## 4 Discussion
However, with the EZSPHERE method alone, the cell proliferation rate was 7.75-fold in our study, which would mean that only nine patients on average could be treated with cells from a single-batch production following EZSPHERE culture methods. Our data in the present study show that growth factor supplementation can markedly increase the NP cell proliferation rate. These findings are consistent with previous work showing that FGF2 has a Tie2 expression-enhancing effect on NP cells [14]. In our study, FGF2 supplementation had positive effect by increasing the cell proliferation rate by up to 62.5%. This would make it possible to manufacture NP cell products for about 77 patients from a single tissue sample. However, cells cultured with other growth factors showed a marked decrease expression of _COL2A1_ and _ACAN_, which may be a disadvantage compared with the original colony assay culture. Further analysis and in vivo experimentation are needed to elucidate these effects. \begin{table}
\begin{tabular}{c c c c c c c} \hline  & **Age** & **Sex** & \begin{tabular}{c} **IVD Tissue Amount** \\ **(g)** \\ \end{tabular}  & \begin{tabular}{c} **Number of Collected** \\ **NPC** \\ \end{tabular}  & \ \begin{tabular}{c} **Number of Cultured** \\ **NPC** \\ \end{tabular}  & \begin{tabular}{c} **Cultured Cell** \\ **Recovery Time (min)** \\ \end{tabular}  \\ \hline
1 & 21 & M & 8.2 & \(1.25\times 10^{6}\) & 7.2 \(\times\) 10\({}^{6}\) & 115 \\
2 & 28 & M & 4 & \(0.93\times 10^{6}\) & 5.85 \(\times\) 10\({}^{6}\) & 113 \\
3 & 28 & M & 10.9 & \(3.42\times 10^{6}\) & 10.80 \(\times\) 10\({}^{6}\) &The different types of cryopreservation solutions and exposure times used in the cryopreservation of cellular products are thought to affect the product quality [24]. Especially in undifferentiated cells such as MSCs, the maintenance and prevention of phenotype loss and changes in metabolism during storage are important. Reports have suggested inadequate therapeutic results can arise because of the effects of cryopreservation in clinical trials using MSCs [31; 32]. Given our aim to deliver multipotent, undifferentiated NP progenitor cells with high Tie2 positivity, concerns were raised about the effects of cryopreservation on NP cells and their potency. In the trial reported by [PERSON] et al. [16], the collection of cultured cells before their administration in the operating room was performed in a similar fashion as in our experiments; that is, the NP cells were cryopreserved before transplantation. The purpose of the current study was to identify the most appropriate cryopreservation solutions and freezing procedures by investigating their effects on NP cells over time. Of the 10 patients treated in the previous clinical trial [16], an average of 4.6 \(\times\) 10\({}^{6}\) NP cells were administered following harvest from their respective coculture conditions. At the time of surgery, the cryostored NP cells were recovered, thawed, and transported from a cell-processing center to the operating room, and the waiting time before transplantation was on average 122.9 min, which meant that the cells were exposed to the cryopreservation agents for about 2 h. Our current results show that NP cells subjected to cryopreservation agents for up to 3 h had survival rates of \(\leq\)70% in SCB and SCBD-Free. Interestingly, cells kept on ice in CB1 retained a cell viability of 90% for up to 5 h.  
Although CB1 has been shown to be optimal, for clinical translation, it is desirable to preserve and administer cells in solutions devoid of animal-derived serum or DMSO. Therefore, we examined six additional commercial cryopreservation preparations. We found that cell viability after thawing was maintained at \(\geq\)80% with CB1, CS10, and CP-1. CS10 was also able to maintain a high Tie2-expressing section. In addition, CS10 is an animal component-free GMP-certified formula, making it potentially ssafer to administer directly to patients than CB1 when used as an OTS [23].