# Production of Alternative Fat from Adipose-Derived Stem Cell from Bovine in 3D Culture
## 3 Results
### bADSC Is Capable of Differentiating into Fat Tissue with Diverse Fatty Acids  
bADSC differentiation was induced on 6-well plate with a standard adipogenic cocktail for 21 days in vitro, as shown in Figure 2A. Lipid droplet formation commenced on the 4 days post adipogenic induction, (Figure S2), with a subsequent increase in droplet size. Multiocular lipid droplets developed, and the presence of intracellular lipid was confirmed through Oil Red O staining (Figure 2B). The quantification of the stained area revealed approximately a 4-fold higher absorbance compared to undifferentiated cells (Figure 2C,D). Adiponectin, an adipokine released by mature adipocytes, stands out as a distinctive marker of adipocyte. Studies have reported its capability to stimulate cell proliferation and the differentiation of pre-adipocytes into mature adipocytes. Quantification of adiponectin in the supernatant was conducted using ELISA at 21 days (Figure 2E). The control group exhibited negligible adiponectin production, whereas bADSC subjected to adipogenic induction showed a significant increase in adiponectin levels.  
Next, the fatty acid composition of adipocytes derived from bADSCs was analyzed and compared with intact bovine adipose tissue (Figure 2F). The ratio of saturated fatty acids (SFA) to unsaturated fatty acids (UFA) in relation to total fats was found to be similar in both adipocytes and adipose tissue (Figure 2F and Table 1). The content of oleic acid, which allows for good flavor, was high in both samples. In addition, the lower the SFA content, the better it is for health, because SFA is known to be a major cause of hyperlipidemia and hypercholesterolemia (Table 1).  
Figure 1: **Characterization of bovine ADSC.** (**A**) Diagram to obtain SVF from fat. (**B**) Morphology of ADSCs from SVF culture. (**C**) Cumulative cell number of bovine ADSC starting from SVF to passage 3. (**D**–**F**) Analysis of MSC markers by FACS. (**D**) Immunofluorescence staining (**E**) and Western blot (**F**). Note: The Western blot data (**F**) shows duplicated results from the same sample loaded in different lanes. Scale bar: 50 \(\upmu\)m.  
Figure 2: **Adipogenic induction of bADSCs in 2D culture.** (**A**) Diagram for adipogenesis in vitro. (**B**) Cellular morphology and Oil Red O staining. (**C**,**D**) Quantification of Oil Red O. (**E**) Measurement of adiponectin in supernatant. (**F**) Comparative analysis of fatty acid profile in adipose tissue and cultured fat. *** \(p\) < 0.005.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Common Name** & **Fame** & **Abbreviation** & **bAdipocyte** & **Fat Tissue** \\ \hline \multicolumn{5}{c}{**Saturated Fatty Acids**} \\ \hline Stearic acid & Methyl stearate & C18:0 & 16\% & 9\% \\ Palmitic acid & Methyl palmitate & C16:0 & 15\% & 24\% \\ Eicosanoic Acid & Methyl arachid & C20:0 & 2\% & 1\% \\ Myristic acid & Methyl myristate & C14:0 & 2\% & 4\% \\ Docosanoic acid & Methyl behenate & C22:0 & 2\% & N/D \\ Lignoceir acid & Methyl lignocerate & C24:0 & 2\% & N/D \\ Heptadecanoic Acid & Methyl heptadecanoate & C17:0 & 2\% & 1\% \\ Pentadecanoic acid & Methyl pentadecanoate & C15:0 & 1\% & 1\% \\ \hline \hline \end{tabular}
\end{table}
Table 1: **The analysis of the composition of the fatty acid.** The fatty acid compositions in induced adipocytes and adipose tissue from bovine were compared, focusing on the differences between saturated and unsaturated fatty acids. bAdipocyte: bovine ADSC. N/D: not detected.