CN115873797A - Amplification culture medium and culture method for retinal pigment epithelial cells - Google Patents
Amplification culture medium and culture method for retinal pigment epithelial cells Download PDFInfo
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Abstract
The application relates to a culture medium and a culture method for retinal pigment epithelial cells, in particular to a culture medium containing a myostatin II ATPase inhibitor, a kit and a reagent combination containing the myostatin II ATPase inhibitor, and a method for culturing the retinal pigment epithelial cells by using the culture medium.
Description
Technical Field
The application relates to a retinal pigment epithelial cell culture technology, in particular to a culture medium and a culture method of retinal pigment epithelial cells. In particular, the application relates to a culture medium containing a myostatin II ATPase inhibitor, a kit containing the myostatin II ATPase inhibitor, a reagent combination, and a method for culturing retinal pigment epithelial cells using the culture medium.
Background
Dry age-related macular degeneration (AMD), which is caused by degeneration or damage to the Retinal Pigment Epithelium (RPE) cells, is one of the leading causes of blindness in the elderly, and, by incomplete statistics, severely affects the quality of life of 3-5 million people worldwide and cannot be effectively treated by traditional pharmaceutical or surgical methods.
Clinical studies have shown that RPE cell transplantation is a very effective means of treating AMD. In 2012, the RPE cell obtained by hESC induced differentiation was applied to clinical treatment of AMD for the first time by Steven Schwartz in the retina department of Ocular institute of Oceania, university of California, los Angeles, and a certain effect was obtained, so that a way was opened up for clinical application of the RPE cell obtained by differentiation of pluripotent stem cells. The university of heaven and earth labor, 6 months and 2013, approved the application of professor gaqiao yao (Masayo Takahashi) in clinical research on retinal regeneration using Induced Pluripotent Stem Cells (iPSCs). The first patient was followed up for 22 months in the high bridge group and the best corrected vision was improved in some patients. The research obtains a culture medium and a culture method which can expand the RPE cells, provides more possibilities for the expansion and the culture of the RPE cells, and widens the seed resource cells for the clinical application of the RPE cells.
Disclosure of Invention
The inventor discovers through a great deal of creative work that the amplification efficiency of retinal pigment epithelial cells can be obviously improved by adjusting the components of a culture medium and adding a myostatin II ATPase inhibitor Blebbistatin. The method lays a foundation for the subsequent cell therapy of stably obtaining high-quality retinal pigment epithelial cells and realizing diseases caused by the defects of various retinal pigment epithelial cells such as macular degeneration and the like.
Culture method
Accordingly, in one aspect, the present application provides a method for culturing a retinal pigment epithelial cell, comprising the step of culturing the retinal pigment epithelial cell in a medium containing a Myosin II ATPase inhibitor;
wherein the concentration of the Myosin II ATPase inhibitor is less than 5.0. Mu.M.
In certain embodiments, the myostatin II ATPase inhibitor is selected from Blebbistatin, a derivative of Blebbistatin (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof.
In certain embodiments, the myostatin II ATPase inhibitor is Blebbistatin.
In certain embodiments, the concentration of the Myosin II ATPase inhibitor is not less than 0.1. Mu.M, e.g., not less than 0.5. Mu.M, not less than 1.0. Mu.M.
In certain embodiments, the concentration of the Myosin II ATPase inhibitor is 0.1-4.0. Mu.M (e.g., 0.1-1.0. Mu.M, 0.2-4.0. Mu.M, 0.2-1.0. Mu.M, 0.5-4.0. Mu.M, 0.5-1.0. Mu.M, 0.5. Mu.M, 1.0. Mu.M).
In certain embodiments, the myostatin II ATPase inhibitor is Blebbistatin, and the concentration of Blebbistatin is 0.1-4.0. Mu.M (e.g., 0.1-1.0. Mu.M, 0.2-4.0. Mu.M, 0.2-1.0. Mu.M, 0.5-4.0. Mu.M, 0.5-1.0. Mu.M, 0.5. Mu.M, 1.0. Mu.M).
