Method for extracting human stromal vascular fraction

A technology of matrix components and extraction methods, applied in vascular endothelial cells, cell dissociation methods, biochemical equipment and methods, etc. Viscosity, damage-reducing effect

Inactive Publication Date: 2018-12-18
上海赛肽生命科学研究有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in this tissue digestion method, the enzyme digestion is too long and ...

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  • Method for extracting human stromal vascular fraction

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Embodiment 1

[0031] A method for extracting human adipose vascular matrix fraction (SVF), comprising the following steps:

[0032] Step 1, collecting adipose tissue, adding pH 7.2 phosphate buffer for washing;

[0033] Step 2, remove the washing solution from the washed adipose tissue, and add an equal volume of extracting solution, which contains collagenase I (mass concentration is 0.1%) and trehalose (mass concentration is 2%) configured in DMEM medium and gelatin (mass concentration: 0.05%); digest at 150 rpm for 30 minutes at 33°C; centrifuge at 800 rpm for 10 minutes and discard the upper layer of digestive juice, oil and red blood cells;

[0034] Step 3, add normal saline to wash the cells; centrifuge at 800 rpm for 10 min, discard the supernatant, add 10 times the volume of normal saline to the pellet to resuspend the cells, and filter at 100 μm;

[0035] Step 4: Centrifuge at 800 rpm for 10 minutes, add 10 times the volume of normal saline to resuspend the cells, and filter at 40...

Embodiment 2

[0037] A method for extracting human adipose vascular matrix fraction (SVF), comprising the following steps:

[0038] Step 1, collecting adipose tissue, adding pH 7.4 phosphate buffer for washing;

[0039] Step 2, remove the washing solution from the washed adipose tissue, and add an equal volume of extracting solution, which is formulated with DMEM low-sugar medium containing collagenase I (mass concentration is 1%), trehalose (mass concentration is 10 %) and gelatin (mass concentration: 0.15%); oscillating digestion at 280 rpm for 60 min at 40°C; centrifugation at 1200 rpm for 5 min, discarding the upper digestate, oil and red blood cells;

[0040] Step 3, add normal saline to wash the cells; centrifuge at 1200rpm for 5 minutes, discard the supernatant, add 10 times the volume of normal saline to the pellet to resuspend the cells, and filter at 100 μm;

[0041] Step 4: Centrifuge at 1200 rpm for 5 minutes, add 10 times the volume of normal saline to resuspend the cells, and...

Embodiment 3

[0043] A method for extracting human adipose vascular matrix fraction (SVF), comprising the following steps:

[0044] Step 1, collecting adipose tissue, adding pH 7.2 phosphate buffer for washing;

[0045] Step 2, remove the washing solution from the washed adipose tissue, and add an equal volume of extracting solution, which is formulated with DMEM low-sugar medium containing collagenase I (mass concentration is 0.5%), trehalose (mass concentration is 6 %) and gelatin (mass concentration: 0.1%); oscillating and digesting at 200 rpm for 50 min at 37°C; centrifuging at 1100 rpm for 5 min and discarding the upper layer of digestive juice, oil and red blood cells;

[0046] Step 3, add normal saline to wash the cells; centrifuge at 1100rpm for 5 minutes, discard the supernatant, add 10 times the volume of normal saline to the pellet to resuspend the cells, and filter at 100 μm;

[0047] Step 4: Centrifuge at 1100 rpm for 5 minutes, add 10 times the volume of normal saline to resu...

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Abstract

The invention discloses a method for extracting human stromal vascular fraction (SVF). The method mainly comprises the following steps: collecting adipose tissues, washing and centrifuging the adiposetissues, adding an SVF extract liquid containing a DMEM low-sugar medium, collagenase I, trehalose and gelatin, carrying out oscillation digestion, performing centrifuging to obtain a precipitate, and washing and centrifuging the precipitate to obtain SVF cells. The trehalose can protect the cell membrane structure; and the gelatin can promote cell proliferation and migration, and can improve theviscosity of a solution and reduce the damages of a shear force to the cells in order to achieve the purposes of protecting the cells and improving the survival rate of the cells in the adipose tissues. The method is adopted to make the number of the SVF cells reach 4.21 * 10<6>/ml and the viability rate reach 95% and make the number of adipose stem cells in the SVF reach 10.67 * 10<6> and the viability rate reach 95% on the ninth day of culture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting human adipose blood vessel matrix components. Background technique [0002] The adipose vascular fraction (stromal vascular fraction, SVF) is adipose tissue digested, centrifuged to remove mature adipose tissue and connective tissue, to obtain a composition containing mesenchymal stem cells (6.7%), endothelial precursor cells (2%) and A mixture of various cells such as monocytes / macrophages (10%). SVF cells can be extracted from adipose tissue and bone marrow, but in adipose tissue, 1% to 10% of nucleated cells are considered stem cells, while only 0.0001 to 0.01% of nucleated cells in bone marrow are stem cells. The number of stem cells that can be obtained from 1g of adipose tissue is about 5,000-200,000 cells. [0003] Stem cells in SVF cells can secrete various cytokines, chemokines, growth factors and exosomes. These substances have positive effects on...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0775C12N5/0786
CPCC12N5/0645C12N5/0667C12N5/069C12N2509/00
Inventor 鲁南施琳邱伟勤
Owner 上海赛肽生命科学研究有限公司
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