Chemistry: molecular biology and microbiology – Differentiated tissue or organ other than blood – per se – or...
Reexamination Certificate
2000-08-25
2001-09-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Differentiated tissue or organ other than blood, per se, or...
C435S370000, C435S400000, C435S405000, C435S387000, C210S321890, C604S006070, C623S017120
Reexamination Certificate
active
06284451
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for forming a multicellular aggregate applicable to the production of useful cellular products by the use of animal cells or a hybrid (or biological) artificial organ, and to a module into which the multicellular aggregate has been incorporated. More particularly, this invention relates to a technique for inducing the formation of an aggregate of cultured cells by the use of a centrifugal force, as a method for forming a multicellular aggregate which can maintain the cells for a long time without losing their inherent functions, to a module for cell culture which reproduces a sinusoid-like structure of a living body by the use of hollow fibers, and to a method for cell culture using the module and the aggregate inducing technique in combination.
This invention further relates to a culture medium suitable for culturing the multicellular aggregate and a culture method of the multicellular aggregate using the medium.
BACKGROUND OF THE INVENTION
Culture devices for multiplying animal cells for the production of useful cellular products, or for using the cells as a component of a hybrid artificial organ, have been conventionally known. Such known culture devices employ agitation tank culture using a micro carrier for high-density culture of cells (W. R. Tolbert et al., Ann. Rep. Ferment. Proc., 6, 35 (1983), etc.), flow bed or fixed bed culture using a porous carrier (JP 7-46988 B, etc.), and culture using a porous hollow fiber membrane to immobilize cells in the lumen or the extra-fiber space (Naka et al., Artificial Organs, 28(1), 68-73 (1999), etc.). Of these, the culture using a hollow fiber membrane is superior in that it provides the protection against shear stress and prevention of leakage, because the cells are separated from the flow of a medium by the hollow fiber membrane. In addition, existing dialysis devices and plasma separation devices can be used as the culture device to be applied to the hollow fiber membrane, because they contain a multitude of hollow fibers in a bundle. In such hollow fiber type culture device, however, the hollow fibers are not uniformly arranged within the culture tank (module). This in turn poses a problem in that, when cells are to be immobilized in the extra-fiber space of the module, the distance of mass transfer to the surface of the hollow fibers differs depending on the position in the module, thereby producing variation in the viability and growth of the cells.
The anchorage-dependent animal cells have been dominantly cultured by a two-dimensional monolayer culture method. This method is associated with a problem in that the cultured cells rapidly lose functions. For example, highly differentiated primary hepatocyte loses its functions during several days of culture under the monolayer culture conditions. In view of such defects, an attempt has been made to develop a cell aggregate culture for the expression and maintenance of highly differentiated functions. For example, culture of spherical multicellular aggregate (spheroid) of cultured cells using a porous polyurethane foam (PUF) has been utilized in the development of a hybrid artificial liver required to express high functions (Matsushita et al., Artificial Organs, 21(3), 1050-1054 (1992)).
When an aggregate is contained in a module, the cell aggregate has an extremely high cell density sufficient to enable high-density culture of the cells, which in turn reduces the size of the module. On the other hand, the consumption of oxygen and nutrients in the module is also striking. When cells with high oxygen consumption, such as primary hepatocytes, are to be cultured, conventional devices allow only the cell density of about 1×10
7
cells/cm
3
, because insufficient supply of oxygen and nutrients for the culture of the highly dense cells results in necrosis of the cells in the aggregate, demonstrating degraded function of the device. Due to such problems, it is difficult for the cells to maintain viability for a long time under the high-density culture conditions without impairing inherent cell functions.
For the maintenance of the functions of the primary hepatocytes, the importance of the composition of the culture medium has been emphasized along with the importance of cell morphology, and there have been documented reports on culture media containing various supplemental factors. For example, there are reports on enhanced viability of hepatocytes by the addition of high concentration L-alanine to a culture medium (JP 5-336959 A), maintenance of albumin secretion by hepatocytes by the use of a culture medium containing ascorbic acid (JP 7-274952 A), maintenance of albumin secretion by spheroid culture hepatocytes for about 60 days by the use of William's E medium (WEM) supplemented with dexamethasone, glucagon, insulin and the epidermal growth factor (EGF) (J. Z. Tong et al., Exp. Cell Res., 189, 87-92, 1990), maintenance of albumin secretion ability of collagen sandwich culture hepatocytes for about one month by the use of Dulbecco's modified Eagle's medium supplemented with proline, insulin, glucagon, hydrocortisone and EGF (J. Lee et al., Biotech. Bioeng., 40, 298-305, 1992), and others.
However, maintenance of the function of detoxifying foreign compound, ammonia produced in the body and drug, as possessed by the hepatocytes in addition to the protein (e.g., albumin) synthesizing ability, is not feasible, where the ammonia metabolizing ability can be maintained in a conventional culture for only about 2 weeks.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for forming an aggregate that allows various animal cells to survive for a long time without losing their inherent functions, as well as a module incorporating such aggregate at high density, which module is useful for the production of advantageous cellular products and is applicable to a hybrid artificial organ.
Another object of the present invention is to provide a culture medium suitable for a long-term maintenance of various physiological functions, particularly at least ammonia metabolizing ability in addition to the albumin secretion ability, of primary hepatocytes in an artificial liver.
A further object of the present invention is to provide a culture technique that can be applied to a hybrid artificial liver and as an alternative method of animal experiments (safety test of drug etc. on cells instead of animals for protection of animals) over an extended period of time by the use of this medium.
The present inventors have found that a multicellular aggregate having high functions can be formed by forcibly immobilizing cells in the lumen or extra-fiber space of the module, or in other culture substratum, by the aid of centrifugal force, thereby to increase the contact frequency between cells. They have also found that a tissue-like structure can be constructed by immobilizing cells at high density in the extra-fiber space or lumen of hollow fibers having a semipermeable membrane, and supplying, via the hollow fiber membrane, oxygen and nutrients necessary for the survival of the cells, wherein the hollow fibers constitute a module incorporating a sinusoid-like structure similar to the biological capillary network, together with at least two spacers having small pores regularly formed therein, the hollow fibers passing through the small pores of the spacers to provide a structure comprising hollow fibers arranged regularly at a very small distance. It has been also found that the use of DMEM rich in amino acid, vitamins and energy source, or a mixture of about 60 to 90% DMEM and about 10 to 40% of other medium, supplemented with proline, EGF, insulin, hydrocortisone, selenious acid, linoleic acid, copper sulfate and zinc sulfate for the culture of liver multicellular aggregate leads to a marked elongation of the maintenance of the typical functions of primary hepatocytes including the ammonia metabolizing ability.
When this medium is used in combination with the above-mentioned module and t
Funatsu Kazumori
Ijima Hiroyuki
Mizumoto Hiroshi
Nakazawa Kouji
Ketter James
Leydig , Voit & Mayer, Ltd.
Toyo Boseki Kabushiki Kaisha
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