WO1997016527A1 - Cultureware for bioartificial liver - Google Patents

Abstract

A disposable cultureware (i.e., bioreactor support module (11) and tube set (12)) assembly (10) for use with a bioartificial liver employing a hollow fiber bioreactor (24 and 26) having an extracapillary flow path connected to both a culture (media) circuit (14) and a hemoperfusion (blood) circuit (16). The assembly (10) includes a gas exchange cartridge (20) operably connected to both the culture (14) and hemoperfusion (16) circuits for controllably gassing the media and blood prior to delivery to the bioreactor (24 and 26). The use of a single gas exchange cartridge (20) within both the media (14) and blood (16) circuits provides advantages in terms of both function and performance as compared to alternative approaches.

Description

CULTUREWARE FOR BIOARTTFICIAL LIVER TECHNICAL FIELD The present invention relates to methods and systems for bioartificial liver support. In another aspect, the invention relates to cultureware useful for supporting the function of hollow fiber bioreactors. In yet another aspect, the invention relates to dialysis and patient support systems, and particularly those that employ means for exchanging gases in the blood or other fluids.

BACKGROUND ART A number of alternative approaches have been proposed to provide extracoporeal liver assistance for patients in acute hepatic failure. Such approaches are intended to sustain liver function during brief critical periods of inadequate liver failure, in order to enable the patient's own organ to begin the regenerative process.

Recently, N. Sussman and J. Kelly have described progress made in the course of artificial liver development using cloned human hepatocytes within a hollow fiber cartridge ("The Artificial Liver", Scientific American. May /June 1995). In particular, the authors describe an approach that involves the use of a cloned liver cell line (C3A). These cells are grown in the extracapillary space of a hollow fiber bioreactor as medium is pumped through the fibers themselves. During cell growth the cells are also fed with dissolved oxygen by the use of an in-line oxygenator and high flow rate. During actual treatment, the authors envision the use of cartridges having continuous function, although they recognize the "problem of cell oxygenation during treatment".

A related approach was developed by Scholz and Hu, and is described for instance in Patent Cooperation Treaty Application No. PCT/US91/07952. In this approach, hepatocytes are suspended in a collagen solution and inoculated into the lumen of a hollow fiber assembly. The cell/collagen solution is then incubated with media for approximately 20 hours under conditions that promote collagen gel contraction. This results in the formation of a biomatrix containing the collagen and cells, with the formation of an open intraluminal space within the fibers.

Once the biomatrix is formed, blood is allowed to flow through the extraluminal shell space, with the semi-permeable hollow fibers serving to separate the blood from the hepatocyte-containing matrix. An intraluminal stream of medium is allowed to flow through the newly created lumen space, to carry the growth factors and nutrients necessary to support cell growth. The intraluminal stream can also be used to remove toxins and metabolic products.

While not admitted to be prior art against the present application, Sielaff et al. , in the article "Gel-Entrapment Bioartificial Liver Therapy in Galactosamine

Hepatitis", J. Surg. Res. 59, 179-184 (1995) describe recent progress made in the use of such a bioreactor.

At Figure 2, the Sielaff et al. article provides a functional diagram of the cell culture hardware and software used in regulating pH, oxygen, and shell flow at both the prehemoperfusion and hemoperfusion phases. The diagram depicts the use of a gas exchanger ("GEX") to provide a blend of air and CO₂.

Gas exchangers were developed and continue to be used for such purposes as open heart surgery, where the patient's own heart and lungs are shut down during surgery. The function of such organs is primarily mechanical, as compared to the function of the liver, which is tremendously complex and primarily metabolic.

Gas exchangers have typically not, however, been suggested or used in the course of hemoperfusion using bioreactors, or more particularly using hollow fiber bioartificial livers. To the contrary, in the above-cited article of Sussman et al. (at p. 73, column 1), the authors address the "problem of cell oxygenation during treatment" by offering only two alternative, and significantly different approaches - either "perfusion with oxygenated plasma, or perfusion with whole blood".

Accordingly, Applicants are unaware of any prior art that suggests, let alone teaches the use of a hollow fiber bioreactor having a two-circuit, two-phase extraluminal flow path, wherein a single gas exchange cartridge is provided to serve both circuits.