In certain embodiments, the medium is a medium suitable for the culture of retinal pigment epithelial cells.
In certain embodiments, the culture medium further comprises a basal medium supplemented with an agent selected from the group consisting of: one or more serum-free substitutes, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, a NEAA additive, KOSR, and any combination thereof.
In certain embodiments, the basal medium is selected from the group consisting of: KO-DMEM, DMEM/F12, neurobasal, neural industry Media.
In certain embodiments, the medium further comprises KO-DMEM medium supplemented with NEAA supplement, KOSR and the stabilized dipeptide of L-alanyl-L-glutamine.
In certain embodiments, the medium further comprises: 70% -90% KO-DMEM medium, 10% -30% KOSR, 0.5% -2% NEAA and 0.5% -2% L-alanyl-L-glutamine stabilized dipeptide.
In certain embodiments, the medium further comprises: 70% -90% CTS KnockOut DMEM, 10% -30% CTS KOSR, 0.5% -2% NEAA, and 0.5% -2% CTS-GlutaMAX-I.
In certain embodiments, the medium further comprises: 78% CTS KnockOut DMEM +20% CTS KOSR +1% NEAA +1% CTS-GlutaMAX-I.
In certain embodiments, the medium further comprises EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
Reagent kit
In another aspect, the present application also provides a kit comprising an inhibitor of myostatin II ATPase, and a culture medium.
In certain embodiments, the medium is suitable for the culture of retinal pigment epithelial cells.
In certain embodiments, the myostatin II ATPase inhibitor is selected from Blebbistatin, a Blebbistatin derivative (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof;
preferably, the myostatin II ATPase inhibitor is Blebbistatin.
In certain embodiments, the kit further comprises an EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
In certain embodiments, the medium is a basal medium supplemented with an agent selected from the group consisting of: one or more serum-free substitutes, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, a NEAA additive, KOSR, and any combination thereof.
In certain embodiments, the basal medium is selected from the group consisting of: KO-DMEM, DMEM/F12, neurobasal, neural indication Media.
In certain embodiments, the medium is KO-DMEM medium supplemented with a NEAA supplement, KOSR, and a stabilized dipeptide of L-alanyl-L-glutamine.
In certain embodiments, the medium comprises: 70% -90% CTS KnockOut DMEM, 10% -30% CTS KOSR, 0.5% -2% NEAA, and 0.5% -2% CTS-GlutaMAX-I.
In certain embodiments, the medium comprises: 70% -90% KO-DMEM medium, 10% -30% KOSR, 0.5% -2% NEAA and 0.5% -2% L-alanyl-L-glutamine stabilized dipeptide.
In certain embodiments, the medium comprises: 78% CTS KnockOut DMEM +20% CTS KOSR +1% NEAA +1% CTS-GlutaMAX-I.
Culture medium
In another aspect, the present application also provides a culture medium comprising an inhibitor of Myosin II ATPase, wherein the concentration of the inhibitor of Myosin II ATPase is less than 5.0 μ M.
In certain embodiments, the concentration of the Myosin II ATPase inhibitor is not less than 0.1. Mu.M, e.g., not less than 0.5. Mu.M, not less than 1.0. Mu.M.
In certain embodiments, the concentration of the Myosin II ATPase inhibitor is 0.1-4.0. Mu.M (e.g., 0.1-1.0. Mu.M, 0.2-4.0. Mu.M, 0.2-1.0. Mu.M, 0.5-4.0. Mu.M, 0.5-1.0. Mu.M, 0.5. Mu.M, 1.0. Mu.M).
In certain embodiments, the myostatin II ATPase inhibitor is selected from Blebbistatin, a derivative of Blebbistatin (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof.
Preferably, the myostatin II ATPase inhibitor is Blebbistatin.
