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Neural crest cell culture fluid, preparation method of neural crest mesenchymal stem cells and application of neural crest mesenchymal stem cells

A technology of stem cells and cell culture, applied in the field of stem cells and regenerative medicine, can solve the problems of long-term cell survival, heterogeneity and functional instability, and limit the wide application of MSCs in clinical practice

Pending Publication Date: 2019-09-17
SHENZHEN RES INST THE CHINESE UNIV OF HONG KONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, we must also realize that there are still many problems to be solved for the widespread clinical application of MSCs: such as the timing of MSCs transplantation, transplantation route, composition and quantity of transplanted cells, whether the transplanted cells can survive for a long time, tissue-directed differentiation, etc.
In addition, the difficulty of large-scale expansion, heterogeneity and functional instability of MSCs has become the three major obstacles that limit the wide application of MSCs in clinical practice.

Method used

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  • Neural crest cell culture fluid, preparation method of neural crest mesenchymal stem cells and application of neural crest mesenchymal stem cells
  • Neural crest cell culture fluid, preparation method of neural crest mesenchymal stem cells and application of neural crest mesenchymal stem cells
  • Neural crest cell culture fluid, preparation method of neural crest mesenchymal stem cells and application of neural crest mesenchymal stem cells

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Experimental program
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Embodiment 1

[0044] Example 1 Induction and subculture of neural crest mesenchymal stem cells

[0045] (1) Human embryonic stem cells or IPS cells were cultured on the wall with StemPro (Gibco, A3349401) medium, and the medium was changed every day to maintain their undifferentiated state;

[0046] (2) Digested with Accutase (Gibco, A1110501) (placed at 37°C for 3 minutes) and passaged totipotent stem cells at a ratio of 1:10;

[0047] (3) When starting neural crest differentiation, the cells should be 6×10 per square centimeter 4 The density of cells was suspended and seeded on Geltrex (1:100, Gibco, A10480-02) coating plate;

[0048] (4) After the cells adhere to the wall, the culture medium is replaced with a neural crest cell culture medium, and the neural crest cell culture medium includes the following components: bovine serum albumin (Probumin) (20% (vol / vol)), 1% Penicillin / streptomycin, 1% L-alanyl-L-glutamine, 1% non-essential amino acids (MEM non-essential amino acids) , 0.1%...

Embodiment 2

[0054] 1. Differentiation culture of neural crest stem cells (NCSC):

[0055] Human embryonic stem cells (H7-hESC or H9-hESC) were 9.2×10 4 cells / cm 2 The density was inoculated in a Petri dish coated with Geltrex (1:100), added StemPro medium (Life Technologies) and cultivated in an incubator at 37°C with a carbon dioxide concentration of 5% for 24 hours. Then the culture medium in the culture dish was discarded, neural crest stem cell medium was added to induce differentiation, and the medium was changed every two days. When the fusion state was close to 80%, it was digested with Accutase (Gibco), passaged, and then 6×10 4 cells / cm 2 density, inoculated in Geltrex-coated Petri dishes. After continuous culture with neural crest stem cell medium for 15 days, the cell morphology has been clearly differentiated into neural crest stem cells. When the proliferation is close to 80% confluence, it is digested and passaged. Repeated passages in this way achieve the purpose of pur...

Embodiment 3

[0060] The establishment of embodiment 3 neonatal rat HIE model

[0061] According to the Rice method, the operation was carried out under strict aseptic conditions. Select 7-day-old neonatal SD rats, lie supine on a 37°C constant temperature board, anesthetize with 1% isoflurane inhalation under a ventilator, fix, expose the neck, separate and ligate the right common carotid artery (5-0 silk), 7-0 Suture the skin. After 2 hours, put it into a closed container, place it on a 37°C water bath, pass a mixed gas of 92% nitrogen and 8% oxygen into the closed container, and monitor the oxygen concentration in the container with a digital oxygen meter to make it Keep at 8% for 2 hours.

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Abstract

The invention relates to the field of stem cells and regenerative medicine, and particularly relates to neural crest cell culture fluid, a preparation method of neural crest mesenchymal stem cells and an application of the neural crest mesenchymal stem cells. Experimental results of the invention show that mesenchymal stem cells of the neural crest stem cell lineage, induced and differentiated by human totipotent stem cells, can obviously improve neurological dysfunctions, reduce an area of an ischemic region, inhibit immune reactions and promote endogenous repairs of brain tissues in a neonatal rat hypoxic-ischemic model, thereby proving that human neural crest-derived mesenchymal stem cells have effects of significantly improving the inflammatory reaction of neonates with ischemic and anoxic encephalopathy and effectively promoting the neural function recovery. The invention also provides a test index for identifying the activity of the neural crest mesenchymal stem cells.

Description

technical field [0001] The invention relates to the field of stem cells and regenerative medicine, in particular to a neural crest cell culture medium, a preparation method of neural crest mesenchymal stem cells and an application of neural crest mesenchymal stem cells. Background technique [0002] Neonatal hypoxic-ischemic encephalopathy (Hypoxic-ischemic Encephalopathy, HIE) is the brain damage caused by hypoxia and ischemia caused by various perinatal factors, and is the most common cause of neurological disability in children. In the past 20 years, due to the advancement of obstetrics and newborn care technology, on the one hand, the survival rate of newborns has increased due to successful rescue, and on the other hand, the incidence of HIE and its sequelae has increased significantly. There are 18 million to 20 million live births in my country every year, and the incidence rate of suffocation is 13.6%, of which 15.6% are disabled. As a result, about 300,000 disabled ...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N5/0797A61K35/28A61P25/00
CPCC12N5/0618C12N5/0623A61K35/28A61P25/00C12N2500/32C12N2500/33C12N2500/05C12N2500/44C12N2500/24C12N2500/38C12N2501/105C12N2501/10C12N2501/999C12N2501/998
Inventor 蒋晓华黄嘉维
Owner SHENZHEN RES INST THE CHINESE UNIV OF HONG KONG
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