WO1993022427A1

WO1993022427A1 - Method of culturing viable cells and method of regulating the level of a compound in a body fluid

Info

Publication number: WO1993022427A1
Application number: PCT/US1993/003843
Authority: WO
Inventors: Robert S. Ward; John Monahan; Robert Kuhn
Original assignee: Somatix Therapy Corp; Polymer Technology Group Inc
Current assignee: Somatix Therapy Corp; DSM Biomedical Inc
Priority date: 1992-04-24
Filing date: 1993-04-23
Publication date: 1993-11-11
Anticipated expiration: 1994-10-24
Also published as: CA2134088A1; EP0640126A1; JPH07505786A; AU4114493A

Abstract

A method of culturing live viable cells comprises culturing the cells under the conditions effective for cells to survive in the presence of a non-porous, semi-permeable biocompatible film of predefined characteristics having a tensile strength of 350-10,000 psi, an ultimate elongation of 300-1,500 %, a water absorption such that the sum of the volume fraction of absorbed water in the hydrophilic volume fraction of the soft segment is 100-2,000 % of the dried polymer volume and 50-95 % of the wet polymer volume. The film permeability can be changed to have different cut-off molecular weights while being substantially impermeable to cells and particulate matter as well as high molecular weight molecules. A method of regulating the level of a compound in a body fluid of the subject afflicted with an endogenous defect resulting in abnormal levels of the compound in the body fluid, in the substantial absence of a detrimental immunological reaction comprises: enclosing cells lacking the endogenous defect of the patient's cells in a biocompatible, implantable device, wherein at least one portion thereof comprises a non-porous, semi-permeable, biocompatible film substantially enclosing the cells, the film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300 % and up to about 1,500 % and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100 % and up to about 2,000 % of the dry polymer volume and exceeds about 50 % and up to about 95 % of the wet polymer volume, and the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; implanting the device comprising the cells into a site in the subject's body where the cells are in contact with the subject's body fluid; and allowing the cells to grow at the implantation site where they are in direct interactive contact with the compound and act to regulate its level in the body fluid.

Description

METHOD OF CULTURING VIABLE CELLS AND METHOD OF REGULATING THE LEVEL OF A COMPOUND IN A BODY FLUID.

BACKGROUND OF THE INVENTION

Field of the Invention This invention relates, in general, to culturing viable cells in the presence of a non-porous, semi- permeable, biocompatible film formed from a copolymer of specific tensile strength, ultimate elongation and water absorption characteristics which can permeate molecules of up to 6,000 - 600,000 molecular weight while being impermeable to cells and particulate matter. In the method of the invention the nutrients and the medium from without are permeated through the film into the cells' environment, and any cell products from within are permeated through the film away from the cells' environment.

Description of the Background

Standard immune suppression poses acute risks for, e.g., a diabetic patient such as nephrotoxicity from the use of cyclosporin, and is extremely- difficult to justify in the case of, e.g., young diabetic patients.

Four methods of immunological isolation used up to the present time are as follows:

(1) . Extravascular diffusion chambers. (2) . Intravascular diffusion chambers. (3) . Intravascular ultrafiltration chambers. (4) . Microencapsulation. All these approaches have detrimental features that are summarized below. I. They produce host fibrotic response to the implant and material's instability, e.g., in alginate microencapsulation.

II. There are limitations to the diffusion of nutrients across semi-permeable membranes of the prior art with decreasing permeability as protein deposition, blood clotting or fibrous ingrowth block the passage of nutrients through the pores of the membrane. • III. A lag time is observed in the permeability and diffusion of glucose and insulin across prior art semi-permeable membrane barriers, resulting in a delay of a reaction by the cells to the host's glucose levels in blood. Membranes used in prior art implants and methods, with the possible exception of microencapsulation with friable gels, have employed microporous semi-permeable membranes. Such membranes have been fabricated from impermeable polymers with pores being introduced into the material through processing conditions and/or leachable additives. MILLIPORE^® and NUCLEOPORE^®, or polycarbonate microporous membranes utilized by the prior art are made from inherently impermeable polymers and do not support long-term cell viability. Other semi- permeable membranes demonstrate poor blood compatibility as well as low permeability proficiency for the transport of glucose and insulin across the membranes. It should be noted that a microporous membrane may have acceptable high permeant flux in a pressure-driven process such as ultrafiltration, but, at the same time, have very low permeability in a concentration-driven process such as the in vivo method of this invention. When the microporous membranes are placed in direct contact with a body fluid such as blood, they accumulate a fibrin layer which becomes a major barrier to mass transportation through the membrane.

In general, polyetherurethane block or segmented copolymers exhibit good biocompatibility along with high strength and elastomeric properties. This unique combination of properties is due in part to the two-phase morphology of the polyurethane molecule. In a typical polyurethane, aggregated aromatic or aliphatic urethane or urea segments constitute a hard glassy or semicrystalline phase, while low glass transition temperature (Tg) oligomeric segments comprise the liquid-like, rubbery soft phase or segment. The morphology of a polyurethane depends on many factors, including hard and soft segment chemistry, segment polarity differences, hard segment content, and hard and soft segment molecular weights.

In both polyurethaneureas and polyurethanes, the chemistry of the soft segment affects the degree of phase separation in the polymer, which in turn affects its bulk and surface properties and subsequent biocompatibility. Polyurethaneureas, similar to the ones disclosed in this patent only as to their hard segment compositions, have been shown to be resistant to degradation in several applications (Paynter, et al. , "The Hydrolytic Stability of Mitrathane, a Polyurethaneurea - An X-ray Photoelectron Spectroscopy Study", J. Biomed. Mater. Res. 22:687-698 (1988); Szycher, et al. , "Blood Compatible Polyurethane Elastomers", J. Biomater. Appl. 2:290-313 (1987)).

The application of natural and synthetic polymer membranes to the separation of gaseous and liquid mixtures of low molecular weight has been reported in a number of reviews. Many studies of membrane permeability to simple low molecular weight (MW) permeants have been reported in which the composition of glassy-rubbery or crystalline-rubbery copolymers are varied. A polyurethane multipolymer membrane different from the one disclosed herewith has been shown to be water and salt permeable. In thermoplastic segmented block copolymers where one block or segment is glassy or crystalline (hard segment) and another is rubbery or liquid-like (soft segment) , the permeation of molecules occurs primarily through the soft segment. The relatively impermeable hard segment, provides physical integrity to the polymer by virtue of its strong intermolecular interactions with like segments on adjacent molecules, even under conditions which may cause swelling of the soft segment.

Okkema, et al., discloses a series of polyether polyurethanes based on polyethylene oxide (PEO) , polytetramethylene oxide (PTMO) and mixed PEO/PTMO soft segments suitable as blood contacting surfaces, but with a hard segment content of 55 wt%, too high to be useful in the present invention. (Okkema et al., "Bulk, Surface, and Blood-Contacting Properties of Polyurethanes Modified with Polyethylene Oxide", J. Biomater. Sci. Polymer. Edn.l(1) :43-62 (1989)). Takahara, et al., discloses the preparation of

Segmented Poly (etherurethaneureas) (SPUU) with hydrophilic and hydrophobic polyether components. (Takahara et al., ^,rSurface Molecular Mobility and Platelet Reactivity of (SPUUS) with Hydrophilic and Hydrophobic Soft Segment Components", J. Biomater. Sci. Polymer. Edn. l(l):17-29 (1989)). Platelet adhesion and dynamic contact angle measured after adsorption of bovine serum albumin revealed that the SPUUs with hydrophilic soft segments had a non-adhesive surface. Chen, et al. , examines the relationship between structure and properties of polyether based polyurethanes. (Chen et al. , "Synthesis, Characterization and Permeation Properties of Polyether Based Polyurethanes", J. Appl. Poly . Sci. 16: 2105-2114 (1972)) . Of particular interest is the testing of the transport of water and low molecular weight salt through polymeric membranes made of elastomers that are block copolymers consisting of hard and soft segments, with the former acting as physical crosslinks.

U.S. Patent 3,804,786 to Sekmakas discloses water-dispersible cationic resins, particularly polyurethane resins prepared by reaction of a resinous polyepoxide with a polyisocyanate to provide an hydroxy-functional polyurethane with tertiary amine functionality. These resins are useful for electrode position at the cathode.