In certain embodiments, the myostatin II ATPase inhibitor is Blebbistatin, and the concentration of Blebbistatin is 0.1-4.0 μ M (e.g., 0.1-1.0 μ M, 0.2-4.0 μ M, 0.2-1.0 μ M, 0.5-4.0 μ M, 0.5-1.0 μ M, 0.5 μ M, 1.0 μ M).
In certain embodiments, the medium is a medium suitable for the culture of retinal pigment epithelial cells.
In certain embodiments, the culture medium further comprises a basal medium supplemented with an agent selected from the group consisting of: one or more serum-free substitutes, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, a NEAA additive, KOSR, and any combination thereof.
In certain embodiments, the basal medium is selected from the group consisting of: KO-DMEM, DMEM/F12, neurobasal, neural industry Media.
In certain embodiments, the medium further comprises KO-DMEM medium supplemented with NEAA supplement, KOSR and the stabilized dipeptide of L-alanyl-L-glutamine.
In certain embodiments, the medium further comprises: 70% -90% KO-DMEM medium, 10% -30% KOSR, 0.5% -2% NEAA and 0.5% -2% stabilized dipeptide of L-alanyl-L-glutamine.
In certain embodiments, the medium further comprises: 70% -90% CTS KnockOut DMEM, 10% -30% CTS KOSR, 0.5% -2% NEAA, and 0.5% -2% CTS-GlutaMAX-I.
In certain embodiments, the medium further comprises: 78% CTS KnockOut DMEM +20% CTS KOSR +1% NEAA +1% CTS-GlutaMAX-I.
In certain embodiments, the medium further comprises EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
Reagent combination
In another aspect, the present application also provides a combination of reagents comprising: (II) a Myosin II ATPase inhibitor, (II) a basal medium, and (iii) an additive selected from the group consisting of: one or more serum-free substitutes, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, a NEAA additive, a KOSR, and any combination thereof.
In certain embodiments, the myostatin II ATPase inhibitor is selected from the group consisting of Blebbistatin, blebbistatin derivatives (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof. In certain embodiments, the myostatin II ATPase inhibitor is Blebbistatin.
In certain embodiments, the basal medium is selected from the group consisting of: KO-DMEM, DMEM/F12, neurobasal, neural indication Media.
In certain embodiments, the combination of agents further comprises (iv) an EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
Use of
In another aspect, the present application also provides the use of the kit, medium or reagent combination as described above for culturing retinal pigment epithelial cells.
In certain embodiments, the present application further provides the following exemplary aspects:
in a first aspect, the present application provides a composition comprising one or more additives that maintain and/or enhance the activity and/or function of retinal pigment epithelial cells, EGF, bFGF, and a Myosin II ATPase inhibitor.
In certain embodiments, the compositions contain a Myosin II ATPase inhibitor, optionally further containing a ROCK inhibitor;
in certain embodiments, the compositions contain an EGF, myosin II ATPase inhibitor, optionally further containing a ROCK inhibitor;
in certain embodiments, the compositions comprise bFGF and a Myosin II ATPase inhibitor, optionally further comprising a ROCK inhibitor;
in certain embodiments, the compositions comprise EGF, bFGF, and a Myosin II ATPase inhibitor, optionally further comprising a ROCK inhibitor;
in embodiments of the present application, the Myosin II ATPase inhibitor is a Myosin II ATPase inhibitor. In certain embodiments, the myostatin II ATPase inhibitor is selected from Blebbistatin and derivatives thereof. For example, blebbistatin, i.e., (S) - (-) -Blebbistatin, having a structural formula
Such as (S) - (-) -Blebbistatin O-Benzoate having the formula->
In a second aspect, the present application provides a culture medium comprising the aforementioned composition, and a basal medium.
In certain embodiments, the basal medium is suitable for the culture of retinal pigment epithelial cells.
