Method for sorting mature RPE cells from iPSC differentiated cells and application thereof

Abstract

The invention relates to the technical field of biology, and provides a method for sorting mature RPE cells from iPSC differentiated cells and application of the mature RPE cells. The method comprises the following steps: firstly inducing iPSC to differentiate into mature RPE cells, then mildly and effectively separating the cells into a single-cell suspension by adopting a Papain and Accutase combined enzyme, then ensuring that the cells are dispersed single cells or small cell clusters with proper sizes by using a cell filter screen, and finally obtaining the high-purity mature RPE cells through a flow cytometry gating strategy and R780-H channel setting. According to the invention, the cells can normally grow and rapidly proliferate through a combined enzyme method, and the damage to the cells is small; r780-H autofluorescence is utilized, so that the sorting accuracy and reliability are improved; the cells can be directly subjected to flow sorting after being digested and filtered, the operation is simple and convenient, damage to the cells is reduced, cell culture amplification is facilitated, and the experiment efficiency is improved; and sorting is carried out according to optical characteristics of mature RPE cytochrome, expensive antibody labeled cells are not needed, and the method has important application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for sorting mature RPE cells from cells differentiated from iPSCs and an application thereof. Background Art

[0002] Induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC) have shown great application potential in the field of regenerative medicine due to their strong differentiation potential. They can differentiate into a variety of cell types, bringing new hope for the treatment of many serious diseases that currently lack effective drugs. Specifically, by reprogramming the patient's own somatic cells into iPSCs, then directing the differentiation of iPSCs into specific target cells, and transplanting them into the patient's body, it is expected to achieve cell replacement therapy for the disease. However, in this process, although the induction and initial sorting of iPSCs are relatively feasible, during the differentiation stage into target cells, even with strict standardized processes, a large number of non-target cells will inevitably be produced. Therefore, how to efficiently and accurately separate the target cells has become a key link in the success of subsequent transplantation therapy.

[0003] Among numerous cell therapy studies, the use of retinal pigment epithelial (RPE) cells differentiated from iPSC / ESC for the treatment of age-related macular degeneration (AMD) has become a research hotspot. At present, a number of preclinical studies and clinical trials have been actively carried out in this field, and certain positive results have been achieved. These studies have adopted a variety of different processes for the differentiation of iPSC / ESC into RPE cells, in which the separation of RPE cells from differentiated cells is a key step. Among the current technical means for sorting RPE cells, taking the flow cytometer sorting method as an example, the operation process is: first, the cells are digested to obtain a single cell suspension, and then the single cell suspension is incubated with a fluorescent-coupled antibody that can recognize specific antigens on the surface of RPE cells. After the incubation is completed, a washing operation is performed to remove unbound antibodies, and finally the cells are sorted by flow cytometry according to the fluorescence intensity emitted by the cells. This method shows an extremely high level of sorting purity, which can make the proportion of RPE cells reach more than 90%. However, this method has obvious defects. Since it relies on specific antibodies, this directly leads to high sorting costs. At the same time, during the long-term incubation of cells with antibodies, and during the process of cells flowing with the liquid in the narrow liquid path of the flow cytometer, the cells will suffer a great degree of damage, which will cause the mortality rate of cells after sorting to increase significantly.

[0004] In view of the above-mentioned problems with existing sorting methods, in the field of regenerative medicine, especially in application scenarios based on iPSC / ESC differentiation cell therapy technology, such as in the treatment of retinal diseases (especially AMD), there is an urgent need to develop a separation method with higher efficiency, higher accuracy, lower cost and less damage to cells, so as to effectively promote the further development of this technology and provide better 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 cells differentiated from iPSCs and its application.

