Composition for coating support for preparation of cell sheet, support for preparation of cell sheet and process for producing cell sheet

A manufacturing method and support technology, applied in cell culture support/coating, biochemical equipment and methods, animal cells, etc., can solve problems such as difficult thinning, and achieve a stable success rate

Inactive Publication Date: 2006-10-25
KEIO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when using the above-mentioned method for producing a cell sheet in a temperature-sensitive culture dish, the cell sheet can be produced relatively stably when the primary culture is performed in a strict manner so that the culture in this special culture dish is optimized. It is difficult to thin the cells when the method usually performed in each unit is directly applied

Method used

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  • Composition for coating support for preparation of cell sheet, support for preparation of cell sheet and process for producing cell sheet
  • Composition for coating support for preparation of cell sheet, support for preparation of cell sheet and process for producing cell sheet
  • Composition for coating support for preparation of cell sheet, support for preparation of cell sheet and process for producing cell sheet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] [Example 1] Petri dish coating using human fibrin

[0126] Mix 90 mg of human fibrinogen, 0.4 U of thrombin, 0.59 mg of calcium chloride dihydrate, 3000 U of anti-protein peptidase (a 1 ml kit from Baxter Co., Ltd. was used above), 16 ml of normal saline, and put on each sheet of 3.5 cm. Quickly spread 0.3ml on the bottom surface of the Petri dish, and keep it at room temperature for 1 hour while keeping it horizontal. Then, the petri dish in which fibrin was formed and the Teishil dilution on the bottom surface was solidified was stored at 4° C. while maintaining a sterile state. By storing in this state, it can be used for up to 2 months. 40 to 50 3.5cm petri dishes can be made with TEISIL 1ml kit.

Embodiment 2

[0127] [Example 2] Cell culture in fibrin-coated culture dish

[0128] (1) Rat cardiomyocytes

[0129]Ventricles were removed from 1-day-old Wistar rat neonates, treated with 0.03% trypsin, 0.03% collagenase, and 20 μg / ml DNase I to isolate ventricular myocytes. Inject each fibrin-coated 3.5 cm dish with medium 199 / DMEM containing 10% FBS and penicillin (50 U / mL) / streptomycin (50 μg / ml) / amphotericin B (25 μg / ml) 2ml and 2×10 6 individual cells at 37°C, 5% CO 2 cultured in an incubator.

[0130] (2) C2C12 cells used by mouse skeletal muscle cells

[0131] Use DMEM medium containing 10% FBS in 5% CO 2 1. The mouse skeletal muscle cell line C2C12 purchased from ATCC was cultured in an incubator at 37°C, and passaged at 80% saturation.

[0132] (3) Mature skeletal muscle-like cells derived from mouse C2C12 cells

[0133] Seed 1×10 in a 75cm flask 7 20ml of DMEM containing 5% horse serum was added to the C2C12 cells subcultured by the method of (2). While observing the sta...

Embodiment 3

[0136] [Example 3] Thinning of cells

[0137] (1) Fabrication of Rat Cardiomyocyte Thin Sections

[0138] On the 4th day after the primary culture, the cells became saturated and pulsated on the bottom of the culture dish. Aspirate the culture solution, and use a scraper to slowly peel off the cardiomyocytes that have been combined into a membrane from the periphery of the culture dish to the center without destroying the cells. After complete dissection, the cell sheet can be stretched on the Petri dish by slowly dripping fresh medium onto the shrunken cell sheet. The lamellae of rat cardiomyocytes are shown in figure 1 .

[0139] (2) Using C2C12 cells, mature skeletal muscle cells and bone marrow mesenchymal cells to make cell sheets

[0140] Using the method of Example 2, continue to culture the cells to saturation on the fibrin-coated culture dish. Then, after continuing the culture for 3 to 4 days, the membranous cell sheet was peeled off using a scraper in the same ...

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Abstract

Disclosed are a method for manufacturing a cell sheet, comprising culturing cells on a fibrin-coated surface of a substrate until the cells reach confluency, continuing the cultivation of the cells for a sufficient time period to cause the degradation of fibrin at the bottom of the cells, and detaching the cultured cells from the substrate surface in a sheet-like form to give a cell sheet; a substrate for cell sheet preparation, a surface of which is coated with fibrin; and a composition for use in coating with fibrin a surface of a substrate for cell sheet preparation, the composition comprising fibrinogen and thrombin. The invention enables to cell sheets to be manufactured by a simple manipulation using a substrate coated with a commercial, commonly available material of which safety has been confirmed.

Description

technical field [0001] The present invention relates to a composition for coating a support used in producing a cell sheet, a support for producing a cell sheet, and a method for producing a cell sheet. Background technique [0002] Cell transplantation using various stem cells is being attempted as a treatment method in place of heart transplantation in which donors are insufficient for a heart that has entered marked insufficiency. In recent years, a method of tissue transplantation in which myocardial tissue is three-dimensionally constructed in vitro starting from such cell transplantation has become popular. For example, it has been reported that the temperature-sensitivity sensor obtained by coating poly(N-isopropylacrylamide) (abbreviated as "PIAAm") with electron beams on the surface of commercially available polystyrene petri dishes has been successfully Petri dishes are used to make various cell sheets. Among them, for cardiomyocytes, a myocardial tissue block tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N5/07A61L27/00A61L27/22A61L27/34A61L27/38C12N5/077C12Q1/02
CPCC12N5/0068C12N2533/56C12N5/00C12N1/00C12N5/06
Inventor 福田惠一板桥裕史坪田一男
Owner KEIO UNIV
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