Author : Anil Khuttan, Kapil Dev, Birbal Singh and Sanjeev K. Gautam

The current study was carried out to isolate, culture and cryopreserve the putative amniotic fluid stem (AFS) cells in sheep. AFS cells were cultured without feeder cells in DMEM containing 16% FBS, 1% penicillin/streptomycin and 1% L-glutamine in 5% CO2 in humidified air at 37±0.5˚C. After 6 days of culture different morphologies of cells were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. The putative AFS cells were cryopreserved using slow freezing method using DMSO as cryoprotectant. After freezing the cells for one week, it was observed that 70% of the cells were viable after trypan blue staining. This study shows that the putative amniotic fluid stem cells in sheep can be isolated, cultured and cryopreserved successfully under in vitro conditions.

Keywords: Amniotic fluid, stem cells, cryopreservation, sheep.