[0006] The present invention first utilizes specific induction differentiation conditions and environment to induce iPSC cells to differentiate and accumulate related features in the direction of mature RPE cells. And the combined enzyme (Papain and Accutase) of digesting cells is used to gently and effectively separate and disperse cells into single cell suspensions from culture plates to ensure the uniformity and operability of cell samples. Then, using a cell strainer, it is further ensured that the cells entering subsequent analysis are relatively dispersed and single cells or small cell clusters of suitable size. Finally, using flow cytometer gating strategy and R780-H channel settings, highly purified mature RPE cells are obtained. Therefore, the present invention can efficiently and accurately separate mature RPE cells, improve the accuracy and reliability of sorting, help obtain highly purified target cell populations, and provide strong protection for in-depth study of the function and characteristics of RPE cells and the drug development of related disease treatment fields.

[0007] The technical solution of the present invention is achieved in this way:

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

[0009] S1. Differentiating iPSC cells without passaging the cells during the differentiation process until the cells differentiate to day 50-84, so that the iPSC cells differentiate into RPE cells;

[0010] S2. After washing the cells with DPBS, use Papain (papain) to digest the cells so that the cells no longer adhere to the wall; after digestion, remove the Papain enzyme 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 35-45 μm cell strainer to obtain a single cell suspension; centrifuge and discard the supernatant, add EDTA solution to resuspend the cells;

[0012] S4. Use a flow cytometer to sort the cells. First, set the gating operation on the FSC / SSC graph to eliminate the interference of cell debris and impurities; then set the R780-H channel parameters; the parameters specifically include using 638nm wavelength excitation light to irradiate the cells, and detecting the autofluorescence intensity in the wavelength range of 750-810nm through a bandpass filter with a central wavelength of 780nm and a bandwidth of 60nm; sort the cells according to the strength of the fluorescence intensity of this channel to obtain mature RPE cells.

[0013] The description of the "strength" of fluorescence detected by flow cytometry in the present invention is based on the following judgment, that is, "relatively strong" or "relatively high fluorescence" in the present 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 high fluorescence intensity;

[0015] (2) When the cells were not divided into two groups under the R780-H fluorescence channel, the cell population with higher fluorescence intensity than the non-pigmented control cells.

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

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

[0018] The amount of enzyme used can be adjusted adaptively according to the specific circumstances of implementation. Because the catalytic action of the enzyme is highly efficient, the reaction can be started and maintained as long as the concentration reaches a certain level (e.g., 5-20 U / mL Papain) when there is sufficient substrate. Those skilled in the art can reasonably determine the volumetric addition amount of the enzyme based on experience according to factors such as the amount of substrate and the size of the reaction system.

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

[0020] Preferably, in some specific embodiments, the step S3 further includes: sucking the cells into a pipette, blowing through a 40 μm cell strainer, removing severely adhered cells and impurities, and obtaining a single cell suspension; flushing the cell strainer with 1 mL of DPBS, collecting all the cell suspensions that pass through the strainer into a centrifuge tube, centrifuging at 300 g for 3 min, and after the centrifugation is completed, removing the supernatant, and resuspending the cells using a DPBS solution with 5 mM EDTA added.

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

[0022] Preferably, in some specific embodiments, the step S4 further includes: dividing the cells of 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] In a second aspect, the present invention provides the application of the method for sorting mature RPE cells from iPSC differentiated cells described in any of the above. The application is included in one of the following applications: screening of ophthalmic disease therapeutic drugs, ophthalmic drug toxicity assessment, retinal disease pathogenesis research, environmental factors on the retina effect research, retinal development research or cell differentiation regulation mechanism research.

[0024] Furthermore, the application includes one of the following applications: 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] The present invention uses Papain and Accutase to sequentially treat iPSC-differentiated RPE cells, which can effectively digest the cells into a single cell state. After Papain digestion, the cell morphology is complete, the boundaries are clear, and there are very few fragments, which has little effect on the cells; after further digestion with Accutase, the sorted cells can still grow normally and proliferate rapidly, indicating that the digestion method has little damage to the cells, high cell viability, and strong growth ability, which is conducive to the subsequent cell culture and research.

[0027] The present invention uses 638nm wavelength excitation light to irradiate cells and detects autofluorescence within the wavelength range of 750-810nm, which can clearly divide the cells into two groups. It has strong resolution ability and can efficiently and accurately separate mature RPE cells, thereby improving the accuracy and reliability of sorting, helping to obtain a high-purity target cell population, and providing a strong guarantee for in-depth research on the functions and characteristics of RPE cells.

