WO1986002379A1

WO1986002379A1 - Hollow fiber culture device for improved nutrient perfusion and product concentration and method of operation

Info

Publication number: WO1986002379A1
Application number: PCT/US1985/001949
Authority: WO
Inventors: Robert D. Walker; Michael L. Gruenberg; Ray F. Cracauer
Original assignee: Endotronics, Inc.
Priority date: 1984-10-09
Filing date: 1985-10-09
Publication date: 1986-04-24
Also published as: EP0198033A1; EP0198033A4; JPS62500356A

Abstract

A hollow fiber culture device includes a first hollow fiber cartridge (12) and a second hollow fiber cartridge (14), the capillaries of which (18) are fluidly connected in series by a fluid connection (36) having a flow restriction therein (38). The flow restriction (38) provides a pressure drop between the capillaries of the first (18) and second (28) cartridges and provides back pressure to the cappilaries of the first hollow fiber cartridge so that medium is perfused under ultrafiltrative conditions. The extracapillary space (34) of the first hollow fiber cartridge is fluidly connected to the extracapillary space of the second hollow fiber cartridge (14) so that waste- and product-containing medium is conveyed to the extracapillary space (34) of the second hollow fiber cartridge wherein the product is concentrated and the waste-containing medium is diffused into the lumen of the capillaries.

Description

HOLLOW FIBER CULTURE DEVICE FOR IMPROVED

NUTRIENT PERFUSION AND PRODUCT

CONCENTRATION AND METHOD OF OPERATION

Reference is hereby made to the following co- pending U.S. patent application Serial No. 658,549 filed October 9, 1984 and entitled, "Improved Hollow Fiber Cell Culture Device and Method of Operation," and assigned to the same assignee as the present application.

BACKGROUND OF THE INVENTION 1. Field of the Invention.

The present invention relates to cell culturing devices, and in particular, it relates to cell culturing devices having a plurality of hollow fiber membran.es that efficiently transfer oxygen, nutrients and other chemical stimuli and remove waste products for the growth and maintenance of cells in vitro at high cell densities to provide high yields of product formation per unit reactor volume.

2. Description of the Prior Art. Cell culture devices for culturing cells in vitro having a shell with a plurality of hollow fiber membranes have been known for quite some time. Medium containing oxygen, nutrients and other chemical stimuli is transported through the lumen of the hollow fiber membranes and undergoes a pressure drop resulting in an outward radial convective flow at the entry port of the device and an inward flow at the exit port of the device. Cells are grown in the fluid space between the fibers and the shell wall.

Hollow fiber culture devices have been proven to be ideal for the maintenance of many types of cells at high densities in vitro. The mass transfer characteristics of hollow fiber culture devices provide an efficient means of delivering nutrients and removing waste products from a culture. The semi-porous hollow fiber membranes can be selected with various pore sizes. With proper pore size selection, the cellular product can be maintained on the outside of the fibers, while waste products and contaminating proteins will pass through the membrane pores into the lumen of the hollow fibers where they can be subsequently removed from the culture.

Secreted cell products will become concentrated in the hollow fiber culture devices. The combination of high cell densities per unit volume and the production of a concentrated and purified product gives hollow fiber culture devices great potential for economically producing cell-derived products on a commercial scale.

Hollow fiber culture devices have many advantages over other culture technologies in economically producing cell-derived products. This is because the efficient mass transfer of nutrients and removal of waste products allows high cell densities to be achieved in a minimal space. In addition, the molecular weight cut-offs of the fibers can be selected to maintain the cell product on the outside of the fibers, while lower molecular weight contaminates are removed by diffusing to the lumen of the fibers. In this manner, cell products concentrate in the fluid outside the fibers in a semi-pure state.

A major disadvantage of prior art hollow fiber culture devices is that the hollow fiber molecular weight cut-offs are generally specified under ultrafiltrative conditions and as a result, products of much larger molecular weights will pass through the lumen of the fibers. This problem becomes worse as the concentration of the product increases, the diffusion rates increase and a significant loss of product can occur.

Some examples of prior art hollow fiber culturing devices are described in the following patents:

Inventor Patent No.

