Method for isolating adherent culture retinal pigment epithelial cells

Abstract

The invention provides a method for separating and purifying adherent culture RPE cells and application of the retinal pigment epithelial cells obtained by the method in manufacturing medicines for treating or preventing retinal degenerative diseases.

Description

Technical Field

[0001] The invention relates to a method for separating adherent cultured retinal pigment epithelial cells and related uses. Background Art

[0002] Retinal pigment epithelium (RPE) cells are a single layer of pigmented cells between the choroid and the retina. They play an important role in maintaining visual function and regulating retinal metabolism. Disorder or damage of RPE cells can lead to retinal degeneration, loss of visual function and blindness.

[0003] Under physiological conditions, human RPE does not proliferate, and the RPE of deletion cannot be obtained by cell regeneration. The dysfunction, damage and loss of RPE cells are factors of many eye diseases and diseases (including age-related macular degeneration (AMD), hereditary macular degeneration (including Best's disease) and retinitis pigmentosa). The potential treatment for these diseases is to transplant RPE cells into the retina of people who need this treatment. It is speculated that supplementing RPE cells by transplantation can delay, prevent or reverse degeneration, improve retinal function and prevent blindness caused by this situation. The use of human embryonic stem cells and induced pluripotent stem cells to differentiate and produce RPE cells has brought new hope for the treatment of related diseases. However, existing research mostly focuses on how to improve the differentiation efficiency of RPE cells, and the separation of RPE cells produced by differentiation sources has not been studied in depth.

[0004] There is a need in the art for a method for effectively separating adherently cultured RPE cells, especially a separation method that can simultaneously maintain the integrity of RPE cells and do not damage the cells, and a method suitable for effectively separating RPE cells in the presence of foreign cells. Summary of the invention

[0005] The present invention adopts a single digestion reagent to achieve digestion separation to obtain high-purity RPE cells. The present invention proposes a digestion time and a two-step digestion method suitable for the passage of P0 generation RPE cells. The purity of the separated RPE cells can be effectively improved, and a certain impurity removal effect is achieved.

[0006] In one aspect, the present invention provides a method for isolating adherently cultured retinal pigment epithelial (RPE) cells, comprising:

[0007] (a) performing a two-step digestion process, wherein in the first step of digestion, the RPE cells are incubated with an enzyme solution comprising a digestive enzyme for a first period of time, the enzyme solution is then removed by aspiration, and in the second step of digestion, the RPE cells are incubated with a newly added enzyme solution comprising a digestive enzyme for a second period of time, wherein the first period of time and the second period of time are 15 min to 40 min in total;

[0008] (b) adding a neutralizing solution to neutralize the enzyme solution comprising the digestive enzyme;

[0009] (c) Aspirate the cells and collect the RPE cell suspension.

[0010] In some embodiments, the RPE cells are obtained by natural differentiation, induced differentiation, or passage.

[0011] In some embodiments, the RPE cells have been cultured adherently for 10-20 days after seeding.

[0012] In some embodiments, the digestive enzyme is trypsin. In some embodiments, the digestive enzyme is selected from TrypLE, trypsin and Dispase, preferably TrypLE. The enzyme solution containing the digestive enzyme can be commercially available or self-prepared, for example, it can be CTS TM TrypLE TM Select enzyme solution (purchased from Gibco, product number A1285901).

[0013] In some embodiments, the incubation is performed at 37°C.

[0014] In some embodiments, the first period of time is longer than or equal to the second period of time. In some embodiments, the first period of time is no more than 20 min. In some embodiments, the first period of time and the second period of time are both within the range of 5 min-15 min. In some embodiments, the first period of time is within the range of 10 min-15 min, and the second period of time is within the range of 5 min-10 min. In some embodiments, the first period of time and the second period of time are 15 min and 5 min, 5 min and 10 min, or 10 min and 10 min, respectively.

[0015] In some embodiments, cell aspiration is not performed during the first and second digestion steps.

[0016] In some embodiments, step (b) of the method neutralizes the enzyme solution comprising the digestive enzymes by adding DPBS solution.

[0017] In some embodiments, the method further comprises washing the cultured RPE cells prior to step (a).

[0018] In some embodiments, the method further comprises centrifuging and resuspending the cell suspension of step (c).

[0019] In some embodiments, the method further comprises filtering the cell suspension of step (c) to further remove foreign cells.