[0028] The present invention can directly use flow cytometry for sorting after cell digestion and filtration, which is easier to operate than conventional flow sorting methods, has less cell processing flow and shorter time, reduces damage to cells, is more conducive to further culture and expansion of enriched cells, improves experimental efficiency and helps to maintain the good state of cells. The present invention is sorted according to the optical properties of mature RPE cell pigments, does not need to use expensive antibodies to label cells, greatly reduces costs, broadens the scope of application of the method, enables more laboratories or research projects to use the technology for cell sorting research, and has obvious advantages in large-scale experiments or cost-sensitive research.

[0029] Explanation of terms :

[0030] Induced pluripotent stem cells (iPS cells or iPSCs): A type of cell with pluripotency similar to that of embryonic stem cells that is 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 the retina, closely adjacent to the retinal photoreceptor cells, and play an important role in maintaining the normal physiological function of the retina.

[0032] Embryonic stem cells (ES cells or ESC) are stem cells with differentiation pluripotency obtained by in vitro culture, isolation and cloning from the inner cell mass of the early blastocyst.

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

[0034] Histogram: It is a method of graphically representing data distribution. In flow cytometry, the horizontal axis of the histogram represents the values ​​of parameters such as fluorescence intensity or scattered light intensity, and the vertical axis represents the frequency or relative number of these values.

[0035] R780-H histogram: A graph drawn with the fluorescence intensity value detected by the R780 fluorescence channel as the horizontal axis and the number of cells or events with this fluorescence intensity value as the vertical axis.

[0036] FSC (Forward Scatter): Forward scattered light refers to the light scattered forward after the laser irradiates the cells.

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

[0038] FSC-H: The "H" here stands for Height.

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

[0040] The R780-H / FSC-H contour map uses FSC-H signal intensity as the horizontal axis and R780-H fluorescence intensity as the vertical axis, and uses contour lines to show the distribution of cells or particles in these two parameters. This contour map can help researchers simultaneously observe the relationship between cell size (reflected by FSC-H) and the expression level of R780 marker substances (reflected by R780-H). For example, it can analyze the expression difference of R780 markers in cell subpopulations of different sizes, or study the expression dynamics of R780 markers during cell size changes, etc., thereby providing more information for further understanding of the biological characteristics and functions of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0042] Figure 1 It is the result of flow cytometry analysis in Example 2 of the present invention; Figure A is a FSC / SSC contour map, in which cell debris and impurities are removed by P1 gating; Figure B is a R780-H / FSC-H contour map, 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; Figure D is a cell-to-debris ratio bar graph, the red part represents normal cells, the blue part represents cell debris, the horizontal axis All represents before P1 gating removal, P1 represents after gating removal, and the vertical axis represents the proportion.

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

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

[0045] Figure 4 The results of flow cytometer analysis in Example 4 of the present invention; Figures AC are control cells: the FSC / SSC graph of Figure A is gated by P1 to remove cell debris and impurities; the R780-H / FSC-H contour graph of Figure B shows that the control cells are a single population; the R780-H histogram of Figure C shows that the control cell fluorescence has only one main peak, most of which are selected by P2 gating, and the part with higher fluorescence is set as P3 gate; Figures DF are differentiated cells: the FSC / SSC graph of Figure D is gated by P1 to remove cell debris and impurities; the R780-H / FSC-H contour graph of Figure E shows that the differentiated cells appear to be a single population, but the fluorescence of some cells is higher than that of the control cells; Figure F maintains the P2 and P3 gating settings of Figure C, and the P3 gate contains about 56% of the cells;

[0046] Figure 5 The cells after flow cytometry sorting in Example 4 of the present invention are shown; R780-Low represents cells sorted by P2 gate, and R780-High represents cells sorted by P3 gate; the upper part is a cell image under a microscope bright field, and the lower part is an image of a cell sediment after centrifugation, and it can be seen that the cells sorted by P3 gate have obvious pigments;