Matsumura 3,734,851

Knazek et al 3,821,087

Knazek et al 3,883,393 Osborne et al 3,911,140

Delente 3,997,396

Feder et al 4,087,327 Knazek et al 4,184,922 Knazek et al 4,200,689 Feder et al 4,201,845 -

Knazek et al 4,206,015 Knazek et al 4,220,725 Chick et al 4,242,460 Yoshida et al 4,391,912 Itsei 4,396,510

Michaels et al 4,440,853 Michaels et al 4,442,206 SUMMARY OF THE INVENTION' The present invention includes a cell culturing device for growing and maintaining cells and collecting product. The device includes first and second hollow fiber cartridges connected to each other such that the cells are grown and maintained in the first hollow fiber cartridge and product is conveyed to the second hollow fiber cartridge where it is concentrated. Flow of medium from the lumen of the capillaries of the first hollow fiber cartridge to the capillaries of the second hollow fiber cartridge is restricted by a restriction that creates a back pressure in the flow of medium in the capillaries of the first hollow fiber cartridge. The restriction permits medium to be delivered to the capillaries at a pressure that subjects the medium in the capillaries • to ultrafiltrative conditions. Subjecting the medium to ultrafiltrative conditions increases the mass transfer capability of the first hollow fiber cartridge and minimizes product loss by diffusion into the capillaries. Product and metabolic waste are transferred from the first hollow fiber cartridge to the second hollow fiber cartridge. The second hollow fiber cartridge has a molecular weight cut-off such that the product is selectively retained while the waste-containing medium is allowed to pass through. Due to the flow restriction between the first and second hollow fiber cartridges, the second hollow fiber cartridge is operated at a lower pressure than the first hollow fiber cartridge thereby aiding in the diffusion of waste contaminated medium. The product in the second hollow fiber cartridge becomes concentrated over time and can be removed for further processing.

The device of the present invention has several advantages over prior art hollow fiber culture devices. First, the first hollow fiber cartridge is operated under ultrafiltrative conditions using a larger molecular weight cut-off which permits better mass transfer. Second, since the product is continuously removed from close proximity of the cells, feedback inhibition problems caused by the product are eliminated. Third, the product is concentrated and purified in the second cartridge on a continuous basis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagrammatical view of the apparatus of the present invention. Figure 2 is a diagrammatical view of an alternative embodiment containing an expansion chamber fluidly connected to and between the extracapillary units of the hollow fiber cartridges.

Figure 3 is a diagrammatical view of an alternative embodiment of the apparatus of Figure 2 further including a pump between the expansion chamber and the extracapillary space of the second hollow fiber cartridge.

Figure 4 is another alternative embodiment including sensors in the expansion chamber and a supply source for replenishing the medium in the expansion chamber.

Figure 5 is a diagrammatical view of still another alternative embodiment wherein the expansion chamber is fluidly connected to the capillaries of the second hollow fiber cartridge.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The apparatus of the present invention is generally indicated at 10 in Figure 1. The apparatus 10 includes a first hollow fiber cartridge 12 and a second hollow fiber cartridge 14.

The first cartridge 12 includes a shell 16 and a plurality of capillaries 18 which are typically disposed in a bundle. The capillaries 18 extend through the shell from an input end 20 to an output end 22. The space between the shell 16 and the capillaries 18 is defined as a cell culturing space 24. Cells, such as mammalian cells, are maintained, grown and produce product in the cell culturing space 24. The capillaries have selectively permeable wall membranes that allow oxygen, Cθ2» nutrients and other chemical components to diffuse through the walls into the cell culturing space 24. The molecular weight cut-off of the capillaries is preferably less than approximately 150,000 Daltons and more than 15,000 Daltons, although 5,000 Daltons has been found sufficient to effect ultrafiltrative conditions, as will be discussed subsequently. The oxygen, ^co ' nutrients and other chemical components are carried within a medium that is transported through the lumen of the capillaries.

The second cartridge 14 is of a similar construction as the first cartridge 12. The cartridge 14 includes a shell 26 and a plurality .of capillaries 28 disposed in a bundle extending from an input end 30 to an output end 32 of the shell. The capillaries 28 also have selectively permeable wall membranes, preferably, less than 15,000 Daltons. A product collection space 34 is defined by the space between the capillaries 28 and the shell 26.

