Impact of cell seeding density and serum starvation on VEGF expression in canine adipose tissue derived mesenchymal stromal cells

Optimization of cell culture protocols in terms of serum percentage as well as seeding density can affect the angiogenic potential of mesenchymal stromal cells. Present study demonstrates the impact of seeding density and serum starvation on the expression of VEGF gene in canine adipose tissue derived mesenchymal stromal cells (cAD-MSCs). Canine adipose tissue derived mesenchymal stromal cells were successfully isolated and expanded in vitro demonstrating typical sigmoid pattern of growth curve. The population doubling time of in vitro expanded cAD-MSCs was found to be 40.04 hrs. and around 40-45% of initial MSCs were observed to show clonogenicity in colony forming unit assay. We demonstrated that higher seeding density i.e. 6x104/cm2 showed significantly (P = 0.005; N=4) better expression (2.495±0.35fold) of VEGF in cAD-MSCs compared to the initial seeding of 1x104/cm2 (1.000±0.00) and 3x104/cm2 (1.393±0.30) cells in the in vitro cAD-MSCs culture. Further, we could not find any statistically significant change in the relative expression of VEGF in cAD-MSCs while serum depriving the culture for 12hrs (1.11±0.43), 24hrs (1.64±0.53) and 36hrs (0.83±0.58) compared to serum rich media (1.000±0.00). Further research is required to optimize the cell culture protocols thereby creating rapid expansion of MSCs without compromising the cell characteristics and making cell-based therapies more cost effective.