Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an...
Reexamination Certificate
1998-11-27
2001-04-10
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an...
C424S093700, C435S182000, C435S382000, C435S395000, C435S396000, C435S397000
Reexamination Certificate
active
06214593
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for the encapsulation of viable animal cells, suitable for research and industrial applications, including production of artificial organs, tissue and cell transplantation and production of cell derived substances.
BACKGROUND OF THE INVENTION
It is known that loss or failure of organs and tissues can be treated by the development of functional substitutes made by cells placed on or within matrices which can be implanted or used as extracorporeal devices.
Some reviews on this topic are: R. Langer and J. P. Vacanti;
Science
, 260, 920 (1993); P. E. Lacy,
Scientific American
, (1995) 40; W. W. Gibbs,
Scientific American
, (1993) 16.
Some literature reports relevant to the problem are T. R. Shockley and M. L. Yarmush,
Biotechnol. Bioeng
., 35 (1990) 843; M. Taya, M. Yoshikawa, and T. Kobayashi,
J. Ferment. Bioeng
., 67 (1989) 138; Y. Shirai, H. Heshimoto, and H. Kawahara,
Appl. Microbiol. Biotechnol
., 29 (1988) 113; Y. Ho and T. M. S. Chang,
Artif. Organs
, 16 (1992) 442; A. A. Demetriou et. al.,
Science
, 233 (1986) 1190; F. Lim and A. M. Sun,
Science
, 210 (1980) 908; E. J. A. Pope,
J. Sol
-
Gel Sci. Tech
., 4 (1995) 225; E. J. A. Pope et al. “Sol-Gel Science and Technology”, Volume 55 (1955) pages 33-49.
In most cases the encapsulation is performed by hydrogels, in particular polysaccaride alginate, acrylonitrile-vinyl chloride copolymers, hollow fibers, carrageenan gel, agar rods and sol-gel derived Sio
2
from hydrolysis of silicon alkoxides in solution.
These approaches are affected by severe shortcomings such as reduction of mass transfer with the medium, insufficient stiffness to avoid cell release, chemical incompatibility with cell viability, production of severe poison byproducts, as for cell encapsulation by sol-gel obtained by hydrolysis and condensation of inorganic alkoxides in solution.
These problems can be solved by reacting supported cells and cell aggregates with gas-phase inorganic alkoxides suitable to react with the cell surface, resulting in a thin porous deposit of inorganic oxides in accordance with PCT application No. PCT/IT95/00083 the content of which is incorporated herewith as reference.
OBJECTS OF THE INVENTION
The aim of the present invention is therefore to avoid the disadvantages of mentioned encapsulation procedures by means of a process which provides a definite encapsulation of viable animal cells by a continuous and permanent layer of inorganic oxides with a pore size distribution ensuring free exchange of nutrients and metabolic products and avoiding antibody and immune-cell invasive action.
Another object of the invention is to provide a general immobilization method for animal cells and cell aggregates without limitations to defined organs, species, and cell functions with preservation of cell viability and metabolic functions.
Still another object of the invention is to provide a method involving simple operations under mild conditions of temperature and pressure which can be performed with industrial-scale devices and production equipments under sterile environmental conditions.
A further object of the invention is the maintenance of viable animal cell and of their specific functions also for use in extra-corporeal devices and the supply of immobilized cell aggregates for transplantation into the body.
DESCRIPTION OF THE INVENTION
These and other objects are achieved, according to the invention by a process for encapsulation of viable animal cells suitable for research and industrial applications, such as production of artificial organs, tissue and cell transplantation and production of cell derived substances, comprising the steps of:
a) providing sterilized supports made of organic and/or inorganic compounds and with suitable geometry to immobilize the desired loads of cells;
b) incubating a cell suspension with the supports in order to ensure adhesion to the support surface;
c) encapsulating the cells with a permanent layer formed by investing the supports with a reactive gas current composed of a carrier gas saturated by inorganic alkoxides for time intervals variable in function of cell nature and load, support geometry and porosity;
d) treating the encapsulated viable cells with steam under mild conditions to perform total hydrolysis of residual alkoxide groups;
e) storing the cells encapsulated on the supports by immersion into appropriate culture media.
Preferably, incubation step b) is accomplished by growing actively replicating cell lines in order to fill most of the available volume of the supports.
Furthermore, the reactive gas of step c) may be composed of a gas carrier saturated by Si(OR)
4
and/or SiX
x
(OR)
4−x
, where x=1,2; X=H, alkyl or halide; R=alkyl.
It has been surprisingly and conclusively found that it is possible to encapsulate animal cells, in accordance with the present invention.
The supports may be formed from foam of organic polymers, polymeric or glass or ceramic fiber textures, natural products, rock wool, organic or inorganic membranes.
The supports may be shaped as sheets, disks, plates, cones, tubes or corrugated solids with void/middle ratios in the interval 0.1-0.9 due to open pores ranging from 1 &mgr;m to 200 &mgr;m, in diameter.
Supports of inorganic materials, after sterilization, can be dipped into a solution of inorganic-oxide precursors, for example silicon alkoxides, suitable of hydrolysis and condensation. The solution viscosity ranges from 0.1 and 100 Pas, the extraction rate is between 1 and 103 mm/s, the nominal oxide concentration is in the interval 1-100 g/dm
3
, providing a definite increase of stiffness and mechanical strength, for example of textured glass fibers.
The cell load may be extended up to the available void volume; supports extracted from culture are mounted in a rack and tranferred into a closed reaction chamber. The items are invested by a sterile air flux saturated by reactive alkoxides, preferably a mixture of HSi(CH
3
)(OC
2
H
5
)
2
and Si(OC
2
H
5
)
4
, at room temperature. Saturation is obtained by bubbling the air flux into the alkoxide mixture kept at temperatures in the interval 10-90° C. The reactive gas flux is variable in function of cell load. The treatment is prolonged for some minutes, then steam is introduced at room temperature for appropriate time intervals.
The treatment with reactive gas, followed by steam reaction can be repeated several times during which the composition of the reactive gaseous species can be modified, for example changing the alkoxides or their concentrations.
These changes can be used to modify the specific surface area and pore size distribution of the deposited layer providing a variable permeability thus affecting the mass transfer as a function of bulkiness and molecular weight.
Further characteristics and advantages of the invention will be come apparent from the description of four examples, illustrated hereinafter only by way of non-limitative examples with reference to the accompanying
FIGS. 1
to
11
.
REFERENCES:
patent: WO 96/36703 (1996-11-01), None
G. Carturan et al., “Entrapment of viable microorganisms by SiO2sol-gel layers on glass surfaces: Trapping, catalytic performance and immobilization durability ofSaccharomyces cerevisae”, Journal of Biotechnology,30 (1993) 197-210.
Carturan Giovanni
Dal Monte Renzo
Muraca Maurizio
Biosil A.G.
Coleman Henry D.
Naff David M.
Sudol R. Neil
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