Vitrification of water buffalo (<i style="">Bubalus bubalis</i>) fetal skin fibroblast cells

Abstract

A method was developed for the cryopreservation of buffalo fetal skin fibroblast cells by vitrification. Skin cells were isolated from 1-2-month-old fetuses obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in DPBS) for 15-20 min. They were washed 5-6 times with DPBS and then once with RPMI-1640 + 10% FBS by centrifugation at 600 rpm. The cells were then cultured in the same medium in a CO₂incubator (5% CO₂in air)at 38.5°C for 3 d. After 3 days, the fibroblast cells from the monolayer were harvested by treating with trypsin-versene solution. They were equilibrated and loaded into 0.25 mL French straw, containing 0.5 <i style="">M</i> sucrose solution in DPBS. The straws were sealed and precooled by keeping them in liquid nitrogen (LN₂) vapour for 1-2 min, following which these were plunged into LN₂. After 5 days of storage the straws were warmed quickly by transferring them to a water bath at about 20ºC for 15-20 sec. The cells were collected by centrifugation and plated in 25 cm² culture flasks with 5-6 mL of culture media and incubated at 38.5º±1ºC with 5% CO₂in humidified (95%) incubator. The fibroblast cells growth started within 3 h of culture set-up and on day 3 the monolayer formation occurred.