
    In one embodiment, at least one of steps (a '), (b), (c), (d) or (e) is carried out at a temperature between 0 and 8 ° C, preferably all steps (a '), (b), (c), (d) and (e). Maintaining a temperature substantially equal to 4 ° C makes it possible to put the biological processes of dormant cells, including the transduction of signals from the external environment. It is thus possible to limit the phenomenon of cell death, which could be induced by cell detachment.
    In another embodiment, the method for preparing a cellular microcompartment according to the invention comprises the steps consisting in (a) mixing differentiated human cells with an extracellular matrix and non-permeable cellular reprogramming agents screw of the hydrogel layer;
    (b) encapsulating the mixture of step (a) in a layer of hydrogel;
    (c) cultivate the capsules resulting from step (b) for 10 to 40 days, and optionally recover the cellular microcompartment obtained.
    In another embodiment, the method for preparing a cellular microcompartment according to the invention comprises the steps consisting in (a) mixing differentiated human cells with an extracellular matrix;
    (b) encapsulating the mixture of step (a) in a layer of hydrogel;
    (c) incubate the capsules from step (b) with permeable cell reprogramming agents vis-à-vis the hydrogel layer and cultivate the capsules for 10 to 40 days, and optionally recover the cellular microcompartment obtained.
    For example, the differentiated cells used are fibroblasts.
    A person skilled in the art knows how to reprogram a differentiated cell into a stem cell by reactivating the expression of genes associated with the embryonic stage by means of specific factors, designated in the present invention as "reprogramming agents". Examples include the methods described in Takahashi et al., 2006 ("Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors" Cell, 2006 Vol 126, pages 663-676), Ban et al., 2009 (“Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome” Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85 ( 8): 348-62) and in international application W02010 / 105311 having the title "Production of reprogrammed pluripotent cells".
    The reprogramming agents are advantageously co-encapsulated with the differentiated cells, so as to concentrate the product and to promote contact with all of the cells. In the case of reprogramming agents permeable to the hydrogel layer, it is possible to add said agents to the culture medium after the encapsulation step.
    Reprogramming agents allow cells to undergo a succession of phenotypic changes up to the pluripotent stage. Advantageously, step (a) of reprogramming is carried out using specific culture media, favoring these phenotypic changes. For example, the cells are cultured in a first medium comprising 10% human, or bovine serum, in a minimum essential medium of
    Eagle (DMEM) supplemented with a serine / threonine protein kinase receptor inhibitor (such as the product SB-431542 (C22H16N4O3)), one or more inhibitors of the RHO / ROCK pathways ("Rho-associated protein kinase"), such as thiazovivin and / or Y-27632, fibroblast growth factors, such as FGF-2, ascorbic acid, and antibiotics, such as Trichostatin A (C17H22N2O3). Then the culture medium is replaced by medium favoring the multiplication of pluripotent cells, such as the mTeSR®1 medium.
    Advantageously, the capsules resulting from step (b) each contain between 1 and 500 differentiated cells, preferably between 50 and 200.
    In one embodiment, at least one of steps (a), (b), (c) or (d) is carried out at a temperature between 0 and 4 ° C, preferably all of the steps (a) , (b), (c) and (d). Maintaining a temperature below or equal to 4 ° C makes it possible to put the biological processes of dormant cells, including the transduction of signals from the external environment. It is thus possible to limit the phenomenon of cell death, which could be induced by cell detachment.
    The micro-compartments obtained during step (d) can be sorted so as to isolate the cellular micro-compartments having the desired cyst shape. Such a sorting step can be carried out continuously, so as to separate the cellular micro-compartments already having the desired cyst shape, from the micro-compartments still being formed. Such a sorting step can be done by simple morphological analysis, without disturbing the other micro-compartments in which the reprogramming is still in progress and / or the organization into a cyst not yet completed.
    In general, the cellular micro-compartments obtained according to the methods of the invention can then be frozen before any use. Indeed, the cyst shape promotes the survival of cells within the micro-compartment, and after thawing, the survival rate is greater than 80%. Advantageously, freezing is carried out using liquid nitrogen allowing the micro-compartments to be vitrified quickly and to limit the risks of crystal formation within the lipid membranes of the cells. Cellular micro-compartments can be suspended in a freezing buffer which promotes cell survival. It is for example possible to use the freezing buffers conventionally used for freezing embryos.
    The cellular microcomputers thus frozen can then be thawed as required.
