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Culture medium and method for inducing adipose-derived stem cells to differentiate into tendon cells

An adipose stem cell and culture medium technology, applied in the field of cell culture, can solve the problems of weak ability of adipose stem cells to differentiate into tenocytes, complex plasma composition, and difficulty in controlling the differentiation of directional tenocytes, so as to reduce risks and immunogenicity, promote Cell proliferation, reducing effects of complex ingredients

Inactive Publication Date: 2016-07-20
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adding plasma or plasma clot to tenocytes cultured in vitro can stimulate tenocytes to proliferate and secrete collagen; however, the composition of plasma is complex, and it is difficult to control and induce directional tenocyte differentiation by adding platelet-rich plasma to adipose stem cells cultured in vitro
Moreover, the ability of adipose-derived stem cells to differentiate into tenocytes in the existing induction culture system is weak

Method used

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  • Culture medium and method for inducing adipose-derived stem cells to differentiate into tendon cells
  • Culture medium and method for inducing adipose-derived stem cells to differentiate into tendon cells
  • Culture medium and method for inducing adipose-derived stem cells to differentiate into tendon cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Primary isolation of adipose-derived stem cells

[0045] After the adipose tissue and swelling fluid are separated naturally, absorb and discard the lower layer of liquid, add an equal volume of normal saline to wash the adipose tissue twice, and aspirate and discard the lower solution; divide the adipose tissue into 50mL centrifuge tubes, 20mL in each tube, Add an equal volume of 0.5% type Ⅰ collagenase (final concentration is 0.25%), mix well, seal, transfer to a constant temperature air shaker, digest at 37°C, 200R for 50min; centrifuge at 1500rpm for 10min after digestion; discard For the centrifuged fat and fat oil, add 40mL normal saline to each tube to resuspend the cells, centrifuge at 11500rpm for 5min, discard the supernatant, resuspend the cells with ordinary medium (DMEM / F12+10%FBS), and inoculate them into a culture dish , when the confluence of the cells reaches 80%-90%, subculture is carried out. Morphological pictures of adipose stem cells see figu...

Embodiment 2

[0066] 1. Experimental grouping

[0067] See Table 3 for test groupings:

[0068] Table 3 Test grouping

[0069] group

Medium composition

Control group 1

DMEM / F12+10%FBS

Control group 2

Commercial tendon induction medium group

Experimental group 1

DMEM / F12+30ng / mL GDF-7

Experimental group 2

DMEM / F12+0.5mg / mL Rehmannia glutinosa water extract

Experimental group 3

DMEM / F12+30ng / mL GDF-7+0.5mg / mL Rehmannia glutinosa water extract

[0070] 2. GDF-7 and rehmannia glutinosa water extract jointly induce the differentiation and proliferation of adipose stem cells into tenocytes

[0071] The ADSCs of passage 4 were taken, and after aspirating the medium, they were digested by adding 0.25% trypsin + 0.02% EDTA digestion solution, and then the digestion was terminated with an appropriate amount of serum. Centrifuge at 1200rpm for 5min, resuspend the adipose stem cell in complete medium, and adjust the density to...

Embodiment 3

[0083] 1. Experimental grouping

[0084] See Table 5 for test groupings:

[0085] Table 5 Test grouping

[0086] group

Medium composition

Control group 1

DMEM / F12+10%FBS

Control group 2

Commercial tendon induction medium group

Experimental group 1

DMEM / F12+40ng / mL GDF-7

Experimental group 2

DMEM / F12+1mg / mL Rehmannia glutinosa water extract

Experimental group 3

DMEM / F12+40ng / mL GDF-7+1mg / mL Rehmannia glutinosa water extract

[0087] 2. GDF-7 and rehmannia glutinosa water extract jointly induce the differentiation and proliferation of adipose stem cells into tenocytes

[0088] The ADSCs of passage 5 were taken, and after aspirating the medium, they were digested by adding 0.25% trypsin + 0.02% EDTA digestion solution, and then the digestion was terminated with an appropriate amount of serum. Centrifuge at 1200rpm for 5min, resuspend the adipose stem cell in complete medium, and adjust the density to 1×1...

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Abstract

The invention relates to the technical field of cell culture, and in particular relates to a culture medium and a method for inducing adipose-derived stem cells to differentiate into tendon cells. The culture medium comprises GDF-7, prepared rehmannia root aqueous extract and a serum-free culture medium. The culture medium provided by the invention can remarkably improve the ability of differentiating the adipose-derived stem cells into tendon cells; the prepared rehmannia root aqueous extract is utilized to culture the adipose-derived stem cells, so that the phenotype and the characteristics of the adipose-derived stem cells further can be kept while cell proliferation is promoted; a growth differentiating factor GDF-7 is used as an inductor, so that the effect is single, and the complex components of blood plasma are greatly reduced, and therefore, the adipose-derived stem cells are differentiated into the tendon cells in a directional manner, and the risk and the immunogenicity of animal serum application can be reduced.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a medium for inducing fat stem cells to differentiate into tenocytes and a method thereof. Background technique [0002] Muscle and tendon injury (muscle and tendon, injury of) is a common motor system injury. The complete or partial rupture of the muscle or the transitional part of the muscle belly caused by direct violence is called muscle rupture. Sudden and violent muscle contraction caused by external force can cause complete or partial tearing of the tendon starting and ending points, which is called tendon rupture. If the tendon is repeatedly subjected to minor trauma for a long time, or the tendon itself has chronic wear and tear, resulting in degeneration and thinning of the tendon fibers, a slight sprain in the future can cause tendon rupture, which is called spontaneous tendon rupture. Chronic muscle strain is the disorder of muscle balance caused by excessive m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/066C12N2500/76C12N2500/90C12N2501/19C12N2506/1384
Inventor 葛啸虎陈海佳王一飞马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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