BRIEF DESCRIPΗON OF THE DRAWING In the Drawing; Figure 1 shows a disposable cultureware assembly of the present invention, in combination with two bioreactors, in operation in the course of the culture circuit;

Figure 2 shows the assembly and bioreactors of Figure 1 in operation in the course of the hemoperfusion circuit; Figure 3 shows a perspective view of a preferred switching valve for use in an apparatus of the present invention; and

Figures 4 A and 4B show, respectively, front and rear perspective views of an operable hemoperfusion system incorporating the assembly and bioreactors of Figures 1 and 2.

SUMMARY OF THE INVENTION

The present invention provides a disposable cultureware (i.e., bioreactor support module and tube set) assembly for use with a bioartificial liver employing a hollow fiber bioreactor having an extracapillary flow path and an intraluminal flow path, the assembly comprising: a) a culture circuit for delivering media to the extracapillary flow path of a bioreactor; b) a hemoperfusion circuit for delivering blood to the extracapillary flow path of a bioreactor; c) switching means operably connected to both the culture circuit and hemoperfusion circuit, for controlling between the delivery of media and blood to the extracapillary flow path; d) a gas exchange cartridge operably connected to both the culture circuit and the hemoperfusion circuit, for controllably gassing the media and blood prior to delivery to a bioreactor; e) a lumen circuit for delivering fluids to the lumen flow path of a bioreactor; and f) bioreactor support and attachment means for operably connecting the extracapillary and lumen flow paths of a bioreactor to the culture, hemoperfusion and lumen circuits.

In a preferred embodiment, hepatocytes or other suitable cells are grown and maintained within the lumen of the bioreactor hollow fibers, as opposed to the extracapillary space. In the media phase of operation, the collagen/cell combination is able to constrict in order to entrap the cells within a gel-like matrix, thereby opening up a media flow path within the lumen. In both the culture circuit and the hemoperfusion circuit, oxygenated media or blood, respectively, are delivered to the cell/collagen matrix across the semi-permeable membrane forming the hollow fibers.

In another aspect, the invention provides a hollow fiber bioreactor operably connected to a cultureware assembly as described herein. The cultureware of the present invention is used to support one or more bioreactors for operation within a cell culture system that provides for the basic mammalian cell functions required for cell sustainment. Such an instrument typically provides an incubator to ensure a 37°C temperature environment and a circulation system (including pumps, tubing, controls, etc.) to provide the cells with nutrients and oxygen. Its integral gassing system supplies oxygen as an essential nutrient and carbon dioxide for pH control. The system has a control panel for user interface to the processor for pump management and for input of sensor and alarm settings. A customized software program allows for varied instrument set up parameters to accommodate varying patient requirements.

In yet another aspect, the invention provides a complete system comprising a cultureware assembly of the present invention, in combination with the bioreactors, pumps, power supply, and other components for its operation in hemoperfusion.

The cultureware is particularly unique in view of its use of a single gas exchange cartridge within both the media and blood circuits. The ability to employ a single cartridge is surprising from the perspective of both engineering and performance functions, and provides significant benefits and options, in terms of production costs, operation and the like.

DETAILED DESCRIPTION The cultureware of the present invention will be further described with reference to the Drawing.

Figures 1 and 2 provide alternative flow diagrams employing a preferred embodiment of the disposable cultureware assembly 10, including both the bioreactor support module 11 and tube set 12. In both Figures 1 and 2, assembly 10 includes two fluid circuits useful for serving the extracapillary flow path of a bioreactor; a culture circuit 14 and a hemoperfusion circuit 16. Culture and hemoperfusion circuits 14 and 16 are each operably connected to switching valve 18. The switching valve can be controlled to select either the culture or hemoperfusion path to be used in operation. Assembly 10 also provides a lumen circuit, for serving the lumen flow path of bioreactor. Figure 3 provides a perspective view of a preferred switching valve for use within an assembly of the present invention. Depicted in Figure 3 is a mechanical valve suitable for manual operation. Those skilled in the art, given the present description, will understand the manner in which automated valve means could similarly be provided, to be operated by control means located, for instance, on the instrument panel.

