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Subject: Veterinary Physiology × Author: Das, Prerana × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    Strategy for development of stem cell like embryonic fibroblast cells
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Das, Prerana; Nath, Nikhil Ch
    Fibroblast cells are the type of cells that play an important role in the formation of connective tissue. The use of fibroblast cell is versatile, for e.g., demonstration of avian viruses, feeder cells, production of vaccines, preservation of genetic resources etc. In this present study, duck embryonic fibroblast cells were isolated, cultured, and sub-cultured up to six passages. The cells were grown in four culture media i.e., Medium 1(MEM), Medium 2(MEM+IGF-1), Medium 3 (MEM+10% FBS), Medium 4 (MEM+10% FBS+IGF-1). In serum and serum-free media the time required for the cells to attain 70% confluence in primary culture was 84.667±.0.152 hours and 111.867±0.161 hours respectively. The cells grown in medium containing serum showed better results than cells grown in serum-free medium. The time taken to reach 70% confluence in 6th passage in Medium 2 and Medium 4 which are IGF-1 supplemented are 94.583±0.217 hours and 62.167±0.096 hours respectively whereas time taken in Medium 1 and Medium 3 which are IGF-1 free media are 95.350±0.039 hours and 62.667±0.152 hours respectively. Therefore, the cells grown in IGF-1 supplemented media showed significant difference compared than the rest of the culture media (p≤0.01). Morphologically, the cells showed characteristic spindle shape, turgor vitalis cytoplasm, centrally located nuclei and flame-like pattern up to the sixth passage. The viability assessment was carried out in first, second, third, fourth, fifth and sixth sub-culture and the viability percentage of the cells in six different sub-cultures were 89.843±0.108, 91.427±0.082, 91.228±0.081, 91.867±0.079, 92.231±0.073, 93.431±0.069 in the case of Medium 1, 90.425± 0.085, 92.358± 0.124, 93.692±0.084, 93.982 ±0.282, 94.625 ±0.089, 94.892 ±0.096 in the case of Medium 2, 89.145 ±0.263, 90.482±0.09, 91.643±0.143, 92.713±0.186, 93.460±0.079, 94.543±0.074 in Medium 3, and 88.597±0.132, 89.387±0.143, 90.552±0.101, 91.423±0.078, 93.077±0.140, 93.077±0.140 in Medium 4. The viability percentage between the passages was significantly different (p≤0.01). However, the viability of the cells was better from the second subculture compared to primary cultures. The pluripotency of the cells was observed by immunostaining using NANOG antibody, a pluripotent marker that is expressed in embryonic stem cells. It was observed that cells showed positive for NANOG at every subculture depicting their pluripotent nature.