Long-term cultivation of cells derived from a Xenopus laevis tumor

1986 Proceedings of the Japan Academy. Series B, Physical and biological sciences  
Communicated by Kiyoshi TAKEWAKI, M. J. A., Oct. 13, from a TAKUMA 1986) Among amphibian tumors, renal adenocarcinoma in Rana pipiens and papilloma in Cynops pyrrhogaster have been studied at some length.1)-3),10),11> However, our knowledge on tumors in other amphibians including Xenopus laevis is restricted.6)12),15) Tweedell and Williams16) succeeded in in vitro culture of tumor cells from Lucke renal adenocarcinoma occurring in Rana pipiens and reported the characterization of the cell lines
more » ... and some nuclear transplantation experiments in these cells.5) ,8) In this paper we report a long-term cultivation of cells derived from a tumor of X enopus laevis. Materials and methods. Tumor o f X enopus laevis. The sarcoma-bearing specimen of X enapus laevis used in this study was one of about 20,000 frogs reared from eggs over a period of 2 years in a pond of a dealer in Hamamatsu, Shizuoka Prefecture, Japan. This frog was found in May 1983, and then kept for nine months in a plastic aquarium (24X33X10 cm) in this laboratory at 20°C. It was a female about 8 cm in length, carrying a tumor of 24 mm in diameter on the dorsal side. Primary culture : After the frog was washed with running fresh water for 2 hrs, it was killed by destroying the hindbrain and pithing the spinal cord. The skin over the tumor was disinfected and cleaned with 70% ethanol, the tumor tissue was then excised under sterile conditions in a clean room. The tumor pieces were washed several times with autoclaved phosphate buffered saline solution (PBS) at pH 6.8 with 1 g kanamycin sulphate (Meijiseika Co.)/liter, before transferring into amphibian culuure medium (ACM),4),14) containing 50% Leibovitz's medium (L-15 medium, Gibco Co.), 10% fetal bovine serum (Flow Labo. Co., Australia), 40% distilled water and 0.2 mg kanamycin sulphate/ml. The pieces were further cross-sliced into small fragments (about 2 mm3) with two sharp scalpels. The fragments were washed with ACM in a 35-mm culture dish (Falcon 3801; Primaria Co., U.S.A.) and finally 8 or 12 fragments were placed in each of several culture flasks having a 25-cm2 growth area (Falcon 3813; Primaria Co., U.S.A.), using Pasteur capillary pipettes. After incubation at 25°C for one hour, 5 ml of ACM was added into each flask, the cap of each flask being sealed hermetically. The culture was continued at 25°C in an incubator (Sanyo Incubator MIR-151), the ACM being changed every 5 days. Subculture : The cells grown out from the tumor fragments were dissociated from the bottom of culture flask with Ca-Mg-free PBS containing 0.1% trypsin (Difco 1:250) and 0.02% EDTA (Wako Dotite Co.). A three-fold volume of ACM was added to stop the action of trypsin, and aliquots of the suspension were transferred to centrifuge tubes (Falcon 2095 Tube) and centrifuged at 1,000 rpm
doi:10.2183/pjab.62.307 fatcat:jhzqxanaynhhdn5n7zq4igkxt4