A method for sorting mature RPE cells from iPSC-differentiated cells and its application

Abstract

This invention relates to the field of biotechnology, and proposes a method for sorting mature RPE cells from iPSC-differentiated cells and its applications. In this method, iPSCs are first induced to differentiate into mature RPE cells. Then, a combination of Papain and Accutase enzymes is used to gently and efficiently separate the cells into single-cell suspensions. A cell filter is then used to ensure that the cells are dispersed and appropriately sized single cells or small cell clusters. Finally, high-purity mature RPE cells are obtained through flow cytometry gating strategies and R780-H channel settings. This invention enables normal cell growth and rapid proliferation with minimal cell damage through a combination of enzymes; utilizes R780-H autofluorescence to improve sorting accuracy and reliability; allows for direct flow cytometry sorting after cell digestion and filtration, simplifying the operation, reducing cell damage, facilitating cell culture and expansion, and improving experimental efficiency; and sorts based on the cytochrome optical properties of mature RPE cells, eliminating the need for expensive antibody labeling, thus possessing significant application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for sorting mature RPE cells from iPSC-differentiated cells and its application. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have demonstrated immense potential in regenerative medicine due to their powerful differentiation potential. They can differentiate into various cell types, offering new hope for treating many serious diseases for which effective drugs are currently lacking. Specifically, by reprogramming a patient's own somatic cells into iPSCs, then directing the differentiation of these iPSCs into specific target cells, and finally transplanting them into the patient, cell replacement therapy for diseases is expected to be achieved. However, in this process, although the induction and initial sorting of iPSCs are relatively feasible, a large number of non-target cells are inevitably generated during the differentiation into target cells, even with strict standardized procedures. Therefore, how to efficiently and accurately isolate target cells becomes a crucial step for the success of subsequent transplantation therapy.

[0003] In numerous cell therapy studies, the use of retinal pigment epithelial (RPE) cells differentiated from iPSCs / ESCs for the treatment of age-related macular degeneration (AMD) has become a research hotspot. Currently, several preclinical and clinical trials are actively underway in this field, achieving positive results. These studies employ various procedures for differentiating iPSCs / ESCs into RPE cells, with the isolation of RPE cells from the differentiated cells being a crucial step. Among current techniques for sorting RPE cells, flow cytometry is used as an example. The procedure involves: first, digesting the cells to obtain a single-cell suspension; then, co-incubating this single-cell suspension with a fluorescently conjugated antibody that recognizes specific antigens on the RPE cell surface; washing to remove unbound antibodies after incubation; and finally, sorting the cells using flow cytometry based on the intensity of fluorescence emitted by the cells. This method exhibits extremely high sorting purity, achieving an RPE cell percentage of over 90%. However, this method has a significant drawback: its reliance on specific antibodies directly leads to high sorting costs. Meanwhile, during the long-term incubation of cells and antibodies, and during the flow of cells through the narrow liquid path of the flow cytometer, cells suffer significant damage, which leads to a significant increase in the mortality rate of cells after sorting.

[0004] Given the aforementioned problems with existing sorting methods, in the field of regenerative medicine, especially in applications based on iPSC / ESC differentiated cell therapy, such as the treatment of retinal diseases (particularly AMD), there is an urgent need to develop a separation method that is more efficient, more accurate, lower cost, and causes less cell damage. This would strongly promote the further development of this technology and provide higher-quality cell resources and technical means for the treatment of related diseases. Summary of the Invention

[0005] In view of this, the present invention proposes a method for sorting mature RPE cells from iPSC-differentiated cells and its application.

[0006] This invention first utilizes specific induction differentiation conditions and environment to induce iPSC cells to differentiate into mature RPE cells and accumulate relevant characteristics. A combination of cell-digesting enzymes (papain and accutase) is then used to gently and efficiently separate and disperse the cells from the culture plate into single-cell suspensions, ensuring the homogeneity and manipulability of the cell sample. Next, a cell filter is used to further ensure that the cells entering subsequent analysis are relatively dispersed and appropriately sized single cells or small cell clusters. Finally, a flow cytometry gating strategy and R780-H channel settings are used to obtain high-purity mature RPE cells. Therefore, this invention can efficiently and accurately separate mature RPE cells, improving the accuracy and reliability of sorting, and contributing to the acquisition of high-purity target cell populations. This provides strong support for in-depth research on the function and characteristics of RPE cells and drug development in the treatment of related diseases.

[0007] The technical solution of this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for sorting mature RPE cells from iPSC-differentiated cells, comprising the following steps:

[0009] S1. Differentiate iPSC cells without passage during the differentiation process until the cells are differentiated into RPE cells on days 50-84.

[0010] S2. After washing the cells with DPBS, digest the cells with Papain to prevent them from adhering to the wall. After digestion, remove the Papain solution, add Accutase to further digest the cells, add DPBS after digestion, centrifuge and discard the supernatant, then add DPBS to resuspend the cells to obtain a cell suspension.

