Method for in-vitro construction of tissue engineered nerves

A tissue engineering and neural technology, applied in the field of biomedical engineering, can solve different problems and achieve the effect of large surface area

Inactive Publication Date: 2013-08-07
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the mechanism of microgravity has not yet been elucidated, and different types of cells may respond quite differently to microgravity, and there is no literature on the attachment of different types of cells to complex structures under mi

Method used

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  • Method for in-vitro construction of tissue engineered nerves
  • Method for in-vitro construction of tissue engineered nerves
  • Method for in-vitro construction of tissue engineered nerves

Examples

Experimental program
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Example Embodiment

Example 1. Isolation, culture, purification and identification of precursor cells derived from rat skin

Take out the SPF SD rats 1-3 days after birth. After disinfection with 75% alcohol, they are decapitated and put to death under aseptic conditions. The back skin tissue is cut, and the subcutaneous tissue is removed mechanically until the skin is more transparent. Rinse with PBS 3 times. Cut skin tissue to 1mm 2 Digest the small tissue pieces with 1mg / ml type XI collagenase at 37℃, pipette repeatedly every 15min, digest for 30-60min, add 20ml DMEM / F12(3:1) medium to clean the tissue, centrifuge at 1200rpm for 10min and discard Then add 1ml of fresh DMEM / F12 medium and repeatedly blow the tissue with a 1ml pipette tip to separate the cells from the tissue. This process is repeated 3-4 times, and 1ml of medium can be added each time. Finally, centrifuge at 1200 rpm for 10 min, discard the supernatant, resuspend the cells in DMEM / F12 (3:1) medium containing FGF2 (40ng / ml), EGF (...

Example Embodiment

Example 2. Induced differentiation, expansion and identification of rat skin-derived precursor cells into Schwann cells.

The obtained P2-P4 generation rat skin-derived precursor cells were digested with 1 mg / ml type XI collagenase at 37°C for 10-20 minutes, and gently pipetted repeatedly to mechanically separate the cell pellets. The single cell suspension was divided into 5×10 4 / ml density is planted on 75cm pre-coated with Laminin (0.02mg / ml) and Poly-D-lysine (0.2mg / ml) 2 Cultivate in a petri dish, use DMEM / F12 (3:1) medium containing FGF2 (40ng / ml), EGF (20ng / ml), B27 (2%) and FBS (5%) for 3 days, and after 3 days The medium was replaced with Forskolin (5μM), Heregulin-1 (50ng / ml), N 2 (2%) and FBS (2%) in DMEM / F12 (3:1) culture medium for 14-21 days to obtain Schwann cell colonies. The Schwann cell colonies are picked, expanded, and frozen for later use. See the results of immunofluorescence detection of Schwann cells induced by skin-derived precursor cells figure 2 .

Example Embodiment

Example 3. Preparation of nerve catheter.

With reference to the patent application number CN201110324474.8, the chitosan nerve catheter is prepared by the method described in "Tissue Engineering Nerve Graft and Its Use", and the polylactic acid-glycolic acid copolymer fiber prepared by electrospinning is passed through Put it into the chitosan nerve duct (200 pieces per mm inner diameter), and coat it with a mixture of laminin (0.02mg / ml) and polylysine (0.2mg / ml) overnight for later use. The stent material is sterilized by Co60 irradiation or ethylene oxide.

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Abstract

The invention provides a method for in-vitro construction of tissue engineered nerves. The method adopting a three-dimensional gravity bioreactor comprises the following steps of: culturing seed cells with nerve conduits under a three-dimensional micro-gravity environment, wherein the culture conditions are as follows: the seed cell suspension and nerve conduits are placed to a rotary culture container which is filled with complete medium, the cell final density is 1*10<6>/ml, the culture is carried out in a CO2 incubator of 37 DEG C, the rotation speed with the first 24 hours is 10rpm, so that the cells sufficiently contact and are adhered with the nerve conduits; adjusting the rotation speed of the three-dimensional micro-gravity bioreactor after 24 hours, so that the nerve conduits are suspended in the culture solution; and stopping the culture after 7-14 days to obtain the tissue engineered nerves. The method disclosed by the invention can be used for ensuring that the seed cells adhered with the nerve conduits are more and more uniformly, so that peripheral nerve regeneration is facilitated; and moreover, schwann cells, which are induced by skin-orientated precursor cells, are closer to the true schwann cells and are more suitable for being used as seed cells of nerve tissue engineering.

Description

technical field [0001] The invention belongs to the field of biomedical engineering, and specifically relates to a method for constructing tissue-engineered nerves in vitro, in particular to a method for constructing tissue-engineered nerves in vitro by using a three-dimensional microgravity bioreactor. Background technique [0002] Every year in the world, there are many cases of peripheral nerve extrusion, stretching, and contusion injuries due to trauma such as car accidents, sharp objects, and bullets. For short-distance peripheral nerve defects, tension-free surgery can be performed using the elasticity and curvature of the nerve itself. The broken end was sutured to repair the defect. When the defect exceeds a certain distance and cannot be sutured without tension, nerve grafting must be performed. At present, autologous nerve transplantation is the most commonly used clinically, and it is regarded as the gold standard of peripheral nerve repair. However, the sources...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/20A61L27/18A61L27/22
Inventor 顾晓松丁斐薛成斌杨宇民顾芸汤欣朱慧
Owner NANTONG UNIVERSITY
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