CN113481158A - Preparation method of non-stent retinal pigment epithelial cell sheet - Google Patents
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Abstract
The invention provides a method for differentiating pluripotent stem cells into retinal pigment epithelial cells through three-dimensional suspension culture, belonging to the technical field of biotechnology. The method comprises the following steps: digesting and resuspending the human induced pluripotent stem cells to obtain monodisperse cells; inoculating the scattered cells to a U/V type low-adhesion culture material, and performing suspension culture by sequentially using a retinal nerve progenitor cell culture solution, an RPE cell differentiation induction solution and an RPE cell maturation differentiation solution to obtain a hollow cell sphere formed by the single-layer retinal pigment epithelial cells; cutting and spreading the obtained hollow cell spheres to obtain the non-stent retinal pigment epithelial cell sheet. The RPE cell sheet obtained by the method has curvature and carries basement membrane tissues which are differentiated together with the RPE cells, so that no external scaffold material is needed when the RPE cell sheet is applied to clinical transplantation in the future, and immunological rejection can be avoided. The method has the advantages of good operability, simple induction process and high repeatability, and is convenient for accurate application in future clinic.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to a preparation method of a non-support retinal pigment epithelial cell sheet.
Background
The retina is a transparent thin film structure located inside the eyeball wall, is located on the inner surface of the choroid, and can be divided into 10 layers of structures, namely a retinal pigment epithelium layer, a photosensitive layer, an outer limiting membrane, an outer nuclear layer, an outer plexiform layer, an inner nuclear layer, an inner plexiform layer, a ganglion cell layer, a nerve fiber layer and an inner limiting membrane from outside to inside. Wherein the retinal pigment epithelial cells (RPE) are a layer of regularly arranged polygonal melanocytes adhered to retinal nerve cells. RPE cells cannot be regenerated and, once they die due to disease or injury, can be treated only by cell transplantation. However, the source of donor RPE cells is very limited, and the transplantation of RPE cells in sheets requires the support of exogenous materials, which is likely to cause immune rejection and complications.
Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells formed by somatic cells undergoing reprogramming dedifferentiation by transfer of transcription factors. With a strong self-renewal capacity and differentiation potential, iPSCs became a favorable tool for modeling and treatment of retinal diseases. Somatic cells from skin, blood or urine of a patient can be separated, reprogrammed to iPSCs, and then differentiated to retinal cell types for autologous transplantation of the patient to treat retinal diseases or establishment of individualized disease models, so that accurate treatment and mechanism exploration of the diseases are realized.
Organoids are aggregates of cell differentiation derived from induced pluripotent stem cells or embryonic stem cells that can recapitulate the structure, function, and genetic properties of body organs, and are an advantageous tool for studying and understanding organ development and the development of disease. Therefore, the retina organs have important application values in the aspects of retinal disease modeling, drug screening, cell therapy, gene editing and the like. In 2011, Sasai et al utilize embryonic stem cells to simulate the development process of the retina of an organism under the specific differentiation condition in vitro to obtain the tissue with the cup-shaped structure, and find that the embryonic stem cells can be self-assembled into the neuroepithelial tissue in polar distribution in a culture solution containing matrigel, then form the alveolar structure of the original retinal tissue, which is very similar to the alveolar structure in the development of the retina of the organism, and then the alveolar structure is turned outwards or inwards to form the cup-shaped structure, so that the differentiated retinal pigment layer and the neuroepithelial layer are distributed at corresponding positions and have biological characteristics. In 2013, Xiufeng et al found that human-derived induced pluripotent stem cells can differentiate to generate retina-like organs, which can further differentiate and develop photoreceptors with complete inner and outer segment structures and RPE cells capable of secreting melanin.