The valve shown in Figure 3 provides a rigid box-like container 70 having walls 72 and 74 supporting a removable cover portion 76 and forming opposite ends 78 and 80. Cover portion 76 provides access to the interior of the container. Walls 72 and 74 are provided with matching inlet 82 and outlet ports 84, provided through the walls to allow pairs of tubes 86 and 88 to enter and exit the container, traversing the interior. The pairs of tubes are part of the media and blood lines, respectively, serving the extracapillary circuit.

A shaft 90 is rotatably retained within and substantially through the container, the shaft having a plurality of circumferential grooves 92 and 94 configured to simultaneously open and close each pair of tubes. The grooves are provided in the form of recessed regions each having a cam-like appearance when viewed in Figure 3B along the axis of the shaft. The regions are recessed slightly (i.e., the cam portion having the greater axis), to allow a tube to be retained in a pinched (i.e. , closed) configuration. The regions are recessed more deeply on the opposite side of the shaft, to allow the tube to return to its original dimensions. In this region of greater depth, the walls of the groove are also preferably tapered 96 slightly inward (i.e., toward each other). This enables the walls of the groove to participate in re-opening the tubes by gently pinching the tubes along an axis parallel to the axis they were pinched closed.

The shaft is then positioned longitudinally within the container so that, with the cover closed, a tube of appropriate outer diameter can be pinched between the raised cam portion and an interior surface. A pair of tubes serving a circuit can therefore be simultaneously closed by rotating the shaft in such a matter that the raised cam portion pinches the tubes closed against the cover or base.

Simultaneously, on the opposite side of the shaft, as the recessed cammed groove allows and assists the previously pinched tubes to re-open. The beveled edges of the recessed groove serve to gently re-open the tubes and reform them to their original shape and dimensions. The shaft is rotatably positioned through apertures 98 and 100 in the ends of the container. The shaft extends beyond one end, where it can be attached on the exterior of the container to a handle 102 that can be grasped by the user and rotated, e.g. , 180 degrees to close the previously opened circuit and open the previously closed one. Both the "culture" and "hemoperfusion" fluid paths can be selectively operated by use of the switching valve for circulating fluids to the shell (i,e. , extracapillary) side of the hollow fiber bioreactors. This allows for mass transfer of gases, nutrients and impurities through the semipermeable membrane to the lumen side which contains the hepatocytes.

Systems of the present invention, wherein both extracapillary phases are performed within the same assembly, have progressed to the stage of clinical trial. It is anticipated that future systems may not require the use of both a culture circuit and a hemoperfusion circuit. Instead, pre-conditioned bioreactors may be prepared separately or at a remote location, and shipped to the location of use.

Patient treatment would then require the use of only a hemoperfusion circuit, perhaps with some initial start up phase. Systems having both circuits are clearly operational, however, and are presently preferred for situations in which an initial pre-hemoperfusion step and circuit, of any sort, may be desired. Completing the circuits are a gas exchange cartridge (GEX) 20 and blood gas monitoring means, here in the form of a pH probe 22. For the purpose of these Figures, the cultureware is depicted with two hollow fiber bioreactor devices 24 and 26, respectively, already in their operable positions.

Gas exchange cartridges suitable for use in the assembly of the present invention are available from a number of sources. Examples of suitable cartridges include the "Capiox II" brand hollow fiber oxygenator available from Terumo Corporation (Japan), the "Safe-1" brand hollow fiber oxygenator from Polystan A/S (Denmark), the "Maxima Plus" brand oxygenator available from Medtronic Cardiopulmonary (Anaheim, CA), and the "Affinity" brand hollow fiber oxygenator and the ECMO brand membrane oxygenator, both available from

Avecor Cardiovascular (Plymouth, MN).

Those skilled in the art, given the present description, will be able to select and employ an oxygenator in a suitable style and providing an optimal combination of such characteristics as the choice of membrane material, membrane pore size and surface area, fiber inside diameter and wall thickness, capillary lumen volume and extracapillary volume, port placement and dimensions, recommended maximum media or blood flow rate.

