A method for separating and culturing rabbit adipose stem cells

An adipose stem cell, separation and culture technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve problems such as inability to completely remove tissue blocks, unstable culture medium preservation, and a large number of adipose stem cells. High-quality adipose stem cell source, good shape, and complete digestion effect

Active Publication Date: 2017-11-28
深圳尤尼智康医疗科技有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing methods for separating adipose stem cells mainly use type I collagenase or mixed digestion of type I, type II and IV collagenase, and filter the digested product filter to remove undigested tissue pieces. Although some adipose stem cells can be separated, they cannot be completely separated. Removing tissue pieces and obtaining a large number of adipose stem cells, the separation efficiency is low
In addition, when the cells are inoculated into culture flasks for culture, the culture medium used is not very stable, and the cell proliferation rate is slow

Method used

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  • A method for separating and culturing rabbit adipose stem cells
  • A method for separating and culturing rabbit adipose stem cells
  • A method for separating and culturing rabbit adipose stem cells

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preparation example Construction

[0031] 6) Preparation of adipose tissue extract: cut the adipose tissue to 0.8-1.2mm 3 For small pieces of fat, add DMEM / F12 culture medium equal to the volume of fat tissue in the centrifuge tube and mix well. The DMEM / F12 culture medium contains 15% volume fraction of FBS (fetal bovine serum) Let stand in a biochemical incubator for 1 hour, discard the upper tissue, and filter the lower supernatant with a 0.22 μm filter membrane to obtain the adipose tissue extract.

[0032] 7) Collection of adipose stem cells: Collect the lower layers obtained by centrifugation in steps (4) and (5), add adipose stem cell culture medium, mix well, and count. The fat stem cell culture fluid refers to the DMEM / F12 culture fluid containing 15FBS-5% adipose tissue extract (that is, the DMEM / F12 culture fluid containing the FBS of 15% volume fraction and the fat tissue extract of 5% volume fraction ).

[0033] The FBS (fetal bovine serum) used in the present invention contains rich nutrients an...

Embodiment 1

[0039] A method for isolating and culturing fat stem cells, comprising the steps of:

[0040] (1) Adipose tissue was aseptically obtained: Bilateral inguinal adipose tissue of New Zealand white rabbits was aseptically obtained in an ultra-clean workbench.

[0041] (2) Removal of blood vessels and other tissues: remove visible blood vessels and other tissues in the ultra-clean workbench, and wash with PBS for 3-5 times until the eluate is clear and translucent;

[0042] (3) Preparation of small pieces of fat: use ophthalmic scissors to cut the fat tissue to 1mm 3 Small pieces of small size, washed 3-5 times with PBS;

[0043] (4) Trypsin digestion: add 0.15% trypsin solution equal to the volume of adipose tissue, mix well, digest at 37°C for 30 minutes, centrifuge at 1000r / p for 10 minutes, and transfer the upper layer to another new centrifuge tube;

[0044] (5) Mixed collagenase digestion: add an equal volume of 0.25% mixed collagenase to the separated upper layer, digest a...

Embodiment 2

[0052] A method for isolating and culturing fat stem cells, comprising the steps of:

[0053] (1) Aseptically obtain adipose tissue: aseptically obtain bilateral inguinal adipose tissue from New Zealand white rabbits in an ultra-clean workbench;

[0054] (2) Removal of blood vessels and other tissues: Remove visible blood vessels and other tissues in the ultra-clean workbench, wash with PBS 3-5 times, until the eluate is clear and translucent;

[0055] (3) Preparation of small pieces of fat: use ophthalmic scissors to cut the fat tissue to 1mm 3 Small pieces of small size, washed 3-5 times with PBS;

[0056] (4) Trypsin digestion: Add 0.5% trypsin solution equal to the volume of adipose tissue, mix well, digest at 37°C for 10 minutes, centrifuge at 1000r / p for 10 minutes, and transfer the upper layer to another new centrifuge tube;

[0057] (5) Mixed collagenase digestion: add an equal volume of 0.1% mixed collagenase to the separated upper layer, digest at 37°C for 1 hour, ...

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Abstract

The invention discloses a method for separating and culturing rabbit fat stem cells. It includes the following steps: (1) aseptically obtain rabbit adipose tissue; (2) prepare a small piece of fat tissue; (3) digest the adipose tissue with trypsin, and separate the upper layer solution and the lower layer precipitate; (4) the upper layer solution Digest by time gradient method; (5) collect adipose stem cells, mix with adipose stem cell culture medium, inoculate and culture; (6) in vitro culture of adipose stem cells; (7) detect and freeze. In the cell separation method, the tissue digestion is not complete enough, and the cell separation efficiency is low. The separation method of adipose stem cells is optimized, which makes the adipose tissue digestion more thorough, improves the cell separation efficiency, and thus provides a better source of adipose stem cells.

Description

technical field [0001] The invention belongs to the fields of biology and medicine, and in particular relates to a method for separating and culturing rabbit fat stem cells. Background technique [0002] Stem cells are primitive cells with the potential of self-replication and multidirectional differentiation, the origin cells of life, and the primitive cells that form various tissues and organs of the human body. Stem cell technology is the cutting-edge technology of biological treatment, which is called regenerative medicine. Stem cells are primitive cells with self-replication and multi-directional differentiation. They are pluripotent cells with self-renewal ability. Under certain conditions, they can differentiate into various functional cells or tissues and organs. Stem cells refer to undifferentiated cells that exist in early embryos, placenta and their appendages, bone marrow, peripheral blood and adult tissues. They can be cultivated into more than 200 kinds of hum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 王红杨洪收张莉
Owner 深圳尤尼智康医疗科技有限公司
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