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Method for inducing and differentiating mature fat cell from adult rabbit into cardiomyocyte

A technique for mature adipocytes and cardiomyocyte-like cells, which is applied in the field of cardiomyocyte-like cells to achieve the effects of simple culture and induction methods, simplified and practical culture steps and methods

Active Publication Date: 2013-08-21
SHANGHAI BIOMED UNION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are no reports about the establishment of rabbit DFAT cell lines and the induction of differentiation into cardiomyocytes

Method used

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  • Method for inducing and differentiating mature fat cell from adult rabbit into cardiomyocyte
  • Method for inducing and differentiating mature fat cell from adult rabbit into cardiomyocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Culture of rabbit dedifferentiated adipocytes

[0032] Adult Dutch White Rabbit (Experimental Animal Center, Second Military Medical University, about 3kg)

[0033] 1. In a relatively sterile state, remove the white adipose tissue from the groin of adult rabbits;

[0034] 2. After washing three times with 0.1M phosphate buffer solution (PBS), cut the tissue into minced shape;

[0035] 3. Digest with collagenase I (Worthington Company) of equal volume (equal to the volume of minced tissue) at 37°C for 40 minutes, and shake properly during the period to ensure uniform digestion. The digestion process can also be placed in a shaker at 37°C.

[0036]4. Use 20% complete medium [20% fetal bovine serum (FBS, Gibico company), high glucose medium (DMEM, PAA company), 1% double antibody (penicillin-streptomycin) (Shanghai Sangong)] and collagenase activity and assists in filtration.

[0037] In order to save, you can choose 5% complete medium [5% fetal bovine serum (FBS, G...

Embodiment 2

[0045] 1. Culture of rabbit dedifferentiated adipocytes

[0046] Adult Dutch White Rabbit (Experimental Animal Center, Second Military Medical University, about 3kg)

[0047] 1. In a relatively sterile state, remove the white adipose tissue from the groin of adult rabbits;

[0048] 2. After washing three times with 0.1M phosphate buffer solution (PBS), cut the tissue into minced shape;

[0049] 3. Digest with collagenase I (Worthington Company) of equal volume (equal to the volume of minced tissue) at 37°C for 45 minutes, and shake properly during the period to ensure uniform digestion. The digestion process can also be placed in a shaker at 37°C;

[0050] 4. Use 20% complete medium [20% fetal bovine serum (FBS, Gibico company), high glucose medium (DMEM, PAA company), 1% double antibody (penicillin-streptomycin) (Shanghai Sangong)] and collagenase activity and assists in filtration. In order to save, you can choose 5% complete medium [5% fetal bovine serum (FBS, Gibico co...

Embodiment 3

[0058] 1. Culture of rabbit dedifferentiated adipocytes

[0059] Adult Dutch White Rabbit (Experimental Animal Center, Second Military Medical University, about 3kg)

[0060] 1. In a relatively sterile state, remove the white adipose tissue from the groin of adult rabbits;

[0061] 2. After washing three times with 0.1M phosphate buffer solution (PBS), cut the tissue into minced shape;

[0062] 3. Digest with an equal volume (equal volume to the minced tissue) of collagenase I (Worthington Company) at 37°C for 50 minutes, shaking properly during the period to ensure uniform digestion. The digestion process can also be placed in a shaker at 37°C;

[0063] 4. Use 20% complete medium [20% fetal bovine serum (FBS, Gibico company), high glucose medium (DMEM, PAA company), 1% double antibody (penicillin-streptomycin) (Shanghai Sangong)] and collagenase activity and assists in filtration.

[0064] In order to save, you can choose 5% complete medium [5% fetal bovine serum (FBS, Gi...

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Abstract

The invention relates to the technical field of dedifferentiation and induced dedifferentiation in cell biology and discloses a method for inducing and differentiating a mature fat cell from an adult rabbit into cardiomyocyte. A dedifferentiated fat cell (DFAT) can be obtained from a white fat tissue; and the DFAT can be differentiated into multiple mesenchyma cell systems through a certain induced culture method and has ossification, lipid formation, gristle formation and muscle formation capacities. The invention aims to culture the DFAT of the rabbit from the mature fat cell of the adult rabbit and induce and differentiate the DFAT into the cardiomyocyte. According to the invention, the white fat tissue of the adult rabbit is selected, isopyknic digestion is carried out through collagenase I, a 20% complete culture medium is adopted, the mature fat cell is dedifferentiated into a DFTA cell through a ceiling culture method, and the morphology of the cardiomyocyte and the expression of an identification molecular are formed. By using the method disclosed by the invention, a seed cell is provided for tissue engineering, convenience is brought for basic research, and a prospect is brought for clinical application.

Description

technical field [0001] The invention relates to the technical field of dedifferentiation and induced differentiation of cell biology, in particular to a method for dedifferentiating mature adipocytes of adult rabbits into pluripotent stem cells and then redifferentiating into cardiomyocytes. Background technique [0002] Adipose tissue is rich in sources, safe and convenient to obtain, and can be used as a practical and promising source of mesenchymal cells. The presence of non-adipocytes in adipose tissue is called the stromal-vascular fraction (SVF). Adipose tissue is separated by mechanical separation or enzyme digestion (after fluorescence-activated cell sorting or cell sorting) to obtain SVF. The cultured SVF is named adipose-derived mesenchymal stem cells (ASC S / ADSC). Studies in vitro and in vivo have shown that human ADSC cells can differentiate into various cell lines, such as adipocytes, osteoblasts, chondrocytes, cardiomyocytes, neuronal cells, endothelial cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077C12N5/071
Inventor 张传森李晓童张喜
Owner SHANGHAI BIOMED UNION BIOTECHNOLOGY CO LTD
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