ISOLATION AND CULTURE OF CELL COLONIES FROM FOETAL MOUSE INNER CELL MASS

The inner cell mass of blastocyst is able to proliferate unlimitedly in vitro and remain as undifferentiated cell population. If these cells occulated in vivo to mouse tissue or transferred to blastocyst, they can be induced to differentiate to derivatives of three germ layers and subsequently to teratoma or chimeric animals respectively. The modification of procedures of colony harvesting from inner cell mass was the aim of this study. In this study 118 embryos transferred to inactivated rat embryonic fibroblastic feeder layer (REF) and DMEM media containing Leukemia Inhibitory Factor (LIF). Two days later the embryos underwent outgrowth stage, and inner cell mass was removed from the dishes by trypsinization or mechanical methods (disaggregated stage) and then were transferred to dishes contained inactivated REF layer or dishes coated with collagen. The cell were cultured were checked for the formation of cell colonies every day for four days. The dishes with the positive colony were passaged in every 2 to 3 day and subcultured up to the sixth passages. The cell colonies were evaluated for morphological and speed of cell doubling criteria. Only 3 colonies were obtained from 30 disaggregated cell mass by mechanical method REF feeder layer. It seems that the mechanical method of disaggregation may damage the cells less than the trypsinization method and will increase the chance of cell colony formation.