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Purifying method of adipose-derived stem cells and application of stem cells on osteogenic induction and chondrogenesis

A technology of adipose stem cells and purification methods, applied in the field of stem cell purification, can solve the problems of weak differentiation ability of seed cells, unsatisfactory repair effect, and different content of stem cells, so as to improve the expression of stem cell surface antigens, improve the use efficiency and repair effect , The effect of high stem cell differentiation potential

Active Publication Date: 2016-10-26
ZHEJIANG UNIV
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  • Claims
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Problems solved by technology

However, primary cultured unpurified ADSCs have problems such as weak differentiation ability and unsatisfactory repair effect when used as seed cells for tissue engineering.
At present, most of them are directly used in primary culture after isolation of cells, without purification. The defect is the same method. Different batches of culture, due to the different content of vascular matrix components, the content of stem cells in the cultured ADSCs is different. Differentiation is the purpose. The efficiency of cells is different, and the effect of tissue engineering repair is uneven
At present, the methods used for purification mainly include microporous filtration membrane method (polyurethane (PU) membrane, PLGA / silk membrane), magnetic bead enrichment method and flow sorting method. Most of the methods used to purify adipocytes are microporous filtration membranes method, the disadvantage is that the pore size of the membrane is difficult to grasp

Method used

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  • Purifying method of adipose-derived stem cells and application of stem cells on osteogenic induction and chondrogenesis
  • Purifying method of adipose-derived stem cells and application of stem cells on osteogenic induction and chondrogenesis
  • Purifying method of adipose-derived stem cells and application of stem cells on osteogenic induction and chondrogenesis

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Experimental program
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Embodiment 1

[0025] (1) ADSCs primary cell culture: Take one New Zealand white rabbit, male, for 3-4 weeks, kill it by intravenous injection of air needle, take the fat block (with fascia) in the groin and place it in the culture medium containing 100U / ml penicillin-streptomycin In double antibody PBS (pH7.2-7.4), remove fascia and blood vessels, add collagenase II, cut into pieces, digest at 37°C for 2 hours, centrifuge, and culture the pellet in low-glucose medium containing 10% serum (DMEM-L , GIBCO) resuspended at 37°C, 5% CO 2 Incubator culture. The medium was changed every 3 days, and the cells were passaged when the cells grew to 80%, and the cells before P5 were used for sorting to obtain the culture medium of primary adipose stromal cells.

[0026] (2) Sorting and purification of CD90+ cell population: Discard the culture medium of primary adipose stromal cells, wash with PBS once, then add PBS, then scrape the cells with a cell scraper, collect them in a 15ml centrifuge tube, ce...

Embodiment 2

[0029] Detection of stem cell surface markers: In order to compare the content of stem cell surface marker-positive cells in the cell population before and after sorting, cells (primary adipose stromal cells cultured in step 1 and CD90+ cells screened in step 2) were obtained with a cell scraper, centrifuged, and washed with CD14, CD105, CD34 labeled primary adipose stromal cells and CD90+ cells, incubated at 4°C for 1-2 hours, washed with PBS, and incubated with fluorescent secondary antibody (Molecular probes Alexa Fluor 488 goat anti mouse IgG(H+L)) for 30 minutes, Washed with PBS, and then detected by flow cytometer.

[0030] The expression of stem cell surface marker antibodies also represents one of the characteristics of stem cells. The expression of stem cell surface marker antibodies CD14 and CD105 also changed greatly before and after sorting, as shown in Figure 4 shown. Before sorting, the expression levels of CD14 and CD105 were 20.7% and 22.2%; after sorting, th...

Embodiment 3

[0032] Detection of three-line differentiation potential: In order to compare the three-line differentiation ability before and after cell sorting, the primary adipose stromal cells prepared in step 1 and the CD90+ cells obtained in step 2 were mixed at the same cell concentration of 5×10 5 Cells / ml were planted in a 24-well plate. (1) Osteocyte induction: When the cells grew to 80%, they were digested with 0.25% trypsin at 37°C for 3 minutes. The supernatant was discarded, and the cells were resuspended and placed in DMEM-L medium containing 10% serum by volume, at a cell concentration of 5×10 5 Cells / ml were seeded in 24-well plates, and induced with bone induction medium (purchased from GIBCO) 24 hours after attachment, and the medium was changed every 3 days for a total of 2 weeks of induction, and detected with Alizarin Red. The composition of osteoinductive fluid: dexamethasone (10-7M), β-glycerol phosphate (10mM), ascorbic acid (50ug / ml).

[0033] (2) Chondrocyte induc...

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Abstract

The invention provides a purifying method of adipose-derived stem cells. The method comprises the following steps: primary adipose-derived stem cell culture and CD90+ cell population purification. The invention further provides application of the purified adipose-derived stem cells in the aspects of osteogenic induction and chondrogenesis. Obtained CD90+ cell population in ADSCs has high stem cell differentiative potential, using efficiency and repairing effect of ADSCs in tissue engineering can be improved, and under the same induction condition, osteogenesis and chondrogenesis are improved by 3-5 times and 5-7 times respectively.

Description

technical field [0001] The invention relates to a method for purifying stem cells, in particular to a method for purifying adipose stem cells derived from rabbits. Background technique [0002] Adipose tissue is the richest and most accessible source of MSCs. Adipose stem cells (ADSCs), or mscs derived from adipose tissue, are an important cell source for regenerative applications. ADSCs are isolated from the stromal vascular fraction (SVF) of adipose tissue, which contains various types of cells, such as smooth muscle cells, endothelial cells, fibroblasts, etc. Recent studies have shown that ADSCs have the ability to differentiate into multiple cell types (adipocytes, osteoblasts, chondrocytes), stimulate angiogenesis, and reduce inflammation and apoptosis. However, primary cultured unpurified ADSCs used as seed cells for tissue engineering have problems such as weak differentiation ability and unsatisfactory repair effect. At present, most of them are directly used in p...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/077
CPCC12N5/0654C12N5/0655C12N5/0667C12N2500/35C12N2500/38C12N2501/15C12N2501/39C12N2501/599C12N2506/1384
Inventor 宋兴辉沈婷洪朝阳李艳伟王佳佳王琳琳
Owner ZHEJIANG UNIV
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