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Separation and culturing method of gastric antrum interstitial cells of SD (Sprague Dawley) rats

A culture method and cell technology, applied in the direction of cell dissociation method, cell culture active agent, gastrointestinal cells, etc., to achieve the effect of avoiding loss, avoiding damage, and increasing the acquisition rate

Inactive Publication Date: 2018-11-23
THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims at the defects of the existing ICC cell separation method, adopts SD rats aged 4-5 weeks (110±10g) as experimental animals, adopts the principle of cell contact inhibition and superiority competition to purify, and adopts the method containing stem cell factor (SCF ) culture medium, can obtain stable, continuous and high-efficiency ICC cells with high expression rate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The method for separating and culturing SD rat gastric antrum interstitial cells comprises the following steps:

[0030] S1, preliminary separation

[0031] 4-week-old SD rats were selected, fasted for 12 hours, anesthetized by ether inhalation, and sacrificed by cervical dislocation; the abdomen was moistened with 75% ethanol, the abdominal cavity was opened under aseptic conditions, the gastric antrum was taken out, and cut along the lesser curvature of the stomach , placed in a sterile glass dish, rinsed repeatedly three times with PBS solution at a temperature of 3°C, quickly replaced the sterile glass dish and placed it on ice, added an appropriate amount of PBS solution, and sharpened it with ophthalmic scissors and ophthalmic tweezers under a dissecting microscope. Separate the gastric mucosa and submucosa, place the stripped tissue in a test tube containing M199 complete medium, and put it under ice to maintain cell activity; the requirements for stripping the t...

Embodiment 2

[0038] The method for separating and culturing SD rat gastric antrum interstitial cells comprises the following steps:

[0039] S1, preliminary separation

[0040]5-week-old SD rats were selected, fasted for 12 hours, anesthetized by ether inhalation, and sacrificed by cervical dislocation; the abdomen was moistened with 75% ethanol, the abdominal cavity was opened under aseptic conditions, the gastric antrum was taken out, and cut along the lesser curvature of the stomach , placed in a sterile glass dish, rinsed repeatedly three times with PBS solution at a temperature of 5°C, quickly replaced the sterile glass dish and placed it on ice, added an appropriate amount of PBS solution, and sharpened it with ophthalmic scissors and ophthalmic forceps under a dissecting microscope. Separate the gastric mucosa and submucosa, place the stripped tissue in a test tube containing M199 complete medium, and put it under ice to maintain cell activity; the requirements for stripping the tis...

Embodiment 3

[0047] The method for separating and culturing SD rat gastric antrum interstitial cells comprises the following steps:

[0048] S1, preliminary separation

[0049] 4.5-week-old SD rats were selected, fasted for 12 hours, anesthetized by ether inhalation, and sacrificed by cervical dislocation; the abdomen was moistened with 75% ethanol, the abdominal cavity was opened under aseptic conditions, the gastric antrum was taken out, and cut along the lesser curvature of the stomach , placed in a sterile glass dish, rinsed repeatedly three times with PBS solution at a temperature of 4°C, quickly replaced the sterile glass dish and placed it on ice, added an appropriate amount of PBS solution, and sharpened it with ophthalmic scissors and ophthalmic tweezers under a dissecting microscope. Separate the gastric mucosa and submucosa, place the stripped tissue in a test tube containing M199 complete medium, and put it under ice to maintain cell activity; the requirements for stripping the...

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Abstract

The invention provides a separation and culturing method of gastric antrum interstitial cells of SD (Sprague Dawley) rats. The separation and culturing method comprises the following steps: selecting4-5-week-old SD rats as elementary bodies; stripping gastric antrum tissues, and performing centrifugal operation under the effect of II-type collagenase and separating out ICC cells; performing culturing in an M199 complete culture medium, and then performing digestion operation and centrifugation treatment to obtain the separated ICC cells. According to the method disclosed by the invention, byusing the 4-5-week-old SD rats (110 plus / minus 10g) as an experimental animal, the defect of low cell acquisition ratio caused by highly differentiation of the ICC cells of adult rats is avoided; meanwhile, according to the separation and culturing method disclosed by the invention, the concentration of the II-type collagenase is adjusted, and damage of low-concentration long-time digestion or high-concentration short-time digestion on cells is avoided; purification is carried out by adopting the principles of cell contact inhibition and advantage competition, so that the damage of gradient centrifugation on the fragile cells is avoided.

Description

technical field [0001] The invention belongs to the field of cell extraction methods, in particular to a method for separating and culturing SD rat gastric antrum interstitial cells. Background technique [0002] Antrum interstitial cells (ICCs) are pacemaker cells for gastrointestinal motility, which can generate autonomous rhythmic slow-wave potentials, and act on smooth muscle cells in the gastrointestinal tract through protrusion and gap junctions to promote rhythmic relaxation and contraction of smooth muscles. And a variety of gastrointestinal diseases such as chronic transit constipation, functional dyspepsia, diabetic gastroparesis, etc. are related to the pathological changes of ICC. The existing ICC primary cell extraction technology mostly uses adult SD rats, digests them with type II collagenase, and purifies them by gradient centrifugation. However, this method cannot avoid the high differentiation of adult rat ICC cells, and cannot avoid the damage and loss of...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0679C12N2501/125C12N2509/00
Inventor 谭人千凌江红鞠静罗高标文一惠
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIV
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