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Mesenchymal stem cells, as well as preparation method and application thereof

A technology of stem cells and adherent cells, applied in the field of cell engineering, can solve the problems of infection with bovine virus, increase allergic reactions, affect cell viability, etc., and achieve the effect of reducing the probability of occurrence, low cytotoxicity, and increasing safety.

Inactive Publication Date: 2012-06-27
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a small amount of bovine serum albumin and porcine pancreatin are mixed in the mesenchymal stem cell products prepared by the existing method, both of which are definite high allergens, and the mesenchymal stem cell suspension prepared by this method will produce corresponding antibodies, Reduce the efficacy of mesenchymal stem cell therapy and increase the occurrence of allergic reactions, and there is a potential risk of infection with bovine viruses including bovine cerebral spongiform disease
[0005] In addition, studies by Furlani D et al. have shown that more than 10% of the mesenchymal stem cells cultured with fetal bovine serum have a cell diameter greater than 45 μm, and the larger the cell diameter, the more obvious the blocking effect on the microcirculatory capillary bed. High-dose intravenous infusion test conditions Severe cases can lead to death of mice, which poses a potential threat to patients using mesenchymal stem cells for regenerative therapy
At the same time, during subculture, pig trypsin digests more targets, which is highly toxic to mesenchymal stem cells, which in turn affects the viability of cells

Method used

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  • Mesenchymal stem cells, as well as preparation method and application thereof
  • Mesenchymal stem cells, as well as preparation method and application thereof
  • Mesenchymal stem cells, as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Preparation of mesenchymal stem cells using the preparation method of the present invention

[0031] Umbilical cord delivery: Select cesarean section umbilical cords that are negative for hepatitis, syphilis, AIDS and other infectious diseases and have no obstetric complications. The informed consent of the puerpera is obtained before the operation and the informed consent form is signed. A 250ml sterile polystyrene storage bottle was prepared before the operation as an umbilical cord transport bottle, which contained 100ml αMEM medium (Gibco company product) with 1000U heparin sodium and 100U penicillin / streptomycin. The 10-30cm long umbilical cord is bottled and sent to the laboratory within 2 hours at 4°C.

[0032] Umbilical cord digestion: After the umbilical cord was transported to the laboratory, it was washed twice with phosphate buffered saline PBS (Gibco company product) to remove blood and amniotic fluid. Umbilical cord processing was performed in ...

Embodiment 2

[0035] Example 2: Immunophenotype detection of mesenchymal stem cells

[0036] Prepare P0 generation mesenchymal stem cell suspension, adjust the cell density to 4×10 5 cells / ml, add 0.5ml cell suspension to each tube, add PE-labeled anti-CD29, CD105, CD73, CD44, HLA-DR, HLA-ABC, CD34, CD45, CD31 monoclonal antibodies to each tube, FC500 flow cytometer test, see the result figure 1 . Depend on figure 1 It can be seen that the immunophenotype of the mesenchymal stem cells prepared in the present invention fully conforms to the recognized phenotype of mesenchymal stem cells.

Embodiment 3

[0037] Example 3: Detection of differentiation potential of mesenchymal stem cells

[0038] 1. Adipocyte differentiation

[0039] P0 generation of mesenchymal stem cells by 5 × 10 3 piece / cm 2 The cell density was inoculated into 6-well plates, and the complete medium for inducing differentiation was low-sugar DMEM medium supplemented with 1 μM dexamethasone, 500 μM isobutylmethylxanthine, 60 μM indomethacin, 5 mg / L insulin and 10% fetal bovine serum. Change the induction differentiation medium every 3 days. Induce the culture for 21 days, drain the culture solution, fix with 10% neutral formaldehyde for 1 hour, take 1ml of 0.16% Oil Red O and stain for 20 minutes. Aspirate the dye solution, wash quickly with 70% ethanol once, add 1ml PBS and observe under the microscope. Microscopic examination results showed that the prepared mesenchymal stem cells could differentiate into adipocytes.

[0040] 2. Osteoblast differentiation

[0041] P0 generation of mesenchymal stem cel...

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Abstract

The invention relates to the field of cell engineering and discloses mesenchymal stem cells, as well as a preparation method and an application thereof. The method comprises the following steps of cutting an umbilical cord into small pieces after removing an umbilical artery and umbilical veins, digesting with an alpha MEM (minimum essential medium) culture medium containing 0.1% of type IV collagenase and a complete medium, then centrifugating cell suspension obtained after digestion, removing supernatant liquid, then washing, further performing resuspension with the complete medium, transferring suspension after resuspension into a culture bottle coated by a wall-adhered matrix for culture till the emergence of shuttle-shaped wall-adhered cells, washing, and replacing the new complete medium for continuing the culture so as to get the shuttle-shaped wall-adhered cells after the culture, namely the mesenchymal stem cells. According to the preparation method disclosed by the invention, the culture medium without serum or foreign protein is used for preparation, a recombinase with a single component is used for passage, and the prepared mesenchymal stem cells are smaller in diameter, have no sensibiligen and can improve the vitality of the cells and increase the safety in regeneration treatment of a patient.

Description

technical field [0001] The invention relates to the field of cell engineering, and specifically provides a mesenchymal stem cell and a preparation method and application thereof. Background technique [0002] Mesenchymal stem cells were originally discovered in the bone marrow, and have attracted increasing attention because of their multi-lineage differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells. It has multi-directional differentiation potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and disease. [0003] In recent years, it has been reported that human umbilical cord Wharton's (transliteration: Wharton's) jelly is also rich ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61L27/38
Inventor 吴德沛陈广华孙爱宁
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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