[0047] Figure 6 Phase contrast images (PH) and bright field images (BF) of cells at various stages under a microscope in Example 4 of the present invention; the first column is an image of cells on the 54th day of differentiation; the second column is an image of cells on the 68th day of differentiation and after Papain digestion for 45 minutes; the third column is an image of cells sorted by P3 gate after further culture for 1 day; the fourth column is an image of cells in the third column after further culture for 1 day;

[0048] Figure 7The results of transcriptome sequencing analysis in Example 4 of the present invention; wherein: Figure A represents a cluster heat map, R1, R2 represent two independent repetitions. P2 represents the cell sample sorted by the P2 gate, P3 represents the sample sorted by the P3 gate, and the color represents the relative expression level, red represents high expression, and blue represents low expression; Figure B represents a volcano map of gene expression differences between cells sorted by the P3 gate and cells sorted by the P2 gate; the horizontal axis represents the multiple of expression difference, and the vertical axis represents the significance of difference, red represents genes with relatively high expression, and blue represents genes with relatively low expression; dark red gene names indicate marker genes for mature RPE cells;

[0049] Figure 8 This is the GO enrichment analysis of the biological functions (green) and cellular components (blue) of the genes in Example 4 of the present invention; wherein: Figure A represents the GO enrichment analysis of genes with relatively high expression levels in cells sorted by the P3 gate, including components that are closely related to vision and are related to cell polarity, proving that mature RPE cells are enriched; Figure B represents the GO enrichment analysis of genes with relatively low expression levels in cells sorted by the P3 gate. DETAILED DESCRIPTION

[0050] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. The methods used in the following embodiments are all conventional methods unless otherwise specified.

[0051] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0052] Example 1 Differentiation of iPSC cells

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

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

[0055] 100 mL of DMEM / F-12 (purchased from Gibco, item number C11330500BT) was added with 1 mL of 100×N2 (purchased from Gibco, item number 17502048) additive, 2 mL of 50×B27 (purchased from Gibco, item number 17504044) additive and 1 mL of 100×non-essential amino acids (NEAA, purchased from Gibco, item number 11140050) to obtain 100 mL of RDM. 1.221 g of nicotinamide (NIC, purchased from Sigma, item number N0636) was dissolved in 8 mL of sterile water, vortexed and fixed to 10 mL to obtain 1 M NIC solution, and sterile filtered.

[0056] 1.2. Growth factors and small molecules preparation:

[0057] Resuspend recombinant mouse noggin (purchased from R&D Systems, Catalog No. 1967-NG-025), human dickkopf WNT signaling pathway inhibitor 1 (DKK-1, purchased from R&D Systems, Catalog No. 5439-DK-010) and IGF-1 (purchased from R&D Systems, Catalog No. AFL291) to 100 μg / mL in PBS, store at -20°C after aliquoting. Resuspend FGF-basic (purchased from primegene, Catalog No. 104-02) to 10 μg / mL in PBS containing 0.1% BSA, and resuspend recombinant human / mouse / rat ActivinA (purchased from R&D Systems, Catalog No. 338-AC / CF) to 100 μg / mL, store at -80°C after aliquoting. 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 in DMSO and stored in aliquots at -20°C.

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

[0059] Prepare 1× EDTA solution (0.2 g EDTA / L PBS, purchased from Zhongsheng Suyuan, product number RP01007), 1× PBS without calcium or magnesium (pH 7.4), 1× trypsin-like dissociating enzyme (TDE, purchased from Gibco, product number 12563011), Dulbecco's phosphate buffer solution (DPBS, purchased from Gibco, product number C14190500BT) and 10 μM Y-27632 dihydrochloride (purchased from Taosu, product number T1725).

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

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

[0062] 2.1. Cultivate stem cell colonies to 80% confluence under feeder-free, serum-free conditions.

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

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

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

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

[0067] 2.6. Finally, subculture one well in the 6-well plate to 4 wells of a 12-well plate at a ratio of 1:4 (aspirate the culture medium and wash the cells with pre-warmed PBS- / -. Rinse the cells once with EDTA and incubate with EDTA for 3-5 minutes. Aspirate the EDTA, add an appropriate amount of RDM culture medium, disperse the cells by blowing, and then inoculate them into an ECM-coated 12-well plate, place them in a 37°C, 5% CO2 incubator, and record the time and change the medium on time).

