Method for inducing in vitro directional differentiation of stem cells by cell co-culture

A culture method and stem cell technology, applied in the field of cell co-culture induced stem cell directional differentiation in vitro, can solve the problems of induced cell and stem cell contamination, high price, and inability to apply clinically, avoid expensive materials and processes, and improve the differentiation effect. The effect of differentiation efficiency

Active Publication Date: 2014-09-17
源创吉因(重庆)细胞应用技术研究院有限公司
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Problems solved by technology

Induced cells and stem cells are mixed and cultured in a direct contact co-culture system. Although this system is closer to the microenvironment of in vivo cells, the induced cells and stem cells are contaminated with each other and cannot be used in clinical practice.
Although the Transwell co-culture system uses a porous flat membrane to separate the two types of cells, which avoids the problem of cell separation and reflects the interaction between cells and cells and between cells and factors, it lacks the influence of extracellular matrix components and stiffness. Moreover, the concentration distribution of soluble factors between the two types of cells cannot be adjusted, thus limiting the differentiation efficiency of stem cells and the improvement of the fun

Method used

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  • Method for inducing in vitro directional differentiation of stem cells by cell co-culture
  • Method for inducing in vitro directional differentiation of stem cells by cell co-culture
  • Method for inducing in vitro directional differentiation of stem cells by cell co-culture

Examples

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Example Embodiment

[0040] Example 1: Cell co-culture promotes the directional differentiation of human mesenchymal stem cells into neural precursor cells

[0041] The 4th generation human umbilical cord mesenchymal stem cells were divided into 2×10 4 cells / cm 2 Inoculate the inner area of ​​the ring gasket with an inner diameter of 35mm, the thickness of the gasket is 300μm, and add DMEM culture medium containing 10% FBS to culture overnight.

[0042] The agarose powder was added to water, and the pressure was kept at 121 degrees Celsius for 20 minutes to obtain a 3% agarose storage solution (W / V, g / ml). Sodium alginate (molecular weight 430kDa, guluronic acid to mannuronic acid ratio 1.5) powder was dissolved in physiological saline to obtain a 1.5% (W / V, g / ml) solution. Dilute the preheated 3% (W / V, g / ml) agarose storage solution and 1.5% (W / V, g / ml) sodium alginate solution with water to prepare the final agarose concentrations of 0.35, 0.7 and 1.4%, respectively (W / V, g / ml) mixed solution, the f...

Example Embodiment

[0044] Example 2: Cell co-culture promotes the directional differentiation of human embryonic stem cells into cardiomyocyte progenitor cells

[0045] The 32-generation human embryonic stem cell line H9 cells were inoculated into the inner area of ​​a circular gasket with an inner diameter of 35mm, the thickness of the gasket was 100, 500, and 1000 μm, respectively, and DMEM culture medium containing 10% FBS was added to culture overnight.

[0046] Add the agarose powder to the water and press at 121 degrees for 20 minutes to obtain a 3% agarose storage solution (W / V, g / ml). Sodium alginate (molecular weight 430kDa, guluronic acid to mannuronic acid ratio 1.5) powder was dissolved in physiological saline to obtain a 1.5% (W / V, g / ml) solution. Dilute the preheated 3% (W / V, g / ml) agarose storage solution and 1.5% (W / V, g / ml) sodium alginate solution with water to prepare the final agarose concentration of 0.7 (W / V, g / ml), the final concentration of sodium alginate is 0.4% (W / V, g / ml)...

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Abstract

The invention relates to a method for inducing in vitro directional differentiation of stem cells by cell co-culture. An annular gasket is arranged on a bottom of a culture dish. Firstly, stem cells are inoculated in an annular zone and are cultured in an incubator. When the stem cells attach a culture dish wall, a mixed solution containing agarose and sodium alginate is added to the annular zone. When the mixed solution is solidified, a calcium chloride solution is added for a carrying out a calcification reaction and a chitosan is added for carrying out a reaction to form an agarose/sodium alginate/chitosan flat composite gel layer on the stem cells. Inductive cells are inoculated on a surface of the composite gel. By means of the co-culture of the inductive cells and the stem cells, interactions among in vivo cells and between cells and soluble factors can be simulated. By means of the polysaccharide-based agarose/sodium alginate/chitosan flat composite gel, an in vivo extracellular matrix can be simulated. The directional differentiation of the stem cells can be adjusted and controlled through rigidity and thickness of the composite gel. Meanwhile, isolated co-culture of the stem cells and the inductive cells can be achieved so that differentiated cells are easy to collect. The method has an important effect in application of regenerative medicine.

Description

technical field [0001] The invention relates to the field of regenerative medicine, and relates to a method for co-cultivating cells to induce directional differentiation of stem cells in vitro. Background technique [0002] Stem cells are ideal seed cells for regenerative medicine due to their ability to self-renew and differentiate into various types of cells such as nerve, cardiac muscle, liver, islet, osteoblast, cartilage or fat. However, due to the uncontrollable spontaneous differentiation of stem cells, when stem cells are implanted into damaged tissues in the body, stem cells not only differentiate into the required cell types and participate in tissue repair, but also differentiate into other types of cells, and even have high tumorigenicity. sex. Pre-differentiation of stem cells in vitro, so that they can be pre-differentiated into the desired type of cells or their precursor cells before transplantation, can improve the efficiency of directed differentiation, r...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/0775C12N5/0797C12N5/0789C12N5/071C12N5/079C12N5/077
Inventor 马小军刘洋连建春孙广炜贺欣
Owner 源创吉因(重庆)细胞应用技术研究院有限公司
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