[0020] In one aspect, the invention provides a composition comprising retinal pigment epithelial cells obtained by the methods disclosed herein.

[0021] In one aspect, the present invention provides use of retinal pigment epithelial cells obtained by the method disclosed herein in the preparation of a drug for treating or preventing retinal degenerative diseases.

[0022] In some embodiments, the retinal degenerative disease is age-related macular degeneration or retinitis pigmentosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The growth of cells around the pigment foci at different culture times is shown (obvious distribution of miscells can be seen in the box area).

[0025] Figure 2 It shows the growth observation of P1 cells on the 4th day of culture (the box area indicates some visible miscellaneous cells).

[0026] Figure 3 Shows the purity detection chart of P1 generation cells by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0028] RPE cells themselves have strong adhesion, and their culture is usually adherent culture. Separating the RPE cells of adherent culture ideally needs to remove the adhesion in a non-damaged and convenient way for passage and proliferation. In the traditional method, the RPE cells of adherent culture still need to be blown and aspirated 5 to 10 times after digestion treatment during passage to collect the digested cells. On the other hand, due to the presence of miscellaneous cells usually in the RPE cells of differentiation source, it is expected to reduce the presence of miscellaneous cells while separating RPE cells (for example, for downstream passage). The present application utilizes the characteristics that the adhesion of RPE cells is stronger than miscellaneous cells, adopts a two-step digestion treatment method to separate the RPE cells of adherent culture, and obtains significantly improved RPE cell purity and viability.

[0029] RPE cells

[0030] The source of the RPE cell used in the present invention is not particularly limited. In some embodiments, RPE cells are differentiated from pluripotent stem cells, such as embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). In some embodiments, ESC is a mammalian ESC, and the mammal may be selected from people, cats, dogs, monkeys, pigs, cattle, horses, goats, sheep, rabbits, mice, rats, etc. The RPE cells may be naturally differentiated or induced to differentiate.

[0031] Differentiation obtains the process of RPE cell and is well known in the art.For example, obtaining RPE cell from pluripotent stem cell differentiation generally comprises: first pluripotent stem cell is differentiated into the pigmented focus with non-pigmented area around in RPE differentiation medium, separates pigmented focus (also referred to as " picking pigmented focus ") from non-pigmented area, and then the pigmented focus picked is further differentiated into RPE cell in RPE differentiation medium. Obvious pigmented focus feature can appear in RPE cell, and the RPE cell in the pigmented focus (preferably picking complete pigmented focus) can be picked by the mode of artificial mechanical separation. The RPE cell formed in the later stage of differentiation can gather to form pigmented focus, and obvious black (part immature RPE cells may not possess black) occurs, but miscellaneous cells often do not have color, and therefore can select the RPE cell in black area to go down to posterity as far as possible by the mode of selecting pigmented focus.

[0032] The RPE cells picked can be cultured in a variety of suitable culture media. The composition of these culture media is well known in the art, and can also be obtained commercially. The composition, culture method and culture conditions of the culture media used in this application are not particularly limited. Any culture media suitable for cultivating RPE cells known in the art can be used for the method of the present invention. The culture media can include DMEM, non-essential amino acids, serum substitutes, etc. that are often used in cell culture. The culture media can further add common supplements, such as vitamin C, glucose, etc.

[0033] In some embodiments, the selected pigment foci are inoculated into a culture plate with 10 to 30 pigment foci / well, for example 15 to 25 pigment foci / well, for example 20 pigment foci / well for culture. The culture plate is preferably pre-incubated with vitronectin (VTN-N) to promote the adherent culture of RPE cells.

[0034] In some embodiments, the RPE cells picked are cultured in culture medium for about 15 to 25 days, for example, about 18 to 22 days, for example, about 20 days, and then digested.Preferably, every 1 to 3 days, for example, every 1 to 2 days, for example, the culture medium is replaced with fresh culture medium every other day.

[0035] In some embodiments, the RPE cells are cultured in culture medium at a temperature of about 36.5°C-37.5°C, such as at 37°C.

[0036] Digestion separation method

[0037] A unique two-step digestion process is used in this application, which includes:

[0038] (a) a first digestion step: incubating the RPE cells with an enzyme solution comprising a digestive enzyme for a first period of time;

[0039] (b) removing the enzyme solution by suction;

[0040] (c) Second digestion step: The RPE cells are incubated with a newly added enzyme solution containing digestive enzymes for a second period of time.

