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Bioreactor for inducing three-dimensional directional differentiation in vitro of stem cells by virtue of non-contact coculture

A bioreactor, a technology for inducing cells, applied in specific-purpose bioreactors/fermenters, bioreactor/fermenter combinations, methods of supporting/immobilizing microorganisms, etc., can solve the problems of scale, efficiency and cost. The actual growth conditions of the cells are quite different, and the cells lose their normal biological properties, so as to avoid expensive materials and processes, improve the induction effect, and reduce the cost.

Inactive Publication Date: 2014-06-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cells in the Transwell system grow in a two-dimensional manner, which is quite different from the real growth of cells in vivo. A large number of studies have shown that the two-dimensional culture system in vitro makes the cells lose their normal biological properties in vivo, while the three-dimensional culture in vitro is better. maintain cell function
In addition, the Transwell system is based on a 24-well plate or a 6-well plate. The scale of the system is small, the price is expensive, the action area of ​​the semi-permeable membrane is limited, there is no fluid flow inside, and there is a serious substance concentration gradient, which makes the existing Transwell system in The scale, efficiency and cost cannot meet the large demand of future clinical applications

Method used

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  • Bioreactor for inducing three-dimensional directional differentiation in vitro of stem cells by virtue of non-contact coculture
  • Bioreactor for inducing three-dimensional directional differentiation in vitro of stem cells by virtue of non-contact coculture
  • Bioreactor for inducing three-dimensional directional differentiation in vitro of stem cells by virtue of non-contact coculture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Non-contact dynamic co-culture induces three-dimensional directed differentiation of mesenchymal stem cells into neural precursor cells in vitro

[0053] First, prepare arginyl-glycyl-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine (IKVAV), tyrosine-isoleucine Sodium alginate modified with acid-glycine-serine-arginine (YIGSR) polypeptide. Sodium alginate (molecular weight 430kDa, ratio of guluronic acid to mannuronic acid 1.5) was dissolved in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing 0.5M NaCl (pH value 6.5), to obtain a 1% (W / V) sodium alginate solution. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and a single type of polypeptide, and stir at room temperature for 24 Hour. The molar ratio of EDC to sodium alginate is 1:20, the molar ratio of EDC to sulfo-NHS is 2:1, and the mass ratio of polypeptide to sodium alginate is 1:1000. Then dialyzed and freeze-dried to o...

Embodiment 2

[0059] Example 2: Non-contact dynamic co-culture induces three-dimensional directed differentiation of mesenchymal stem cells into myocardial progenitor cells in vitro

[0060] First, arginyl-glycyl-aspartic acid (RGD) modified sodium alginate powder was prepared according to the method in Example 1. Then the RGD-modified sodium alginate powder was dissolved in normal saline, and the concentration of the solution was 1.5% (W / V).

[0061] Mix C2C12 cells with 1.5% (W / V) RGD-modified sodium alginate solution, and adjust the cell density to 2×10 6 cells mL -1 , the cell suspension was dripped into 100mmol·L via a syringe pump -1 CaCl 2 The solution was calcified for 20 minutes to obtain calcium alginate microgel beads. The calcium alginate microgel beads were reacted with 0.5% (W / V) chitosan with a molecular weight of 60,000 to form a film for 10 minutes to form a non-liquefied sodium alginate / chitosan microcapsule film, and then mixed with 0.15% (W / V ) sodium alginate solu...

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Abstract

The invention relates to a bioreactor for inducing three-dimensional directional differentiation in vitro of stem cells by virtue of non-contact coculture. The bioreactor is a container for cell culture; one or more than two separation meshes are arranged in the container so that the container is divided into more than two chambers; inducing cell embedded sodium alginate microcapsules and stem cell embedded calcium alginate microgel beads are arranged in different chambers, so that non-contact dynamic coculture of two or more than two types of cells under a three-dimensional growth condition in the same system is realized; the reactor is used for promoting the directional differentiation of the stem cells under the three-dimensional growth condition by virtue of an inducing factor secreted by the inducing cells in the microcapsules; after the completion of the differentiation, the microgel beads are dissolved by virtue of a sodium citrate solution, and finally, the differentiated cells can be obtained. The bioreactor is simple to operate and low in cost; the separation meshes in the bioreactor enable the microcapsules and the microgel beads to be separated from each other thoroughly, and then the two carriers are prevented from being mixed together.

Description

technical field [0001] The invention relates to the field of stem cells and tissue engineering, and is a bioreactor for inducing three-dimensional directional differentiation of stem cells in vitro by non-contact co-culture. Background technique [0002] Stem cells are stem cells with self-renewal ability and multi-directional differentiation potential. They can differentiate into nerve, cardiac muscle, liver, chondrogenic and other cell types. They are ideal seed cells for repairing damaged tissues in the body. They are used in cell therapy, plastic surgery and tissue engineering. It has important research and clinical application value. Studies have shown that when stem cells are directly implanted into damaged tissues, the repair effect is not ideal, and often accompanied by complications, such as arrhythmia. This is because the microenvironment of damaged tissue has been destroyed, the ability of stem cells to recognize this microenvironment is reduced, and the efficien...

Claims

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Application Information

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IPC IPC(8): C12M3/00C12M1/04
CPCC12M23/34C12M25/14C12M25/16C12M29/12
Inventor 马小军刘洋刘畅孙广炜于炜婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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