[0011] S3. Filter the cell suspension using a 35-45μm cell filter to obtain a single-cell suspension; centrifuge, discard the supernatant, and add EDTA solution to resuspend the cells;

[0012] S4. Cells are sorted using flow cytometry. First, gating is set on the FSC / SSC plot to exclude interference from cell debris and impurities. Then, the R780-H channel parameters are set. Specifically, the parameters include irradiating cells with 638nm excitation light and detecting the autofluorescence intensity in the 750-810nm wavelength range through a bandpass filter with a center wavelength of 780nm and a bandwidth of 60nm. Cells are sorted according to the intensity of the fluorescence in this channel to obtain mature RPE cells.

[0013] The descriptions of fluorescence "intensity" in flow cytometry detection in this invention are all based on the following judgment, namely, "relatively strong" or "relatively high fluorescence" in this invention means one of the following (1)-(2):

[0014] (1) When cells are divided into two groups under the R780-H fluorescence channel, the cell group with higher fluorescence intensity;

[0015] (2) When the cells were not divided into two groups under the R780-H fluorescence channel, the fluorescence intensity was higher than that of the cell population of pigment-free control cells.

[0016] Preferably, in some specific embodiments, in step S1, the cells differentiate to day 60-84.

[0017] Preferably, in some specific embodiments, in step S2, 5-20 U / mL Papain is added, and digestion is carried out at 37°C for 30-50 min.

[0018] The amount of enzyme used can be adjusted according to the specific circumstances of the implementation. Because enzymes are highly efficient at catalysis, as long as the concentration reaches a certain level (e.g., 5-20 U / mL Papain) when the substrate is sufficient, the reaction can be initiated and maintained. Those skilled in the art can reasonably determine the volume of enzyme to be added based on factors such as the amount of substrate and the size of the reaction system, using their experience.

[0019] Preferably, in some specific embodiments, step S2 further includes: adding Accutase to further digest the cells, and digesting at 37°C for 30-50 minutes.

[0020] Preferably, in some specific embodiments, step S3 further includes: aspirating cells into a pipette, blowing them through a 40μm cell filter to remove severely adhered cells and impurities, and obtaining a single-cell suspension; rinsing the cell filter with 1mD PBS, collecting all cell suspensions that have passed through the filter into a centrifuge tube, centrifuging at 300g for 3min, removing the supernatant after centrifugation, and resuspending the cells in a DPBS solution with 5mM EDTA.

[0021] Preferably, in some specific embodiments, step S4 further includes: setting a first gate P1 to select cells on the FSC / SSC contour map of the flow cytometer software according to the FSC / SSC signal, and filtering out impurities and cell debris.

[0022] Preferably, in some specific embodiments, step S4 further includes: separating the cells with the two main peaks from the R780-H channel histogram, selecting the cell population with relatively low fluorescence using the P2 gate, and selecting the cell population with relatively high fluorescence using the P3 gate, sorting the cells through the P2 and P3 gates, and the cells collected by the P3 gate are mature RPE cells.

[0023] Secondly, the present invention provides applications of the method for sorting mature RPE cells from iPSC-differentiated cells as described in any of the above-described methods. These applications include, in one of the following contexts: screening of drugs for the treatment of ophthalmic diseases, assessment of the toxicity of ophthalmic drugs, research on the pathogenesis of retinal diseases, research on the effects of environmental factors on the retina, research on retinal development, or research on cell differentiation regulation mechanisms.

[0024] Furthermore, the application includes one of the following: drug development for age-related macular degeneration, drug development for retinitis pigmentosa, or drug development for macular dystrophy.

[0025] The beneficial effects of the present invention include at least the following:

[0026] This invention employs sequential treatment of iPSC-differentiated RPE cells with Papain and Accutase, effectively digesting the cells into a single-cell state. After Papain digestion, the cells retain intact morphology, clear boundaries, and minimal debris, minimizing cellular impact. Further digestion with Accutase results in sorted cells still growing normally and proliferating rapidly, indicating that this digestion method causes minimal cell damage, resulting in high cell viability and strong growth capacity, which is beneficial for subsequent cell culture and research.

[0027] This invention uses 638nm wavelength excitation light to irradiate cells and detects autofluorescence in the 750-810nm wavelength range. This clearly separates cells into two groups with strong resolution, enabling efficient and accurate separation of mature RPE cells. This improves the accuracy and reliability of sorting and helps obtain a high-purity target cell population, providing a strong guarantee for in-depth research on the function and characteristics of RPE cells.

[0028] This invention allows for direct sorting of cells using flow cytometry after digestion and filtration. Compared to conventional flow cytometry sorting methods, it is simpler to operate, involves fewer cell processing steps, and is faster, reducing cell damage and facilitating further culture and expansion of enriched cells. This improves experimental efficiency and helps maintain the optimal cell condition. Based on the optical properties of mature RPE cytochromes, this invention eliminates the need for expensive antibody labeling, significantly reducing costs and broadening the application scope of the method. It enables more laboratories and research projects to use this technology for cell sorting research, offering significant advantages, especially in large-scale experiments or cost-sensitive studies.

[0029] Terminology Explanation :

[0030] Induced pluripotent stem cells (iPS cells or iPSCs): A type of cell with pluripotency similar to embryonic stem cells, obtained by introducing specific transcription factors into differentiated somatic cells or by treating differentiated somatic cells with small molecule compounds to reprogram them.

[0031] Retinal Pigment Epithelium Cells (RPE Cells): RPE cells are located in the outermost layer of cells in the retina, closely adjacent to the photoreceptor cells of the retina, and play an important role in maintaining the normal physiological function of the retina.

