• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Biopolymer composition, procedure for its preparation and use thereof. The present invention relates to a composition based on biopolymers that mimics the natural extracellular matrix of the retinal pigment epithelium (RPE) and that is useful for the cultivation of induced pluripotent cells and the retinal pigment epithelium. The invention also relates to the process for the preparation of said composition and to the use thereof. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2823398A1
    申请号:ES201930963
    申请日:2019-11-05
    公开日:2021-05-06
    发明作者:La Cerda Haynes Berta De；Corrales Francisco Javier Díaz；Delgado Ana Belén García；González Sara Borrego；Cuenca Aranzazu Díaz
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Fundacion Publica Andaluza Progreso y Salud；
    IPC主号:
    专利说明:

    [0002] Biopolymer composition, procedure for its preparation and use of the same
    [0004] The present invention relates to a composition based on biopolymers that mimics the natural extracellular matrix of the retinal pigment epithelium (RPE) and that is useful for culturing pluripotent cells, as well as cells of the retinal pigment epithelium. The invention therefore falls within the fields of medicine, tissue engineering and ophthalmology.
    [0006] BACKGROUND OF THE INVENTION
    [0008] Macular degeneration affects the portion of the retina responsible for central and detailed vision, causing significant disability in people who suffer from it. The most common form is age-related macular degeneration (AMD), a complex disease in which both genetic predisposition and lifestyle intervene and which affects a growing number of patients, being the main cause of vision loss of people over 70 years of age (Bhutto I. et al. Mol Aspects Med. 2012; 33 (4): 295-317). Another macular dystrophy is Stargardt's disease, also known as juvenile macular dystrophy, in this case a recessive hereditary disease caused by mutations mainly in the ABCA4 gene, although there are other genes that are also associated with this disease, and that affects individuals in the second decade of life. In both cases, the degeneration begins in the retinal pigment epithelium (RPE) and secondarily affects the photoreceptors, which are the cells responsible for the perception of visual stimuli (Strauss O. Physiol Rev. 2005; 85 (3): 845- 81). When degeneration involves the macular region, the ability to read, drive, recognize faces, etc. is lost. Currently there are few therapeutic options for patients with macular degeneration, so RPE replacement cell therapy is being tested at the preclinical and clinical level as a possible intervention to protect the photoreceptors from degeneration, with the aim of stopping the progression of the disease. disease or even provide visual improvement to affected people (Nazari H. et al. Prog Retin Eye Res. 2015; 48: 1-39). Different laboratories (Schwartz SD. Et al. The Lancet.
    [0009] 2015; 385 (9967): 509-16; Liu Y. et al. Cell Discov. 2018; 4:50; Tezel TH. Investig Opthalmology Vis Sci. 2004; 45 (9): 3337; da Cruz L. et al. Nat Biotechnol.
    [0010] 2018; 36 (4): 328-37. Mandai M. et al. N Engl J Med. 2017; 377 (8): 792-3) are developing preclinical and clinical trials of cell therapy using RPE cells. These epithelial cells can be derived by differentiation in vitro from embryonic cells or from induced pluripotency cells (iPSCs) from the patients themselves, which would also allow an autologous transplant. EPR is grown in the laboratory on a substrate that mimics the extracellular matrix. The natural extracellular matrix of RPE is Bruch's membrane, which forms an interface between RPE and the vascular layer under the epithelium, called choriocapillaris. Bruch's membrane is structured in five layers and its composition is very complex, with different types of collagen, which form a reticular matrix, fibrins, elastin, laminins, proteoglycans, fibronectin, etc. and is determined by the contributions of fibroblasts, RPE and endothelial cells.
    [0012] The optimal substrate for RPE adherence and culture should mimic the behavior of Bruch's membrane. The most widely used matrix to date for culturing human RPE in the laboratory has the trade name Matrigel, and it is a solubilized basement membrane prepared from an extract of Engelbreth-Holm-Swarm mouse sarcoma by the Corning company. Corning® Matrigel® (hereinafter Matrigel), is composed of a heterogeneous and variable mixture of structural proteins and growth factors. On the other hand, the iPSCs that are used to differentiate autologous RPE also need an adhesion and growth substrate, which can be provided by a layer of inactivated fibroblasts, called "feeders" or by a commercial matrix, among which Matrigel is the most widespread substrate. This commercial Matrigel matrix, being very useful for cell culture, has the drawbacks that its composition is not defined as it is produced by cells in culture, so that each batch entails certain variability. In the field of cell therapy, for use in clean room conditions (sterile conditions), it is preferable to have a cell growth and adhesion material that maintains a defined and standardized formulation and whose composition mimics the mixture of structural proteins. of the natural extracellular matrix of RPE. Matrigel is also a costly product economically and complicated to handle since it is very sensitive to temperature, gelling from 100C. For this reason, it must be kept frozen, thawed very slowly and kept cold during the preparation of the culture plate coating to prevent it from gelling. These drawbacks have prompted the inventors of the present invention to develop and test a substrate alternative both for the culture of human PSCs and the RPE derived from them with the aim of providing an accessible, inexpensive and useful material for work in research laboratories and for RPE replacement cell therapy. In short, based on the composition of the first layers of Bruch's membrane where the RPE rests in the fundus, it is intended to produce a matrix that mimics said membrane with a defined composition, easy handling and storage, and a cost of competitive manufacturing for its application in cell cultures in cell biology laboratories and, in the future, for its application in a clean room in pre-clinical experimentation and / or clinical trials of cell therapy for diseases involving RPE degeneration.
    [0014] DESCRIPTION OF THE INVENTION
    [0016] The inventors of the present invention have found that a composition comprising structured collagen fibrils (with D-stepping), gelatin and laminin is an optimal substrate for adhesion and cultivation of pluripotent cells and RPE, exhibiting a behavior similar to Bruch's membrane. .
    [0018] Then, a first aspect of the invention refers to the composition characterized by comprising: structured collagen fibrils (with D-stepping), gelatin and laminin.
    [0020] It is a biopolymeric composition, since it comprises biopolymers. By "biopolymer" in the present invention is meant a polymer synthesized by or derived from living beings. In the present invention the biopolymers are structured collagen fibrils (with D-stepping), gelatin and laminin.
