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A method for preparing tissue-engineered spinal cord using skin-derived progenitor cells

A technique for tissue engineering spinal cord and skin, applied to animal cells, vertebrate cells, artificial cell constructs, etc., can solve problems such as poor repair effect, inability of transplanted cells to play their role effectively, inappropriate timing of transplantation, etc., to achieve The effect of promoting cell proliferation

Active Publication Date: 2016-02-10
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 skin-derived progenitor cells
  • A method for preparing tissue-engineered spinal cord using skin-derived progenitor cells
  • A method for preparing tissue-engineered spinal cord using skin-derived progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 separates SKPs

[0023] 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, the dermal tissue was cut into cell sheets, crushed and blown into a cell suspension, filtered and inoculated into a culture bottle containing DMEM medium. After 2 hours, the suspended cells were centrifuged and transferred to a medium suitable for the growth of SKPs (DMEM-F12, 3:1, containing 40ng / mlFGF2, 20ng / mlEGF). After culturing for about 7 days, SKPs cells growing like suspension clones could be seen. , figure 1 .

[0024] Cell immunofluorescence histochemistry was used to detect whether SKPs differentiated into neurons and oligodendrocytes after induction, and the p...

Embodiment 2

[0025] Embodiment 2 SKPs rapid expansion

[0026] 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 SKPs 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-2 (FGF-2), epidermal growth factor (epidermal growth factor, EGF), leukemia inhibitory factor (leukemia 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 (Plated derived growth factor, PDGF), brain-derived Neurotrophin (brainderivedneurotrophicfactor, BDNF), neurotrophin-3 (neurotrophin-3, NT-3), nerve growth factor-β (nervegrowthfactor-β, NGF-β), bone morphogenetic protein-4 (bonemorphogenticprotein4, BM...

Embodiment 3

[0031] Example 3 Optimization of SKPs differentiation neuron and oligodendrocyte differentiation conditions

[0032] 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.

[0033] In vitro experiments were performed to observe the conditions for SKPs to differentiate 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 probability of...

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Abstract

The invention discloses a method for preparing a tissue engineering spinal cord by using ancestral cells originated from skin. The method comprises the following steps: 1) separating SKPs, and passing to obtain SKPs primary cells; 2) immigrating the SKPs primary cells separated to an amplification culture medium for amplification; and 3) preparing the tissue engineering spinal cord, and dropwise adding saturated aqueous liquor of a tissue engineering spinal cord material to physiological saline liquor containing Neurotrophin-3(NT3), retinoic acid and Neuregulin; infecting the amplified SKPs by a brain-derived neurenergen adenovirus expression vector, and inoculating the cells to the tissue engineering spinal cord material to cultivate. In the method disclosed by the invention, the tissue engineering spinal cord can effectively promote cell proliferation, and the differentiating proportions of the tissue engineering spinal cord toward nerve cells and oligodendroglia cells are about 4.8% and 1.5% which are far greater than 1.8% and 0.5% of a pure support material.

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 progenitor cells derived from skin. 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 and p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071A61L27/38
Inventor 宗兆文陈思旭
Owner ARMY MEDICAL UNIV
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