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Separation culture method for cartilage stem cells

A technology of chondrocytes and stem cells, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of no sorting process, cell aging, limited quality, etc., and achieve high purity, accurate definition, and good expansion. empowering effect

Inactive Publication Date: 2012-04-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current treatment of osteoarthritis has a short-term symptomatic effect and uncertain long-term efficacy, and the autologous chondrocyte transplantation therapy based on the principle of tissue engineering also has the defects of insufficient source and limited quality of seed cells: the donor site of adult chondrocytes is extremely Limited; chondrocytes have limited ability to expand during in vitro culture; after passage, the cells age, "dedifferentiation" occurs, and the ability to form cartilage is lost, etc.
However, in such a separation process, there is no standard sorting process that can guarantee the sorting efficiency

Method used

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  • Separation culture method for cartilage stem cells
  • Separation culture method for cartilage stem cells
  • Separation culture method for cartilage stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 (the reagents in this example were purchased from Invitrogen Company except for special instructions):

[0032] 1) Chondrocyte culture: take 0.20g of cartilage tissue (discarded tissue, donated by Sir Run Run Shaw Hospital Affiliated to Zhejiang University School of Medicine), cut it into 1mm×1mm×1mm pieces, PBS buffer (containing 100U each of penicillin and streptomycin) / mL) after washing, single chondrocytes were isolated under sequential digestion with hyaluronidase and type II collagenase. Filter, centrifuge at 1500r / min for 5min with a centrifugal radius of 4.5cm, and wash the precipitated cells twice with PBS. Low-sugar DMEM (20% FBS) to make cell suspension, with 10 cells / cm 2 Density inoculation in 10em culture dish, medium change once every 3d, 0.25% trypsin-EDTA for subculture, in vitro monolayer culture, low sugar DMEM (10% FBS) to the third generation.

[0033] 2) In vitro monolayer culture and CD146 immunofluorescence staining.

[0034] The in...

Embodiment 2

[0042] Example 2: The three-lineage differentiation ability of the cells obtained in Example 1 was identified.

[0043] (1) Identification of skeletal differentiation:

[0044] Induction process: cartilage stem cells were injected at 100 cells / cm 2 Inoculate with a 24-well culture plate, using osteoinductive medium (solvent: H-DMEM, add: 10% FBS, 100U / ml penicillin, 100U / ml streptomycin, 50μM ascorbic acid, 10mM β-sodium glycerophosphate, 0.1uM ground dexamethasone) for 14 days.

[0045] Osteogenic differentiation identification results: positive expression of ALP alkaline phosphatase (see Figure 6 A)

[0046] (2) Identification of adipose differentiation:

[0047] Cartilage stem cells were harvested at 100 cells / cm 2 Inoculated with 24-well culture plate, using adipose induction medium (solvent: H-DMEM, added: 10% FBS, 100U / ml penicillin, 100U / ml streptomycin, 0.5mM 3-isobutyl-1-methyl ( IBMX), 10uM insulin, 0.5uM dexamethasone) were induced for 14 days.

[0048] Adip...

Embodiment 3

[0052] After culturing the cells obtained in Example 1 and the existing bone marrow-derived mesenchymal stem cells to full growth, plant 2.5×10 6 Cells (50ul, 5×10 7 / ml) in sodium alginate gel to construct tissue engineered bone.

[0053] 1) Cells were marked with Dil fluorescent dye, implanted into the vacancy of the scaffold material, the cell and material complex was wrapped with sodium alginate gel, BMP4 was added as an osteogenic induction factor, and implanted subcutaneously in nude mice.

[0054] 2) Take it out after 8 weeks, track its fluorescence, and observe the cell survival, which shows that the cell survival is good. Cartilage stem cells (CSPC) were superior to bone marrow-derived mesenchymal stem cells (BMSC) by X-ray detection of calcification. The comparison results of cartilage stem cells and MSCs in vivo osteogenic ability are shown in Figure 7 , suggesting that it has better ectopic osteogenesis ability.

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Abstract

The invention provides a separation culture method for cartilage stem cells. In the method, the cartilage stem cells are screened from cartilage tissue cells; fibroblast-like cells maintained under the culturing condition of a specific culture medium have the capabilities of differentiating bones, cartilages and fat; and compared with adult cells, the cartilage stem cells have good amplification capabilities, and can pass on for over 15 generations without changing the original characteristics after being obtained from a primary generation. In the method, the cartilage stem cells are obtained by screening and purifying under a specific culturing condition, so that the content of the cartilage stem cells is over 95 percent. A technology disclosed by the invention contributes to harvesting cartilage tissue specific stem cells, and can be applied to bone and cartilage tissue engineering.

Description

(1) Technical field [0001] The invention relates to a method for isolating and culturing cartilage stem cells. (2) Background technology [0002] Cartilage defects are a common disease, and severe articular cartilage defects can lead to osteoarthritis. In my country, the incidence rate of osteoarthritis is 3%, and there is an increasing trend year by year. However, the current treatment of osteoarthritis has a short-term symptomatic effect and uncertain long-term efficacy, and the autologous chondrocyte transplantation therapy based on the principle of tissue engineering also has the defects of insufficient source and limited quality of seed cells: the donor site of adult chondrocytes is extremely Limited; chondrocytes have limited ability to expand during in vitro culture; after passage, cells age, "dedifferentiation" occurs, and the ability to form cartilage is lost. Therefore, it is difficult to use a small amount of adult chondrocytes to expand in vitro to obtain a lar...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 姜洋子欧阳宏伟
Owner ZHEJIANG UNIV
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