    [0022] Structured (D-staggered) collagen fibrils, also called "D-structured collagen fibrils", are nanofibers about 100-150 nm in diameter made up of atelocollagen molecules self-assembled or associated with each other in parallel but staggered fashion. This staggering, characteristic of native collagen tissues, gives rise to a contrast pattern that is observed by electron microscopy techniques as a consequence of the periodicity in the organization of the forming molecules (of atelocollagen in this case). The typical D-stepping pattern is a 67 nm periodicity constant, which means that, over their entire length, the fibrils show transverse striations at 67 nm intervals.
    [0023] As is known to the person skilled in the art, by atelocollagen is understood collagen obtained from tropocollagen fibers by breaking their ends or telopeptides, which are the non-helical ends that exist in tropocollagen molecules, by treatment with protease enzymes. or peptidases. Atelocollagen is therefore devoid of telopeptides, since they are eliminated with the enzymatic treatment and it is soluble in acidic aqueous medium.
    [0025] An important property of atelocollagen is that it can reshape itself into the ordered fibrillar structures found in native collagen fibrils. This fibril formation process occurs despite the removal of telopeptides. The process can be used as an advantage for the synthesis of synthetic collagen biomaterials since the purified soluble precursor material, whose immunogenicity is substantially reduced, can be formed into fibrils, which acquire the native packaging that is necessary for many molecular processes essential for interactions. cell-biomaterial
    [0027] Tropocollagen is the basic unit of a collagen fiber, it is a triple helix of three equal polypeptide chains, of about 1,400 amino acids each, which are each forming a left-handed helix.
    [0029] In a preferred embodiment, the composition further comprises water, said composition being a liquid. The proportion of water (% by weight) in the composition can vary between 94 and 97%. The rest up to 100% of the weight would be composed of gelatin, structured collagen fibrils and laminin.
    [0031] In another preferred embodiment, the composition consists of gelatin, structured collagen fibrils, and laminin. In this case, the composition would be solid.
    [0033] The solid composition (sponge type) of biopolymer presents a porous structure that presents cavities or pores between 1-2 p, m in diameter (measured by scanning electron microscopy with a field emission equipment, FEG-SEM, for its acronym in English Field Emission Gun-Scanning Electron Microscopy).
    [0034] In a preferred embodiment, the ratio of each component (biopolymer) to the total sum of the three biopolymers (% by weight) in the composition is between: 95.8 and 99.5 gelatin,
    [0035] 0.33 and 3.50 collagen fibrils (with D-stepping),
    [0036] 0.16 and 0.65 laminin,
    [0037] being 100% the total sum of the percentages of the three biopolymers
    [0039] Then if the composition consists of gelatin, D-stepping collagen fibrils and laminin, the preferred weight composition is that described above. If the composition also comprises water, the above percentages refer to the total of the biopolymers in the composition (without taking into account the water,% dry).
    [0041] Therefore, gelatin is preferably the major component in which collagen fibrils and laminin are integrated.
    [0043] In a more preferred embodiment, the proportion of each component with respect to the total biopolymers (% by weight) in the composition is:
    [0044] 97.2 gelatin,
    [0045] Collagen fibrils 2.6 (D-stepping),
    [0046] 0.2 laminin.
    [0048] The composition thus described mimics the natural extracellular matrix of RPE, which makes it especially useful for culturing RPE cells, as well as pluripotent cells from which RPE cells are generated. Unlike existing conventional compositions for this purpose, the composition of the invention has a defined composition so it is not subject to variability, which would lead to a lack of reproducibility due to the existence of uncontrolled compositional parameters. In addition, it presents easy handling and storage and a competitive manufacturing cost for its application in cell cultures.
    [0050] In a second aspect, the present invention refers to a process for the preparation of the biopolymeric composition described in the first aspect comprising the following steps: i.
    [0052] i. preparation of collagen fibrils with D-stepping and
    [0053] I. Preparation of a solution in water or in a mixture of water and ethanol of the collagen fibrils with D-stepping obtained in the previous step, laminin and gelatin.
    [0054] In a preferred embodiment of the process of the invention, the preparation of the collagen fibrils with staging D comprises the following steps:
    [0056] a) mixing atelocollagen with a saline solution until reaching a concentration of atelocollagen in the solution of between 0.50 and 2.48 mg m L1;
    [0058] b) adjusting the pH of the atelocollagen solution obtained in step a) to a value between 7.0 and 8.0;
    [0060] c) incubation of the atelocollagen solution from step b) at a temperature between 250C and 340C for a period of time between 4 h and 48 h;
    [0062] d) washing the suspension of collagen fibrils with staging D obtained in step c).
    [0064] Carrying out steps a) -c) results in the self-assembly of atelocollagen molecules, forming D-structured collagen fibrils.
    [0066] In a preferred embodiment of the process of the invention, the preparation of the solution in water or in a mixture of water and ethanol of the collagen fibrils with staging D, laminin and gelatin, is prepared with the following concentration of each biopolymer component:
    [0067] between 30 and 60 mg mL'1 of gelatin
    [0068] between 0.20 and 1.10mg mL'1 of collagen fibrils (with D-stepping)
    [0069] between 0.10 and 0.20 mg mL'1 of laminin.
    [0071] The process steps are carried out under sterile conditions.
    [0073] The procedure as described would result in a liquid composition due to the presence of water or water and ethanol.
    [0075] Atelocollagen is commercially available, for example, in the form of vials of the molecule solubilized in an acid medium.
    [0076] The atelocollagen is preferably of bovine origin, although any atelocollagen, such as that of porcine origin, could be used in the process of the invention.
    [0078] The gelatin is preferably of porcine origin, although any gelatin could be used in the process of the invention. It can be purchased commercially.
    [0080] Laminins, as is well known, are glycoproteins of high molecular mass (approximately 140 to 400 kDa). Any laminin could be used in the process of the invention. They can be purchased commercially. Laminins could also be synthesized, for example, in the manner described in: Aisenbrey S, et al. Invest Ophthalmol Vis Sci. 2006 Dec; 47 (12): 5537-44 (for example, the laminins named as laminin 1,5, 10 and 11 that are described in that article).
    [0082] In a preferred embodiment, in step a) the mixture of atelocollagen with a saline solution has an atelocollagen concentration of 2.48 mg mL'1 and the pH is adjusted in step b) to 7.4.
    [0084] The saline solution used in step a) is an aqueous solution preferably comprising phosphate salts, more preferably comprising a mixture of Na2HP04 and KH2PO4, even more preferably, an equimolecular mixture of Na2HP04 and KH2PO4 and even more preferably, a mixture Na2HP04 IM / KH2PO4 1M. In order to increase the ionic strength in the saline solution, another salt, such as sodium chloride, can be added in amounts between 20 and 35 mg mL'1, more preferably 35 mg mL'1. The ionic strength favors the self-assembly process of atelocollagen molecules.
