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Culture method, group of mature adipocytes, and drug screening method

A technology of mature fat cells and culture methods, which is applied in the field of fat cell culture and can solve problems such as difficulties in differentiation and culture

Inactive Publication Date: 2014-02-26
KURARAY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, long-term differentiation culture, observation until final differentiation, or experiments on physiologically active substances released only by mature adipocytes are difficult.

Method used

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  • Culture method, group of mature adipocytes, and drug screening method
  • Culture method, group of mature adipocytes, and drug screening method
  • Culture method, group of mature adipocytes, and drug screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-3

[0099] [Examples 1-3, Comparative Example 1]

[0100] 1. Culture container

[0101] As a culture surface formed with a wall perpendicular to the culture bottom, it will have the above-mentioned figure 1 , A 1 cm x 1 cm membrane on the culture surface shown in 2 was pasted on the bottom of a well plate-shaped culture container to culture preadipocytes (3T3-L1).

[0102] In the examples, the part of the film was used as the culture surface, and in the comparative example, the flat part without the film was used as the culture surface.

Embodiment 1 and 2

[0104] As the culture surface having the walls 12 perpendicular to the culture bottom surface 14 used in Examples 1 and 2, two types of membranes having the partitioned regions 11 described below were prepared. In embodiment 1, figure 1 The distance a is 200μm, figure 2 The height c is 50 μm, in Example 2, figure 1 The distance a is 200μm, figure 2 The height c is 100 μm.

[0105] 1-2. Comparative example 1

[0106] In Comparative Example 1, the flat portion other than the 1 cm×1 cm membrane was used as the culture surface.

[0107] 2. Cell Culture and Observation

[0108] 2-1. Embodiments 1 and 2

[0109] Strain preadipocytes (3T3-L1) with 25cm 2 The flasks were subcultured, and when 70% confluency was reached, the cells were seeded into each well.

[0110] Here, confluence refers to the ratio of the area occupied by cells to the entire culture surface. For example, using a 25cm 2 If the cells occupy 50% of the area of ​​the flask, it is said to be 50% confluent. ...

Embodiment 1

[0125] Figure 11 Photographs of cells differentiated for 20 days in Example 1 are shown. Figure 11 In , the part surrounded by a circle is a cell. For the part enclosed by the quadrilateral, cells aggregated to form colonies. After 20 days of differentiation, cells were observed to aggregate near the center of the compartmentalized area.

[0126] In the observation up to 20 days after the differentiation, cells existed not only near the center of the microvessel but also near the wall (side wall) and filled the bottom. However, as the cells matured, the cells continued to aggregate near the center of the microvessels. Figure 11 In the photo taken at 40X magnification, since the fat droplets are overlapped and visible, it is observed that cells gather one by one near the center to form a block. This is presumed to be the self-organized formation of cell aggregates.

[0127] In addition, the size of one cell tended to be smaller than that of Comparative Example 1. This ...

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Abstract

In a culture method for adipocytes, seeded adipocytes are cultured while adhering the adipocytes to a culture bottom surface (14) and walls (12) positioned perpendicularly to the culture bottom surface (14), and mature adipocytes are obtained by generating spherical, enlarged fat droplets in cells without suspending the adipocytes in a culture medium. In the method, a culture vessel (10) is used in which the culture walls (12) are formed perpendicularly to the culture bottom surface (14). With cells or cell aggregates adhered in at least two places, namely the walls (12) and the culture bottom surface (14), spherical mature adipocytes having spherical enlarged fat droplets in cells are cultured. The adipocytes with a form close to in-vivo adipocytes are thus obtained.

Description

technical field [0001] The present invention relates to the culture of adipocytes, and in particular to a method for culturing adipocytes having spherical fat droplets in cells, a mature adipocyte population, and a drug screening method in a manner that can be easily used in experiments and the like. Background technique [0002] In recent years, it has gradually become clear that adipose tissue not only stores and releases nutrients, but also is an important organ that releases various physiologically active substances such as leptin. It is also gradually being recognized as an important causative organ of diabetes and various adult diseases. Therefore, adipocytes are being investigated as targets of drugs for the treatment of the above diseases. In the development or screening tests of such drugs, it is necessary to utilize cultured adipocytes. [0003] In the body, fibroblast-like precursor cells or preadipocytes contained in adipose tissue first form tiny fat droplets ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12Q1/02C12M3/00
CPCG01N33/5044C12M23/12C12M25/06C12N2513/00C12N5/0653C12M23/20C12M23/22C12N2501/33C12N2501/39C12N2503/02
Inventor 一丁田洋子细田雅也福田始弘永山昌史
Owner KURARAY CO LTD
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