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Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells

A technology of pluripotent stem cells and chondrocytes, which is applied in the field of differentiation of induced pluripotent stem cells into chondrocytes, can solve the problems of high price, complicated induction system, and poor repeatability of the operating system, so as to reduce the probability of pollution, advanced and simple technology, Effect of reducing cell damage

Inactive Publication Date: 2013-02-13
川北医学院第二临床医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] US patent US2011229440-A1 reported that iPS stem cells have the potential to differentiate into chondrocytes, but it uses bone morphogenetic proteins (BMPs) as an inducer, which is expensive, and the operating system is not repeatable, and the induction system is too complicated , therefore, new approaches need to be developed to differentiate induced pluripotent stem cells (iPSCs) into fully functional chondrocytes

Method used

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  • Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells
  • Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells

Examples

Experimental program
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Effect test

Embodiment 1

[0016] (1) Preparation of primary embryonic mouse fibroblasts

[0017] Under sterile conditions, take the fetuses of BABL / C mice at 13-14 days of pregnancy, remove the embryo head and viscera, cut the fetuses into paste with ophthalmic scissors, put them in 0.25% trypsin, and put them in a water bath at 37°C Digest for 15 minutes, pour the upper cell suspension into a 10ml centrifuge tube, and centrifuge at 1000r / min for 5 minutes to collect the cells. The obtained cells are made into a cell suspension with 10% fetal bovine serum DMEM medium, and 1×10 6 Inoculated into 100mm Petri dishes at a density of 37°C, 5% CO 2 Cultured in an incubator, about 2-3 days after the cells are full, the cells can be collected and frozen in liquid nitrogen. The high-sugar DEME medium is a disclosed medium composition, and commercially available products, such as Gibco or Sigma, can be used.

[0018] (2) Preparation of feeder cells

[0019] Resuscitate the embryonic mouse fibroblasts prepared...

Embodiment 2

[0034] (1) Preparation of primary embryonic mouse fibroblasts

[0035] Under sterile conditions, take the fetuses of BABL / C mice at 13-14 days of pregnancy, remove the embryo head and viscera, cut the fetuses into paste with ophthalmic scissors, put them in 0.25% trypsin, and put them in a water bath at 37°C Digest for 15 minutes, pour the upper cell suspension into a 10ml centrifuge tube, and centrifuge at 1000r / min for 5 minutes to collect the cells. The obtained cells are made into a cell suspension with 10% fetal bovine serum DMEM medium, and 1×10 6 Inoculated into 100mm Petri dishes at a density of 37°C, 5% CO 2 Cultured in an incubator, about 2-3 days after the cells are full, the cells can be collected and frozen in liquid nitrogen. The high-sugar DEME medium is a disclosed medium composition, and commercially available products, such as Gibco or Sigma, can be used.

[0036] (2) Preparation of feeder cells

[0037] Resuscitate the embryonic mouse fibroblasts prepared...

Embodiment 3

[0046] (1) Preparation of primary embryonic mouse fibroblasts

[0047] Under sterile conditions, take the fetuses of BABL / C mice at 13-14 days of pregnancy, remove the embryo head and viscera, cut the fetuses into paste with ophthalmic scissors, put them in 0.25% trypsin, and put them in a water bath at 37°C Digest for 15 minutes, pour the upper cell suspension into a 10ml centrifuge tube, and centrifuge at 1000r / min for 5 minutes to collect the cells. The obtained cells are made into a cell suspension with 10% fetal bovine serum DMEM medium, and 1×10 6 Inoculated into 100mm Petri dishes at a density of 37°C, 5% CO 2 Cultured in an incubator, about 2-3 days after the cells are full, the cells can be collected and frozen in liquid nitrogen. The high-sugar DEME medium is a disclosed medium composition, and commercially available products, such as Gibco or Sigma, can be used.

[0048] (2) Preparation of feeder cells

[0049] Resuscitate the embryonic mouse fibroblasts prepared...

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Abstract

The invention discloses a method for differentiating induced pluripotent stem (iPS) cells into cartilage cells. The method is characterized in that the iPS cells are subjected to induction culture by use of a conditioned medium in a cell micell mode, wherein in the medium, the high-glucose DMEM (Dulbecco Modified Eagle Medium) contains 5-10% of fetal bovine serum, 6-6.5mug / ml insulin, 10-20ng / ml transforming growth factor beta1, 95-110mumol / ml dexamethasone, 37-38mg / ml ascorbic acid, 0.8-1.2mumol / ml sodium pyruvate and 6-6.5mug / ml transferrin. The method disclosed by the invention is advanced in technology, simple in operation and strong in repeatability, and provides a chance for implementing the cell replacement therapy and finally realizing regeneration and replacement of the tissue and organ; and a cell platform for large-scale production is expected, and then a brand new research prospect is developed for the fields of development biology, medicine and genetics.

Description

technical field [0001] The invention belongs to the field of cell tissue engineering, in particular to a method for inducing pluripotent stem cells to differentiate into chondrocytes. Background technique [0002] Articular cartilage damage and degenerative diseases caused by trauma and conditions such as osteoarthritis remain one of the major health threats worldwide. Despite the rapid progress in the intervention of surgical and non-surgical measures, the repair of cartilage damage is still a very difficult problem. In recent years, the emergence of tissue engineering technology has brought dawn to the repair of cartilage defects, and seed cells are one of the important factors. At present, both embryonic stem cells and mesenchymal stem cells have been used in cartilage tissue engineering research, but embryonic stem cells have limited sources, face ethical justice issues, and have strong immunogenicity, while mesenchymal stem cells have Insufficient and limited differen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
Inventor 冯刚刘康陈竹
Owner 川北医学院第二临床医学院
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