Mesenchymal stem cell induction differentiation medium and method

A technique for inducing differentiation of stem cells, applied in the field of biomedicine, can solve the problems of short survival time, lack of nerve cell function, long induction period, etc., and achieve the effect of simple induction method, good cell growth and simple method.

Inactive Publication Date: 2014-06-25
曾因明
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Problems solved by technology

Although the above-mentioned commonly used chemical inducers can quickly induce the generation of neural-like cells, they have poor cell viability, and death or apoptosis occurs in an average of 4 days, while the cell growth factor induction method has low induction efficiency, long induction cycle, and short survival time, etc. Defects, even some literatures believe that the nerve-like cells generated by th

Method used

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  • Mesenchymal stem cell induction differentiation medium and method
  • Mesenchymal stem cell induction differentiation medium and method
  • Mesenchymal stem cell induction differentiation medium and method

Examples

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preparation example Construction

[0032] (1) Preparation of human autologous bone marrow plasma

[0033] Aseptically collect 20-30 mL of the patient's bone marrow, anticoagulate with 15 IU / mL heparin, centrifuge horizontally at 1025-1450 g for 15-20 min at room temperature, remove the cell layer for later use, and obtain human bone marrow plasma.

[0034] (2) Preparation of autologous BMSCs medium

[0035] The human body's autologous bone marrow plasma that step (1) makes is added in the basal culture medium with the volume percentage of 2-5% [the concrete formula of this basal culture medium is identical with the formula listed in embodiment 1 (except not containing vitamin H , containing 25mg / L (60mM) phytohemagglutinin PHA-I), pH6.8±0.3, osmotic pressure 299±5%] mixed.

[0036] (3) Extraction and culture of BMSCs

[0037] Inoculate the cell layer obtained in step (1) on 25cm 2 In a culture bottle (containing the medium prepared in step (2)), place in 5% CO at 37°C and 100% saturated humidity 2 In the in...

Embodiment 1

[0040] This embodiment provides a method for inducing differentiation of mesenchymal stem cells into neural precursor cells, comprising the steps of:

[0041] (1) Preparation of induction culture medium

[0042] Under aseptic conditions, take 2-5 mL of cerebrospinal fluid from the patient and add it to the basal medium (the composition of the basal medium is as follows, pH6.8±0.3, osmotic pressure 299±5%), so that the content of cerebrospinal fluid in the culture medium is 10~30ng / mL.

[0043]

[0044]

[0045]

[0046] (2) Induction of neural precursor cells

[0047] During the most vigorous period of stem cell division, that is, 80-90% mesenchymal stem cells confluent after purification (the second and third passages after expansion), discard the culture medium, and cover the cells with the induction culture medium prepared in step (1). Days later, the desired neural precursor cells were obtained.

[0048] (3) Morphological identification of neural precursor cell...

Embodiment 2

[0061] The experiments provided in this example show that the application of the neural precursor cell transplantation prepared in this example 1 to treat the rat model of middle cerebral artery ischemia shows that the effect is significantly better than that of the stem cell transplantation group induced by cytokines (using EGF, bFGT respectively). 10mL cultured in neurooasal serum-free medium for 3 days).

[0062] (1) Scoring results of behavioral experiments

[0063] The rats with right middle cerebral artery infarction showed different degrees of neurological deficits, such as flexion of the left limb, circular movement to the paralyzed side, and falling. There was no difference in the modified NSS scores of the two groups of rats before transplantation, but the neurological deficit scores of each group decreased significantly on day 4 and day 32 after transplantation (p<0.05). The modified NSS score of the CSF-induced BMSC-Ns transplantation group was significantly lower...

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Abstract

The invention provides a mesenchymal stem cell induction differentiation medium and method. The mesenchymal stem cell induction differentiation medium is prepared by adding 10-30ng/mL cerebrospinal fluid into a basic medium. The invention also provides the method for induction differentiation of mesenchymal stem cells into neural precursor cells by use of the medium. According to the medium and the culture method, in consideration of time, the growth cycles of neural stem cells and neurons can be greatly shortened, cells grow well, differentiated cells are mainly neuronal cells and are long in service life is, an efficient xenogeneic animal component-free clinical neural precursor cell transplantation technology platform is provided, and the method is simple, and less in spend.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a medium for inducing differentiation of mesenchymal stem cells and a method thereof. Background technique [0002] Stem cells, as the progenitor cells that form various tissues and organs in the body, have the ability of self-replication and multi-lineage differentiation under specific conditions. Therefore, stem cell research has unlimited potential to bring new treatments for a large number of serious diseases and injuries. [0003] Mesenchymal stem cells (MSCs) are an important branch of stem cell research. They were first discovered in bone marrow, so they are collectively called bone marrow mesenchymal stem cells (BMSCs). It is found in various connective tissues and interstitium of organs, such as fat tissue and skin. [0004] Mesenchymal stem cells have multi-directional differentiation ability, and have the ability to differentiate into mesoderm cells such as oste...

Claims

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Application Information

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IPC IPC(8): C12N5/0797A61K35/30A61P25/00A61P25/28A61P25/02A61P9/10A61P25/16
Inventor 万美蓉高恒耿德勤
Owner 曾因明
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