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

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

[0070] Day 2: Replace 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: Replace the medium with RDM containing 100 ng / mL activin A, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. Add 1 mL to each well. At this point, the cells are already confluent.

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

[0073] Days 8, 10, and 12: Replace 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 day 0 of passage

[0075] 4.1. Coat a 6-well plate with ECMH containing reduced growth factors and leave at room temperature for 1 hour.

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

[0077] 4.3. Y-27632 was added to RSM at a concentration of 10 μM to improve adhesion from day 1 to day 4.

[0078] 4.4. Aspirate the old medium and add 1 mL of pre-warmed growth factor-free RDM to each well.

[0079] 4.5. Manually remove non-RPE cells using a stereo microscope and a P10 pipette tip.

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

[0081] 4.7. Add 0.5 mL TDE to each well of the 12-well plate, incubate at 37°C for 5 min, scrape the cells with a scraper, and pipette 3-4 times with a P1000 pipette to form a uniform 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 strainer, count and calculate the cell concentration.

[0085] 4.11. Cells were cultured at 1×10 5 cells / cm 2 The cells were plated at a density of 100 μg / cm2 on growth factor-reduced ECM-coated plates in RSM containing 10 μM Y-27632.

[0086] 4.12. 48 hours after cell inoculation, the medium was replaced with RSM containing 10 μM Y-27632, and then the medium was replaced every 3-4 days (after 4-7 days, the medium containing 10 μM Y-27632 was no longer replaced).

[0087] 4.13. Grow cells at 37°C and 5% CO2 for 28-35 days, replacing RSM every 3-4 days.

[0088] 5. Maturation: RPE passages 1 and 2 (In the following steps, the amounts in brackets refer to a single well of a 6-well plate or a T75 culture flask)

[0089] 5.1. On the 28th to 35th day of passage 0, coat a 6-well plate (T75 culture flask) with ECMH. Prepare DPBS and RSM and preheat them at 37°C, and return TDE 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, 5% CO2 for 5 min, and confirm cell shrinkage and detachment under a microscope.

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

[0092] 5.4. Use a P1000 pipette to pipette up and down the cell / TDE suspension 3-4 times to form a uniform suspension.

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

[0094] 5.6. Centrifuge the cell suspension at 173×g for 5 minutes.

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

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

[0097] 5.9. Cells were cultured at 1×10 5 cells / cm 2 The density of the cells was seeded on ECMH-coated plates in 4 mL (15 mL) of RSM.

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

[0099] 5.11. Repeat the above steps (5.2-5.11) on day 30 to pass the cells from passage 1 to passage 2.

[0100] 6. Creation of intermediate cell bank: Cryopreservation of 2nd generation 3-5 day RPE (cryopreservation of cells that have not fused (about 50%) and have not recovered pigmentation)

[0101] 6.1. Calculate the amount of cryopreservation medium containing 10% DMSO based on the number of cells to achieve a concentration of 3×10 6 cells / mL.

[0102] 6.2. Follow steps 5.2-5.8 and resuspend the cell pellet to 3×10 6 cells / mL, and take 1 mL of the suspension into a 1.2 mL cryovial.

[0103] 6.3. Place the cryovials in a freezing container cooled at -1°C / min, store at -80°C overnight, and then transfer to liquid nitrogen for storage. (These cells were cultured for the third generation after thawing and identified after 30 days of culture. After thawing, the cells were cultured at 1.5×10 5 cells / cm 2 The density of inoculation (3rd generation RPE)

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

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

[0106] A. Specific settings of cell differentiation process: In combination with the method in Example 1, specific induction differentiation conditions and environment are used to guide iPSC cells to differentiate into specific cells, and the consistency and stability of cells in the differentiation process are maintained by suppressing passage operation, so that cells gradually obtain the characteristics of mature RPE cells during long-term differentiation. Obtain a cell colony in a specific differentiation stage (day 65) and with certain characteristics, provide basic samples for subsequent processing and analysis, and promote iPSC cells to differentiate into mature RPE cells and accumulate related characteristics.