    [0086] The pH adjustment of step b) can be carried out by adding the necessary amount of acid (eg HCl) or base (eg NaOH, KOH, etc.).
    [0088] The washing of step d) preferably comprises the addition of water to the solution resulting from step c) followed by centrifugation and subsequent removal of the supernatant.
    [0089] In a preferred embodiment, the solution of the three biopolymers is prepared with the following concentration of each component:
    [0090] 40 mg mL'1 of gelatin,
    [0091] 1.07 mg mL'1 of collagen fibrils (with D-stepping),
    [0092] 0.10 mg mL'1 of laminin.
    [0094] In one embodiment of the invention, the solution of the three biopolymers is prepared in water, giving rise to a liquid composition (in the present invention, this liquid composition is called Colamigel-L).
    [0096] In another embodiment of the invention, the solution of the three biopolymers is prepared in a mixture of water and ethanol, with water / alcohol ratios (v / v) between 45/55 and 65/35. In a preferred embodiment, the water: ethanol (v / v) ratio is 60/40.
    [0098] In another more preferred embodiment of the invention, in case the solution is prepared in a mixture of water and ethanol, the process additionally comprises the following steps:
    [0100] - Cooling process of the biopolymer solution in the mixture of water and ethanol at -80oC for a period of between 5 and 24 h,
    [0102] - Washing treatment with ethanol at -20 ° C between 10 and 24 hours,
    [0104] - Lyophilization of the ethanol-washed solution obtained in the previous step, where the lyophilization is carried out at a pressure of 0.02-0.04 mbar for 10-24h so that water and ethanol are eliminated,
    [0106] -Vacuum heat treatment of the resulting solid in the lyophilization stage at 5 10 mb and 140-160 ° C for 20-48h.
    [0108] In the step of cooling the solution to -80oC the biopolymers are preferably arranged in the water, so that ethanol microdomains are created in which preferably there are no biopolymers or they are arranged in a lower proportion than with respect to water. As this solution is lyophilized, the solvents evaporate and the areas or microdomains of ethanol will give rise to cavities or pores, thus forming a sponge-like structure.
    [0110] After the heat treatment, a composition in solid state, sponge type, is obtained. (called in the present invention, Colamigel-S) formed by a gelatin matrix that is the major component, collagen fibrils and laminin.
    [0112] In a preferred embodiment, the ethanol wash treatment at -20 ° C is carried out for 20 h.
    [0114] In a preferred embodiment, the lyophilization is carried out at a temperature between -30 ° C and 20 ° C. In a preferred embodiment, lyophilization is carried out at a pressure of 0.04 mbar for 10 hours.
    [0116] In another preferred embodiment, the heat treatment is carried out at 10mbar and 150 ° C for 20 h.
    [0118] The present invention also relates to the composition obtained by the process described in the second aspect of the invention. The composition obtained by the described procedure has the characteristics defined in the first aspect of the invention.
    [0120] Another aspect of the invention relates to the use of the composition defined above for the cultivation and maintenance of pluripotent cells and / or for the cultivation and maintenance of cells of the retinal pigment epithelium (RPE).
    [0122] The cells of the retinal pigment epithelium (RPE) are cells that are derived, in turn, from pluripotent cells.
    [0124] The stem cells include induced stem cells (iPSCs).
    [0126] For the cultivation and maintenance of said cells, the composition of the invention is deposited on culture plates and the same is conditioned with the appropriate culture medium. In the case of using the composition in solid state, it is previously immersed in absolute ethanol, followed by washing with a saline buffer solution (for example, a Na 2 HP 04 / KH 2 P 04 solution) and subsequent hydration thereof. .
    [0127] The composition of the invention has proven useful in the culture of human iPSCs cultured on Colamigel L and S (composition of the invention). Colony fragments of iPSCs were seeded on the substrates and their growth and maintenance of pluripotency were studied by studying their morphology, markers by in situ immunostaining and alkaline phosphatase staining. In all cases, a similar growth of the iPSCs colonies was observed on the new material compared to the control substrate (Matrigel), the cells maintaining all the characteristics that are their own in terms of morphology and adequate expression of pluripotency markers, for What Colamigel can be considered a substitute material for Matrigel for the cultivation and maintenance of human iPSCs, providing as main advantages the one that presents a defined and stable composition, is cheaper and more comfortable handling for the user.
    [0129] EPR cells derived from human iPSCs were cultured on Colamigel-L and after one month of culture the mature cell morphology was evaluated by light microscope and marking with phalloidin and DAPI for the observation of F-actin filaments and cell nuclei, respectively. In all cases, a similar growth of RPE cells derived from human iPSCs was observed on the new material compared to the control substrate (Matrigel), maintaining all the characteristics of their own in terms of morphology and adequate expression of specific RPE markers. Therefore, Colamigel-L can be considered a substitute material for Matrigel, suitable as a basal substrate for the cultivation and maintenance of EPR derived from human iPSCs, providing as main advantages that it presents a defined and stable composition, is more economical and handling more comfortable for the user.
    [0131] A final aspect of the invention relates to a cell culture comprising pluripotent cells or RPE cells and the composition of the present invention.
    [0133] Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0134] BRIEF DESCRIPTION OF THE FIGURES
    [0136] FIG. 1: (a) Representation of the synthesis route of Colamigel-L, consisting of the mixture of fibrils with stepped structure D of 67 nm periodicity, previously formed by the process of molecular self-assembly from a precursor solution of atelocollagen, and its Mix with the gelatin and laminin solutions. (b) Image of an Eppendorf tube containing the final product Colamigel-L and (c) image of the characterization of the D nanostructure of the fibrils of the collagen component obtained by means of transmission mode scanning electron microscopy (STEM) prior preparation of the sample using a negative stain.
    [0138] FIG. 2: (a) Scheme of the synthesis of the pieces of Colamigel-S, consisting of the conformation of the mixture of gelatin, collagen fibrils with structure D, laminin, water and ethanol; cooling of the mixture; lyophilization and heat treatment, also called dehydrothermal treatment (DHT). (b and c) Characterization images of Colamigel-S by FEG-SEM scanning electron microscopy. (d) Graph of nitrogen physisorption, isotherm and (e) estimation of the parameters of specific surface and mesopore volume. (f) X-ray diffraction analysis and (g) infrared spectroscopy in comparison with control samples prepared by the same procedure (a) but consisting of a single component, gelatin solution (NfGel) or dispersion of structured collagen fibrils D (DCol).
