EFFECT OF CELL DENSITY AND SERUM STARVATION ON VASCULAR ENDOTHELIAL GROWTH FACTOR GENE EXPRESSION IN CANINE ADIPOSE TISSUE DERIVED CELLS
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Date
2021-09-20
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Kerala Veterinary and Animal Sciences University,Pookode, Wayanad
Abstract
Present experiment was conducted to study the effect of cell density and serum starvation on
Vascular Endothelial Growth Factor (VEGF) gene expression in canine adipose tissue derived
mesenchymal stromal cells (MSCs). Subcutaneous fat tissue sample was collected from adult
dog presented at the Department of Veterinary Surgery and Radiology, CV & AS, Pookode. The
tissue pieces were washed and digested using 0.2 % collagenase type 3 and filtered using 40μm
filter to isolate the adipose tissue derived stromal cells which were seeded in
DMEM
(Dulbecco's Modified Eagle's Medium) supplemented with 15 % FBS (Fetal Bovine Serum) for
the primary cell culture. As primary culture attained 70-80 % confluency, subculture was done
till fourth passage. Growth curve and population doubling time of the in vitro expanded canine
adipose tissue derived MSCs of cells were calculated by using 0.4 % trypan blue exclusion test.
Fourth passage cells were seeded in different seeding densities (10 4 /cm 2 ; 3x10 4 /cm 2 ; 6x10 4 /cm 2 )
within six well cell culture plates and were grown for 72 hrs (pre- incubation) in normal serum
rich (+) growth media. After pre-incubation, cells were further incubated for 36 hrs replacing
with either serum rich (+) or serum free (-) media at respective time intervals (Zero h; 12 h; 24 h;
36 h). At the end of incubation period, treated/adherent cells were harvested from each well and
total RNA was isolated and cDNA was synthesized. Expression of VEGF in different groups was
analyzed by real time PCR using specific primers designed for VEGF keeping the expression of
GAPDH as reference. Among the different seeding density and serum starvation combination
experimented, we could not find any significant impact on the expression of VEGF, but we
observed a significant increase in VEGF expression in canine adipose tissue when used at a
higher cell seeding density (6x10 4 /cm 2 ). These findings suggest that cell therapy using stromal
cells derived from subcutaneous canine ASCs, regulated with high seeding density may be a
novel therapeutic option to enhance angiogenesis thereby provides therapeutic benefits in
pathological vascular conditions including wound repair, ischemic damage, microvascular
permeability and diabetes.
Description
Submitted in partial fulfillment of the requirement for the degree of Master of Veterinary Science in Veterinary Physiology