[0107] B. Usage of the combined enzymes (Papain and Accutase) for digesting cells: Papain can decompose extracellular matrix proteins to separate cells from the surface of the culture dish; Accutase further acts on the adhesion structure between cells to disperse the cells (Papain alone cannot separate RPE cells from each other and cannot obtain a single cell suspension; Accutase alone cannot effectively separate cells from the surface of the culture dish, and the cells will still adhere to the surface of the culture dish). Specific digestion time and temperature can effectively separate cells while reducing damage to cells. Gently and effectively separating cells from the culture plate and dispersing them into a single cell suspension facilitates subsequent centrifugation, filtration, and flow cytometer analysis operations, ensuring the uniformity and operability of the cell sample.

[0108] C. Use and operation details of the cell strainer: In the operation of the cell strainer, the 40μm filter can filter out severely adhered cell clumps and larger impurities according to the cell size, ensuring that the cells entering the subsequent analysis are relatively dispersed and suitable-sized single cells or small cell clusters. Improve the purity and uniformity of cell samples, avoid interference of large cell clumps or impurities on flow cytometer detection and sorting, and ensure the accuracy and reliability of the analysis results.

[0109] D. Flow cytometer gating strategy and analysis method: After cell digestion, there is no need to stain the cells, and flow cytometry can be used for sorting directly. Sorting is based on the optical properties of the pigments of mature RPE cells, using red light excitation and detecting fluorescence in the red to near-infrared band. According to the forward scattered light (FSC) and side scattered light (SSC) characteristics of the cells, the larger parts 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, and a relatively pure cell population can be screened out for further analysis. Preliminary purification of the cell population improves the accuracy and pertinence of subsequent analysis.

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

[0111] 1. According to the differentiation process in Example 1, iPSC cells were differentiated in a 12-well plate. The cells were not passaged during the differentiation process until the cells were differentiated to day 65.

[0112] 2. After removing the culture medium with a pipette, add 1 mL of DPBS (purchased from Gibco, product number C14190500BT) buffer to wash the cells, discard the supernatant after washing, and repeat the washing 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 digestion for 45 min.

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

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

[0116] 6. After digestion is complete, 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 300 g for 3 min.

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

[0119] 9. Pipette the cells into a pipette and blow through a 40 μm cell strainer to remove severely adherent cells and impurities to obtain a single cell suspension.

[0120] 10. Rinse the cell strainer with 1 mL of DPBS, collect all the cell suspension that passes through the strainer 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. Prepare for flow cytometry (Beckman The cells were analyzed by using a CytoFLEX SRT cell sorter. The R780-H channel parameters were set, which specifically included irradiating the cells with 638 nm wavelength excitation light, and detecting the autofluorescence intensity in the wavelength range of 750-810 nm through a bandpass filter with a central wavelength of 780 nm and a bandwidth of 60 nm.

[0122] 12. On the FSC / SSC contour map of the flow cytometer software, select the part with larger FSC-A (forward scattered light area) and SSC-A (side scattered light 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, it can be clearly seen that the cells are divided into two groups along the R780-H channel. In the histogram of the R780-H channel, it can be seen that the histogram forms two main peaks.

[0125] It can be seen that cell debris and impurities can be effectively excluded through gating operation (P1 gating in Figure A), which account for 10.11% of the total number of particles (the blue part of All in Figure D), and a relatively pure cell population is obtained for analysis. The fluorescence characteristics of the R780-H channel (Figures B and C) indicate that there are two different subpopulations of cells. These subpopulations may represent different stages of cells in the differentiation path or different cell types. This shows that the present invention can successfully differentiate iPSC cells and detect changes in cell characteristics during differentiation by flow cytometry analysis.

[0126] Example 3

[0127] (I) Methods and Steps

[0128] 1. According to the differentiation process in Example 1, iPSC cells were differentiated in a 12-well plate. The cells were not passaged during the differentiation process until the cells were differentiated to day 74.

