WO1990013639A1

WO1990013639A1 - Cell culture system

Info

Publication number: WO1990013639A1
Application number: PCT/US1990/002490
Authority: WO
Inventors: William R. Tolbert; Mark F. Baumgartner
Original assignee: Invitron Corporation
Priority date: 1989-05-05
Filing date: 1990-05-04
Publication date: 1990-11-15
Also published as: KR920701420A; CA2054210A1; AU5639490A; EP0471747A1; EP0471747A4; JPH04506149A

Abstract

A cell culture apparatus (10) is provided which permits perfusion of medium into a cell culture chamber (12) and continuous harvest of a product wherein gases entrapped in the medium supply (18) or in the cell culture chamber (12) are eliminated from the system (10).

Description

IMPROVED CELL CULTURE SYSTEM

Technical Field

The present invention relates to an apparatus and method for maintaining eucaryotic, especially animal, cells in culture and for continuous recovery of secreted products from these cells.

Background The technology associated with growing and maintaining cells in culture has been widely applied to the production of peptides, hormones, growth factors, antibodies and other biologically active substances. A great variety of cells has been adapted to growth in culture in order to exploit the secretion of important biomolecules which are of significant biomedical interest. While viral vaccines have been the traditional commercial cell culture product, other products such as interferon and monoclonal antibodies are generating a renewed interest in commercial cell culture. Many recombinant products, such as human tissue type plasminogen activator and blood factor VIII, as well as other products with great potential value as human therapeutics or in vivo diagnostics, are produced from genetically modified animal and plant cells grown in culture.

Consequently, there is a need to develop methods and apparatuses of commercial cell culture which will allow large quantities of cells to be grown to high density and will provide for efficient recovery of the product of interest. The present invention accomplishes these goals and represents a significant improvement over the prior art.

Traditionally, cell culture and cell derived protein production have focused on cell growth and maximizing cell proliferation. However, many valuable proteins produced by cultured cell lines are made more efficiently and abundantly during the non-proliferative phase. In the body, for instance, most cells are not actively growing or proliferating but instead expend their energy making and secreting proteins or performing other functions. By simulating such an in vivo environment, this invention enables cells which are maintained at high density in a non-proliferative state to expend less energy on growth and more on production of desired cell derived products. The reduction of proliferative activity also lowers the risk of mutation as a result of cell division which could otherwise occur in growing systems.

U.S. Patent No. 4,537,860, which describes a cell culture maintenance system developed by the inventor of the invention herein, provides a general background for the present invention. Other perfusion cell culture systems have been described in U.S. Patent No. 4,661,458 and by Rainen, American Biotechnology Laboratory (1988) :20-24; and Kle ent et al, Develop Biol Standard (1987) 6:221-226. Perfusion culture and maintenance systems for animal, and more specifically, mammalian cells have also been described in U.S. Patent 4,201,845 and in papers by Tolbert, et al, in Large Scale Mammalian Cell Culture, pages 97-123, Acadmic Press 1987; by Van Brunt, Bio/Technology 4:505-510, 1986 and by Tolbert et al in Animal Cell Biotechnology, Academic Press. Nilsson et al, Nature (1983) 302:629-630 has described the use of agarose beads to entrap mouse hybridoma cells while maintaining production of monoclonal antibodies.

The use of large scale cell culture apparatuses for the production of biomolecules in plant cells has been described by Shuler and Hallsby in Biotechnology In Plant Science, pages 191-205, Academic Press 1985, and by Hallsby and Shuler in Biotechnology and Bioengineering Symposium No. 17, pages 741-746, John Wiley & Sons 1986.

The method and apparatus described in U.S. Patent No. 4,537,860 are particularly pertinent. In this system, cells are suspended in the interstices of an inert matrix material which uniformly fills the space between various porous tubes. These tubes are used for the introduction of fresh medium and for the removal of conditioned medium containing secreted cellular products and waste material. (Also contained between the porous tubes and in close proximity to the cells is a semi-permeable tubular membrane for the diffusion of oxygenated gases into the culture and removal of carbon dioxide.) The porous tubes penetrating the matrix material provide for the radial outflow of fresh medium, which results in a non-uniform distribution of nutrient medium. This is because, as medium moves out from the surface of the porous tube, its concentration decreases with increased distance from the tube. This is in part remedied by the cross-over patterns from adjacent supply tubes, but the distribution of fresh nutrients to the cells is not completely uniform. In addition, air bubbles collecting within the porous tubes or within the cell/matrix mixture can dramatically affect the flow of medium. Uniformity of gas distribution within the cell culture is also a problem. The apparatus described in the above mentioned U.S. Patent No. 4,537,860 does not provide a means for removing air bubbles which may become trapped in the medium supply tubes, and does not provide a means from removing air bubbles which may become trapped in the cell chamber itself.

