Three-dimensional co-culture induction method of mesenchymal stem cells

A stem cell and co-culture technology, applied in the field of cell engineering, can solve the problems of separation of two co-culture cells, poor cell activity, long induction period, etc., and achieve the effect of increasing transformation rate, enhancing cell activity, and promoting growth and differentiation

Inactive Publication Date: 2015-12-09
深圳华毓造血干细胞研究有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a method for the induction of two-dimensional co-cultured mesenchymal stem cells in the prior art, which has defects such as long induction period, low induction rate, poor cell activity, and difficulty in separating two types of co-cultured cells. A three-dimensional co-culture induction method for mesenchymal stem cells that can improve the induction transformation rate of mesenchymal stem cells, enhance cell activity, and easily separate co-cultured cells

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  • Three-dimensional co-culture induction method of mesenchymal stem cells
  • Three-dimensional co-culture induction method of mesenchymal stem cells

Examples

Experimental program
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Embodiment 1

[0049] The three-dimensional co-culture induction method of mesenchymal stem cells provided by the present invention comprises the following steps:

[0050] 1. Isolation of mesenchymal stem cells and chondrocytes;

[0051] Air was injected into the marginal ear vein of New Zealand white rabbits to make them die. Use 75% alcohol to smear the limbs of the white rabbit to disinfect and reduce the pollution of rabbit hair.

[0052] 1) Isolation of mesenchymal stem cells;

[0053] Under sterile conditions, cut off the femur and coelom of the rabbit limbs, remove the skin tissue and retain the muscle connective tissue at the joint, and cut off the muscle tissue in the rest of the body. Soak the treated tissue in 75% alcohol, take it out after 30 min, wash it with sterile PBS, and place it in an ultra-clean bench. Expose the joint by cutting away the muscle at the joint with a sterile scalpel. Cut the joint with bone scissors, exposing the bone cavity. Use a disposable syringe t...

Embodiment 2

[0073] The difference from Example 1 is that Step 3 in this Example 2 is:

[0074] 1) Contains Fe 3 0 4 Preparation of granular sodium alginate solution A

[0075] Take magnetic nanoparticles Fe with a diameter of 20nm 3 0 4 Mix with sodium alginate in a 50ml centrifuge tube, initial Fe 3 0 4 The concentration is 10mg / ml. The mixed solution is shaken in an ultrasonic cleaner for 30 minutes, and then sterilized at 105°C for 20 minutes. It is prepared and used immediately.

[0076] 2) Embedding

[0077] Take the P3 generation mesenchymal stem cells in step 2, and resuspend to adjust the cell density to 2×10 7 each / ml, use a syringe to draw an equal volume of Fe-containing solution prepared in step 1). 3 0 4 Granules of sterile 20% W / V sodium alginate, mixed evenly with P3 generation mesenchymal stem cell suspension, at this time the cell density is 1×10 7 pc / ml, the final concentration of sodium alginate is 10% W / V, Fe 3 0 4 The concentration is 5mg / ml. Use a syring...

Embodiment 3

[0081] The difference from Example 1 is that step 3 in this example is:

[0082] 1) Contains Fe 3 0 4 Preparation of granular sodium alginate solution A

[0083] Take magnetic nanoparticles Fe with a diameter of 20nm 3 0 4 Mix with sodium alginate in a 50ml centrifuge tube, initial Fe 3 0 4 The concentration is 4mg / ml. The mixed solution is shaken in an ultrasonic cleaner for 30 minutes, and then sterilized at 105°C for 20 minutes. It is prepared and used immediately.

[0084] 2) Embedding

[0085] Take the P3 mesenchymal stem cells in step 2, and resuspend to adjust the cell density to 1×10 7 each / ml, use a syringe to draw an equal volume of Fe-containing solution prepared in step 1). 3 0 4 Granules of sterile 8% W / V sodium alginate, mixed with the suspension of P3 mesenchymal stem cells, and the cell density at this time was 5×10 6 pc / ml, the final concentration of sodium alginate is 4% W / V, Fe 3 0 4 The concentration is 2mg / ml. Use a syringe to absorb the mixtu...

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Abstract

The invention relates to a three-dimensional co-culture induction method of mesenchymal stem cells, wherein the induction method comprises the following steps: respectively acquiring mesenchymal stem cells and a target cell source; covering the mesenchymal stem cells and a magnetic oxide in gel beads A; covering the target cell source in gel beads B; and co-culturing the mesenchymal stem cells in the gel beads A and the target cell source in the gel beads B by virtue of an inducer so that the mesenchymal stem cells are converted into target cells, and then isolating the gel beads A and the gel beads B in a magnetic field, wherein the magnetic oxide particles are 1-100nm in diameter. The induction method disclosed by the invention not only can achieve a purpose of simplifying the isolation of co-cultured cells, but also can expand a growth space of the mesenchymal stem cells as well as substance signal exchange between a culture solution and the cells, promote the growth and the differentiation of the mesenchymal stem cells, improve a transformation rate and enhance cell activity by virtue of the three-dimensional spatial structure of an alginic acid gel body.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a three-dimensional co-culture induction method of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are a kind of cells with multi-lineage differentiation potential, which can be directional induced into various cells such as islet-like cells and chondrocytes. Moreover, mesenchymal cells exist in various tissues of the human body, especially mesenchymal stem cells derived from umbilical cord, placenta, bone marrow, and adipose tissue. Target cells required for treatment, such as chondrocytes, islet cells, etc. However, at present, the method of static two-dimensional co-cultivation of mesenchymal stem cells and target cells is often used to transform mesenchymal stem cells into target cells. The surrounding mesenchymal stem cells differentiate, but this induction method is difficult to effectively identify the source of the target cells in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077
Inventor 曾宪卓鲁菲
Owner 深圳华毓造血干细胞研究有限公司
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