In certain embodiments, the basal medium is selected from KODMEM medium, DMEM/F12 medium.
In certain embodiments, the basal medium is KODMEM medium.
In certain embodiments, the basal medium is a basal medium supplemented with: one or more serum-free substitutes, glutamine or stabilized dipeptides of L-alanyl-L-glutamine.
In certain embodiments, the basal medium is a basal medium supplemented with: n2 additive and a stabilized dipeptide of L-alanyl-L-glutamine.
In certain embodiments, the basal medium consists of: 78% CTS TM KnockOut TM DMEM+20％CTS TM KOSR+1％CTS TM NEAA+1％CTS-GlutaMAX TM -I。
In certain embodiments, the concentration of the EGF, bFGF and Myosin II ATPase inhibitor may be 5-20 μ M, but is not limited thereto. In some embodiments, the concentration of the myostatin II ATPase inhibitor (e.g., blebbistatin) is 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M, 11 μ M, 12 μ M, 13 μ M, 14 μ M, 15 μ M, 16 μ M, 17 μ M, 18 μ M, 19 μ M, or 20 μ M. The preferred concentration is 10. Mu.M. In some specific embodiments, the concentration of the EGF is 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, 9. Mu.M, 10. Mu.M, 11. Mu.M, 12. Mu.M, 13. Mu.M, 14. Mu.M, 15. Mu.M, 16. Mu.M, 17. Mu.M, 18. Mu.M, 19. Mu.M, or 20. Mu.M. The preferred concentration is 10. Mu.M. In some embodiments, the concentration of bFGF is 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, 9. Mu.M, 10. Mu.M, 11. Mu.M, 12. Mu.M, 13. Mu.M, 14. Mu.M, 15. Mu.M, 16. Mu.M, 17. Mu.M, 18. Mu.M, 19. Mu.M, or 20. Mu.M. The preferred concentration is 10. Mu.M.
In certain embodiments, the concentration of the ROCK inhibitor can be from 1 to 50 μ M, but is not limited thereto. In certain embodiments, the concentration of the ROCK inhibitor is 1 to 30 μ M, e.g., 5 to 30 μ M. In some embodiments, the ROCK inhibitor (e.g., Y-27632) is at a concentration of 1 μ Μ, 2 μ Μ, 3 μ Μ, 4 μ Μ,5 μ Μ, 6 μ Μ, 7 μ Μ, 8 μ Μ, 9 μ Μ, 10 μ Μ, 11 μ Μ, 12 μ Μ, 13 μ Μ, 14 μ Μ, 15 μ Μ, 16 μ Μ, 17 μ Μ, 18 μ Μ, 19 μ Μ, 20 μ Μ, 21 μ Μ, 22 μ Μ, 23 μ Μ, 24 μ Μ, 25 μ Μ, 26 μ Μ, 27 μ Μ, 28 μ Μ, 29 μ Μ or 30 μ Μ. The preferred concentration is 10. Mu.M.
In certain embodiments, the medium is suitable for culture, e.g., expansion culture, of retinal pigment epithelial cells.
In certain embodiments, the medium is suitable for culture of retinal pigment epithelial cells that expand retinal pigment epithelial cells by at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold.
In certain embodiments, the basal media is DMEM/F12 and KODMEM.
In certain embodiments, the medium contains 78% KO-DMEM/F12, 20% KOSR,1% NEAA,1% Glutmax additive, and 5-20 μ M Blebbistatin.
In certain embodiments, the medium contains 78% KO-DMEM/F12, 20% KOSR,1% NEAA,1% Glutmax additive, 5-20 μ M EGF, and 5-20 μ M Blebbistatin.
In certain embodiments, the medium contains 78% KO-DMEM/F12, 20% KOSR,1% NEAA,1% Glutmax additive, 5-20 μ M bFGF, and 5-20 μ M Blebbistatin.