In conclusion, the method for preparing RPE cells in vitro by iPSCs remains to be improved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a non-stent retinal pigment epithelial cell sheet. The method induces the differentiation of the pluripotent stem cells into pigment epithelial cells by a three-dimensional suspension culture retina organoid technology, provides a new source approach for an autologous transplantable RPE cell sheet, and the RPE cell sheet does not need an exogenous scaffold material, improves the safety and effectiveness of clinical RPE cell transplantation, solves the potential safety hazard problem caused by the transplantation of the RPE cell sheet formed by the scaffold material in the prior art, and provides a new technical scheme for treating the RPE defect eye diseases by utilizing the RPE cell sheet transplantation.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of a non-bracket retinal pigment epithelial cell sheet comprises the following steps:
digesting and resuspending induced pluripotent stem cells to obtain monodisperse cells;
inoculating the monodisperse cells obtained in the step one to a U/V type low-adhesion culture material, adding a retinal nerve progenitor cell culture solution, and performing suspension culture to obtain cell balls formed by inducing the self-assembly of the pluripotent stem cells;
transferring the cell balls obtained in the step II to a flat-bottom low-adhesion culture plate, adding an RPE cell differentiation inducing liquid, and continuing suspension culture to obtain cell balls formed by immature retinal pigment epithelial cells;
transferring the cell balls obtained in the step (III) to a new flat-bottom low-adhesion culture plate, adding RPE cell maturation differentiation liquid, and continuing suspension culture to obtain hollow cell balls formed by single-layer retinal pigment epithelial cells; the retinal pigment epithelial cells are black, regularly arranged, and polygonal, and the inner surface of the retinal pigment epithelial cell layer carries basement membrane tissue.
Fifthly, cutting and spreading the hollow cell spheres obtained in the step IV to obtain the non-support retinal pigment epithelial cell sheet.
Further, in the step (i), the induced pluripotent stem cells are human induced pluripotent stem cells.
Furthermore, in the step I, a reagent used for digestion is an Ethylene Diamine Tetraacetic Acid (EDTA) solution of 0.4-0.6 mmol/L. The ethylene diamine tetraacetic acid is an excellent calcium and magnesium ion chelating agent solvent, and can act on cells and an extracellular matrix to digest human induced pluripotent stem cells.
Furthermore, in the step (i), the reagent used for digestion is 0.5mmol/L of ethylenediamine tetraacetic acid solution.
Further, in the step (i), the reagent used for resuspending is a retinal neural progenitor cell culture solution.
Further, in the second step, the inoculation amount of the inoculation is (1-2) multiplied by 105cells/mL broth meter.
Furthermore, in the second step, the inoculation amount is 120000 cells/mL culture solution.
Further, in the second step, the retinal neural progenitor cell culture solution is Ham's F12 culture solution containing 10% by volume of serum replacement (KSR), 1% by volume of lipid concentrate, 1% by volume of streptomycin mixture and 450 μmol/L of thioglycerol.
Further, in the second step, the suspension culture time is 15-20 days, and BMP4 human recombinant protein is added when the culture is carried out for 5-7 days; the addition amount of the BMP4 human recombinant protein is 50-60 ng/mL.
Furthermore, in the second step, the suspension culture time is 18 days, and BMP4 human recombinant protein is added when the culture is carried out till the 6 th day; the addition amount of the BMP4 human recombinant protein is 55 ng/mL.
Further, in the third step, the RPE cell differentiation-inducing solution is DMEM/F12 medium containing 1% by volume of N2 cell culture supplement and 1% by volume of streptomycin mixed solution.
Further, in the third step, the suspension culture time is 5-10 days, and CHIR99021 and SU54026 are added when the suspension culture is carried out for 18-24 days; the addition amount of the CHIR99021 is 2-4 mu mol/L of the final concentration of the CHIR99021 in the system, and the addition amount of the SU5402 is 4-6 mu mol/L of the final concentration of the SU5402 in the system.
Furthermore, in the third step, the suspension culture time is 6 days, and CHIR99021 and SU54026 are added when the suspension culture is carried out until the 18 th day; the addition amount of the CHIR99021 is 3 mu mol/L according to the concentration of the CHIR99021 in the system, and the addition amount of the SU5402 is 5 mu mol/L according to the concentration of the SU5402 in the system.
Further, in the fourth step, the RPE cell maturation differentiation solution is DMEM/F12-Glutamax medium containing 1% by volume of N2 cell culture additive, 1% by volume of streptomycin and 1% by volume of Fetal Bovine Serum (FBS).
Further, in the fourth step, the suspension culture time is 8-15 days, and CHIR99021 is added when the suspension culture is carried out for 24-34 days; the addition amount of the CHIR99021 is 2-4 mu mol/L according to the final concentration of the CHIR99021 in a system.
