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Improved immortalized human skin cell lines and novel serum-free medium useful for the production thereof

a technology of immortalized human skin and serum-free medium, which is applied in the direction of cell culture active agents, artificial cell constructs, genetically modified cells, etc., can solve the disadvantages of their use, achieve moderate effect on keratinocyte cell growth, improve both the attachment of keratinocytes, and enhance normal keratinocyte growth

Inactive Publication Date: 2002-01-31
NESTEC SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0030] (iii) obtaining keratinocytes and / or melanocytes from said prepared skin sample and seeding said keratinocytes and / or melanocytes into a serum-free growth medium, preferably either the NR-3 medium or NR-4 (for melanocytes) (described infra) onto culture plates provided with a coating which facilitates cell attachment and cell growth, said coating comprising fibronectin, type 1 collagen and BSA.
[0085] A significant potential application of primary keratinocytes or melanocytes produced according to the invention, given their availability and method of production, is for skin grafting. Because these primary keratinocytes and melanocytes are produced under serum-free conditions, they should pose minimal risk of being contaminated by pathogens (e.g. viruses) and infectious agents. Moreover, because the subject melanocytes and keratinocytes may be derived from an autologous host, i.e., a patient having a large wound, this should minimize or eliminate the risk of rejection of the skin graft, or other adverse immunological reaction, as well as minimizing the risk of infection.

Problems solved by technology

However, while numerous groups have reported immortalized keratinocyte cell lines, and their usage in in vitro assays, previous immortalized keratinocyte cell lines and melanocyte cell lines have typically exhibited one or more properties which render their usage disadvantageous.

Method used

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  • Improved immortalized human skin cell lines and novel serum-free medium useful for the production thereof
  • Improved immortalized human skin cell lines and novel serum-free medium useful for the production thereof
  • Improved immortalized human skin cell lines and novel serum-free medium useful for the production thereof

Examples

Experimental program
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Effect test

example 2

[0090] 1) Characterization of Keratinocytes Growth in Serum-free Media: primary cell cultures were cultivated in modified MCDB 153 [Boyce et al., J. Tissue Cult. Meth., 9:83-93 (1985); and Pittlekow et al., J. Invest. Dermatol., 86:410-417, 1986] and NR-3 media. The best cell growth has been observed in NR-3 medium (FIG. 1). Improved cell growth has been also observed in fully defined NR-3 medium (NR-3 without bovine pituitary extract, BPE) compared to modified MCDB 153 without BPE.

[0091] FIG. 1 comprises cell growth in NR-3 and epinephrine-supplemented modified MCDB 153 (keratinocyte growth medium) after 6 days. The modified MCDB 153 medium refers to modified MCDB 153. Keratinocytes were harvested in trypsin / EDTA (0.05% / 0.01%) and counted by using a hemocytometer. The results shown in FIG. 1 are the mean of triplicates.

[0092] 2) Effect of Coating on Cellular Attachment and Cell Growth: the coating of culture plates was found to improve the cell attachment and the cell growth of nor...

example 3

[0093] 1) Immortalization of Keratinocytes: a cell suspension produced from skin samples described in example 1, which contains dissociated melanocytes, keratinocytes and fibroblasts, are cultured in the subject NR-3 medium. This is effected by seeding such cells onto culture dishes which are continuously coated with the "cocktail" coating previously described for bronchial cells (Lechner et al., J. Tiss. Cult. Meth., 9:43-48 (1985)). During culturing the primary cell cultures when they reach or substantially reach confluence, the cells are treated for 4 min with trypsin / EDTA (0.025% / 0.01%). During this treatment, the melanocytes detached from the keratinocytes culture, and they are collected separatly. Primary melanocytes and keratinocytes are thus separated at this stage. After the primary keratinocytes have been cultured and expanded to desired cell numbers in NR-3 serum-free medium using the described coated culture dishes (promotes the growth of keratinocytes versus melanocytes...

example 4

[0108] 1) Immortalization of melanocytes: a cell suspension produced from skin sample DKO-NR described in example 1, which contains dissociated melanocytes, keratinocytes and fibroblasts, are cultured in the subject NR-3 medium. This is effected by seeding such cells onto culture dishes which are continuously coated with the "cocktail" coating previously described for bronchial cells (Lechner et al., J. Tiss. Cult. Meth., 9:43-48 (1985)). During culturing the primary cell cultures when they reach or substantially reach confluence, the cells are treated for 4 min with trypsin / EDTA (0.025% / 0.01%). During this treatment, the melanocytes detached from the keratinocytes culture, and they are collected separatly. Primary melanocytes and keratinocytes are thus separated at this stage. The collected primary melanocytes are then seeded in NR-4 serum-free medium which specifically inhibits the growth of keratinicytes. After the primary melanocytes have been cultured and expanded to desired ce...

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Abstract

The present invention relates to improved continuous (immortalized) cell lines, in particular keratinocytes and melanocytes derived from normal human skin tissue. The present invention also relates to novel serum-free media for isolating, producing and maintaining said improved continuous keratinocyte and melanocyte cell lines. The present invention also relates to methods for producing primary melanocytes and keratinocytes under serum-free conditions without any feeder cells.

Description

[0001] The present invention relates to improved continuous (immortalized) cell lines derived from normal human skin tissues, in particular keratinocytes and melanocytes which retain the ability to express differentiation proteins characteristic of differentiated melanocytes or keratinocytes, even in high passages. The present invention also relates to novel serum free media which do not require the use of feeder cells.[0002] The production of immortalized cell lines derived from human skin tissues has been previously described. In general such methods comprise transfection or transformation of human skin cells, e.g., keratinocytes and melanocytes, cultured in vitro with agents which provide for immortalization.[0003] Immortalization refers to the production of cells which are able to be cultured for prolonged time periods in vitro, ideally indefinitely. These cells are also referred to as continuous cell lines. By contrast non-immortalized cells are only capable of growing for a fi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12A61K35/36C12N15/09C12N5/00C12N5/071C12N5/10C12N5/22
CPCA61K35/12C12N5/0629C12N2500/20C12N2500/25C12N2500/32C12N2500/38C12N2500/40C12N2500/44C12N2500/46C12N2500/99C12N2501/11C12N2501/115C12N2501/39C12N2501/81C12N2501/999C12N2510/04C12N2500/90C12N5/0602C12N5/00
Inventor BAUR, MARKUSMACE, CATHERINEMALNOE, ARMANDPFEIFER, ANDREAREGNIER, MARCELLE
Owner NESTEC SA
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