## 4 Discussion  
In this study, we aimed to tackle some practical issues that potentially limit the market and clinical translation of NP progenitor cells as a regenerative cell transplantation product. First, we examined the ability of EZSPHERE cultures plates to allow large-scale colony-forming culture of NP progenitor cells. Second, we examined the effects of cryopreservation solution on NP cell viability and potency under real-world conditions. Spheroid formation rates were highest using EZSPHERE plate type 4000-903, which has wells with a diameter of 800 \(\upmu\)m and a depth of 400 \(\upmu\)m. The proliferation rates were also the highest for this plate type. 3D cell cultures are used widely in research in various tissues, such as the vascular endothelium and cartilage, in an attempt to simulate the in vivo microenvironment. Better differentiation potential has been demonstrated in vitro compared with monolayer cultures of MSCs in spheroid cultures [26]. Previous work has shown that spheroid-forming NP cells have a greater ability to produce ECM [9; 27] and are thus considered as promising cell types for regenerative and cellular therapeutics. Our data support these previous findings, as EZSPHERE cultured cells exhibited a markedly higher _COL2A1_ expression (on average 836-fold) compared with monolayer cultured NP cells. By contrast, _COL1A2_ expression, as a negative NP marker [28], was markedly downregulated (on average 0.6-fold). The ECM produced by NP cells in human IVD tissue is classified mainly as type II collagen and proteoglycans, and IVD is not formed normally in type II collagen-deficient mice [29]. Therefore, type II collagen is  
Figure 7: Cells (\(n=3\)) were suspended in CellBanker-1 (CB-1) and CryoStor-10 (CS10) and kept for up to 5 h on (B,D) room temperature or (A,C) on ice. Cell viability tested at multiple time points either before or after 1 day of cryopreservation. thought to play an important role in the formation of the IVD, which suggests that NP cells that can be collected by this culture method should have excellent functionality. This supports our previously stated belief that the ability for colonization is an indicator of NP functionality [7]. We were particularly interested in the potential application of EZSPHERE plates as an alternative to the current standard of methylcellulose-based CFU-s cultures [13]. Culturing in EZSPHERE plates has an additional potential advantage because the diffusion gradient of the cultured cells and solution can be controlled more closely, which should improve the reproducibility and quality control for commercialization [30]. Conversely, methylcellulose-based culture leads to the formation of spheroid colonies of different sizes and, because of the high viscosity of the culture medium, cell recovery can be complicated. By contrast, the size of colonies obtained using EZSPHERE culture relies on the well's dimension, which produces relatively uniform colony sizes (Figure 1A). Moreover, the colonies are not maintained in a hydrogel, and the recovery of large quantities of colonies is much easier. The EZSPHERE culture systems offer a promising culture device that could be scalable for production of CFU-s NP cells. As a goal for commercialization, for example, cells from one sample must be extracted and available for at least 100 people. This is important because IVD cell therapy products generally involve the transplantation of a total of 1-100 \(\times\) 10\({}^{6}\) cells per disc (although an optimal concentration has not been determined) (15). For example, in our previous clinical trial [16], we obtained an average of 1.24 \(\times\) 10\({}^{6}\) primary cells/7.92 g tissue per patient donor material (Table 3). Assuming that a cell therapy product requires 1 \(\times\) 10\({}^{6}\) NP cells, \(\sim\)100 times the cell proliferation effect is required.