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Culture medium for preparing neural stem cells and application thereof
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A technology of neural stem cells and culture medium, applied in the field of culture medium for preparing neural stem cells, which can solve the problems that neural stem cells need to be improved
Active Publication Date: 2012-07-25
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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[0004] However, the current research on neural stem cells still needs to be improved
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Embodiment 1
[0058] Example 1: Preparation of neural stem cells
[0059] 1. Isolation of human urine exfoliated cells (Urine cells, UC)
[0060] Follow the steps below to isolate urine exfoliated cells:
[0061] (1) Collect 150-200ml of mid-section urine, put it in a sterile container, and add double antibodies (penicillin / streptomycin);
[0062] (2) Then transfer the above urine into a 50ml centrifuge tube and centrifuge at 400g for 10min;
[0063] (3) Suck off the supernatant so that about 5ml of urine remains;
[0064] (4) Add about 10-30ml of PBS containing double antibody to the above remaining urine, mix gently, and then centrifuge at 400g for 10min;
[0065] (5) Suck off the supernatant until the remaining liquid is less than 0.5ml;
[0066] (6) Then add 1ml of urine cell culture medium to the above remaining urine to resuspend the pellet, and collect the cells;
[0067] (7) Inoculate the above-mentioned collected cells on a 60mm culture dish (or six-well plate) pre-coated with...
Embodiment 2
[0091] Example 2: Identification of Induced Neural Stem Cell (iNSC) Phenotypes
[0093] (1) Put Matrigel-coated glass slides in a 24-well plate, take 5-10 small iNSC neurospheres (i.e. iNSC neural stem cells) prepared in Example 1 and stick them on the glass slides, Then add 100 μl of neural stem cell culture medium to the slide, culture overnight, and add 500 μl of neural stem cell culture medium to each well the next day, and culture for 1-2 days;
[0095] (3) Fully rinse the fixed cells with PBS for 3 times, 5 min each time;
[0096] (4) Add primary antibodies (Pax6, Nestin, Sox1, Sox2), 1% BSA, 10% normalgoat serum, 0.3% Triton X-100 and PBS to the washed cells, and then culture overnight at 4°C;
[0097] (5) Wash the cells cultured overnight with PBS for 3 times, ea...
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Abstract
The invention relates to a culture medium for preparing neural stem cells and an application of the culture medium. The culture medium for preparing the neural stem cells comprises a culture medium suitable for the growth of stem cells, and a cellsignal pathway inhibitor which is selected from at least one of the following agents: a GSK (Glaxo Smith Klein) inhibitor, an MEK (Methyl Ethyl Ketone) inhibitor, a TGF (Transforming Growth Factor)-beta inhibitor, a ROCK inhibitor and a BMP inhibitor. The somatic cells are cultured by using the culture medium, especially the somatic cells for expressing the transcription regulator are cultured, so as to effectively transdifferentiate the somatic cells into neutral stem cells and the transdifferentiation time is greatly shortened.
Description
technical field [0001] The invention relates to the field of biomedicine. Specifically, the present invention relates to a culture medium for preparing neural stem cells and its use. Background technique [0002] Stem cells (stem cells) are the initial source of the human body and its various tissue cells, and its most notable biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multidirectional differentiation. Stem cells are divided into somatic stem cells (somatic stem cells) and embryonic stem cells (embryonic stem cells, ES cells) according to different sources. Adult stem cells include neural stem cells, bone marrow mesenchymal stem cells, and hematopoietic stem cells. At present, in addition to the research on hematopoietic stem cells and mesenchymal stem cells, the research on neural stem cells is also relatively in-depth. [0003] In 1992, Reynolds and Weiss first successfully isolated neural stem ce...
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