[0041] As will be appreciated by those skilled in the art, the digestion time cannot be too long to avoid damage to cell viability. Preferably, the first period of time and the second period of time are 15 min-40 min in total, for example, 20 min, 30 min or 35 min. In some embodiments, the first period of time and the second period of time are both within the range of 5 min-15 min.

[0042] Due to the fact that a small amount of non-RPE cells will inevitably be carried when using a puncher and other artificial pigmented foci, the RPE cells picked usually have miscells. Miscells are produced in the differentiation process, and there is no color basically. According to its cell morphology, it is generally based on neuronal cells or fibroblasts. Miscells have a lower adhesion to the culture plate. Therefore, the intention of the first step digestion is to utilize the adhesion difference between miscells and RPE cells to remove miscells as much as possible, wherein miscells are delaminated and digested to a greater extent because of the lower adhesion. In contrast, the RPE cell itself has a very strong wall adhesion ability and is not easily digested. Therefore, when miscells are more, the length of the first period of time can be appropriately increased. In some embodiments, the first period of time is longer than the second period of time, such as the first period of time is in the range of 10min-15min, and the second period of time is in the range of 5min-10min. In a preferred embodiment, the first period of time is about 15min, and the second period of time is about 5min.

[0043] The digestive enzyme can be selected from, for example, trypsin, Dispase and TrypLE. TrypLE is a recombinant fungal trypsin-like protease, a recombinant enzyme obtained by microbial fermentation. The digestion kinetics and cleavage specificity of Tryple are similar to those of trypsin. TrypLE is a non-animal-derived digestive enzyme that can dissociate cultured cells and causes less damage to cells than trypsin. Dispase, also known as dispersase, is a non-specific metalloprotease that can be used to digest extracellular matrix from different tissues or organs, release and prepare primary single cells; or for cell harvesting or inoculation transfer in cell culture; it is also used to prevent accidental cell agglomeration during suspension cell culture. The enzyme solution containing the digestive enzyme can be self-prepared, for example, by dissolving trypsin in a suitable buffer, or it can be commercially available. In some embodiments, the digestive enzyme used is TrypLE. In some embodiments, the enzyme solution containing TrypLE used is CTS TM TrypLE TMSelect enzyme solution (purchased from Gibco, product number A1285901).

[0044] After completing the first step digestion of step (a), white suspension will be produced, which is mainly the cell debris produced after the assorted cells are digested. In some embodiments, the enzyme solution with this white suspension will be sucked off from the RPE cell in step (b). When sucking off the enzyme solution comprising digestive enzyme, the residue after the cell digestion will also be sucked off, thereby the efficient of the next step digestion will be increased. Preferably, when carrying out step (b), the blowing and suction operation of the cell is not adopted to reduce the damage to the RPE cell.

[0045] In step (c), the newly added enzyme solution containing digestive enzymes may be the same or different from the enzyme solution of step (a), preferably the same as the enzyme solution of step (a). The digestive enzyme may be, for example, TrypLE, trypsin, or Dispase. The enzyme solution containing digestive enzymes may be self-prepared, for example, by dissolving trypsin in a suitable buffer, or may be commercially available. In some embodiments, the newly added enzyme solution containing digestive enzymes is also CTS TM TrypLE TM (Gibco, Cat. No. A1285901).

[0046] Method of the present invention may further include after completing two-step digestion, adding neutralizing solution (also referred to as diluent) to neutralize or dilute the enzyme solution comprising digestive enzyme. This neutralization step can minimize the impact of residual digestive enzyme on cells, such as causing damage to cells, cell membrane rupture, affecting cell viability and activity, etc. The neutralizing solution can adopt conventional safe inorganic salt solution, such as PBS, DPBS (Dulbecco's Phosphate Buffered Saline), EBSS (Earle's Balanced Salt Solution), HBSS (Hank's Balanced Salt Solution), 0.9% NaCl injection, etc. In some embodiments, the neutralizing solution is DPBS solution. Preferably, after adding neutralizing solution to neutralize, gently pipetting cells to obtain RPE cell suspension.

[0047] Evaluation of RPE cells

[0048] Retinal pigment epithelial (RPE) cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually identified by their cobblestone morphology and the initial appearance of pigment.