A suitable hollow fiber cartridge is made by Amicon Corporation of Danvers, Massachusetts. The capillaries of the cartridges 12 and 14 are fluidly connected in series such that the output end 22 of the cartridge 12 is fluidly connected to the input end 30 of the cartridge 14. The cartridges 12 and 14 are fluidly connected by a fluid connection 36. The fluid connection 36 is made of tubing and includes a restriction 38, such as produced by a pinch clamp, that restricts flow from the capillaries of the first cartridge 12 to the capillaries of the second cartridge 14. The restriction 38 produces a pressure drop between the first cartridge 12 and the second cartridge 14. The restriction 38 aids in presentation of medium within the capillaries 18 under ultrafiltrative conditions. In addition, the restriction 38 aids in removal of waste-containing medium from product, as will be discussed subsequently.

A delivery system 40 delivers a primary medium supply containing oxygen, nutrients or_> other chemical stimuli through supply conduit 42 to the lumen of the capillaries 18. The delivery system 40 delivers the primary medium supply at a selected rate and pressure. Suitable delivery systems are well known in the art. Any suitable delivery system that is capable of pumping medium at a controlled rate and concentration to the capillaries 18 is includable within the present invention. A suitable delivery system is sold by Endotronics, Inc. of Minnesota under the trademarks "ACUSYST" and "APS10" . Suitable delivery systems are also described in application Serial No. 350,135 filed on February 19, 1982, application Serial No. 388,136 filed on June 14, 1982, and application Serial No. 483,284 filed on April 8, 1983, all assigned to the same assignee as the present application and which are hereby incorporated by reference. The medium is returned to the delivery system 40 by flow through the capillaries 18, through the fluid connection 36 with the restriction 38, into the capillaries 28, and through a recirculation line 44.

The shell 16 includes first and second ports 46 and 48 which fluidly connect tubing sections 50 and 52 with the extracapillary space 24. Likewise, the shell 26 of the cartridge 14 includes first and second ports 54 and 56. Tubing 58 fluidly connects the tubing sections 50 and 52 with the port 54 such that the extracapillary space 24 is fluidly connected to the extracapillary space 26. The port 56 fluidly connects an outlet conduit 60 with the extracapillary space 26. The conduit 60 preferably includes a valve 62 that is positionable between an open and a closed position.

In operation, medium from delivery system 40 is delivered to the capillaries 18 at a pressure such that the medium is subjected to ultrafiltrative conditions which results in substantially even diffusion along the length of the capillaries within the cartridge 12. As waste material is secreted by the cells in the extracapillary space 24 and product produced, the product and the waste materials and other components are forced to flow within the capillary space in the general direction of arrows 64 and 66 causing circulation within the extracapillary space 24. The product and the waste materials flow out of the extracapillary space 24 into tubing 50 and 52, and then into tubing 58 and into the extracapillary space 34 of the cartridge 14. The capillaries 28 generally have a molecular weight cut-off which is smaller than the molecular weight cut-off of the capillaries 24 to retain product in the extracapillary space 34. The molecular weight cut-off is such that waste products and medium are allowed to diffuse into the capillaries 28 while product is retained. For example, if the product being produced is a monoclonal antibody, the molecular weight cut-off of the capillaries 18 is approximately 50,000 Daltons and the molecular weight cut-off of the capillaries 28 is approximately 6,000 to 10,000 Daltons to minimize diffusion of the monoclonal antibody into the capillaries 28. Diffusion of waste products and medium from the extracapillary space 34 into the capillaries 28 is enhanced by the flow restriction 38 since the pressure in the capillaries 28 is less than the pressure in the extracapillary space 34. As product accumulates in the extracapillary space 34, the concentration of the product with respect to any medium in the extracapillary space increases. The increase in the concentration or quantity of product results in a purer product for extraction. Once a selected concentration of product is achieved, the valve 62 is opened and the product is pumped out of the extracapillary space 34.

