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A medium for culturing and/or expanding mesenchymal stem cells and its culturing method
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A technology of stem cells and culture medium, applied in the field of biomedicine, can solve the problems of complicated operation and increased cost, and achieve the effects of low mortality, high cell number and great clinical application value.
Inactive Publication Date: 2017-02-08
曾因明
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For example, Chinese patent application CN101760445A discloses a method of cultivating bone marrow mesenchymal stem cells using low-concentration autologous serum, but various cell growth factors and proteins such as insulin, transferrin, and albumin need to be added to the medium. components, resulting in increased costs and cumbersome operations
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Embodiment 1
[0044] This embodiment provides a method for culturing bone marrow mesenchymal stem cells in vitro by the semi-bone marrow method, which specifically includes the following steps:
[0045] (1) Preparation of human autologous plasma
[0046] By aseptically collecting 20-30ml of the patient's bone marrow, anticoagulating with 15IU / ml heparin, centrifuging horizontally at 1025-1450g for 15-20min at room temperature, removing the cell layer for later use, to obtain human autologous plasma.
[0047] (2) Preparation of autologous BMSCs medium
[0048] Add the human autologous plasma prepared in step (1) to the basal medium at a concentration of 20-50ml / l (the specific formula of the basal medium is as follows, pH6.8±0.3, osmotic pressure 299±5%) and mix.
[0049]
[0050]
[0051]
[0052] (3) Extraction and culture of BMSCs
[0053] Inoculate the cell layer obtained in step (1) on 25 cm 2 In a culture bottle (containing the medium prepared in step (2)), place in 5% CO a...
Embodiment 2
[0065] This example confirms that the BMSCs prepared in Example 1 have the ability to differentiate into osteoblast-like cells and hepatoblast-like cells as an example.
[0066] 1. Identification of osteogenic induction of BMSCs:
[0067] BMSCs by 1.5×10 5 Cells / mL were inoculated into 6-well culture plates (cover slips pretreated with 100mg / mL polylysine), added to Tiancheng No. 1 medium containing bone marrow plasma, and observed that the cells were 80-90% confluent. Aspirate the medium. Add conditioned medium (add 10nmol / L dexamethasone, 10mmol / L β-glycerophosphate sodium, 50μmol / L vitamin C to the basal medium in step (2), place at 37°C and 5% CO in saturated humidity 2 The culture was induced in the incubator, and the medium was changed every 3 days.
[0068] (1) Morphological observation after induction
[0069] After being cultured under an inverted microscope for 72 hours, the cells changed from a single fibrous shape to a cubic triangular polygon after mineralizat...
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Abstract
The present invention provides a culture medium and a culture method for culturing and / or amplifying mesenchymal stem cells. According to the culture medium, 2-5% of by volume of an autologous bone marrow blood plasma is added to a base cell culture medium to form the culture medium for culturing and / or amplifying mesenchymal stem cells. The present invention further provides a culture method for culturing and / or amplifying mesenchymal stem cells by using the culture medium. With the culture medium and the culture method, patient pain (additionally requiring serum drawing) can be reduced, no zoonosis can be achieved, the heterologous protein reaction (fetal bovine serum) can be avoided, the unnecessary expense (stimulating factor) can be reduced, and the culture medium and the culture method are completely safe and effective, and meet requirements of clinical cell transplantation.
Description
technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a medium for cultivating and / or expanding mesenchymal stem cells and a culturing 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. Certain stem cell-based treatments, such as hematopoietic stem cell transplantation for leukemia and epithelial stem cell therapy for burns and corneal disease, have established clinical standards. In recent years, advances in stem cell research have greatly expanded the range of applications for stem cell therapy. As of October 18, 2010, there were 3,248 stem cell therapy clinical trials officially registered at the National Institutes of...
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