[0049] When assessing cell purity, any marker that can distinguish RPE cells from other cells can be used.RPE cell-specific markers are selected from, for example, but not limited to, ZO-1 protein, RPE65 protein, CRALBP protein and PMEL17 protein.Any molecule that can specifically identify the above-mentioned RPE cell-specific marker can be used.In some embodiments, the molecule that can specifically identify the above-mentioned RPE cell-specific marker is an antibody for the above-mentioned marker, for example, commercially available antibodies.

[0050] When assessing cell viability, any method that can distinguish between live cells and apoptotic cells can be used. In some embodiments, the method can be an immunofluorescence-based method or a flow cytometry-based method.

[0051] Uses of RPE cells

[0052] The retinal pigment epithelium is a basic component of the retina and plays a vital role in visual function. Damage to the structure and function of the retinal pigment epithelium can lead to a variety of retinal diseases. The retinal pigment epithelium is composed of a layer of regular polygonal cells, namely RPE, arranged in the outermost layer of the retina. The structure and function of the RPE are essential for normal vision, and changes in the RPE can impair function and lead to retinal lesions. For example, retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt disease (STGD) are all retinal degenerative diseases, in which RPE dysfunction is related to their pathogenesis. Therefore, RPE cells can be widely used in various fields. RPE cell transplantation has become one of the important clinical methods for the treatment of retinal degenerative diseases. The purpose is to replace the RPE cells that support photoreceptors and provide nutrition and support to surrounding tissues. Transplanted RPE cells are mainly derived from autologous, allogeneic and different types of stem cells. For example, retinal degenerative diseases can be treated or prevented by transplanting artificially differentiated and separated RPE cells into patients with RPE damage.

[0053] Beneficial effects of the present invention

[0054] 1. It has the effect of reducing or removing foreign cells. It is not only suitable for digesting and separating RPE cells with high purity, but also suitable for separating RPE cells mixed with a large number of foreign cells due to the two-step digestion.

[0055] 2. It can effectively dissociate the adherent RPE cells.

[0056] 3. The cell viability is high, at least above 89%, and can even reach above 95%.

[0057] 4. Achieve higher cell viability while reducing foreign cells and improving the purity of RPE cell passage. Example

[0058] The following examples are provided for a better understanding of the present invention and are not intended to be limiting.

[0059] Instruments and reagents

[0060]

[0061]

[0062] Example 1: Isolation of P0 generation pigment focus cells

[0063] In this embodiment, the total digestion time of RPE cells is fixed at 20 min, and different two-step digestion time combinations are designed for processing P0 generation pigment focus cells, namely 5+15 min, 10+10 min, and 15+5 min. After passaging, the purity of P1 generation cells is detected by flow cytometry to evaluate the digestion method suitable for P0 generation pigment focus cells.

[0064] VTN-N pre-incubation

[0065] Take three groups of 6-well plates, dilute vitronectin (VTN-N) at a ratio of 1:100, add 1 mL of diluted VTN-N to each well and incubate in a 37°C incubator (Thermo, HERA cell 240i) for 60 min. After incubation, remove VTN-N and add 2 mL of fresh RPE cell culture medium (KO-DMEM 78%, KOSR 20%, GlutaMax 1%, NEAA1%, vitamin C 5 μg / mL by volume) to each well and incubate at 37°C for use.

[0066] Picking out pigmented foci of RPE cells

[0067] After the induction and differentiation process, RPE cells were obtained. After obvious pigment foci characteristics appeared, the pigment foci were picked by manual mechanical separation. According to the inoculation of 20 pigment foci per well, they were subcultured and inoculated in a 6-well plate containing 2 mL / well fresh culture medium. The cells after the induced differentiation pigment foci were manually separated and transferred to the 6-well plate were defined as P0 cells.

[0068] The medium was changed every other day, and 3 mL of medium was changed per well. The medium could be adjusted to 4 mL per well according to the cell growth. The growth of cells around the pigment foci at different culture times was observed under a microscope during the culture period. Figure 1 ), it can be clearly observed that a large number of RPE cells began to crawl out from around the P0 generation pigment focus after inoculation, and the cells expanded with the extension of culture time, and the color gradually deepened, which is an important sign of RPE cell maturity. However, the presence of surrounding miscellaneous cells became an obstacle to the harvest of high purity RPE cells.

[0069] First step digestion

[0070] Three groups of 6-well plates containing P0 generation pigment foci cells grown under the same culture conditions (37°C, 5% CO2) for 20 days were taken out from the incubator, the culture medium was aspirated, 2 mL of CTS DPBS was added to each well to wash the residual culture medium and then aspirated, 1 mL of CTS TrypLE was added to each well and placed in a 37°C incubator for digestion. According to the experimental design, the first step digestion time corresponding to the three groups of 6-well plates taken out was 5 min, 10 min, and 15 min, respectively.