    [0140] FIG. 3: Characterization of the growth and maintenance of the pluripotent state of human PSCs grown on the different substrates. In the upper row is presented the in situ immunostaining for TRA 1-60, which is a cell surface antigen specific and characteristic of pluripotent cells; in the bottom row, alkaline phosphatase activity.
    [0142] FIG. 4: Detailed characterization of the maintenance of the pluripotent state of human PSCs cultured on Colamigel-L and Matrigel, by means of immunofluorescence to detect the presence of specific and characteristic markers of pluripotent cells; such as the surface antigens TRA 1-81 and SSEA4 and the nuclear localization transcription factors NANOG and OCT4. In the figure you can see positive marking and proper location in all cases.
    [0143] FIG. 5: Detailed characterization of the maintenance of the pluripotent state of human iPSCs cultured on Colamigel-S and Matrigel, by immunofluorescence to detect the presence of specific and characteristic markers of pluripotent cells; such as the surface antigens TRA 1-81 and SSEA4 and the nuclear localization transcription factors NANOG and OCT4. In the figure you can see positive marking and proper location in all cases.
    [0145] FIG. 6: Morphology of EPR derived from human iPSCs grown for 30 days on Colamigel-L compared to standard conditions on Matrigel.
    [0147] FIG. 7: Characterization of the growth and differentiation of RPE from human iPSCs cultured for 60 days on Colamigel-L and Matrigel reference material, by immunofluorescence. Phalloidin (Phalloidin) was used for marking the actin filaments of the cytoskeleton, which allowed us to observe the characteristic polygonal morphology of mature RPE cells. The presence and correct subcellular localization were also detected for two characteristic RPE proteins: RPE65, which corresponds to an enzyme of the regeneration cycle of visual pigments, located in the cytoplasm, and bestrophin (BEST1), which corresponds to a transmembrane chlorine channel. located in the basal-lateral portion of RPE cells, one of the main characteristics of which is the marked apico-basal polarity.
    [0149] EXAMPLES
    [0151] Next, the prepared compositions are described and their usefulness in cell culture of both EPR and human iPSCs and their ability to sustain the growth and maintenance of the cellular characteristics of both cell types are shown, which will allow the whole process to be carried out. culture and differentiation of autologous RPE on the same material.
    [0152] Example 1: Composition of the invention with liquid consistency (Colamigel-L): Preparation and characterization
    [0154] The Colamigel matrix is composed of collagen fibrils with a staggered D structure self-assembled from an atelocollagen precursor that is mixed with a gelatin solution and a laminin solution. In this way, a biopolymer matrix of defined, cheap and safe composition is achieved, with a component majority of gelatin chains, with low organization, but which incorporates the necessary organization for cell recognition through functionalization with collagen fibrils provided with supramolecular domains provided by the quaternary structure formed by the self-assembly of soluble collagen molecules. The manufacture of Colamigel-L was carried out under sterile conditions, therefore, each of the steps described here has been carried out in a laminar flow cabinet and using sterile material.
    [0155] The first stage for the synthesis of Colamigel consists of the process of fibrillogenesis or self-assembly of atelocollagen monomers to obtain a gel containing fibrils with a stepping structure D. A solution with a concentration of 3.1 mg mL'1 of atelocollagen was used. bovine skin type I in hydrochloric acid, 0.01M HCl (BD Biosciences, 354231). For this, 500 pL of this acidic collagen precursor solution was pipetted into a plastic tube (Eppendorf) that was kept at 4 ° C in a thermoblock (CH-100, Biosan). Then, 125 pL of the buffer Na2HP04 (1M) 6.23 mL / KH2P04 (1M) 1.26 mL were added with an ionic strength of 1000 mM (1.75 g of NaCl) and a phosphate concentration of 150 mM that had been previously cooled. The final reaction concentration of the collagen molecules was 2.48 mg mL'1. The mixed solution was vigorously stirred for 20 seconds using a vortex mixer. The pH of the solution was adjusted to 7.4 by adding 1.7 pL of NaOH 0.1 g mL'1. At this point, the solution was clear and colorless. The next step consisted of transferring the neutralized solution to a thermoblock (AccuBlockTM Digital Dry Baths, 20 Labnet) at the appropriate temperature 34 ° C or room temperature (25 ° C) for its reaction at different times of 4 or 24 h. After incubation, a cloudy gel was obtained that was subjected to a washing treatment to remove the salts from the buffer solution. The protocol consisted of dispersing the fibrils with vortexing for 2 minutes followed by centrifugation at 5000 rpm for 60 seconds. After centrifugation, 200 pL of the supernatant was removed and 1 mL of dH 2 Ü (deionized water) was added, vortexed for 1 minute, centrifuged again and 1 mL of supernatant removed. The process was repeated with another 1 mL of dH20. Finally, it was vortexed for another 2 minutes. The possible loss of collagen fibrils was determined by Bradford test in the excess water from the wash. This analysis determined a final concentration of fibrillar collagen in the precursor dispersion of 3.57 mg mL'1. To check the structure of the fibrils, the gel obtained was observed by scanning electron microscopy in the STEM transmission mode. Figure 1 shows a micrograph where the bands are observed with light-dark contrast with the characteristic pattern of 67 nm (stepping D). After the wash protocol, the structured collagen suspension was transferred from the Eppendorf tube to a glass container with a lid and pre-conditioned (shaken) for 1 hour using a magnetic stirrer at 1300 rpm and 34 ° C.
    [0157] To carry out the next step, a solution of gelatin (Sigma-Aldrich, CAS No. 9000-70-8) in water at 66.7 mg mL'1 was needed, which was sterilized in an autoclave and kept at 40C. For this preparation, the gelatin solution was heated in an oven at 50oC until a homogeneous solution was obtained. 0.6 mL were added to a glass container with a capacity of 2 mL at 350 ° C and stirred at 1100 rpm. Then, 0.1 mL of the laminin solution (Sigma, L2020) (1 mg mL'1) was added. . The mixture was stirred for 10 minutes and then 0.3 mL of the dispersion of the collagen fibrils (with a concentration of 3.57 mg mL'1) were added, obtaining a gel with the composition gelatin / collagen D / laminin = 97.2 / 2.6 / 0.2; expressed as the total sum of the three biopolymers (percent by weight). For maintenance, the gel was stored at -20 ° C until use.
