## 3 Results
### Cell Potential after Cryopreservation  
Next, NP cells were exposed to cryostorage solutions (CB1, SCB, or SCBD-Free) for 3 h on ice before freezing. The cells were thawed and subsequently cultured. The cells frozen in CB1 had an average cell viability of 95.7% (Figure 4A), but the other two cryopreservation solutions resulted in considerable cell loss (Figure 5A). The average growth rate of cells frozen in CB1 was 5.9 times (\(p\) = 0.53), which was lower than the growth rate of cells frozen in SCB (8.93 times; Figure 5B). The Tie2- and CD24-positive rates in flow cytometry analysis showed a trend toward reduced Tie2- and CD24-expressing fractions for NP cells kept in CB 1 compared with SCBD-Free, although this difference was not significant (Figure 5C,D).  
Additional investigation with a wider range of cryopreservation solutions (CB1, SCB, SCBD-Free, CS10, CP-1, and PF) revealed viability ranging from 63.6% (PC) to 84.6% (CS10) (Figure 6A). Interestingly, the Tie2-positivity rates of NP cells was 12.6% in cells in CS10, suggesting a higher Tie2 retention rate in cells in CS10 than in CB1 (Figure 6B).  
Figure 4: Cells (\(n=1\)) were suspended in CellBanker-1 (CB-1), STEM-CELLBANKER (STEM-CB), or STEM-CELLBANKER without DMSO (STEM-CB\({}_{\text{DMSO}}\) freeze) and kept for up to 5 h on (**A**,**B**) room temperature or kept (**C**,**D**) on ice. Cell viability tested at multiple time points either before or after 1 day cryopreservation.  
Finally, CS10 and CB1 were used to resuspended primary NP cells and the effects on cell viability in cells with time left at room temperature or on ice were examined. The average cell viability was 95.2% (\(\pm\)1.8%) for cells in CS10, and this value was maintained even after 5 h on ice (Figure 7A) and at 89.9% (6.5%) in cells stored at room temperature  
Figure 5: Cells (\(n=3\)) were suspended in CellBanker-1 (CB-1), STEM-CELLBANKER (STEM-CB), or STEM-CELLBANKER without DMSO (STEM-CB\({}_{\text{DMSO}}\) freeze) and kept for on ice for 3 h and frozen for 1 day. Subsequently (**A**) cell viability was assessed. Following cryostorage and thawing cells were seeded cultured for 5 days. The (**B**) fold-increase in cell numbers was analyzed, as well as the fraction of (**C**) Tie2 expressing and (**D**) CD24 expressing cells as analyzed by flow cytometry. Data is presented as mean values with error bars presenting standard deviation. * \(p\) < 0.05.  
Figure 6: Comparison of 6 types of cryopreservation solutions, namely CELLBANKER 1 (CB-1), STEM-CELLBANKER GMP grade (STEM-CB), STEM-CELLBANKER DMSO Free GMP grade (STEM-CB\({}_{\text{DMSO}}\) freeze), CryoStor\({}^{\text{\it[B]}}\) CS10 (CS10), and Pro FreezeTM-CDM (ProF). Nucleus pulposus cells suspended in the cryopreservation solutions and kept on ice for 3 h before cryostoring them for 1 day. The cells were recovered and cultured for 5 days. Subsequently, the (**A**) viability rate and the (**B**) fraction of Tie2 positive cells were determined. Data is presented as mean values with error bars presenting standard deviation.  
(Figure 7B). CB1 produced similar results (Figure 6B). The average cell viability after cryopreservation and thawing also remained high: 88.4% (\(\pm\)4.6%) in cells on ice in CS10 or 87.7% (\(\pm\)6.0%) in CB1 (Figure 7C). However, when kept at room temperature, the survival rate after cryopreservation and thawing appeared to decrease to 67.5% (\(\pm\)29.9%) after 3 h in CS10 compared with 82.1% (\(\pm\)6.5%) in CB1 (Figure 7D).