U.S. Patent No. 4,201,845 and the references of Shuler and Hallsby, and Hallsby and Shuler, describe flat chambers with cells contained between membranes or porous plates. These apparatuses are essentially three- chambered devices with nutrient medium in the first chamber flowing through the middle or cell culture chamber into the third or product collection chamber. In the apparatus of U.S. Patent No. 4,201,845, porous hollow fibers are used within the cell chamber both for gas exchange and for attachment of anchorage dependent cells. Such a system is severely limited by the amount of surface area available on the hollow fibers for cell attachment and by the inappropriateness of the device for cells that are anchorage independent. The devices described by Hallsby and Shuler, and Shuler and Hallsby suffer from the same deficiency and in addition, do not provide a means for gas exchange. Disclosure of the Invention

The invention provides an improved apparatus and method for the culture of animal cells, especially mammalian cells. The improved apparatus and method are particularly useful in culturing cells which -are not actively or normally proliferating but maintained in a "static" or greatly slowed condition with respect to growth, and actively secreting a desired biological product. The invention method and apparatus provide a means of medium supply to such cells which resists the accumulation of gas bubbles, thus assuring an even and uninterrupted supply of medium. The invention apparatus also provides means for release of any gas accumulation in the culture itself. The method and apparatus are applicable both to anchorage independent and anchorage dependent cells.

In one aspect, the invention is directed to an apparatus which comprises a housing having, within it, a cell culture chamber flanked by a medium supply cartridge and a product removal cartridge. The cell culture chamber has two vertical faces approximately opposite each other, one interfacing the medium supply cartridge and the other interfacing the product removal cartridge. The vertical faces are porous surfaces of suitable pore size for delivery of the medium from the medium supply cartridge and for product removal into the product removal cartridge and are of appropriate relative size to assure flow from medium supply cartridge to product removal cartridge.

(The cell culture chamber also contains an inlet or port for the introduction of matrix and suspended cells, and a number of generally tubular semi-per eable membranes for diffusion of oxygen into and removal of carbon dioxide from the body of the cell culture chamber. These membranes are disposed so that a finely divided or porous matrix and cells included within the cell culture chamber have no volumes which are too distant from the supply of oxygen or ability to remove carbon dioxide to prevent effective diffusion to and from the cells.)

The cell culture chamber may also include, at its top, a gas outlet means to provide for release of any gas bubbles which may accumulate in the cell culture chamber.

The product removal cartridge has an outlet means for the medium containing secreted product which outlet means may optionally be in communication with a refrigerated reservoir for immediate cooling of the product. The medium supply cartridge has an inlet and outlet means spaced preferably diagonally in the faces of the cartridge perpendicular to the porous face contiguous with the cell culture chamber. The inlet means is disposed at the bottom of the medium supply cartridge; the outlet means is disposed proximal to the top of the cartridge in a manner which assures the medium will permeate the entire volume of the cartridge. This can be accomplished by having the outlet diagonally opposite the inlet at the top of the opposing face, or by providing a means for circulating the medium throughout the cartridge, in which case the outlet can be on the same vertical face as the inlet but at the ^toP-

The medium entering the apparatus may thus be partially removed at the outlet means along with any accumulated gas bubbles. During operation, a percentage of the medium input flow between 0-75%, preferably 1-20%, is thus removed through the outlet means. Temperature control means may also be utilized upstream of the inlet or downstream of the outlet means for this cartridge.

In preferred embodiments, the housing may be provided with a multiplicity of cell culture chambers arranged in parallel and separated by alternating medium supply cartridges having inlet and outlet means as described, and product removal cartridges.

In these preferred embodiments, the medium supply cartridges and product removal cartridges will have two parallel vertical faces which are porous to serve the adjacent cell culture chambers, except for the medium supply cartridge or product removal cartridge at one end of the parallel array and the cartridge at the opposite end, which will have only one vertical porous face. Thus, in one embodiment, a medium supply cartridge, having a single porous vertical face, and an opposite impermeable face, shares the porous face as a vertical face of a cell culture chamber whose opposite porous face is in common with a product removal cartridge having two porous vertical faces, one of which forms the vertical face of the next cell culture chamber which is in turn bounded at the opposite face by the porous face of an additional medium supply cartridge which has an impermeable opposite face. Alternatively, this cartridge has a permeable opposite face contiguous with still another cell culture chamber which is, in turn, faced by a product removal cartridge again either having an opposite face which is impermeable or a porous face which forms the vertical face of still another cell culture chamber and so forth.