In Figure 2, an alternative embodiment, generally indicated at 70, is diagrammatically illustrated. The embodiment 70 is similar in many respects to the apparatus 10 illustrated in Figure 1 and like reference characters are used to indicate like elements. An expansion chamber 72 is preferably fluidly connected to the ports 46 and 48 of the first cartridge 12 by tubing 74 and 76, respectively. The expansion chamber 72 contains a supplementary supply of medium 78 that is pressurized by a gas pressurization system 80. The gas pressurization system provides a substantially constant pressure level within the chamber 72. The medium within the chamber 72 is fluidly connected by conduit 82 to the port 54 of the second cartridge 14.

First and second valves 84 and 86 are operably disposed with respect to the tubing 74 and 76, respectively, to selectively restrict flow between the extracapillary space 24 and the expansion chamber 72. In one working example, the tubing 74 and 76 was flexible tubing and the valves 84 and 86 were pinch-type valves which pinch the tubing to stop flow therethrough. The valves 84 and 86 are operated in an alternating fashion, that is, one valve is kept open while the other valve is kept closed, to effect flow through the cell culturing space in ^• a predetermined direction.

For example, when valve 84 is closed and valve 86 is opened and the pressure within the capillaries 18 is at a level that is below the level of pressure within the chamber 72, medium flows from the chamber 72 through conduit 76 into the extracapillary space as indicated by arrow 85. Then valve 86 is closed and valve 84 is opened and the pressure within the capillaries 18 is increased to a level greater than the pressure in the chamber 72 so that medium flows from the extracapillary space, as indicated by arrow 87 through tubing 74 into the chamber 72. The cycle is repeated . causing a circulation effect within the extracapillary space.

The circulation of medium between the cell culturing space and expansion chamber results in product, metabolic waste material produced by the cells, and medium to be taken from the extracapillary space 24 into the expansion chamber 72. From the expansion chamber 72, product-containing medium flows from the expansion chamber through tubing 82 into the extracapillary space 34 due to the pressure differential between the expansion chamber and the capillaries 28 of the second cartridge 14. Product is concentrated and collected in the same manner as was previously described with respect to the embodiment 10 in Figure 1.

Still another alternative embodiment 90 is diagrammatically illustrated in Figure 3. The embodiment 90 is quite similar to the embodiment described with reference to Figure 2 and like elements are designated with like reference characters. The embodiment 90 additionally includes a pump 92 operably disposed with respect to tubing 82 to provide motive force for transporting medium from the chamber 72 into the extracapillary space 34. The pump 92 is preferably a peristaltic pump which provides a positive pressure pumping action and with which the medium does not come in contact with.

The pump 92 minimizes the need for balancing the pressures between the chamber 72 and the capillaries 18 to effect flow of product and waste-containing medium from the first cartridge 12 to the second cartridge 14. Another alternative embodiment 100 of the present invention is diagrammatically illustrated in

Figure 4. The embodiment 100 includes a first hollow fiber cartridge 102, a second hollow fiber cartridge 104, an expansion chamber 106, a first medium delivery system 108 and a second medium delivery system 110.

The first cartridge 102 is similar to the cartridge 12 and has an exterior shell 112 and a plurality of capillaries 114 extending between an input end 116 and an output end 118 of the shell. The capillaries 114 have selectively permeable membrane walls that permit diffusion of oxygen, C0₂, nutrients and other chemical components through the membrane walls. An extracapillary space 120 is defined between the shell 112 and the capillaries 114. The capillaries 114 preferably have a pore size that permits medium to flow under ultrafiltrative conditions, such as 50,000 Daltons.^' The second cartridge 104 is similar to the . cartridge 14 and also includes an exterior shell 122 and a plurality of capillaries 124 disposed in a bundle extending from an input end 126 to an output end 128 of the shell 122. The capillaries 124 have selectively permeable membrane walls preferably having a molecular weight cut-off of 6,000 to 10,000 Daltons. An extracapillary space 130 is defined between the shell 122 and the capillaries 124.

