Title:  In vivo induction for enhanced function of isolated hepatocytes

United States Patent:  6,699,716

Issued:  March 2, 2004

Inventors:  Sullivan; Susan J. (Newton, MA); Gregory; Paul G. (Shrewsbury, MA); DiMilla; Paul A. (Dover, MA)

Assignee:  Organogenesis Inc. (Canton, MA)

Appl. No.:  036593

Filed:  December 21, 2001

Abstract

The invention features a liver cell culture comprising hepatocytes that have increased detoxification enzyme activity when isolated from a liver of a donor that had been administered at least one induction agent prior isolation of hepatocyte cells. The induced hepatocytes are used in a bioreactor and cultured to produce hepatocyte cell products or metabolize toxins added to the culture. The bioreactor is, or is an integral part of, a liver assist device used to treat a patient in need of liver assist.

SUMMARY OF THE INVENTION

The invention features a liver cell culture comprising hepatocytes that have increased functional enzyme activity when isolated from a liver of a donor that had been administered at least one induction agent in vivo prior to isolation of hepatocyte cells from the liver. The induced hepatocytes are used in a bioreactor and cultured to produce hepatocyte cell products or metabolize toxins added to the culture, or both. In the preferred embodiment, the bioreactor is, or is an integral part of, a liver assist device used to treat a patient in need of liver assist. In another preferred embodiment at least two cultures of hepatocytes from different isolations induced by different induction agents may be mixed or used together in a bioreactor to have a bioreactor that exhibits a wider range of increased functional enzyme activity.

DETAILED DESCRIPTION OF THE INVENTION

Heretofore, cell cultures from liver procured from induced donors have not been incorporated in a bioreactor, particularly for use in a liver assist device.

In the method to obtain induced cells, a liver donor is selected and screened for appropriate age and health necessary to obtain healthy cells from the donor's organs. The liver donor for obtaining hepatocytes is preferably a normal or transgenic animal donor of either mammalian or rodent species, more preferably of equine, canine, porcine, bovine, ovine, or murine species; and most preferably, a porcine donor. Due to the ease of handling smaller animals and liver organs, pigs between about 5 kg to about 20 kg are used, preferably about 8 kg, but any size donor may be used as a source for liver organs.

Induction is preferably performed by administering at least one induction agent to an animal donor via direct injection to the bloodstream, intraperitoneally, or intramuscularly; however, induction agents may also be administered to a donor using other routes such as orally, transdermally, or by inhalation. One or more agents may be administered at one time in a single dose or over a time as separated doses of different induction agents. The donor may be dosed with a combination of two or more induction agents to upregulate certain desired detoxification enzymes to create a hepatocyte culture having a customized enzyme activity profile. The dosing of the induction agent may be administered in a single day or over a time, such as over a number of hours or days, before isolating the hepatocyte cells from the donor liver. For example, some induction agents such as phenobarbital are relatively unstable molecules after injection to a donor and are, therefore, more effective if provided at multiple intervals prior to procuring the organ. The amount of the induction agent in the dose depends on (1) the induction agent or agents used, (2) the species, sex, and size of the donor, (3) the mode of administration of at least one induction agent, and (4) the frequency of dose administration. Typically, when the induction agent is administered over a series of doses, the dosage of induction agent may be less. One of skill in the art would be able to successfully determine how to manipulate these dosing parameters in order to obtain in vivo induced hepatocyte cultures for use in the method and bioreactor of the invention.

"Induction agent" means an agent that is capable of increasing or upregulating hepatocyte cell functions, particularly those enzymes involved with detoxification, particularly cytochrome P450 or the conjugative reactions involved in detoxification. It is also useful if the induction agent maintains or improves other hepatocyte cell functions including metabolic functions such as ammonia clearance and synthetic functions such as albumin and transferring production.

