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Method for increasing extraction rate of CD34 positive stromal vascular fraction (SVF) in high-fat tissue

A fat tissue extraction rate technology, applied in tissue culture, cell dissociation methods, bone/connective tissue cells, etc., can solve the problem of low yield of stem/progenitor cells

Pending Publication Date: 2017-01-18
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The single mechanical method or enzymatic method currently used to separate SVF generally has the problem of low yield of stem / progenitor cells

Method used

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  • Method for increasing extraction rate of CD34 positive stromal vascular fraction (SVF) in high-fat tissue
  • Method for increasing extraction rate of CD34 positive stromal vascular fraction (SVF) in high-fat tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: preparation of collagenase solution

[0020] According to the enzyme activity unit of collagenase (purchased from sigma company), add an appropriate amount of normal saline (purchased from Sangon Biotechnology Co., Ltd.) to make it reach 120-180 units per milliliter of normal saline, and the solution obtained after filtering through a 0.22 μm filter is collagenase solution.

Embodiment 2

[0021] Example 2: Isolation of SVF from adipose tissue

[0022] Put 10ml of human adipose tissue obtained by liposuction into a 50ml centrifuge tube, add appropriate amount of normal saline, mix well, centrifuge at 4500rpm for 30s, remove the oil layer, take out the tissue and move it to a new centrifuge tube, repeat twice. Put the cleaned tissue into a centrifuge tube, cut it up with sterile scissors or suck it into a chyle-like shape with a 20ml syringe. Put the tissue into a 50ml centrifuge tube, add an equal volume of the collagenase solution prepared in Example 1, and shake and digest at 37°C for 30min. After digestion, centrifuge at 130 g for 10 min to collect the precipitate. Repeat 2 times, and the precipitate obtained is SVF. After adding 1 ml of normal saline to suspend the cells, samples were taken to detect the proportion of CD34 positive cells by flow cytometry. The results are shown in Table 1 and figure 1 .

[0023] Table 1 The number of living cells, the p...

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Abstract

The invention discloses a method for increasing the extraction rate of the CD34 positive stromal vascular fraction (SVF) in high-fat tissue. The method includes the steps that fat tissue is subjected to suspension with normal saline, centrifugation is carried out to remove an oil layer, sediment is repeatedly centrifuged, sediment obtained in the last time of centrifugation is crushed, and crushed tissue is obtained; then, a collagenase aqueous solution is added into the crushed tissue, oscillation is carried out at 37 DEG C for digestion, centrifugation is carried out after digestion is finished, and sediment, namely, the CD34 positive SVF is taken. By means of the method, low-concentration collagenase can be used for digestion, and the SVF containing high-ratio CD34 positive stem / progenitor cells is obtained from the fat tissue; the ratio of CD34 positive stem / progenitor cells in the SVF is larger than 24%, and more than 6.0*10<5> CD34 positive cells can be obtained from each milliliter of fat tissue.

Description

[0001] (1) Technical field [0002] The invention relates to the field of biotechnology, in particular to a method for efficiently preparing SVF from adipose tissue. [0003] (2) Background technology [0004] Current studies have found that adipose tissue is one of the sources of pluripotent cells, and these pluripotent cells exist in the stromal vascular fraction (SVF) of adipose tissue. SVF is a group of heterogeneous cell populations obtained after removing mature adipocytes from adipose tissue, and CD34-positive stem / progenitor cells are the most important functional cell population. Studies have shown that adipose-derived SVF not only has the multidirectional potential to differentiate into osteogenic, cartilage, fat, blood vessel and neuron-like cells, but also secretes various factors such as VEGF, bFGF, HGF, angiogenesis factor, angiopoietin 1, etc. This heterogeneity makes it uniquely advantageous in participating in the repair of tissue damage and promoting regenera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2509/00C12N2509/10
Inventor 潘若浪张强戴玲华王金福
Owner HANGZHOU S EVANS BIOSCI LTD
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