U.S. Patent 3,826,768 to Suzuki and Osonol discloses a process for preparing polyurethane compositions by dispersion of polyurethane-containing isocyanates made from polyols and organic isocyanates in water under specified conditions.

U.S. Patent 3,852,090 to Leonard et al. , discloses the utilization of a urethane film for waterproofing a breathable textile substrate. U.S. Patent 4,124,572 to Mao relates to thermoplastic polyurethanes prepared by a specified method. The thus produced elastomers are useful for automotive products, applications such as cattle ear tags, coatings and coated fabrics.

U.S. Patent 4,183,836 to Wolfe, Jr. discloses a water-based polyurethane dispersion and its preparation by reacting an aliphatic diisocyanate with three critical active hydrogen compounds to form a pre-polymer containing carboxyl and free isocyanate groups, and then dispersing the pre-polymer in an aqueous medium with a tertiary amine and a diamine. These dispersions are useful in coating applications such as textile materials. U.S. Patent 4,190,566 to Noll et al., relates to non-ionic, water dispersible polyurethanes with substantially linear molecular structure and lateral polyalkylene oxide polyether chains containing ethylene oxide units of specified content. U.S. Patent 4,202,880 to Fildes et al. , discloses sustained release delivery means comprising a biologically active agent, i.e., a drug, a linear hydrophilic block polyoxyalkylene-polyurethane copolymer, and optionally a buffer. A single hydrophilic soft segment is used. Only the hard segment is hydrophobic.

U.S. Patent 4,202,957 to Bunk, et al., discloses polyurethane polyether-based elastomers which are thermoplastic and recyclable, and have increased high temperature resistance that makes them suitable for injection molding.

U.S. Patent 4,224,432 to Pechhold et al., discloses a polyurethane comprising a reaction product of a polymerizate of tetrahydrofuran and an alkylene oxide, an organic polyisocyanate and a chain extender which is an aliphatic polyol or a polyamine. U.S. Patent 4,367,327 to Holker et al., relates to a breathable polyurethane film for coating fabrics to make them waterproof. The polyurethane film comprises in stoichiometric amounts a hard segment made of a low molecular weight diisocyanate with a difunctional compound, and a soft segment comprising polyethylene glycol. The mechanical properties of the film are improved by crosslinking with a triisocyanate. U.S. Patent 4,849,458 to Reed et al. , discloses a hydrophilic, segmented polyether polyurethane-urea exhibiting increased tensile strength and elongation when wet with water. The polymers form clear films that are permeable to water vapor.

Many of the afore-mentioned materials are segmented polyurethane elastomers. Some of them, moreover, have found biomedical applications virtually without being modified. However, despite their widespread use, many biomaterials were originally developed for nonmedical uses. In fact, most polyurethane materials were developed to satisfy high volume, industrial needs. A most notable example is DuPont's LYCRA SPANDEX^®, a polyurethane utilized in the fabrication of circulatory support device components. This material was later sold under the trade name BIOMER* Segmented Polyurethane. AVCOTHANE-51^® resulted from the combination of two commercially available polymers, a silicone and a polyurethane, both of which are widely used as fabric coatings. AVCOTHANE-51^® is utilized in biomedical devices such as an intra-aortic balloon. The sole improvements introduced for its biomedical applications were the use of highly purified starting materials, the filtration of the product solution and clean conditions for the fabrication of blood-contacting surfaces. Another.biomedical polyurethane, AVCOTHANE-610^®, also called CARDIOMAT-610^®, and ANGIOFLEX^® are presently being used in blood pumps and trileaflet heart valves.

The thermoplastic material PELLETHANE^® was first applied to the manufacture of cannulae for blood vessels, and later of catheters. This material had originally been developed as an extrusion molding resin exhibiting superior hydrolytic stability over their polyester-based counterparts. Although many polyurethanes and polyurethaneureas are available commercially-, some of which were discussed above, none forms membranes of permeability, strength, flexibility, and biocompatiblity required for growing cells by permitting the passage of nutrients, cell products and cell waste materials while preventing the passage of immunological or microbiological substances that might be detrimental to cell growth and the manufacture of cell products.

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily perceived as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying figures.

Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion.

SUMMARY OF THE INVENTION This invention relates to a method of culturing live viable cells, that comprises: culturing live viable cells under conditions effective to grow the cells in the presence of a non- porous, semi-permeable, biocompatible film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or a phipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500%, and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and is up to about 2,000% of the dry polymer volume and exceeds about 50% and is up to about 95% of the wet polymer volume; the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter and wherein the nutrients and the medium's components being permeated through the film into the cells' environment, and any cell products being permeated . through the film outside of the cells' environment.

This invention also relates to a method of regulating the level of a compound in a body fluid of a subject afflicted by an endogenous defect resulting in abnormal levels of the compound of the body fluid, in the substantial absence of a detrimental immunological reaction, comprising: enclosing cells lacking the endogenous defect of the patient's cells in a biocompatible device wherein at least one portion thereof comprises a non-porous, semi-permeable, biocompatible film substantially enclosing the cells, the film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction- of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume, and the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; implanting the device comprising the cells into a site in the subject's body where the cells are in contact with the subject's body fluid; and allowing the cells to survive at the implantation site where they are in direct interactive contact with the compound and act to regulate its level in the body fluid.

Also provided herein is a kit for correcting a metabolic defect in a subject, comprising: a biocompatible implantable device, wherein at least one portion thereof comprises a non-porous, semi-permeable, biocompatible, implantable film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about- 95% of the wet polymer volume and being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; syringe; needles; and instructions for use of the kit to insert preselected cells lacking the metabolic defect into the device and implant the device in the body of a subject afflicted with the metabolic defect so that the cells may interact with the subject's body fluids and alleviate the symptoms associated with the defect.

This invention provides a method of culturing cells in the presence of a device comprising at least one portion of a film comprising a biocompatible, hydrophilic, segmented block polyurethane copolymer. In one of the aspects of this invention, the in vivo method is applied to the remediation of a metabolic defect that produces a detrimental or undesirable compound in a body fluid such as blood, that when in direct interactive contact with the cells is transformed into a harmless or desirable compound and then returned to the body fluid. This is the case of phenylketonuria, where a genetically- engineered cell may transform phenylalanine that builds up in an infant's blood stream into L-tyrosine or phenylpyruvic acid; harmless products.

In another aspect, the method utilizes cells that comprise product-producing cells. An example of this type of method is that for countering high blood levels of glucose in diabetic patients. Once insulin producing cells are implanted in accordance with this invention, and when activated by increased glucose levels in blood, the cells produce insulin which is permeated into the blood stream and distributed to target cells where they aid in the incorporation of glucose into the cells.

Still part of this invention is a kit for correcting a metabolic defect in a subject, that comprises: a biocompatible device wherein at least one portion thereof comprises a non-porous, semi- permeable, biocompatible implantable film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume and being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; syringe; needles; and instructions for use of the kit to insert preselected cells lacking the metabolic defect into the device and implant of the device in the body of a subject afflicted with the metabolic defect so that the cells may interact with the subject's body fluids and alleviate the symptoms associated with the defect.

In addition to the above components, the kit may also comprise cell culture medium, either in powdered form, or in liquid form.

With regard to all of the aforementioned embodiments of the present invention, a further embodiment relates to the use of a hydrogel to immobilize cells to insure an even distribution of cells. The hydrogel of the present invention is comprised of greater than about 35% water. It is preferably that the hydrogel be an alginate.

Brief Description of the Drawings

Figure 1 is a graph showing the results of a glucose tolerance test in mice that compares various treatment groups .

DESCRIPTION OF THE PREFERRED EMBODIMENTS This invention arose from a desire of the inventors to improve on prior art methods to treat a variety of diseases, such as diabetes, clotting disorders, organ failure and brain disfunction, among others.

Up to the present time, other technologies that have attempted to restore the function of faulty biological systems in the human body have relied on the administration of a missing or defective product such as the administration of insulin or other hormones by various methods, the ex vivo treatment of a human body fluid such as blood, and the implantation of a device such as a catheter that remains connected through the skin with an outside ex vivo system that effects a therapeutic action on the fluids . The present invention provides a novel and unobvious method for the continuous treatment of human diseases of genetic origin. In the more general method of the invention, the method comprises : a method of culturing live viable cells, comprising culturing live viable cells under conditions effective to grow in the presence of a non-porous, semi-permeable, biocompatible film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment,and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500%, and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up- to about 95% of the wet polymer volume and the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; wherein the nutrients and the medium's components from without are permeated through the film into the cells' environment, and any cell products from within are permeated back through the film outside of the cells^■ environment.