In use, those skilled will similarly be able to determine the optimal operating conditions for the oxygenation of both media and blood in an assembly of the present invention. Typically, gas exchange using a hollow fiber oxygenator will be achieved and controlled by adjusting such factors as the gas composition and flow rate and the liquid (media or blood) pressure and flow rate. Media or blood are able to flow through the lumen of the hollow fibers as gas flows across the outside of the lumen (extracapillary space). In addition to the oxygenator, a preferred system of the present invention also includes means for monitoring the blood gas values in the extracapillary circuit during hemoperfusion. Variations in pO₂, pCO₂ and pH levels are each useful in determining blood gas values during hemoperfusion. Variations in these parameters can occur due to patient condition, control settings and equipment performance. Variations can be regulated by suitable control of oxygenator function (e.g. , gas composition, flow rate) and other parameters. Intermittent sample techniques can be used, but may miss or mask dramatic fluctuations. As a result, real time status of hemoperfusion management, in the form of continuous monitoring, is preferred. The pH probe can take any suitable form, e.g. , including those that make direct contact with the media or blood, and those that make only indirect contact. Examples of suitable pH probes include the "TH" line of pH electrodes available from Ingold, the "ASI" line of electrodes available from Analytical Sensors, Inc. (Houston, TX), and the "GX" line of electrodes available from pHoenix Electrode Company (Houston, TX). Preferred electrodes provide an optimal combination of such properties as accuracy, safety, storage/regeneration properties, temperature compensation, and compatibility with the patient's blood within the temperature, flow and other conditions of use.

The manner of selecting a suitable electrode will become apparent to those skilled in the art in light of the present description. See, for example "pH Measurement: The State of the Art", G.K. McMillan, Intech pp 35-39 (1993), the disclosure of which is incorporated herein by reference. Electrodes are available in a number of sizes (e.g., 3mm- 20mm), sensor types (e.g., gold, antimony, platinum, silver), connector types (E.G., U.S. Standard, BNC, pin-type, plug- type), electrode types (e.g., metallic, reference, conductivity), and electrode body construction (e.g., PVC, teflon, glass, epoxy/polymer), and shapes (flat surface, dome- shaped, hemispherical, bulb).

As an alternative to, or in addition to the use of a pH monitor, the system of the present invention can employ other suitable means to monitor and/or alter the blood gas composition (and pH) in the course of operation. Extracorporeal blood gas monitoring systems such as the "Cardiomet 4000" system available from Biomedical Sensors Limited (England), and the "CDl System 400" system available from 3M Health Care (Valencia, CA) are examples of such systems.

Turning to Figures 4 A and 4B, it can be seen that the cultureware can be used to form a complete hemoperfusion system 50, including the assembly of the present invention and first and second bioreactors, all housed within instrument cabinet 52. Also seen within the system are blood monitor panel 54, a universal power supply 56, an IV pole 58, pressure gauges 60, a media bag hangar 62, as well as supporting cart 64 itself. As seen from the rear view provided in Figure 4B, the system also includes an on/off switch 66 controlling the bioreactor support module, a printer port 68, an oxygen tank and regulators 70 as well as a CO₂ tank and regulators 72, and a power cord 74.

First and second bioreactors can be prepared and used in the manner described in PCT Patent Application No. PCT//US91/07952 , the disclosure of which is incorporated herein by reference. Suitable bioreactors for use in a system of the present invention can be constructed using available materials and methods.

A suitable gas blending system is capable of operable attachment to the gas exchange cartridge 20 and preferably includes supplies of medical grade oxygen, medical grade carbon dioxide, and room air gas. The air, oxygen, and carbon dioxide gas flows are controlled and mixed to provide an oxygen-enriched and variable carbon dioxide supply to the gas exchange cartridge. The oxygen is needed to provide additional oxygen to the liver cells by means of oxygenating the media solution during the culture phase and oxygenating the patient's venous blood during the treatment mode. The carbon dioxide gas flow is controlled to modulate the pH of the media during the culture phase and the patient's blood during the hemoperfusion phase.

