Method for extracting human bone marrow mesenchymal stem cells by using implant cavity bone fragments
A bone marrow mesenchymal and stem cell technology, applied in the medical field, can solve the problems of high technical sensitivity, cumbersome steps, and great influence on the extraction results, and achieve the effect of low technical sensitivity, wide range of sources, and promotion of jaw defect repair
Pending Publication Date: 2022-04-12
AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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Problems solved by technology
These extraction methods are cumbersome, and the density gradient centrifugation method and the adherence method all involve multiple filtration steps and centrifugation steps, which greatly increase the probability of contamination and the loss of cells, and a large number of red blood cells and stem cells are mixed together after centrifugation. Affects the attachment behavior of stem cells, which may reduce the proportion of stem cells in primary cells
In addition, the technical sensitivity of extraction using magnetic bead sorting method is high, and individual operation differences have a greater impact on the extraction results
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Embodiment 1
[0019] A method for extracting human bone marrow mesenchymal stem cells by using bone chips in planting holes, comprising the steps of:
[0020] 1. Apply for the approval of the Ethics Committee, select dental implant patients without systemic diseases, and sign the informed consent.
[0021] 2. During the implantation operation, aseptically collect the bone debris from the implantation cavity brought out by the tap drill, immediately place it in 4 mL of α-MEM culture medium containing 2% double antibody and 10% FBS, and seal it. Culture solution formula: Mix 50mL of α-MEM medium, 5mL of fetal calf serum, and 1mL of penicillin / streptomycin double antibody solution in a clean bench.
[0022] 3. Transfer the bone debris in the centrifuge tube together with the culture medium to the petri dish in the ultra-clean bench, and place it at 37°C, 5% CO 2 Incubate in a constant temperature incubator with saturated humidity, and record it as P0.
[0023] 4. Change the medium for the fi...
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The invention relates to a method for extracting human bone marrow mesenchymal stem cells by using implant cavity bone scraps, which comprises the following steps of: aseptically collecting the implant cavity bone scraps attached to the walls of a reamer and a tapping drill, immediately putting the implant cavity bone scraps into an alpha-MEM culture solution, and sealing; transferring the crushed bone scraps in the centrifugal tube together with the culture solution into a culture dish in a super clean bench, and incubating in a constant-temperature incubator; after 3 days, half of the fresh culture solution is replaced for the first time, and then full of the culture solution is replaced every 3 days, and non-adherent cells are removed; after the long fusiform cells are observed to climb to the periphery by taking the bone scraps as the center, replacing the culture solution with an alpha-MEM culture solution for continuous culture; when the cells climb to the dish bottom and reach 80% of density, adding 1 mL of 0.25% trypsin / EDTA for digestion, performing passage at 1: 3, recording as P1, and performing passage at 1: 3 to P3, so as to obtain the human mesenchymal stem cells.
Description
technical field [0001] The invention belongs to the field of medicine, and in particular relates to a method for extracting human bone marrow mesenchymal stem cells by using bone scraps in planting holes. Background technique [0002] Mesenchymal stem cells have self-renewal ability, multi-directional differentiation potential and immunoregulatory properties, and are widely used in experimental research in the fields of tissue defect repair, immune regulation, gene therapy, and drug screening. Mesenchymal stem cells can be isolated from various tissues, such as umbilical cord, endometrial polyps, menstrual blood, bone marrow, adipose tissue, etc. [Ding D-C, Shyu W-C, Lin S-Z. Mesenchymal Stem Cells [J]. Cell Transplantation, 2011 , 20(1): 5-14.]. Among them, bone marrow-derived mesenchymal stem cells are easily isolated and can differentiate into various cell subsets such as osteoblasts, adipocytes, chondrocytes, and nerve cells [Sasaki H, Takeuchi I, Okada M, et al. Labe...
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IPC IPC(8): C12N5/0775A61K35/28A61P19/08
Inventor 邱憬朱文卿汤泽华陈蔚
Owner AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV