FIGURE 7.

Post-thaw survival and function of cryopreserved D7 SN under varying nucleation temperatures and cooling rates. Data are represented as mean ± 95% confidence interval. *p < 0.05; n.s.: p > 0.05. Red box: best-case parameters tested with the highest recovery and reattachment. (A) Post-thaw recovery of D7 SN showing no significant change with greater undercooling and the best cooling rate at −3°C/min with statistical significance. n = 4. (B) Post-thaw reattachment of D7 SN showing similar trends as recovery but more distinct cell damage with lower nucleation temperature (−8°C). n = 4. (C) Calcein AM-stained culture 24 h after replating dissociated (fresh) or cryopreserved (best-case) D7 SN showing high confluence and normal morphology of immature neurons. Scale bar: 100 µm. (D) Calcium imaging of post-thaw culture after 7-day maturation of D7 SN cryopreserved with −4°C nucleation and −3°C/min cooling rate, showing little to no response to 0.1% DMSO (negative control), positive response to 1 µM veratridine that was inhibited by TTX, and positive response to 30 mM KCl that was unaffected by TTX. Red line: responder cell; black line: nonresponder cell. Proportion of responder cell population indicated per graph. Range of n = 417–662.