Umbilical cord mesenchymal stem cell preparation and preparation method and application thereof
A stem cell preparation and stem cell technology, applied in the field of preparations containing umbilical cord mesenchymal stem cells and their preparation, can solve the problems of insufficient utilization of umbilical cord mesenchymal stem cells, low yield of active ingredients, limited types and quantities, etc., and achieve effective factor Enrich, optimize product quality, and ensure the effect of cell state
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Embodiment 3
[0051] Example 3 MTT detects the effects of ordinary medium and special medium on the proliferation of human umbilical cord mesenchymal stem cells
[0052] After recovery and culture of human umbilical cord mesenchymal stem cells, 3000 cells / well were plated into 96-well plates, 2 groups were plated, each group had 5 duplicate wells, and 4 96-well plates were plated in total. The cells in the two groups were cultured with ordinary medium and special medium, and MTT was added every 24 hours, and the OD value in the wells at a wavelength of 570nm was detected with a microplate reader. The ordinary medium did not add the above-mentioned 20 kinds of amino acids and 8 kinds of vitamins. The test results are shown in Table 1 and figure 1 As shown, the transfer culture medium adopted in the present invention can obviously promote the proliferation of human umbilical cord mesenchymal stem cells and maintain the viability and better cell state of stem cells, and after 72 hours of cult...
Embodiment 4
[0055] Example 4 Immunofluorescence Detection of CD44 Expression in Human Umbilical Cord Mesenchymal Stem Cells
[0056] Human umbilical cord mesenchymal stem cells of P3, P10, and P11 generations were divided into 5×10 4 The density of cells / well was spread into a 24-well plate. After the cells adhered to the wall, the cells were fixed with paraformaldehyde, blocked with BSA, then coated with CD44 antibody and fluorescent secondary antibody, and finally stained with DAPI and observed under a fluorescent microscope. ,Photograph. Test results such as figure 2 As shown, under the same experimental and microscopic examination conditions, the P3-P11 generation umbilical cord mesenchymal stem cells can express CD44 molecules, but the CD44 expression level decreased significantly since the P11 generation, and the P3 generation was the best. It can be seen that the umbilical cord mesenchymal stem cells The selection of young passages is necessary for cell viability, maintenance of...
Embodiment 5
[0057] Example 5 Preparation of Umbilical Cord Mesenchymal Stem Cell Extract Liquid Powder
[0058]Select the umbilical cord mesenchymal stem cells of passage 3-10, and inoculate them into T75 culture flasks, add 10ml of special medium to the culture flask, collect the medium after 24 hours, store it at 4°C, and replace it with 10ml of fresh special medium. Then collect again at 24 hours until the cell fusion rate in the T75 culture flask reaches 90%, the cells are digested and collected.
[0059] The collected cells were suspended in PBS, lysed by low-temperature ultrasonication, and then centrifuged to remove cell debris, and the supernatant was collected, and the remaining supernatant contained cell lysates.
[0060] Mix all cell culture supernatants and cell lysate supernatants collected and stored at 4°C, add mannitol, trehalose, and dextran as freeze-drying protective agents, filter and sterilize at 0.22 μm, and add them to freeze-drying bottles,- After pre-freezing at ...
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