This method of culturing viable live cells permits the passage of nutrients and substrates from the medium outside of the film or membrane into the cell area and, vice versa, any cell products are transported out of the cell environment into the medium on the other side of the film or membrane. The characteristics of the present non-porous, semi- permeable biocompatible film and membranes permits the passage of molecules of varying molecular weights while, at the same time, the characteristics of the film remains substantially impermeable to cells and other particulate matter. Thus, the film may be tailored, by varying its composition, to have a predefined cut-off molecular weight above which no molecules can be transported into the cell environment. Thus, the film may be custom tailored to preempt the passage of immunological molecules such as complement and the like which normally are produced by a patient upon implantation of foreign cells.

Any type of cell that is capable of "curing" an endogenous functional defect of biochemical origin may be used in the practice of the present invention. Substrates, proportions thereof, polymers, methods of preparation and forms of custom-tailoring the characteristics of the film for different applications are disclosed in a co-filed, co-pending U.S. application entitled "Copolymers and Non-Porous, Semi-Permeable Membrane Thereof and Its Use For Permeating Molecules of Predetermined Molecular Weight Range" by Robert Ward and Kathleen White, (Attorney Docket No. SOM 20011) the text of the portions disclosing such information being incorporated herein by reference.

The preferred polymers to be used in the method of the present invention may be synthesized to have a specific permeability to a given permeant and/or to have a specific molecular weight cutoff, by implementing an empirical, yet systematic approach.

The empirical nature of the method is mandated by the nature of the phenomenon of permeability through dense membranes, the properties of specific permeants or non-permeants, including their solubility properties, molecular size and conformation. The inventors provide herein a systematic approach to the production of membrane polymers in accordance with the present invention, which may be used to tailor membrane properties for specific applications. This is described briefly in the following paragraphs. The permeation of solutes through dense polymeric membranes is determined for the most part by the diffusivity and solubility of the permeants in the membrane polymer. If the membrane polymer absorbs a significant amount of the solvent, then the permeation of the solutes will be determined by the diffusivity and solubility of the permeants in the solvent-swollen membrane polymer.

The absorption of a solvent, e.g., water, by the membrane polymer requires that the polymer have some affinity for the solvent. In addition, by definition, the solvent must be capable of dissolving ^"the solute/permeant. It follows, thus, that the absorption of the solvent by the membrane may increase the contribution of the solubility factor to the permeability coefficient by making the environment within the membrane polymer more like the pure solvent than it was in the dry state. In general, in addition to enhancing the solubility of the permeant in the membrane polymer, a low molecular weight solvent will often act as a plasticizer for the membrane polymer. Plasticization involves a degree of dissolution of the polymer by the plasticizer. Furthermore, as the level of plasticizer/solvent increases, the glass transition temperature of the mixture will generally decrease. A decreased glass transition temperature suggests that the plasticizer may facilitate the relative movement of macromolecular chains by inserting themselves between adjacent chains to increase the intermolecular spacing there between. In addition to the above, plasticizer/solvents may reduce the degree of possible polymer-polymer interactions through specific interactions between the polymer and the plasticizer/solvent. A reduction in the soft segment crystallinity upon ydration, which occurs with certain membrane polymers of the present invention, is an example of the latter mechanism.

In the case of an isotropic polymer membrane, significant solvent absorption/swelling will produce a measurable increase in the physical dimensions of the membrane, e.g., along each of the x, y and z axes, by an amount approximately equal to the cube root of the volume fraction of the solvent absorbed therein. This provides direct evidence that the polymer chains have increased intermolecular distance in the swollen state since the same number of polymer molecules are now contained in a larger total volume. This increased spacing and facilitated movement of polymer chains may increase permeability by . increasing the diffusivity contribution to the permeability coefficient.

Thus, the absorption of a solvent by a membrane polymer may enhance the membranes permeability by increasing both the diffusivity and the solubility of a particular permeant. One method of tailoring the membrane of the present invention to obtain a specific permeability rate and/or molecular weight cutoff, is to vary the composition and morphology of the membrane. This will effect an enhancement of the amount of solvent absorbed, and of the extent of solubility and diffusivity that results from greater solvent absorption.

Although in some instances it may not always be possible to make exact quantitative predictions of the permeation characteristics of the resulting membrane, the inventors have found that certain qualitative and quantitative relationships exist which guide the process. Furthermore, the permeability of candidate membranes may be performed with the methods described by the inventors herein. The structure vs. property relationships provided herein may be used to adjust the permeability properties of the membrane through an iterative process of synthesis, membrane casting and permeability measurement, until the desired values for the intended use are attained.

In the examples provided below it is assumed that the permeant is a water-soluble macromolecule and that the solvent is water or an aqueous fluid. Those skilled in the art will know that similar approaches may be applied that are suited for other solvent/permeant systems by modifying the soft segment to facilitate the absorption of a non aqueous solvent, for example.

Table 1: Membrane Polymer Structure Versus Property Relationships

Variable Effect

Increasing Soft Segment Molecular • Increases water absorption at Weight constant soft segment hydrophilicity and constant soft segment content (++) •Increases permeability rate (+ + +) •Increases molecular weight cutoff (++) •Increases (dry) soft segment crystallinity

(++) •Decreases (dry) tensile modulus unless soft segment crystallizes (-) •Increases ultimate tensile elongation unless soft segment crystallizes (+)

Increasing Soft Segment •Increases water absorption at constant Hydrophilicity soft segment molecular weight and constant soft segment content (+ + +) •May increase soft segment crystallinity if hydrophilic segments crystallize (++)

Increasing Hard Segment Content •Decreases permeability rate ( )

•Increases tensile strength (++) •Increases tensile modulus (+ + +) •Increases wet strength (++)

Increased Hard Segment Domain •Increases permeability rate at constant Size hard segment content (+)

Mixing Two or More Soft •Increases permeability rate when it Segments decreases soft segment crystallinity (++) •Can be used to increase solubility of permeant in polymer (by adding groups which have an affinity for permeant) and therefore increases permeability (++) Crosslinking At Low Crosslink Density •Increases permeability if used to obtain strength by significantly reducing hard segment content. (++) •Can decrease permeability rate and molecular weight cutoff at higher crosslink density ( )

(+) and (-) refer to the nature of the effect and its intensity: (+ + +) — Strong positive effect. (-) = Weak negative effect, etc. The hard segment of the copolymer of the invention may preferably have a molecular weight of about 160 to 10,000, and more preferably about 200 to 2,000. Its components also have preferred molecular weights as shown in Table 2 below.

Table 2: Preferred Molecular Weights for Hard

Segment Component Hard Seg. Component Most Preferred MW Preferred MW Aromatic Diisocyanates 150-270 100-500

Aliphatic Diisocyanates 150-270 100-500

Chain Extenders 60-200 18-500

Although both the hard and soft segments may be utilized in a broad range of molecular weights, Table 3 below shows typical useful molecular weight ranges and preferred molecular weight ranges for some exemplary components of the soft segment.

Table 3: Preferred Molecular Weights for Soft Segment Components Soft Segment Component Most Preferred MW Preferred MW

Polyethylene oxide 1000-9,000 200-1,000,000

Polytetramethylene oxide 1000-9000 500-50,000

Polypropylene oxide-polyethylene oxides 1000-5,000 500-50,000

Polytetramethylene oxide-polyethylene oxides 1000-2,000 500-50,000

Amine-capped polypropylene-polyethylene oxides 600-6,000 200-1,000,000 200-50,000

200-50,000

100-20,000 200-1,000,000 200-50,000

500-10,000

The content of hard segment of the copolymer is typically about 5 to 45 wt%, the remainder of the polymer consisting of soft segment, which may be a combination of hydrophilic, hydrophobic and amphipathic oligomers .

In one preferred embodiment, the copolymer comprises about 9 to 30 wt% of the hard segment, and more preferably 10 to 28 wt% thereof. Similarly, a typical content of the soft segment is about 91 to 70 wt%, and more preferably about 90 to 72 wt%. However, other proportions of hard and soft segments are also suitable for practicing this invention.

