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Method for amplifying mensenchymal stem cell under three-dimensional dynamic condition

A bone marrow mesenchymal, three-dimensional dynamic technology, applied in medical science, prosthesis, etc., can solve the problems of reducing the quality of seed cells, uneven nutrient supply, cell aggregation, etc., to promote contact, increase the number of cell expansion, reduce The effect of mass transfer resistance

Inactive Publication Date: 2007-07-11
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the seed cells attached to the surface of the microcarriers will be damaged due to the shear force and the collision between the microcarriers, and the adsorption of the microcarriers to the components of the culture medium and metabolites will easily cause cell aggregation and cell differentiation (Yoo JU et al., J Bone Joint Surg Am, 1998, 80(12); Johnstone B et al., Clin Orthop, 1999, (367 Suppl): S156), reduced seed cell quality
Although the rotating wall culture system has low shear force, which reduces the damage to the cells caused by the shear force, there is a certain concentration gradient and the supply of nutrients is not uniform.

Method used

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  • Method for amplifying mensenchymal stem cell under three-dimensional dynamic condition
  • Method for amplifying mensenchymal stem cell under three-dimensional dynamic condition
  • Method for amplifying mensenchymal stem cell under three-dimensional dynamic condition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] This example is the culture of rabbit bone marrow mesenchyme and cells in type I collagen gel under static conditions.

[0029] In order to verify that cells can grow well in type I collagen gel, rabbit bone marrow mesenchymal stem cells were embedded in type I collagen gel for in vitro culture and expansion. Specifically, set the number of cells to 10 x 10 5 cells of rabbit bone marrow mesenchymal stem cells containing 3ng·mL -1 The concentration of bFGF is 3mg·mL -1 1 mL of collagen type I solution was mixed evenly, and inoculated into a 96-well culture plate, 50 μL per well. Set at 37°C, 5% CO 2 , saturated humidity CO 2 In the incubator for 2 hours to form a gel containing cells, add 20% fetal bovine serum, 3ng·mL -1 100 μL of DMEM culture solution of bFGF, at this time, the density of cells in each well is 5×10 5 cells mL -1 , set at 37°C, 5% CO 2 , saturated humidity CO 2 Cultured in an incubator. Another portion of bone marrow mesenchymal stem cell susp...

Embodiment 2

[0035] This example is the harvest rate of cells when enzymatically hydrolyzing collagen from rabbit bone marrow mesenchymal stem cells embedded in type I collagen gel.

[0036] Use 3mg·mL -1 Type I collagen gel was used to embed rabbit bone marrow mesenchymal stem cells, and the harvest rate of cells was determined by enzymatically hydrolyzing type I collagen with type I collagenase. Specifically, set the number of cells to 25 x 10 5 cells Rabbit bone marrow mesenchymal stem cells with 0.5mL concentration is 3mg·mL -1 The type I collagen solution was mixed evenly, inoculated into a 96-well plate, 50 μL per well, and placed at 37 ° C, 5% CO 2 , saturated humidity CO 2 A gel containing cells was formed in the incubator for 2 hours. Add 2.5% type I collagenase for enzymolysis at 37°C for 20 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 1 mL of culture medium, pipette, count, and calculate the cell harvest rate.

[0037] The above experiments were repea...

Embodiment 3

[0040] This example is the cultivation of rabbit bone marrow mesenchymal stem cells embedded in type I collagen gel in a hollow fiber membrane in an airlift loop bioreactor.

[0041] In order to verify that the ALB cell culture system can successfully culture stem cells, in this example, rabbit bone marrow mesenchymal stem cells embedded in type I collagen gel in the hollow fiber membrane were cultured in the ALB culture system.

[0042] Isolation of rabbit bone marrow mesenchymal stem cells: New Zealand white rabbits weighing about 2.5 kg at the age of four weeks were taken, and 3% pentobarbital sodium was used to press 1 mL kg -1 After intravenous anesthesia, under aseptic operation, use a bone marrow puncture needle to puncture the bone marrow cavity of the rabbit femur, and then connect a 10mL syringe containing 3000U·mL -1 0.5mL of heparin, extract 4-6mL of bone marrow, and dilute with low-sugar serum-free DMEM, with a specific gravity of 1.073g·mL -1 Gradient centrifuga...

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Abstract

The invention relates to a method for expanding the stem cell between marrows in three-dimension dynamic condition, wherein it is characterize in that: hollow fiber film uses I-type collagen gel to embed the marrow stem cell; then cultivates said cell in cyclone biological reactor to obtain the marrow stem cell. The invention has the advantages that: I-type collagen gel can provide better three-dimension growth condition for stem cell, to cultivate cell multilayer; the hollow fiber film can protect cell from flow shearing force and impact; the cyclone reactor can reduce the transmission resistance of cultivate system, to provide enough nourishment to cell.

Description

technical field [0001] The invention belongs to the field of biotechnology and tissue engineering, in particular to a method for expanding bone marrow mesenchymal stem cells under three-dimensional dynamic conditions. Background technique [0002] Seed cells are the most basic raw materials for tissue engineering. It is the most important link in tissue engineering research to cultivate, amplify and plant a small amount of cells on the extracellular matrix to form tissues with certain structures and functions. However, the source of most tissue cells is limited, and the separation is difficult, which limits the progress of tissue engineering research. [0003] Bone marrow mesenchymal stem cells are easy to obtain in vitro, and can highly self-proliferate and renew. It not only has the potential to differentiate into osteoblasts, chondrocytes, cardiomyocytes, adipocytes, fibroblasts, reticular cells, etc. (Pittenger MF et al., Science, 1999, 284 (1)), but also in When the b...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/48
Inventor 刘天庆李香琴崔占峰朱琳
Owner DALIAN UNIV OF TECH
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