Pseudosciaena crocea muscle cell line and its establishing method
A technology of muscle cell line and establishment method, which is applied in the field of fish cell culture to achieve the effect of good stability, short passage time and strong reproducibility
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Embodiment 1
[0049] The establishment of large yellow croaker muscle cell line of the present invention:
[0050] (1) Since the large yellow croaker is a crocodile fish and is extremely sensitive to hypochlorite, sterilized seawater containing double antibodies is used instead of hypochlorous acid disinfectant in the body surface disinfection step. The 7-month-old large yellow croaker fry with a length of 9-11 cm were temporarily raised in sterilized seawater containing double antibodies for 1-2 hours. At the end, 3-4 drops of eugenol were dripped until the fish turned over, the abdomen was upward, and there was no stress behavior to the stimulus. Wipe off the mucus on the surface of the fish with a sterilized gauze piece. Wipe the surface of the fish twice with a cotton ball soaked in 75% alcohol. Move the fish body into the ultra-clean bench, take out the muscle tissue with a dissecting instrument, and put it into a petri dish with D-hanks added before rinsing for 3-4 times.
[0051] ...
Embodiment 2
[0055] Cryopreservation and recovery of the large yellow croaker muscle cell line of Example 1.
[0056] (1) Frozen storage: Take a bottle of 75cm 2 The cells of the large yellow croaker muscle cell line covered the bottom of the culture bottle were vigorously grown, and the cell pellet was collected after centrifugation by trypsin digestion, and 3 mL of the prepared cell freezing solution was slowly added, and the cells were gently blown to make them evenly dispersed In cell freezing solution, use a pipette to transfer the liquid into a cryovial. Store the cryopreservation tubes at 4°C for 1 hour, then place them in an ice box, place the ice box at -80°C for 1 day, and finally take out the cryopreservation tubes and immerse them in liquid nitrogen for long-term freezing.
[0057] (2) Take the cryopreservation tube out of the liquid nitrogen and quickly place it in a water bath with adjusted temperature (40°C). During the process of thawing the cells, shake the cryopreservati...
Embodiment 3
[0060] The influence of different passage ratio proliferation curves of the large yellow croaker muscle cell line of embodiment 1 and the population doubling time analysis:
[0061] From the large yellow croaker muscle cell line established in Example 1, select cells with good morphology and vigorous proliferation, digest and pass, and carry out passage to 25 cm at 1:2, 1:3, 1:5 and 1:10 respectively. 2 Cell culture flasks (that is, add 2.5mL, 1.6mL, 1mL, 0.5mL cell suspension to the 4 culture flasks, and then make up the total amount of each bottle to 5mL with culture medium). Photographs were taken every 24 hours from the 24th hour of subculture, and the cultured cells were taken by inverted microscope using the classic five-point cross sampling method to count the number of cells observed in the visual field, and the sum of the number of cells observed at five points in each group was plotted for analysis.
[0062] Such as Figure 8 As shown, for the large yellow croaker m...
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