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Suspension culture method for intestinal cells of stomachless lukewarm water fish

A cell suspension and culture method technology, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problem that the separation method cannot be directly applied to warm water fish without stomach

Inactive Publication Date: 2013-05-01
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The intestinal structure and physiological characteristics of fish with a cold stomach are very different from those of warm water fish without a stomach. Therefore, the existing isolation methods and suspension culture parameters of fish with a cold stomach cannot be directly applied to the fish without a stomach. warm water fish

Method used

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  • Suspension culture method for intestinal cells of stomachless lukewarm water fish

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Embodiment Construction

[0027] The present invention will be described in detail below in conjunction with specific embodiments.

[0028] 1. Select a healthy carp or grass carp with a weight of about 500g, soak it in 2% urethane solution for anesthesia, and cut off the head after the fish body is overturned to destroy the spinal cord. Disinfect the body surface of the fish according to the conventional method, remove the whole viscera aseptically, remove the mesangial tissue on the surface of the intestinal tract, and wash the contents of the intestinal tract with solution-1 (Hanks solution).

[0029] 2. Tie one end of the intestinal tract first, fill the intestinal tract with solution-1, clamp the other end with hemostatic forceps, and incubate at room temperature for 10 minutes. The solution is then discarded to remove food particles and mucus that may remain in the intestines.

[0030] 3. Then fill the intestinal tract with solution-2 (Hanks solution + 0.2mM EDTA), clamp both ends with hemostatic...

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Abstract

The invention discloses a suspension culture method for intestinal cells of stomachless lukewarm water fish. According to the invention, collected intestinal cell suspension is mixed together for refrigeration and centrifugation for 10 min at 1500 Xg; collected cell deposition is suspended with a solution -3 anew and subjected to oscillation incubation at a temperature of 25 DEG C for 15 min, andsuspension of intestinal cells is filtered with a blood nylon filter having a bore diameter of 100 -mu m and subjected to refrigeration and centrifugation for 10 min at 1500 Xg; obtained cell deposition is suspended in a solution -3 anew for subsequent usage in tests; the integrity of cell membranes is detected by using the method of 0.5% typan blue exclusion; cell suspension and dyes are isopyknicly mixed and added on a counting panel drop by drop, a coverslip is covered, and after the counting panel is stood for one minute, the number of dead cells, the total number of cells and the cell viability are calculated; the cell suspension is subjected to refrigeration and centrifugation for 10 min at 1500 Xg, and the activity of a liquid supernatant and cell deposition LDH is determined respectively; the cell suspension is lightly and evenly mixed, a desired amount of the cell suspension is taken for oscillation incubation at a temperature of 26 DEG C, a rotate speed is 70 revolution / min,and total culture time is 240 minutes. The invention brings forward the suspension culture method for intestinal cells of stomachless lukewarm water fish for the first time.

Description

technical field [0001] The invention relates to a method for separating and cultivating animal intestinal cells, in particular to a method for separating and suspending the intestinal cells of fish in warm water without stomach, and belongs to the field of biotechnology. Background technique [0002] Fish enterocytes are the main functional cells of the intestine, and participate in various important physiological processes such as digestion and absorption of intestinal nutrients, immune barrier and stress response. Studying enterocytes in vivo is difficult due to the extensive interactions between nerves, humoral cells, and many cell types. The research model of enterocyte culture in vitro is an ideal model to explore the mechanism of nutrients on enterocyte nutrition, the mechanism of intestinal damage and intestinal repair caused by drugs, heavy metals and other toxic and harmful substances, and the screening of functional substances that are beneficial to fish intestinal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 周小秋姜俊冯琳刘扬胡凯姜维丹
Owner SICHUAN AGRI UNIV
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