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Kerala Veterinary and Animal Sciences University, Wayanad

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20211
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Subject: Veterinary Physiology × Author: HARSH JAMDA × End date: 2022 × Start date: 2020 ×
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    ThesisItemOpen Access
    EFFECT OF CELL DENSITY AND SERUM STARVATION ON VASCULAR ENDOTHELIAL GROWTH FACTOR GENE EXPRESSION IN CANINE ADIPOSE TISSUE DERIVED CELLS
    (Kerala Veterinary and Animal Sciences University,Pookode, Wayanad, 2021-09-20) HARSH JAMDA; M. D. Pratheesh
    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.