A polymer made from this composition will have the properties described in Table 4 below. Table 4: Characteristics of Film of the Invention

Characteristics Range -

Tensile strength > about 350 and up to about 10,000psi

Elongation at Break > about 300 % and up to about 1,500% Water Absorption + > about 100% and up to about 2000% dry wt

Hydrophilic Soft Segment

> about 50% and up to about 95% wet wt or more preferably

Water absorption only > about 100 % and up to about 2000% dry wt

> about 50% and up to about 95% wet wt

Thickness about 5 to 100 microns (when unsupported)

Thickness about 1 to 100 microns

(when supported or reinforced)

This invention also provides a non-porous, semi-permeable, biocompatible film that comprises the block copolymer of the invention. In a preferred embodiment, the film is formed from the copolymer of this invention. In another preferred embodiment the film is coated onto a support. In still another preferred embodiment, the film is an integrated part of the substrate and is made of the same or similar polymer. In particularly preferred embodiments, the non-porous film of the invention is provided in the form of a flexible sheet and a hollow membrane or fiber. . Typically, the flexible sheet may be prepared as a long rollable sheet of about 10 to 15 inches width and 1 to 6 feet length. However, other dimensions may also be selected. Of particular importance is the thickness of the sheet which may be about 5 to 100 microns, and more preferably about 19 to 25 microns when it is to be used without support or reinforcement. The flexible sheet is prepared from the block copolymer of the invention by methods known in the art, typically, by casting, and more preferably by casting on a web or release liner. As already indicated, the composition may be coated as a film onto a substrate. Where permanently supported on a reinforcing web, e.g., a fabric, the film or membrane may be thinner, e.g., as thin as about 1 micron, whereas when used unsupported the thickness may only be as low as about 5 to 10 microns.

When membranes are fabricated, from the polymer of the invention by knife-over-roll casting onto a release paper, web or liner in the form of dry films, they may have an about 1 to 100 micron nominal thicknesses on a continuous coating line. A

20-foot-long continuous web coater may be utilized having, e.g., a maximum web width of 15 inches equipped with two forced-air ovens. In one particular embodiment, the coater may be modified for clean operation by fitting the air inlet ducts with High Efficiency Particulate Air (HEPA) filters. A nitrogen-purged coater box may be used to hold and dispense filtered polymer solutions or reactive prepolymer liquids. However, other set-ups are also suitable.

All but trace amounts of a casting solvent, e.g., dimethylformamide may be removed by coater's hot air ovens fitted with HEPA filters. After membrane casting, membrane and substrate may be further dried to reduce residual solvent content to less than about 100 ppm, as determined by liquid chromatography. The thickness of the fully-dried cast films may be measured by, e.g., using a spring micrometer sensitive to 0.0001 inch (2.5 μM) or visually by using a microscope. The membrane of this invention may have any shape resulting from a process utilizing a liquid which is subsequently converted to a solid during or after fabrication, e.g., solutions, dispersions, 100% solids prepolymer liquids, polymer melts, etc.

Converted shapes may also be further modified using methods such as die cutting, heat sealing, solvent or adhesive bonding or any of a variety of other commonly-used fabrication methods. For example, when in the form of a hollow tube, the membrane is generally prepared with a diameter of about 0.5 to 10 mm , and more preferably about 1 to 3 mm, and a thickness of about 1 to 100 microns, and more preferably about 19 to 25 microns. The hollow membrane may easily be prepared in long rollable form, and be cut to a length of about 0.75 to 31 inches, and more preferably about 0.5 to 6 inches.

Any type of cell that is capable of "curing" an endogenous functional defect of biochemical origin is suitable for use herein. The present method may be practiced with a wide range of genetically-engineered or mutated cell types having many different therapeutic applications.

One example is the application of the present method to the treatment of diabetes. Another example is that of the application of the present method for the treatment of phenylketonuria with cells that are capable of producing an enzyme that transforms phenylalanine into either tyrosine or phenylpyruvic acid which are innocuous to the human body.

The method of this invention will reduce the incidence and severity of microvascular complications associated with type I diabetes, and other metabolic diseases. The present method utilizes a device that contains the pertinent cells for correcting a defect in a subject and provides :

(1) protection to the cultured cells from macrophages and the immune system;

(2) maintains cell viability for extended periods;

(3) permits the free passage of nutrients, secretagogues and cell products; (4) presents a biocompatible surface to the body fluids and tissues;

(5) simple surgical implantation and, if necessary, cell replenishment;

(6) the implementation of the device utilized in the in vivo method provided herein is easily fixed in place and easily removed, when necessary.

The present method overcomes a major obstacle to long-term survival of cell grafts: immunological rejection of the allografted or xenografted tissue. The viable exogenous cells may be introduced in the human body by means of a variety of devices. Examples, of such devices have been disclosed in a U.S. application entitled "Biocompatible, Therapeutic, Implantable Device" by Robert S. Ward, Robert Kuhn and Veronica Jean Chater, (Serial No.

07/874,342) the sections of the text describing the various devices for implanting live viable exogenous cells into the human body being incorporated herein by reference. One method of the invention relies on the in vivo implantation of a device containing viable cells capable of performing a function that is defective in the human subject in which it is implanted, for example, the method of the present invention may be practiced on a diabetic person by implanting insulin- producing cells in a biocompatible, implantable device wherein at least a portion thereof comprises a film or membrane as described herein, the device surrounding the cells and placing them in isolation inside the human body. Thus, the method may be conducted by growing the cells in isolation within the human body, where they are placed in contact with a human fluid such as blood in direct continuous interaction therewith. In the case of a diabetic, the insulin-producing cells are implanted at a site such as a blood vessel, intestinal cavity or in or around an organ, among other sites, and allowed to interact with the person's blood. The protein or ^'hormone, such as insulin produced by the cells is permeated out of the device through the non-poroous, semi-permeable film or membrane and into the blood stream, wherefrom it can reach target cells. Any glucose present in blood will then be capable of entering into the human body's cells for utilization and metabolism. Accordingly, the undesirable high blood levels of glucose suffered by diabetics go down.

In a preferred embodiment, the live viable cells are not only capable of producing insulin but, in addition, the production of the hormone is regulatable in response to the levels of glucose in the blood. However, other cells may also be utilized.

The use of a non-porous, self-supporting, semi- permeable membrane such as the one utilized herein maintains an environmental immuno-isolation for the cells while permitting the passage of nutrients, secretagogues and cell products. The present method uses a strong, dense, water-swollen membrane or film, that is permeable to the body fluid components that must be in contact with the cells and the device, and to the products produced by the cells. In a particular embodiment of the in vitro method of the invention, the cells comprise product- secreting cells. The products may be hormones such as thyroid, pancreatic, and other hormones, or recombinant proteins produced subsequent to genetic modification.

In another preferred embodiment, the cells comprise insulin-producing cells. In a more preferred embodiment, the production of insulin by the cells is regulatable by changes in the level of glucose in the medium.

Examples of cells that may be used for the production of insulin in vitro are unmodified mammalian islets of Langerhans, insulin producing recombinant prokaryotic or eukaroytic cells and glucose-regulated insulin-producing eukaryotic cells arising from homologous recombination or mutation.

In general, to practice the method of the invention, the cells may be selected from prokaryotic and eukaryotic cells. Among these, the cells may be selected from the group consisting of immortalized cells, live tissue cells, and primary culture cells. These are obtained by methods known in the art that need not be further described herein or commercially available sources. See, for example, Gazdar, A.D., Chick, W.L., Oie, H.K. et al. , " Continuous, Clonal, Insulin and Somatostatin-Secreting Cell Lines Established From a Transplantable Rat Islet Cell Tumor. Proc. Natl. Acad. Sci. USA, 77:3519-3523 (1980); and Santerre, R.F., Cook, R.A. , Crisel,

R.M.D. et al. , "Insulin Synthesis In Clonal Cell Line of Simian Virus 40-Transformed Hamster Pancreatic Beta Cells," Proc. Natl. Acad. Sci. USA .78.:4339-4343. (1981) . In a particularly preferred embodiment of the method of the invention, the device utilized here substantially encloses the cells.