In such parallel arrays of multiple chambers and framing cartridges, the inlet and outlet means of the medium supply cartridges may be connected through ports in the housing to manifolds for supply of the medium through the inlet means and removal of medium from the outlet means along with any accumulated gases. Similarly, the outlet means for the product removal chambers may be connected through ports in the housing to a manifold for collective removal of product. Access ports at the base of the cell culture chambers for introduction of suspended cells and a finely divided or porous matrix may be similarly connected through a manifold.

In another aspect, the invention is directed to a method for culturing animal cells, especially mammalian cells using the apparatus of the invention. In this method, fresh medium is supplied through the medium supply cartridge to the cell culture chambers which have been provided with suspensions of the subject cells along with a suspension of a finely divided or porous matrix having suitable interstices for cell growth. Medium is supplied to the cartridge through the inlet means at the lower port and any accumulated gases, and part of the supplied medium, if desired, is removed from the outlet means at the top. About 0-75% of the supplied medium is removed through the outlet means, preferably 1-20%. The remainder of the medium passes through the porous face of the cartridge into the cell culture chamber. In culturing cells according to the invention method, oxygen is supplied through the permeable membranes disposed through the cell culture chamber and carbon dioxide is removed through these membranes. Conditioned medium containing secreted product from the cells is removed through the porous face shared by the cell culture chamber and the product removal cartridge and through the outlet means of said cartridge. In preferred embodiments, the medium is stored and maintained at refrigerated temperatures of about 0-10°C and warmed before introduction into the medium supply cartridge to a suitable temperature of ambient-37°C, preferably 37°C. The removed product stream is cooled after exit from the outlet means of the cartridge to refrigerated temperatures of 0-10°C. The conditioned medium may also be subjected to dialysis or other means to concentrate the desired product.

Brief Description of the Drawings

Figure 1 is a top view of the cell culture apparatus of the invention with a single cell culture chamber.

Figure 2 is an end view along the line 1-1' of the cell culture apparatus of Figure 1.

Figure 3 is a side view of the medium supply cartridge.

Figure 4 is a side view of product removal cartridge.

Figure 5 is a top view of the apparatus of the invention with multiple cell culture chambers and alternating medium supply and product removal cartridges. Figure 6 shows the apparatus of the invention in a product recovery system with a product flow-through configuration.

Figure 7 shows the apparatus of the invention in a product recovery system with a medium recycle configuration.

Modes of Carrying Out The Invention Preferred Embodiments

In one embodiment the apparatus may be constructed as a single cell culture chamber flanked by one medium supply cartridge having a single porous face and one product removal cartridge, also having a single porous face. The porous surface of the medium supply cartridge has a pore size of 0.1-10 microns, preferably 0.5-5 microns; the product removal cartridge has a porous surface having a pore size of 1-200 microns, preferably 10-100 microns, and, in any case, a pore size at least slightly greater than that of the face of the medium supply cartridge. A top view of this embodiment having a single cell culture chamber is shown in Figure 1.

As shown in Figure 1, the apparatus comprises a housing 10, preferably rectangular, which contains a cell culture chamber 12 bounded on two vertical parallel opposing sides by the vertical faces 14 and 16 of a medium supply cartridge 18 and a product removal cartridge 20 which may be either removable or fixed in place, and which extend to the vertical walls of the housing 10. At least one port (not shown) preferably disposed proximal to the bottom of the cell culture chamber 12 is provided for the introduction of a thick slurry of cells at high density, mixed with a non-toxic matrix material 26 in which the cells are held. The cells are generally eucaryotic cells, preferably animal cells, and most preferably mammalian cells. The cells may be cultured in suspension, or may be anchorage- dependent cells using the matrix as an anchor.

The matrix material 26 may be any non-toxic finely divided or porous substance which can be used for separation and retention of anchorage-independent cells or for the attachment of anchorage-dependent cells. For example, commercially available microcarriers such as Cytodex® 1, 2, or 3, or microcarriers marketed by Pharmacia Company, or collagen (gelatin) based micro¬ carriers from KC Biological or Ventrex Corp. , or porous microcarriers such as those described by Nilsson, K. , et al, Nature (1983) 302:629-630 may be used. Many other types of material including finely divided glass, stainless steel, or polymerics may also be used. The matrix material itself may be solid, porous, or permeable to the medium.

