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Cytokine-producing cell sheet and method for using same

A cytokine and cell sheet technology, applied in biochemical equipment and methods, animal cells, vascular endothelial cells, etc., can solve the problems of cell necrosis, outflow, and inability to carry out effective transplantation, and achieve high production capacity and cardiac function recovery. high-capacity effect

Inactive Publication Date: 2013-12-25
TOKYO WOMENS MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cell transplant sheets disclosed in Patent Documents 2 and 3 are all methods of injecting a cell suspension, and the transplanted cells have the following problems: the transplanted cells flow out from the transplanted site, and it is difficult to control the transplanted site; Effective transplantation, etc., expect further improvement
However, until now, there has been no method for quantitatively and conveniently evaluating the vascular endothelial cell network constructed in three-dimensional living tissues when constructing three-dimensional living tissues including cell sheet stacks.
Therefore, no method has been reported for the optimal design of cell sheet laminates with vascular endothelial network

Method used

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  • Cytokine-producing cell sheet and method for using same
  • Cytokine-producing cell sheet and method for using same
  • Cytokine-producing cell sheet and method for using same

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Construction of a co-culture system of vascular endothelial cells and myoblast sheets (patent document: International Publication No. 2010 / 101225). figure 1 A method for quantitatively evaluating the formation of a vascular endothelial cell network constructed in a cell sheet is shown. The vascular endothelial cell (green) network constructed in the cell sheet is image-processed to deduce the length (L) of the network and the number of endpoints of the network (N T ). The system is calculated by dividing the length of the network (L) by the number of endpoints (N T ) (L / N T ), enabling comparison of the degree of construction of the vascular endothelial network.

[0076] (Analysis of the behavior of vascular endothelial cells in high-density and low-density myoblast sheets)

[0077] Using the above-mentioned system, cell sheets with different cell densities were co-cultured with endothelial cells, and the effects of different cell densities on the formation of endo...

Embodiment 2

[0098] (cytokine production capacity of myoblasts dependent on culture state)

[0099] The five-layer cell sheet is a three-dimensional tissue formed by three-dimensionally overlapping single-layer sheets formed by confluence, and it can be said that it is also in a state of dense confluence in three dimensions. Cells temporarily lose proliferative properties due to contact inhibition. In order to understand the cytokine production of sheet-forming cells cultured three-dimensionally in a confluent state, the characteristics of cytokines in a more simple system, that is, normal planar culture, were compared. The culture state can be regarded as a layered structure according to low-density, high-density, sheet, laminated sheet, dense culture of cells, flat and three-dimensional culture. Cytokine production per unit cell was investigated for this hierarchical structure.

[0100] Dilute myoblasts at 1.0×10 4 Cell number / cm 2 , 8.0×10 4 Cell number / cm 2 , 2.3×10 5 Cell numbe...

Embodiment 3

[0124] (for the network formation of endothelial cells in mixed sheets of myoblasts and fibroblasts)

[0125] A myoblast sheet and a myoblast / fibroblast mixed sheet were mounted on endothelial cells for co-culture. It is known that the properties of the cell sheet itself, such as sheet fluidity and cytokine secretion pattern, are adjusted by mixing of fibroblasts, and these are considered to impart adjustments to the surrounding environment to vascular endothelial cells. Therefore, research on the influence of hybrid slices on the formation of the network structure ( Figure 10 ).

[0126] Figure 11 Results of network formation using mixed sheets of skeletal muscle myoblasts and dermal fibroblasts are shown. At 48 hours of co-culture (i.e., 48 hours after the start of the co-culture of skeletal muscle myoblasts and dermal fibroblasts with vascular endothelial cells), 50% of myoblasts were mixed with 100% of myoblasts. % flakes can also form a dense multi-branched homogene...

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Abstract

The production of an angiogenesis-promoting cytokine can be optimized and the formation of a vascular endothelial network can be optimized by altering the kinds of cells constituting a layered cell sheet, the mixing ratio thereof and the number of the disseminated cells to thereby change the conditions of the cells in the layered cell sheet.

Description

technical field [0001] The present invention relates to a cell sheet that produces cytokines related to angiogenesis, a cell sheet that constructs a high-density vascular endothelial cell network, a method for producing the same, and a method for using the same, which are useful in the fields of drug research and development, pharmacy, medicine, and biology . This application claims priority based on Japanese Patent Application (Japanese Patent Application No. 2011-043198) filed on February 28, 2011. Background technique [0002] In recent years, various regenerative medical technologies have attracted attention in order to regenerate damaged living tissue. Regarding these, many studies have been carried out taking the regeneration of myocardial tissue as an example, such as: a method of directly injecting cardiomyocytes, skeletal myoblasts, and mesenchymal stem cells into myocardial tissue to regenerate; as described later, in special A method of culturing myocardial tiss...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/00C12M3/04
CPCA61L27/3826A61L27/3886A61L2300/64A61L2430/20A61L27/3804A61L2300/414A61L2300/426C12N5/0691C12N2502/1335C12N2502/28C12N2533/52C12N2502/1323A61P9/00A61P9/04A61P9/10A61K35/33A61K35/34
Inventor 纪之冈正博斋藤充弘泽芳树清水达也冈野光夫
Owner TOKYO WOMENS MEDICAL UNIV
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