    [0159] Example 2: Colamiqel matrix with solid consistency, Colamiqel-S: Processing and characterization
    [0161] To obtain Colamigel-S matrices (Figure 2a), a mixture of the same final gelatin / collagenD / laminin composition as for Colamigel-L was prepared but in which ethanol was incorporated to carry out the thermally induced phase separation process. for obtaining microporous matrices. We proceeded by mixing 0.04 g of gelatin in 200 pL dH 2 Ü in a 2 mL glass container and stirring at 500 rpm at 45 ° C for 45 minutes. Then, 200 pL of ethanol (UN1170, Anala® Normapur) was added to the gelatin mixture and stirred for another 30 minutes. After this time, the temperature was lowered to 34 ° C and 100 pL of laminin (Sigma, L2020) was added and stirred for 10 minutes. 300 pL of the preconditioned dispersion of atelocollagen D-structured fibrils and 200 pL of ethanol were added to the mixture which was kept under magnetic stirring for 45 min at 500 rpm. Considering the volume of the 300 pL of the preconditioned dispersion of collagen fibrils and the laminin solution as water, a final water / ethanol (v / v) ratio of 60/40 was estimated. Finally, 100 pL of the mixture were formed in the wells of a 96-well polystyrene plate (655161, Greiner Bio-one) or 300 pL for a 24-well plate, placed and preheated in a thermoblock. at 36 ° C. The mixture formed in the wells was then subjected to a thermally induced phase separation process at -80 ° C for 5 h. Then, 200 pL of ethanol was added to each well and the plates were kept for 20 h in a freezer. at -20 ° C. The next step consisted of draining the ethanol from the plates and lyophilizing the pieces using the program detailed in Table 1.
    [0163] Table 1. Freeze-drying treatment program.
    [0165] EL1 EL2 EL3 EL4 EL5 EL6 EL7 EL8 EL9 Temperature (° C) -30 -30 -30 -20 -20 0 0 20 20 Pressure (mbar) 0.120 0.04 0.04 0.04 0.04 0.04 0, 04 0.04 0.04 Time (hh: mm) 3:20 0:20 10:00 1:00 4:00 0:20 0:30 0:20 0:30 to EL: Freeze Drying Stage
    [0167] Finally, the already dry materials were subjected to a crosslinking process using a vacuum heat treatment ( DeHydroThermal, DHT treatment) consisting of the program presented in Table 2.
    [0169] Table 2. DHT treatment program .____________________________________
    [0170] Time Temperature Pressure (hh: mm) (° C) (mbar) Stage 1 00:05 Ta. environment (20-24) 10
    [0171] Stage 2 02:00 150 10
    [0172] Stage 3 20:00 150 10
    [0173] Stage 4 03:00 43 10
    [0174] Stage 5 00:05 43 1000
    [0176] Figure 2 b-g presents the characterization of the Colamigel-S matrices using the techniques of Field Emission Gun Scanning Electron Microscopy (FEG-SEM), nitrogen fission, X-ray diffraction and infrared spectroscopy. The FEG-SEM micrographs (Figure 2 b-c) show the porous microstructure of the material in which interconnected cavities of between 1 and 2 pm can be seen, which are formed by interweaving the biopolymer fibers with a diameter between 70 and 100 nm. The BET fit from the nitrogen adsorption isotherm measures a specific surface area of 17 ± 2 m2g-1 (Figure 2 d).
    [0177] Figure 2f presents the diffraction diagrams under routine conditions (Wide Angle X-ray Diffraction, WAXRD), for the Colamigel-S matrix in comparison with two control materials prepared in a similar way to the procedure described but constituted by a single component, gelatin solution (NfGel) or dispersion of collagen fibrils with structure D ( DCol). The Colamigel-S diffractogram shows, like DCol, the characteristic collagen peak with the lowest angle at 20 = 5.5 and several more with different intensity superimposed on the broad band between 20 = 12-27, the only signal observed in amorphous materials such as gelatin. This analysis therefore confirms that the procedure described for the preparation of Colamigel-S preserves the supramolecular structure corresponding to collagen fibrils with structure D. Although the infrared spectrum for Colamigel-S in Figure 2g is less decisive than the WAXRD analysis on the structural characteristics of the material, the comparison with the spectrum of gelatin NfGel, the majority component in Colamigel-S, allows the identification of variations in the components of the bands corresponding to the functional groups Amide A (~ 3300 cm_1) and Amide I (relative intensity of the bands at 1660 and 1630 cm_1) suggesting the identification of collagen in the material.
    [0179] Example 3: Conditioning of Colamiqel-L and Colamigel-S for cell culture with induced pluripotent human cells (PSCs) and retinal pigment epithelium (RPE)
    [0181] Colamigel-L previously autoclaved is kept at -20 ° C and thawed at room temperature for use. To obtain a homogeneous gel, it was heated at 37 ° C for 2 min. A 1:10 dilution was prepared in DMEM-F12 culture medium and 0.5 mL of said dilution was deposited per well in a 24-well plate. After one hour at room temperature, a DMEM-F12 culture medium was washed, removed and replaced with the appropriate culture medium for the cell type. As a positive control, wells were prepared in a 24-well plate with the commercial reference material Matrigel Corning (cat # 356277, Life Sciences), diluted according to the supplier's instructions and kept for 1 hour at room temperature. Before use, it was washed with DMEM-F12 (GIBCO, 11330057) twice and left in the appropriate culture medium for the cell type until use. As negative control, wells without adhesion substrate were used, only with culture medium.
    [0183] Colamigel-S matrices need to be sterilized. To do this, they were introduced into absolute ethanol for 3 hours, and then they remained 16 hours in 70% ethanol. The following day, 3 washes with PBS (GIBCO, 10010023) of 1 hour duration each were carried out. Once prepared and sterilized, the Colamigel-S matrices were placed at the bottom of the culture wells and hydrated in an appropriate medium for cell culture for 1h before depositing the cells.
    [0185] Culture media:
    [0186] - For human PSCs cells, mTeSR ™ 1 medium (Stemcell Technologies) is used. - For culture of EPR cells derived from human PSC, HESC-15% medium (KO DMEM; KSR 15%; Glutamax 2mM; non-essential amino acids 0.1 mM; pmercaptoethanol 0.23mM; Penicillin / streptomycin, all from GIBCO) is used. .