The housing 10 may also contain a port 27 contiguous to an opening disposed in a side proximal to the top of the housing or on the top surfaces of the cell culture chamber for removal of gases which may otherwise become trapped.

The cell culture chamber 12 is also traversed by a number of selectively permeable generally tubular membranes 44 which provide for diffusion of oxygenated gases into, and carbon dioxide out of the cell-filled matrix 26. The positioning of the membranes is spaced such that the majority of cells are located within an effective distance from the diffusion surface. Thus disposition is such that all volumes in the chamber are within about 3 mm, preferably 1 mm, of a diffusing surface of the permeable membrane. This tubular membrane is attached to a gas inlet 46 and outlet 48 means disposed in one or more faces of the cell culture chamber and corresponding ports 50 in the housing 10. The tubular membrane may be arranged horizontally along the length of the cell culture chamber as shown in Figure 1, or may be attached to a separate framework placed in the chamber, or may be wrapped around the medium or product cartridge, or both and in contact with the chamber if the chamber is sufficiently narrow. Various configurations, including a randomly twined configuration, consistent with the necessity for the proximity of the diffusion surface to all locations in the cell culture, can be used.

The flanking cartridges are of two types, one 18 for the supply of a fresh nutrient medium to the cell chamber 12 and the other 20 for the removal of product containing medium. The medium supply cartridge 18 is disposed generally vertical in the housing 10 with the side 14 which faces the cell chamber 12 consisting of a porous material such as a membrane filter, sintered plate, screen, or woven material with a pore size generally between 0.1-10 μ, but in any case, smaller than the pore size of the opposite wall shared by the cell culture chamber with the product removal cartridge described below. The medium supply cartridge 18 contains at least one inlet means 32 and at least one outlet means 34. The inlet means 32 is located near the bottom of the cartridge for the introduction of fresh nutrient medium and is adjacent to a port 36 disposed in a wall proximal to the bottom of the housing 10. The outlet means 34 is disposed near the top of the cartridge, and adjacent to a port 38 disposed in a wall proximal to the top of the housing, and is for removal of trapped gas bubbles and a percentage of the input nutrient medium of between 0 and 75% of the flow of input medium, preferably between 1 and 20%. The remaining flow of medium exits the cartridge 18 through the porous face 14 into the cell chamber 12 containing the cells and matrix. As medium perfuses, generally horizontally, around and through the interstices of the matrix, the desired cell product is secreted into the medium.

The product-conditioned medium from the cell culture chamber 12 enters the product removal cartridge 20, disposed as shown generally vertically opposite the medium supply cartridge 18, through the face of the cartridge 16 common to the chamber 12. The side of the cartridge 16 is composed of porous flat material such as a membrane filter, sintered plate, screen or woven material, with a pore size between 1-200 μ, preferably 10-100 μ and, in any case, larger than the pore size of the medium supply cartridge face. The product removal cartridge has at least one outlet means 42 adjacent to a port 40 disposed in the wall of the housing which provides for removal of product containing conditioned medium.

Figures 2-4 show alternate views of the apparatus of the invention and of its parts. Figure 2 shows a side view of the apparatus along the line 1-1'. The cell culture chamber contains the inlet port for introduction of cells and matrix at 24 and an exit port for trapped gases 27. At the opposite wall, shown by dotted lines, are the inlet and exit means for the gas diffusion membranes disposed throughout the chamber 46 and 48. The medium supply cartridge shown on the left shares the face 14 with the cell culture chamber and has an inlet means for medium 32 and an outlet means diagonally opposite (in this embodiment) at the top of the cartridge 34 for the trapped gases and a portion, if desired, of the medium. At the right, the product removal cartridge 20 shares a face 16 with the cell culture chamber and contains an outlet means, 42 for the conditioned medium. The position of the outlet means in the vertical face shown in Figure 2 is not critical.

Figures 3 and 4 are side views of the medium supply cartridge and product removal cartridge, respectively, where the porous faces 14 and 16 are shown along with the inlet and outlet means 32 and 34 for the medium supply cartridge and the outlet means 42 for the product removal cartridge.

