Isolation and culture method for primary rat colonic smooth muscle cells

A smooth muscle cell, separation and culture technology, applied in the field of separation and culture of primary rat colon smooth muscle cells, can solve the problems of excessive blowing and beating during the separation process, uncontrollable pollution, impure cell types, etc., to achieve no perfusion dead angle, avoid cell viability, The effect of high cell viability

Inactive Publication Date: 2016-08-31
王晓冰 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantages are that, firstly, due to the presence of colonic contents, the culture of colonic smooth muscle cells is very susceptible to various contaminations. Simple cleaning and tissue block culture methods cannot control the contamination of various bacteria and fungi at all; secondly, The cell type is impure, simply using tissue pieces to digest and separate cultures, resulting in the mixing of many other cells other than colonic smooth muscle cells, such as various connective tissue cells; finally, using a single collagenase to digest cells, resulting in more blowing during the separation process , poor cell separation and reduced activity

Method used

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  • Isolation and culture method for primary rat colonic smooth muscle cells
  • Isolation and culture method for primary rat colonic smooth muscle cells
  • Isolation and culture method for primary rat colonic smooth muscle cells

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Embodiment 1

[0040]In this example, neonatal SD rats were used as the source of cells. First, they were sacrificed by cervical dislocation, soaked in alcohol for 5 minutes, opened their laparotomy, and removed the colon from 1 cm above the anus to the cecum until the appendix was seen. Take care to peel off the mesentery when collecting materials. Soak the removed colon tissues in PBS containing 2% penicillin and streptomycin, and store them on ice. The isolated rat colon was washed with 37°C pre-warmed D-Hanks solution containing 2% bis-antibody. Then take the smallest scalp needle and smooth the front end with sandpaper to prevent the needle tip from piercing the colon tissue. Perfuse the scalp needle into the colon with preheated preperfusate at 37°C, the preperfusion speed is 5mL / min, the preperfusion time is 5 minutes, and the preperfusion solution formula is 15mM / L HEPES, 127.8mM / L NaCl, 3mM / L KCl, 0.7mM / LNa 2 HPO 4 12H 2 O, EGTA of 0.6mM / L, 2% double antibody and 1% amphoteric...

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Abstract

The invention provides an isolation and culture method for primary rat colonic smooth muscle cells. With the isolation and culture method provided by the invention, the acquired cells are great in gross quantity and high in both isolation degree and motility rate; the probability of contamination by bacteria, fungi and other cells is low; and the method can realize subculturing, so a good cell culture scheme is provided for related research based on culture of rat colonic smooth muscle cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for separating and culturing primary rat colonic smooth muscle cells. Background technique [0002] Smooth muscle contraction is the basic movement mode in gastrointestinal peristalsis. Studies have shown that intestinal smooth muscle cells secrete IL-6 under the stimulation of IL-1b and TNF-a, and IL-6 can significantly induce systemic inflammatory response. The use of colonic smooth muscle cell cultures can help to understand the contraction, proliferation, and response of the connective tissue of the gastrointestinal tract to smooth muscle cells. Most of the colonic smooth muscle culture techniques currently used are obtained from the colon tissue of adult mice or rats, and the cells cultured in this way greatly increase the probability of contamination. However, due to the particularity of the sampling technique, the colon tissue of newborn mice or rats is not...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0679
Inventor 王晓冰杨国峰
Owner 王晓冰
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