    [0188] Example 4: Application of Colamiqel-L and Colamiqel-S in the culture and maintenance of growth in the form of colonies and the pluripotent state of human PSCs cells
    [0190] Human PSCs cells were grown on Colamigel-L and Colamigel-S. Colony fragments of PSCs were seeded on the substrates, the positive control (Matrigel) and the negative control (culture medium only). The comparative evolution of the growth of colonies of PSCs on the matrices of the invention was followed throughout 7 days of culture in medium without serum (mTeSR ™ 1). Similar growth was observed in the three types of matrices tested. In the negative control wells in which no adhesion matrix was available, there was no growth of PSCs.
    [0192] Figure 3 shows images taken under a fluorescence microscope of the colonies that grew on the different substrates, stained with a specific surface antibody that is used as a pluripotency marker (TRA 1-60). The images were taken directly from the culture plate after incubating the cells in culture with a conjugated antibody for "in situ" marking (Mouse anti-human TRA 1-60, Stemgent, cat n0: 09-0010) according to the following methodology : Cells were grown at 37 ° C and 5% CO 2 until colonies were of adequate size for imaging. The culture medium was removed from the well and replaced by a dilution of the primary antibody to a final concentration of 2.5 pg / ml in fresh culture medium, which was kept in contact with the cells for 30 minutes at 37 ° C and 5% CO 2 . The medium with the antibody was aspirated and the culture was washed twice with culture medium. cool. Immediately images were taken on an Olimpus Ix71 fluorescence microscope and a DP72 camera directly from the cutive plate. It can be seen that in both Colamigel-L and Colamigel-S the iPSCs form flat and compact colonies of cells with a high nucleus / cytoplasm ratio and that show positive labeling for TRA 1-60 (pluripotency marker). The specificity of the surface marking is seen in the magnification in the upper left quadrant of each image. Additionally, the result of the reaction for the detection of alkaline phosphatase activity (reddish coloration) is presented, which is specifically elevated in pluripotent cells. To reveal said activity, the Alkaline Phosphatase II kit, Stemgent Cat N0: 00-0055, was used, following the manufacturer's instructions; specifically: The culture medium was aspirated and washed twice with PBST (PBS 0.05% Tween-20). Cells were incubated with fixation solution for five minutes, fixation solution was aspirated, and again two PBST washes were performed. The cells were immediately covered with freshly prepared “AP Substrate Solution” (0.5 mL solution A, 0.5 mL solution B, incubated for 2 min at room temperature, 0.5 mL solution C) and incubated in the dark at room temperature. environment between 5 and 15 minutes. The cells were observed periodically and the reaction was stopped once the reddish coloration appeared, aspirating the substrate and washing twice with PBS to stop the reaction and avoid nonspecific signal. Zeiss Axiovert 40C light microscope images were taken with an Axiocam ERc5c camera directly from the culture plates. All colonies of human iPSCs grown on both Colamigel-L and colamigel-S were positive for alkaline phosphatase.
    [0194] Figure 4 presents a more complete characterization of the pluripotent state of iPSCs cultured on Colamigel-L compared to growth on Matrigel as a control. Immunofluorescence has been performed for specific markers of pluripotent cells. These images have greater definition than those of Figure 3 as they were taken from cell repairs grown on glass slides (Microscope cover Glasses High Precision, Paul Marienfeld GmbH cat N0 01117530) on which the adhesion matrices were deposited in the same way as it was done on the bottom of the culture wells before growing the cells. The method begins with fixing the cells, removing the culture medium, washing twice with PBS, incubating 15 minutes in 4% paraformaldehyde, (Aldrich 16005) and washing twice with PBS. Once fixed, the preparations were incubated with permeabilization and blocking solution (PBS 1% bovine serum albumin (Milliopre 12659) TRITON X-100 0.2% (Sigma T-9284)) for 1h at room temperature. The primary antibodies were incubated overnight at 40C and were diluted in blocking solution in the following proportions (rabbit anti-OCT4 (Cell Signaling C30A3) 1: 400; rabbit anti-NANOG (Cell Signaling D73G4) 1: 400; mouse anti -TRA 1-81 (Stemgent 09-0068) 1: 100 and anti-SSEA4 mouse (BD Pharmingen 560073) 1: 100). 3 washes of 10 minutes were carried out at room temperature with PBS and incubated for 2 h at room temperature and protected from light with secondary antibodies conjugated with fluorophore diluted in PBS 1: 500 (goat anti-rabbit 594 (Invitrogen A11001) and goat anti -mouse 488 (Invitrogen A11012)). After 3 washes of 10 minutes with PBS, the preparation was finished by placing the glass slides on a glass microscope slide on a drop of "anti-fade" mounting medium with DAPI (Diamidino-2-Phenylindoline) to mark the nuclei. cells (Vectashield Mounting Medium, Vector Lab H-1200). Images were taken with a Leica TCS SP5 fluorescence confocal microscope.
    [0195] It is clearly appreciated that iPSC colonies present a characteristic morphology of this type of culture, with flat colonies formed by a compact mass of small cells with positive and specific labeling for the pluripotency markers TRA 1-81, NANOG, SSEA4 and OCT4, which make up a standard pluripotent cell characterization panel.
    [0197] In the same way, a complete characterization of the pluripotency of iPSCs cells cultured on Colamigel-S was carried out following the same methodology and using the same materials explained for Figure 4. The result is presented in Figure 5. It can be seen that, in In all cases, iPSCs cells express nuclear and cytosolic pluripotency markers in the correct cellular location, confirming the ability of the substrate Colamigel-S to provide the conditions for the undifferentiated growth of human iPSCs and the maintenance of their pluripotency characteristics.
    [0199] In conclusion, we can say that Colamigel-L and Colamigel-S have been shown to promote the growth of human iPSCs under feeder-free conditions, as well as the maintenance of cell pluripotency.
    [0201] Example 5: Application of Colamigel-L in the culture and maintenance of the differentiated state of RPE cells derived from human iPSCs
    [0202] From human iPSCs, RPE cells were obtained by standard in vitro differentiation techniques. 60,000 cells were seeded per well of 24-well plate after applying Matrigel as a reference control material as culture substrate and Colamigel-L according to preparation and conditioning already explained in Example 3 of this document. The evolution of growth and re-acquisition of the characteristic morphology of RPE was followed, for this the cells were cultured for 60 days on the different substrates. RPE cells, being a monostratified epithelium, have a very characteristic morphology like the “cobblestone” of a road. The function of these cells and the health of the tissue they compose are directly related to the maintenance of this morphology, since they line the bottom of the retina and are the main component of the blood-retinal barrier.
