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Medium for preparing feeder cells for embryonic stem cells and feeder cells

A technology of embryonic stem cells and culture medium, applied in the field of feeding cells for embryonic stem cells

Inactive Publication Date: 2007-02-14
ASAHI TECHNO GLASS CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, most of these use primary cells. Since materials from multiple sources are required to produce embryonic stem cells in large quantities, it takes time to check each source one by one.
In addition, since the culture medium for cultivating these feeder cells from humans uses serum such as fetal bovine serum (FBS), there is also the risk of spreading an unknown source of infection or an unknown pathogen of prion from the serum. sex

Method used

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  • Medium for preparing feeder cells for embryonic stem cells and feeder cells
  • Medium for preparing feeder cells for embryonic stem cells and feeder cells
  • Medium for preparing feeder cells for embryonic stem cells and feeder cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] With the MEM medium supplemented with FBS 10v / v%, culture to 41.77PDL (population doubling level, cell group doubling value (Japanese Society for Tissue Culture "Technique of Tissue Culture" 3rd Edition (Basic Edition) 1996 p42)), will be obtained by Human normal 2 times lung fibroblast cell line (MRC-5) from the cell bank, use the following feeding cell culture medium (A) to make the number of cells suspended to 2.4×10 6 The cells were subcultured once to 42.99 PDL on gelatin-coated Petri dishes.

[0052] Composition of feeding cell culture medium (A):

[0053] RITC80-7 medium with bovine serum albumin (BSA) 5 g / L, EGF 10 μg / L, insulin 1 mg / L, and cortisol (hydrocortisone) 1 mg / L was added.

[0054] Next, the MRC-5 cultured above was cultured for 2 to 3 hours using the feeding cell culture medium (A) containing Mitomycin-C (MMC) at a final concentration of 10 μg / mL to inert cell division. . Thereafter, the feeding cell culture medium (A) containing MMC was removed, ...

Embodiment 2

[0056] The MRC-5 distributed from the same cell bank as in Example 1 was suspended using the following feeding cell culture medium (B) to make the number of cells suspended to 2.1×10 5 The cells were subcultured 4 times to 53.47PDL on gelatin-coated Petri dishes.

[0057] Next, the above-cultured MRC-5 was cultured for 2 to 3 hours using a feeding cell culture medium (B) containing MMC at a final concentration of 10 μg / mL to inert cell division. Thereafter, the feeding cell culture medium (B) containing MMC was removed, and the cells were washed 3 times with PBS to prepare feeding cells. Put the feeder cells on a gelatin-coated petri dish with a diameter of 60 mm, at a rate of about 4×10 per piece. 5 The number of viable cells was seeded and attached.

[0058] Composition of feeding cell culture medium (B):

[0059] Serum substitute containing 83g / L of albumin and 100mg / L of insulin (Knock out Serum Replacement, Inpidolujian Company (Patent Document 1)) 20v / v%, MEM non-esse...

Embodiment 3

[0068] With the MRC-5 allocated from the same cell bank as in Example 1, the number of cells was suspended to 2.4×10 using the feeding cell culture medium (B). 6The cells were subcultured 4 times on gelatin-coated Petri dishes to 53.68 PDL. Next, the above-cultured MRC-5 was cultured for 2 to 3 hours using a feeding cell culture medium (B) containing MMC at a final concentration of 10 μg / mL to inert cell division. Thereafter, the feeding cell culture medium (B) containing MMC was removed, and the cells were washed 3 times with PBS to prepare feeding cells.

[0069] Put the feeder cells on a gelatin-coated petri dish with a diameter of 60 mm, at a rate of about 4×10 per piece. 5 The number of viable cells was seeded and attached. Thaw the cryopreserved monkey embryonic stem cells on the culture dish to which the aforementioned feeding cells have been attached, and then suspend the number of living cells to about 2×10 with medium for monkey embryonic stem cells (A). 5 The con...

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Abstract

A culture medium for preparation of feeder cells for embryonic stem cells, which can efficiently establish feeder cells for use in culture of embryonic stem cells including human's from limited donor-derived materials and culture them in a condition of a reduced risk of infection, is provided. Further, a preparation method of feeder cells, which is relatively safe even when subjected to coculture with embryonic stem cells including human's, and the resulting feeder cells therefrom are provided. With the culture medium for preparation of feeder cells for embryonic stem cells comprising at least a serum albumin and insulin in a basal medium, a cell population comprising at least one kind of cells selected from fetal skin fibroblasts, fetal myofibroblasts, fetal lung fibroblasts, fetal epithelial cells, fetal endothelial cells, adult skin fibroblasts, adult lung fibroblasts, adult epithelial cells and endothelial cells which can become feeder cells for embryonic stem cells can be stably proliferated.

Description

technical field [0001] The present invention relates to a culture medium for feeding cells used for culturing embryonic stem cells including human embryonic stem cells, a method for producing feeding cells using the medium, and feeding cells for embryonic stem cells produced thereby. Background technique [0002] Embryonic stem cells are undifferentiated cells derived from the inner cell group in the blastocyst. They have the diversity of being able to differentiate into all tissues, and are expected to be used in the fields of cell culture, tissue transplantation, new drug research, and gene therapy. Furthermore, it has also been known that embryonic stem cells can be induced from cloned embryos obtained by transplanting somatic cell nuclei from adults. In recent years, the technology of introducing embryonic stem cells into various tissues including nervous tissue has been reported. Since the tissues induced by embryonic stem cells are genetically equivalent tissues withou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06A61K35/36A61K48/00A61L27/00C12N5/00C12N5/0735C12N5/077
CPCC12N2501/113C12N2502/1323C12N2501/11C12N2500/44C12N5/0606C12N5/0652C12N2502/13C12N2533/54C12N5/00
Inventor 中辻宪夫末盛博文浅香勋菅井晴美冈本玲子
Owner ASAHI TECHNO GLASS CORP