In a most preferred embodiment of the method of the invention, the cells that are enclosed within the device are immobilized in a hydrogel that is comprised of greater than about 35% water. The hydrogel maintains an even distribution of cells within the device. This insures the optimal diffusion of the intracellular products out of the implanted device. Suspending the cells within a hydrogel has also been found to provide enhanced cell viability.

When the method of the invention is practiced in vivo, it further comprises implanting the device comprising the cells into a subject's body; and wherein the culturing step is conducted in vivo and in the substantial absence of a detrimental immunological response. As already indicated, the film or membrane utilized in the present method prevents the passage into the cell environment of immunological molecules that could be elicited by the foreign cell's presence in the subject^rs body. One method of the invention relies on the in vivo implantation of a device containing live viable cells capable of performing a function that is defective in the human subject in which it is implanted. For example, the method of the present invention may be practiced on a diabetic person by implanting insulin-producing cells in a biocompatible, implantable device, wherein at least a portion thereof comprises a film or membrane as described herein, the device surrounding the cells and placing them in isolation inside the human body. Thus, the method may be conducted by growing the cells in isolation within the human body, where they are placed in contact with a human fluid such as blood in direct continuous interaction therewith. In the case of a diabetic, the insulin-producing cells are implanted at a site such as a blood vessel, intestinal cavity, or in or around an organ, among other sites, and allowed to interact with the person's blood. The protein, or hormone, e.g., insulin produced by the cells is permeated out of the device through the non-porous, semi-permeable film or membrane and into the blood stream, wherefrom it can reach target cells. Any glucose present in blood will then be capable of entering into the human body's cells for utilization and metabolism. Accordingly, the undesirable high blood levels of glucose suffered by diabetics go down.

In a preferred embodiment, the live viable cells are not only capable of producing insulin but, in addition, the production of the hormone is regulatable in response to the levels of glucose in blood. This is the case of cells such as recombinant, insulin producing, mammalian cells, and the like, and in particular the use of retroviral vectors capable of expressing proteins such as insulin. However, other cells may also be utilized. According to recent estimates, approximately 400,000 Americans have insulin-dependent type I diabetes that is characterized by deficient insulin production and/or release. The method of this invention is helpful in the treatment of diabetes by implantation of an "artificial pancreas" comprising a biocompatible device having at least one permeable area permitting the passage of molecules up to a predetermined molecular weight but keeping out cells and other particulate matter, the device containing insulin-producing cells capable of providing approximate normal glycemia through insulin release in response to changing glucose concentrations in the area of the device that is in contact with the semi- permeable film or membrane. In one particularly preferred embodiment, the cells implanted are the subject's own defective cells and they are genetically engineered to overcome the defect prior to implantation.

The method of the invention may be practiced by planting the device loaded with the cells in a multiplicity of sites in the subject's body. Examples of the sites are in and around an organ, in and around the omental pouch,, intravaginally, intradermally, subcutaneously, intracavitarily, intraperitoneally and intravascularly, among others.

When the method is practiced by implanting the device subcutaneously, the implantation may be conducted anywhere in the subject's body, depending on the size and nature of the product produced by the cells or the substance present in a body fluid that needs to be exposed to the cells to affect their release of the product(s) . One example is in the area adjacent to where lymph returns to the circulation. The administration of hormones, such as growth hormone or other proteins products, may be practiced in accordance with the method of the invention by implanting the device subcutaneously, e.g., under the arm. Other areas of implantation could be in the fat pad under the epidermis, in the intestinal cavity, and the like.

One preferred embodiment is where the method of the invention is practiced by implanting a device such as a catheter having at least a portion thereof made of the film of the invention, in a vessel, such as an artery of a subject. Examples of cells that can be utilized, although nonregulatable, in the case of production of insulin are pancreatic tumour cell lines such as hamster insulinoma, and rat insulinoma. Cell lines such as these may be selected and/or mutated repeatedly in order to transform them into glucose-regulatable insulinomas.

The present method may counter a metabolic fault in a subject in multiple ways. One example is that wherein the subject's cells do not produce sufficient amounts of a certain compound, e.g., a hormone such as insulin, or thyroxine, or clotting factors as in treating hemophilia. A second example is where undesirable levels of metabolites accumulate in the circulation due to the patients inability to alter them. Another example is that where the medium comprises a detrimental or undesirable component that when in direct interactive contact with cells are transformed into harmless or desirable components that are returned to the medium. Thus, e^'.g., an enzyme present in this cell metabolizes a medium component and renders it harmless .

In another aspect, this invention provides a method of regulating the level of a compound in a body fluid of a subject afflicted by an endogenous defect resulting in abnormal levels of the compound in a body fluid, in the substantial absence of a detrimental immunological reaction, the method comprising the steps of: enclosing cells lacking the endogenous defect of the patient's cells in a device, wherein at least one portion thereof comprises a non-porous, semi- permeable, biocompatible film substantially enclosing the cells, the film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume, and the film being permeable to molecules of up^* to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; implanting the device comprising the cells into a site in the subject's body where the cells are in contact with the subject's body fluid; and allowing the cells to survive at the implantation site where they are in direct interactive contact with the compound and act to regulate its level in the body fluid.

In a most preferred embodiment of the method, the body fluid comprises blood and the endogenous defect is a substantially higher than normal level of the compound in blood. In another embodiment the endogenous defect is a substantially lower than normal level of a compound in blood. These are cases of hormonal imbalances whether an excess of hormone or deficiency exists. An example where the endogenous defect is a substantially higher than normal level of a compound in blood is that where glucose's levels in blood increase due to the lack of insulin. A case where the endogenous defect is a substantially lower than normal level of a compound in blood is that where there is a hormonal deficiency that needs to be replenished, such as the case of thyroxine, growth hormone, and the like.

The types of cells that are preferred for the practice of this method are those where the compensation of the enzyme effect of the compound levels being regulated is inversely regulatable by the level of the compound present therein.

As in the case of the in vitro method, examples of cells suitable for practicing this invention are immortalized cells, live tissue cells, and primary culture cells. In the case of a method for treating insulin deficiency cells such as unmodified mammalian islets of Langerhans, or glucose-regulated insulin- producing eukaryotic cells arising from transgenic techniques; homologous recombination or mutation can be used. These cells are known in the art, may be produced by cloning of wild type genes from the same or different species or by, transgenic techniques. In addition, the cells may be produced by mutation by means other than cloning. Examples of these are radiation, mutagenesis and selection.

In one aspect of this invention, the method is applied to a patient that is a diabetic and the cells comprise glucose-regulatable, insulin-producing cells. The cells may be the subject's own insulin- defective cells, and they may be isolated from the subject's body and then genetically engineered or otherwise mutated to produce glucose-regulatable, insulin-producing cells and then implanting them in this same subject.

Typically, cells used in the case of the diabetic are mammalian islets of Langerhans which contain glucose-regulated, insulin-producing cells. Having now generally described this invention, the same will be better understood by reference to specific examples, which are included herein for purposes of illustration only, and are not intended to be limiting of the invention or any embodiment thereof, unless so specified.

EXAMPLES In order to serve as an implantable immunologic barrier, an appropriate membrane material should meet a number of criteria.

(1) The material must possess sufficient strength and be sufficiently resistant to biodegradation to function well for at least one year in the body. (2) The material must be nontoxic to any living cells lodged within it and to tissue surrounding- it.

(3) The material must allow the passage of sufficient nutrients and oxygen through the membrane to support! the maintenance and/or growth of the cells there within.

(4) The membrane must allow the rapid transport of physiologic signals in both directions, e.g., glucose for nourishing islet cells, and cell products of interest, e.g., insulin produced by islets.

(5) The membrane must protect any cells lodged therewithin from the cellular immune system, and optionally, prevent the passage of immunoglobulins such as IgG, thus ensuring that complement mediated cell lysis can not occur.

STUDIES ON TRANSPORT OF MOLECULES ACROSS PTG MEMBRANES The experiments described below were designed to test the transport of molecules across a polyurethane membrane according to the invention. The diffusion of glucose, insulin, and serum components of varying chemistry across a membrane was examined.

