Epinephelus coioides intestinal cell line ECGI-21 and application thereof
A technology of ECGI-21 and oblique-banded grouper, which is applied to animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of no reports of intestinal cell lines of seawater fish, not many, etc., and reach the growth state Good, stable cell proliferation effect
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Embodiment 1
[0027] Embodiment 1: the construction method of slanted grouper intestinal cell line
[0028] (1) Fish body selection: select healthy cultured slanted grouper, and the experimental fish is 20cm long.
[0029] (2) Preparation of rinse solution and cell culture solution:
[0030] Basal medium: Leibovitz's-15 (L-15) medium is a product of Gibco Company. The medium is prepared according to the company's product instructions, and 0.266% NaCl and 5mM HEPES are added, filtered through a 0.22μm filter, aliquoted, and stored at 4°C for later use; when used, add 15-30% fetal bovine serum, 1-4 times the double antibody Mixture (penicillin, streptomycin).
[0031] Rinsing Solution: Basal medium L-15 contains 400 IU / mL penicillin and 400 μg / mL streptomycin, pH 7.2-7.4.
[0032] Primary cell culture medium: basal medium L-15 contains 30% fetal bovine serum, 0.266% NaCl, 5 mM HEPES, 400 IU / ml penicillin and 400 μg / ml streptomycin, and the medium pH is 7.2-7.4.
[0033] The reagents and s...
Embodiment 2
[0045] Example 2 Detecting the effect of different FBS concentrations on the growth of intestinal cells of grouper oblique
[0046] The growth curve of the cells under different serum concentrations was detected using the oblique grouper intestinal cell line ECGI-21. The specific operations were as follows: aspirate the medium in the culture flask, wash it with trypsin once, and add 2 ml of The cells were digested with 0.25% trypsin until they were completely detached, and the medium was added to the cell suspension by gently pipetting to make the cells into single cells. Count the cell density with a hemocytometer.
[0047] After the cells were counted, 1.2 × 10 4 The cells were seeded in 24-well plates, and 0.5 ml of L-15 medium was added to each well, and the serum concentration in the L-15 medium was 5%, 10%, 15% or 20%, respectively. Cultured in a constant temperature incubator at 28°C. The cells were continuously cultured for 6 days, and the cells were counted with a ...
Embodiment 3
[0050] Example 3 Verification of the cryopreservation and resuscitation ability of the cells of the grouper intestinal cell line of the present invention
[0051] 1. Cell cryopreservation
[0052] The intestinal cell line ECGI-21 of grouper was taken and digested with trypsin to obtain a single cell suspension, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cell pellet was resuspended with 4°C pre-cooled freezing solution (L-15 medium containing 20% fetal bovine serum and 10% DMSO), and 1 ml of each tube was transferred to a 2 ml cryogenic tube. The cryopreservation procedure is: 4°C, 30min; -20°C, 2h; -80°C overnight, and transferred to liquid nitrogen for long-term storage the next day.
[0053] 2. Recovery of cryopreserved cells
[0054] The cryopreserved cells were recovered after 5 days of cryopreservation. Cells were removed from -80°C and quickly thawed in a 37°C water bath. The thawed cell suspension was centrifuged at 1200 rpm for 5 m...
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