Differentiation of porcine stromal vascular cells in primary culture Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/3t945v122

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  • The objective of this study was to examine optimum conditions for primary culture of porcine preadipocytes. Dorsal subcutaneous adipose tissue was obtained from biopsies of 40-50 kg pigs and digested with collagenase in Krebs Ringer Bicarbonate buffer. Isolated stromal vascular cells were cultured in different test media. Stromal vascular cells (2-3 X10⁴ cells/cm²) were plated in DME medium containing 10% FCS for 24 hours (unless stated otherwise). After plating, the cells were washed with DME medium without serum three times to remove excess FCS. For the next 11 days, cells were grown in various test media as assigned. The results of the study agree with previous observations that indicate serum-containing medium fails to stimulate porcine preadipocyte differentiation. Serum-free medium (ITTC medium) has been shown to induce a relatively high rate of porcine preadipocyte differentiation. The plating time in serum-containing medium was found to be crucial in order to achieve a high rate of cell differentiation. A plating time of 24 hours in the serum-free culture system resulted in a higher number of differentiated cells (P<.05) compared to 48, 72, and 96 hours. Maintained in serum-free medium (ITTC), approximately 40% of the stromal vascular cells underwent differentiation characterized by numerous cytoplasmic lipid droplets and a positive reaction to a histochemical test for lipogenic enzymes (esterase, glycerol-3-phosphate dehydrogenase and glucose-6- phosphate dehydrogenase). The number of differentiated cells was significantly higher (P<.05) in serum-free medium compared to serum-containing medium. Addition of calf serum (2.5%, 5%, and 10%) reduced (P<.05) the number of differentiated adipocytes and increased (P<.05) total cell number (both adipocytes and non-differentiating cells). The effect of fibroblast growth factor (FGF) in stimulating the proliferation of porcine stromal vascular cells had not previously been investigated. The results of this study show that FGF stimulates proliferation of both preadipocytes and non-differentiating cells. The number of adipocytes as well as non-differentiating cells increased (P<.05) in ITTC medium plus FGF compared to ITTC medium alone. The results of this study also show that insulin is an important factor in ITTC medium since exclusion of insulin caused a 73% reduction (P<.05) in the number of differentiated cells. T3 appeared to be a minor factor in this serum-free system. Transferrin, a binding protein of iron, which is used in other serum-free systems, appears to be important in supporting porcine preadipocyte differentiation, since exclusion of transferrin reduced (P<.05) the number of adipocytes by 29%. Hydrocortisone also seems to play an important role since its elimination from the medium reduced (P<.05) differentiation by 71%. A serum-free medium (ITTC) which allows a high rate of porcine preadipocyte differentiation has been established. This serum-free culture system for porcine preadipocytes will allow investigation of factors that regulate the formation of adipocytes.
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