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

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

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

[0132] (II) Results

[0133] like Figure 2 As shown, the present invention obtains a relatively pure cell population through gating operation, removes 10.11% of the total cell debris or impurities, and improves the reliability of the experiment. The cells are obviously grouped in the 780nm fluorescence characteristics, which may be related to the differentiation state of the cells or the intracellular substances. The number of cells without melanin enrichment and melanin enrichment is similar, which is of great value for studying biological processes related to cytochromes.

[0134] like Figure 3As shown in Figure A, before flow sorting, the cell pellets were mixed together, with no obvious pigment distinction. As shown in Figures B and C, the cell pellet images collected by gate P2 showed that the cells had less or no melanin enrichment, while the cell pellet images collected by gate P3 showed that the cells had obvious melanin. It can be seen that according to the changes in cell pigments before and after flow sorting, we can further understand the differences in the degree of cell pigment enrichment, cell number, cell morphology, etc.

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

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

[0137] In Example 2, on the FSC / SSC contour map of the flow cytometer software, the larger part of FSC-A and SSC-A is framed and set to the first gate (P1). The main purpose is to exclude cell debris and impurities, and to initially obtain a relatively pure cell population for analysis, with emphasis on the initial purification of the overall cell population and the initial observation of the two subpopulations. In this embodiment, the cells are further divided from the peaks and depressions of the two main peaks on the R780-H channel histogram, and the area where the low fluorescence main peak is located is set to the P2 gate, and the area where the high fluorescence main peak is located is set to the P3 gate, and then the cells are sorted and collected by the P2 and P3 gates. Example 3 focuses more on the more refined subpopulation division and sorting of cells according to the fluorescence characteristics of cells, to study the differences between cell subpopulations with different fluorescence characteristics (which may be related to the degree of melanin enrichment).

[0138] It can be seen that the focus of Example 2 is to prove that cell debris and impurities can be effectively excluded by gating operation (P1 gating), and a relatively pure cell population is obtained for analysis, and it is found that the presence of two different subpopulations of cells in the R780-H channel may be related to the cell differentiation path and the influence of growth factors, emphasizing the detection of characteristic changes during cell differentiation. This example 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, highlighting the research value of cytochrome-related biological processes, and paying more attention to the characteristic differences of cell subpopulations in pigments.

[0139] Example 4

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

[0141] 1. According to 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 the cells were differentiated until day 68.

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

[0143] 3. Take ARPE-19 cells (RPE cell line, no pigment production in normal culture) from 12-well plate.

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

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

[0146] 6. Add 1 mL of EDTA solution to resuspend ARPE-19 cells (referred to as NC group)

[0147] (I) Flow cytometric analysis

[0148] The obtained cells were placed on the FSC / SSC dot plot of the flow cytometer software, and the dot with larger FSC-A and SSC-A was selected as the P1 gate. Flow cytometry analysis was performed on the cells in the NC group.

[0149] like Figure 4 , it was observed on the FSC-H / R780-H contour map that the cells in the NC group were a single group, and there was only one main peak on the R780-H channel histogram. On the R780-H channel histogram, the main peak was selected from small to large fluorescence and set as P2 gate, and the rest of the high fluorescence was set as P3 gate. The differentiated cells (recorded as the iRPE group) were put into the instrument for analysis, and it was found that the cells were not obviously grouped on the FSC-H / R780-H contour map, but more than half of the cells on the R780-H channel histogram had stronger fluorescence than the NC group (P3 gate contained 56.33% cells)

[0150] (II) Cell sorting and observation

[0151] The cells were sorted and collected by flow cytometry through the P2 and P3 gates. The collected cell suspension was centrifuged and it was observed that the cells collected by the P2 gate were colorless and the cells collected by the P3 gate were black (e.g. Figure 5 Resuspend the collected cells in culture medium and observe some of the cells under a microscope. The cells collected by gate P3 are black. Take some of the cells collected by gate P3 and continue to culture them. After one day of culture, the cells adhere to the wall. After two days, the number of cells increases (e.g. Figure 6 ).