Induction agents are selected from the group including but not limited to: beta-naphthoflavone (BNF), phenobarbital, 3-methylcholanthrene (3MC), ethanol, dexamethasone, arochlor 1254, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenothiazine, chlorpromazine, isosafole, .gamma.-chlordane, allylisopropylacetamide (AIA), trans-stilbene oxide, kepone, acetone, isoniazid, pyridine, pyrazole, 4-methylpyrazole, pregnenolone 16.alpha.:-carbonitrile (PCN), troleandomycin (TAO), clotrimazole, clofibrate, clobuzarit, di(2-ethylhexyl)phthalate (DEHP), or mono-(2-ethylhexyl)phthalate (MEHP). It should be noted that the aforementioned terms in parentheticals are abbreviations known in the art for the chemical names that precede them. The most preferred induction agents of the group are: beta-naphthoflavone, phenobarbital, and 3-methylcholanthrene. In the most preferred method, the induction agents are administered to a donor by injection to the intraperitoneal area. It should be noted that dosages recited herein are in terms of milligrams of induction agent per kilogram of donor bodyweight. Phenobarbital is administered preferably up to about 125 mg/kg, more preferably between about 40 to about 80 mg/kg. Beta-naphthoflavone is administered preferably up to about 180 mg/kg, more preferably between about 10 to about 15 mg/kg. 3-methylcholanthrene is preferably administered up to about 25 mg/kg, more preferably between about 5 to about 10 mg/kg. Some chemicals that are either functionally or structurally similar to those listed may be identified by one of skill in the art for practicing the invention. While not wishing to be bound by theory, many of the chemicals listed are customarily classified together in the same chemical classes with a number of other aromatic or barbituate compounds and are able to upregulate functional metabolic activity of hepatocytes. Carrier agents, adjunct agents, encapsulation means, or a combination thereof may also be added with the induction agent in the dose to regulate uptake and absorption rates of induction agent. Carriers may be aqueous, such as water or saline, and may be buffered with phosphate, borate, or citrate, for example. Non-aqueous carriers may also be used, such as dimethylsulfoxide (DMSO) or benzene. The induction agent may also be released from an encapsulation means.

One or more induction agents may be used in vivo to upregulate the enzymatic activity of the hepatocytes prior to isolation. A single induction agent may be administered to a donor one or more times prior to isolation. Induction agents may be combined, meaning as a mixture or `cocktail` at the same time, or serially, meaning separately at different times, when administered to upregulate a profile of target enzymes. The amount of induction agent contained in the dose should be enough to induce the hepatocytes to increase their functional metabolic activity but not so much as to be lethal to the liver organ or to the donor. The time that the induction agent is provided to a donor should be long enough to result in upregulation of enzymatic detoxification activity, preferably at least about 24 hours prior isolation.

In vivo induction initiates upregulation of several functional detoxification enzymes such as cytochrome P450 isozymes and conjugating enzymes so that the hepatocytes, after isolation and incorporation in a bioreactor, sustain measurable detoxification activity for about a week. Non-induced hepatocyte cultures are not upregulated to the levels of activity found in in vivo induced hepatocyte cultures and do not sustain such levels for as long, only about 3 or 4 days.

To date, much of the cytochrome P450 isozyme work has been performed on either rat or human hepatocytes and therefore many of the known cytochrome P450 isozymes have been identified and named based on the correlation between the induction agents and the isozymes they upregulate. Extending that knowledge to porcine hepatocytes, however, one will find both similarities and differences between the induction agent and isozyme activity. The induction agents have effect on the expected isozyme or its species-specific homolog. In the preferred embodiment, hepatocytes are isolated from porcine liver so the induction agent or agents used will have effect on the expected isozyme or its porcine homolog.

Table 1 summarizes the induction activity of the most preferred induction agents for use in the invention along with their target isozymes, and the substrates that the isozymes convert. Induced hepatocytes initially express increased P450 isozyme activity on alkoxyresorufin substrates, converting them to resorufin, at a level higher than that of noninduced hepatocytes. A preferred level of targeted P450 isozyme activity increase of in vivo induced hepatocytes over non-induced hepatocytes is at least about two (2)-fold for use in the bioreactor of the invention. Certain induction agents are chosen to target and upregulate particular isozymes responsible for conversion of alkoxyresorufin substrates that may concomitantly upregulate conversion activity on other substrates. This upregulation may occur by the same or different pathways.