Example 1: Transport of Glucose Across the Membrane

Two methods were tested to examine the diffusion of substances across a membrane. In the first method, membrane sheets were cast from the copolymer as disclosed and claimed in co-pending, co-filed patent application of Robert S. Ward and Kathleen White (Ser. No. 07/874,336) incorporated herein by reference. The membranes were then placed into a two-compartment diffusion chamber of 5 ml volume each to separate the two compartments. Phosphate buffered saline (PBS) was placed on one side and a test solution containing the test compound was placed on the other side. A 10 mg/ml solution of glucose in PBS was utilized for this study of glucose diffusion experiment.

The diffusion of glucose across the membrane occurs within a short time. Glucose rapidly crosses the membrane and reaches equilibrium within about 2 hours. If enough glucose can cross the membrane within a period of time of up to 0.5 hr. the membrane being examined was considered suitable. The tested membrane permitted the passage of more than 1500 μg glucose/ml in the first half hour. This is a fast transport of glucose through a membrane.

Example 2: Transport of Proteins Through a Membrane Tube Membrane tubes instead of planar membranes were cast as described in a co-pending, co-filed application of Robert S. Ward and Kathleen A. White (Ser. No. 07/874,336) incorporated herein by reference. The tube was filled with a 1 mg/ml solution of test protein containing a trace amount of the protein labeled with 125-1. After sealing both ends of the tube with a heat sealing device, it was placed into 1 ml PBS in a 1.5 ml Eppindorf centrifuge tube. Aliquots of PBS were removed at various times and the amount of radioactivity in each sample determined. To each sample was added an equal volume of 20% TCA, the sample was mixed, and the precipitate collected as a pellet by centrifugation. The radioactivity present in the pellet was determined. Only TCA-precpitable, and hence protein bound, counts were used for calculating the rates of diffusion. The amount of protein crossing the cross-sectional area of a known membrane in a given time can be calculated based on the original specific activity of the protein placed in the tube. The permeability coefficients, p, are shown in Table 5. As can be seen, the permeability is not simply a function of molecular size.

TABLE 5: Permeability of Membrane Used In In Vivo Studies: Tubes and Insulets PERMEANT MOLECULAR WEIGHT (Daltons) P g.cm7sQcm*dav/g/ml

9650 2300 400

90 25 10

0

Example 3: Insulin Transport Across A Membrane Tube

For this experiment, the membrane tubes described and used in Example 2 were filled with 0.5mg/ml insulin in PBS containing a trace amount of insulin labeled with 125-1. The tubes were cut and sealed and placed into a 1.5 ml centrifuge tube containing 1.0 ml PBS. Samples were collected and analyzed as in Example 2. The amount of insulin crossing the membrane was calculated on the basis of its original specific activity at a rate of 2-11 x 10^" ¹⁰ moles/cm² within the first 30 minutes, and to reach equilibrium at about 60-120 minutes, as evidenced by a flattening of the insulin curve as a function of time. Thus, the membrane of the invention was shown to be suitable for the transport of insulin. The membrane also showed good strength and handling characteristics.

In summary, the membrane of the invention showed diffusion characteristics that prove it suitable for uses where molecules of the size of both glucose and insulin must be transported therethrough at a rapid rate. One example is the replacement of insulin levels outside of the membrane in the presence of an increase in glucose levels. In this case, both glucose and insulin are transported across the membrane at reasonable rates, and both glucose and insulin reach equilibrium across this membrane between 1 and 2 hours.

Cell Sources:

Studies aimed at examining the ability of cells to survive and function in either tubes or Insulets™ composed of the dense membraned material were performed with either established cell lines or isolated porcine islets. Two cell lines have been obtained from ATCC and used for these studies. These are: RAJI cells (a lymphoblast-like human cell established from a Burkitt lymphoma. These cells grow in suspension culture and are absolutely dependent upon the presence of serum for their maintenance and growth. These cells are routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) ) , and MOPC-31C cells (These are an IgG secreting mouse plasmacytoma which grow in suspension culture. They are routinely cultured in RPMI 1640 medium supplemented with 10% FBS) . Porcine islets were prepared by a modification of the methods of Crowther et al. , (Crowther, N.J., Gotfredson, C.F., Moody, A.J., and Grene I.e. "Porcine Islet Isolation, Cellular Composition and Secretory Response". Hor . Metabol. Res. 21: 590 - 595, (1989) and Ricordi et al. , (Ricordi, C. Finke, E.IL, and Lacy, P.E. A Method for The Mass Isolation of Islets from the Adult Pig Pancreas". Diabetes 35: 649-653, (1986) ) . Following isolation, islets are routinely cultured in vitro in RPMI containing 10% horse serum and 11.5 mM glucose. Cell lines were cultured at 37°C while islets were cultured at room temperature. All cultures were performed under 5% C0₂.

In Vitro Culture Studies: I. Toxicity Studies:

RAJI or MOPC cells (3 x 10₄ per well) were placed into each well of 6 well tissue culture plates. To each well was added 5 ml of RPMI 1640 medium containing 10% FBS. Into each well was placed either nothing (control) , 10 inches of a hollow tube composed of HWU 22866 (test) , or a rod of USP negative control plastic reference standard which contained an identical surface area. This latter material is a well recognized nontoxic material. At 1, 5, 7, and 16 days after seeding the number of cells in each well were determined. A plot of number of cells versus days in culture was generated and used to determine the growth rate of the cells. No difference was seen with any of the conditions demonstrating that HWU 22866 membrane material is not toxic to the cells in culture. Islets do not proliferate in culture. When islets were cultured for periods of up to 2 weeks, no decrease in either islet number or the secretion of insulin was observed in wells containing the membrane as compared to those which did not. These studies clearly demonstrate that the membrane material is not toxic either to cell lines or to isolated islets.

II. Cell Survival:

Studies were designed to look at the ability of cell lines and islets to survive and grow within membranes when their only source of nutrients was the media outside the membrane device. For these studies, both heat sealed tubes and "insulet" devices were utilized. Cells were loaded into the devices as follows. Tubes were filled with media containing cells, and the ends were then heat sealed after the removal of all air. If experiments required shorter lengths of tubing, the long tube could be compartmentalized into smaller segments simply by heat sealing at the appropriate intervals. With Insulets, two 26 gauge needles were inserted through the thick membrane "O-ring" at approximatley 30° angles to each other. Using a syringe, the cell suspension is placed into the insulet. The second needle allows the removal of all air from the device. Tube segments or insulets were then placed into cell culture chambers in 6 well plates and 5 ml of culture medium was added. Culture medium was replaced weekly. The survival and growth of the cells was noted visually (these tubes are optically clear) . While this method does not give quantitative results, growth of cells is apparent. Cell number increased markedly with time. By 6 weeks, the tubes were nearly confluent with cells. Cells continued to grow for as long as 4 months (the longest we have carried these out) . At the end of this time period, the tubes contained a nearly solid mass of cells and were observed to be "bulging" with the cell mass. In all of these studies, the only nutrients required for growth and maintenance were supplied by diffusion through the membrane. MOPC cells synthesize and secrete immunoglobins. At no time were we able to detect immunoglobin in the outside culture media. This confirms our earlier diffusion studies which showed that immunoglobins do not cross the membrane barrier. A buildup in IgG conentration within the devices could be detected.

Islets do not proliferate in culture. Therefore, one does not see an increase in cell number in islets preparation placed inside membrane devices. Survival of the islets, however, can be continuously monitored. These experiments established that islets can survive for at least 6 months in in vitro culture with fresh media being supplied only outside the tube. The external media was assayed for the presence of insulin and such assays found that insulin secretion and diffusion occurred throughout this entire period.

Example 4: Cell line studies

Membrane tubes 10 inches in length were prepared as described in Example 1, autoclaved, and filled with 7 x 10⁸ Raji or MOPC cells, the membranes were then heat scaled at 1 inch intervals. Five one inch segments were implanted mtraperitoneally into either Swiss-Webster or Nude mice. At 2, 4, and 12 weeks, three mice were sacrificed, their implants removed, and the viability of the cells within the membrane determined. Upon explanation, the membranes appeared intact. No alterations in structure could be observed with either normal or microscopic examination. By four weeks, the membrane was entirely surrounded by a large well vascularized fat pad. No abnormalities in the surrounding tissue was noted. Similarly, no physiologic changes in the mouse were observed as a consequence of the implant. The explanted membranes were opened with a scalpel and the cells contained within examined for viability. The cells retrieved from within the membrane were determined to still be viable by their ability to exclude trypan blue and their ability to grow under in vitro culture conditions following retrieval. Examination of the cell in the tubes prior to implantation and subsequent to their removal revealed that they had not only survived but had proliferated extensively. This occurred in both immunocompromised (nude) and normal mice demonstrating that the membrane had successfully protected the cell lines from the host mouse's immune system. In addition, if the Raji cells has escaped from the membrane device, a lymphoma would have been observed in the nude mouse Study. Since this was not found, it can be concluded that the membrane not only protects the cells within it from the host immune system but that it also protects the host from cells within.

