Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation

Abstract

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post-thaw culture, the presence of 10 microM ROCK inhibitor (Y-27632) and 1 microM pifithrin-mu together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells.

Figure 1. Cell viability immediately after thawing

Results are expressed as the mean of independent experiments ± the standard deviation (n = 3), Statistical analysis was performed using one-way ANOVA. *P < 0.05, compared with the results obtained in 10% DMSO.

Figure 2. Cell growth after cryopreservation for feeder-independent and feeder-dependent culture

A: For feeder-independent culture. B: For feeder-dependent culture. R(+)/R(−): with or without Rock inhibitor; P53(+)/P53(−): with/without p53 inhibitor in the subsequent culture medium. Results are expressed as the mean of 3–4 independent experiments ± the standard deviation. Statistical analysis was performed using one-way ANOVA, *P < 0.05. Compared with the results obtained in 10% DMSO. **P<0.05, compared with the results between 10% DMSO in the group of R(+)P53(−) PEG, ***P < 0.05, compared with the results between R(+)p53(−) and R(+)p53(+).

Figure 3. Apoptosis rate after 2 h plating for feeder-independent culture

A: Apoptosis rate for the cells frozen at the different conditions, B: Apoptosis rate for the cells frozen by 10% DMSO and then cultured at different conditions after thawing, R(+)/R(−): with or without Rock inhibitor. P53(+)/P53(−): with or without pifthrin μ inhibitior. Results are expressed as the mean of independent experiments ± the standard derivation (n ≥ 3). Statistical analysis was performed using one-way ANOVA.

Figure 4. Caspase-9 activity after 2 h and 1 day of plating after cryopreservation for feeder-independent culture

A: Caspase-9 activity after 2 h of plating, B: Caspase-9 activity after 1 day of plating. Rock inhibitor present/absent R(+)/R(−) at the first day of culture after cryopreservation, pifithrin-μ present/absent P53(+)/P53(−) at the first day of culture after cryopreservation. Results are expressed as the mean of 3–4 independent experiments ± the standard deviation. Statistical analysis was performed using one-way ANOVA. *P < 0.05, compared with the results in R(−)p53(−) for 10% DMSO and 7.5% DMSO +2.5% PEG groups. **P < 0.05, compared with the results in R(+)p53(−) for 10% DMSO and 7.5% DMSO +2.5% PEG groups.

Figure 5. Intracellular superoxide anion and the amount of F-actin from feeder-independent culture before and after cryopreservation at different freezing conditions

A: Intracellular superoxide anion. B: The amount of F-actin. Results are expressed as the mean of 3–4 independent experiments ± the standard derivation. Statistical analysis was performed using one-way ANOVA, *P < 0.05, compared with the results from regular passage. **P < 0.05 compared with the results from 10% DMSO. D10: 10% DMSO, D7.5Peg: 7.5% DMSO +2.5% PEG, D7.5: 7.5% DMSO.

Figure 6. Expression of undifferendiated marker, nuclear markers, OCT4 and Nanog, and surface marker, SSEA4, for hES cells at passage two after cryopreservation using 10% DMSO and 7.5% DMSO +2.5% PEG

The cells after cryopreservation were cultured in the presence of ROCK inhibitor, or in the presence of ROCK inhibitor and pifithrin-μ. A: Feeder-independent culture, B: Feeder-dependent culture. Scale bar: 50 μm for feeder-dependent culture and 25 μm fro feeder-independent culture, respectively.