In a preferred embodiment, the gas delivery system includes a number of components to ensure proper gas delivery timing and pressures. Such components include, but are not necessarily limited to, the use of in-line pressure relief valves, diaphragm air pumps rated for continuous service, in-line coalescent filters to remove particulate material, and separate precision regulators, e.g., separate precision needle valves, to provide separate control for each gas.

A preferred gassing system further includes medical grade oxygen and carbon dioxide gas cylinders, high pressure regulators, and monitoring gauges.

The gas cylinders provided are of sufficient size to provide for continuous operation of the gassing system of the instrument for ten days without a cylinder change.

Also included with each system are containers for fiuid storage, including containers for storing extracapillary media, lumen-in media, lumen-out media, and a waste bottle, together with appropriate tubing, valves, holders, and the like.

The system also provides several pumps for moving fluids through the system. These are of two types: peristaltic pumps and bellows pump. A blood pump, described in greater detail below, is designed to operate on human blood. Other pumps in the system are typically employed for delivery or moving the various solutions (i.e., media, waste, etc.). A bellows pump is preferably used during culture phase to propel media through the shell side of the bioreactors. Peristaltic pumps are intended to deliver quantities of media to the hepatocytes over several days without loss of accuracy. Since, in a preferred embodiment, the system employs two bioreactors, with their extracapillary flow paths connected in series (and their lumen flow paths connected in parallel), four of the peristaltic pumps are "ganged" in pairs, a configuration envisioned by the manufacturer of these pumps and their drive motors.

The system also provides an incubator, preferably in the form of a forced draft convection system, designed to keep the cultureware at a controlled temperature. A fan on the incubator circulates heated air from a coil heater to provide pre-set ambient air conditions. Inside the incubator are typically rows of bulkhead fittings used for electrical cable interface and gas supply to the cultureware. A variety of blood pump modules are suitable for use in the system of the present invention, in order to control and supervise the extracorporeal blood circuit. A preferred blood pump module will include with the pump, an arterial and venous pressure sensor/alarm, a drip chamber air detector, arterial and venous solenoid clamps for blood line clamping for certain alarms, and an optional heparin pump. Such a module can be used to circulate the blood from the patient to the tube set and return it to the patient during the hemoperfusion phase of the procedure.

A suitable uninterruptible power system ("UPS") is used to provide battery backed A.C. line voltage for the bioreactor support and blood monitoring modules; A.C. line power conditioning for the two modules; and A.C. line isolation which brings patient leakage current to an acceptable level.

The instrument and consumable supplies are preferably installed on a cart to provide a mobile self-contained platform for operation. Assorted docking grids, hangers and holders can be provided, e.g., for hanging media bags and waste containers on the cart, and for docking and securing the blood pump and power supply, and bioreactor support module.

Those skilled in the art will appreciate the manner in which the various components, including the materials used to make them, need to be selected, qualified or validated, inspected, labeled and approved in a rigorous manner in view of the applications intended. The assembly and system is typically used in a clean, and optionally sterile environment, and must therefore itself be sterilized or cleansed, as well as inventoried and used appropriately.

Those skilled in the art will also appreciate the manner in which suitable software can be developed to control the operation of the cultureware and bioreactors. Preferably, the software provides the following functions: 1) user interface via a keypad, display, audio alarm, and printer, 2) pump controls, 3) maintenance of incubator temperature, 4) control of O₂ and CO₂ gassing and maintenance of pH during the culture phase, and 5) provision of timing and sequential control of operations. In operation, viable primary hepatocytes can be recovered from pigs and prepared in the manner described in their above-captioned article of Sielaff et al. This article goes on to describe the need for liver support devices to provide a substantial viable hepatocyte mass exhibiting differentiated phenotype, and biotransformation function. The article describes the use of a bioartificial liver based on the cell-entrapment hollow fiber bioreactor of Scholz and Hu. See, for example, Patent Cooperation Treaty Application No. PCT/US91/07952, the disclosure of which is incoφorated herein by reference.

Suitable media, including culture media, lumen media, shell media, and cell perfusion media can be prepared and sterilized using known techniques. As described previously, the recovered cells can be combined with type I collagen in buffer and the resulting solution used to charge (i.e. , load) one or more bioreactors.

