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Method for inducing in vitro directed differentiation of stem cells through non-contact coculture

A technology for inducing cells and stem cells, applied in the field of non-contact co-culture induced stem cells in vitro directed differentiation, which can solve the problems of difficult separation and purification of stem cells, limited area of ​​semi-permeable membrane, and increased risk of clinical application, etc. Directed differentiation, easy suspension effect

Inactive Publication Date: 2011-06-22
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods have the following problems: (1) the chemical reagents contained in the conditioned medium have certain toxicity, which can cause stem cells to mutate and increase the risk of clinical application; (2) direct contact with co-culture will induce cells and stem cells to be in the same Mixed together in the culture system, the stem cells cannot be completely separated from the induced cells, so there are problems such as difficulty in separation and purification of stem cells, poor immune safety, and easy virus transmission, which is not suitable for clinical application; (3) Although the Transwell membrane separation co-culture method has passed The semipermeable membrane separates the induced cells from the stem cells, and the two cells are not in direct contact. However, the Transwell system is based on a 24-well plate or a 6-well plate. Not suitable for large-scale induction of directed differentiation of stem cells in vitro

Method used

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  • Method for inducing in vitro directed differentiation of stem cells through non-contact coculture
  • Method for inducing in vitro directed differentiation of stem cells through non-contact coculture
  • Method for inducing in vitro directed differentiation of stem cells through non-contact coculture

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Non-contact co-culture induces bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro

[0032] Sodium alginate-polylysine microcapsules embedded with BMP-2 gene transfected CHO cells were prepared by electrostatic droplet method. The prepared microcapsules were added to the cell culture medium and cultivated in an incubator (see literature: Optimization of the Seeding Density in Microencapsulated Recombinant CHO Cell Culture. Ying Zhang, Jing Zhou, Xulang Zhang, Weiting Yu, Xin Guo, Wei Wang , Xiaojun Ma.Chemical and Biochemical Engineering Quarterly, 2008, 22(1): 105-111), the culture medium was regularly replaced every 3 days.

[0033] Microencapsulated BMP-2 transgenic cells were mixed with 1×10 4 cells / cm 2 Bone marrow mesenchymal stem cells seeded at a density of two-dimensional adherent culture were co-cultured according to the above culture conditions, and bone marrow mesenchymal stem cells cultured alone were used as a control...

Embodiment 2

[0034] Example 2: Non-contact co-culture induces embryonic stem cells to differentiate into cardiomyocytes in vitro

[0035] Sodium alginate-chitosan microcapsules embedded with human cardiac muscle cell line HCM were prepared by air jet method. The prepared microcapsules were added to the cell culture fluid, and cultivated in an incubator, and the culture fluid was regularly replaced every 3 days, and the culture conditions were the same as in Example 1.

[0036] Microencapsulated HCM cells were mixed with 1 × 10 4 cells / cm 2 Two-dimensional adherent cultured mouse embryonic stem cells inoculated at a density of 100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000ss to h See the experimental results Figure 5 . The results showed that the mouse embryonic stem cells in the co-culture group had differentiated into cardiomyocytes, which was significantly different from that in the control group.

Embodiment 3

[0037] Example 3: Non-contact co-culture in a bioreactor induces bone marrow mesenchymal stem cells to differentiate into three-dimensional neural tissues in vitro

[0038] Sodium alginate-chitosan microcapsules embedded with SD rat olfactory ensheathing cells were prepared by electrostatic droplet method. The prepared microcapsules were added to the olfactory ensheathing cell culture medium, cultured in an incubator, and the culture medium was regularly replaced every 3 days. The culture conditions are the same as in Example 1.

[0039] Microencapsulated olfactory ensheathing cells were mixed with 2×10 6 cells / cm 3 SD rat bone marrow mesenchymal stem cells seeded at a density of 3D chitosan scaffolds were co-seeded into a rotary bioreactor for dynamic co-culture. On the 20th day of dynamic co-culture, the cells in the scaffold material Perform βIII-tubulin / Hoechst33342 immunofluorescence detection. See the experimental results Figure 6 . The results showed that the bon...

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Abstract

The invention relates to the field of stem cell and tissue engineering, and discloses a method for inducing in vitro directed differentiation of stem cells through non-contact coculture. The method comprises the following steps of: embedding inducing cells with function of inducing differentiation in biological microcapsules; and coculturing the microcapsules and the stem cells, so that the stem cells are subjected to directed induction and differentiation through the directed induction function of the inducing cells in the microcapsules. The method is easy to operate; and one or more kinds of dioecious and heterogeneous inducing cells are allowed to be used for promoting the in vitro directed differentiation of the stem cells, namely microcapsule films can make inducing factors secreted by the inducing cells permeate the microcapsules to act on the external stem cells, and the inducing cells and the stem cells can be subjected to imunoisolation and do not contact each other directly to facilitate the separation and harvest of different cells. By the method, in vitro directed induction and differentiation of the stem cells can be realized under the condition of conventional static culture, an effective induction method can be provided for dynamically inducing directed differentiation of the stem cells to form three-dimensional tissues in a biological reactor, and the method has important significance for clinical therapeutic research of damaged tissues.

Description

technical field [0001] The invention relates to the field of stem cell tissue engineering, in particular to a non-contact co-culture method for inducing directional differentiation of stem cells in vitro. Background technique [0002] Stem cells are a type of cells that come from embryos, fetuses or adults and have certain self-renewal, proliferation and differentiation capabilities under certain conditions. This type of cells can not only produce daughter cells that are identical in phenotype and genotype to themselves, but also It can produce specialized cells that make up body tissues and organs, and can also differentiate into progenitor cells. Therefore, stem cells are ideal seed cells for repairing damaged tissues in the body, and are extremely important in cell therapy, surgical plastic surgery, and tissue engineering. research and clinical application value. [0003] Stem cells can be classified into embryonic stem cells (ESC) and adult stem cells (ASC) according to...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/077C12N5/079
Inventor 马小军刘洋王为于炜婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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