Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-01-07
2001-10-09
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S395000, C435S397000, C436S008000
Reexamination Certificate
active
06300128
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a matrix for tissue culture, a method for culturing tissue, a method for fixing cultured tissue and an artificial skin fixed.
BACKGROUND OF THE INVENTION
In the field of medicine, food, cosmetics, etc., a number of products has been developed and sold in every year. In order to develop such products directly applied to a human body, confirmation of safety thereof is essential. An animal testing mainly conducted as a safety check test has drawbacks in expense, long test period and incomplete equivalence to human. In addition, improvement of an animal testing is demanded from the viewpoint of prevention of cruelty to animals.
A method for measuring an intensity of response by candidate substances comprising applying a tissue-specific substance to a tissue model reconstructed in vitro with cells or substances of said tissue is recently attempted as one of alternatives.
On the other hand, conventional artificial organs composed of plastics and metals as raw material, artificial organs further combined with materials from organism and so-called hybrid-type artificial organs further combined with cultured cells have been developed in a clinical field.
A cultured skin is a comparatively well-developed example in the field of tissue models and artificial organs. A cultured skin includes skin prepared by culturing human fibroblast in collagen gel, followed by inoculating and culturing human keratinocyte on the gel when the gel is shrunk (U.S. Pat. No. 4,485,096); skin prepared by inoculating and culturing human fibroblast on nylon mesh, followed by inoculating and culturing human keratinocyte thereon when pores of the mesh is filled up with secreted materials from fibroblast (Slivka, S. R., L. Landeen, Zimber, M., G. K. Naughton and R. L. Bartel, J. Invest. Dermatol., 96: 544A, 1991); and skin prepared by inoculating and culturing human fibroblast in collagen sponge, followed by laminating collagen gel or film anoculating and culturing human keratinocyte thereon (J.Jpn.P.R.S., 10, 165-180 (1990); Japanese Examined Patent Publication No. 47043/1995).
The most important problem in producing tissue models, typically cultured skins, and artificial organs is to reconstruct a three-dimensional structure of tissues or organs as quickly as possible. For example, a skin mainly comprises keratinocyte in epidermis, fibroblast in dermis and inter-cellular substances such as collagen, which are not existed in a mixed form. A skin comprises a dermis layer formed by three-dimensional proliferation of fibroblast in collagen fiber matrix and an epidermal layer formed thereon by laminating keratinocyte over and over again in a complex differentiation manner from basal layer to corneous layer by way of several steps.
However, conventional tissue models and artificial organs do not have a desired three-dimensional structure and have a problem that in spite of progress of development with respect to a cultured skin, in particular, it takes more than one month to prepare a conventional cultured skin from inoculation of cells to completion of skin reconstruction, and that keratinocyte laminates in only several layers and is low in differentiation stage in comparison with an actual human skin.
In order to overcome the problems mentioned above, it is an object of the invention to provide a matrix for tissue culture and a method for culturing tissue which make it possible to obtain tissue culture having a desired three-dimensional structure within a short period of time by culturing heterogenous or homogenous cells individually or simultaneously in suitable conditions thereof.
It is another object of the invention to provide a method for fixing cultured tissue and artificial skin fixed, suitable for preservation and transportation, which inhibit spill of liquid from container thereof and destruction of cultured tissue (especially cultured skin) when vibrating and upsetting container thereof.
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“Composite Skin Substitute Composed of Cultured Keratinocytes and Fibroblasts Combined in Collagen Matrix ” Ohtake et al., J. Jpn. P.R.S., 10: 165-180, 1990.
“Characterization of a Three Dimensional Co-Culture of Neonatal Human Fibroblasts and Keratinocytes ” Slivka, et al. The Journal of Investigative Dermatology, 96: 544A, 1991.
M.V. Nermut, et al. Method for the Study of the Underside of Cultured Cell Monolayers,, Experimental Cell, Research (Jan. 1991) 192 (1), 311-4.
Morita Shinichiro
Morota Katsuyasu
Gunze Limited
Knobbe Martens Olson & Bear LLP
Lankford , Jr. Leon B.
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