# Production of Alternative Fat from Adipose-Derived Stem Cell from Bovine in 3D Culture
## 4 Discussion  
Cultivated meat relies on the inherent capacity of stem cells to proliferate to achieve a sufficient cell number in vitro and to undergo specific differentiation in response to biological stimuli. Proliferation and differentiation potential of stem cells is obviously affected by their cellular activity. Numerous studies have endeavored to optimize the in vitro establishment of a mass culture system to obtain a substantial supply of healthy/highly proliferative stem cells. The differentiation process can commence once a stem cells is acquired. Taste, flavor, and texture comparable to conventional bovine tissue are essential traits to attain post-differentiation of stem cells in vitro. This underscores the importance of fat tissue, since the fat content is recognized as a critical determinant for meat flavor. The objective of this study was to generate fat tissue from bADSCs within a plant-based scaffold. To achieve this goal, it was imperative to establish a stable culture system of bADSCs in vitro. We undertook the isolation and characterization of ADSCs from the subcutaneous fat of the bovine back. The culture of SVF revealed the presence of fibrobl colony-forming cells, which demonstrated robust proliferation up to passage 8. Notably, approximately 2 g of fat tissue was used for cell isolation, resulting in a substantial yield of cells from the culture. The isolated cells displayed the characteristic spindle-shaped morphology and expressed CD73, CD90, and CD105, while lacking CD45. After confirmation of the presence of stem cell markers, bADSCs were induced to undergo adipocyte differentiation, as evidenced by the observation of lipid droplets. Comparison between bovine fat and adipocytes derived from bADSCs corroborated a similar composition of fatty  
Figure 3: **Adipogenic induction of bADSCs in TVP**. (**A**) Schematic diagram to culture and induce adipogenesis in scaffold. (**B**) Gross view of TVP after adipogenic induction. (**C**) Oil Red O staining in adipocyte in TVP. acids. This suggests that in vitro adipogenesis of bADSCs can lead to the production of fat, potentially serving as a substitute for conventional meats. This finding implies the successful establishment of in vitro adipogenic induction in bovine ADSCs with characteristics comparable to intact tissue in terms of fatty acid. In order to use cultured meat as a food source, it is essential to integrate fat tissue with other tissue components, such as proteins. bADSCs were plated on TVP as a scaffold and exposed to adipogenic stimuli for a period of 2 weeks. Adipogenesis progressed in the scaffold, and lipid droplets were observed in the TVP containing bADSCs. These results showed that bADSCs are capable of differentiating into adipocytes in a 3D culture system, and TVP stands out as a promising candidate scaffold for the development of hybrid cultured meat. Cell-cultured meat presents a promising alternative to conventional meat production, with the potential to substantially reduce environmental impact, achieving reductions of 45% in energy consumption, 96% in greenhouse gas emissions, 99% in land use, and 96% in water usage [28]. In addition to its environmental benefits, this approach offers a shorter production timeline and enhanced efficiency in both resource utilization and scalability [28]. Advanced food technologies are increasingly being explored by the space industry to ensure a safe, sustainable food supply during long-duration missions in extreme environments [29]. Cell-culture meat technology could offer viable solutions to many of the current limitations faced in space food systems. Nevertheless, several technical hurdles must still be addressed to realize its full potential [28]. One limitation in the application of this study is the use of FBS, which increases production costs compared to conventional TVPs. Therefore, further research is needed to minimize or replace FBS with cost-effective, serum-free alternatives.