Method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing

An adipose tissue and stem cell technology, applied in the field of biomedicine, can solve the problems of long operation time, fever, easy to cause inflammation, etc., and achieve the effect of reducing pain

Inactive Publication Date: 2011-02-16
王泰华
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  • Summary
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  • Description
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  • Application Information

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Problems solved by technology

[0004] At present, the general steps of extracting adipose-derived mesenchymal stem cells from fat include anesthesia liposuction to obtain a large amount of human adipose tissue, digestion with collagenase, gradient centrifugation, filtration, and then expansion and culture in an in vitro culture system, wherein the in vitro culture The system contains fetal bovine serum; and a large area of ​​local anesthesia is required during the liposuction process, and an incision is made at the liposuction site, causing certain trauma and pain to the patient. In order to obtain a large amount of fat tissue, a large area of ​​liposuction is required Liposuction surgery will lead to a larger area of ​​liposuction, longer operation time, more bleeding or fever. If the post-care is not good, it will easily lead to inflammation

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  • Method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing
  • Method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing
  • Method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing

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Embodiment

[0022] 1. Reagents

[0023] STEMPRO MSC SFM serum-free medium, penicillin-streptomycin, gentamicin, glutamine, platelet-derived growth factor, basic fibroblast growth factor, type I collagenase, trypsin, phosphate buffer, oil Red O, Alizarin Red.

[0024] 2. Isolation and extraction of adipose-derived mesenchymal stem cells

[0025] Soak in phosphate buffer, wash the adipose tissue by centrifugation at 1500r / min, digest with 0.1% collagenase type Ⅰ in a 37°C water bath for about 30min, add phosphate buffer to dilute the collagenase and centrifuge, suspend the lower layer of sediment, filter through a 100-mesh sieve, and filter at 1500r Centrifuge at 1000r / m for 10min to remove suspended adipocytes and lipid droplets, discard the supernatant, add serum-free culture medium, pipette evenly, and centrifuge at 1000r / m for 5min; discard the supernatant, add complete medium A, pipette evenly, and set Pan blue staining, after counting with a counting board, press 1×10 5 Cells were ...

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Abstract

The invention belongs to the field of biomedicine, and in particular discloses a method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing. The method for extracting mesenchymal stem cells from trace human fatty tissues comprises the steps of: locally narcotizing in small area by injecting lidocaine, extracting trace fatty tissues of a patient by using a micro draining needle; then soaking with a phosphate buffer, centrifugally washing and obtaining fatty tissues in a sterile state; and digesting with collagenase, centrifuging with gradient and filtering and then culturing in an in-vitro culturing system. The invention effectively reduces the pain of the patient, eliminates the barrier that the thin patient can not obtain the fatty tissues, avoids using animal products in the whole cell culturing system, especially serum-free products, and meets the demands on future clinical safety application.

Description

(1) Technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for extracting mesenchymal stem cells from a small amount of human adipose tissue and cultivating them on a large scale. (2) Background technology [0002] Mesenchymal stem cells are a group of pluripotent stem cells derived from mesoderm with self-renewal and multilineage differentiation potential. Mesenchymal stem cells not only have the common characteristics of stem cells, that is, the ability of self-renewal, multidirectional differentiation and homing, but also have the ability to differentiate into various types of cells, which can be differentiated into fat, bone, cartilage, muscle, tendon, ligament, nerve , liver, myocardium, endothelial and other tissue cells still have multi-directional differentiation potential after continuous subculture and cryopreservation. Animal experiments and preliminary clinical studies have shown that mesenchymal stem cells have low im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667
Inventor 王泰华孙涛李荣荣
Owner 王泰华
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