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Construction method and application of tissue engineering nerve seed cell

A technology of seed cells and tissue engineering, which is applied in the construction of tissue engineering nerve seed cells and in the field of tissue engineering nerve construction, can solve the problems of in vitro proliferation and culture, limited sources of Schwann cells, etc., and achieve strong in vitro expansion ability and wide application Prospect and market value, effect of reducing usage

Pending Publication Date: 2019-07-02
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many kinds of nerve guides, mainly including various biological materials; the cells required for tissue engineering nerve construction are mainly Schwann cells, but the defect is that the source of Schwann cells is limited, and the source is mainly to destroy other parts of the tissue nerve. , such as obtained from normal nerves from other parts, and in vitro proliferation and culture is extremely difficult, and the number of its in vitro expansion has obvious limitations. Although researchers in this technical field have carried out research on various types of stem cells, such as using umbilical cord Mesenchymal stem cells, adipose stem cells, bone marrow stem cells, etc. are used as seed cells to construct tissue engineered nerve grafts, but problems such as tumorigenicity and ethics often limit their application

Method used

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  • Construction method and application of tissue engineering nerve seed cell
  • Construction method and application of tissue engineering nerve seed cell
  • Construction method and application of tissue engineering nerve seed cell

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Construction of neural seed cells for tissue engineering

[0031] In this example, the test animals used are female or male mice born 5-7 days old, wild-type c57BL / 6J (B6 mice; Shanghai Shrek Animal Co., Ltd., Shanghai, China). After routine treatment, 75 % Alcohol soak for 15min;

[0032] Ultra-clean Taichung (Suzhou Antai Air Technology Co., Ltd., Suzhou, China), remove the bilateral sciatic nerves, and routinely treat them with 0.2% Dispase II (neutral protease, grade II) (Godo Shusei, Tokyo, Jpan) and 0.2% collagenase Digested in NB4 (Serva, Heidelberg, Germany) mixed digestion solution in equal proportions, placed in a 37 degree shaker for 50 minutes, centrifuged in a 1500-r, 5 minutes, 37 degree centrifuge (Thermo, USA), and resuspended , And inoculated into a petri dish, placed at 37℃, 5% CO 2 Culture in an incubator;

[0033] After three days of primary culture, high-purity Schwann cells were purified by patent (application number 200910195924.0), that is, ...

Embodiment 2

[0035] The neural fibroblasts prepared in Example 2 are used in the construction of nerve regeneration

[0036] The prepared fibroblasts and Schwann cells were mixed 1:1, centrifuged, and the matrigel (BD BioSCience, Franklin Lakes, NJ, USA) was placed in a 4℃ environment overnight. On ice, the cell suspension and matrigel1: 1 Mix well; use a disposable sterile insulin syringe (BD, USA) to suck the mixed solution on ice for later use. In this example, the Schwann cells used for replantation are taken from female or male B6 children aged 5-7 days. Mouse; Sciatic nerve fibroblasts are derived from male or female C57BL / 6-Tg(CAG-EGFP)C14-Y01-FM131Osb mice (EGFP mice; Nanjing Institute of Model Animals, Nanjing, China) 5-7 days old;

[0037] After the experimental mice were induced to anesthetize, the skin of each mouse’s right hind limb buttocks was conventionally treated, and then the sciatic nerve was freed; a 7mm-long PUR catheter (internal diameter 0.9mm, external diameter 10mm) (B...

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Abstract

The invention belongs to the biology field / tissue engineering and relates to a construction method and an application of a tissue engineering nerve seed cell. Schwann cells and fibroblasts are separated and purified by Dispase II, and the constructed seed cell is applied to construction of tissue engineering nerves to be implanted in a nerve conduit to be composited into a nerve transplant and further used for repairing nerve deficiency. The invention provides seed cell acquisition method which is simpler and good in effects, peripheral nerve fibroblasts are easy to obtain, have high in-vitroamplification capacity and have good nerve generation promotion effect after being combined with Schwann cells and implanted, and the amount of the Schwann cells is obviously reduced; regenerated nerves after repair have thicker diameters and no neuroma is formed. The method is particularly applicable to seed cells added to tissue engineering nerve biomaterials and has application prospects and market values.

Description

Technical field [0001] The invention belongs to the technical field of biology / tissue engineering, and relates to a method for constructing tissue engineering nerve seed cells and uses thereof. The tissue engineering nerve seed cells constructed by the invention can be further used to construct tissue engineering nerves. Background technique [0002] The industry knows that peripheral nerve defect is a very difficult problem in clinical practice. Practice has shown that in peripheral nerve defect diseases, even if some short-distance defects can be recanalized, the nerve structure function of the regenerated part is extremely poor. At present, autologous nerve transplantation is regarded as the "gold standard" for the treatment of nerve defects, but its clinical application is severely restricted due to the insufficient source of "nerve supply materials" and the functional defect of the donor site. In recent years, tissue-engineered nerve grafts have gradually become the main res...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/079A61L27/38
CPCC12N5/0656C12N5/0622A61L27/383A61L27/3878A61L2430/32C12N2509/00
Inventor 王洋金羽青沈华沈尊理
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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