    [0204] For culturing these cells in the laboratory under standard conditions, they are seeded on Matrigel and allowed to reach confluence for two to three weeks. Before reaching confluence, RPE cells have a spindle-shaped morphology similar to that of fibroblasts. Once the confluence is reached, the re-acquisition of the morphological characteristics of the RPE begins, which is associated with the re-acquisition of its physiological characteristics. From there, the cultivation can be extended for several months in which a greater degree of maturity and pigmentation is reached. In our case, to test whether the developed matrices serve to support the growth and differentiation towards the mature morphology of RPE derived from human iPSCs, we have evaluated the morphology after 30 days of culture taking images with a Zeiss axiovert 40C optical microscope with an Axiocam camera. ERc5c directly from the culture plates.
    [0206] Figure 6 shows how the RPE morphology after one month in culture on Colamigel-L substrate does not differ from the control on Matrigel. Presenting an appearance of monostratified epithelium with cells that, as they mature, acquire a cuboidal morphology.
    [0208] The RPE culture was allowed to mature and, at 60 days, immunostaining with specific RPE markers was performed to verify that the epithelium maintains its specific physiological characteristics. This immunostaining was performed with cells that had been cultured on glass slides (Microscope cover Glasses High Precision, Paul Marienfeld GmbH cat N001117530). The method begins with fixing the cells, removing the culture medium, washing twice with PBS, incubating 15 minutes in 4% paraformaldehyde, (Aldrich 16005) and washing twice with PBS. Once fixed, the preparations were incubated with permeabilization and blocking solution (PBS bovine serum albumin 1% (Milliopre 12659) TRITON X-100 0.2% (Sigma T-9284)) for 1h at room temperature. The primary antibodies were incubated overnight at 4 ° C, being diluted in blocking solution in the following proportions (rabbit anti-BEST1 (Novus biological NB300-164) 1: 100; rabbit anti-RPE65 (Bioss, bs-9575R) 1: 100). 3 washes of 10 minutes were carried out at room temperature with PBS and incubated for 2 h at room temperature and protected from light, with secondary antibody conjugated with fluorophore, diluted in PBS 1: 500 (goat anti-rabbit 594 (Invitrogen A11001)) .
    [0209] For staining with rhodamine fluorophore conjugated phalloidin (Phalloidin Sigma P1951), the fixed and permeabilized cells were incubated with a 1: 500 diluted phalloidin solution (6.6pg / mL) for 30 minutes at room temperature and in the dark, without using secondary antibody.
    [0211] After 3 washes of 10 minutes with PBS, the preparation was finished by placing the glass slides on a glass microscope slide on a drop of "anti-fade" mounting medium with DAPI (Diamidino-2-Phenylindoline) to mark the nuclei. cells (Vectashield Mounting Medium, Vector Lab H-1200). Images were taken with a Leica AF6000 fluorescence microscope and a Leica DFC350 FX camera.
    [0212] Figure 7 shows the correct marking and cellular localization of the RPE65 enzyme, a specific cytosolic protein of RPE and a participant in the regeneration cycle of visual pigments and bestrophin (BEST1), a basolateral membrane channel located in RPE. . On the other hand, phalloidin binds to actin filaments and allows the cytoskeleton that is placed in these cells to be visualized according to their polygonal morphology. Therefore, it has been possible to show the maturation of RPE epithelial cells with their characteristic “cobblestone” shape of cuboidal cells closely joined together forming a monolayer, both in the microscopy images with visible light and in the microscopy images of fluorescence. Additionally, the positive labeling and the correct cellular localization of the RPE65 and BEST1 proteins confirm the maintenance of the functional identity of the RPE grown on Colamigel-L.
    [0213] In conclusion, the histological study of RPE grown on Colamigel-L allows defining this matrix as a suitable substrate for culturing and maintaining the differentiation characteristics of RPE obtained from human iPSCs.
    权利要求:
    Claims (20)
    [1]
    1. Composition characterized by comprising the following biopolymers: D-structured collagen fibrils, gelatin and laminin.
    [2]
    2. Composition according to claim 1 wherein the D-structured collagen fibrils have a periodicity of 67 nm.
    [3]
    Composition according to any of claims 1 to 2, wherein said composition consists of D-structured collagen fibrils, gelatin and laminin.
    [4]
    4. Composition according to claim 3, having a solid structure in the form of a sponge with a pore size of between 1-2 pm in diameter.
    [5]
    Composition according to any one of claims 1 to 2, wherein said composition further comprises water.
    [6]
    6. Composition according to claim 5, where the percentage by weight of water in the composition varies between 94 and 97%.
    [7]
    7. Composition, according to any of the preceding claims, wherein the proportion of each biopolymer with respect to the total of the three biopolymers in the composition is between:
    95.8% and 99.5% by weight of gelatin,
    0.33 and 3.50% by weight of D-structured collagen fibrils
    0.16-0.65% by weight of laminin,
    100% being the total sum of the percentages of the three biopolymers.
    [8]
    8. Composition according to claim 7, where the proportion of each component with respect to the total biopolymers in the composition is as follows:
    97.2% by weight of gelatin,
    2.6% by weight of D-structured collagen fibrils,
    0.2% by weight of laminin. 9
    [9]
    9. Process for the preparation of a composition described in any of the preceding claims 1 to 8 comprising or the following steps: i) Preparation of D-structured collagen fibrils and
    ii) preparation of a solution in water or in a mixture of water and ethanol of the following biopolymers: collagen fibrils with D-grading obtained in the previous step, laminin and gelatin.
    [10]
    10. Process according to claim 9, wherein the preparation of D-structured collagen fibrils comprises the following steps:
    a) Mixing atelocollagen with a saline solution until reaching a concentration of atelocollagen in the solution of between 0.50 and 2.48 mg m L1,
    b) adjustment of the pH of the atelocollagen solution obtained in step a) to a value between 7.0 and 8.0,
    c) incubation of the atelocollagen solution from step b) at a temperature between 250C and 340C for a period of time between 4 h and 48 h,
    d) washing the suspension of collagen fibrils with staging D obtained in step c).
    [11]
    11. Process according to claim 9 or 10, where the solution of biopolymers in water or in a mixture of water and ethanol is prepared with the following concentration of each biopolymer:
    between 30 and 60 mg mL'1 of gelatin,
    between 0.20 and 1.10mg mL'1 of D-structured collagen fibrils,
    between 0.10 and 0.20 mg mL'1 of laminin.
