624 



H. MOSER, I. HADJI-AZIMI AND S. SLATKINE 



GROWTH MEDIA 



The unavailability of a C0 2 -flow cabinet suggested to us the use of the 

 Leibovitz-L15 nutrient médium as the basai (molecular) component of our 

 growth média for Xenopus cells. L15 has been described (A. Leibovitz, 1963) as 

 a nutrient liquid for the propagation and maintenance of mammalian cell and 

 tissue cultures in free gaseous exchange with the atmosphère, using free base 

 amino acids, notably L-arginine, instead of sodium bicarbonate as the bufTer. 

 L15 contains, in addition to the essential amino acids, vitamins and electrolytes 

 for growth of mammalian cells, D(-f-)-galactose and sodium pyruvate (instead 

 of D(+)-glucose), several non-essential amino acids and amides (for example 

 glycine, L-serine, L-asparagine), CaCl 2 , and the antibiotics penicillin, strepto- 

 mycin and Fungizone (SQUIBB). When supplemented with fetal calf sérum at 

 final concentrations of from 10% to 20% and when diluted with H 2 to amphi- 

 bian osmolaiity (80% of mammalian osmolarity), L15 is an excellent growth 

 médium for monolayer and organ cultures derived from Xenopus laevis (Daudin). 



For the maintenance of permanent cell strains of Xenopus laevis 1 and for 

 the maintenance of already well initiated primary-explant cultures of Xenopus 

 we are using a growth médium (maintenance médium TCM-A1, see Table IV) 

 which is composed of 70% (by volume) of L15, 20% (by volume) of H 2 and 

 10% (by volume) of fetal bovine sérum. 



Table IV 



Composition of growth média for eells of Xenopus laevis 





Growth média (1000 ml) 





TCM-A1 



TCM-A2 



Leibovitz-L15 basai médium 



(HYLAND LABORATORIES) .... 



700 ml 



600 ml 



H 2 



200 ml 



200 ml 



Fetal Bovine Sérum 

 (HYLAND LABORATORIES) .... 



100 ml 



200 ml 



Sterilized by filtration through Millipore filter, type GS, 0.22 \x pore size, under positive pressure. Stored frozen 

 in 100 ml aliquots in screw capped bottles at — 25° C, and protected from exposure to light, until used. 



Our expérience has shown that the increase of the concentration of fetal 

 bovine sérum from 10% to 20% greatly improves the efficiency of L15 in initiating 



x The permanent heteroploid cell strains derived by K A. Rafferty, Jr., from adult 

 Xenopus laevis liver, cell strain A8, and from adult Xenopus laevis kidney, cell strain A6. 



