Culture and three-line differentiation induction methods of adipose tissue-derived mesenchymal stem cells

An adipose stem cell and adipose tissue technology, applied in the field of cell biology, can solve problems such as time-consuming, unfavorable clinical application, and order of magnitude instability of adipose stem cells, and achieve the effects of promoting cell differentiation, improving differentiation efficiency, and shortening differentiation time.

Inactive Publication Date: 2020-02-28
山东省齐鲁细胞治疗工程技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Preliminary studies have found that traditional experimental methods to obtain adipose-derived stem cells are unstable in magnitude and time-consuming, which is not conducive to clinical application

Method used

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  • Culture and three-line differentiation induction methods of adipose tissue-derived mesenchymal stem cells
  • Culture and three-line differentiation induction methods of adipose tissue-derived mesenchymal stem cells
  • Culture and three-line differentiation induction methods of adipose tissue-derived mesenchymal stem cells

Examples

Experimental program
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Embodiment 1

[0031] Example 1. Primary and subculture of adipose stem cells

[0032] (1) Solid fat Remove the fascia, skin, blood vessels and other tissues above the fat, clean the fat block, and cut it into pieces with scissors.

[0033] (2) Afterwards, transfer the shredded fat or liquid fat from liposuction to a 250mL centrifuge tube, add an appropriate amount of saline to it, blow and blow it 20 times with a straw, then let it stand for 2 minutes, and suck out the liquid in the lower layer of the tissue with a straw. Repeat 3 times until the sucked liquid is transparent without blood cells.

[0034](3) Add 0.1% collagenase type I / PBS digestion solution with a volume ratio of 1:1 to the centrifuge tube containing adipose tissue; seal the centrifuge tube and place it in a constant temperature shaker at 37°C, at 90rpm Digest with shaking for 60min.

[0035] (4) Use a 10ml pipette to suck out the lower liquid in the centrifuge tube, filter it through a 100µm filter into a new 50ml centri...

Embodiment 2

[0044] Example 2. Three-line differentiation induction and identification of adipose stem cells

[0045] (1) Adjust the cell density of the subcultured adipose-derived mesenchymal stem cells to 1×10 5 / mL, 1×10 7 / mL.

[0046] (2) Take 2mL with a density of 1×10 5 Adipose-derived mesenchymal stem cells per mL were inoculated into 6-well plates, and after overnight culture, the medium was replaced with adipogenic induction medium (Dayou MSCBM + 5% ultroGRO + 10mg / mL insulin + 1µM dexamethasone + 100µM indole domethacin + 500µM IBMX), osteogenic induction medium (Dayou MSCBM + 5% ultroGRO + 0.1µM dexamethasone + 50µM ascorbic acid + 10 mM β-glycerophosphate).

[0047] (3) Take 10µL density as 1×10 7 / mL adipose-derived mesenchymal stem cells were inoculated in a 24-well plate, cultured statically for 2 h, and 1 mL of chondrogenic induction medium (Dayou MSCBM + 110 µg / mL sodium pyruvate + 10 ng / mL TGF-β3 + 0.1 µM dextrose metasone + 50µg / mL L-ascorbic acid-2-phosphate + 40µ...

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Abstract

The invention belongs to the technical field of cell biology and particularly relates to culture and three-line differentiation induction methods of adipose tissue-derived mesenchymal stem cells. Theculture method specifically includes: placing adipose tissue into a centrifugal tube, adding normal saline, pipetting with a pipet, standing, sucking out liquid on the lower layer of the tissue, adding collagenase type I/PBS digestive juice with the concentration being 0.1% to perform digestion, and placing the culture bottle into a culture tank of 37 DEG C and with 5% CO2; taking out cells when the cells grow to 80% and merge, adding TrypLE digestive juice, terminating the digestion when the cells start retracting and rounding, and performing subculture and multiplication culture. The differentiation induction method has the advantages that differentiation time is shortened, the method needs 10 days to differentiate the cells into adipose and bones and needs 14 days to differentiate the cells into cartilage, and differentiation efficiency is increased greatly as compared a common induction method which needs 14 days to differentiate cells into adipose and needs 21 days to differentiate the cells into bones and cartilage.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for culturing and three-line differentiation induction of adipose tissue-derived mesenchymal stem cells. Background technique [0002] Adipose-derived stem cells (hADSc) are a type of cells derived from human adipose tissue that can proliferate in vitro and have multi-lineage differentiation potential. These cells have similar expression and multi-lineage differentiation potential to bone marrow mesenchymal stem cells (BMSCs) , providing lifelong self-renewal of adipose tissue and maintaining the balance of fat metabolism. Compared with stem cells from other sources, hADSc has unique advantages such as adipose stem cells have sufficient sources, are convenient to obtain materials, cause less trauma to the human body, can be obtained repeatedly, are easy to separate, have a large amount of cells obtained, and can be used in adipose tissue. The number of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2509/00C12N2501/33C12N2500/38C12N2500/32
Inventor 谭毅张剑慧马贺然张慧慧韩镇
Owner 山东省齐鲁细胞治疗工程技术有限公司
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