In certain embodiments, the medium is contained 78% KO-DMEM/F12, 20% KOSR,1% NEAA,1% Glutmax additive, 5-20 μ M EGF,5-20 μ M MbFGF and 5-20 μ M Blebbistatin.
In another aspect, the present application provides a method for maintaining or increasing the number of cells in vitro, comprising the step of culturing said cells in the aforementioned culture medium; or the present application provides the use of the aforementioned culture medium for maintaining or increasing the number of cells in vitro.
In certain embodiments, the method of culturing is: preparing the cells into a single cell suspension at 2X 10 4 /cm 2 -6×10 4 /cm 2 Is inoculated to the culture medium and cultured for 7 to 14 days for 1 passage, wherein the culture conditions of each passage are the same. In certain embodiments, at 1 × 10 5 /cm 2 Is inoculated at a density of (a).
In certain embodiments, the adherent culture conditions are: preparing the cells into a single cell suspension at 2X 10 4 /cm 2 -6×10 4 /cm 2 Is inoculated to the culture medium and is passaged for 1 time in 7 to 14 days in adherence, wherein the culture conditions of each generation are the same. In certain embodiments, at 1 × 10 5 /cm 2 Is inoculated at a density of (a).
In certain embodiments, the cell is a retinal pigment epithelial cell.
In certain embodiments, the amplified cells are identical to the primary cell genome.
In another aspect, the present application provides a cell or population of cells, the method of preparation and expansion of which is as any of the methods described above. In another aspect, the application provides a cell or population of cells, wherein >60%, >65%, >70%, >75% >, 80%, >85%, >90%, >95%, >98%, or >99% of the cells specifically express at least one or more of the following markers: RPE65, OTX2, MITF, ZO-1, BEST1, and CRALBP.
In certain embodiments, the method of preparing said cell or population of cells comprises the steps defined in the method of maintaining or increasing the number of cells in vitro as described in the previous aspect.
In another aspect, the present application provides a cell or population of cells prepared by the method of:
(1) Culturing the retinal pigment epithelial cells obtained by amplification in a first culture medium for 7-14 days;
(2) Culturing the cells obtained in step (1) in a second culture medium to obtain the cells or cell groups; wherein the content of the first and second substances,
the first culture medium is a basal culture medium supplemented with: NEAA, glutmax, serum replacement, and Y-27632; preferably, the first culture medium is a basal medium supplemented with: 0.5% -2% NEAA, 0.5% -2% Glutmax, 10-20% KOSR and 1-30mM Y-27632; preferably, the first culture medium is a basal medium supplemented with: 1% NEAA,1% Glutmax, 20% KOSR and 10mM Y-27632;
the second culture medium is a basal culture medium supplemented with: NEAA, glutmax, serum replacement, blebbistatin, and Y-27632; preferably, the second medium is a basal medium supplemented with: 0.5% -2% NEAA, 0.5% -2% Glutmax, 10-20% KOSR, 5-20 μ M Blebbistatin and 1-30mM Y-27632; preferably, the second medium is a basal medium supplemented with: 1% NEAA,1% Glutmax, 20% KOSR, in combination with EGF or bFGF containing 10. Mu.M Blebbistatin, and 10mM Y-27632;
in certain embodiments, the retinal pigment obtained by amplification is applied toMaking single cell suspension of the skin cells at 1 × 10 4 /cm 2 -1×10 6 /cm 2 (e.g., 1X 10) 4 /cm 2 、5×10 4 /cm 2 、1×10 5 /cm 2 、5×10 4 /cm 2 Or 1X 10 6 /cm 2 ) Is inoculated to the first medium.
In another aspect, the present application also provides a pharmaceutical composition comprising a cell or cell population according to any one of the above.
In another aspect, the present application also provides the use of a cell or population of cells according to any one of the above aspects in the manufacture of a medicament for the treatment of a retinal disease (e.g. macular degeneration).