[0032] Embryonic stem cells (ES cells or ESCs): pluripotent stem cells obtained from the inner cell mass of early blastocysts through in vitro culture, isolation, and cloning.

[0033] Flow cytometry (FACS) can be used to detect the expression levels of cell surface markers, analyze cell cycle and apoptosis, identify and sort different types of immune cells, detect the heterogeneity of tumor cells, and conduct cell function studies and drug screening.

[0034] Histogram: A method of representing the distribution of data graphically. In flow cytometry, the horizontal axis of the histogram represents the numerical value of parameters such as fluorescence intensity or scattered light intensity, while the vertical axis represents the frequency or relative quantity of these values.

[0035] The R780-H histogram is a graph plotted with the fluorescence intensity value detected by the R780 fluorescence channel on the x-axis and the number of cells or events with that fluorescence intensity value on the y-axis.

[0036] FSC (Forward Scatter): refers to the light that is scattered forward after a laser beam hits a cell.

[0037] SSC (Side Scatter): refers to side-scattered light, which is the scattered light measured at 90 degrees to the laser beam.

[0038] FSC-H: Here, "H" stands for Height.

[0039] FSC-A / SSC-A: Here, "A" stands for Area.

[0040] R780-H / FSC-H contour plots use FSC-H signal intensity as the x-axis and R780-H fluorescence intensity as the y-axis, employing contour lines to illustrate the distribution of cells or particles across these two parameters. This type of contour plot allows researchers to simultaneously observe the relationship between cell size (reflected by FSC-H) and the expression level of R780-labeled substances (reflected by R780-H). For example, it can analyze differences in R780 marker expression in cell subpopulations of different sizes, or study the dynamics of R780 marker expression during cell size changes, thus providing more information for further understanding the biological characteristics and functions of cells. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 The following are the flow cytometry analysis results in Example 2 of this invention; Figure A is the FSC / SSC contour plot, showing cell debris and impurities removed by P1 gating; Figure B is the R780-H / FSC-H contour plot, showing that the cells are divided into two groups; Figure C is the R780-H histogram, showing that the cell fluorescence has two main peaks; Figure D is a bar chart of cell to debris ratio, where the red part represents normal cells and the blue part represents cell debris. The horizontal axis All represents before P1 gating removal and P1 represents after P1 gating removal, and the vertical axis represents the percentage.

[0043] Figure 2The results of flow cytometry analysis in Example 3 of this invention are shown in Figure A, which is an FSC / SSC contour plot, with cell debris and impurities removed by P1 gating; Figure B is an R780-H / FSC-H contour plot, showing that the cells are divided into two groups; Figure C is an R780-H histogram, showing that the cell fluorescence has two main peaks, with the lower fluorescence peak set as P2 gate and the higher fluorescence peak set as P3 gate.

[0044] Figure 3 The figures show the cytochrome status before and after sorting in Example 3 of the present invention; wherein, Figure A is a cell precipitate obtained by centrifugation before flow cytometry sorting; Figure B is an image of the cell precipitate collected by P2 gate and P3 gate respectively after flow cytometry sorting; Figure C is an image of the cells collected by P2 gate and P3 gate under a bright field microscope after flow cytometry sorting.

[0045] Figure 4 The following are the flow cytometry analysis results in Example 4 of this invention; Figures A and C show control cells: the FSC / SSC plot in Figure A was gated with P1 to remove cell debris and impurities; the R780-H / FSC-H contour plot in Figure B shows that the control cells are a single population; the R780-H histogram in Figure C shows that the control cells have only one main fluorescence peak, with most cells selected using P2 gating and the higher fluorescence portion set to P3 gating; Figures D and F show differentiated cells: the FSC / SSC plot in Figure D was gated with P1 to remove cell debris and impurities; the R780-H / FSC-H contour plot in Figure E shows that the differentiated cells appear to be a single population, but some cells have higher fluorescence than the control cells; Figure F shows that with the P2 and P3 gating settings in Figure C maintained, the P3 gate contains approximately 56% of the cells;

[0046] Figure 5 This is the cell condition after flow cytometry cell sorting in Example 4 of the present invention; R780-Low represents cells sorted by the P2 gate, and R780-High represents cells sorted by the P3 gate; the upper part is the cell image under bright field microscopy, and the lower part is the cell precipitate image after centrifugation, showing that the cells sorted by the P3 gate have obvious pigmentation.

[0047] Figure 6 The images shown are phase-contrast (PH) and bright-field (BF) images of cells at various stages in Example 4 of this invention. The first column shows cell images on day 54 of differentiation; the second column shows cell images on day 68 of differentiation after digestion with Papain for 45 minutes; the third column shows cell images of P3 gate sorted cells after culturing for another day; and the fourth column shows cell images of the cells in the third column after culturing for another day.

[0048] Figure 7The transcriptome sequencing analysis results in Example 4 of this invention are shown below. Figure A represents a clustering heatmap, where R1 and R2 represent two independent replicates. P2 represents cell samples sorted by the P2 phylum, and P3 represents samples sorted by the P3 phylum. Colors indicate relative expression levels: red for high expression and blue for low expression. Figure B shows a volcano plot of gene expression differences between cells sorted by the P3 phylum and those sorted by the P2 phylum. The horizontal axis represents the fold change in expression, and the vertical axis represents the significance of the difference. Red indicates genes with relatively high expression, and blue indicates genes with relatively low expression. Dark red gene names indicate marker genes for mature RPE cells.

