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Kit and method for fast separating bone marrow mesenchymal stem cells

A bone marrow mesenchymal and stem cell technology, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of long primary culture time, complicated culture medium preparation, and low cell purity, so as to ensure Effect of Growth and Purification Effects

Inactive Publication Date: 2011-05-04
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some deficiencies in the existing experiments, such as the preparation of the culture medium is too complicated, the primary culture time is still too long, and the purity of the cells is not high.

Method used

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  • Kit and method for fast separating bone marrow mesenchymal stem cells
  • Kit and method for fast separating bone marrow mesenchymal stem cells
  • Kit and method for fast separating bone marrow mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Preparation of a kit for isolating bone marrow mesenchymal stem cells

[0030] 1. Washing solution: take 7.9g NaCl, 0.2g KCl, 0.24g KH 2 PO 4 , 1.8g K 2 HPO 4 , the volume was adjusted to 1000ml with deionized water, 0.01M phosphate buffer was prepared by autoclaving, the pH was adjusted to 7.2-7.4, and it was placed in a 4°C refrigerator for later use.

[0031] 2. Cell culture medium: mix α-MEM liquid medium and sterile fetal bovine serum culture medium according to the volume ratio of 9:1 to prepare the cell culture mixture used in cell culture.

[0032] 3. Cell digestion solution: prepare a trypsin solution with a mass / volume percentage concentration of 0.25%.

Embodiment 2

[0033] Example 2. Packing of the kit for isolating bone marrow mesenchymal stem cells

[0034] The specification of the kit is 1 time / box, and the amount of each component in each box: 1 bottle of washing solution (100ml / bottle), 1 bottle of cell culture solution A (90ml / bottle), 1 bottle of cell culture solution B (10ml / bottle) bottle), 1 bottle of cell digestion solution (100ml / bottle). Each component in the kit is packaged independently according to the above dosage to obtain a kit for rapidly isolating bone marrow mesenchymal stem cells.

Embodiment 3

[0035] Example 3, Rapid Separation of Bone Marrow Mesenchymal Stem Cells

[0036] (1) Isolation and primary culture of bone marrow mesenchymal stem cells

[0037] Take 1 clean-grade SD rat aged 4 weeks, and kill it by cervical dislocation. Separate the rat femur and tibia under aseptic conditions, cut them off from both ends of the femoral shaft and tibial shaft, and rinse the bone marrow cavity repeatedly with washing liquid. The washing liquid passed through 200 mesh A stainless steel standard sieve was used to filter out large clumps, and then the cell suspension was obtained by pipetting and mixing thoroughly. Collect the cell suspension and transfer it to a 15ml centrifuge tube at 1200r / min, centrifuge for 5min. Discard the supernatant, add 3ml of cell culture medium to resuspend, and inoculate at 25cm 2 placed in a culture flask at 37°C with a volume fraction of 0.05 CO 2 cultured in a humidified incubator. Half of the medium was changed 24 hours after inoculation. ...

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Abstract

The invention provides a kit and method for fast separating bone marrow mesenchymal stem cells. The kit comprises a cleaning solution, a cell culture solution A, a cell culture solution B and a cell dissociation solution, wherein the cleaning solution is a phosphate buffer solution; the cell culture solution A is an alpha-MEM liquid culture medium; the cell culture solution B is fetal calf serum; and the cell dissociation solution A is a trypsin solution with the mass / volume percentage concentration of 0.25%. Compared with the traditional separation culture method, the method comprises the following steps of carrying out adherent culture for 24 hours with the culture solutions by adopting the kit and utilizing bone marrow emigrated cells and then culturing and purifying in a half solution changing way, therefore, obtained cells have high purity and are faster to proliferate and primary cell culture can be completed for 7 days generally; and in addition, primary cells are subjected to passage and cultured by utilizing the culture solutions, thus the cell density can reach 90 percent after 2-3 days, and rich sources are provided for carrying out further experimental research by utilizing the bone marrow mesenchymal stem cells.

Description

technical field [0001] The invention belongs to the field of tissue engineering, and in particular relates to a kit for rapidly isolating bone marrow mesenchymal stem cells, an application of the kit, and a method for rapidly isolating bone marrow mesenchymal stem cells. Background technique [0002] Bone marrow mesenchymal stem cells are a type of adult stem cells with multi-directional differentiation potential, which can be differentiated into osteoblasts, chondrocytes, adipocytes, nerve cells, liver cells and other tissue cells. They are clinically used in tissue engineering, gene therapy, cytokine replacement therapy and other fields. However, bone marrow mesenchymal stem cells account for only 0.001% to 0.01% of the total number of nucleated cells in the bone marrow, and decrease with age. The prerequisite for further experiments using bone marrow mesenchymal stem cells is to quickly achieve the expansion and isolation of bone marrow mesenchymal stem cells. [0003] ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 杨仕明秦贺赵立东翟所强郭维维孙建和杨伟炎
Owner GENERAL HOSPITAL OF PLA
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