[0071] Second step digestion

[0072] After the first step of digestion treatment corresponding to each group is completed, it is taken out of the incubator and the plate is shaken slightly. White cell suspension can be seen, which is mainly the digestion product of miscellaneous cells. Use a 1mL pipette to gently aspirate CTS TrypLE and the white cell suspension without any neutralization or blowing and suction treatment. After completing the above steps, add 1mL CTS TrypLE reagent to each well again and put it into the incubator to continue digestion. The second step digestion time corresponding to the three groups of 6-well plates was 15min, 10min, and 5min, respectively, and the two-step digestion of each group was 20min in total. Group 4 was a single-step digestion for 20min as a control.

[0073] Neutralization of digestive juices

[0074] After 20 minutes of digestion, each group of 6-well plates was taken out of the incubator, and 1 mL of CTS DPBS was added to each well to neutralize the digestion reagent. After neutralization, a 1 mL pipette was used to gently pipette 5 to 10 times to obtain an RPE cell suspension.

[0075] Centrifuge, resuspend, and filter cells

[0076] The cell suspensions from each group were collected and transferred into 15 mL centrifuge tubes and centrifuged at 800 g / 5 min. The supernatant was discarded after centrifugation. The cell pellets collected from each group of 6-well plates were resuspended in 3 mL of fresh cell culture medium. The resuspended cell suspensions were filtered through a 40 μm mesh into new 15 mL centrifuge tubes to remove incompletely digested pigment foci and some miscellaneous cells.

[0077] Cell counting and viability determination

[0078] After filtration, 100 μL of each group of cell suspension was taken. For viable cell determination, AO-PI staining was used and the cell count was performed using a fluorescent cell analyzer. The cell viability represents the proportion of viable cells in the population, and the viable cell concentration represents the number of viable cells per unit volume at the same resuspension volume. The results are shown in the following table:

[0079] Table 1. P0 cell counting results

[0080]

[0081]

[0082] Through the above results analysis, it can be obtained that the cell viability after the 15+5min two-step digestion treatment is better than that of the other two groups, and the 5+15min digestion group has the highest live cell concentration. In the 5+15min digestion group, the number of impurity cells that have not lost their activity in the early stage is relatively large, and the impurity removal effect of the first step of digestion is lower than that of the 15+5min digestion group. The 15+5min digestion group has the highest cell viability, which proves that the first step of digestion has played an effective role in removing impurity cells. The cell viability and live cell concentration of the 10+10min digestion group are both poor. The possible reason is that although the impurity cells are digested and lose their activity after the first step of digestion, they are not completely detached from the wall. The more impurity cells that have lost their activity reduce the overall cell viability. After the 20min one-step digestion is completed, the cell viability is low, and the live cell concentration is not significantly different from the above groups. The reason may be that more impurity cells die after too long of digestion, and RPE cells themselves have good tolerance, so the overall cell viability is significantly reduced, and the live cell concentration is roughly the same.

[0083] Cell passaging

[0084] The cells of groups 1-3 were cultured at 1E5 / cm 2 The cells were subcultured into three groups of 6-well plates incubated with VTN-N. The volume of cell suspension to be added was calculated based on the cell counting results. The cells were cultured for 10 days and the medium was changed every other day.

[0085] P1 cell harvest

[0086] Take out 3 groups of 6-well plates of P1 generation RPE cells grown under the same culture conditions (37°C, 5% CO2) for 10 days from the incubator, remove the culture medium, add 2mL CTS DPBS to each well to wash the residual culture medium and then remove it, add 1mL CTSTrypLE to each well and put it into the 37°C incubator for digestion for 20min. After digestion, add 1mL CTS DPBS to neutralize CTSTrypLE, use a 1mL pipette to blow and aspirate 5 to 10 times to collect the cells in the well plate, centrifuge at 800g / 5min to obtain the P1 generation RPE cell precipitate, and add fresh RPE cell culture medium at 5mL / well for resuspending. After resuspending, the cell suspension is filtered through a 40μm mesh into a new 50mL centrifuge tube to further remove the miscellaneous cells.