[0049] Figure 8 Figure 4 shows the GO enrichment analysis of gene biological function (green) and cellular components (blue) in Example 4 of the present invention; wherein: Figure A shows the GO enrichment analysis of genes with relatively high expression levels in cells selected by the P3 phylum, including components closely related to vision and cell polarity, proving that the enriched cells are mature RPE cells; Figure B shows the GO enrichment analysis of genes with relatively low expression levels in cells selected by the P3 phylum. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0051] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0052] Example 1: Differentiation of iPSC cells

[0053] 1. Reagent Preparation (Day 0-14)

[0054] 1.1. Preparation of retinal differentiation medium (RDM):

[0055] 1 mL of 100×N2 (Gibco, catalog number 17502048), 2 mL of 50×B27 (Gibco, catalog number 17504044), and 1 mL of 100×Non-essential amino acids (NEAA, Gibco, catalog number 11140050) were added to 96 mL of DMEM / F-12 (Gibco, catalog number C1133050BT) to obtain 100 mL LRDM. 1.221 g of nicotinamide (NIC, Sigma, catalog number N0636) was dissolved in 8 mL of sterile water, vortexed, and brought to a final volume of 10 mL to obtain a 1 MNIC solution, which was then sterilely filtered.

[0056] 1.2. Preparation of growth factors and small molecules:

[0057] Recombinant mouse head protein (noggin, R&D Systems, catalog number 1967-NG-025), human Dickkopf WNT signaling pathway inhibitor 1 (DKK-1, R&D Systems, catalog number 5439-DK-010), and IGF-1 (R&D Systems, catalog number AFL291) were resuspended to 100 μg / mL in PBS, aliquoted, and stored at -20°C. FGF-basic (primegene, catalog number 104-02) was resuspended to 10 μg / mL in PBS containing 0.1% BSA, and recombinant human / mouse / rat Activin A (R&D Systems, catalog number 338-AC / CF) was resuspended to 100 μg / mL, aliquoted, and stored at -80°C. SU5402 (purchased from Santa Cruz Biotechnology, catalog number sc-204308) and CHIR-99021 (GSK-3β inhibitor, purchased from Sigma, catalog number SML1046) were resuspended to 10 mM with DMSO, aliquoted and stored at -20°C.

[0058] 1.3. Obtain the reagents needed for Day 0 and / or Day 14:

[0059] Prepare 1×EDTA solution (0.2g EDTA / LPBS, purchased from Zhong Sheng Su Yuan, catalog number RP01007), 1× PBS without calcium or magnesium (pH 7.4), 1× trypsin dissociation enzyme (TDE, purchased from Gibco, catalog number 12563011), Dulbecco phosphate buffer solution (DPBS, purchased from Gibco, catalog number C14190500BT) and 10μM Y-27632 dihydrochloride (purchased from Taosu, catalog number T1725).

[0060] RPE support medium (RSM): 100 mL of L-glutamine substitute (GlutaMAX, Gibco, catalog number 35050061), 1 mL of NEAA, 1 mL of sodium pyruvate (Gibco, catalog number 11360070), 1 mL of penicillin / strep antibiotic (Gibco, catalog number 15140122), 1 mL of 1 MNIC, and 2 mL of heat-inactivated fetal bovine serum (FBS, NewZerum, catalog number FBS-E500) were added to 93 mL of DMEM / F-12.

[0061] 2. Day 0: Passaging of PSCs (pluripotent stem cells) for differentiation

[0062] 2.1. Culture stem cell colonies to 80% confluence under conditions of no feeder layer and no serum.

[0063] 2.2. Coat 12-well plates with extracellular matrix hydrogel (ECMH, purchased from Corning, catalog number 356237) and incubate at room temperature for 1 hour.

[0064] 2.3. Prepare RDM and PBS- / - for Day 0, preheat in a 37°C water bath, and allow EDTA to return to room temperature.

[0065] 2.4. Add growth factors to the preheated RDM: 10 mM NIC, 50 ng / mL noggin, 10 ng / mL LDDKK-1 and 10 ng / mL IGF-1.

[0066] 2.5. Remove differentiated cells using a P10 pipette.

[0067] 2.6. Finally, passage one well of the 6-well plate into four wells of the 12-well plate at a 1:4 ratio (aspirate the culture medium, wash the cells with pre-warmed PBS- / -, rinse the cells once with EDTA, incubate with EDTA for 3-5 min, aspirate the EDTA, add an appropriate amount of RDM medium, disperse the cells by pipetting, and seed them into ECM-coated 12-well plates. Incubate at 37°C in a 5% CO2 incubator, record the incubation time, and change the medium as needed).

[0068] 3. Days 1-14: Add growth factors

[0069] Day 1: Change the medium with RDM containing Day 0 growth factor combination, adding 1 mL to each well.

[0070] Day 2: Change the medium with RDM containing 10 mM NIC, 5 ng / mL FGF-basic, 10 ng / mL noggin, 10 ng / mL DKK-1 and 10 ng / mL IGF-1, adding 1 mL to each well.