    Applications
    The cellular micro-compartments which are the subject of the present invention can be used for numerous applications. Indeed, the cells which they contain can be easily recovered, by simple hydrolysis and / or dissolution of the external layer in hydrogel. In addition, it is possible to differentiate pluripotent cells inside the hydrogel capsule or after hydrolysis / dissolution of said hydrogel capsule, as required, so as to obtain large quantities of cell lines of interest. . Advantageously, the cells are differentiated into one or more cell types of interest, within the micro-compartment, that is to say before hydrolysis of the outer layer in hydrogel.
    Cellular micro-compartments, and more precisely the cells which they contain, can be used for research and development purposes, both in the form of a 3D cellular network and more conventionally in 2D culture. It is also possible to use them for therapeutic purposes, in particular for cell therapy, tissue engineering applications, etc.
    EXAMPLES
    Example 1: Protocol for obtaining cellular micro-compartments from pluripotency-induced human cells.
    Solutions used:
    Solution 1, DMEML12 medium base supplemented with 2μΜ Thiazovivin
    Solution 2, PBS without magnesium and without calcium supplemented with ΙμΜ 2μΜ Thiazovivin
    Solution 3, non-enzymatic cell detachment buffer: RelesR ™ supplemented with 2μΜ Thiazovivin.
    Solution 4, pluripotent stem cell culture medium: MTeSRl ™ hES / hIPS cell medium STEMCELL ™).
    Solution 4+, Solution 4 supplemented with 2μΜ Thiazovivin.
    Solution 5, Matrigel ™.
    Solution 6, sorbitol 300mM with 2μΜ Thiazovivin.
    Cellular solution:
    A petri dish of human IPS cells (obtained from Primary Dermal Fibroblast; Normal, Human, Adult ATCC® PCS-201-012 ™ and CytoTune ™ -iPS 2.0 Sendai Reprogramming Kit (ref A16517) using the technology presented in Example 2) of
    25cm 2 at 90% confluence is used thereafter to correspond to the recommended volumes. All the following steps are carried out at 4 ° C. until the hydrogel shell is crosslinked in the calcium bath.
    Step 1: Rinse the cells with solution 1. Wait 10 minutes to 1 hour.
    Step 2: Rinse twice with 4 mL of solution 2.
    Step 3: Gently aspirate the solution.
    Step 4: Incubate the cells with 4 mL of solution 3 for 5-10 minutes.
    Step 5: Detach the cells with 2mL of 4+ solution with a large cone pipette to reduce shear stress.
    Step 6: Centrifuge the cell suspension at 360 xg for 5 minutes.
    Step 7: Aspirate the supernatant.
    Step 8: Resuspend with 0.5 mL of 4+ solution.
    Step 9: Centrifuge again at 360g and aspirate the supernatant.
    Step 10: Resuspend the cell pellet in 70pL of solution 5 and 100pL of solution 6 (the volume of the pellet should be 30pL). The cell solution is ready.
    Encapsulation:
    The encapsulation device is prepared as described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, p. 1593-1604).
    In summary, the different parts of the device are sterilized (by autoclave); The three necessary solutions are loaded onto three syringe pumps, i) alginate solution (PRONOVA®SLG100 2% in a mass in distilled water), ii) intermediate solution (sorbitol at 300mM), iii) cellular solution ( prepared in the previous step); The three solutions are co-injected concentrically using a microfluidic injector which makes it possible to form a jet which splits into drops, the outer layer of which is the alginate solution and the core the solution of cells; These drops are collected in a calcium bath (at 100 mM) which stiffens the alginate solution to form the shell.
    To improve the mono-dispersity of the cellular microcompartments, the alginate was charged with a direct current at + 2KV. A ring with a mass of 2cm in diameter is placed at 500pm from the tip in the plane perpendicular to the axis of the jet leaving the microfluidic injector to generate the electric field.
    Note that under these encapsulation conditions, the Matrigel® layer forms spontaneously.
    Treatment after encapsulation:
    Step 1: The capsules are collected with a 40 µm cell sieve and then after rinsing with solution 1 they are stored in a 75 cm 2 flask with 20 ml of solution 4+.
    Step 2: The flask is kept for 12 hours in the incubator at 37 ° C and 5% CO2.
    Step 3: Change the medium for solution 4 to allow the formation of cysts.
    Step 4: After 24 to 72 hours, cysts of a few tens of cells form in the capsules. The cellular microcomputers are mature after 5 to 10 days.
