Standardization of a common protocol for establishment and cryopreservation of ibroblast cell lines from different indigenous livestock species

Abstract

The majority of precious livestock genetic resources remain exposed to the vagaries of genetic erosion due to 
limitations of in situ conservation programs. Fibroblast bank offers a practical approach to preserve 
germplasm due to advancement of cell culture techniques. The objective of current study was to biologically 
develop, evaluate and cryopreserve the skin ibroblast cells of different Indian livestock species obtained using 
an ef icient, common protocol. Ear marginal tissues from Manipuri and Marwari horses (n=8) and Kutchi camel 
(n=6) were collected for this purpose. Primary culture and irst passage was done using ibroblast speci ic 
media (HiFibroXL™). Epithelial-like and ibroblast-like cells emerged from the tissue explant margins within 
10-14 days of culture in both the species. Subsequent passaging of cells for both horse and camel was continued 
using DMEM+Ham's F12 (1:1) media with 10% FBS. Fibroblast cells showed typical fusiform morphology with 
centrally located oval nuclei. Cells exhibited radiating, lame like or whirlpool like migrating patterns and 
density dependent inhibition during cell proliferation. The growth curve at passage-4 represented typical S
shape as the cell population passed through a lag phase, a logarithmic phase and a plateau phase. Population 
doubling time varied between 27.9 to 31.37 hrs with multiplication rate of 0.76- 0.86 population doubling/24
 hrs. The cells were cryopreserved from 3 to 6 passage stocking at least 75 cryogenically-preserved vials
 (1×10⁶cells/ml) per animal. The protocol was further validated in six more species, mithun, yak, buffalo, goat, 
sheep and donkey. It can be concluded that ibroblast cell culture can be established from eight different 
livestock species at a faster rate and in a cost effective manner following a single common protocol described in 
this paper.