METHOD FOR DIGESTION OF RPE CELLS

Abstract

The present invention disclose a method for digesting RPE cells to dissociate adherent cells, comprising steps: (1) using 1 recombinant enzyme TrypLE to cover RpE cells evenly; (2) using neutral protease Dispase II solution to cover RPE cells evenly, in which steps (1) and (2) are applied step by step.

Claims

1. A digestive enzyme composition for dissociating adherent retinal pigment epithelium (RPE) cells, comprising a recombination enzyme TrypLE and a neutral proteinase Dispase II, wherein the concentration of the neutral protease Dispase II is from about 0.5 mg/mL to about 4.0 mg/mL.

2.-3. (canceled)

4. The composition of claim 1, wherein the concentration of the neutral protease Dispase II is from about 2 mg/mL to about 4.0 mg/mL.

5. A method for digesting RPE cells to dissociate adherent RPE cells, comprising the steps of: (1) covering RPE cells evenly with 1 recombinant enzyme TrypLE and incubating; (2) discarding the TrypLE; and (3) covering RPE cells evenly with neutral protease Dispase II solution and incubating; wherein steps (1) to (3) are applied step by step.

6. The method of claim 5, wherein the concentration of the neutral protease Dispase II is from abut 0.5 mg/mL to about 4.0 mg/mL.

7. The method of claim 5, wherein the RPE cells are cultured for about 10 to about 17 days.

8. The method of claim 5, wherein step (1) comprises incubating the RPE cells and the recombinant enzyme TrypLE for about 14 to about 16 minutes at 37 C.

9. The method of claim 7, wherein step (3) comprises incubating the RPE cells and the neutral protease Dispase II for about 10 to about 15 minutes at 37 C.

10. The method of claim 5, wherein the RPE cells are cultured for over 17 days.

11. The method of claim 10, wherein step (3) comprises incubating the RPE cells and the neutral protease Dispase II for about 20 to about 22 minutes at 37 C.

12. The method of claim 5, further comprising washing the cells with calcium and magnesium free DPBS and discarding the DPBS before step (1) or step (3).

13. The method of claim 5, wherein the concentration of the neutral protease Dispase II is from about 2.0 mg/mL to about 4.0 mg/mL.

14. The method of claim 5, wherein the RPE cells are cultured for about 10 days or longer.

15. The method of claim 14, wherein step (3) comprises incubating the RPE cells and the neutral protease Dispase II for about 10 to about 15 minutes at 37 C.

Description

DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows the experimental flow chart of a specific embodiment of the present invention.

[0029] FIG. 2 shows the counting result after digestion with different concentrations of digestive enzymes.

[0030] FIG. 3 shows the cell viability after digestion with different concentration of digestive enzymes.

[0031] FIG. 4 shows the cell viability after digestion with different concentration of digestive enzymes.

[0032] FIG. 5 shows the images of cells before or after digestion.

[0033] FIG. 6 shows the quantitation of cell number and cell viability after digestion with TrypLE+Dispase II group and trypsin+Dispase II group, separately.

[0034] FIG. 7 shows the quantitation of cell adherent rate after TrypLE+Dispase II group and trypsin+Dispase 11 group digestion, separately.

[0035] FIG. 8 shows the images of RPE cells after TrypLE digestion only.

[0036] FIG. 9 shows the images of RPE cells after Dispase II digestion only.

DETAILED DESCRIPTION

[0037] The present invention will be further illustrated in detail below with reference to the specific examples. These examples are only used to describe the invention, and shall not be construed as limiting the scope or content of the invention in any way.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0039] As described herein, terms TrypLE, recombinant enzyme TrypLE, and Tryple are interchangeable.

[0040] As described herein, terms Dispase II, neutral protease II, and disperse enzyme II are interchangeable, and refers to a non-specific matrix metalloproteinase.

[0041] As described herein, term adherent rate refers to the percentage of adherent and survival cells after digestion, freeze-thaw, recovery centrifugation of the same number of cells, and plate culture of the same number of cells after centrifugation precipitation. Therefore, the adherent rate used in the invention reflects the cell viability after cryopreservation. Recovery rate is the ratio of the count value of the recovered cells after washing to that of before cryopreservation.

EXAMPLES

Example 1: Study of Digestive Solution on the Cell of P2 Generation

[0042] FIG. 1 shows the experimental flow chart, cells in different groups are digested according to table 1. For each group, four wells of cells are digested. Count after digestion and freeze the cells, 110.sup.6 of live cells per tube.