[0071] Day 4: Change the medium with RDM containing 100 ng / mL activin A, 10 ng / mL DKK-1 and 10 ng / mL IGF-1, adding 1 mL to each well. At this point, the cells have fused.

[0072] Day 6: Change the medium with RDM containing 100 ng / mL activin A and 10 μM SU5402, adding 1 mL to each well.

[0073] Days 8, 10, and 12: Change the medium with RDM containing 100 ng / mL activin A, 10 μM SU5402, and 3 μM CHIR99021, adding 1 mL to each well.

[0074] 4. RPE enrichment to passage day 0

[0075] 4.1. Coat a 6-well plate with ECMH of reduced growth factor and incubate at room temperature for 1 hour.

[0076] 4.2. Prepare DPBS and 1 mL of RDM per well and preheat to 37°C. Heat TDE, RSM and Y-27632 to 37°C.

[0077] 4.3. Add 10 μM of Y-27632 to RSM to improve adhesion on days 1 to 4.

[0078] 4.4. Remove the old culture medium and add 1 mL of preheated growth factor-free RDM to each well.

[0079] 4.5. Remove non-RPE cells manually using a stereomicroscope and a P10 pipette tip.

[0080] 4.6. After scraping off non-RPE cells, aspirate RDM and debris, and wash each well twice with 1 mL of pre-warmed DPBS.

[0081] 4.7. Add 0.5 mL LTDE to each well of a 12-well plate, incubate at 37°C for 5 min, scrape off the cells with a scraper, and pipette 3-4 times to form a homogeneous suspension.

[0082] 4.8. Dilute the cell / TDE suspension 1:10 with pre-warmed RSM without Y-27632 and collect the cells by centrifugation.

[0083] 4.9. Aspirate the culture medium and resuspend the cell pellet in RSM containing 10 μM Y-27632 (1 mL per well).

[0084] 4.10. Filter the cells using a 40 μm nylon mesh cell filter, count the cells, and calculate the cell concentration.

[0085] 4.11. The cells were loaded at a rate of 1×10⁻⁶. 5 cells / cm 2 The cells were seeded at a density reduced from growth factor on ECM coated plates and then seeded in RSM containing 10 μM Y-27632.

[0086] 4.12. 48 hours after cell seeding, change the medium with RSM containing 10 μM Y-27632, and then change the medium every 3-4 days (after 4-7 days, stop changing the medium containing 10 μM Y-27632).

[0087] 4.13. Cells were grown at 37°C and 5% CO2 for 28-35 days, with the RSM replaced every 3-4 days.

[0088] 5. Maturation: RPE Generation 1 and 2 (In the following steps, the amounts shown in parentheses are for a single well of a 6-well plate or a T75 culture flask)

[0089] 5.1. On days 28–35 of generation 0, coat 6-well plates (T75 culture flasks) with ECMH. Prepare DPBS and RSM and preheat at 37°C, allowing TDE to return to room temperature. Aspirate the culture medium and wash twice with 2 mL (10 mL) of preheated DPBS.

[0090] 5.2. Aspirate DPBS, add 1 mL (5 mL) TDE, incubate at 37°C and 5% CO2 for 5 min, and confirm cell shrinkage and detachment under a microscope.

[0091] 5.3. Use a cell scraper to scrape cells from the bottom of the well (flask).

[0092] 5.4. Use a P1000 pipette to agitate the cell / TDE suspension 3-4 times to form a homogeneous suspension.

[0093] 5.5. Dilute the cell suspension with RSM at a ratio of 1:10, reserve 2 mL (5 mL) of RSM for rinsing and adding to the suspension.

[0094] 5.6.173×g centrifuge the cell suspension for 5 min.

[0095] 5.7. Aspirate the supernatant and resuspend the cells in 1 mL (5 mL) RSM.

[0096] 5.8. Filter the cells using a 40μm nylon mesh cell filter, count the cells, and calculate the cell concentration.

[0097] 5.9. The cells were loaded at a rate of 1 × 10⁻⁶. 5 cells / cm 2 The cells were inoculated at a density of 4 mL (15 mL) of RSM onto ECMH-coated plates.

[0098] 5.10. Allow cells to mature for 30 days, and replace the RSM every 3-4 days.

[0099] 5.11. Repeat the above steps (5.2-5.11) on day 30 to passage the cells from the first generation to the second generation.

[0100] 6. Create an intermediate cell bank: Second-generation cells are cryopreserved for 3-5 days after regeneration (for cells that have not fused (approximately 50%) and have not recovered pigmentation).

[0101] 6.1. Calculate the amount of cryopreservation medium containing 10% DMSO based on the cell count, to achieve a concentration of 3 × 10⁻⁶ cells / year. 6 Cells / mL.

[0102] 6.2. Follow the steps in 5.2-5.8, resuspending the cell pellet to 3 × 10⁻⁶ using cryopreservation medium containing 10% DMSO. 6 Cells / mL, take 1 mL of suspension and put it into a 1.2 mL cryovial.