Definition of terms
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
When the terms "for example," "such as," "such," "including," "containing," or variants thereof are used herein, these terms are not to be construed as limiting terms, but rather are to be construed to mean "without limitation" or "without limitation".
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
As used herein, the term "retinal pigment epithelial cells" refers to hexagonal single-layered epithelial cells containing pigment particles that are obtained by primary isolation or differentiation, transdifferentiation, etc. of human retina and can form tight junctions.
As used herein, the term "inhibit", "block" or "block" refers to a decrease in the activity of a particular signaling pathway of a cell treated with a compound (i.e., inhibitor).
Advantageous effects of the invention
According to the application, the components of the culture medium are adjusted, and the myostatin II ATPase inhibitor Blebbistatin is added, so that the amplification efficiency of the retinal pigment epithelial cells can be obviously improved, better cell resources are provided for scientific research or commercialization, and a foundation is laid for realizing the cell therapy of diseases caused by macular degeneration and other various retinal pigment epithelial cell defects.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention and do not constitute a limitation of the invention. In the drawings:
FIG. 1 shows the procedure of obtaining retinal pigment epithelial cells by differentiation from human embryonic stem cells and performing amplification culture.
FIG. 2 shows the effect of different concentrations of Blebbistatin (0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M) on retinal pigment epithelial cell proliferation.
FIG. 3 shows the state of retinal pigment epithelial cells cultured at various concentrations of Blebbistatin (0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
The embodiment is as follows: effect of Blebbistatin on retinal pigment epithelial cells
1. Reagents used for the experiment:
(1) Blebbistatin solution: working concentrations were 1. Mu.M and 10. Mu.M. Can be stored at 4 deg.C for two weeks.
(2) Preparing a basic culture medium:
78％CTS TM KnockOut TM DMEM+20％CTS TM KOSR+1％CTS TM NEAA+1％CTS-GlutaMAX TM -I
2. the experimental method for the amplification of the retinal pigment epithelial cells comprises the following steps:
inducing and differentiating the human embryonic stem cells and obtaining retinal pigment epithelial cells for subsequent amplification and cryopreservation experiments. Human embryonic stem cells are derived from national stem cell resources, and commercially available human embryonic stem cell lines can also be used. Methods for inducing retinal pigment epithelial Cells from human embryonic stem Cells can be referenced to the prior art, such as Marutotti J, wahlin K, gorrell D, bhutto I, lutty G, zack DJ (2013) A simple and scalable process for the differentiation of retinal pigment epithelial Cells, stem Cells Transl Med 2.
(1) The specific acquisition method of the retinal pigment epithelial cells comprises the following steps:
1) Preparing a Vitronectin substrate: at room temperature, 6mL of DPBS was pipetted into a 15mL centrifuge tube, 60. Mu.L of Vitronectin (purchased from Gibco, cat # A14700) was pipetted into the DPBS, and the mixture was blown and beaten 10 times by a pipette to obtain Vitronectin matrix, which was prepared as it is. Adding 1ml of prepared matrix into each hole of a six-hole plate, and incubating for 1h at room temperature for later use.
2) Human embryonic stem cells were seeded into stromal-coated well plates. Charging at 37 deg.C, 5% 2 Culturing in an incubator. Culturing for 7-10 days, changing into RPE differentiation culture medium when the clone fusion degree reaches 95%, and obtaining human embryonic stem cell differentiated retinal pigment epithelial cells after several days.
(2) Retinal pigment epithelial cell expansion experiment:
1) Preparing a Vitronectin substrate: at room temperature, sucking 6mL of DPBS and adding into a 15mL centrifuge tube, sucking 60 microliter of Vitronectin and adding into the DPBS, blowing and beating for 10 times by a pipetting gun and mixing uniformly to obtain a Vitronectin matrix, wherein the Vitronectin matrix is prepared at present. Adding 1ml of prepared matrix into each hole of a six-hole plate, and incubating for 1h at room temperature for later use.