    [12]
    12. Process according to claim 10 or 11, where, in step a), the mixture of atelocollagen with a saline solution has an atelocollagen concentration of 2.48 mg mL'1 and the pH is adjusted in step b) to 7.4.
    [13]
    13. Process according to any of claims 10 to 12, wherein the washing of step d) comprises the addition of water to the solution resulting from step c), followed by centrifugation and subsequent removal of the supernatant.
    [14]
    14. Process according to any of claims 9 to 13, wherein the solution of biopolymers in water or in a mixture of water and ethanol is prepared with the following concentration of each component
    40 mg mL'1 of gelatin,
    1.07 mg mL'1 of D-structured collagen fibrils,
    0.10 mg mL'1 of laminin.
    [15]
    15. Process according to any of claims 9 to 14, wherein the biopolymer solution is prepared in water.
    [16]
    16. Process according to any of claims 9 to 14, wherein the biopolymer solution is prepared in a mixture of water and ethanol.
    [17]
    17. Process according to claim 16 where the mixture of water and ethanol is prepared with water / alcohol ratios (v / v) comprised between 45/55 and 65/35.
    [18]
    18. Process according to claim 16 or 17, which also includes the following steps:
    - Cooling process of the biopolymer solution in the mixture of water and ethanol at -80oC for a period of between 5 and 24 hours,
    - Washing treatment with ethanol at -20 ° C between 10 and 24 hours,
    - Lyophilization of the ethanol-washed solution obtained in the previous stage where the lyophilization is carried out at a pressure of 0.02-0.04 mbar for 10-24h so that water and ethanol are eliminated,
    - Vacuum heat treatment of the resulting solid in the lyophilization stage at 5 10 mb and 140-160 ° C for 20-48h.
    [19]
    19. Use of the composition described in any of claims 1 to 8 for the in vitro cultivation and differentiation of induced pluripotent cells and / or cells of the retinal pigment epithelium.
    [20]
    20. Cell culture comprising pluripotent cells or cells of the retinal pigment epithelium and the composition defined in any of claims 1 to 8.
    类似技术:
    公开号 | 公开日 | 专利标题
    US10179194B2|2019-01-15|Self-assembling peptides, peptidomimetics and peptidic conjugates as building blocks for biofabrication and printing
    Chen et al.2018|Biomaterials for corneal bioengineering
    Kuo et al.2015|Incorporation of chitosan in biomimetic gelatin/chondroitin-6-sulfate/hyaluronan cryogel for cartilage tissue engineering
    Chung et al.2008|Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis
    Harkin et al.2011|Silk fibroin in ocular tissue reconstruction
    Madden et al.2011|Human corneal endothelial cell growth on a silk fibroin membrane
    Akiyama et al.2013|In situ tissue engineering with synthetic self-assembling peptide nanofiber scaffolds, PuraMatrix, for mucosal regeneration in the rat middle-ear
    ES2423731T3|2013-09-24|Biomaterial based on a hydrophilic polymeric support
    Ghorbani et al.2017|Injectable natural polymer compound for tissue engineering of intervertebral disc: In vitro study
    Kennedy et al.2019|Poly-ε-lysine based hydrogels as synthetic substrates for the expansion of corneal endothelial cells for transplantation
    Kwak et al.2019|Formation of a keratin layer with silk fibroin-polyethylene glycol composite hydrogel fabricated by digital light processing 3D printing
    US20210244659A1|2021-08-12|Compositions and methods for in situ-forming tissue constructs
    Salinas-Fernandez et al.2020|Genetically engineered elastin-like recombinamers with sequence-based molecular stabilization as advanced bioinks for 3D bioprinting
    Wang et al.2017|Self‐assembled peptide‐based hydrogels as scaffolds for proliferation and multi‐differentiation of mesenchymal stem cells
    Ruan et al.2019|Biofunctionalized self-assembly of peptide amphiphile induces the differentiation of bone marrow mesenchymal stem cells into neural cells
    Hilderbrand et al.2020|Hierarchically structured hydrogels utilizing multifunctional assembling peptides for 3D cell culture
    Lee et al.2007|Injectable gel with synthetic collagen-binding peptide for enhanced osteogenesis in vitro and in vivo
    Oelker et al.2011|Synthesis, characterization, and in vitro evaluation of a hydrogel-based corneal onlay
    ES2823398B2|2021-10-15|Biopolymer composition, procedure for its preparation and use of the same
    JP6903299B2|2021-07-14|Extracellular matrix-containing composition and method for producing the same, three-dimensional tissue, three-dimensional tissue forming agent
    CN105504069B|2019-12-13|Fusion peptide for promoting osteoblast adhesion and osteogenic differentiation and preparation method and application thereof
    WO2019208831A1|2019-10-31|Extracellular-matrix-containing composition, temporary scaffold for three-dimensional tissue formation, three-dimensional tissue formation agent, and method for recovering cells from three-dimensional tissue
    KR20220023958A|2022-03-03|Biomaterials and methods related thereto
    Makuloluwa2017|Development of a Synthetic Substrate for Conjunctival Cell Transplantation
    AU2020219676A1|2021-09-30|Biomaterials and methods related thereto
    同族专利:
    公开号 | 公开日
    ES2823398B2|2021-10-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2007127172A2|2006-04-27|2007-11-08|The Trustees Of Columbia University In The City Of New York|Layered bio-adhesive compositions and uses thereof|
    US20140341965A1|2013-03-14|2014-11-20|Georgetown University|Compositions and Methods Comprising Biodegradable Scaffolds and Retinal Pigment Epithelial Cells|
    EP3246394A1|2015-01-15|2017-11-22|Osaka University|Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells|
    WO2019099552A1|2017-11-14|2019-05-23|The J. David Gladstone Institutes|Methods of generating retinal pigment epithelium |
    法律状态:
    2021-05-06| BA2A| Patent application published|Ref document number: 2823398 Country of ref document: ES Kind code of ref document: A1 Effective date: 20210506 |
    2021-10-15| FG2A| Definitive protection|Ref document number: 2823398 Country of ref document: ES Kind code of ref document: B2 Effective date: 20211015 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201930963A|ES2823398B2|2019-11-05|2019-11-05|Biopolymer composition, procedure for its preparation and use of the same|ES201930963A| ES2823398B2|2019-11-05|2019-11-05|Biopolymer composition, procedure for its preparation and use of the same|
    [返回顶部]