[0103] 6.3. Place the cryovials in a freezing container cooled at -1℃ / min, incubate at -80℃ overnight, then transfer to liquid nitrogen for storage. (These cells were thawed and cultured for the third generation. Identification was performed after 30 days of culture. After thawing, the cells were cultured at 1.5 × 10⁻⁶ cells / mL.) 5 cells / cm 2 (Density of inoculation with 3rd generation RPE)

[0104] Example 2: Isolation of mature RPE cells from iPSC-differentiated cells

[0105] (I) This embodiment Design Process Including the following ADs:

[0106] A. Specific Setup of Cell Differentiation Process: Combining the method in Example 1, specific induction differentiation conditions and environment are used to guide iPSC cells to differentiate into specific cell types. By inhibiting passage operations, the continuity and stability of cell differentiation are maintained, allowing cells to gradually acquire the characteristics of mature RPE cells over a long period of differentiation. A cell population at a specific differentiation stage (day 65) with certain characteristics is obtained, providing a basic sample for subsequent processing and analysis, promoting the differentiation of iPSC cells towards mature RPE cells and accumulating relevant characteristics.

[0107] B. Method of using the combined enzymes (Papain and Accutase) for cell digestion: Papain breaks down extracellular matrix proteins, allowing cells to separate from the culture dish surface; Accutase further acts on the cell adhesion structures, dispersing the cells (Papain alone cannot separate RPE cells from each other, making it impossible to obtain a single-cell suspension; Accutase alone cannot effectively separate cells from the culture dish surface, and the cells will remain attached). Specific digestion times and temperatures can effectively separate cells while minimizing cell damage. This method gently and effectively separates and disperses cells from the culture plate into single-cell suspensions, facilitating subsequent centrifugation, filtration, and flow cytometry analysis, ensuring the homogeneity and operability of the cell sample.

[0108] C. Cell Filter Usage and Operation Details: During cell filter operation, the 40μm filter can filter out severely aggregated cell clumps and larger impurities based on cell size, ensuring that the cells entering subsequent analysis are relatively dispersed and appropriately sized single cells or small cell clusters. This improves the purity and homogeneity of cell samples, avoids interference from large cell clumps or impurities on flow cytometry detection and sorting, and ensures the accuracy and reliability of analytical results.

[0109] D. Flow Cytometry Gating Strategy and Analytical Methods: Cells are sorted directly using flow cytometry without staining after digestion. Sorting is based on the pigment optical characteristics of mature RPE cells, using red light excitation and detecting fluorescence in the red to near-infrared band. Based on the forward scattered light (FSC) and side scattered light (SSC) characteristics of cells, larger portions of FSC-A and SSC-A represent intact cells rather than debris or impurities. By setting a gate (P1) on the FSC / SSC contour map, interfering factors such as cell debris can be excluded, screening out relatively pure cell populations for further analysis. This preliminary purification of the cell population improves the accuracy and specificity of subsequent analyses.

[0110] (II) This embodiment Detailed steps as follows:

[0111] 1. Following the differentiation process in Example 1, iPSC cells were differentiated in a 12-well plate without passage of the cells until day 65 of differentiation.

[0112] 2. After removing the culture medium with a pipette, add 1 mL of DPBS (purchased from Gibco, catalog number C14190500BT) buffer to wash the cells. Discard the supernatant after washing and repeat the washing process twice.

[0113] 3. After washing, add 500 μL of 15 U / mL Papain (purchased from Worthington, catalog number LS003119) to the wells and place the cells in a 37°C incubator for 45 min to digest.

[0114] 4. After digestion is complete, gently aspirate the enzyme solution to avoid aspirating it into the cells.

[0115] 5. Add 500 μL of Accutase (purchased from Gibco, catalog number A1110501) at a 1x concentration to the wells, gently pipette the cells to disperse them, and continue to digest in a 37°C incubator for 45 min.

[0116] 6. After digestion, add 1 mL of DPBS and gently pipette the cells to disperse them.

[0117] 7. Transfer the cells to a centrifuge tube and centrifuge the cells at 300g for 3 minutes.

[0118] 8. After centrifugation, remove the supernatant and resuspend the cells in 1 mL of DPBS to obtain a cell suspension.

[0119] 9. Aspirate the cells into a pipette and pipette through a 40μm cell filter to remove severely clump-together cells and impurities, obtaining a single-cell suspension.

[0120] 10. Rinse the cell filter with 1 mL of DPBS, collect all the cell suspension that has passed through the filter into a centrifuge tube, and centrifuge at 300 g for 3 min.

[0121] 11. After centrifugation, remove the supernatant and resuspend the cells in DPBS solution with 5 mM EDTA added, ready for flow cytometry (Beckman). Cells were analyzed using a CytoFLEX SRT cell sorter. The R780-H channel parameters were set, specifically including irradiating cells with 638nm excitation light and detecting autofluorescence intensity in the 750-810nm wavelength range using a bandpass filter with a center wavelength of 780nm and a bandwidth of 60nm.

[0122] 12. On the FSC / SSC contour map of the flow cytometer software, select the area with larger FSC-A (forward scattering area) and SSC-A (side scattering area) and set it as the first gate (P1).

[0123] (III) Analysis and Conclusion

[0124] like Figure 1As shown in the FSC-H / R780-H contour plot, the cells are clearly divided into two groups along the R780-H channel. The histogram of the R780-H channel shows two main peaks.