2) Digesting retinal pigment epithelial cells into single cell suspension with TrypLE, adding 10 μ L of the suspension into 10 μ LMixing the AO-PI solution evenly, and counting by using a Countstar cell analyzer; then, the Vitronectin substrate was aspirated and discarded at a density of 1X 10 5 /cm 2 Re-inoculation with 2D into 24-well plates containing different concentrations of amplification medium of Blebbistatin (purchased from MedChenexpress, MCE, cat.: # HY-13441/CS-4983), at 37 ℃ with 5% CO 2 And (5) culturing in an incubator.
3) After culturing for a certain number of days, observing the cell morphology of each group by a microscope; then, a single cell suspension was obtained by digesting a part of the cells of each group with TrypLE, 10. Mu.L of the suspension was added to 10. Mu.L of AO-PI solution and mixed, and the mixture was counted by a Countstar cell analyzer to draw a cell proliferation curve.
3. Results of retinal pigment epithelial cell expansion experiments:
the effect of Blebbistatin on the proliferation efficiency of retinal pigment epithelial cells is shown in FIGS. 2-3. Among them, FIG. 2 shows the effect of various concentrations of Blebbistatin (0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M) on retinal pigment epithelial cell proliferation; FIG. 3 shows the state of retinal pigment epithelial cells cultured at various concentrations of Blebbistatin (0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M).
As can be seen from the results of FIGS. 2-3, RPE cells proliferated more rapidly in the amplification medium containing a relatively low concentration of Blebbistatin (e.g., 0.1. Mu.M, 0.5. Mu.M, or 1. Mu.M Blebbistatin) relative to the amplification medium without Blebbistatin; in expanded media containing a relatively high concentration of Blebbistatin (e.g., 5. Mu.M or 10. Mu.M Blebbistatin), RPE cell proliferation is slowed.
Furthermore, in the amplification medium containing a relatively low concentration of Blebbistatin (e.g., 0.1. Mu.M, 0.5. Mu.M, or 1. Mu.M Blebbistatin), the RPE cell morphology was consistent with the control group; in contrast, in the amplification medium containing a relatively high concentration of Blebbistatin (e.g., 5. Mu.M or 10. Mu.M Blebbistatin), RPE cell morphology changed significantly, cells became smaller, and cell boundaries became gradually blurred.
Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.
Claims (15)
1. A method for culturing a retinal pigment epithelial cell, comprising the step of culturing the retinal pigment epithelial cell in a medium containing a myostatin IIATPase inhibitor;
wherein the concentration of the Myosin IIATPase inhibitor is less than 5.0 μ M.
2. The method of claim 1, wherein said myostatin atpase inhibitor is selected from Blebbistatin, a Blebbistatin derivative (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof;
preferably, the myostatin IIATPase inhibitor is Blebbistatin.
3. The method of claim 1 or 2, wherein the concentration of the myosiniatpase inhibitor is not less than 0.1 μ Μ, e.g., not less than 0.5 μ Μ, not less than 1.0 μ Μ;
preferably, the concentration of the Myosin IIATPase inhibitor is 0.1-4.0. Mu.M (e.g., 0.1-1.0. Mu.M, 0.2-4.0. Mu.M, 0.2-1.0. Mu.M, 0.5-4.0. Mu.M, 0.5-1.0. Mu.M, 0.5. Mu.M, 1.0. Mu.M).
4. The method of any one of claims 1-3, wherein the culture medium is a medium suitable for the culture of retinal pigment epithelial cells;
preferably, the medium further comprises a basal medium supplemented with a substance selected from the group consisting of: one or more serum-free substitutes, stabilized dipeptides of glutamine or L-alanyl-L-glutamine, NEAA additive, KOSR, and any combination thereof;
preferably, the basal medium is selected from: KO-DMEM, DMEM/F12, neurobasal, neural indication Media;
preferably, the medium further comprises KO-DMEM medium supplemented with NEAA supplement, KOSR and the stabilized dipeptide of L-alanyl-L-glutamine.