The capillaries 114 and the capillaries 124 are fluidly connected by a fluid connection 132, preferably a section of tubing, having a restriction 134. The restriction 134 is similar to the restriction 38, as discussed with respect to Figures 1-3, and provides back pressure to the capillaries 114 so that an ultrafiltrative condition in the capillaries 114 is created. The restriction 134 also produces a pressure drop between the capillaries 114 and the capillaries 124.

The delivery system 108 is similar to the delivery system 40 that was discussed previously with reference to Figures 1-3 and delivers a primary supply of medium, preferably containing oxygen, ^co2' nutrients and other chemical components, to the capillaries 114 and 124. A recirculation line 136 circulates the medium back to the delivery system 108 for replenishment. The expansion chamber 106 is fluidly connected to the extracapillary space 120 by tubing 138 and 140, through ports 142 and 144, respectively. First and second valves 146 and 148 are operably disposed to control flow within the tubing 142 and 144, respectively, so that flow can be selectively restricted in either tubing 138 and 140. Preferably, the tubing 138 and 140 is a medical grade flexible tubing and the valves 146 and 148 are pinch-type valves that pinch the tubing to stop flow therethroug .

The expansion chamber 106 contains a supply of medium 150. The medium 150 preferably contains oxygen, ^co2' nutrients and other chemical components for use by the cells in the extracapillary space 120. The chamber 150 is pressurized by a pressure system 152 that provides a relatively constant level of pressure to the chamber 106. The pressurizing system 152 is preferably a gas system similar to the gas pressurizing system 80 described with reference to Figures 2 and 3. - 14 -

The expansion chamber 106 is fluidly connected to the cartridge 104 by a product transport line 154 to a port 156 of the cartridge 104. The port 156 fluidly connects the line 154 with the extracapillary space 130. A pump 158 provides motive force to transport medium from the expansion chamber 106 to the extracapillary space 130.

A second port 160 is provided at a rearward position in the shell 122 as an outlet for the extracapillary space 130. An outlet conduit 162 and a valve 164 provide a discharge exit from the extracapillary space 130.

Temperature,. pH and oxygen are monitored within the expansion chamber 106 by a temperature probe 166, a pH electrode 168, and an oxygen sensitive electrode 170. The probe and electrodes 166, 168 and 170 provide signals to the second delivery system 110. The second delivery system 110 adjusts the oxygen level and pH so that a preselected level is maintained by addition of further medium through a transport line 172 and a pump 174. As will be understood, the pH and oxygen level within the expansion chamber 106 is reflective of the pH and oxygen level of the medium within the extracapillary space 120 since the medium is being circulated between the expansion chamber 106 and the extracapillary space 120.

Another alternative embodiment generally indicated at 200 is illustrated in Figure 5. The embodiment 200 includes a first hollow fiber cartridge 202, an expansion chamber 204, a second hollow fiber cartridge 206 and a medium delivery system 208. The first hollow fiber cartridge 202 is similar to the cartridge 12 and has an outer shell

210 with a plurality of capillaries 212, preferably disposed in a bundle that extends from a forward end 214 to a rearward end 216 of the shell 210. An extracapillary space 218 is defined as the space between the shell 210 and the capillaries 212.

Tubing 220 and 222 fluidly connect the expansion chamber 204 to the extracapillary space through ports 224 and 226, which are disposed proximate the forward end 214 and the rearward end 216 of the shell, all respectively. Valves 228 and 230 are operably disposed with respect to tubing 220 and tubing 222, respectively, to selectively restrict flow between the expansion chamber and the extracapillary space 218 to effect circulation in the extracapillary space 218.

The second cartridge 206 is similar to cartridge 14 and includes an outer shell 232 and a plurality of capillaries 234, preferably disposed in a bundle, extending from a forward end 236 of the shell to a rearward end 238. An extracapillary space 240 is defined as the space between the shell 232 and the capillaries 234. The expansion chamber 204 is fluidly connected to the capillaries 234 of the cartridge 206 by a transport conduit 242. A pump 244, preferably a peristaltic pump, is disposed operably with respect to the conduit 242 to provide motive force to transport liquid from the chamber 204 to the capillaries 234.

