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Human fat mesenchymal stem cell extract and lyophilized powder and application thereof

A technology of mesenchymal stem cells and extracts, which is applied in the field of human adipose-derived mesenchymal stem cells in biomedicine or medical cosmetology, can solve the problems of expensive cytokines and high cost of stem cell application, and achieve simple preparation methods and easy preservation and transport effects

Active Publication Date: 2014-05-14
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, although there have been studies using specific induction conditions to induce stem cells to secrete supernatants in vitro for medical treatment, cosmetics, etc., this conditional medium is to use specific cytokines to induce stem cells to secrete some specific factors to achieve curative effect. The factor itself is expensive, making the application cost of stem cells abnormally high

Method used

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  • Human fat mesenchymal stem cell extract and lyophilized powder and application thereof
  • Human fat mesenchymal stem cell extract and lyophilized powder and application thereof
  • Human fat mesenchymal stem cell extract and lyophilized powder and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Primary isolation, culture and identification of human adipose-derived mesenchymal stem cells

[0054] 1. Primary isolation and culture of human adipose-derived mesenchymal stem cells

[0055] Centrifuge 800g of human abdominal subcutaneous fat tissue extract obtained through liposuction (endocrine diseases and infectious diseases have been ruled out before surgery), rinse with PBS buffer, soak, and then wash 3 times at 800g×4min; remove visible Blood vessels and hoof tissue are fully shredded with ophthalmic scissors and put into a 50ml centrifuge tube. Add 2 times the volume of 0.2% collagenase, 1 times the volume of 1% BSA and double antibody, mix well, seal, and put it in a shaker Digest at 37°C for 45 minutes until it becomes a paste;

[0056] Add an equal volume of low-sugar DMEM containing 10% fetal bovine serum to stop digestion, centrifuge at 1800r / min for 10min, and divide into 3 layers after centrifugation, the upper layer is oil and undigest...

Embodiment 2

[0060] Example 2 Human adipose-derived mesenchymal stem cell extract

[0061] The human adipose-derived mesenchymal stem cell extract of this embodiment is prepared by the following method:

[0062] step 1.

[0063] The identified subcultured human adipose-derived mesenchymal stem cells of the P3-P30 generation were used in the cell growth medium (the cell growth medium was added to the commercially available basic DMEM / F12 culture medium with a final concentration of 10% fetal bovine serum and penicillin with a final concentration of 200 U / ml and streptomycin with a final concentration of 250 U / ml) when the cells were 80% confluent, the culture medium was discarded, and the phosphate buffer (the phosphate The buffer solution is PBS solution with a pH value of 7.0-7.2), and the cells were washed three times, and then commercially available serum-free medium was added to continue culturing for 72 hours, and the serum-free cells obtained from P3-P30 generations of human adipo...

Embodiment 3

[0068] Example 3 Human adipose-derived mesenchymal stem cell extract freeze-dried powder

[0069] The BCA method was used to measure the protein concentration of the human adipose-derived mesenchymal stem cell extract solution prepared in Example 2, and the absorbance of A570 was measured by a microplate reader to obtain the protein concentration of the human adipose-derived mesenchymal stem cell extract solution prepared in Example 2, Then according to the protein concentration, add the excipient mannitol (mannitol accounts for 5% of the total weight of the human adipose-derived mesenchymal stem cell extract) and ultrapure water at 50-60°C according to the final mass of the lyophilized solution, and mix well. and adjusted to a final protein concentration of 50.0±0.5 μg / ml, sterilized by filtration, and then freeze-dried at a freeze-drying temperature of -20 to -35°C, a vacuum of 50 to 200Pa, and a freeze-drying time of 48 hours to obtain The required freeze-dried powder of ...

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Abstract

The invention discloses a human fat mesenchymal stem cell extract and lyophilized powder and applications thereof. The human fat mesenchymal stem cell extract is prepared by the steps: collecting subcultured P3-P30 generations of human fat mesenchymal stem cells, culturing with cell growth liquid until 80% of cells are fused, removing culture liquid, washing the cells with a phosphate buffer solution for three times, adding into a serum-free culture solution, continuously culturing for 72 hours, collecting the serum-free culture solution, digesting uncollected cells with a digestion solution, crushing the cells with an ultrasonic crushing device, centrifuging, and then collecting supernatant; and mixing the collected serum-free culture solution and the collected supernatant, filtering, collecting filtrate, concentrating, and sterilizing. The human fat mesenchymal stem cell extract and the lyophilized powder thereof have multiple biological activities, can be used for preparing wound healing or cell repair medicaments and other biopharmaceutical products, and can also be used for preparing whitening products or skin anti-wrinkle products and other fine chemical products.

Description

technical field [0001] The present invention relates to the technique of using stem cells in biomedicine or medical cosmetology, in particular to the use of human adipose mesenchymal stem cells in biomedicine or medical cosmetology. Background technique [0002] Adipose tissue has a wide range of sources, is easy to obtain, has high cell separation efficiency, causes less trauma to patients, and does not involve ethical issues, so it has received increasing attention. [0003] Human adipose-derived mesenchymal stem cells are a type of pluripotent stem cells in adipose tissue, which can secrete a variety of biologically active substances, including proteins, peptides and cytokines, such as stem cell growth factor (SCGF), vascular endothelial growth factor (VEGF ), insulin-like growth factor (IGF), hepatocyte growth factor (HGF) and transforming growth factor, etc. These cell growth factors can control or maintain injured tissue cells and guide their own repair. In addition, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/34A61K8/98A61P17/02A61Q19/08A61Q19/02A61K35/35
Inventor 陈海佳王一飞舒辉萍刘秋英马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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