In another preferred embodiment of the apparatus the housing 10 contains multiple cell culture chambers as shown in top view in Figure 5. The horizontal dimension of the housing is increased along one axis 52 relative to the embodiment described in Figure 1. In order to prevent the depletion of nutrients in the medium as it diffuses through the chamber, and to maximize the recovered biological activity of the secreted biomolecules, the cell culture chambers 12 are defined by multiple alternating medium supply cartridges 18' and product removal cartridges 20' « These subdividing cartridges 18' and 20' differ from the medium supply cartridge 18 and the product recovery cartridge 20 as shown in Figure 1 and from those at opposite walls of the housing in that both parallel vertical walls are porous, so that medium flows out of each medium supply cartridge 18' into cell culture chambers 12 flanking it through vertical faces 14'. Conditioned medium flows through the walls 16' of the product removal cartridges 20' . Thus, the internal cartridges have two parallel porous sides and the end cartridges 18 and 20 have single porous faces as described in Figure 1. This provides for the flow of medium, in parallel, through the multiple cell chambers 12 and the concomitant increase in the volume and capacity of the cell culture chambers 12 without significantly changing the effective geometric relationship between the medium supply cartridges 18 and 18', the cell culture and the product removal cartridges 20 and 20' .

In other respects, the cell culture chambers and the medium supply cartridges 18 and 18' and product removal cartridges 20 and 20' are constructed as described for the single cell culture chamber embodiment described in Figure 1. The cell culture chambers optionally contain a gas outlet port proximal to the top of the chamber, as well as a means for introduction of the cells and matrix, as well as an internally disposed generally tubular gas diffusion membrane. The product removal cartridge contains an outlet means for the conditioned medium containing product; the medium supply cartridges have an inlet means for medium at approx¬ imately the bottom of the cartridge and an outlet means for entrapped gas and, optionally, unperfused medium diagonally opposite and proximal to the top of the chamber.

It will be recognized that in order to provide each cell culture chamber with fresh medium and with a means for withdrawal of product, only certain configurations are permissible. If there are two cell culture chambers in the housing, there will be a single double walled medium supply cartridge or product removal cartridge between them, and two single walled product removal cartridges or medium supply cartridges at either side. Thus, for n cell culture chambers there will be two cartridges with single porous walls and n-1 cartridges with double porous walls. Medium supply cartridges and product removal cartridges will alternate across the array of cell culture chambers. The single walled cartridges will be of the same type for even values of n, and of opposite types for odd values of n. When multiple cell culture chambers are used, it is convenient to connect the various inlet and outlet means in corresponding chambers or cartridges in a manifold system. A typical such system is also shown in Figure 5. Thus, as shown in the figure, medium is supplied to the medium supply cartridges 18 and 18' through a common manifold 90 exterior to the housing and in fluid communication with the inlet means 32. Similarly, the outlet means 34 of the medium supply cartridges are in fluid communication with a manifold 92. The outlet means 42 of the product removal cartridges 20 and 20' are in fluid communication with a manifold 94 exterior to the housing. In the same fashion, the gas inlet means 46 are preferably supplied by an exterior manifold 96 and the gas outlet means 48 collect into a manifold 98.

The apparatus described in Figures 1 or 5 can be incorporated into an overall system that operates in a flow-through or recycling mode and provides for the culture of cells at high density for extended periods of time and for the continuous recovery of secreted cell products. A flow-through configuration is shown in Figure 6. Medium is pumped through the system by a series of sterile pumps 58, preferably peristaltic pumps. The pump pressure is regulated such that the pressure differentials are maintained within the system; generally, the liquid pressure in the cell culture chambers is maintained at a level in relation to gas pressure in the diffusion membrane disposed in the chambers to optimize diffusion of gases through the wall of the tubular membrane with or without gas bubble formation, and to permit the diffusion back of carbon dioxide.

The nutrient medium is pumped from a reservoir 60 kept at 4°-10°C, through flexible tubing 61 through heat exchanger 62 to warm the medium to the desired temperature. The warmed medium then passes through a gas removal vessel 64 which permits gas evolved from the medium due to the change in temperature to be dispelled, and into the invention apparatus 66, for example, into the medium supply manifold 90 of Figure 5. The condi¬ tioned medium, for example from the product recovery manifold 94 of Figure 5 passes out of the apparatus. The product-conditioned medium is returned to a refrigerated environment via flexible tubing 61 and then to a product cooler 68 and finally to a product reservoir 70.