Example 5: Islet System

Porcine islets (15,000; 40,000; or 50,000 each device) in either RPMI media or RPMI media containing Matrigel were placed into either membrane tubes or

Insulets as described in Example 1. The devices were then implanted either mtraperitoneally or subcutaneously into normal BALB/C mice. After 3 months, the devices were removed and tested for the viability of the islet cells. Quantitation of the number of islet cells present at the end of the experiment was not possible. We were, however, able to demonstrate the presence within the device of live islet cells. In addition, when such a device was placed into culture in vitro we were able to measure insulin in the culture media. This could only occur if the islet cells had survived and continued to function after the three month period in the animal. Since these studies were performed in normal mice, these studies demonstrated the xenografted cells were protected from the host immune system.

Example 6: Treatment of diabetic mice

To demonstrate the ability of porcine islets to function in vivo within the membrane, diabetic mice were implanted with devices prepared as described in Example 1. Diabetes was induced in BABL/C mice by the injection of Streptozotocin (STZ) . Animals were considerred to be diabetic when their fasting blood glucose levels were greater than 300 mg/dl for three consecutive measurements. Devices containing 50,000 porcine islets each were implanted either mtraperitoneally or subcutaneously into diabetic mice. These animals were considered to be cured if their fasting blood glucose levels returned to 200 mg/dl or less. Control diabetic animals received implanted devices with no islets. Blood glucose levels were monitored weekly. Within two weeks following implantation, animals began to show a reduction in blood glucose levels. Figure shows typical results for animals implanted for either 4 or 7 weeks. The 0 time point represents the fasting blood glucose levels. As can be seen, diabetic "controls" had fasting blood glucose levels greater, than 300 mg/dl while those or normal or implanted mice are about lOOmg/dl. The presence of porcine islets within the device has clearly regulated the blood glucose levels of the implanted diabetic mice. A glucose tolerance test more closely demonstrates the ability of the animal to respond to the physiologic stress of increased glucose levels. These are performed by injecting the mice intrapcritoncally with 3 mg of glucose per gram body weight at time 0. At 30, 60, 90, 120, and 180 minutes blood glucose levels were determined. As can be seen in Figure 1 the normal mice show a slight rise in glucose levels and rapidly return to normal by 120 min post injection. In contrast, diabetic mice increase and show no tendancy to return to pre- challenge levels during the course of the experiment. The diabetic animals implanted with devices containing porcine islets show an initial rise similar to that observed in the diabetic mice. In contrast, however, these animals return to more normal levels of glucose during the course of the experiment. This experiment clearly demonstrates that porcine islets survive in the membrane device in normal mice, that they continue to function and can ameliorate their diabetic state. In a true sense, this device is functioning as an artificial pancreas. It has been demonstrated that the membrane material used in the practice of this invention is not toxic to cells and that serum dependent cell lines can survive and grow within the membrane for extended periods of time so long as serum is kept in the medium outside the membrane devices. Two serum dependent cell lines, Raji and MOPC-31C have been grown inside the membranes for up to six months. Not only have they survived but they have proliferated normally. Devices containing these cells have also been placed into normal mice. These cells survived and grew for longer than 4 months. They obviously protected the cells from the host immune system. In addition, they protected the host from the cells within the membrane as no evidence of invasion or tumor formation was observed. Islets have been cultured inside devices composed of this membrane material and have survived in vitro for periods greater than six months. Throughout this time period they continued to function normally releasing insulin both basally and in response to secretagogue challenges.

Example 7: Improved Product Transport and Cell

Viabilty Through the Use of Hvdrogels It has been found that optimal diffusion of the intracellular products put of the membrane implant is achieved, in part, by maintaining an even distribution of the cells within the membrane. It has been known in the art to use hydrophilic natural polymers to encapsulate mammalian cells. Generally, mammalian cells have been microencapsulated through crosslinking using, for example, alginate and polylysine. One drawback to microencapsulation using certain crosslinked hydrophilic polymers is the tendency of crosslinked polymers to biodegrade. It is also known that alginates and some vegetable gums are capable of forming high water content gels suitable as encapsulating agents. Gels where the water content is higher than 99.5% have been produced. The high water content of these gels provides these gels with high permeability to the various permeants important to hybrid artificial organs. Even at very high water contents, these gels are very viscous and thus capable of immobilizing cells dispersed within the gel. However, one drawback to the use of some acrylic hydrogels is their biodegradability, particularly when the gels are implanted as microcapsules directly into tissue.

By placing these high water content hydrogels within the membranes of the instant invention, it has been found that the hydrogel compositions do not rapidly biodegrade once implanted. Thus a preferred embodiment of the instant invention is the use of a hydrophilic gel with a water content ≥ of about 35% water but preferably ≥ 90% water inside of a dense, semi-permeable polymer membrane. The dense membranes of the instant invention provide immunoisolation and necessary selective permeability while also providing an absolute barrier to cells. The water swollen gel fixes the position of the cells within the.dense membrane (e.g. in the shape of a hollow fiber) to prevent bunching. In addition, the high water content of the water swollen gel provides low resistance to the permeability of species leaving or arriving at the contained cells. Further, it has been observed that cells fixed in a hydrogel exhibit improved viability within the implantable device.

The dense membrane may also provide a biostable protective layer to the water-swollen gel, thus preventing or reducing biodegradation of the gel. Suitable dense membranes include but are not limited to hydrophilic or amphipathic polyurethanes having ≥ about 20% water but preferably ≥ 50% water but less equilibrium water content than the water- swollen gel. Water content is measured in water @

37°C and expressed as a percentage of the wet weight of the sample after maximum weight gain has occurred. This value is determined by weighing the wet polymer and again after the polymer has been dried to equilibration at standard laboratory conditions, e.g. -23°C and 50% relative humidity. Suitable water-swellable gels are alginates (e.g. sodium alginate, ammonia alginates, potassium alginates, propylene glycol alginates, algins) , guar gum, gum tragacanth, locust bean gum, methocel, xanthan gum, polyethylene oxide, polypropylene oxide, dextrans, acrylates, methacrylates, polyvinyl alcohol, polyvinyl pyrolidone and combinations of the above.

Those gums or resins capable of "crosslinking" may be used crosslinked or linear. For example, sodium alginate may be used "as is" or converted to its insoluble calcium form.

The most preferred hydrogel is calcium alginate wherein the water content is greater than about 90%. With regard to all of the embodiments of the present invention, it is preferred that a hydrogel, that comprises greater than about 35% water, be used to suspend the cells. The hydrogel serves to immobilize the cells within the membrane, thus insuring an even cell distribution within the membrane.

The most preferred embodiment of the instant invention comprises the implantation of a dense membrane in the form of hollow fibers where the hollow fiber is filled with calcium alginate with a water content greater than about 90%. However, it is understood that the geometry of the dense membrane can be in any form including sheets, larger diameter tubes, etc. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without- departing from the spirit or scope of the invention as set forth herein.

Claims

WHAT IS CLAIMED IS:

1. A method of culturing live viable cells, comprising culturing live viable cells under conditions effective for the continuous survival of cells in the presence of a non-porous, semi- permeable, biocompatible film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment,and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500%, and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume and the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; wherein the film is in the presence of a nutrient medium, the medium comprising nutrients and medium's components, the nutrients and medium's components being permeated through the film into the cells' environment, and any cell products from within are permeated through the film outside of the cells' environment.

The method of claim 1, wherein the cells comprise product-secreting cells.

3. The method of claim 2, wherein the cells comprise insulin-producing cells.

4. The method of claim 3, wherein the production of insulin by the cells is regulatable by changes in the level of glucose in the medium.

5. The method of claim 3, wherein the cells are selected from the group consisting of unmodified mammalian islets of Langerhans, insulin producing recombinant prokaryotic cells, and glucose-regulated insulin-producing eukaryotic cells arising from homolgous recombination or mutation.

