Effects of nutrient media on gastrula ectoderm of Rana pipiens cultured in vitro

Abstract

It has been suggested that the molecular basis of the induction of cellular differentiation might depend upon the provision of materials which stimulate protein synthesis in the reacting cells (Flickinger, '58, '60, '62). One way to test this speculation is to culture explants of undifferentiated embryonic cells in the presence of substrates and cofactors which are necessary for protein synthesis to ascertain if they will stimulate differentiation. The present report deals with experiments in which explants of the ventral mesoderm-ectoderm and ectoderm alone of the early gastrula of the frog embryo, Rana pipiens, were cultured in various nutrient media. MATERIALS AND METHODSVentral mesoderm-ectoderm and ectoderm areas were isolated from stage 10 gastrulae ( fig. 1) and were cultured on millipore filter assemblies ( fig. 2), according to the method of Grobstein ('56), or in plastic petri dishes. The explants were cultured in a CO, incubator (5% C0295% air) at a temperature of 23"C, under humid conditions, for a period of eight days. The cultures were examined daily and were processed for histological examination at the termination of the culture period. The medium used in the control series of experiments was Niu-Twitty solution (Flickinger, '49), which is comprised solely of inorganic salts.The nutrient media used were the amino acid and vitamin mixtures of Eagle's basal medium and NCTC 109 (Paul, '59); the latter medium contains amino acids, vitamins, nucleotides, and coenzymes. Since NCTC 109 was designed for the culture of mammalian cells, it was diluted with an equal volume of deionized water, and each liter was fortified with NaHC03 (1,100 mg) and Na2HP04 (70 mg) so as to maintain a pH of 7.5 during culture in the presence of 5% CO,. A preparation containing 5,000 units each of penicillin-streptomycin was employed at a 1% level to give a final concentration of 50 units/ml of each antibiotic in all the media. The nutrient media were also supplemented with 1% glutamine, 0.2% dextrose, 5 or 7% horse serum, and 1-7% chick embryo extract, and these fractions were used in various combinations in the experimental series. Horse serum, embryo extract, NCTC 109, Eagle's vitamin and amino acid mixtures, glutamine and penicillin-streptomycin were obtained commercially; the embryo extract and horse serum being supplied in a frozen state. The millipore-filter culture dishes were filled with 1.5 ml of medium, while the small, plastic petri dishes were filled with 1.2 ml. The media were changed every two days. In both the petri dishes and the filter assemblies the explant was covered by a very thin film of fluid, but in the latter case the tissues were also exposed to culture fluid diffusing up through the millipore filter.