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Previous experiments using cultures of Mexican axolotl cells have been reported for skeletal myoblasts (Duprat et al., 1975), mesenchymal blastema cells (Boilly and Albert, 1988), and for various tissues including heart (Frost et al., 1989). We have developed the following procedure for culturing cells from stage 38-41 embryonic hearts. To enhance sterility, pre-hatchingembryoswere washed while still in their jelly coats, twice in filter-sterilized modified Steinberg's solution (Zackson and Steinberg, 1986), once in 95% ethanol, and then twice in filtered Steinberg's solution. The embryos were then dejellied and washed four more times in filtered Steinberg's solution. Post-hatching embryos were only washed four times in filtered Steinberg's solution. Embryos were dissected in modified Steinberg's solution in a 60 mm Petri dish previously lined with three layers of parafilm and sterilizedby ultraviolet light for 30 minutes. Hearts were removed with dry-heat sterilizedwatchmaker's forceps under a dissecting microscope and placed into calciumand magnesium-free Steinberg's solution (CMF-Steinberg's= 59 mM NaCI, 0.67 mM KCI, 0.40 mM EDTA, 9.99 mM HEPES, pH to 7.4, with 1% antibiotic/antimycotic;GibeD stock composed of penicillin G sodium 10,000 unitslml, 10 ~glml streptomycin sulfate, 25 ~glml amphotericin B). The CMF-Steinberg's solution was contained in a 1.5 ml microfuge tube. When all of the hearts were collected, they were centrifuged at 91 g for 60 sec. The supernatant was replaced with a CMF-Steinberg's solution containing 774 unitslml collagenase (Sigma type II: C. hlstolyticum) and 1760 unitslml hyaluronidase (Sigma type I-S from bovine testis) for 20 min at room temperature on an orbital shaker. The heart tissue was centrifuged at 91 g for 60 see, after which the supernatant was ROBERT W. ZAJDEL, YONGZE ZHU. MARGARET E. FRANSEN and LARRY F. LEMANSKI*

A method for the isolation and culture of embryonic cardiomyocytes from Mexican axolotl.