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Primary dissociated culture method for spleen telocytes

A technology for separating and culturing cells, applied in cell dissociation methods, tissue culture, animal cells, etc., can solve the problems of small number, unfavorable cell experiments, low purity, etc., and achieve high viability, high cell purity, and good viability.

Inactive Publication Date: 2016-12-21
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key is that the number and purity of TCs from each tissue isolated and cultured by these methods are small, which is not conducive to carrying out related cell experiments.
At present, there is no report on the isolation and culture of TCs in the spleen

Method used

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  • Primary dissociated culture method for spleen telocytes
  • Primary dissociated culture method for spleen telocytes
  • Primary dissociated culture method for spleen telocytes

Examples

Experimental program
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Embodiment Construction

[0022] Take rat spleen samples under aseptic conditions, rinse with pre-cooled PBS containing 1% penicillin-streptomycin, and cut the spleen tissue into about 1 mm with ophthalmic scissors 3 For small pieces, add an equal volume of digestive enzyme mixture to the tissue block: 0.1% collagenase I (purchased from Roche), 0.125% trypsin (purchased from Sigma), and 0.002% type I deoxyribonuclease (purchased from Sigma) ); Digestive enzyme configuration: Weigh 100mg of powdered type I collagenase, 12.5mg of trypsin and 0.2mg of type I deoxyribonuclease, dissolve in 100ml of PBS (pH 7.2), stir and mix well, filter with 0.22 micron Sterilize by filtration, subpackage in 1mL cryopreservation tubes, store at 20°C for later use; place the tissue pieces and an equal volume of mixed digestive enzymes at 37°C, pipette 100 times within 5 minutes, let stand at 37°C for 5 minutes, and absorb the supernatant; repeat The above steps are performed 3-5 times until the tissue block is completely d...

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Abstract

The invention belongs to the technical field of animal cell culture and relates to a primary dissociated culture method for spleen telocytes. The method includes steps: spleen sample pretreatment; sample enzymolysis for obtaining suspension of the telocytes; cell inoculation of the suspension of the telocytes to prepare primary cells; primary cell passage enlarged culture for preparing passage cells; passage cell immobilization for completing primary dissociated culture of rat spleen telocytes. Aiming at structural characteristics of spleen tissues, 0.1% of collagenase I, 0.125% of trypsin and 0.002% of type-I deoxyribonuclease are adopted for isolated culture of spleen TCs (telocytes) according to a digestion process. The method has advantages that high cell purity and activity are achieved, cell morphology stability and high activity after passage are realized, and the method can be widely applied to effective development of follow-up relevant TCs morphological and functional experiments.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and relates to a primary separation and culture method of spleen terminal septal cells. Background technique [0002] Telocytes (TCs), a type of mesenchymal cells newly discovered and named in recent years, are widely distributed in skin, heart, kidney, gastrointestinal tract, bladder, parotid gland, placenta, lung and bronchi. It has been confirmed to exist in the myocardium, endocardium, connective tissue interstitium of epicardium, interstitium of myocardial stem cell pool, and pulmonary vein muscle sleeve. Septal terminal cells have typical morphological features: small cell body, one or more elongated processes (telopodes, TPs) of different lengths protruding from the cell body, with constriction (podomer) and enlargement (podom) on the protrusions Arranged alternately, showing a rosary-like structure. For the research on TCs, the current focus is mainly on the morphological ob...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/077
CPCC12N5/0648C12N5/0652C12N2509/00
Inventor 常玉巧郭康马洁郭志坤李辞霞付海鹏郭永龙
Owner XINXIANG MEDICAL UNIV