[0152] (III) Transcriptome analysis

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

[0154] like Figure 7 As shown in A, the transcriptome results were analyzed using a cluster heat map, showing that the two repeated P2 gate sorting samples and P3 gate sorting samples were clustered separately, indicating that the sorting was stable.

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

[0156] like Figure 8 , biological function and cellular component GO enrichment analysis was performed on the 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), and the cells sorted by the P2 gate were likely muscle cells (not related to RPE), indicating that the sorting method can correctly sort RPE cells and the effect is good.

[0157] It can be seen that the difference in fluorescence characteristics between control cells and differentiated cells can be clearly observed through flow cytometric analysis, and this difference provides a basis for subsequent cell sorting. In addition, observation and transcriptome analysis of cells collected by gates P2 and P3 after cell sorting further verified that cells sorted by gate P3 have the 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 by gate P2 may not be related to RPE cells (possibly muscle cells), indicating 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 in the protection scope of the present invention.

Claims

1. A method for sorting mature RPE cells from cells differentiated from iPSCs, characterized in that, The steps include: S1. Differentiating iPSC cells without passaging the cells during the differentiation process until the cells differentiate to day 50-84, so that the iPSC cells differentiate into RPE cells; S2. After washing the cells with DPBS, use Papain to digest the cells so that they no longer adhere to the wall; After digestion, remove the Papain enzyme 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; S3. Filter the cell suspension using a 35-45 μm cell strainer to obtain a single cell suspension; centrifuge and discard the supernatant, add EDTA solution to resuspend the cells; S4. Use a flow cytometer to sort the cells. First, set the gating operation on the FSC / SSC graph to eliminate the interference of cell debris and impurities; then set the R780-H channel parameters; the parameters specifically include using 638nm wavelength excitation light to irradiate the cells, and detecting the autofluorescence intensity in the wavelength range of 750-810nm through a bandpass filter with a central wavelength of 780nm and a bandwidth of 60nm; sort the cells according to the strength of the fluorescence intensity of this channel to obtain mature RPE cells.

2. The method according to claim 1, characterized in that In step S1, the cells are differentiated to day 60-84.

3. The method according to claim 1, characterized in that In step S2, 5-20 U / mL Papain is added and digested at 37° C. for 30-50 min.

4. The method according to claim 1, characterized in that The step S2 specifically further includes: adding Accutase to further digest the cells, and digesting at 37° C. for 30-50 minutes.

5. The method according to claim 1, characterized in that The step S2 specifically includes: adding 1 mL of DPBS after digestion to disperse the cells, transferring the cells to a centrifuge tube, centrifuging the cells at 300 g for 3 min, removing the supernatant after centrifugation, and resuspending the cells with 1 mL of DPBS to obtain a cell suspension.

6. The method according to claim 1, characterized in that The step S3 specifically includes: sucking the cells into a pipette, blowing through a 40 μm cell strainer, removing severely adhered cells and impurities, and obtaining a single cell suspension; using 1 mL of DPBS to rinse the cell strainer, collecting all the cell suspensions that pass through the strainer into a centrifuge tube, centrifuging at 300 g for 3 minutes, and after the centrifugation is completed, removing the supernatant, and resuspending the cells using a DPBS solution with 5 mM EDTA added.

7. The method according to claim 1, characterized in that The step S4 specifically includes: on the FSC / SSC contour map of the flow cytometer software, setting a first gate P1 to select cells according to the FSC / SSC signal to screen out impurities and cell debris.

8. The method according to claim 1, characterized in that The step S4 specifically also includes: separating the cells of 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.

9. Use of the method according to any one of claims 1 to 8, characterized in that The application includes one of the following applications: screening of drugs for treating ophthalmic diseases, evaluation of toxicity of ophthalmic drugs, study of the pathogenesis of retinal diseases, study of the effects of environmental factors on the retina, study of retinal development or study of the regulatory mechanism of cell differentiation.

10. Use of the method according to any one of claims 1 to 8, characterized in that The application includes one of the following applications: age-related macular degeneration drug development, retinitis pigmentosa drug development or macular dystrophy drug development.

Images