A medium delivery system 208 provides a first medium supply to the capillaries 212 of the cartridge 202 through a feed line 246. At the outlet end 216 of the cartridge 202, a recirculation line 248 transports the medium back to the delivery system 208 for replenishment. The extracapillary space 240 of the cartridge 206 is fluidly connected to the recirculation line 248 by conduit 250 and 252 which are fluidly connected to the recirculation conduit 248 by conduit 254. Conduit 250 is fluidly connected to the extracapillary space 240 through port 256 and conduit 252 is fluidly connected to the extracapillary space 240 through port 258. A section of flexible tubing 251 is fluidly disposed in the recirculation line 248 downstream of the capillaries 212 and upstream of the connection of conduit 254. The tubing 251 includes a flow restriction 253 that provides back pressure to the capillaries 212.

A discharge conduit 260 is fluidly connected to the capillaries 234 of the cartridge 206 at the outlet end 238. The discharge conduit is preferably a flexible piece of tubing and includes a restriction 262 which is preferably caused by a pinch-type clamp pinching the tubing. The restriction 262 causes back pressure within the capillaries 234. in operation, medium from the delivery system

208 is delivered to the capillaries 212 at a pressure sufficient to result in ultrafiltrative conditions,

' similar to what was described with reference to the cartridge 12 in Figure 1. Circulation in the extracapillary space 218 is effected in the same manner as previously described. The waste- and product-containing medium is pumped from the expansion chamber 204 through the line 242 into the capillaries 234. The flow restriction 262 creates a back pressure within the capillaries 234 causing waste materials along with the medium to diffuse under ultrafiltrative conditions into the extracapillary space 240 for transport to the recirculation line 248. Product in a concentrated and purified form exits the capillaries 234 past the flow restriction 262, as generally indicated by arrow 264. The result is a continuous stream of concentrated product that was produced by cells located in the extracapillary space 218 of the cartridge 202.

Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS;

1. A cell culture apparatus used for producing and collecting product from iri vitro cell growth being supplied a first supply of medium from a first medium delivery system, the apparatus including: a first cell unit having a shell and a plur¬ ality of capillaries extending between an input end and an output end of the shell with at least some of said capillaries having selectively permeable walls, the capillaries and the shell defining a cell culturing space therebetween and the shell having first and second spaced-apart ports in fluid communication with the cell culturing space and said capillaries defining a plurality of first lumens for transport of the first medium; a second cell unit having a shell and a plur¬ ality of capillaries extending from an input end to a output end of the shell with at least some of said capillaries having selectively permeable walls, the capillaries and the shell defining a product collection space therebetween and the shell having first and second spaced-apart ports in fluid communica¬ tion with the cell culturing space and said capillaries defining a plurality of second lumens for transport of the first medium; first fluid connection means for connecting the output end of the capillaries of the first cell unit to the input end of the capillaries of the second unit and including flow restriction means for restricting flow of the medium and causing a pressure drop between the capillaries of the first and second units resulting in back pressure in the capillaries of the first cell unit; second fluid connection means for fluidly connecting the first and second ports of the first cell unit with the first port of the second cell unit; and wherein components of the medium are diffused through the capillaries of the first cell unit under ultrafiltrative conditions and product- and waste- containing medium is conveyed from the cell culturing space to the product collection space.

2. The apparatus of claim 1 wherein the capillaries of the first cell unit have a molecular weight cut-off greater than the capillaries of the second cell unit.

3. The apparatus of claim 2 wherein the molecular weight cut-off of the capillaries of the first cell unit is between approximately 15,000 ^"and 150,000 Daltons.

4. The apparatus of claim 3 wherein the molecular weight cut-off of the capillaries of the second cell unit is less than approximately 15,000^' Daltons.

5. The apparatus of claim 1 and further including an expansion chamber with a second supply of medium fluidly connected to the first and second ports of the first cell unit and fluidly connected to the first port of the second cell unit by the second fluid connection means.

6. The apparatus of claim 1 wherein the second fluid connection means includes first and second conduits fluidly connecting the expansion chamber to the first and second ports, respectively, and a third conduit fluidly connecting the chamber to the first, port of the second cell unit.

7. The apparatus of claim 6 and further including means for maintaining a relatively constant pressure within the expansion chamber.

