Establishment and optimization of an isolation and culture system for Sertoli cells of Nile tilapia

Abstract:

The aim of this study to establish and optimize an isolation and culture system for Sertoli cells of Nile tilapia. Fresh testis tissues from Nile tilapia in development stage Ⅲ were obtained and then rinsed with phosphate-buffered saline. The tissue was dissected into segments and digested with 0.5 mg/ml collagen for 30 min, followed by 0.25% trypsin–0.04% EDTA for 5 min, and the digestion was terminated with L-15 culture medium with 10% newborn bovine serum (NBS). Sertoli cells were selected using the characteristic that they adhered more quickly than germ cells to the culture vessels. Sertoli cells were cultured in 96-well plates in L-15, M199, or F12 culture medium, supplemented with 10% NBS, or L-15 culture medium supplemented with 5%, 10%, and 15% NBS, or L-15 culture medium supplemented with 10% NBS and 1% Nile tilapia serum. For each treatment group, Sertoli cells were collected from six culture wells every 2 days; the number of cells in each well was counted using a hemocytometer, and a growth curve was drawn for the Sertoli cells. Compared with F12 or M199 culture medium, more rapid growth of Sertoli cells occurred in the L-15 culture medium (P<0.01). Compared with supplementation with 5% or 15% NBS, the proliferation of Sertoli cells was accelerated (P<0.05) by supplementation with 10% NBS in the culture medium. Comparatively, the effect of the addition of 1% Nile tilapia serum was greater (P<0.05) than the absence of serum. The proliferation of Nile tilapia Sertoli cells can be improved by supplementation with 10% NBS and 1% Nile tilapia serum in L-15 cell culture medium.