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Non-contact three-dimensional co-culture method for cells

A non-contact, co-culture technology, applied in the field of cell and tissue engineering, can solve the problems of limiting the research and development of cell co-culture, the inability to control the molecular weight cut-off of the membrane, and the difficulty of expanding the scale, so as to achieve a wide range of applications, avoid expensive materials and Process, the effect of ensuring biosafety

Inactive Publication Date: 2013-05-15
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

[0005] In addition, cells grow in a three-dimensional state in vivo. Studies have shown that cells under traditional in vitro two-dimensional culture conditions cannot truly reflect the shape and function of cells in vivo. It can be seen that it is necessary to develop a variety of cells that can achieve three-dimensional An effective method for non-contact co-culture to regulate multiple behaviors of target cells in co-culture
[0006] However, at present, the common method of non-contact co-culture is to use transwell chamber, which has obvious disadvantages: 1) it is expensive, and the experimental cost increases; 2) it can only realize the co-culture of cells under two-dimensional conditions; 3) The molecular weight cut-off of the membrane cannot be controlled; 4) The transwell system is based on a 24-well plate or a 6-well plate, and it is not easy to expand the scale
Due to the defects of existing cell co-culture methods, which limit the development of cell co-culture research, it is urgent to develop a convenient and practical non-contact three-dimensional cell co-culture method

Method used

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  • Non-contact three-dimensional co-culture method for cells

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Co-culture system regulates three-dimensional differentiation of bone marrow mesenchymal stem cells into osteoblasts

[0034] First, prepare arginyl-glycyl-aspartic acid (RGD) modified sodium alginate. 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, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and RGD polypeptide were added, and the reaction was stirred at room temperature for 24 hours. 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 RGD to sodium alginate is 1:1000. Then, it is dialyzed and freeze-dried to obtain RGD-modified sodium alginate.

[0035] After that, the fifth-generation vascular endothelial cells from SD rats were mixed with 1.5% (W / V)...

Embodiment 2

[0039] Example 2: Co-culture system improves renal epithelial cell activity after treatment with chemotherapeutics

[0040] The preparation of RGD modified sodium alginate is the same as in Example 1.

[0041] The 5th generation of SD rat bone marrow mesenchymal stem cells were mixed with 200 μL of physiological saline solution of 1.5% (W / V) RGD modified sodium alginate, and the cell density was adjusted to 5×10 6 cells·mL -1 , 200 μL of the cell suspension was slowly added to the culture wells of the 24-well plate to form a liquid film. After that, slowly add 100mmol·L -1 CaCl 2 500 μL of the solution was calcified for 20 minutes to obtain a calcium alginate gel layer. Then, add 500 μL of 0.5% (W / V) chitosan (molecular weight 150kDa, degree of deacetylation 90%) solution, and react to form a film for 15 minutes. After that, wash 3 times with normal saline and add 0.5μmol·L containing chemotherapy drugs -1 200μL of 1.5% (W / V) RGD-modified sodium alginate solution of renal tubular ...

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Abstract

The invention relates to the fields like cells and tissue engineering and provides a non-contact three-dimensional co-culture method for cells. According to the method, a calcium alginate gel layer, a chitosan film layer and a calcium alginate gel layer are successively prepared in culture holes of a culture plate, so a sandwich type composite gel is formed; different cells are in a three-dimensional growth state in the calcium alginate gel layer at two sides of a chitosan film with controllable molecular weight cut off in the composite gel, a culture solution is added, so non-contact three-dimensional co-culture of different cells in a same culture system is realized, thereby allowing behaviors like propagation and differentiation of a target cell in the co-culture system to be regulated and controlled. The method provided by the invention has the advantages of simple operation and low cost, can realize three-dimensional co-culture of different cells which may influence each other and furthest simulates a complex in-vivo cell survival environment.

Description

Technical field [0001] The invention relates to the fields of cells, tissue engineering and the like, and is a non-contact cell three-dimensional co-cultivation method. Background technique [0002] Cell co-cultivation is an in vitro culture method established through some special culture techniques. It can simulate the in vivo tissue environment in vitro to the greatest extent, maintain the characteristics of a variety of cells in vivo, observe the interaction between cells, and regulate cell behavior. Because it retains the structure and function basis of the internal tissue microenvironment and reflects the controllability of in vitro culture conditions, it is a hotspot in tissue engineering research and has broad research and application prospects in scientific research and clinical fields. [0003] The methods to realize cell co-cultivation mainly include direct contact co-cultivation and non-contact co-cultivation. Direct contact co-cultivation is to mix different types of c...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/071C12N5/077C12N5/0775
Inventor 马小军宋益哲刘洋张英孙广炜王为于炜婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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