Furthermore, in the step (iv), the suspension culture time is 10 days, and CHIR99021 is added when the suspension culture is carried out till the 24 th day; the addition amount of the CHIR99021 is calculated according to the concentration of the CHIR99021 in the system, which is 3 mu mol/L.
Further, in the second step, the third step and the fourth step, suspension culture is carried out in a carbon dioxide incubator at 37 ℃, and fresh culture solution is replaced by half every 2 days in the culture process.
Compared with the prior art, the invention has the following advantages and effects:
the invention utilizes the suspension culture scheme of three-dimensional retina organoid to induce human iPSCs to be differentiated into the retina pigment epithelial cell sheet without the exogenous scaffold, and the technical method has the following beneficial effects:
1) in the differentiation process, a suspension three-dimensional retina organoid culture scheme is adopted to simulate the embryonic retina development process, so that the induction efficiency and the cell quality of RPE cells are improved;
2) the RPE cell slices obtained by induction have curvature and carry basement membrane tissues which are differentiated together with the RPE cells, so that the safety and operability of the future clinical transplantation application are improved;
3) the retinal pigment epithelial cell sheet obtained by induction does not need an exogenous scaffold material, so that immune rejection reaction of future clinical transplantation can be avoided;
4) the induction method has good operability, simple induction process and high repeatability, and is convenient for accurate application in future clinic.
Drawings
FIG. 1 is a schematic diagram of a method for inducing pluripotent stem cell iPSCs to differentiate into human RPE sheets by three-dimensional suspension; wherein, A is a small molecule induced differentiation flow chart; b is the cell bright field map at different culture time points.
FIG. 2 is a diagram of the specific protein expression result of iPSCs differentiation source RPE cell sheet identified by immunofluorescence.
FIG. 3 is a diagram of the result of identifying the specific gene expression of the iPSCs differentiation-derived RPE cell sheet by real-time fluorescent quantitative PCR.
FIG. 4 is a schematic diagram of a comparative example for inducing differentiation of pluripotent stem cell iPSCs into human RPE sheets; wherein A is a bright field image of the RPE cell sheet obtained by the method of comparative example 1; b is a bright field image of the cell sheet obtained by the RPE method of comparative example 2; c is the bright field pattern of the RPE cell sheet obtained in comparative example 3.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.
Some of the additives used in the examples below:
CHIR 99021: from seleck, molecular weight: 465.34, chemical name: 6((2((4- (2, 4-dichlorophenyl) -5- (4-methyl-1 h-imidozol-2-yl) pyrimidin-2-yl) amino) ethyl) amino) nicotinonitrile, formula: c22H18Cl2N8CAS number: 252917-06-09;
SU 5402: from seleck, molecular weight: 465.34, chemical name: 2- [ (1, 2-dihydro-2-oxo-3H-indol-3-ylidene) methyl]-4-methyl-1H-pyrrole-3-propionic acid of formula: c17H16N2O3CAS number: 215543-92-03.
Some of the biomaterials used in the examples below:
human Induced Pluripotent Stem Cell (iPSCs) cell line CJ 127-iPS: registration numbers for human pluripotent Stem Cell registry KLRMMEi001-A, http:// hPSwreg.eu have been disclosed in the literature "Guo Y, Zeng Q, Liu S, et al.
BMP4 human recombinant protein: product designation PHC9534 available from ThermoFisher;
rabbit polyclonal MITF antibody: purchased from Abcam, product cat No. ab 122982;
rabbit polyclonal Tyrosinase antibody: purchased from Abcam, product cat No. ab 170905;
murine monoclonal RPE65 antibody: purchased from Abcam, product cat No. ab 13826;
murine monoclonal ZO-1 antibody: product Cat #33-9100 from ThermoFisher;
murine monoclonal CRALBP antibodies: purchased from Abcam, product cat No. ab 15051;
FITC-labeled goat anti-rabbit IgG antibody: product designation a27034, available from ThermoFisher;
cy3 goat anti-mouse IgG antibody: available from ThermoFisher, product catalog number A28175.
The culture media and formulations used in the following examples:
retinal neural progenitor cell culture fluid: ham's F12 medium containing 10% by volume of KSR serum replacement (GIBCO, cat # 10828028), 1% by volume of lipid concentrate (chemical refined lipid concentrate) (ThermoFisher, 11905031), 1% by volume of penicillin mixed solution (100IU/mL penicillin and 100. mu.g/mL streptomycin), 450. mu. mol/L thioglycerol.
