Method for preparing photoreceptor cells
A technology of photoreceptor cells and cells, which is applied in the fields of stem cell biology and regenerative medicine, can solve the problems of low purity, low differentiation efficiency, and disadvantages of stem cell transplantation, and achieve the effects of reducing rejection, improving integration efficiency, and reducing tumorigenicity
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Embodiment 1
[0095] Example 1: Induction of pluripotent stem cells (hPSCs) into fibroblasts (FLCs)
[0096] 1. Before resuscitating hPSCs cells (which can be purchased commercially), prepare the diluent, resuscitation solution, and coat the culture flask with Geltrex.
[0097] 2. Culture hPSCs with mTeSR1 or E8 medium, and change the medium every day;
[0098] 3. When the cells grow to 80%, wash twice with 1×PBS, add MEF medium;
[0099] 4. Change the medium every day for the first four days, and change the medium every two days thereafter, and cultivate for 6 to 12 days;
[0100] 5. Digest cells with 0.05% Trypsin-EDTA, centrifuge, cell density 1×10 6 / well, the cells were plated in a 6-well plate coated with Geltrex;
[0101] 6. When the cells grow to about 5 days, digest the cells with 0.05% Trypsin-EDTA, and spread the cells on a 6-well plate coated with 0.1% gelatin after centrifugation. The cell density is 2×10 5 ;
[0102] 7. After 3 to 5 days of cell culture, the cell shape is...
Embodiment 2
[0104] Example 2: Transfection of FLCs with lentivirus carrying Nrl-tRFP gene
[0105] 1. Culture hFLCs in a 6-well plate coated with 0.1% gelatin gel at a cell density of 2×10 5 ;
[0106] 2. When the cell density reaches 50-60%, transfect hFLCs with lentiviral particles carrying Nrl-tRFP gene;
[0107] 3. After 24 hours of transfection, remove lentiviral particles and add MEF medium;
[0108] 4. After 48 hours of transfection, add puromycin (final concentration 1ug / mL) MEF medium, if the cells grow to 100%, digest the cells with 0.05% Trypsin-EDTA, and then re-spread the cells according to the ratio of 1:2 in a 6-well plate coated with 0.1% gelatin;
[0109] 5. Replace the MEF medium containing puromycin every other day and culture for 4-6 days;
[0110] 6. Collect the cells treated with puromycin;
[0111] 7. Transfer the cells to a 6-well plate coated with 0.1% gelatin gel and culture for 3-5 days;
[0112] 8. Nrl-tRFP FLCs transfected with lentivirus are passaged ev...
Embodiment 3
[0113] Example 3: Induction of Nrl-tRFP FLCs cells into chemically reprogrammed photoreceptor cells;
[0114] Note: During the whole process of cell induction, all small molecules or media should be replenished daily according to the shape of the cells.
[0115] 1. Nrl-tRFP hFLCs were cultured in 12-well plates coated with 0.1% gelatin glue, the medium was the fifth medium, and the cell density was 50-80×10 3 ;
[0116] 2. Day 1: Replace the culture medium with the first culture medium;
[0117] 3. Day 3: replace the first medium with the second medium;
[0118] 4. Day 4-7: Replace the second medium according to the state of the cells;
[0119] 5. Day 8: Replace the second culture medium with the third culture medium;
[0120]6. The 10th to 11th day: can be observed and collected;
[0121] 7. Day 15-16: The third medium can be further replaced with the fourth medium to further increase the differentiation ratio and differentiation efficiency of photoreceptor cells.
[01...
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