A carrier for the cultivation of animal and/or human cells in a
fermenter, comprises a polymer base body of a solid formed into
a pressure-resistant matrix, with a capillary system for carrying
the liquid cell culture medium therethrough, with no cell growth
occurring therein, and with interstices for three-dimensional cell
growth therein, growth factors for the cells being covalently bonded
to the boundary surfaces of the interstices. The carrier can provide
a high level of cell density of the order of magnitude of 10.sup.9
cells/ml, by three-dimensional cell growth.
1. A carrier for the cultivation of animal cells in a fermenter,
comprising a plurality of porous polymer beads, said beads having
a diameter of from 50 to 300 .mu.m and said pores having a diameter
from 0.1 to 3.0 .mu.m, transmissive for liquid cell culture medium
and in which no cell growth occurs, formed into a pressure-resistant
matrix with interstices within which cell growth takes place, growth
factors for the cells being attached to the boundaries defined by
wherein said beads are arranged on a sieve means in the fermenter,
such that cell culture medium flows downwardly therethrough,
wherein said fermenter is of a hollow cylindrical configuration
and a plurality of said beads is arranged in a plurality of horizontal
layers in superposed relationship in the fermenter, and wherein
said fermenter comprises means for transporting the cell culture
medium through the fermenter centrally along the axis of the cylindrical
configuration thereof, then radially outwardly over said horizontal
layers and then downwardly through said layers due to flow pressure
of said medium.
2. A carrier as set forth in claim 1 wherein said growth factors
are covalently bound.
3. A carrier as set forth in claim 1 wherein said beads are arranged
in stationary relationship in the fermenter and in operation said
cell culture medium flows therethrough.
4. A carrier as set forth in claim 3 wherein said beads are arranged
in the form of one or more layers in the fermenter and in operation
said cell culture medium flows therethrough in the direction of
the thickness of said layers.
5. A carrier as set forth in claim 1 wherein the bonding of said
growth factors is effected by oxirane groups.
6. A carrier as set forth in claim 1 wherein said growth factors
7. A carrier as set forth in claim 1 wherein said growth factors
are selected from blood serum, blood serum constituents, fibronectin
and fetal calf serum.
8. A carrier as set forth in claim 1 wherein said growth factors
are a combination of cell metabolism products and biochemically
modified natural products.
9. A carrier as set forth in claim 1 wherein spaces are provided
on respective sides of said superposed layers, a said space above
a respective layer serving to receive the radially outward flow
of the cell culture medium and the other space beneath said respective
layer serving to receive the flow of medium through said layer for
discharge of said medium.
10. In a fermenter comprising an at least substantially cylindrical
casing; means for introducing a flow of liquid cell culture medium
into said casing; means for passing said flow of liquid cell culture
medium substantially axially centrally through said fermenter; means
for directing said flow as a plurality of branch flows in radially
outward directions; perforate support means; means for passing said
branch flows of cell culture medium downwardly through said perforate
support means; and means for discharge of said cell culture medium
from said casing; carriers on said support means, for the cultivation
of animal cells, comprising a plurality of porous polymer beads,
transmissive for liquid cell culture medium and within which no
cell growth occurs, formed into a pressure-resistant matrix with
interstices within which three-dimensional cell growth takes places,
growth factors for the cells being bonded to the boundaries defined
by said interstices.
BACKGROUND OF THE INVENTION
The present invention relates generally to a carrier for the cultivation
of animal cells in a fermenter. In this specification any reference
to animal cells is intended also to embrace human cells, so that
the carrier is thus suitable for the cultivation of human cells
and/or animal cells.
In order to provide for growth of adherent animal cells on the
fixed surface of a substrate, the substrate must have a surface
nature such as to permit the cells satisfactorily to adhere thereto.
Surfaces with a positive or a negative electrical charge have been
found to be suitable substrates for that purpose. Adhesion and growth
factors are also known, for example bivalent cations, fibronectin
or serum, which are applied to plastic surfaces in order to promote
adhesion and growth of the cells thereon. The electrical charge
on the surface of the substrate causes absorption of the adhesion
and growth factors on which the cells thus grow. However certain
types of cells can themselves synthesize fibronectin and of themselves
are therefore capable of growing on charged plastic surfaces, for
example diploid fibroblasts. Other cells however necessitate the
addition of serum or fibronectin to the culture medium. As will
be appreciated however, the addition of serum or fibronectin to
the culture medium as adhesion or growth factors give rise to a
considerable increase in the cost of cell cultivation ant it is
undesirable in many cases, for example in the production of therapeutics.
