Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Solid support and method of culturing cells on said solid...
Reexamination Certificate
2000-12-27
2003-10-14
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Solid support and method of culturing cells on said solid...
C424S093700, C435S177000, C435S325000, C435S375000, C435S384000, C435S385000
Reexamination Certificate
active
06632668
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a monolayer sheet structure of primary hepatocytes, and a method of manufacturing it, and more particularly, to a method of forming a confluent monolayer sheet structure of primary hepatocytes so as to allow a dramatic improvement in the adhesion (attachment) and extension (spreading) of primary hepatocytes on to the culture substrate, and to the monolayer sheet structure formed by such a method.
This invention falls into the category of elemental technology for the systems engineering needed to construct artificial internal organs and bioreactors using cells, and is particularly useful as a technique for forming cell aggregates.
2. Description of the Related Art
The most widely used conventional technique for forming monolayers of primary hepatocytes is the simple method which consisting of suspending isolated primary hepatocytes in animal cell culture medium containing various bioactive hormones (such as dexamethasone) or serum, and spreading them on a culture substrate (such as a plastic laboratory dish coated with collagen, etc.), after which they are subjected to standing culture for a few hours to 24 hours. However, the monolayer structure of hepatocytes formed by this method becomes intermittently linked patch-like discontinuous monolayers of one to tens of cells, rather than a so-called confluent monolayer sheet structure, which is continuous and without gaps. Even from this state, a confluent state might be achieved through cell proliferation in the case of other cells, but primary hepatocytes do not proliferate easily, making it impossible to subsequently form a confluent sheet structure from the monolayer sheet structure formed during the first 24 hours.
Under these circumstances, and reflecting on prior art as described above, the present inventors discovered as a result of exhaustive research that the adhesion and extension of primary hepatocytes on to a culture substrate could be dramatically enhanced by treatment thereof with a suitable amount of a protein-phosphorylation inhibitor having an indolo [2,3-a] carbazole skeleton (typical example: staurosporine) during formation of the primary hepatocytes monolayers according to prior art, and achieved this invention by exploiting this effect.
SUMMARY OF THE INVENTION
The present invention provides a monolayer sheet structure of primary hepatocytes that could not be formed by conventional cell layer formation technology, along with a simple method of forming such a monolayer sheet structure.
This invention is a monolayer sheet structure formed as a confluent monolayer of primary hepatocytes cultured on a culture substrate, said monolayer sheet structure is formed by treating the hepatocytes with a protein-phosphorylation inhibitor having an indolo [2,3-a] carbazole or other similar structure in the monolayer formation stage.
This invention is aimed at providing a confluent monolayer sheet structure of primary hepatocytes, that could not be formed with conventional cell monolayer formation technology, and also providing a simple method of forming such a structure.
Furthermore, this invention is also aimed at providing a method of manufacturing a monolayer sheet structure of primary hepatocytes using a protein-phosphorylation inhibitor (compound) with an indolo [2,3-a] carbazole or other similar skeleton, and also providing a monolayer sheet structure manufactured according to said method.
This invention, which is to solve the subjects described above, comprises the following technological means.
(1) A monolayer sheet structure consisting of a confluent monolayer formed from primary hepatocytes cultured on a culture substrate,
said monolayer sheet structure of primary hepatocytes is produced by treating the hepatocytes with a protein-phosphorylation inhibitor having an indolo [2,3-a] carbazole or similar skeleton in the step of formation of the monolayer.
(2) The monolayer sheet structure of primary hepatocytes according to (1) above, wherein the monolayer sheet structure is produced by treating the hepatocytes with staurosporine.
(3) A method of manufacturing a monolayer sheet structure by culturing primary hepatocytes on a culture substrate to produce a confluent monolayer, which comprises treating the hepatocytes with a protein-prosphorylation inhibitor having an indolo [2,3-a] carbazole or similar skeleton in the step of formation of the monolayer to form the monolayer sheet structure of primary hepatocytes.
(4) The method of manufacturing a monolayer sheet structure of primary hepatocytes according to (3) above, wherein the hepatocytes are treated with staurosporine to form the monolayer sheet structure of primary hepatocytes.
REFERENCES:
Sumio Maeda, et al. “Staurosporine Promotion of Formation of Continuous Monolayers of Primary Rat Hepatocytes by Improving Attachment and Spreading”, Biosci. Biotechnol. Biochem., vol. 64, No. 9, 2000, pp. 1985-1987.
Inagaki Hidetoshi
Maeda Sumio
Saito Takao
Japan as represented by the Secretary of Agency of Industrial Sc
Naff David M.
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