5. The method of any one of claims 1-4, wherein the culture medium further comprises EGF, bFGF and/or a ROCK inhibitor (e.g., Y-27632).
6. A kit comprising an inhibitor of myostatin atpase, and a culture medium;
preferably, the medium is suitable for the culture of retinal pigment epithelial cells.
7. The kit of claim 6, wherein said MyosinIIATPase inhibitor is selected from the group consisting of Blebbistatin, a Blebbistatin derivative (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof;
preferably, the myostatin IIATPase inhibitor is Blebbistatin.
8. The kit of claim 6 or 7, further comprising an EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
9. The kit according to any one of claims 6 to 8, wherein the culture medium is a basal medium supplemented with a substance selected from the group consisting of: one or more serum-free substitutes, stabilized dipeptides of glutamine or L-alanyl-L-glutamine, NEAA additive, KOSR, and any combination thereof;
preferably, the basal medium is selected from: KO-DMEM, DMEM/F12, neurobasal, neural indication Media;
preferably, the medium is KO-DMEM medium supplemented with NEAA supplement, KOSR and a stabilized dipeptide of L-alanyl-L-glutamine.
10. A culture medium comprising an inhibitor of Myosin IIATPase, wherein the concentration of the inhibitor of Myosin IIATPase is less than 5.0 μ Μ;
preferably, the concentration of the Myosin II ATPase inhibitor is not less than 0.1. Mu.M, e.g., not less than 0.5. Mu.M, not less than 1.0. Mu.M;
preferably, the concentration of the Myosin II ATPase inhibitor is 0.1-4.0. Mu.M (e.g., 0.1-1.0. Mu.M, 0.2-4.0. Mu.M, 0.2-1.0. Mu.M, 0.5-4.0. Mu.M, 0.5-1.0. Mu.M, 0.5. Mu.M, 1.0. Mu.M).
11. The culture medium of claim 10, wherein the myostatin atpase inhibitor is selected from the group consisting of Blebbistatin, a Blebbistatin derivative (e.g., (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof;
preferably, the myostatin IIATPase inhibitor is Blebbistatin.
12. The medium according to claim 10 or 11, wherein the medium is a medium suitable for the culture of retinal pigment epithelial cells;
preferably, the culture medium further comprises a basal medium supplemented with an agent selected from the group consisting of: one or more serum-free substitutes, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, a NEAA additive, KOSR, and any combination thereof;
preferably, the basal medium is selected from: KO-DMEM, DMEM/F12, neurobasal, neural industry Media;
preferably, the medium further comprises KO-DMEM medium supplemented with NEAA supplement, KOSR and the stabilized dipeptide of L-alanyl-L-glutamine.
13. The culture medium of any one of claims 10-12, further comprising an EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
14. A combination of reagents comprising: (ii) a MyosinIIATPase inhibitor, (ii) a basal medium, and (iii) an additive selected from the group consisting of: one or more serum-free substitutes, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, a NEAA additive, KOSR, and any combination thereof;
preferably, the MyosinIIATPase inhibitor is selected from Blebbistatin, a Blebbistatin derivative (e.g. (S) - (-) -Blebbistatin O-Benzoate), and any combination thereof; preferably, the myostatin IIATPase inhibitor is Blebbistatin;
preferably, the basal medium is selected from: KO-DMEM, DMEM/F12, neurobasal, neural indication Media;
preferably, the combination of agents further comprises (iv) an EGF, bFGF and/or ROCK inhibitor (e.g., Y-27632).
15. Use of a kit according to any one of claims 6 to 9 or a culture medium according to any one of claims 10 to 13 or a combination of reagents according to claim 14 for culturing retinal pigment epithelial cells.
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