    Example 2: Protocol for Obtaining Cellular Microcompartments from Human Fibroblasts.
    Solutions used:
    Solution 1, DMEML12 medium base
    Solution 2, PBS without magnesium without calcium supplemented
    Solution 3, trypsin EDTA cell detachment buffer
    Solution 4, fibroblast culture medium: 10% human serum in a base of DMEM medium
    Solution 4+, Solution 4 supplemented with 2μΜ Thiazovivin.
    Solution 5, Matrigel ™.
    Solution 6, sorbitol 300mM with 2μΜ Thiazovivin.
    Cellular solution:
    A Petri dish of human fibroblasts (Primary Dermal Fibroblast; Normal, Human, Adult (ATCC® PCS-201-012®) of 25cm 2 with low confluence density is then used to correspond to the recommended volumes (1 to 2 million All the following steps are carried out at 4 ° C. until the crosslinking of the shell in the calcium bath.
    Step 1: Rinse the cells with solution 2.
    Step 2: Gently aspirate the solution.
    Step 3: Incubate the cells with 4 mL of solution 3 for 5-10 minutes.
    Step 4: Detach the cells with 2mL of 4+ solution with a wide cone pipette to reduce shear stress.
    Step 6: Centrifuge the cell suspension at 360 xg for 5 minutes.
    Step 7: Aspirate the supernatant.
    Step 8: Resuspend with 0.5 mL of 4+ solution.
    Step 9: Centrifuge again at 360g and aspirate the supernatant.
    Step 10: Resuspend the cell pellet in 90pL of solution 5 and 100pL of solution 6 (the volume of the pellet should be 10pL).
    Step 11: Add 1/10 of the contents of the “CytoTune® -IPS 2.0 Sendai Reprogramming Kit” kit (containing a reprogramming virus) intended for a 6-well plate. The cell solution is ready.
    Encapsulation:
    The encapsulation is carried out in accordance with the protocol of Example 1.
    Treatment after encapsulation:
    Step 1: The capsules are collected with a 40pm cell sieve and then after rinsing with solution 1 they are stored in a 75cm 2 flask with 20mL of solution 4+.
    Step 2: The flask is kept for 24 hours in the incubator at 37 ° C and 5% CO 2 .
    Step 3: Change the medium every day. Each capsule contains 1 to 10 fibroblasts to form the capsules. The reprogramming virus has a transformation efficiency of around 0.2%. The majority of capsules will therefore contain little or no reprogrammed cells. Cysts begin to form after 15 to 40 days. Fibroblasts have an elongated shape and do not form cysts. Thus, all the cysts that form are formed from IPS.
    i
    权利要求:
    Claims (11)
    [1" id="c-fr-0001]
    1. Cellular micro-compartment comprising successively, organized around a light:
    - at least one layer of human pluripotent cells not derived from a human embryo;
    - a layer of extracellular matrix;
    - an outer hydrogel layer.
    [2" id="c-fr-0002]
    2. A cellular micro-compartment according to claim 1, wherein said micro-compartment is closed.
    [3" id="c-fr-0003]
    3. Cellular micro-compartment according to claim 1 or 2, wherein the outer layer comprises alginate.
    [4" id="c-fr-0004]
    4. Cellular micro-compartment according to one of the preceding claims, in which said micro-compartment has a spherical or elongated shape.
    [5" id="c-fr-0005]
    5. Cellular micro-compartment according to one of the preceding claims, in which said micro-compartment has a diameter or a smaller dimension of between 10 μm and 1 mm, preferably between 50 μm and 500 μm, more preferably less than 500 μm, even more preferably less than 400 pm.
    [6" id="c-fr-0006]
    6- Cellular micro-compartment according to one of the preceding claims, in which the cell density is between one and several thousand cells, preferably 50 to 1000 cells per micro-compartments.
    [7" id="c-fr-0007]
    7. Process for the preparation of a cellular micro-compartment according to one of claims 1 to 6, comprising the steps consisting in:
    (a) incubating human pluripotent stem cells in a culture medium containing an inhibitor of the RHO / ROCK pathways;
    (b) mixing the pluripotent stem cells from step (a) with an extraceilular matrix;
    (c) encapsulating the mixture of step (b) in a layer of hydrogel;
    (d) cultivating the capsules obtained in step (c) in a culture medium containing an inhibitor of the RHO / ROCK pathways;
    (e) rinse the capsules from step (d), so as to eliminate the inhibitor of the RHO / ROCK pathways;
    (f) cultivate the capsules resulting from step (e) for 3 to 20 days, preferably 5 to 10 days, and optionally recover the cellular microcompartments obtained.