[0087] P1 cell counting and purity detection

[0088] The viability and concentration of P1 cells after resuspension were detected by fluorescence cell analyzer, and the purity of cells was detected by flow cytometry. The CRALBP of RPE cells was calculated. + PMEL17 + Positive cell ratio. The results of P1 cell count and purity test are shown in the following table:

[0089] Table 2. P1 cell count and cell purity test results

[0090]

[0091]

[0092] The cell viability and live cell concentration of cells harvested by the same digestion time in the P1 generation were slightly different among the groups, and there was no obvious difference between the groups. However, according to experimental observations and the purity detection chart of the P1 generation cell flow analyzer, it was found that the purity of cells digested by 15+5min in the P0 generation was better than that of the other two groups. At the same time, the reasons for the differences in cell viability and live cell concentration between the P0 generation cells were verified. The live cell concentration of 5+15min was higher but the cell purity was the lowest, indicating that active impurity cells accounted for a considerable proportion. The cell purity of 10+10min was improved, which confirmed that its digestion had a certain impurity removal effect but did not reach the optimal digestion time. The two-step digestion of 15+5min can effectively remove impurity cells and improve the purity of RPE cell passage.

[0093] The same or similar effects may be achieved by using a longer digestion time according to the two-step digestion method. For example, the total digestion time is 40 minutes, and the two-step digestion of 10+30 minutes, 20+20 minutes, and 30+10 minutes is used for treatment. As shown in Table 3 below, the cells still maintain a high cell viability after 40 minutes of digestion. It can be predicted that the two-step digestion of 30+10 minutes has a better effect, because the longer digestion treatment time in the early stage can more effectively remove foreign cells.

[0094] Table 3. Cell viability with prolonged digestion time

[0095]

[0096] Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosed subject matter.

Claims

1. A method for isolating adherently cultured retinal pigment epithelial (RPE) cells, comprising: (a) performing a two-step digestion process, wherein in the first step of digestion, the RPE cells are incubated with an enzyme solution comprising a digestive enzyme for a first period of time, the enzyme solution is then removed by aspiration, and in the second step of digestion, the RPE cells are incubated with a newly added enzyme solution comprising a digestive enzyme for a second period of time, wherein the first period of time and the second period of time are 15 min to 40 min in total; (b) adding a neutralizing liquid to neutralize the enzyme solution comprising the digestive enzymes; and (c) Collect RPE cell suspension.

2. the method for claim 1, wherein said RPE cell is obtained by natural differentiation, induced differentiation or going down to posterity.

3. The method of claim 1 or 2, wherein the RPE cells have been adherently cultured for 10-20 days prior to step (a).

4. The method according to any one of claims 1 to 3, wherein the digestive enzyme is selected from the group consisting of TrypLE, Trypsin and Dispase, preferably TrypLE.

5. The method of any one of claims 1 to 4, wherein the incubation is performed at 37°C.

6. The method of any one of claims 1-5, wherein the first period of time is longer than or equal to the second period of time, and the first period of time does not exceed 20 minutes.

7. The method of claim 6, wherein the first period of time and the second period of time are both in the range of 5 min to 15 min.

8. The method of claim 6, wherein the first period of time is in the range of 10 min-15 min, and the second period of time is in the range of 5 min-10 min.

9. The method of claim 7, wherein the first period of time and the second period of time are 15 min and 5 min, 5 min and 10 min, or 10 min and 10 min, respectively.

10. The method of any of the preceding claims, wherein no cell aspiration is performed during the first and second digestion steps.

11. The method according to any one of the preceding claims, wherein step (b) neutralizes the enzyme solution comprising the digestive enzyme by adding a neutralizing solution selected from the group consisting of DPBS solution, PBS solution, EBSS solution, HBSS solution and 0.9% NacL injection solution.

12. The method of any one of the preceding claims, further comprising washing the cultured RPE cells before step (a).

13. The method of any one of the preceding claims, further comprising pipetting the cells in step (c), and / or centrifuging and resuspending the cell suspension.

14. The method according to any one of the preceding claims, further comprising filtering the cell suspension of step (c) to further remove foreign cells.

15. A composition comprising retinal pigment epithelial cells obtained by the method of any one of claims 1 to 14.

16. Use of the retinal pigment epithelial cells obtained by the method according to any one of claims 1 to 14 in the preparation of a medicament for treating or preventing retinal degenerative diseases.

17. The use according to claim 16, wherein the retinal degenerative disease is age-related macular degeneration or retinitis pigmentosa.

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