In the cytochrome P450 pathway, in vivo induction of a donor using phenobarbital upregulates CYPIIB1 and CYPIIB2 isozymatic activity of hepatocytes, or the activity of their porcine homologs, on benzyloxyresorufin (BROD) and pentoxyresorufin (PROD) substrates, respectively. Beta-naphthoflavone is specific for upregulation of CYPIA2 and CYPIA1 isozymatic activity, or the activity of their porcine homologs, on methoxyresorufin (MROD) and ethoxyresorufin (EROD) substrates, respectively. Methylcholanthrene upregulates CYPIIB1 isozymatic activity, or its porcine homolog, to PROD; CYPIA2 isozymatic activity, or its porcine homolog, on MROD; and CYPIA1 isozymatic activity, or its porcine homolog, on EROD. Another widely used substrate to assess hepatic enzymatic activity is 7-ethoxycoumarin (7-EC). This substrate is O-deethylated to yield a fluorescent product and is also indicative of oxidative metabolism of the cytochrome P450 enzymes. The results from these assays suggest that increases in isozymatic function are obtained following in vivo induction. Furthermore, HPLC analysis of the detoxification processes in the liver show that drugs, such as lidocaine and diazepam, which are metabolized in the liver, are cleared at a much greater rate than in the noninduced state. This finding is clinically significant as drug overdoses are a major cause of hepatic failure.

The conjugation reaction pathway is another induction pathway for increased conversion activity by hepatocytes. There are several known conjugation reactions that may be upregulated by in vivo induction methods, such as the glucoronidation and sulfation conjugation reaction pathways. Glucuronidation is a primary mechanism for producing polar metabolites of xenobiotics for excretion. Phenobarbital is involved with not only cytochrome P450 isozyme activity but also conjugation enzymes. Alcohol, phenol, N-hydroxylamine, and carboxyl groups undergo O-glucoronidation; alkylamine, arylamine, amide, sulfonamide, and tertiary amine groups undergo N-glucoronidation; sulfhydryl groups undergo S-glucoronidation; and tetrahydrocannabinol groups undergo C-glucoronidation. Enzymatic glucuronidation is accomplished by the enzyme UDP-glucuronyltransferase. Another conjugation pathway for the reduction of foreign compounds and drugs bearing a hydroxyl group is sulfation. The class of sulfotransferase enzymes that may be upregulated by in vivo induction include alcohol sulfotransferase, amine N-sulfotransferase, and phenol sulfotransferase.

If a recipient patient is in need of liver assist treatment for an indication where the expression of detoxification enzyme activity is low, a liver assist device may be prepared using a mixture of cell isolates having a profile of hepatocytes with a number of enzyme activities upregulated to achieve the greatest range of detoxification activity and provide a tailor-made culture for treatment of acute failure.

After the induction stage, the cells are isolated using a modification of the Seglen method as described in Seglen, P O. Preparation of isolated rat liver cells. In Methods in Cell Biology (D M Prescott, ed.) vol. 13. Academic Press (NY, N.Y.), 1976, incorporated herein. The animal is anesthetized, opened, and the exposed liver is cannulated and perfused in situ with cold lactated Ringers solution before excision to rinse blood and excess induction agent from the liver tissue. The excised liver is transported to a biological safety cabinet where the remainder of the procedure may be performed under aseptic conditions. The extracellular matrix that provides the physical structure of the liver is then digested by quickly perfusing the organ with warmed EDTA, preferably at 37^oC., followed by perfusion of 1 mg/ml collagenase at 37^oC. until digestion appears complete (mean digestion time is about 22 minutes). Further digestion is then stopped with the addition of cold Hank's Balanced Salt Solution (HBSS) supplemented with calf serum. Digestion of liver matrix releases cells and cell aggregates from the matrix structure to create a suspension of cells. Undigested tissue and gallbladder are excised and the remainder of the tissue is passed through 200 micron and 100 micron stainless steel sieves to release cells and cell aggregates. The cell suspension is washed twice by centrifugation and decanting of rinse media and the cell pellet resuspended in media preferably after the third rinse. At this point, cells may be cultured in culture medium or cryopreserved in a cryopreservation medium for long-term storage for future use.

