Method for preparing medium-sized spinous process neurons from non-neuronal cell transformation and application

A neuron cell and neuron technology, applied in the field of cell transformation, can solve the problems of low cell survival rate, difficulty in industrialized preparation, market demand, and difficult realization of cryopreservation recovery utilization, etc., to achieve high cell viability and cryopreservation Recovery utilization, high-purity effect

Active Publication Date: 2022-01-18
宁波易赛腾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is mainly based on the direct transdifferentiation technology mediated by mir-9 / 9*-124, which generally takes a long time (over 30 days) to obtain fully transformed mature neurons; It is difficult for nerve cells to be processed by conventional methods such as trypsin digestion, resulting in low cell survival rate after digestion, and it is difficult to achieve cryopreservation recovery and utilization
Therefore, this technology is limited to a few laboratories with mature technology, and it is difficult to realize industrial production in a certain period of time to meet market demand.

Method used

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  • Method for preparing medium-sized spinous process neurons from non-neuronal cell transformation and application
  • Method for preparing medium-sized spinous process neurons from non-neuronal cell transformation and application
  • Method for preparing medium-sized spinous process neurons from non-neuronal cell transformation and application

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preparation example Construction

[0058] Based on the above preparation method, the specific preparation method of the present invention may include the following steps:

[0059] Step S01, determining the cell transdifferentiation gene combination, wherein the cell transdifferentiation gene combination comprises a combination of the following genes: (1) at least one gene selected from NGN2 and ASCL1; (2) selected from DLX1, DLX2, LHX6, CTIP2 and (3) at least one gene selected from SOX4 and SOX11;

[0060] Step S02, constructing the genes in the above-mentioned cell transdifferentiation gene combination as a single, two, or three (connecting two different genes with 2A sequence or IRES sequence) into a commercially available or self-owned retroviral vector, Lentiviral vectors or AAV viral vectors, the promoters that regulate expression levels in each vector can be any one, two or more combinations of CMV, CAG, EF1α, PGK, TRE Tight, and TRE3G. No restrictions. At the same time, green or red fluorescent reporte...

Embodiment 1

[0087] A method for preparing medium-sized spinous neurons from non-neuronal cells. The materials, equipment and reagents used in the process of preparing medium-sized spinous neurons in this embodiment are as follows:

[0088] 1. Materials

[0089] Cells: 293T cells were purchased from American ATCC (CRL-3216); human skin fibroblasts were purchased from American Sciencell (product number #2310, #2320). The cells were cultured with high-glucose DMEM medium containing 10-20% FBS and 1×P / S double antibody.

[0090] 2. Instruments and reagents:

[0091] (1) CO2 cell incubator (ESCO CLM-170B-8-CN); ultra-clean bench (ESCO AC2-5S1); fluorescence microscope (Thermo EVOS M5000); ultra-low temperature refrigerator (Haier DW-86L388J); liquid nitrogen tank ( Haier YDS-175-216-F); high-speed refrigerated centrifuge (Baiyang BY-R20); room temperature high-speed centrifuge (Xiangyi H1650-W);

[0092] (2) Intelligent high-pressure steam sterilizer (Shanghai Shenan LDZM-80KCS); American S...

Embodiment 2

[0119] 1. Materials, equipment and reagents

[0120] Human lung fibroblast MRC-5 (CCL-171) was purchased from ATCC Corporation of the United States. Other materials, equipment and reagents used in the preparation of medium-sized spiny neurons in this example are the same as those in Example 1.

[0121] 2. Preparation method

[0122] A method for preparing medium-sized spinous neurons from non-neuronal cells, the non-neuronal cells are human lung fibroblasts, so embodiment 2 provides a method for preparing medium-sized spinous neurons from lung fibroblasts, the The method includes the following steps:

[0123] Step S21, construction of plasmids: Ascl1, Dlx1, Dlx2, LHX6, CTIP2, and Sox4 genes were respectively constructed into retroviral vectors, the promoters regulating gene expression levels in the vectors were CAG and CMV, and a green fluorescent reporter was introduced through the IRES sequence at the same time Gene, in order to determine the quality and titer of virus pac...

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Abstract

The invention discloses a method for preparing medium-sized spinous process neurons from non-neuronal cells through transformation and application, and the method comprises the following steps: constructing a virus vector containing a cell transdifferentiation gene combination, and carrying out virus packaging through a transfection reagent; culturing donor cells, and infecting the donor cells with the packaged virus; transdifferentiating the donor cells into medium-sized spinous process neurons in an induced differentiation culture solution, wherein the induced differentiation culture solution comprises a small molecule compound and a growth factor for promoting transdifferentiation; separating and purifying medium-sized spinous process neurons; wherein the cell transdifferentiation gene combination comprises the following combinations: at least one gene selected from NGN2 and ASCL1; selecting at least three genes from DLX1, DLX2, LHX6 and CTIP2; and selecting at least one gene from SOX4 and SOX11. According to the method disclosed by the invention, the medium-sized spinous process neurons with high conversion efficiency and high purity can be prepared, the converted nerve cells are easily treated by conventional methods such as trypsin digestion and the like, the cell viability after digestion is high, and cryopreservation, resuscitation and utilization are favorably realized.

Description

technical field [0001] The invention relates to the technical field of cell transformation, in particular to a method and application for preparing medium-sized spinous neurons from non-neuron cell transformation. Background technique [0002] Huntington's disease (HD) is a rare neurodegenerative disease. The main subtype of nerve cells that are invaded and undergo continuous degeneration and death in the brain of the patient is the medium-sized spiny neuron. This pathological process is related to various symptoms of HD patients. closely related. The use of medium-sized spiny neurons as HD cell pathology models is of great significance for in-depth study of pathogenesis, screening and development of effective therapeutic drugs and development of gene therapy, and has a very considerable market demand. [0003] The in vitro separation and culture technology of animal nerve cells is relatively mature, but it is mainly limited to embryonic or primary mouse cells, which have p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0793C12Q1/02
CPCC12N5/0619G01N33/5058C12N2501/60C12N2501/999C12N2500/40C12N2501/15C12N2501/727C12N2500/38C12N2501/115C12N2501/13C12N2533/52C12N2533/54C12N2533/90C12N2503/02C12N2510/00G01N2500/10
Inventor 刘猛六柳明杰沈雁飞
Owner 宁波易赛腾生物科技有限公司
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