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Temperature-responsive substrate for cell culture and method for producing same

A technology for cell culture and manufacturing methods, applied in general culture methods, cell culture supports/coatings, tissue cell/virus culture devices, etc., to achieve the effect of simple production

Active Publication Date: 2013-05-01
TOKYO WOMENS MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the techniques has been able to achieve the same culture of cells as the conventional substrates for cell culture, and the same temperature-responsive substrates for cell culture produced using the above-mentioned electron beams. It is detached, or the technical level of detaching it as a cell sheet when cultured cells become confluent

Method used

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  • Temperature-responsive substrate for cell culture and method for producing same
  • Temperature-responsive substrate for cell culture and method for producing same
  • Temperature-responsive substrate for cell culture and method for producing same

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1: Preparation and Characterization of Temperature Responsive Surfaces

[0066] In this example, a temperature-responsive block copolymer comprising segments of poly-N-isopropylacrylamide (PIPAAm) as a temperature-responsive polymer and a water-insoluble Polymer segments of poly(n-butyl methacrylate) (PBMA) are spin-coated on the substrate surface. For the produced temperature-responsive block copolymer-coated surface, physical properties such as the amount of polymer incorporated into the surface and surface wettability were evaluated, and differences in cell adhesion to the substrate surface due to temperature changes were studied.

[0067] (1) Preparation of temperature-responsive block copolymers

[0068] Specifically, PBMA as a water-insoluble polymer synthesized by RAFT polymerization was used as a macromolecular RAFT agent to prepare a PBMA-b-PIPAAm block copolymer ( figure 1 ). In addition, the analysis values ​​of PBMA and PBMA-b-PIPAAm synthesized ...

Embodiment 2

[0072] Example 2: Research on grafting amount and cell behavior

[0073] According to the description of Example 1, the substrates coated with TCPS with various concentrations of B79-IP120 were prepared, and on the prepared substrates, 1.0×10 4 cells / cm 2 Vascular endothelial cells (BAEC) derived from bovine carotid artery were seeded at a concentration, and the adhesiveness of the cells on the surface of the polymer-coated substrate at 37°C and the detachment behavior of the cells by low temperature treatment at 20°C were evaluated. Figure 5 The black dots in the figure represent the TCPS substrate without polymer coating, and the black diamonds and white diamonds represent the changes of cell behavior on the substrates coated with B79-IP120 on TCPS at 0.3 and 0.5w / v%, respectively. Figure 5 BAEC cells are seeded on each substrate and cultured for 72 hours, and the change in the cell adhesion rate (%) is shown when the culture temperature is changed from 37°C to 20°C. Mor...

Embodiment 3

[0075] Example 3: Detailed Analysis of Cell Behavior Changes Caused by Block Copolymers

[0076] (1) Relationship between PIPAAm molecular chain length and cell behavior

[0077] The number of monomer units (number of repeating units) of BMA in the block copolymer was kept constant, and a block copolymer in which the monomer unit of IPAAm was changed was prepared according to the production method of Example 1. In the same manner as in Example 1, the block copolymer was coated with TCPS, and the change in the adhesion rate of BAEC cells accompanying the temperature change was examined ( Figure 7 ). As in Example 2 above, the cell adhesion rate increased as the amount of PIPAAm coated decreased. In addition, regarding the detachment of cells caused by temperature changes, it can be seen that the substrate with a low coating amount of PIPAAm (1.18 μg / cm 2 ), and substrates with higher coatings (1.44, 1.60, 1.63μg / cm 2 ) compared with the slow removal, the substrate with hig...

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Abstract

To form a temperature-responsive surface for cell culture by simple processes, said temperature-responsive surface for cell culture being capable of efficiently culturing cells. Cultured cells or a cell sheet can be efficiently removed from the temperature-responsive surface for cell culture by merely changing the temperature of a substrate surface. To cover a substrate surface with a block copolymer, which functions as a temperature-responsive polymer component, in an amount of 0.8-3.0 [mu]g / cm2, said block copolymer having a structure wherein a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded with each other.

Description

technical field [0001] The present invention relates to a cell culture substrate useful in fields such as biology and medicine. This application is an application claiming priority based on Japanese Patent Application (Japanese Patent Application No. 2010-208506) filed on August 31, 2010. Background technique [0002] Currently, animal cell culture technology has been significantly advanced, and research and development targeting animal cells is widely carried out in various fields. The method of using the target animal cells is not only to commercialize the cells themselves at the beginning of development, or to commercialize their products, but now to analyze the cells or their surface proteins to design useful drugs and proliferate patients in vitro One's own cells, or the method of improving the function of the cells and returning them to the patient's body for treatment are also being implemented. Now, the technology of cultivating animal cells has attracted the atten...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/04C08F293/00
CPCC08F293/005C12N5/0068C12N2539/10C08F2438/03
Inventor 中山正道冈野光夫
Owner TOKYO WOMENS MEDICAL UNIV
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