Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids

A technology of cell culture and culture substrate, which is applied in the direction of cell culture active agent, cell culture support/coating, tissue cell/virus culture device, etc. It can solve the problem of lack of mass production, inappropriate spheroids, and inability to form spheroids problems such as cells, and achieve the effect of excellent cell survival rate, uniform size and high survival rate

Pending Publication Date: 2021-05-25
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such a cell culture substrate provided with fine unevenness has a problem that it lacks mass productivity and is not suitable for use in forming a large number of spheroids.
In addition, the spheroids that can be formed are limited to spheres, and there is a problem that spheroids of shapes other than spherical cannot be formed.

Method used

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  • Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids
  • Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids
  • Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)thio]valeric acid, 7.11 g (50 mmol) of n-butyl methacrylate, azo 33 mg (0.2 mmol) of bis(isobutyronitrile) was dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the reaction was carried out at 70°C for 24 hours. After the completion of the reaction, the reaction solvent was distilled off under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrated solution was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

[0155]0.9 g (0.3 mmol) of the aforementioned n-butyl methacrylate polymer, 8.14 g (72 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added to the test tube, and dissolved in 1,4 - Dioxane in 15 mL. After degassing by nitrogen bubbling for 30 minutes, the reaction wa...

Embodiment 2

[0161] As the metal mask in Example 1, a metal mask having a plurality of rectangular holes of 0.5 mm×1 mm was used, and a cell culture substrate was produced in the same manner as in Example 1.

[0162] The cells were cultured in the same manner as in Example 1, and 24 hours, 96 hours, and 144 hours after the inoculation of the cells, the state of the cells was observed with a phase contrast microscope, and it was confirmed that the cells adhered along the patterned shape in all cases. Attached to the proliferation, replaced with a new aforementioned medium. After 144 hours from cell seeding and after medium change, the substrate was cooled at room temperature for 20 minutes, and the side of the substrate was tapped by hand. Rolled spheroids that are uniform in size and elongated in shape can be recovered.

Embodiment 3

[0164] As the metal mask in Example 1, a metal mask having a plurality of circular holes having a diameter of 0.5 mm was used, and the same procedure as in Example 1 was used to produce a cell culture substrate.

[0165] The cells were cultured in the same manner as in Example 1, and 24 hours, 96 hours, and 144 hours after the inoculation of the cells, the state of the cells was observed with a phase contrast microscope, and it was confirmed that the cells adhered along the patterned shape in all cases. and proliferation, and replaced with a new aforementioned medium. After 144 hours from cell seeding and medium exchange, the substrate was cooled at room temperature for 20 minutes and incubated again at 37°C for 1 hour. With cooling, the colonies start to peel from the periphery to form spheroids, and it is possible to obtain spheroids adhered to the substrate.

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PUM

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Abstract

Provided are: a cell culture substrate that enables efficient spheroid formation of cells and can form spheroids of uniform size and desired shape at a high cell survival rate; a method for producing the cell culture substrate; and a spheroid production method in which the cell culture substrate is used, the method being such that the cell survival rate inside the spheroids is exceptional. A cell culture substrate having a substrate and a stimulus-responsive polymer coated on the substrate, wherein the cell culture substrate is characterized in that the stimulus-responsive polymer is a block copolymer having a water-insoluble block segment and a stimulus-responsive block segment, and the cell culture substrate has the following two regions (A) and (B). (A) An island-shaped region having cell proliferation properties and stimulus responsiveness, the area of said region being 0.001-5 mm2. (B) A region having no cell proliferation properties, said region being adjacent to region (A).

Description

technical field [0001] The present invention relates to a cell culture substrate capable of efficiently forming spheroids and capable of forming spheroids of uniform size and arbitrary shape; a method for producing the cell culture substrate; and production of spheroids using the cell culture substrate method. Background technique [0002] Pluripotent stem cells such as embryonic cells (ES cells) and artificial cells (iPS cells) are cells that have the ability to differentiate into various tissues of the living body (differentiation totipotency), and are used as a source of cells for regenerative medicine and drug discovery screening received widespread attention. In order to apply pluripotent stem cells to regenerative medicine and drug discovery screening, pluripotent stem cells must be differentiated into target cells, and spheroids (embryoids) of pluripotent stem cells need to be formed. In addition, although pluripotent stem cells can differentiate into various cells,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12N5/0735C12N5/10
CPCC12M23/04C12M23/20C12N5/0696C12N2513/00C12N2533/30C12N2535/00C12N2501/727C08F265/06C08F220/54C12M25/14C12N11/08C12N2539/10
Inventor 久野豪士今泉裕
Owner TOSOH CORP
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