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Method for repairing nerve defects in tissue engineering

An engineering and neurological technology, applied in the field of cell biology, can solve problems such as limiting the clinical application of stem cells, and achieve the effect of avoiding tumorigenicity, broad clinical application prospects and clinical value, and maintaining activity.

Inactive Publication Date: 2015-11-04
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the tumorigenicity of stem cells is currently a thorny defect that severely limits the application of stem cells in clinical practice

Method used

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  • Method for repairing nerve defects in tissue engineering
  • Method for repairing nerve defects in tissue engineering
  • Method for repairing nerve defects in tissue engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 : Isolation, purification, and enrichment of fat-derived Schwann cells

[0047] Newborn 7-day-old C57 mice were put into 75% alcohol and soaked for 5-10 minutes after routine treatment.

[0048] 1. Under sterile conditions, take the adipose tissue of Linti mice, cut it into pieces, and use 0.1-0.2% compound collagenase NB4 and 0.05-0.1% Dispase mixed enzyme solution with a mass volume ratio of 5-20 times the tissue volume Digest the pieces for 60 minutes;

[0049] 2. Centrifuge the dissolved tissue in step 1 at 1500r for 5 minutes, discard the supernatant, and obtain cell pellets;

[0050] 3. Resuspend the cells obtained in step 2 with Schwann cell culture medium (DMEM medium containing 2μM forskolin, 10ng / ml heregulin-β-1, 10ng / ml BFGF), count, and press 1x10 4 -5x10 4 piece / cm 2 Inoculated on Petri dishes. Set at 37°C, 5% CO 2 incubator cultivation;

[0051] 4. After the cells were cultured for 48-72 hours, the cells were purified with 0.2% DIspase...

Embodiment 2

[0054] Example 2: Adipose-derived Schwann cells repairing sciatic nerve defect in mice

[0055] a) After the cells were cultured for 48-72 hours in step 5 in Example 1, the culture medium was sucked off, the Schwann cells were digested with 0.25% trypsin for 1 minute, DMEM neutralized the trypsin, and the cell suspension was collected;

[0056] b) centrifuge the cell suspension obtained in step a at 100 rpm for 5 minutes, and discard the supernatant;

[0057] c) Resuspend the cells obtained in step b in a mixture of Schwann cell culture medium (SCCM) and Matrigel glue, and make a mixture of SCCM and Matrigel glue at a ratio of 1:1;

[0058] d) Pipette the suspension obtained in step c repeatedly for 3-5 minutes to fully mix the cells. Both steps c and d need to be operated on ice at 4°C;

[0059] e) The mice were anesthetized with 5% chloral hydrate (6ml / kg) intraperitoneally, and the right sciatic nerve was exposed;

[0060] f) Use silicone tubes to make artificial nerve...

Embodiment 3

[0064] Example 3 Adipose-derived Schwann cells and adipose-derived stem cells repair sciatic nerve defects respectively

[0065] 1) Acquisition of adipose-derived Schwann cells and adipose stem cells

[0066] Acquisition of Schwann cells: as described in Example 1.

[0067] Obtaining fat stem cells:

[0068] 1. Mice were routinely treated, soaked in 75% ethanol for 10 minutes, and adipose tissue was taken under aseptic conditions, placed in a disposable centrifuge tube, and cut into pieces to a size of 1 mm×1 mm×1 mm;

[0069] 2. Add 10-15ml 0.2% NB4 and 5-10 ml 0.2% Dispase, shake and digest for about 60 minutes;

[0070] 3. Centrifuge the dissolved tissue in step 2 at 1500rpm for 5 minutes, discard the supernatant, and obtain cell pellets;

[0071] 4. Add 8ml of DMEM culture medium to resuspend the cells, press about 1×10 4 / cm 2 Density Inoculate the cells into culture flasks and place them in a 37°C, 5% CO2 incubator for culture;

[0072] 5. When the primary cells ...

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Abstract

The invention belongs to the field of cytobiology, relates to a method for repairing nerve defects in tissue engineering, and in particular relates to a method for repairing artificial peripheral nerve defects by employing schwann cells with a fat source and an application of the method. According to the method, the damaged nerves are repaired by the schwann cells obtained from waste adipose tissues and the nerve defects are repaired by a lot of schwann cells with the fat sources and a few of adipose-derived stem cells in common. An animal experiment proves that regeneration of the damaged nerves can be effectively promoted. The invention provides a novel method for repairing the nerve defects in tissue engineering; a relatively high-quality treatment reference method is provided for repairing of the damaged nerves; and the method provided by the invention has a clinical application prospect and clinical value.

Description

technical field [0001] The invention belongs to the field of cell biology and relates to a method for repairing nerve defects by tissue engineering, in particular to a method for repairing artificial peripheral nerve defects by using adipose-derived Schwann cells and an application thereof. Background technique [0002] The prior art discloses that Schwann cells are glial cells of the peripheral nervous system and play a very important role in nerve injury repair. When nerves are damaged, Schwann cells can secrete many extracellular matrix components and nerve growth factors to promote nerve regeneration. When axonal loss and demyelination occur with Wallerian degeneration, Schwann cells proliferate and dedifferentiate, phagocytose myelin debris and form longitudinally aligned columnar cell processes or characteristic Bungner ribbons within the endoneurial canal to guide axons regeneration. [0003] As the seed cells of artificial nerves, Schwann cells are generally divide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/38A61L27/18
Inventor 陈露露沈尊理秦金保
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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