A small fraction of the medium is removed from the medium supply cartridges 18 (and 18') for example into the exhaust manifold 92. This excess fresh medium may be alternatively combined with the product stream 72, reintroduced into the gas removal vessel 74 or discarded 76, as will be further described below.

A recycling form of the system described in Figure 6 is illustrated in Figure 7. The product flow may be split, so that a portion is returned to the input means of the supply cartridges and the remainder harvested. The product flow is split such that a portion 78 returns to the input side of the apparatus 66 and the remainder 79 is removed to the product vessel 70. Optionally, a filtration system 80 (consisting of prefilter and filter cartridges of appropriate size ranges) is used to remove any particles from the product flow to prevent clogging of the apparatus. A pressure sensing and control device 88 may also optionally be employed. Optionally a hollow fiber dialysis cartridge 84 or other means for concentrating larger proteins in the recycle circuit and for removal of water and low molecular weight components not containing the product may also be included.

The fraction of medium 78 returned to the supply side of the reactor is 0-100%, preferably 50-80%. A means of pH monitoring and adjustment 82 is included in this recycle circuit, as well as automated or manual sampling systems 86. Such information could be used to adjust and/or supplement the incoming nutrient medium stream to replace those components that were being metabolized by the cells.

The foregoing description is illustrative and not limiting. Other variations within the scope of the invention as defined by the appended claims will be obvious to one of ordinary skill in the art.

Claims

1. An apparatus for maintaining animal cells in vitro in a substantially arrested state of proliferation with a means for continuously recovering secreted cell products which comprises: a housing; at least one cell culture chamber within said housing for holding cells in a finely divided or porous matrix with interstices for passage of fluid culture medium; at least one inlet means proximal to the bottom of said chamber for introduction of said cells and matrix material; said chamber having external gas inlet and outlet means in communication with corresponding ports disposed in one or more walls of said housing and said inlet and outlet means in gaseous communication with generally tubular diffusion membranes disposed substantially throughout the interior of said chamber, wherein the majority of said cells are within an effective distance of a diffusing surface of said generally tubular membrane; said chamber bounded on two vertical approxi¬ mately opposite parallel sides by the vertical faces of a medium supply cartridge and a product removal cartridge said faces extending to the vertical walls of said housing, wherein the face of each said cartridge bounding the cell culture chamber is porous; said medium supply cartridge being for the supply of fresh medium and said product removal cartridge being for the removal of secreted cellular product; said medium supply cartridge having a medium inlet means and a medium outlet means in fluid _ communication with the interior of said cartridge, said inlet means being disposed proximal to the bottom of said cartridge and said outlet means disposed proximal to the top of said cartridge in a configuration whereby the medium is provided to substantially all portions of _Q the medium supply cartridge.

2. The apparatus of claim 1 wherein said medium inlet means and medium outlet means are positioned diagonally opposite in said medium supply cartridge. 5

3. The apparatus of claim 1 which comprises an alternating array of n cell culture chambers and n+1 cartridges, wherein the n-1 cartridges disposed between each two of said n chambers has two parallel porous 0 faces and wherein each of the two cartridges disposed at either end of the array has one porous face facing the chamber, wherein every other cartridge is a medium supply cartridge and the remaining cartridges are product removal cartridges.

4. The apparatus of claim 1 which further includes at least one outlet means disposed proximal to the top of the cell culture chamber for removal of trapped gases.

5. The apparatus of claim 3 wherein n is 2.

6. The apparatus of claim 3 wherein n is >2.

7. A method for maintaining animal cells in vitro in a substantially arrested or reduced state of proliferation and continuously recovering secreted cell products, introducing a suspension of said cells and porous matrix into at least one cell culture chamber within a housing, said chamber bounded on two parallel vertical sides by the porous vertical face of a medium supply cartridge for the supply of fresh medium and the porous vertical face of a product removal cartridge for the removal of secreted product; said medium supply cartridge having a medium inlet means and medium outlet means in fluid communication with the interior of said cartridge, said inlet means being disposed proximal to the bottom of said cartridge and said outlet means disposed proximal to the top of said cartridge; supplying fresh medium to the interior of the product supply cartridge through said inlet means and removing gases and optionally a portion of fresh medium from said outlet means; and removing product from the interior of said product removal cartridge through an outlet means disposed proximal to the top of said cartridge.

8. The method of claim 7 wherein said medium inlet means and medium outlet means are positioned diagonally opposite in said medium supply cartridge.

9. The method of claim 7 which further includes periodically expelling entrapped gas from the chamber through a port disposed proximal to the top of said chamber.