Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line

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Abstract
The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.