RPE cell differentiation-inducing solution: DMEM/F12 medium containing 1% by volume of N2 cell culture supplement and 1% by volume of penicillin mixed solution.
RPE cell maturation differentiation liquid: DMEM/F12-GlutaMax medium (DMEM/F12(PM150312) + 1% GlutaMax (PB180419)) containing 1% by volume of N2 cell culture additive, 1% by volume of streptomycin penicillin mixture, and 1% by volume of Fetal Bovine Serum (FBS).
Example 1
The method for inducing the differentiation of the pluripotent stem cells into the retinal pigment epithelial cells by three-dimensional suspension comprises the following steps:
(1) suspension induced differentiation of human-derived induced pluripotent stem cells into retinal pigment epithelial cell sheets:
digesting CJ127-iPS cells of a clone cluster sample by using 0.5mmol/L EDTA, and re-suspending the CJ127-iPS cells into monodisperse cells by using a retinal neural progenitor cell culture solution;
12000 single-dispersed iPSCs are inoculated on a V-shaped low-adhesion culture (Sumitomo Bakelite Co., Ltd., MS-9096V) material through counting, retinal nerve progenitor cell culture solution is used for suspension culture in a carbon dioxide incubator at 37 ℃ according to the proportion of 120000 cells/mL culture solution, and the cells are induced to be differentiated into retinal nerve bulbs; changing fresh culture medium half a day every 2 days, and adding BMP4 human recombinant protein with the final concentration of 55ng on the 6 th day of culture;
③ at the 18 th day of culture, transferring the suspension cell balls to a flat-bottom low-adhesion culture plate, continuing the suspension culture in a carbon dioxide incubator at 37 ℃ for 6 days by using an RPE cell differentiation inducing solution, changing fresh culture medium for half a day every 2 days, and adding CHIR99021 with the final concentration of 3 mu mol/L and SU5402 with the final concentration of 5 mu mol/L at the 18 th day of culture;
selecting cell balls with complete morphological results and black focus structures on the 24 th day of culture, transferring the cell balls to a new low-adhesion culture plate, continuing to perform suspension culture in the RPE cell maturation differentiation solution for 10 days, completely replacing a fresh culture medium every 2 days, and adding CHIR99021 with the final concentration of 3 mu mol/L on the 24 th day of culture.
Finally, a layer of black, regularly arranged, polygonal RPE cells is located on the outer surface of the cell sphere, and basement membrane tissue is located on the inner layer. And cutting off along the central axis of the hollow sphere by using a micro corneal scissors, and tiling to obtain an RPE cell sheet.
The results are shown in FIG. 1: after culturing on a low-adhesion V-shaped low-adhesion culture material for 18 days, iPSCs can be self-assembled to form a cell sphere structure, and after culturing in an RPE cell differentiation inducing liquid for 6 days, the cell sphere has a melanin focus and is complete in structure; after continuously culturing in RPE cell mature differentiation liquid for 10 days, RPE structural tissue gradually grows up and develops into a single-layer vacuole-like structure, the outer surface of the RPE structural tissue is a layer of black RPE cells which are regularly arranged and take the shape of a polygon, and the RPE structural tissue is cut along the central axis of the hollow sphere by using micro corneal scissors and is tiled to form RPE cell slices.
(2) And (3) immunofluorescence staining:
on the 34 th day of suspension organoid differentiation, the hollow RPE spheres were cut along the central axis, laid flat on a glass slide, washed 3 times with PBS for 5 minutes each time, added with 4% by volume paraformaldehyde and incubated for 15 minutes at room temperature; washing with PBS for 3 times, each time for 5 minutes, adding PBS containing 2% by mass and volume of bovine serum, and incubating for 15 minutes; the incubation was continued for 15 minutes with the addition of 0.2% by mass/volume Triton-X100 and 2% by mass/volume bovine serum in PBS. Then add primary antibody: rabbit polyclonal MITF antibody (diluted 1:500 with diluent), rabbit polyclonal tyrrose antibody (diluted 1:500 with diluent), mouse monoclonal RPE65 antibody (diluted 1:500 with diluent), mouse monoclonal ZO-1 antibody (diluted 1:500 with diluent), mouse monoclonal CRALBP antibody (diluted 1:500 with diluent), and incubation at 4 ℃ overnight; PBS washing for 3 times, each time for 5 minutes; adding a secondary antibody: FITC-labeled goat anti-rabbit IgG antibody (diluted 1:200 with diluent), Cy3 goat anti-mouse IgG antibody (diluted 1:200 with diluent) was incubated at room temperature for 2 hours. Then PBS wash 3 times, each time for 5 minutes; DAPI was added to stain cell nuclei for 15 minutes at room temperature, and finally PBS was washed 3 times, and photographs were observed under a fluorescent microscope after 5 minutes each.