Adherent animal, including human, cells, in particular connective
tissue cells, form a cell growth which permits only a single layer
of cells, being what is referred to as a monolayer. That means that
the cells can only grow in two dimensions. It is thus necessary
to use very substantial surface areas in order to achieve high levels
of cell density, which are required in a technical use situation.
That is achieved for example by using spherical carrier bodies,
also referred to as microcarriers, consisting of cross-linked dextran,
with denatured collagen being covalently bonded to the surface thereof.
The glutinous dextran bodies are distributed by stirring in the
culture medium. The soft consistency of the carrier bodies or microcarriers
is intended to reduce damage to or erosion of the grown cells in
the event of collisions between the carrier bodies during the stirring
operation. If the growth-bearing carrier bodies come into contact
with each other and adhere to each other, it is possible to provide
for three-dimensional cell culture (reference may be made in this
respect to the brochure entitled `Microcarrier cell culture principles
& methods` from Pharmacia Fine Chemicals AB, of Uppsala). However,
that procedure does not make it possible to achieve controlled three-dimensional
cell growth, which is a necessary condition if cell densities as
occur in a human or animal body (about 10.sup.9 cells/ml) are to
Also known is the hollow fiber module through which the cell culture
medium is passed. Cell growth takes place in the interstices between
the fibers. When that occurs, a gradient in respect of cell feed
with the cell culture medium is formed in the longitudinal direction
of the hollow fiber module.
Another known arrangement involes using porous ceramic cartridges
through which the cell culture medium is passed. It is also known
to provide for microencapsulation of the cells, with cells being
encapsulated in Na-alginate and the alginate droplets being enclosed
with a polymer network. The alginate is then dissolved out of the
network. Those known processes also can only achieve cell densities
of the order of magnitude of around 10.sup.7 cells/ml.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a carrier for
cultivation of animal cells, including therefrom human cells as
indicated above, which provides a cell growth with cell densities
of up to approximately the cell densities of 10.sup.9 cells/ml to
be found in a human or animal body.
Another object of the invention is to provide a carrier for the
cultivation of animal cells in a fermenter, to permit the cultivation
of even sensitive cells in synthetic medium without serum additive.
Yet another object of the present invention is to provide a carrier
for cultivating animal cells, such as to optimize cell reproduction.
A further object of the invention is to provide a carrier for animal
cell cultivation in a fermenter, which provides a secure basis for
Still a further object of the present invention is to provide an
animal cell cultivation carrier which is such as to provide for
reliable feed of cell culture medium to the cells and removal of
cell metabolism products.
In accordance with the principles of the present invention, these
and other objects are achieved by a carrier for the cultivation
of animal cells, which as defined thus comprises human and/or animal
cells, in a fermenter with a polymer base substance, growth factors
for the cells being covalently bonded to the surface thereof. The
base substance in the fermenter comprises a solid which is formed
into a pressure-resistant matrix with a capillary system transmissive
in respect of the liquid cell culture medium, in which no cell growth
occurs. The matrix solid has spaces or interstices for three-dimensional
cell growth, with the growth factors being bonded to the boundary
Thus, in the carrier in accordance with the present invention,
the base body forms a solid bed in which cell growth occurs, the
bed being inherently stable insofar as being resistant to pressure,
more particularly a flow pressure. The bed has two different internal
inherently stable interstices areas, in one of which the interstices
may be formed by the contours of beads of a bead polymer and serve
for three-dimensional cell growth. The other capillary interstices
in the solid bed may be formed by macropores of the beads of the
bead polymer, the macropores being of such small pore diameters
that cells cannot grow thereinto, while however being such as to
permit the liquid cell culture medium to flow therethrough, thereby
ensuring that the cells are always supplied with the cell culture
medium and cell metabolism products are removed. That thus gives
a continuous cell growth. The carrier which is provided in accordance
with the present invention may accordingly be a component of a tissue
fermenter which is economical in operation and in which animal and/or
human cell lines occur in large-scale or mass culture.