Culture circuit 14 is operated in the course of media circulation for conditioning the cells in the bioreactor prior to the hemoperfusion procedure, when the patient's blood is circulated through the bioreactor. With switching valve placed in the culture mode, a bellows pump 28 pulls media from reservoir 30 and circulates it through the switching valve, GEX, pH probe, first and second bioreactors, and a PO₂ probe 32, before returning to the reservoir. During this phase the shell media is circulated through the bioreactors, e.g., at a flow of about 500 ml/min, for a period of about 20 to about 24 hours. Over this time the cells contract and the collagen gel forms and constricts, thereby opening up the lumen of the hollow fibers. After completion of the conditioning period, lumen media flow can be established, and the system used for hemoperfusion.

A separate lumen-in/lumen-out fluid path 40 provides a dedicated fluid pump 42 for each bioreactor lumen-in and a dedicated fluid pump 44 for each bioreactor lumen-out. This insures equal fluid volume to be pumped in and out of the bioreactors with little fluid loss or gain across the membrane. The effect of this arrangement is to prevent the semipermeable membrane from acting as an ultrafiltration device and consequently to restrict membrane transport of solutes to diffusion controlled processes.

Hemoperfusion circuit 16 is operated by placing the switching valve in the appropriate "Patient" or "Hemoperfusion" mode, in order to terminate the flow of media and begin the flow of blood through the circuit. Prior to beginning the hemoperfusion mode, the flow circuit is optionally, and preferably, primed or cleaned, e.g., by vacuum and/or flushing with the patient's blood or another suitable material. In that mode, a blood pump (not shown) is used to circulate the patient's blood, using standard dialysis blood line 36 through the switching valve, GEX, pH probe, first and second bioreactors, and finally back to the patient via a standard dialysis venous blood line 38. Blood is allowed to flow through the extraluminal shell space, with the semi-permeable hollow fibers serving to separate the blood from the hepatocytes. An intraluminal stream of medium is allowed to flow through the hollow fibers, to carry the growth factors and nutrients necessary to support cell growth and to provide toxin or metabolic product removal. The selectively permeable hollow fibers allow diffusion of toxic waste products, such as ammonia and bilirubin from the blood to the intraluminal biomatrix. The blood composition, including the pH and/or blood gas levels can be continually monitored in order to ensure that they are within suitable limits. Variations of these parameters can be achieved by appropriate control of the gas composition and/or flow rate in the oxygenator. As described in Sielaff et al., using such an approach, the three- dimensional extracellular matrix allows high density cell inoculation and promotes differentiated metabolic function and viability. Further, the hollow fiber membrane molecular weight cut off of 100 kDA provides immunoprotection for device borne xenohepatocytes during whole blood perfusion. Bioartificial liver hemoperfusion using such a system appears to provide a source of differentiated hepatic function, prevent the onset of coma, and improve survival.

The present invention has been described with reference to certain preferred embodiments. These embodiments are given for the purpose of further illustrating, but not limiting the apparatus and system of the present invention.

Claims

CLAIMS What is claimed is:

1. A disposable cultureware assembly for use with a bioartificial liver employing a hollow fiber bioreactor having an extracapillary flow path and an intraluminal flow path, the assembly comprising: a) a culture circuit for delivering media to the extracapillary flow path of a bioreactor; b) a hemoperfusion circuit for delivering blood to the extracapillary flow path of a bioreactor; c) switching means operably connected to both the culture circuit and hemoperfusion circuit, for controlling between the delivery of media and blood to the extracapillary flow path; d) a gas exchange cartridge operably connected to both the culture circuit and the hemoperfusion circuit, for controllably gassing the media and blood prior to delivery to a bioreactor; e) a lumen circuit for delivering fluids to the lumen flow path of a bioreactor; and f) bioreactor support and attachment means for operably connecting the extracapillary and lumen flow paths of a bioreactor to the culture, hemoperfusion and lumen circuits.

2. A cultureware assembly according to claim 1, further comprising two bioreactors, the extracapillary flow paths of which are operably connected in series.

3. A hemoperfusion system comprising a cultureware assembly according to claim 2, and further comprising a blood pump, power supply, and other components for its operation in hemoperfusion.