We believe that the potency of NP cell therapy could be increased by using a cell population with high Tie2-positive rates. However, Tie2-positive cells remain difficult to maintain in culture because they rapidly lose their Tie2 expression and consequently their proliferative capacity and potency [13]. In addition, mass production of these cells is limited by the high viscosity of the methylcellulose medium and unsuitability for industrialization. A variety of studies have investigated ways to improve the culture conditions [10; 14; 20] or isolation methods [21] to increase the number of Tie2-expressing cells. For example, [PERSON] et al. reported beneficial effects of fibroblast growth factor 2 (FGF2) and hypoxic culture conditions [14]. Recent work by [PERSON] et al. [10] showed the benefits of three-dimensional (3D) pellet culture environments for maintaining the multipotent capacity and retaining Tie2 expression in Tie2-positive NP progenitor cells. These findings provide evidence for the potential of culture augmentation to increase the retention of NP progenitor cells. However, an optimized culture method that would be scalable, marketable, and clinically applicable remains to be determined. Another consideration that could hinder the translation of Tie2-positive NP progenitor cells to the market is their preservation. [PERSON] et al. has shown that cell viability, differentiation ability, and ECM production do not change significantly from before to after NP cell cryopreservation [22]. This finding suggests that activated NP cells could be used in a cryopreserved state whenever needed according to the patient's condition, thus increasing their potential as a commercial, mass-produced, off-the-shelf (OTS) regenerative therapeutic product. In a canine study, an OTS osteogenic NP cell product was shown to be as effective in regenerating induced disc degeneration compared with freshly cultured cells [23]. These products are currently undergoing clinical trial evaluations (NCT03347708, NCT03955315). There is a large difference between the actual clinical application and the results in laboratory-based reports. In addition, the time required before and after freezing is rarely considered [24]. The cytotoxicity of animal serum and dimethyl sulfoxide (DMSO) contained in the cryopreservation solutions may also present safety and regulatory problems during clinical applications [13; 25]. Furthermore, during the product management and transportation after mass culture, the process from cell isolation to storage becomes more complicated and time consuming. We wanted to examine the potential for optimization of the practical aspects of NP cell production in terms of cell culture, storage, and logistic handling. Given the needs of future commercialization, we aimed to identify optimal cryopreservation solution(s) that could limit the functional deterioration and maintain the high cell viability of Tie2-positive NP cells. We examined the potential for using specialized nonadherent EZSPHERE(r) dishes, which have been used with induced pluripotent stem cells and cancer cells but not with NP cells. This culture dish supports 3D cell cultures without the need for hydrogel encapsulation to enable mass production of spheroid-forming NP cells.