The cells are cultured as a cell suspension or plated on a surface suitable for animal cell or tissue culture, such as a culture dish, flask, or roller-bottle, which allows for hepatocyte culture and maintenance. The cells may be incorporated in a bioreactor, either in suspension or plated on a culture substrate such as a culture bead or fiber, or on a flat surface or membrane. Suitable cell growth substrates on which the cells can be grown can be any biologically compatible material to which the cells can adhere and provide an anchoring means for the cell-matrix construct to form. Materials such as glass; stainless steel; polymers, including polycarbonate, polystyrene, polyvinyl chloride, polyvinylidene, polydimethylsiloxane, fluoropolymers, and fluorinated ethylene propylene; and silicon substrates, including fused silica, polysilicon, or silicon crystals may be used as a cell growth surfaces. To enhance cell attachment or function, or both, the cell growth surface material may be chemically treated or modified, electrostatically charged, or coated with biologicals such as with extracellular matrix components or peptides. In one embodiment, the hepatocytes are cultured either within or on the surface of extracellular matrix disposed on the culture surface such as collagen in the form of a coating or a gel. In another embodiment, the hepatocytes are cultured on either a liquid-permeable membrane or a gas-permeable membrane. Other cells present in liver may also be included with the induced hepatocytes such as endothelial cells; Kupfer cells, a specialized macrophage-like cell; and, fibroblasts. A co-culture of hepatocytes with one or more of these or other types of cells may be desirable to optimize hepatocyte functioning.

The in vivo induced hepatocytes are preferably seeded in a bioreactor that is used as, or is incorporated into a LAD. Some LAD designs are based on a hollow fiber cartridge design where the hepatocytes are seeded either in the lumen of the hollow fibers or on the outside of the hollow fibers. The hollow fiber serves as a culture substrate that allows for liquid or gas transport across the hollow fiber. Other LAD designs incorporate a flat planar culture substrate. Hepatocyte culture between two collagen gel layers is described in U.S. Pat. Nos. 5,602,026, and 5,942,436 to Dunn, et al. Another design using a planar culture substrate is disclosed in U.S. Pat. No. 5,658,797 and in International PCT Publication No. WO 96/34087 to Bader, et al. Some flat planar substrates may be micropatterned so that two or more cell types may be cultured together, as a co-culture, in discrete regions on a substrate such as those described by Bhatia, et al. The disclosures of these aforementioned patents that disclose culture substrates and methods and their use as a bioreactor device to treat patients in need of liver assist are incorporated herein by reference. A preferred bioreactor design for the culture of hepatocytes incorporates a gas-permeable, liquid impermeable membrane that defines two regions of a bioreactor chamber. Hepatocytes are seeded on the surface of the membrane cultured in a liquid medium while engaging in oxygenation and other gas transfer not only in the culture medium but also across the membrane. In alternative embodiments, the membrane is treated to improve cell adhesion such as by modifying the electrical charge of the membrane, as by corona discharge, or by treating or coating the membrane with extracellular matrix components, peptides, cell-adhesion molecules or other chemicals. A preferred coating for the membrane is collagen.

When cultured, the cells are preferably contacted with a cell culture medium for a time to maintain their metabolic activity and optimal hepatocyte function. Albeit in varying concentrations, cell culture media provide a basic nutrient source for cells in the form of glucose, amino acids, vitamins, and inorganic ions, together with other basic media components. Culture media generally comprises a nutrient base further supplemented with one or more additional components such as amino acids, growth factors, hormones, anti-bacterial agents and anti-fungal agents. One preferred medium for use in the method after hepatocyte isolation comprises: Williams' E medium, newborn calf serum (NBCS), glucose, insulin, glucagon, hydrocortisone, HEPES, epidermal growth factor (EGF), and glutamine. In a more preferred embodiment, the culture medium comprises: Williams' E media supplemented with up to 1% newborn calf serum (NBCS), 4.5 g/l glucose, 0.5 U/ml insulin, 7 ng/ml glucagon, 7.5 .mu.g/ml hydrocortisone, 10 mM HEPES, 20 ng/ml EGF, and 200 mM glutamine. Other concentrations for the aforementioned medium components or their functional equivalents may be determined for use by one of skill in the art of hepatocyte culture.