TABLE-US-00001 TABLE 1 Study groups of the digestion solution on P2 cell Group Concentration of digestion solution Digestion time Experiment 1 TrypLE + 0.5 mg/mL Dispase II 10 + 15 minutes group 1 digestion Experiment 1 TrypLE + 2.0 mg/mL Dispase II 10 + 15 minutes group 2 digestion Experiment 1 TrypLE + 10 mg/mL Dispase II 10 + 15 minutes group 3 digestion

[0043] As shown in FIGS. 2 to 4, the result shows that the cell viability after digestion with high concentration of digestive enzyme (10 mg/mL) is worse. The cell viability after digestion with low concentration of digestive enzyme (0.5 mg/mL) has no significant difference compared to that of the 2.0 mg/mL group, but it requires hard pipetting using pipetman to blow all the cells down. In sun, high-quality RPE cells can be obtained by Dispase II digestion at 2.0 mg/mL. However, the combination of 2.0 mg/mL Dispase II and 1rypLE is not conventionally selected randomly or simply. In fact, the inventor has found that RPE cells are characterized by a strong connection between cells, and even stronger connections between cells and substrate matrix. According to the characteristics of the two different connecting forces, the two-step digestion method of the inventor's invention finally determines the combination of 1TrypLE and Dispase II after a large number of screening and experiments, and find that 2.0 mg/mL of Dispase II exerts the optimal effect.

Example 2: Comparison to Trypsin Combination

[0044] TrypLE select is purchased from Sigma, disperse enzyme Dispase II solution is purchased from ROCHE; RPE cells are from the P2 generation cells of primary dissociated human ocular tissue; DPBS is purchased from Sigma.

[0045] The adherent RPE cells are dissociated according to the steps:

[0046] (1) Pre-heating RPE complete medium, DPBS and TrypLE select in thermostat water bath at 37 C. for 5 to 10 minutes, and then transfer them into UV sanitized biosafety cabinet for use after sanitization by 75% ethanol.

[0047] (2) Obtaining the RPE cells cultured in 6-well plate for 10 days from CO.sub.2 incubator to the biosafety cabinet, discarding the supernatant in 6-well plate, adding in 1 mL of DPBS and washing the cell surface twice.

[0048] (3) Discarding the abovementioned DPBS, adding in TrypLE select at 1.0 mL/well to cover well surface evenly, put it into CO.sub.2 incubator at 37 C. for 10 minutes and discarding TrypLE select, and use trypsin for the control group; Discard the supernatant after wash the cell surface once with DPBS (calcium and magnesium free), adding in 1.0 mL of 2.0 mg/mL Dispase II solution diluted by DPBS (calcium and magnesium free), digesting in CO.sub.2 incubator at 37 C. for 12 minutes.

[0049] (4) After digestion, discarding Dispase II solution, adding in 1 mL of RPE complete medium, aspirating the cells by gently pipetting gently, collecting the cells into 15 mL centrifugation tube and centrifuging at 2000 rpm for 5 minutes, discarding the supernatant, resuspending by DPBS and counting, freezing the cells according to the counting result, and getting them out from the liquid nitrogen tank to test the adherent rate afterwards.

[0050] As shown in FIG. 5, the images of cells before and after trypsin digestion show that the effect of trypsin is not as good as that of TrypLE, but generally equivalent. However, after adding in Dispase II solution again, the digestion effect of the TrypLE group is obviously better, showing the synergistic effect of TrypLE and Dispase II, i.e. in situation without affecting number and viability of RPE cells, the RPE cells have better disperse effect by the synergistic effect of TrypLE and Dispase II, and finally the cells have higher adherent ratio after freezing and recovery.

[0051] In fact, the inventor has found that the cell digestion way has a great influence on the adherent rate of cells after cryopreservation, however, there is no effective method to control cell adherence after cryopreservation and recovery in present techniques. The present invention has for the first time discovered the effect of digestion way on subsequent cell adherent rate for RPE cells, invented the two-step digestion method for RPE cells, and used TrypLE and Dispase II to greatly reduce the cost of culture and injection of RPE cells in clinic, and maintain the cell state and activity at a high level after recovery through the synergistic effect, which has good reproducibility.

Example 3: Digestion Effect by Using TrypLE or DispaseII Only

[0052] When using a single enzyme such as TrypLE to digest cells, as it only works on cell-cell dissociation, it cannot achieve the expected digestion results; By experiments, it is found that cell dissociation time should be prolonged to more than 35 minutes, however, by that time part of the cells have already been damaged because of the over dissociation and will be lysed after pipetting, which resulting in cell number to be significantly lower than the actual number. Even though after hard pipetting, cells still cannot be dissociated from culture matrix completely, as shown specifically in FIG. 8. Therefore, the TrypLE digestion alone is not good.

[0053] As Dispase II is mild and found in the invention that it mainly functions on cell and culture matrix, dissociating cells by Dispase II alone can hardly dissociate cells, even after a very long time, as shown in FIG. 9 for dissociation effect.

[0054] Therefore, it further demonstrates that the RPE cells in Example 2 has much better disperse effect by the synergistic effect of TrypLE and Dispase II.

INCORPORATION BY REFERENCE

[0055] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes

EQUIVALENTS

[0056] The invention may be embodied in other specific forms without departing from the essential characteristics thereof. The foregoing embodiments are therefore to be considered illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and the range of equivalency of the claims are intended to be embraced therein.