[0125] Therefore, gating (P1 gating in Figure A) effectively excludes cell debris and impurities, which account for 10.11% of the total particles (blue portion of All in Figure D), resulting in a relatively pure cell population for analysis. Fluorescence characteristics in the R780-H channel (Figures B and C) indicate the existence of two distinct cell subpopulations. These subpopulations may represent different stages in the differentiation pathway or different cell types. This demonstrates that the present invention can successfully differentiate iPSC cells, and flow cytometry analysis can detect changes in cell characteristics during the differentiation process.

[0126] Example 3

[0127] (I) Methods and Steps

[0128] 1. Following the differentiation process in Example 1, iPSC cells were differentiated in a 12-well plate without passage of the cells until day 74 of differentiation.

[0129] 2. Process according to steps 2-12 of Example 2.

[0130] 3. Divide the cells with the two main peaks from the R780-H channel histogram. Use the P2 gate to select the cell population with relatively low fluorescence and the P3 gate to select the cell population with relatively high fluorescence.

[0131] 4. Sort cells through the P2 and P3 gates and collect the cells sorted by the two gates.

[0132] (II) Results

[0133] like Figure 2 As shown, this invention obtained a relatively pure cell population through gating operations, removing 10.11% of cell debris or impurities, thus improving the reliability of the experiment. The cells exhibited clear clustering in their 780nm fluorescence characteristics, which may be related to the cell differentiation state or intracellular substances. The number of cells with and without melanin enrichment was similar, which is of significant value for studying cytochrome-related biological processes.

[0134] like Figure 3As shown in Figure A, before flow cytometry sorting, the cell pellet was mixed together without obvious pigment differentiation. However, Figures B and C show that the cell pellet images collected by the P2 gate show less or no melanin accumulation in the cells, while the cell pellet images collected by the P3 gate show obvious melanin accumulation. Therefore, based on the changes in cytochrome before and after flow cytometry sorting, we can further understand the differences in the degree of cytochrome enrichment, cell number, and cell morphology.

[0135] (III) Comparison between this embodiment and embodiment 2

[0136] In Example 2, iPSC cells differentiated to day 65; while in this example, iPSC cells differentiated to day 74. This time difference may lead to differences in the degree of differentiation and cell characteristics, thus affecting subsequent experimental results and analysis.

[0137] In Example 2, on the FSC / SSC contour plot of the flow cytometry software, the portion with larger FSC-A and SSC-A values ​​was selected and set as the first gate (P1). The main purpose was to exclude cell debris and impurities, obtaining a relatively pure cell population for preliminary analysis. The focus was on the initial purification of the overall cell population and the initial observation of the two subpopulations. In this example, the cells were further divided from the peak depressions of the two main peaks on the R780-H channel histogram. The region containing the low fluorescence main peak was set as the P2 gate, and the region containing the high fluorescence main peak was set as the P3 gate. Cells were then sorted and collected through the P2 and P3 gates. Example 3 focuses more on the finer subpopulation division and sorting of cells based on their fluorescence characteristics to study the differences between cell subpopulations with different fluorescence characteristics (possibly related to the degree of melanin enrichment).

[0138] Therefore, Example 2 focuses on demonstrating that gating (P1 gating) can effectively eliminate cell debris and impurities, obtaining a relatively pure cell population for analysis. It also reveals that the existence of two distinct subpopulations of cells in the R780-H channel may be related to cell differentiation pathways and growth factor influences, emphasizing the detection of characteristic changes during cell differentiation. This example, however, focuses on observing the enrichment of cytochromes (especially melanin) before and after cell sorting to further understand the differences in cytochrome enrichment, cell number, and cell morphology. It highlights the research value of cytochrome-related biological processes and pays more attention to the differences in pigment characteristics among cell subpopulations.

[0139] Example 4

[0140] This embodiment was repeated twice in parallel. The specific steps of this embodiment are as follows:

[0141] 1. Following the differentiation process in Example 1, iPSC cells were differentiated in a 12-well plate. The cells were passaged on day 30 during the differentiation process and differentiated until day 68.

[0142] 2. Process according to steps 2-12 of Example 2.

[0143] 3. Take ARPE-19 cells (RPE cell line, which does not produce pigment under normal culture) from the wells of a 12-well plate.

[0144] 4. Digest with 200 μL of 0.25% trypsin for 1 min, add 500 μL of culture medium to stop digestion, and centrifuge at 1000 rpm for 3 min.

[0145] 5. Discard the supernatant, resuspend in 1 mL of DPBS, and centrifuge again.

[0146] 6. Resuspend ARPE-19 cells in 1 mL of EDTA solution (designated as NC group).

[0147] (I) Flow Cytometry Analysis

[0148] The obtained cells were plotted on the FSC / SSC scatter plot in the flow cytometry software. Scatter points with larger FSC-A and SSC-A values ​​were selected and set as P1 gates. Flow cytometry analysis was performed on the NC group cells.

[0149] like Figure 4 On the FSC-H / R780-H contour plot, the NC group cells appeared as a single population, with only one main peak on the R780-H channel histogram. On the R780-H channel histogram, the main peak was selected by fluorescence intensity from lowest to highest and set as P2-gated, while the remaining high-fluorescence regions were set as P3-gated. Differentiated cells (denoted as the iRPE group) were then analyzed. The FSC-H / R780-H contour plot showed no obvious cell clustering, but on the R780-H channel histogram, over half of the cells exhibited stronger fluorescence than the NC group (P3-gated cells comprised 56.33% of the cells).