The results are shown in FIG. 2: and (3) performing immunofluorescence staining identification on the RPE cell sheet after suspension differentiation, wherein the result shows that the cell sheet can positively express the specific marker protein of the RPE cell: MITF, ZO-1, Tyrprose, RPE65, and CRALBP.
(3) Real-time fluorescent quantitative PCR:
RPE cell sheet total RNA was extracted using Trizol method as described and OD value was measured to ensure OD of extracted RNA260/OD280The value is between 1.8 and 2.0. Total RNA was reverse transcribed into cDNA using a reverse transcription kit (Toyobo, Japan), reaction temperature was adjusted and system was referred to kit instructions. The product cDNA was used for real-time fluorescent quantitative PCR, and the reaction system was referred to the SYBR product (Toyobo, Japan) manual. The upper stage after the instant centrifugation, the reaction temperature of 94 ℃ for 2 minutes, the reaction for 35 cycles (94 ℃, 30 seconds, 59 ℃, 30 seconds, 72 ℃, 30 seconds), the reaction results by statistical analysis. The primers and their sequences used in the PCR process are shown below:
F-MITF：5’-AGCTTGTATCTCAGTTCCGC-3’；
R-MITF：5’-ATGGCTGGTGTCTGACTCAC-3’；
F-OTX2：5’-CCTCACTCGCCACATCTACT-3’；
R-OTX2：5’-GTTTGGAGGTGCAAAGTCGG-3’；
F-PEDF：5’-AGTGTGCAGGCTTAGAGGGAC-3’；
R-PEDF：5’-CCCGAGGAGGGCTCCAATG-3’；
F-RLBP1：5’-GGAAGTCACAACTTGGCCCT-3’；
R-RLBP1：5’-CCCCTTCTGACATGTTGCCT-3’；
F-RPE65：5’-CCACCTGTTTGATGGGCAAG-3’；
R-RPE65：5’-CAGGGATCTGGGAAAGCACA-3’；
F-TJP1：5’-AGCCATTCCCGAAGGAGTTG-3’；
R-TJP1：5’-ATCACAGTGTGGTAAGCGCA-3’；
F-PMEL17：5’-ACCCAGCTTATCATGCCTGT-3’；
R-PMEL17：5’-TGCTTCATAAGTCTGCGCCT-3’。
the results are shown in FIG. 3: according to the statistical analysis result of real-time fluorescent quantitative PCR data, after differentiation, the specific gene of the human iPSCs positively expresses the RPE cell: MITF, OTX2, PEDF, RLBP1, RPE65, TJP1, and PMEL 17.
In conclusion, the above experimental results suggest that human-derived induced pluripotent stem cells can be differentiated into retinal pigment epithelial cell sheets by a three-dimensional suspension spherical culture method.
Comparative example 1
With reference to the operation of example 1, the only difference is: on the 8 th day of culture, the suspension cell spheres were transferred to a common cell culture plate, the retinal neural progenitor cell culture solution was newly added for culture, and the culture plates used in the subsequent operations were changed to common cell culture plates.
As shown in FIG. 4A, the RPE cells were long fusiform in morphology, free of melanin, and hardly expanded to form a cell sheet-like structure.
Comparative example 2
With reference to the operation of example 1, the only difference is: on the 28 th day of culture, RPE tissues (black parts) in retinal organoid tissues were excised, transferred to a common cell culture plate, and cultured with addition of an RPE cell maturation differentiation medium.
As shown in fig. 4B, the differentiation results showed that RPE cells had a substantially intact morphological structure, but no melanin, and that some of the differentiated cells were apoptotic.
Comparative example 3
With reference to the operation of example 1, the only difference is: BMP4 is not added in the step (2) of human recombinant protein; CHIR99021 and SU5402 are not added in the step (3); in step (4), CHIR99021 was not added.
