A method for preparing tissue-engineered spinal cord using dermal-derived mesenchymal stem cells

A technology for tissue engineering spinal cord and cells, applied to cells modified by introducing foreign genetic material, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of poor repair effect, difficult to achieve neural stem cells and embryonic stem cells Issues such as autologous transplantation and improper transplantation timing can achieve the effect of promoting cell proliferation

Active Publication Date: 2016-08-24
ARMY MEDICAL UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0002] Spinal cord injury repair is one of the difficulties and priorities in clinical treatment. Stem cell transplantation is one of the more promising methods for treating spinal cord injury. However, the main problems currently exist are: 1. Autologous stem cell transplantation has no immunogenicity and is easy to use, etc. Advantages, but it is difficult to achieve autologous transplantation of neural stem cells and embryonic stem cells, while skin-derived stem cells are rich in sources and can be autologously transplanted
2. Seed cells cannot differentiate more into repair cells such as neurons and oligodendrocytes, resulting in poor repair effect, so it is necessary to find suitable means to induce more differentiation into neurons and other cells
3. Improper timing of transplantation causes transplanted cells to fail to play their role effectively

Method used

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  • A method for preparing tissue-engineered spinal cord using dermal-derived mesenchymal stem cells
  • A method for preparing tissue-engineered spinal cord using dermal-derived mesenchymal stem cells
  • A method for preparing tissue-engineered spinal cord using dermal-derived mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation of dMSCs

[0026] After routine disinfection of the inner thigh or back skin of patients with spinal cord injury, anesthetized with 1% lidocaine, the size is about 0.5×4cm 2 full-thickness skin. Alcohol disinfection, cut the skin and put it in 0.25% trypsin to digest overnight. The next day, after washing with Hank's solution, the epidermis and subcutaneous tissue were removed, and the dermal tissue was cut into cell fragments and blown into a cell suspension, which was filtered and inoculated into a culture bottle, and the medium was IMEM / 10% fetal bovine serum. After 6 hours, discard the culture medium and suspended cells, and continue to culture the early adherent cells. Passage repeatedly until colony-like cell populations are formed, inoculate and select cell clones according to conventional methods, and passage for future use. Isolated dMSCs such as figure 1 shown.

[0027] Among them, cell immunofluorescence histochemistry was used to det...

Embodiment 2d

[0028] Embodiment 2d MSCs rapid expansion

[0029] Usually, the growth of primary cells is relatively slow. In order to obtain a sufficient amount of cells within the optimal time window for transplantation, this example studies the promotion of proliferation of dMSCs by different growth factors. According to literature research, a variety of cytokines are involved in the regulation of stem cell proliferation, mainly basic fibroblast growth factor (basic fibroblast growth factor-2, FGF-2), epidermal growth factor (epidermal growth factor, EGF), leukemia inhibitory factor (1eukemia inhibiting factor, LIF), db-cAMP, insulin growth factor-1 (insulin growth factor-1, IGF-1), insulin growth factor-2 (insulin growth factor-2, IGF-2), platelet-derived growth factor (Plate derived growth factor, PDGF), brain derived neurotrophic factor (brain derived neurotrophic factor, BDNF), neurotrophin-3 (neurotrophin-3, NT-3), nerve growth factor-B (nerve growth factor-β, NGF -β), bone morphoge...

Embodiment 3d

[0032] Example 3d Optimization of MSCs differentiation neuron and oligodendrocyte differentiation conditions

[0033] The local microenvironment of spinal cord injury is not conducive to the differentiation of transplanted stem cells into neurons and other repair cells. Therefore, it is necessary to first explore the conditions that increase the differentiation of stem cells into neurons and other repair cells, and then combine these conditions in the tissue engineered spinal cord to improve stem cell transplantation. repair effect.

[0034] In vitro experiments were performed to observe the conditions for the differentiation of dMSCs into neurons and oligodendrocytes. There are three kinds of media for inducing neuron differentiation: (1) 5% FBS; (2) 6ng / ml retinoic acid; (3) 10ng / ml BDNF+10ng / mlNT3+6ng / ml retinoic acid+5%FBS. Two kinds of induction media were used for induction to Schwann cells: (1) 5% FBS; (2) 5% FBS+10 ng / ml Neuregulin. The results showed that the probab...

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Abstract

The invention discloses a method for preparing tissue engineering spinal cords by using mesenchymal stem cells derived from dermis, which comprises the following steps of 1) separating dMSCs, and carrying out passage on the dMSCs so as to obtain dMSCs primary cells; 2) moving the dMSCs primary cells obtained through separating to an amplification culture medium so as to carry out amplification; and 3) dropwise adding an engineering spinal cord saturated water solution into a physiological saline solution containing deep nerve nutriments, retinoic acid and Neuregulin, standing the obtained mixture, carrying out gradient alcohol dehydration on the mixture, and carrying out vacuum drying on the mixture; and infecting the dMSCs subjected to amplification by using a brain-derived neurenergen adenovirus expression vector, and inoculating cells to an engineering spinal cord material for culturing. In the method, tissue engineering spinal cords can effectively promote cell proliferation; and the differentiation rates of the tissue engineering spinal cords to nerve cells and oligodendroglia cells are respectively about 4.8% and 1.5% which are far higher than those of pure scaffold materials (the differentiation rates of pure scaffold materials to nerve cells and oligodendroglia cells are respectively 1.8% and 0.5%).

Description

technical field [0001] The invention relates to a method for preparing tissue-engineered spinal cord, in particular to a method for preparing tissue-engineered spinal cord from dermis-derived mesenchymal stem cells. Background technique [0002] Spinal cord injury repair is one of the difficulties and priorities in clinical treatment. Stem cell transplantation is one of the more promising methods for treating spinal cord injury, but the main problems currently exist are: 1. Autologous stem cell transplantation has no immunogenicity and is easy to use, etc. Advantages, but it is difficult to achieve autologous transplantation of neural stem cells and embryonic stem cells, while skin-derived stem cells are rich in sources and can be used for autologous transplantation. Skin-derived stem cells can differentiate into various types of cells including neurons under the induction of different microenvironments, so their application in spinal cord injury repair has good prospects an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N5/10
Inventor 宗兆文陈思旭
Owner ARMY MEDICAL UNIV
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