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European eel kidney cell line EK and application thereof

A kidney cell and eel technology, applied in the direction of urinary tract/kidney cells, animal cells, vertebrate cells, etc., can solve the difficulty of increasing the establishment of European eel somatic cell lines, the inability to maintain the biological characteristics of eel somatic cell lines for a long time, Instability of passage and other problems, to achieve the effect of simple and easy cultivation method, excellent performance and short passage interval

Active Publication Date: 2019-01-15
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty in obtaining European eel embryos and young larvae, and the import and export restrictions after being listed as an endangered species objectively increase the difficulty of establishing European eel somatic cell lines. At the same time, the established eel somatic cell lines often have the problem of unstable passage , cannot maintain the excellent biological characteristics of the initially established eel somatic cell line for a long time

Method used

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  • European eel kidney cell line EK and application thereof
  • European eel kidney cell line EK and application thereof
  • European eel kidney cell line EK and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Primary and Early Subculture of European Eel Kidney Cells

[0050] 1. Experimental animals:

[0051] Healthy juvenile European eel weighing about 2g.

[0052] 2. Reagents:

[0053] L-15 (purchased from Hyclone); 1000000U / 10mg penicillin-streptomycin mixed solution, absolute ethanol, tincture of iodine (purchased from Shanghai Shenggong); NaCl, Na 2 HPO 4 , KCl, KH 2 PO 4 , NaHCO 3 , trypsin (Trypsin) (purchased from Sigma); fetal bovine serum (Fetalbovine serum, FBS) (purchased from Gibco).

[0054] 3. Instrument:

[0055] Ultra-clean bench (AirTech); inverted fluorescence microscope (Nikon); biochemical incubator (Boxun); ultrapure water machine (Millipore).

[0056] 4. Consumables:

[0057] Ophthalmic scissors, ophthalmic forceps, and Venus scissors for dissection (Liuliu Ophthalmology); 50mL cell culture flask (Corning); 15mL centrifuge tube (BD Falcon); 5mL elbow glass dropper.

[0058] 5. Steps:

[0059] 1) Preparation before the test: During the transpo...

Embodiment 2

[0067] Establishment of European eel kidney cell line:

[0068] 1. Reagent:

[0069] L-15 (purchased from Hyclone); NaCl, Na 2 HPO 4 , KCl, KH 2 PO 4 , NaHCO 3 , trypsin (Trypsin) (purchased from Sigma); fetal bovine serum (Fetal bovine serum, FBS) (purchased from Gibco)

[0070] 2. Instrument:

[0071] Ultra-clean bench (AirTech); inverted fluorescence microscope (Nikon); biochemical incubator (Boxun); ultrapure water machine (Millipore)

[0072] Liquid nitrogen tank (Locator), centrifuge (Beckman)

[0073] 3. Consumables:

[0074] 50mL Cell Culture Flask (Corning), 250mL Cell Culture Flask (Corning); 15mL Centrifuge Tube (BDFalcon); 5mL Elbow Glass Dropper

[0075] 4. Steps:

[0076] 1) Preparation of complete culture medium for routine subculture: prepare a complete culture medium for conventional subculture based on L-15 medium, containing fetal bovine serum at a final concentration of 8% by volume, and excluding antibiotics and other nutrient additives.

[0077...

Embodiment 3

[0081] Determination of growth characteristics of self-established European eel kidney cell line EK

[0082] 1. Reagent:

[0083] L-15 (purchased from Hyclone); Trypsin (purchased from Sigma); Fetal bovine serum (FBS) (purchased from Gibco)

[0084] 2. Instrument:

[0085] Ultra-clean bench (Air Tech); inverted fluorescence microscope (Nikon); biochemical incubator (Boxun); ultrapure water machine (Millipore), liquid nitrogen tank (Locator), centrifuge (Beckman), flow cytometer ( Bio-rad)

[0086] 3. Consumables:

[0087] 50mL cell culture flask (Corning); 15mL centrifuge tube, 2.5mL centrifuge tube (BD Falcon); 5mL elbow glass dropper

[0088] 4. Steps:

[0089] The 63rd generation of EK cells was treated with 2×10 5 The density of each bottle was inoculated into 50mL cell culture flasks, divided into 7 groups, and cultured at 10°C, 15°C, 20°C, 25°C, 30°C, 37°C and 40°C.

[0090] Every other day, take 3 bottles from each group of cells, digest and harvest the cells wit...

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Abstract

The invention provides a European eel kidney cell line EK and application thereof, belonging to the technical field of animal cell culture. The kidney cell line of Anguilla anguilla is deposited as CCTCC NO: C2018160. The cell line is a fibroblast type cell, which is primary cultured by tissue block adherence method and passaged by trypsin digestion method. The kidney cell line EK in L-15 containing 8% of fetal bovine serum has the optimum state during culture at 26 DEG C.. The typical morphology of fibroblasts can be maintained after 65 passages in vitro. The karyotype of fibroblasts is aneuploid. After cryopreservation and resuscitation, the survival rate of fibroblasts is 85%, and the fibroblasts grew rapidly. The cell line EK can be used to construct the expression model of exogenous gene, can also be used as a host cell for viral isolation and other etiological and immunological research.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a European eel kidney cell line EK and an application thereof. Background technique [0002] Since Wolf et al. established the first permanent teleost cell line—the rainbow trout gonad cell line RTG-2 in 1962, more than 300 fish cell lines have been established at home and abroad; fish somatic cell line culture The technology is widely used in many fields such as etiology, physiology, pharmacology, environmental toxicology, cell biology and germplasm resource protection. It plays an especially important role in virology research. Basis for research. [0003] Eel is a popular traditional delicacy in coastal areas of Asia and Europe. Eel aquaculture is an important pillar industry of my country's aquaculture industry. Its annual output accounts for about 2 / 3 of the world's total output, and the export value of a single product ranks first among aquatic prod...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/85C12N7/00C12R1/93
CPCC12N5/0686C12N7/00C12N15/85C12N2710/00051C12N2800/106
Inventor 郑在予陈斌龚晖杨金先池洪树刘晓东黄河
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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