[Researches on the methodology of establishment of BALB/c murine embryonic stem cell lines].
Several methods and processes of establishment and culture of BALB/c mouse ES cell lines were discussed detailedly. A new method to establish and culture ES cell lines derived from BALB/c mouse was set up successfully using mouse embryonic fibroblast feeder layer and rat-heart-cell-conditioned medium (RH-CM). These culture conditions not only maintain the undifferentiated state and normal diploid karyotypes of BALB/c mouse ES cells effectively, but also maintain a series of their characteristics of murine stem cells. two different kinds of digestive methods and Two kinds of digestive juice with different concentrations were designed to dissociate proliferous inner cell mass (ICM) and ES cell colonies derived from dissociative ICM. two different kinds of digestive methods are "single time dissociation method" and "several times dissociation method", two kinds of digestive juice are 0.25% Trypsin-0.04% EDTA and 0.05% Trypsin-0.008% EDTA. At the same time, appropriate dissociated occasion of ICM and the effect of RH-CM on establishment and culture of BALB/c mouse ES cell lines were discussed. The results suggested that it is a reasonable method to establish BALB/c mouse ES cell lines using low concentration digestive juice and "several times dissociation method" to dissociate ICM after 4 days' proliferation. Judged by the form of ES cells and its colonies, proliferous capability, karyotypes examine, alkaline phosphatase activity assay and differentiation capability in vitro and in vivo, the 9 ES cell lines that we established satisfied the all traits of murine ES cell line.
Meng
Guo-Liang
GL
College of Life Sciences, Peking University, Beijing 100871, China. menggl@263.net
Teng
Lu
L
Xue
You-Fang
YF
Shang
Ke-Gang
KG
D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D013234:Stem Cells / Q000166:cytology*