As shown in fig. 4C, the results showed that cell limbal cells began to apoptotic and shed on day 26 of differentiation and were difficult to differentiate into monolayer-like cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (10)
1. A preparation method of a non-bracket retinal pigment epithelial cell sheet is characterized in that: the method comprises the following steps:
digesting and resuspending induced pluripotent stem cells to obtain monodisperse cells;
inoculating the monodisperse cells obtained in the step one to a U/V type low-adhesion culture material, adding a retinal nerve progenitor cell culture solution, and performing suspension culture to obtain cell balls formed by inducing the self-assembly of the pluripotent stem cells;
transferring the cell balls obtained in the step II to a flat-bottom low-adhesion culture plate, adding an RPE cell differentiation inducing liquid, and continuing suspension culture to obtain cell balls formed by immature retinal pigment epithelial cells;
transferring the cell balls obtained in the step (III) to a new flat-bottom low-adhesion culture plate, adding RPE cell maturation differentiation liquid, and continuing suspension culture to obtain hollow cell balls formed by single-layer retinal pigment epithelial cells;
fifthly, cutting and spreading the hollow cell spheres obtained in the step IV to obtain the non-support retinal pigment epithelial cell sheet.
2. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the second step, the retinal nerve progenitor cell culture solution is Ham's F12 culture solution containing 10% by volume of serum substitute, 1% by volume of lipid concentrate, 1% by volume of penicillin streptomycin mixed solution and 450 μmol/L of thioglycerol.
3. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the third step, the RPE cell differentiation inducing solution is DMEM/F12 medium containing 1% by volume of N2 cell culture additive and 1% by volume of streptomycin mixture.
4. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the fourth step, the RPE cell maturation differentiation solution is a DMEM/F12-Glutamax culture medium containing 1% by volume of N2 cell culture additive, 1% by volume of streptomycin and 1% by volume of fetal bovine serum.
5. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the second step, the suspension culture time is 15-20 days, and BMP4 humanized recombinant protein is added when the culture is carried out for 5-7 days; the addition amount of the BMP4 human recombinant protein is 50-60 ng/mL;
in the third step, the suspension culture time is 5-10 days, and CHIR99021 and SU54026 are added when the suspension culture is carried out for 18-24 days; the addition amount of the CHIR99021 is 2-4 mu mol/L of the final concentration of the CHIR99021 in the system, and the addition amount of the SU5402 is 4-6 mu mol/L of the final concentration of the SU5402 in the system;
in the fourth step, the suspension culture time is 8-15 days, and CHIR99021 is added when the suspension culture is carried out for 24-34 days; the addition amount of the CHIR99021 is 2-4 mu mol/L according to the final concentration of the CHIR99021 in a system.
6. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 5, wherein:
in the second step, the suspension culture time is 18 days, and BMP4 human recombinant protein is added when the culture is carried out till the 6 th day; the addition amount of the BMP4 human recombinant protein is 55 ng/mL;
in the third step, the suspension culture time is 6 days, and CHIR99021 and SU54026 are added when the suspension culture is carried out until the 18 th day; the addition amount of the CHIR99021 is 3 mu mol/L according to the final concentration of the CHIR99021 in the system, and the addition amount of the SU5402 is 5 mu mol/L according to the final concentration of the SU5402 in the system;
in the fourth step, the suspension culture time is 10 days, and CHIR99021 is added when the suspension culture is carried out till the 24 th day; the addition amount of CHIR99021 is 3 mu mol/L according to the final concentration of the CHIR99021 in the system.
7. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the first step, a reagent used for digestion is an ethylene diamine tetraacetic acid solution with the concentration of 0.4-0.6 mmol/L;
in the step I, the reagent used for resuspending is a retinal neural progenitor cell culture solution.
8. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the second step, the inoculation amount of the inoculation is 1 multiplied by 105～2×105cells/mL broth meter.
9. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the second step, the third step and the fourth step, suspension culture is carried out in a carbon dioxide incubator at 37 ℃, and fresh culture solution is replaced by half every 2 days in the culture process.
10. The method for preparing a stentless retinal pigment epithelial cell sheet according to claim 1, wherein:
in the step I, the induced pluripotent stem cells are human induced pluripotent stem cells.
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