Method for digesting RPE cells
A technology of cells and digestive enzymes, applied in the field of life sciences, can solve problems such as vision restoration without reversal, and achieve a highly controllable effect
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Embodiment 1
[0046] Example 1: Research on the concentration of the digestive fluid of P2 generation cells
[0047] figure 1 Shown is the experimental procedure. Cells in each group were digested according to Table 1, and cells in four wells on one plate were digested in each group. Count after digestion, and freeze the cells, each tube 1×106 living cells.
[0048] Table 1. Research grouping of concentration of digested solution of P2 generation cells
[0049]
[0050] Such as Figures 2 to 4 As shown in the results, it can be seen that the cell viability is poor after digestion with high concentration (10mg / mL) digestive enzymes. However, the number and viability of cells digested with low concentration (0.5mg / mL) digestive enzymes were not significantly different from those in the 2.0mg / mL group, but it was necessary to use a pipette to forcefully blow the cells to come down. Taken together, the use of 2.0mg / mL Dispase II digestion can obtain high-quality RPE cells. However, the ...
Embodiment 2
[0051] Embodiment 2 compares with trypsin combination
[0052] TrypLE select was purchased from sigma, and dissociative enzyme Dispase II solution was purchased from ROCHE; RPE cells were P2 cells from primary isolated human eye tissue; DPBS was purchased from sigma.
[0053] Follow the steps below to release RPE cells in the adherent state:
[0054] (1) Preheat RPE complete medium, DPBS, and TrypLE select in a constant temperature water bath at 37°C for 5-10 minutes. After preheating, the surface is sterilized with 75% alcohol and transferred to a biosafety cabinet that has been sterilized by ultraviolet light. use;
[0055] (2) Take the RPE cells cultured in six-well plate culture flasks for 10 days out of the carbon dioxide incubator and put them into a biological safety cabinet. Discard the culture supernatant in the six-well plate, and add 1mL DPBS to wash the cell surface twice.
[0056] (3) Discard the above-mentioned DPBS, add 1.0mL / well TrypLE select to evenly cover...
Embodiment 3
[0060] Embodiment 3 uses the digestion effect of TrypLE or DispaseII alone
[0061] When digesting cells with a single enzyme, such as TrypLE, because it only affects the dissociation between cells, it cannot achieve the expected digestion results; the experiment found that the cell dissociation time needs to be extended to more than 35 minutes, but at this time due to excessive dissociation Some of the cells have been damaged, and they will be lysed after being blown, resulting in a significantly lower number of cells than the actual number. Even after strong pipetting, the cells still cannot be completely dissociated from the culture medium. For the specific effect, see Figure 8 shown. So digestion with TrypLE alone is not good.
[0062] Because DispaseII is relatively mild and found in the present invention that it mainly acts on cells and culture substrates, when DispaseII is used alone to dissociate cells, it is difficult to dissociate even if it dissociates for a long...
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