The fact that the carrier body is in the form of a fixed, unmoving
matrix means that it is possible to provide an arrangement for three-dimensional
growth of animal, including human, cells. The carrier bodies are
coated with growth factors which are bonded to the interface between
the fixed matrix with respect to the interstices into which three-dimensional
cell growth takes place.
The matrix has a capillary system, formed for example by the macropores
of a bead polymer, into which the cells cannot grow. However, as
indicated above, the capillary system serves to provide a feed of
cell culture medium for the cells and for removal of cell metabolism
products. The matrix may be formed by bead-like base bodies which,
as already indicated above, may be of a macroporous nature, with
the pores forming the capillary system. The surfaces of the beads
are coated with the growth factors and in the matrix form the boundary
surfaces of the interstices in which three-dimensional cell growth
In an advantageous aspect the invention further provides that,
when using the carrier according to the invention, it is possible
to eliminate the addition of serum of fibronectin or other adhesion
and growth factors, to the culture medium. In a preferred feature
of the invention, the adhesion and growth factors which may be for
example glycoproteins, in particular from serum and preferably in
that case from fetal calf serum, as well as fibronectrin, are preferably
covalently bonded to the surface of the polymer body by way of oxirane
groups. A suitable bead material for the polymer body is for example
the commercially available bead polymer described in `forum mikrobiologie`
8 (1985) page 296.
For the purposes of bonding the adhesion or growth factors to the
carrier body, in particular to the surfaces thereof which define
the above-mentioned interstices in which three-dimensional cell
growth occurs, it is possible to use other covalent bonding modes,
as are disclosed for example in `Angewandte Chemie 94` (1982), page
838 or `Chemiker-Zeitung`, 97th edition (1973) No 11, page 612 including
use of an azide, a carbodiimide, an isothiocyanate, a cyanogen bromide,
or an azo compound.
By charging the carrier bodies according to the invention which
have the adhesion and growth factors at the surface thereof, with
feeder cells, for example macrophages, it is possible to produce
biologically active carrier materials, by means of which it is also
possible to cultivate sensitive cells in a synthetic medium without
serum additive. Cell reproduction but also secondary metabolism
can be optimized by the use of suitable growth factors.
Further objects, features and advantages of the present invention
will be apparent from the following description of a preferred embodiment,
with reference to the accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a diagrammatic view of part of an embodiment of a carrier
which is formed from a bead polymer, and
FIG. 2 is a diagrammatic view of a tissue fermenter using the carrier
of bead polymer in the form of a fixed bed, in a particular arrangement.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring firstly to FIG. 1, shown therein in diagrammatic form
is a small portion of a matrix-like carrier 1 which is formed by
a polymer 2, more particularly a bead polymer. Beads of the bead
polymer, as indicated by reference numeral 2, are from about 50
to 300 .mu.m, more particularly around 200 .mu.m, in diameter. The
beads 2 of the bead polymer have macropores as indicated at 3, the
pore diameter thereof being smaller than around 10 .mu.m, more particularly
from 0.1 to 3.0 .mu.m.
For example, as known in the art as disclosed in forum mikrobiologie
8 (1985), the beads are formed from bead polymers of methacrylamide,
N-methylenebismethacrylamide, allylglycidylether or glycidylmethacrylate.
The density is approximately 1.039 g/ml in water. The surfaces of
the beads 2 are coated with growth factors such as peptides, proteins,
hormones and the like, for animal cells. The surfaces of the beads
2 thus form boundary surfaces for gaps or interstices 4 in which
threedimensional cell growth can occur. The macropores 3 of the
beads form a capillary system into which the cells cannot grow as
the cell diameter is about 20 .mu.m. The capillary system is capable
of passing the liquid growth medium therethrough, so that the cells
can be supplied with the growth medium and cell metabolism products
can be carried away therefrom, not only during the growth phase
but also in the production phase (the stable phase). The metabolic
effect is produced by a given flow pressure which is produced in
Reference will now be made to FIG. 2 which is a diagrammatic view
of part of a fermenter in which carriers 1 as arranged for example
as shown in FIG. 1 are used in layer form, as a component of the
fermenter. The thickness of the layers formed by the carriers 1
may be between 0.5 to 30 mm, depending on the cell type to be cultivated.