    [8" id="c-fr-0008]
    8. A method of preparing a micro-compartment according to claim 7, comprising an intermediate step consisting in (a ') dissociating the pluripotent stem cells originating from step (a) before step (b), preferably by means of an enzyme-free reagent.
    [9" id="c-fr-0009]
    9. A method of preparing a cellular microcompartment according to one of claims 1 to 6, comprising the steps consisting in (a) mixing human differentiated cells with an extracellular matrix and cell reprogramming agents;
    (b) encapsulating the mixture of step (a) in a layer of hydrogel;
    (c) cultivate the capsules resulting from step (b) for 10 to 40 days, and optionally recover the cellular microcompartment obtained.
    [10" id="c-fr-0010]
    10. A method of preparing a cellular microcomputer according to claim 9, in which each capsule resulting from step (b) contains between 1 and 500 differentiated cells.
    [11" id="c-fr-0011]
    11. A method of preparing a cellular microcompartment according to one of claims 7 to 10, comprising a subsequent step consisting in freezing the cellular microcompartment obtained in step (f) according to claim 7, or in step (c ) according to claim 9.
    i
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    同族专利:
    公开号 | 公开日
    US20190330589A1|2019-10-31|
    FR3059009B1|2018-12-07|
    KR20190111013A|2019-10-01|
    AU2017365846A1|2019-05-16|
    MX2019006050A|2019-12-09|
    CL2019001378A1|2020-01-10|
    CN110603319A|2019-12-20|
    BR112019010512A2|2019-09-17|
    IL266777D0|2019-07-31|
    WO2018096277A1|2018-05-31|
    JP2019535326A|2019-12-12|
    CA3040781A1|2018-05-31|
    EP3545080A1|2019-10-02|
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    US20150132847A1|2013-11-11|2015-05-14|Auburn University|ENCAPSULATION AND CARDIAC DIFFERENTIATION OF hiPSCs IN 3D PEG-FIBRINOGEN HYDROGELS|
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    法律状态:
    2017-11-13| PLFP| Fee payment|Year of fee payment: 2 |
    2018-05-25| PLSC| Publication of the preliminary search report|Effective date: 20180525 |
    2019-11-18| PLFP| Fee payment|Year of fee payment: 4 |
    2020-11-27| PLFP| Fee payment|Year of fee payment: 5 |
    2021-09-17| RM| Correction of a material error|Effective date: 20210810 |
    2021-11-29| PLFP| Fee payment|Year of fee payment: 6 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1661377A|FR3059009B1|2016-11-23|2016-11-23|CELL MICROCOMPARTMENT AND METHODS OF PREPARATION|
    FR1661377|2016-11-23|FR1661377A| FR3059009B1|2016-11-23|2016-11-23|CELL MICROCOMPARTMENT AND METHODS OF PREPARATION|
    BR112019010512A| BR112019010512A2|2016-11-23|2017-11-23|cellular microcompartment and preparation processes|
    US16/462,962| US20190330589A1|2016-11-23|2017-11-23|Cellular microcompartment and preparation processes|
    AU2017365846A| AU2017365846A1|2016-11-23|2017-11-23|Cellular microcompartment and preparation methods|
    CN201780071921.9A| CN110603319A|2016-11-23|2017-11-23|Cell microcompartment and method of preparation|
    MX2019006050A| MX2019006050A|2016-11-23|2017-11-23|Cellular microcompartment and preparation methods.|
    PCT/FR2017/053225| WO2018096277A1|2016-11-23|2017-11-23|Cellular microcompartment and preparation methods|
    JP2019547789A| JP2019535326A|2016-11-23|2017-11-23|Cell microcompartment and preparation method thereof|
    EP17816914.0A| EP3545080A1|2016-11-23|2017-11-23|Cellular microcompartment and preparation methods|
    KR1020197016903A| KR20190111013A|2016-11-23|2017-11-23|Cell microcompartment and preparation method|
    CA3040781A| CA3040781A1|2016-11-23|2017-11-23|Cellular microcompartment and preparation methods|
    IL266777A| IL266777D0|2016-11-23|2019-05-21|Cellular microcompartment and preparation processes|
    CL2019001378A| CL2019001378A1|2016-11-23|2019-05-22|Cell microcompartment and preparation methods.|
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