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Cell culture method and cell sheet

A cell culture and cell sheet technology, applied in the field of cell culture and cell sheet, can solve the problem of the troublesome stacking of single-layer sheets one by one, and achieve the effect of high callus effect

Inactive Publication Date: 2006-04-19
本多 裕之 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the method using a temperature-sensitive polymer can construct a multilayer tissue by overlapping single-layer sheets, but it is troublesome to stack the single-layer sheets one by one.

Method used

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  • Cell culture method and cell sheet
  • Cell culture method and cell sheet
  • Cell culture method and cell sheet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] In Example 1, the case where epidermal cells (keratinocytes) were cultured to prepare a cultured epidermal sheet will be described.

[0033] (1) Using cells

[0034] Normal human epidermal keratinocytes are commercially available cells manufactured by Kurabo Company for research. As a medium for epidermal keratinocytes, a serum-free medium HuMedia-KG2 (manufactured by Kurabo) was used as a growth medium; a medium in which calcium chloride was added to HuMedia-KG2 and the total calcium concentration was adjusted to 1.0 mM was used as a differentiation induction culture base.

[0035] (2) Preparation of Positively Charged Liposomes (MCL) Encapsulated with Magnetite

[0036] MCL was prepared according to the method described in Jpn. J. Cancer Res. Vol. 87, pp. 1179-1183 (1996). Specifically, firstly, N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (SOGO Pharmaceutical Co., Ltd.), dilauroylphosphatidylcholine (Sigma Chemicals), dioleoylphosphatidylethanolami...

Embodiment 2

[0044] In Example 1, the case of culturing retinal pigment epithelial cells (hereinafter referred to as RPE) and preparing a cell sheet will be described.

[0045] (1) Using cells, etc.

[0046] Normal human RPE cells were ARPE-19 cells from ATCC (American Type Culture Collection). cultured at 37°C in 5% CO 2 in a humidified air environment. As a medium, a medium in which 10% fetal bovine serum and antibiotics (ampicillin at a concentration of 100 U / m and streptomycin at a concentration of 0.1 mg / ml) were added to DMEM / HAM F12 was used.

[0047] (2) Preparation of MCL

[0048] MCL was prepared in the same manner as in (2) of Example 1 above.

[0049] (3) MCL uptake into RPE cells

[0050] ARPE-19 cells were suspended in the culture medium to prepare a cell suspension, which was inoculated on a tissue culture dish (manufactured by Asahi Techno Glass) at a concentration of 6 × 10 5 piece / cm 2 . After culturing for 24 hours, it was replaced with a medium containing MCL (m...

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PUM

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Abstract

Epidermal keratinocytes not having magnetic fine particles incorporated therein and epidermal keratinocytes having magnetic fine particles incorporated therein are seeded in a 24-well ultralow adhesion plate. A neodymium magnet is disposed outside the well bottoms, and the cells are cultured for one day. As a result, control epidermal keratinocytes not having magnetic fine particles incorporated therein do not adhere to the well bottoms, resulting in non-formation of undifferentiated cell sheet. By contrast, epidermal keratinocytes having magnetic fine particles incorporated therein, while being attracted to the well bottoms by magnetic force, form a multilayered cell sheet. This cell sheet can be easily recovered by removing the neodymium magnet disposed outside the well bottoms, causing a magnet having a hydrophilic film stuck thereto to come close to the cell sheet from upside and attract it through the hydrophilic film, and updrawing them.

Description

technical field [0001] The present invention relates to a culture method for culturing adhesion-dependent cells and a cell sheet obtained therefrom. Background technique [0002] In recent years, the technique of culturing cells in vitro to prepare cultured tissues for transplantation or experimental tissue has attracted attention. In the early 1980s, Howaed Green et al. used inactivated mouse 3T3 cells as feeder cells to culture human epidermal cells. When using this technique to prepare epidermal cell sheets, after the epidermal cells are cultured to form a sheet, the sheet adheres to the bottom of the culture container, so a step of detaching the epidermal cell sheet from the bottom of the culture container by enzymatic treatment such as a decomposing enzyme is performed. However, enzymatic treatment breaks down adhesion proteins on the cell surface, so it may have adverse effects on cells. [0003] In order to eliminate this possibility, various proposals have been mad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00A61K35/12A61L27/00C12M1/00C12M1/42C12M3/00C12M3/04C12N5/071C12N11/14C12N13/00
CPCC12N13/00C12N5/0629C12M25/00C12N5/0075C12N11/14
Inventor 本多裕之井藤彰小林猛上田实各务秀明畠贤一郎寺崎浩子
Owner 本多 裕之
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