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Compound inducing culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by virtue of compound inducing culture medium

A technology of neuron-like cells and induction medium, applied in animal cells, culture process, tissue culture, etc., can solve the problems of carcinogenesis transformation risk, long induction time, immature neurons, etc., to ensure biological safety, induce Short time, good nerve recovery effect

Active Publication Date: 2017-08-18
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although commonly used differentiation schemes can induce the generation of neuron-like cells, there are still deficiencies: (1) Classic high-concentration antioxidants can break and shrink the actin of the cytoskeleton structure in a short period of time, causing MSCs to show cone-like terminal expansion However, it is also accompanied by many problems such as small number of viable cells, limited efficiency, and risk of carcinogenesis transformation, which are closely related to the concentration, time, and inherent cytotoxicity of the inducing components; scholar Hofstetter found that MSCs were treated with 5 mM β - 48 hours after ME induction, the results of patch clamp electrophysiology showed that the induced neuron-like cells did not express sodium and potassium voltage-gated channels, did not generate action potentials, and belonged to immature neurons
(2) A single application of cytokines can rebuild axons and improve the survival of neuron-like cells through target-derived improvement of neurotrophic factor supply, but the induction time is long and the efficiency is low
(3) Scholar Yao Xiaoli believes that there is a phenomenon of reverse transformation in the process of MSC differentiation induced by some traditional Chinese medicines, and the induction time still needs to be explored

Method used

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  • Compound inducing culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by virtue of compound inducing culture medium
  • Compound inducing culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by virtue of compound inducing culture medium
  • Compound inducing culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by virtue of compound inducing culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The compound induction medium of the present invention is made up of three kinds of differentiation culture fluids of DA, DB and DC, and the preparation methods of three kinds of differentiation culture fluids of DA, DB and DC are as follows:

[0031] DA differentiation medium: for every 1000ml of DMEM-LG (HyClone, product number: SH30021.01B) medium, add 40ml of FBS (HyClone, product number: SH30396.03) and 10 μg bFGF (Peprotech, product number: 100-18B);

[0032]DB differentiation medium: for every 1000ml of DMEM-LG (HyClone, product number: SH30021.01B) medium, add 50ml Cerebrolysin (EVER Neuro Pharma GmbH, product number: H20100440) and 4mg Forskolin (AMQUAR, product number: EY0022) according to the characteristics of the raw materials , 5mg Insulin (aladdin, product number: I119752-15mg), 0.36mg Hydrocortisone (Sigma, product number: H0888), 1.86g KCl (Sigma, product number: P5405), 7g NaCl (Sigma, product number: S5886), 2.2g NaHCO 3 (Sigma, Cat. No.: S5761), 190...

Embodiment 2

[0035] The compound induction medium prepared in Example 1 was used to induce the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into neuron-like cells, and the specific steps were as follows:

[0036] 1. Isolation and culture of human umbilical cord mesenchymal stem cells (hUC-MSCs)

[0037] Collection of human umbilical cord tissue samples: The umbilical cord tissue must come from healthy maternal volunteers who delivered full-term cesarean sections, and all relevant indicators of infectivity were tested negative. When collecting, wipe the outer wall of a sterile 50ml centrifuge tube with 75% alcohol, and pre-cool normal saline at 4 degrees for a short period of time after removing blood stains.

[0038] Isolation of human umbilical cord tissue specimens: In a biological safety cabinet, take two sections of fresh umbilical cord specimens, each section is 5 cm, rinse 3 times with pre-cooled normal saline, remove blood stains, and wash clean. In a p...

Embodiment 3

[0053] Identification of neuron-like cells induced by Example 2

[0054] 1. Fluorescent quantitative PCR was used to detect the level of genes related to neural differentiation after induction. The specific scheme is as follows:

[0055] 1. Extract the total RNA of induced group cells, the steps are as follows:

[0056] 1) Collect the induced cells in good condition, centrifuge at 1000rpm, and remove the supernatant after 3-5min; add 1ml Trizol to the cell pellet, pipette quickly, let stand at room temperature for 5min, and transfer to a new 1.5ml enzyme-free EP tube ;

[0057] 2) Add 200 μl / tube of chloroform, turn the EP tube upside down for 20 seconds, and let stand at room temperature for 10 minutes;

[0058] 3) After centrifugation at 4°C and 12000 rpm for 15 minutes, transfer the supernatant to a new 1.5 ml enzyme-free EP tube, add an equal volume of pre-cooled isopropanol, pipette evenly, and place at 4°C for precipitation for 10 minutes;

[0059] 4) Discard the supe...

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Abstract

The invention discloses a compound inducing culture medium. The compound inducing culture medium contains three differentiation culture solutions AD, DB and DC. A method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by virtue of the compound inducing culture medium comprises the following steps: pre-inducing the umbilical cord mesenchymal stem cells for 2 days by virtue of the differentiation culture solution DA; carrying out differentiation culture for 1 day by virtue of the differentiation culture solution DB; finally, carrying out maintenance culture for 1 day by virtue of the differentiation culture solution DC; and observing the morphological characteristics of the induced neuron-like cells, and detecting the mRNA level of induced neural differentiation relevant genes and the expression conditions of neuron migration proteins DCX and neuron specific enolase NSE which induce the neuron-like cells, wherein positive subjects of DCX expression are neural precursor cells, and positive subjects of NSE expression are the neuron-like cells. According to the method, the induction process is divided into three stages, and different culture mediums are adopted in each stage, so that the induction time is short, the induction efficiency is high, and the induced differentiated neuron-like cells can stably live and have no repellence after being transplanted.

Description

technical field [0001] The invention relates to stem cell neural differentiation technology, in particular to a compound induction medium, and also relates to a method for using the medium to induce umbilical cord mesenchymal stem cells into neuron-like cells with a relatively high survival level. Background technique [0002] Clinically, damage to the central nervous system can lead to massive death of nerve cells in the brain and spine, permanent loss of nerve function, and it is difficult to repair itself. In view of the fact that there is still no stable solution for the rapid extraction of neural stem cells, the strategy of inducing stem cells from other sources to obtain a stable number of neuron-like cells with complete functions has placed high hopes on the scientific community. [0003] Compared with other sources of stem cells, human umbilical cord mesenchymal stem cells (HUCMSC) have the characteristics of less pain in collection, faster self-renewal, strong proli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793C12N5/0775
CPCC12N5/0619C12N2500/32C12N2501/01C12N2501/11C12N2501/115C12N2501/33C12N2501/39C12N2501/998C12N2506/1392
Inventor 关方霞马珊珊邢衢王欣欣黄团结孟楠杨波
Owner ZHENGZHOU UNIV
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