6. The method of claim 1, wherein the cells are selected from the group consisting of prokaryotic and eukaryotic cells.

7. The method of claim 6, wherein the cells are selected from the group consisting of immortalized cells, live tissue cells and primary culture cells.

8. The method of claim 1, wherein the device substantially encloses the cells.

9. The method of claim 1, wherein the medium comprises a detrimental or undesirable component that when in direct interactive contact with the cells is transformed into a harmless or desireable component that is returned to the medium.

10. The method of claim 8, further comprising implanting the device comprising the cells into a subject's body; and wherein the culturing step is conducted in vivo and in the substantial absence of a detrimental immunological response.

11. The method of claim 10, wherein the cells are the subject's own defective cells; and the cells are genetically engineered to overcome the defect prior to implantation.

12. The method of claim 10, wherein the device comprising the cells is implanted intradermally, subcutaneously, intracavitarily, mtraperitoneally, mtravascularly, in or around an organ, in or around the omental pouch or intravaginally.

13. A method of regulating the level of a compound in a body fluid of a subject afflicted by an endogenous defect resulting in abnormal levels of the compound in the body fluid, in the substantial absence of a detrimental immunological reaction, comprising: enclosing cells lacking the endogenous defect of the patient's cells in a biocompatible, implantable device, wherein at least one portion thereof comprises a non-porous, semi-permeable, biocompatible film substantially enclosing the cells, the film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume, and the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; implanting the device comprising the cells into a site in the subject's body where the cells are in contact with the subject's body fluid; and allowing the cells to grow at the implantation site where they are in direct interactive contact with the compound and act to regulate its level in the body fluid.

14. The method of claim 13, wherein the body fluid comprises blood; and the endogenous defect is a substantially higher than normal level of a compound in blood.

15. The method of claim 12, wherein the effect of the cells on the level of the compound in the body fluid is inversely regulatable by the level of the compound.

16. The method of claim 13, wherein the cells are selected from the group consisting of immortalized cells, live tissue cells, and primary culture cells.

17. The method of claim 13, wherein the patient is a diabetic; and the cells comprise glucose-regulatable, insulin producing cells.

18. The method of claim 17, wherein the cells are selected from the group consisting of unmodified mammalian islets of Langerhans, and glucose-regulated insulin-producing eukaryotic cells arising from homologous recombination or mutation.

19. The method of claim 17, wherein the cells are the subject's own insulin- defective cells; and the cells are isolated from the subject's body and are genetically engineered to glucose- regulatably produce insulin prior to implantation.

20. The method of claim 13, wherein the body fluid comprises a detrimental or undesirable compound that when in direct interactive contact with the cells is transformed into a harmless or desireable compound that is returned to the body fluid.

21. The method of claim 13, wherein the cells comprise product-producing cells.

22. A kit for correcting a metabolic defect in a subject, comprising a biocompatible, implantable device wherein at least one portion thereof comprises a non- porous, semi-permeable, biocompatible implantable film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the\ sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume and being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; syringe; needles; and instructions for use of the kit to insert preselected cells lacking the metabolic defect into the device and implant of the device in the body of a subject afflicted with the metabolic defect so that the cells may interact with the subject's body fluids and alleviate the symptoms associated with the defect.

23. The insulin-producing kit of claim 22, further comprising cell culture medium.

24. The method of claim 1 wherein the live viable cells are suspending the cells in a hydrogel comprising greater than about 35% water.

25. The method of claim 24 wherein the hydrogel is an alginate.

26. The method of claim 24 where the hydrogel is polyethylene glycol.

27. The method of claim 24, wherein the cells comprise product-secreting cells.

28. The method of claim 27, wherein the cells comprise insulin-producing cells.

29. The method of claim 28, wherein the production of insulin by the cells is regulatable by changes in the level of glucose in the medium.

30. The method of claim 28, wherein the cells are selected from the group consisting of unmodified mammalian islets of Langerhans, insulin producing recombinant prokaryotic cells, and glucose-regulated insulin-producing eukaryotic cells arising from homolgous recombination or mutation.

31. The method of claim 24, wherein the cells are selected from the group consisting of prokaryotic and eukaryotic cells.

32. The method of claim 31, wherein the cells are selected from the group consisting of immortalized cells, live tissue cells and primary culture cells.

33. The method of claim 24, wherein the device substantially encloses the cells.

34. The method of claim 24, wherein the medium comprises a detrimental or undesirable component that when in direct interactive contact with the cells is transformed into a harmless or desireable component that is returned to the medium.

35. The method of claim 33, further comprising implanting the device comprising the cells into a subject^'s body; and wherein the culturing step is conducted in vivo and in the substantial absence of a detrimental immunological response.

36. The method of claim 35, wherein the cells are the subject's own defective cells; and the cells are genetically engineered to overcome the- defect prior to implantation.

37. The method of claim 35, wherein the device comprising the cells is implanted intradermally, subcutaneously, intracavitarily, mtraperitoneally, mtravascularly, in or around an organ, in or around the omental pouch or intravaginally.

38. A method of regulating the level of a compound in a body fluid of a subject afflicted by an endogenous defect resulting in abnormal levels of the compound in the body fluid, in the substantial absence of a detrimental immunological reaction, comprising: suspending cells lacking the endogenous defect of the patient's cells in a hydrogel wherein the hydrogel comprises greater than about 35% water; enclosing the suspended cells in a biocompatible, implantable device, wherein at least one portion thereof comprises a non-porous, semi-permeable, biocompatible film substantially enclosing the cells, the film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume, and the film being permeable to molecules of up to about 6,000 to 600,000 molecular weight and substantially impermeable to cells and particulate matter; implanting the device comprising the cells into a site in the subject's body where the cells are in contact with the subject's body fluid; and allowing the cells to grow at the implantation site where they are in direct interactive contact with the compound and act to regulate its level in the body fluid.

39. The method of claim 38 wherein the hydrogel is an alginate.

40. The method of claim 38 wherein the hydrogel is- polyethylene glycol.

41. The method of claim 38, wherein the body fluid comprises blood; and the endogenous defect is a substantially higher than normal level of a compound in blood.

42. The method of claim 38, wherein the effect of the cells on the level of the compound in the body fluid is inversely regulatable by the level of the compound.

43. The method of claim 38, wherein the cells are selected from the group consisting of immortalized cells, live tissue cells, and primary culture cells.

44. The method of claim 38, wherein the patient is a diabetic; and the cells comprise glucose-regulatable, insulin producing cells.

45. The method of claim 44, wherein the cells are selected from the group consisting of unmodified mammalian islets of Langerhans, and glucose-regulated insulin-producing eukaryotic cells arising from homologous recombination or mutation.

46. The method of claim 44, wherein the cells are the subject's own insulin- defective cells; and the cells are isolated from the subject's body and are genetically engineered to glucose- regulatably produce insulin prior to implantation.

47. The method of claim 38, wherein the body fluid comprises a detrimental or undesirable compound that when in direct interactive contact with the

is transformed into a harmless or desireable compound that is returned to the body fluid.

48. The method of claim 38, wherein the- cells comprise product-producing cells.

49. A kit for correcting a metabolic defect in a subject, comprising: a hydrogel comprising greater than about 35% water; a biocompatible, implantable device wherein at least one portion thereof comprises a non- porous, semi-permeable, biocompatible implantable film formed from a copolymer comprising about 5 to 45 wt% of at least one hard segment, and about 95 to 55 wt% of at least one soft segment comprising at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group consisting of aliphatic polyols, aliphatic and aromatic polyamines and mixtures thereof; the film having a tensile strength greater than about 350 psi and up to about 10,000 psi, an ultimate elongation greater than about 300% and up to about 1,500% and a water absorption such that the sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment exceeds about 100% and up to about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the wet polymer volume and being permeable to molecules of up to about 6,000 to

600,000 molecular weight and substantially impermeable to cells and particulate matter; syringe; needles; and instructions for use of the kit to suspend the preselected cells lacking the metabolic defect in the hydrogel, to insert the cells into the device and implant of the device in the body of a subject afflicted with the metabolic defect so that the cells may interact with the subject's body fluids and alleviate the symptoms associated with the defect.

50. The insulin-producing kit of claim 49, further comprising cell culture medium.