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Larimichthys crocea liver cell line and establishment method thereof

A technology of liver cells and establishment methods, applied in the field of fish cell culture, to achieve the effects of preventing cell pollution, easy digestion, and excellent cell properties

Inactive Publication Date: 2014-08-20
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the establishment of large yellow croaker cell lines is only reported by Sun Ai in 2010, and the three cell lines of adult large yellow croaker fin, snout, and spleen have been established, and no other tissue cell lines have been reported.

Method used

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  • Larimichthys crocea liver cell line and establishment method thereof
  • Larimichthys crocea liver cell line and establishment method thereof
  • Larimichthys crocea liver cell line and establishment method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The establishment of large yellow croaker liver 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 liver tissue with a dissecting instrument, and put it into a petri dish with D-hanks added before rinsing for 3-4 times.

[0051] (2...

Embodiment 2

[0055] Cryopreservation and recovery of the large yellow croaker liver cell line in Example 1.

[0056] (1) Frozen storage: Take a bottle of 75cm 2 The cells of the large yellow croaker liver cell line that are vigorously growing and covered the bottom of the culture bottle were digested with trypsin, and the cell pellet was collected after centrifugation. Slowly add 3mL of the prepared cell freezing solution, and gently blow the cells 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 cryopreservation...

Embodiment 3

[0060] The influence of different passage ratio proliferation curves and population doubling time analysis of the large yellow croaker liver cell line of embodiment 1:

[0061] From the large yellow croaker liver cell line established in Example 1, cells with good morphology and vigorous proliferation were selected, digested and passaged, and passaged 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 liv...

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Abstract

The invention discloses a larimichthys crocea liver cell line and an establishment method thereof. The larimichthys crocea liver cell line is preserved in the China center for type culture collection (CCTCC) On October 31, 2013 and has a preservation number of CCTCC NO: C2013157. The larimichthys crocea liver cell line can realize serial passage, can provide a large amount of larimichthys crocea liver cell lines, can be used for pathogenic characteristic research, vaccine development, functional gene research and breeding research, has excellent properties, comprises fibroblast-like cells and mainly comprises cells having parapodiums stretching out and irregular shapes. The larimichthys crocea liver cell line has the advantages of exuberant division, short passage time, easy digestion, good adherence rate and hunger resistance. The establishment method has good repeatability. The larimichthys crocea liver cell line has good stability.

Description

technical field [0001] The invention belongs to the technical field of fish cell culture, and in particular relates to a large yellow croaker liver cell line and a method for establishing the same. Background technique [0002] Large yellow croaker (Larimichthys crocea), Osteichthyes, Perciformes, Totoabaidae, Yellow croaker. The meat is fresh and tender, the economic value is high, and the market demand is large. Before the 1970s, wild large yellow croaker resources were relatively abundant, and the average annual catch once reached 120,000 tons. In the early 1970s, due to the overfishing of wild large yellow croaker, especially its overwintering groups, the production of wild large yellow croaker was rapidly exhausted. After the 1980s, due to coastal economic development and environmental pollution, the original spawning grounds in the coastal waters were no longer suitable for large yellow croaker spawning, and the catch of wild large yellow croaker is extremely low. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12R1/91
Inventor 王艺磊庄道华张子平李敏
Owner JIMEI UNIV
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