Establishment of cell lines from catfish (Clarias batrachus) and snakeheads (Ophicephalus striatus)

摘要

Primary cell cultures from catfish (Clarias batrachus) and snakeheads (Ophicephalus striatus) were prepared from whole fry and fingerling organ tissues of the brain, fins, gonad, heart, kidney, liver, skin and spleen. Four methods were tried: method A wherein explants were placed onto the surface of 25-cm~2 Primaria flasks (Falcon), allowed to attach for an hour before addition of Leibovitz medium (L-15) supplemented with 15% fetal bovine serum (FBS)(L15-15); Method B wherein explants were inoculated into 25-cm~2 Primaria flasks (Falcon) already containing L15-15; Method C which required forcing minced organ sections through a stainless steel sieve with the aid of a syringe plunger into a petri dish containing L15-15 medium; and Method D wherein immersed sections of minced tissues to 0.5% trypsin-EDTA slowly agitated using a magnetic stirrer for one hour at 25 deg C. Method B was most effective in the establishment of cell cultures from both fish species. Passage numbers of the cells are to date catfish gonad (CFG) P-56, catfish heart (CFH) P-51, catfish kidney (CFK) P-7, catfish liver (CFL) P-8, catfish spleen (CFS) P-54, snakehead gonad (SHG) P-26, snakehead heart (SHH) P-22, snakehead kidney (SHK) P-19, snakehead liver (SHL) P-49 and snakehead spleen (SHS) P-76. Attempts to derive primary cell cultures from organ tissues of the brain, fins, skin and whole fry were unsuccessful. Established cells were fibroblastic. The cells grew rapidly and became confluent 24 h after seeding at 20 and 25 deg C. Both SHS and CFS were susceptible to a virus isolated from EUS-affected fish in the Philippines. The cells were best maintained at 20 deg C and stored in liquid nitrogen or -70 deg C.