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Method for separation and primary culture of intestinal mucosal cells of fishes

A technology of intestinal mucosa and primary culture, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problem of cells, tissue blocks, mixed dead cells, incomplete application of aquatic temperature-changing animals, and difficulty in cell separation Large and other problems, to achieve the effect of good cultivation and growth, good digestion and separation, and good growth

Inactive Publication Date: 2011-08-31
SUZHOU UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] ⑴Using collagenase to separate intestinal mucosal cells is the main technology for animal intestinal mucosal cell separation, but the traditional method is mainly suitable for terrestrial constant temperature animals such as mice, rats, poultry, etc., and is not completely suitable for aquatic variable temperature animals. , first of all, the type and concentration of collagenase suitable for the separation of intestinal mucosal cells of terrestrial homeothermic animals in the prior art are not suitable for the separation of intestinal mucosal cells of fish; Protease digestion treatment, but the obtained cells, tissue pieces, dead cells, etc. are mixed, which makes the separation of cells very difficult. At the same time, broken cells and tissues release a lot of hydrolytic enzymes from lysosomes in the cells, which are harmful to the separated cells. have a greater impact
[0006] (2) In the prior art, rat tail collagen is usually used as a cell support carrier for cell culture and growth, but the effect is not ideal, so it is necessary to screen a cell support carrier suitable for fish cell culture and growth
[0008] (4) In the prior art, the conditions suitable for the primary culture of cells of terrestrial homeothermic animals are not suitable for fish, because, for example, fish are variable-temperature aquatic animals that live in the water environment for a long time, and the intestinal mucosal cells of their Culture conditions such as temperature are very different from terrestrial homeothermic animals; fish live in water environment, and the osmotic pressure required for fish cell growth is different from that of terrestrial animals, so fish cell culture medium should be adjusted appropriately

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  • Method for separation and primary culture of intestinal mucosal cells of fishes
  • Method for separation and primary culture of intestinal mucosal cells of fishes
  • Method for separation and primary culture of intestinal mucosal cells of fishes

Examples

Experimental program
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Embodiment 1

[0032] A method for the isolation and primary culture of fish intestinal mucosal cells, comprising the following steps:

[0033] ⑴Choose the intestinal tract of the test material fish that has been cultivated for 2 to 3 weeks with feed that has strengthened the growth of intestinal mucosal cells. After disinfection, use scissors to mash the brain of the fish body in a sterile room, quickly cut open the belly of the fish, take out the intestine, and remove extra-intestinal fat;

[0034] ⑵The intestinal tract is made into intestinal sacs or turned over and then digested to effectively separate intestinal mucosal cells; then centrifuge at a speed of 300r / min to 500r / min for 3 to 5 minutes to obtain intestinal mucosal cell clusters: the ratio of mucosal cells reaches 1: The mixed cell suspension of 2.5-5 can meet the inoculation concentration requirements of subsequent experiments.

[0035] The process of making the intestinal tract into an intestinal capsule and then digesting i...

Embodiment 2

[0054] Step 1: The surface of the material fish is disinfected with alcohol with a mass fraction of 75%.

[0055] Step 2: Take out the intestinal tract of the fish body by routine dissection, and remove the extra-intestinal fat.

[0056] Step 3: Inject the fish into the intestinal tract with a syringe and rinse with normal saline for 3 to 4 times to clean the contents of the intestinal tract.

[0057] Step 4: Turn the intestine completely over, with the mucosa facing outward and the intestinal wall facing inward. Use antibiotic-containing D-Hanks solution to wash the intestinal mucosal surface to remove mucus and debris on the intestinal mucosal surface, and then ligate both ends of the intestinal tract. The D-Hanks solution containing antibiotics was 200,000 U / L of penicillin, 200,000 U / L of gentamicin, and 200 mg / L of streptomycin.

[0058]Step 5: Submerge the inverted intestinal sac in the mixed digestive solution, digest in a water bath at 25-30°C for 20-40 minutes, then...

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Abstract

The invention belongs to the field of separation and culture of intestinal mucosal cells of animals, and particularly relates to a method for separation and primary culture of intestinal mucosal cells of fishes. The method comprises two steps of preparing materials, namely taking fish intestines out according to the conventional method, removing the fat outside the intestines, and digesting and separating the intestinal mucosal cells of fishes by any of the following two methods to obtain digestive juice containing free mucosal cells and cell mass, and centrifuging the obtained digestive juice containing free mucosal cells and cell mass to obtain the mixture of mucosal cells and cell mass which is used as inoculating material cells for primary culture; and culturing, namely performing primary culture by using skin collagens as a culture support vector, using the improved Dulbecco's modified Eagle medium (DMEM) liquid as a culture medium, and using the improved D-Hanks liquid as balanced salt solution at the culture temperature of between 26 and 28 DEG C and pH value of between 7.2 and 7.4 for more than 24 hours, namely attaching to the surface to ensure that the cells grow. The obtained intestinal mucosal cells of fishes grow well, and the structure and functional marking index system is perfect.

Description

technical field [0001] The invention belongs to the field of separation and cultivation of animal intestinal mucosa cells, and in particular relates to a method for separation and primary culture of fish intestinal mucosa cells. Background technique [0002] Cells are the basic unit of animal structure and function. Cultured cells are used as experimental materials in many research fields, such as basic theoretical research on cell structure and functional damage mechanism, new drug screening, and new feed additive research. Compared with living animals (fish), it has the advantages of short research period, easy control of experimental conditions, large-scale experiments in batches, good repeatability of experimental results, etc., and the use of primary cultured cells is better than that of cell lines The technical difficulty is reduced, the experimental cost is reduced, and it is closer to the physiological conditions of the fish itself. [0003] Fish is a temperature-ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 叶元土蔡春芳张宝彤周志刚姚仕彬秦杰
Owner SUZHOU UNIV
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