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Method for culturing goat precursor fat cells in vitro

A fat cell culture technology in vitro, applied to animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of long time, slow proliferation, and many manpower, and achieve reliable principle, good cell shape, Cultivate a good effect

Inactive Publication Date: 2013-04-24
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the successfully cultured preadipocytes mainly come from human, mouse and pig. Culture method; the tissue block culture method takes a long time and requires a lot of manpower, and the cells reach monolayer fusion in about 10-12 days; the digestion culture method is shorter than the tissue block culture method, and the cell growth and fusion time also takes 7-8 days, and Cells age after multiple passages, and proliferation slows down to death

Method used

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  • Method for culturing goat precursor fat cells in vitro
  • Method for culturing goat precursor fat cells in vitro
  • Method for culturing goat precursor fat cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] This example is based on the traditional digestion and culture method, taking goat intercostal fat tissue, using the improved collagenase I medium to treat the fat tissue, and using the improved DMEM low-sugar culture medium containing fetal bovine serum to treat goat preadipocytes in vitro Cultivate and obtain goat preadipocytes; the specific process steps are:

[0016] (1) Preparation of collagenase I medium: Dissolve 100mg of collagenase I, 3g of bovine serum albumin and 1ml of penicillin-streptomycin solution (PS) in 50ml of HBSS solution, adjust the pH value to 7.2-7.4, pass through 0.22 micron Filter and subpackage, prepare collagenase I medium with a weight percentage of 0.2%, and store it at -20°C for later use;

[0017] (2) Preparation of cell culture medium: Add 15% of its volume of fetal calf serum, 2% NEAA, 1% L-glutamine, 1% penicillin-streptomycin solution (PS) and 20ng / mlEGF, filtered through a 0.22 micron filter and subpackaged, prepared into a cell cu...

Embodiment 2

[0025] In this example, when the primary cultured goat preadipocytes reached 80%-90% confluence, 300 μL of trypsin digestion solution was added to each plate of cells and digested at 37°C for 3 minutes. The preparation method of the trypsin digestion solution was as follows: 0.25 Mix g trypsin with 100ml of PBS buffer to make 0.25% trypsin digestion solution; add the same volume of cell culture medium as the trypsin digestion solution to stop the digestion, centrifuge at 2000r / min for 5min to obtain a precipitate, and mix the precipitate with the cell culture medium Subculture according to the volume ratio of 1:3, inoculate and culture in 60mm culture dish, replace the cell culture medium every 2 days during the culture process; culture until the cells reach 80%~90% confluence and cover the whole culture dish , repeat the above digestion steps; pass down successively until the subculture reaches the 20th generation.

Embodiment 3

[0027] In this example, the oil red O staining method was used to detect the differentiation of goat preadipocytes. After the goat preadipocytes grew to 100% confluence, they were replaced with induction differentiation culture medium for induction and differentiation culture; the preparation method of induction differentiation culture medium was in DMEM Add 0.5% bovine serum albumin (BSA), 10% fetal bovine serum (FBS), 10 μg / mL bovine insulin (INS), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) to the low-sugar medium. ), 0.5 μM dexamethasone (DEX) and 0.1 mM indomethacin (IDM) were mixed, adjusted to pH 7.2-7.4, filtered through a 0.22 μm filter, aliquoted, and stored at 4°C for later use; Small lipid droplets began to appear in the cytoplasm, and on the 6th day, the number of small lipid droplets in the cytoplasm increased significantly, and a few began to fuse into large lipid droplets; after 12 days of induction, the differentiated adipocytes were cultured and identified by th...

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Abstract

The invention belongs to the technical field of cell culture engineering, and relates to a method for culturing goat precursor fat cells in vitro. The method comprises the following steps of: preparing a collagenase I culture medium and a cell culture solution, killing a goat, taking goat precursor intercostal fatty tissues out, quickly soaking the fatty tissues into alcohol, repeatedly flushing the fatty tissues by using sterile normal saline, transferring the fatty tissues to a sterilization phosphate buffer solution (PBS), putting the fatty tissues in a sterile room, rinsing the fatty tissues by using the PBS, removing blood vessels and connective tissues by using ophthalmic scissors to obtain a fatty tissue sample, quickly cutting the sample into small blocks, transferring the small blocks to a centrifugal tube, adding the collagenase I culture medium, mixing uniformly and digesting; and performing filtration by using a cell sieve to remove undigested tissue blocks and fatty cells, transferring the digested cell culture solution to the centrifugal tube, centrifuging, removing the supernate, adding the cell culture solution, cleaning, centrifuging, preparing a cell suspension, inoculating cells to a culture dish, and culturing the cells in an incubator. The method is simple, reliable in principle, controllable in culture, good in culture effect and environment-friendly.

Description

Technical field: [0001] The invention belongs to the technical field of cell culture engineering and relates to a method for in vitro culture of goat preadipocytes. Background technique: [0002] Preadipocytes are a type of adipocytes, which are differentiated from multipotential stem cells (MSC) or embryonic stem cells (ESC). The cytoplasm does not contain lipid droplets, but has the ability to accumulate lipid droplets in the cytoplasm, can divide and proliferate in vivo and in vitro, and accumulate lipid droplets in the cytoplasm to differentiate into mature adipocytes. At present, the successfully cultured preadipocytes mainly come from human, mouse and pig. Culture method; the tissue block culture method takes a long time and requires a lot of manpower, and the cells reach monolayer fusion in about 10-12 days; the digestion culture method is shorter than the tissue block culture method, and the cell growth and fusion time also takes 7-8 days, and After many passages, ...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 曲贞晓潘庆杰沈伟闵令江孙小凤张敏姜颖
Owner QINGDAO AGRI UNIV
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