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Culture substrate, culture sheet, and cell culture method

A technology of cell culture and equipment, applied in cell culture support/coating, 3D culture, tissue culture, etc., can solve the problems of loss, uneven size, high three-dimensional tissue stripping, etc., and achieve efficient culture methods and uniform size Effect

Inactive Publication Date: 2012-11-28
HITACHI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a device for forming a three-dimensional tissue of cells, a sheet for culture (nano-pillar sheet) in which extremely fine uniform protrusions are formed on the surface of a regularly arranged sheet has been developed, but the three-dimensional tissue formed is far from the device. The detachability on the surface is high (Patent Document 1), and there is a problem of loss in the process of medium exchange
In addition, since the diameter of the formed three-dimensional structure cannot be controlled, there are problems of uneven size and performance of each three-dimensional structure, and it is still immature as a practical formation method.

Method used

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  • Culture substrate, culture sheet, and cell culture method
  • Culture substrate, culture sheet, and cell culture method
  • Culture substrate, culture sheet, and cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 shows an example in which a culture sheet is applied to a chamber culture slide as a culture sheet holding member. Hereinafter, a sheet having a partition wall structure forming a culture region of the present invention and a plurality of protrusions formed inside the partition wall structure is referred to as a culture sheet compared to a conventional nanopillar sheet. The culture sheet is made of a material that does not adversely affect the cells, in this example polystyrene. However, the material is of course not limited to polystyrene.

[0060] figure 1 It is a schematic diagram of a scanning electron micrograph of the culture sheet 100 prepared in this example. Also, the structure of one of the wells 101 (hereinafter referred to as wells) constituted by a plurality of partition wall structures 102 existing in each culture sheet is also shown. The inside of the well 101 constitutes a culture area of ​​the cell tissue forming unit.

[0061] The plurali...

Embodiment 2

[0080] Next, according to Figure 4 , Figure 5 The second embodiment will be described. In Example 2, the configuration of a multiwell plate with a culture sheet and its production example are shown. Figure 4 (a) is a bottom view of the frame 111 constituting the perforated plate. The frame body 111 as a culture sheet holding member is formed into a cylindrical hole with a total of 24 holes with 4 holes in a row and 6 holes in a row with a width of about 125 mm, a length of about 80 mm, and a height of about 20 mm. Section 111a. The raw material uses polystyrene.

[0081] The number of holes formed in the frame is usually from 6 holes to 1536 holes depending on the application, so the frame is not limited to 24 holes. In addition, the material of the frame is not limited to polystyrene.

[0082] In the production of the culture equipment, the frame body 111 and the culture sheet 100 are joined by ultrasonic welding.

[0083] The following processing is performed on th...

Embodiment 3

[0109] Example 3 shows an example of application to cell tissue culture using the culture equipment produced in Examples 1 and 2. In the development of new drugs, the construction of three-dimensional tissues reflecting the functions of living bodies is required for various evaluations using cells instead of animal experiments.

[0110] In addition, since induced pluripotent stem cells (Induced pluripotent stem cells: iPS cells) or embryonic stem cells (Embryonic stem cells: ES cells) must be formed into three-dimensional tissues before they are cultured and differentiated into target cells, it is expected to be easily constructed in the field of regenerative medicine Techniques for three-dimensional organization. From this background, here is an example of forming a three-dimensional tissue using a chamber culture slide, but the essential part of cell culture is not particularly changed even in a multi-well plate. In this example, an example using rat hepatocytes was shown, ...

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Abstract

Disclosed is a culture sheet which enables technology in which three-dimensional tissues with uniform diameter are formed without applying chemicals to the surface of a culture substrate. A plurality of holes are formed on the culture sheet of the culture substrate, and nanopillars capable of controlling the adhesiveness or migration of a cell are formed on a culture surface that serves as the bottom surface of each of the holes. The culture surface of each of the holes having a structure in which a partition wall is provided, wherein, by forming the internal nanopillars in the vicinity of the center of each of the holes, the interaction of the disseminated cells can be limited to uniform the size of the three-dimensional structures of the cells to be formed.

Description

technical field [0001] The present invention relates to techniques for culturing animal and plant cells using culture equipment to form spherical tissues (three-dimensional tissues) and monolayer tissues (two-dimensional planar tissues) of cells. Background technique [0002] In the process of drug development, in vitro (in vitro) analysis using cells is required as an alternative to animal experiments. In particular, the screening of candidate drugs and the trend of toxicity and metabolism tests have become active. [0003] Based on this background, many studies using cell replacement methods that replace existing animal experiments have been attempted, but they are often limited in predicting clinical responses. The reason for this is considered to be that cells do not adopt a structure that mimics an actual in vivo structure in these culture methods (Non-Patent Document 1). Therefore, attempts have been made to construct three-dimensional tissues that function closer to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12N5/10
CPCC12N2535/10C12M23/12C12M25/06C12M21/08C12N5/0062
Inventor 高桥亮介久田明子园田浩齐藤拓
Owner HITACHI LTD
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