The fermenter comprises a cylindrical casing 5 and a stirrer or
agitator 6 which is disposed at a stationary location in the lower
part of the fermenter, on the axial center line of the cylindrical
casing 5, as indicated by A. The carriers 1 are arranged in superposed
relationship and in annular layers around the center line A, in
the interior of the fermenter. Disposed between the assembly of
the carriers 1 and the fermenter casing 5 is a semipermeable foil
7. The stirrer 6 which may alternatively be replaced by a pump arrangement
causes cell culture medium to be transported along the center line
A in a flow in the direction indicated by arrow B, centrally and
axially in the fermenter in the upward direction as shown in FIG.
2. The feed of cell culture medium into the fermenter may be in
the direction indicated by the arrow C, between the fermenter casing
5 and the foil 7. It should be noted however that the direction
of flow of the medium along the line A may also be in a downward
The primary flow of the cell culture medium, which occurs centrally
in the fermenter in the direction indicated by the arrow B branches
radially outwardly into the intermediate spaces 8 defined between
the layers of carriers 1. The cell culture medium which is to be
found in the spaces 8 above the carriers 1 is under a flow pressure
which is produced by the pump action of the stirrer 6, or of the
alternative pump arrangement to which reference has been made above.
By virtue of that flow pressure, the cell culture medium passes
into the matrix of the carriers 1 which are arranged in superposed
relationship in layers, and flows downwardly through the carrier
The carriers 1 are arranged on sieve or screen members 10, in a
frame indicated at 9 in FIG. 2. That arrangement ensures that, after
the cell culture medium has passed through the layers of carriers
1, it can then flow downwardly into spaces 11 beneath the respective
layers. While the cell culture medium is passing through the carriers
1, it gives off nutrients for cell growth. Even when the sizes of
the interstices 4 are reduced by three-dimensional cell growth occurring
therein, flow of the cell culture medium through the carriers and
removal of cell metabolism products from the carriers 1 is always
ensured because, as already referred to above in relation to FIG.
1, the matrix has a capillary system which is formed in the illustrated
embodiment by the pores of the beads of the polymer, into which
the cells cannot grow because of the respective dimensions thereof
and which is sufficient to supply the cells with further fresh cell
culture medium which is fed thereto from the spaces 8 above the
layers of carriers 1, and to carry cell metabolism products into
the spaces 11, after having passed through the carriers 1. The cell
culture medium leaves the spaces 11 in a radially outward direction,
as indicated by the flow arrows. Low molecular weight substances
are exchanged through the semipermeable foil 7. The renewed medium
is removed from the fermenter in an upward direction as indicated
by arrow E. Proteins as products of secondary cell metabolism are
retained and enriched.
The cell culture medium may conventionally include amino acids,
carbon sources, in particular glucose and additives such as oxygen
and carbon dioxide and possibly serum. The choice of culture medium
depends on the nature of the cells to be cultivated.
Removal of the consumed nutrient medium in the direction indicated
by the arrow E, upwardly from the fermenter, ensures that the level
of lactate concentration in the fermenter is held at a low value,
so that cell growth is not adversely affected.
It should also be appreciated that the feed of fresh cell culture
medium may also occur within the semipermeable foil 7 which is cylindrical,
like the casing 5.
It will be appreciated that the above-described carrier was set
forth by way of example of the principles of the present invention
and that various modifications may be made therein without thereby
departing from the spirit and scope of the invention, and likewise
in regard to the arrangement of the fermenter. Thus, in the described
embodiment the bead polymer comprises beads which are of a diameter
of from about 50 to 300 .mu.m, but bead diameters of from 30 to
1000 .mu.m could also be employed. Also, as indicated, while the
cell culture medium flows upwardly from the stirrer 6, into the
spaces 8 and downwardly through the horizontally disposed layers
of carriers 1, in the direction therefore of the thickness of the
layers, it would also be possible to use a different flow configuration.
Furthermore, any suitable growth factors may be employed, for example
a combination of cell metabolism products and biochemically modified