In an alternate preferred embodiment, hepatocytes are cryopreserved for storage after isolation until needed for incorporation in a bioreactor. Cryopreservation of cell suspensions, cell monolayers, and engineered tissue constructs are known in the art of cryopreservation. Cryopreservation is useful for long term storage, banking, and shipping. When needed, the cultures are removed from frozen storage, thawed, rinsed of cryopreservative, and ready for use.

After either isolation or removal from cryopreservation storage, the in vivo induced hepatocytes are preferably incorporated and cultured in a bioreactor. Hepatocytes from a single isolation induced with either a single or multiple doses of the same induction agent, or a number of induction agents, may be used. In one alternative embodiment, hepatocytes isolated from a non-induced donor are cultured in a bioreactor with hepatocytes isolated from an in vivo induced donor. In another alternative embodiment, hepatocytes from two or more donor isolations induced by the same induction agent or at least two different induction agents are combined together in the same bioreactor. If the bioreactor has multiple culture chambers or regions, hepatocytes from different donors that have been pre-treated with different induction agents may be segregated but used together for the overall functioning of the bioreactor. Combining in vivo induced hepatocyte cultures that have different enzyme activity profiles in a bioreactor used as a LAD will benefit a patient treated with the cultures in the bioreactor. In one embodiment, the bioreactor may contain several isolations of different in vivo induced hepatocyte cultures to provide the patient with a full profile of upregulated enzymes to achieve the greatest range of detoxification activity. An alternative embodiment is one where the patient may be treated with a bioreactor seeded with one or more isolations of in vivo induced hepatocytes with certain selected enzymatic activities that augment or replace certain levels of enzymatic activity where the patient's liver expresses low levels of a certain detoxification enzyme.

The bioreactor may be used to culture the cells to produce cell products or to functionally act on substances, such as toxins normally metabolized by liver. The bioreactor may serve as, or be an integral part of, a liver assist device to treat a patient in need of liver assist. Hepatocytes having upregulated enzymatic activity may be used in various types of bioreactors used as liver assist devices. Bioreactors suited for this purpose comprise suspension means, hollow fibers, radial flow surfaces and planar substrates as cell culture.

Hepatocytes that have been induced in vivo are useful to treat a patient in need of liver assist when cultured in a bioreactor that is used as, or is incorporated into, a liver assist device. Usually, hepatocyte perfusion medium and the patient's plasma or blood are circulated through the device in separate flow loops. The flow loops contact each other via a membrane for the exchange of gases, toxins, and albumin but also provide an immunological barrier between the hepatocytes and the patient.

Claim 1 of 32 Claims

We claim:

1. A method for increasing detoxification enzyme activity of one or more hepatocyte isolated from a donor liver, comprising:

administering at least one induction agent to a non-human donor in an amount sufficient to increase detoxification enzyme activity of one or more hepatocyte,

isolating the hepatocyte from the donor liver,

incorporating the isolated hepatocyte in a bioreactor,

wherein the induction agent is selected from the group consisting of: beta-naphthoflavone, phenobarbital, 3-methylcholanthrene, ethanol, dexamethasone, arochlor 1254, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenothiazine, chlorpromazine, isosafole, .gamma.-chlordane, allylisopropylacetamide, trans-stilbene oxide, kepone, acetone, isoniazid, pyridine, pyrazole, 4-methylpyrazole, pregnenolone 16.alpha.-carbonitrile, troleandomycin, clotrimazole, clofibrate, clobuzarit, di(2-ethylhexyl)phthalate, and mono-(2-ethylhexyl)phthalate.

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