CULTURE OF CELLS AND TISSUES DERIVED FROM XENOPUS LAEVIS 



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primary explant cultures. Therefore, for explantation procédures we are using 

 now exclusively a growth médium (primary-explant médium TCM-A2, see 

 Table IV) which contains 60% (by volume) of L15, 20% (by volume) of H 2 

 and 20% (by volume) of fetal bovine sérum. Because of the relatively high con- 

 centration of fetal bovine sérum and consequently of the relatively high concentra- 

 tion of the fetuin rich a-globulins, even fairly large tissue fragments attach 

 to the surface of the culture vessels readily and without artificial supporting 

 structures (plasma clot etc.) in this growth médium, and cell prolifération is 

 often initiated already within 48 hours after explantation. 



In order to préserve the potency of the antibiotica and to avoid décomposi- 

 tion of L-glutamine the growth média are stored frozen before being used. 



Culture vessels, incubation température, ph and nutrient changes 



Normally we grow monolayer cultures of Xenopus laevis in stérile plastic 

 tissue culture flasks (FALCON) with a surface area of 25 cm 2 and a capacity 

 for 30 ml. The ideally flat inner surface of thèse flasks facilitâtes observation of 

 explanted cells. Normally cultures grown in thèse flasks are overlaid with 4 ml 

 of growth médium. Monolayer cultures are also grown by us on micro-slides in 

 60 mm diameter Petri-dishes containing 5 ml of growth médium and kept in a 

 humified atmosphère. 



According to our expérience monolayer and organ cultures derived from 

 Xenopus tissues grow with near-optimum rates at températures ranging from 

 20° C to 26° C, and at pH values ranging from 7.0 to 7.5. We have found that 

 primary and established cultures derived from Xenopus laevis can be maintained 

 without appréciable growth and without significant impairment of cell viability 

 at a température of 10° C to 11° C for long periods of time, provided the nutrient 

 médium is replaced with fresh médium once every two weeks. Cultures grown 

 at 25° C or at room température are usually fed at intervais ranging from 4 to 

 5 days. 



Observations with xenopus laevis kidney-cell cultures 



When explanted in our growth média TCM-A1 or TCM-A2 at a tempéra- 

 ture of 25° C Xenopus laevis kidney cells attach within 24 to 48 hours to the glass 

 or plastic surface of the culture vessels. Foci of flattened transparent polygonal 

 shaped cells appear within 48 to 72 hours after explantation, most frequently 

 around the periphery of cell-clumps or of small fragments. Monolayer kidney- 

 cell cultures are normally produced within incubation periods ranging from 7 to 

 10 days, but in several instances cell cultures prepared from adult Xenopus laevis 

 kidneys degenerate spontaneously before monolayer formation is completed 

 (see also Granoff, A., Came, P. E. and Rafferty, K. A. Jr.; 1965). Explanta- 



