Study On Isolation And Culture Of Nile Tilapia And Zebrafish Embryonic Cells

The research of the fish cell culture has rapidly developed.At the present, the technology of the fish cell culture as important research tool has been extensively applied to the genetics, the virology, the toxicology, the cell biology and the environmental protection. The Nile Tilapia (Oreochromis niloticus) is important cultivation fishes, and the Zebrafish (Brachydanio rerio) are frequently used as animal models. However, the researches of the in vitro culture of the embryonic cells (ECs) derived from the Nile Tilapia and the Zebrafish were a few in our country. In the present study, the in vitro isolation and culture conditions of the ECs and the embryonic stem-like cells (ESs-like) had been probed into by isolating and culturing the ECs from the Nile Tilapia and the Zebrafish and the ES-like cells from the Zebrafish. The results were as follows1. The embryos were manually taken from the Nile Tilapia at somite differentiation stage. The ECs derived from the Nile Tilapia were isolated and sub-cultured with the trypsin digestion. The ECs were separately cultured in the L-15 medium, the M199, the MEM or the DMEM medium with conditions in the 28℃, 5% CO2 and saturated humidity. The effects of four cell culture media on the in vitro proliferation of the ECs were researched. The ECs were cultured in the medium supplemented with 5%, 10%, 15%, 20% or 25% new bovine serum (NBS). The effects of the different concentrate NBS on the in vitro proliferation of the ECs were compared. The effects of the different culture temperature at 18℃, 28℃or 37℃on the in vitro proliferation of the ECs were compared. The whole culture medium was used as the control, and the effects of the culture medium without the tilapia perch serum, the basic fibroblast growth factor (bFGF), theβ-Mercaptoethanol (2-ME) or the insulin on the in vitro proliferation of the ECs were researched. The results indicated that the ECs significantly proliferated in the DMEM. Supplemented with 15% NBS in culture medium, the ECs proliferated fastest. The ECs proliferated fast and grew well at the temperature of 28℃. However, the ECs grew slowly at temperature of 18℃, and survived only four day to six day at temperature of 37℃. The ECs proliferated faster in culture medium supplemented with the TPS, the bFGF, the 2-ME and the insulin.2. The ECs derived from the zebrafish at gastrula stage were isolated and sub-cultured with the trypsin digestion. The ECs were separately cultured in the DMEM, the L-15 or the LDF medium with conditions in the 0 or 5% CO2, 28℃and saturated humidity. The effects of different CO2 concentration and three culture media on the in vitro proliferation of the ECs were researched. With conditions in the 5% CO2, 28℃and saturated humidity, the ECs were cultured in the medium supplemented with 5%, 10%, 15%, 20% or 25% NBS. The effects of the different concentrate NBS on the proliferation of the ECs were compared. The whole culture medium was used as the control, the effects of the culture medium without the tilapia perch serum, the bFGF, the 2-ME or the insulin on the proliferation of the ECs were researched. The results showed that the ECs from the zebrafish grew well in the 5% CO2, 28℃and saturated humidity. The ECs significantly proliferated in the DMEM supplemented with 15%NBS. The ECs were in favor of medium supplemented with the bFGF, the 2-ME or the insulin except of the tilapia perch serum.The embryos were manually taken from the zebrafish at blastula stage. The ESCs-like derived from the zebrafish were isolated and sub-cultured with the trypsin digestion. The ESCs-like were cultured on the fibroblast from zebrafish feeder layer. The ESCs-like were identified by the morphology, karyotype analysis, the alkaline phosphatase staining and the in vitro differentiation. The result indicated that the colony of the ESCs-like from the zebrafish was the same using the mechanical method or the trypsine digestion to isolating the primary ESCs-like. Furthermore, using mechanical method was more conducive to the subculture of the ESCs-like. The ESCs-like from the zebrafish were subculture for three generations. The ESCs-like was positive with the alkaline phosphatase staining. Karyotype analysis that the ESCs-like were normal diploid cells. The ESCs-like differentiated into the never-like cells, the fibroblast-like cells, epidermal cells and the melanoma cells with in vitro differentiation culture. These identified that the ESCs-like derived from the zebrafish were the embryonic stem cells.