[0150] (II) Cell sorting and observation

[0151] Cells were sorted and collected using flow cytometry through the P2 and P3 gates. Centrifugation of the collected cell suspension revealed that cells collected through the P2 gate were colorless, while cells collected through the P3 gate were black (as shown in the image). Figure 5 The collected cells were resuspended in culture medium, and a portion of the cells were observed under a microscope. Cells collected from the P3 phylum appeared black. A portion of the P3-collected cells were further cultured; after one day, the cells adhered to the culture vessel, and after two days, the cell number increased (e.g., ...). Figure 6 ).

[0152] (III) Transcriptome Analysis

[0153] RNA was extracted from collected cells using an RNA extraction kit, a transcriptome library was constructed using a library construction kit, and then subjected to next-generation sequencing.

[0154] like Figure 7 As shown in Figure A, the transcriptome results were analyzed using a clustering heatmap, which showed that the P2 gate sorted samples and the P3 gate sorted samples from the two replicates aggregated separately, indicating that the sorting was stable.

[0155] like Figure 7 As shown in B, volcano plot analysis of differentially expressed genes between the two types of samples showed that the cells sorted by the P3 gate had high levels of mature RPE cell marker genes, indicating that more mature RPE cells were sorted.

[0156] like Figure 8 Biological function and cellular component GO enrichment analysis were performed on differentially expressed genes. The results showed that the gene functions of cells sorted by the P3 gate were related to vision and cell polarity (consistent with the characteristics of RPE cells), while the cells sorted by the P2 gate were likely myocytes (unrelated to RPE). This indicates that the sorting method can correctly sort RPE cells and has good results.

[0157] Therefore, flow cytometry analysis clearly reveals the differences in fluorescence properties between control cells and differentiated cells, providing a basis for subsequent cell sorting. Furthermore, observation and transcriptomic analysis of cells collected through the P2 and P3 gates after sorting further validated that cells sorted through the P3 gate possess characteristics of RPE cells (such as pigment production, high expression of mature RPE cell marker genes, and gene functions related to vision and cell polarity), while cells sorted through the P2 gate may not be RPE cells (they may be myocytes). This indicates that the method used in this experiment can effectively sort RPE cells.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for sorting mature RPE cells from iPSC-differentiated cells, characterized in that, Includes the following steps: S1. Differentiate iPSC cells without passage during the differentiation process until the cells are differentiated into RPE cells on days 50-84. S2. After washing the cells with DPBS, add 5-20 U / mL Papain and digest at 37°C for 30-50 min to prevent the cells from adhering to the wall. After digestion, remove the Papain enzyme solution, add Accutase to further digest the cells, digest at 37°C for 30-50 min, add DPBS after digestion, centrifuge and discard the supernatant, then add DPBS to resuspend the cells to obtain a cell suspension. S3. Filter the cell suspension using a 35-45μm cell filter to obtain a single-cell suspension; centrifuge, discard the supernatant, and add EDTA solution to resuspend the cells; S4. Cell sorting is performed using flow cytometry. First, gating is set on the FSC / SSC plot. On the FSC / SSC contour plot in the flow cytometry software, the first gate P1 is set to select cells based on the FSC / SSC signal to exclude interference from cell debris and impurities. Then, the R780-H channel parameters are set. Specifically, the parameters include irradiating cells with 638nm wavelength excitation light and detecting the autofluorescence intensity in the 750-810nm wavelength range through a bandpass filter with a center wavelength of 780nm and a bandwidth of 60nm. Cells with two main peaks are separated from the R780-H channel histogram. Cells with relatively low fluorescence are selected using the P2 gate, and cells with relatively high fluorescence are selected using the P3 gate. Cells are sorted using the P2 and P3 gates. Cells collected by the P3 gate are mature RPE cells. The relatively high fluorescence is one of the following (1)-(2): (1) When cells are divided into two groups under the R780-H fluorescence channel, the cell group with higher fluorescence intensity; (2) When the cells were not divided into two groups under the R780-H fluorescence channel, the fluorescence intensity was higher than that of the cell population of pigment-free control cells.

2. The method according to claim 1, characterized in that, In step S1, cell differentiation occurs from day 60 to 84.

3. The method according to claim 1, characterized in that, In step S2, the following steps are also included: after digestion, add 1 mL of DPBS to disperse the cells, transfer the cells to a centrifuge tube, centrifuge the cells at 300g for 3 min, after centrifugation, remove the supernatant, and resuspend the cells in 1 mL of DPBS to obtain a cell suspension.

4. The method according to claim 1, characterized in that, In step S3, the following steps are also included: aspirating cells into a pipette, blowing them through a 40μm cell filter to remove severely adhered cells and impurities, and obtaining a single-cell suspension; rinsing the cell filter with 1mD PBS, collecting all cell suspensions that have passed through the filter into a centrifuge tube, centrifuging at 300g for 3min, removing the supernatant after centrifugation, and resuspending the cells in a DPBS solution with 5mM EDTA.

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