Embryonic stem (ES) cells have become very important resources in basic medical researches. ES cells are derived from
explanted culture of the inner cell mass (ICM) of blastocyst stage embryos and has definite explicative potential and
differentiate into derivatives of all three germ layers. Initially invitro fertilized embryos were cultured using TCM 199 +20% Fetal
bovine serum + L?Glutamin 2mMol and Penicillin/Streptomycin 100mg/100IU/ml for 3 days. These cell lines were generated
by removing ICM from preimplantation embryos using 0.75% pronase. Es cells are grown on a layer of mitotically inactivated
primary mouse embryonic fibroblast (MEF) feeder layer to promote growth and prevent differentiation using mitomycin ? c,
primary embryonic fibroblast were isolated from 13 to 14 days old fetuses. The cells were disaggregated and trypsinized every
3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase.
The ES cells were cultured in suspension state for 5 days; at the same time Leukaemia Inhibitory Factor (LIF) was removed from
media to form embryoid bodies (EBs). Undifferentiated cells were seen as intensely small cells attached together compactly with
high nucleus/cytoplasm (N/C) ratio. The cells of colonies tend to differentiate by separation from each other and became larger
and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES
colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types
of cells such as nerve and hematopoitic lineages.
P. Iyya Shankar completed her Post graduation in M.Sc Biotechnology from University of Madras in the year 2007. He is currently working as Senior
Executive ? cell culture department in Life cell International Private ltd, Chennai, Tamil Nadu, previously working as Senior Research Fellow in
Department of Animal Biotechnology, MVC, Tanuvas, Chennai ? 600053, Tamil Nadu, India.
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