8. The apparatus of claim 7 and further including valving means for selectively restricting flow of the second medium supply through the first and second conduit lines in an alternating fashion such that circulation is effected in the cell culturing space.

9. The apparatus of claim 8 and further including a pump operably disposed with respect to the third conduit to provide a force for effecting flow therein.

10. The apparatus of claim 5 and further including a second medium delivery system for selectively delivering medium to the expansion chamber.

11. The apparatus of claim 10 and further including means for sensing pH within the expansion chamber and communicating with the second medium delivery system such that medium is added to the expansion chamber.

12. A method for culturing cells and for collecting a product produced by the cells, the method comprising: growing and maintaining cells that produce a secretable product in an extracapillary space of a first hollow fiber cartridge'; providing a first medium supply to the capil¬ laries of the first hollow fiber cartridge under ultrafiltrative conditions; and conveying waste- and product-containing me¬ dium from the first hollow fiber cartridge through a first conduit means to an extracapillary space of a second hollow fiber cartridge for concentrating the product in the extracapillary space of the second hollow fiber cartridge.

13. The method of claim 12 wherein the cells are mammalian cells.

14. The method of claim 12 wherein the cells are hybridoma cells.

15. The method of claim 12 wherein the product produced is an antibody.

16. The method of claim 12 and further including circulating a second medium supply from an expansion chamber within the extracapillary space of the first hollow fiber catridge such that waste- and product- containing medium are transported to the expansion chamber and are then transported to the extracapillary space of the second hollow fiber cartridge.

17. The method of claim 12 wherein the second medium contains oxygen and nutrients for use by the cells in the extracapillary space of the first hollow fiber cartridge.

18. The method of claim 12 wherein the chamber is fluidly connected to the extracapillary space of the first hollow fiber cartridge by first and second conduit lines that include first and second valves, respectively, and alternatively closing and opening the valves of the first and second conduit lines in cooperation with alternatively decreasing and increasing the pressure in the capillaries to effect circulation between the extracapillary space of the first hollow fiber cartridge and the chamber.

19. A cell culture apparatus used for producing and collecting product from in vitro cell growth being supplied a first supply of medium from a first medium delivery system, the apparatus including: a first cell unit having a shell and a plur¬ ality of capillaries extending between an input end and an output end of the shell with at least some of said capillaries having selectively permeable walls, the capillaries and the shell defining an extracapillary space therebetween and the shell having first and second spaced-apart ports in fluid communication with the cell culturing space and said capillaries defining a plurality of first lumens for transport of the first medium; a second cell unit having a shell and a plur¬ ality of capillaries extending from an input end to an output end of the shell with at least some of said capillaries having selectively permeable walls, the capillaries and the shell defining a extracapillary space therebetween and the shell having first and second spaced-apart ports in fluid communica¬ tion with the cell culturing space and said capillaries defining a plurality of second lumens for transport of the first medium; first fluid connection means for fluidly con¬ necting the first and second ports of the first cell unit to the capillaries of the second cell unit for flow of product- and waste-containing medium from the extracapillary space of the first cell unit to the lumens of the capillaries of the second cell unit; first flow restriction means fluidly con¬ nected to an output end of the capillaries of the second unit for creating back pressure within the lumens of the capillaries of the second unit; second flow restriction means fluidly con¬ nected to the capillaries of the first unit creating a back pressure within the capillaries of the first unit; and second fluid connection means for fluidly connecting the ^• extracapillary space of the second cell unit with the output of the capillaries of the first cell unit after the second flow restriction means and recirculating medium flowing therethrough to the first delivery system.

20. The apparatus of claim 19 and further including an expansion chamber having a second medium supply and wherein the first fluid connection means includes first and second conduit lines fluidly connecting the extracapillary ^• space with the expansion chamber and including a third conduit for fluidly connecting the expansion chamber to the input end of the capillaries of the second cell unit.

21. The apparatus of claim 20 and further including a pump for providing force to effect flow in the third conduit.

22. The apparatus of claim 20 and further including valving means operably disposed in the first and second conduits to alternatively restrict the flow in the first and second conduits such that circulation is effected in the extracapillary space by medium flowing from and to the expansion chamber.