Background: 3D (three-dimensional) cell culture technology has been researched steadily because of its high potential of biocompatibility compared to single cells since 1990s, and is being developed to 3D spheroids recently. Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, and stem cells spheroids have been studied extensively in therapeutic transplantation. Stem cells were considered as a method of replacing autologous chondrocyte in regenerative treatment of articular cartilage. Compared to conventional single cells, 3D cell culture is artificially created an environment similar to a living body in vitro so that all cells collectively, a cell culture model that allows growth or interaction with the environment. Therefore, the findings of this study indicate that enhancement of treatment efficiency of stem cells caused by potential of survival and proliferation of hASC spheroid in Osteoarthritis. In conclusion, spheroid positive subpopulation of hASCs has high cell proliferation and survival but not apoptosis and cell death potential, which may contribute to successful cartilage regeneration and the development of stem cell therapies in the future.

Objectives: Studied for 3D spheroids to investigate the mechanism of enhancement of survival and proliferationof hASC (human adipose stem cells) spheroid, which may contribute to successful improvement of therapeutic efficacy of stem cells.

Methods: Cell isolation and culture / 3D cell culture dish preparation / hASCs culture on 3D cell culture dish / Real-time PCR analysis / Western blotting / Alcian blue staining / ACLT + MM (Anterior cruciate ligament transection with Medial meniscectomy) model / In vivo fluorescence for cell tracking / In vivo effects of spheroids in OA joint / Histological analysis / Enzyme-linked immunosorbent assay (ELISA) results for inflamma -tory cytokines in rat synovial fluid / Statistical Analysis

Results: In order to see how the spheroid showed more residual than single, and how effective it was in actual cartilage regeneration, the result of paraffin tissues were confirmed by safranin O staining for each condition. The tendency of cartilage regeneration efficiency was good for spheroid. Although the differences between the single and spheroid groups were small, they reaffirmed that they could somewhat protect cartilage and help regeneration treatment. However, immunohistochemistry of HN(Human nucleic antigen) staining showed that cells of single and spheroid were not observed in the wound but disappeared by the paracrine effect.

Conclusion: Spheroids do not exhibit differentiation characteristics, but they could be seen as a result of expression of related genes such as Bax, Bcl-XL and Alcian blue staining. Spheroids tend to have low potential of cell death rather than proliferation and reduction in the proliferation. So, we conclude the fact that instead of hASCs going directly to the surgical site to regenerate cartilage, they can help catrilage regeneration.

Acknowledgments: This research was supported by the National Research Foundation of Korea (NRF-2019R1H1A2039685 and 2019R1I1A1A01043778).

Disclosure of Interests: None declared