JPH11225752A - Liver cell clone for artificial liver and extracorporeal liver assist device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a set of functional human liver cell clones (DYD) exhibiting enhanced detoxification activities as characterized by increased GSH content and elevated GST activities and capable of readily detoxicating a toxin and useful for production of an extracorporeal liver assistant device. SOLUTION: This set of functional human liver cell clones is derived from normal hepatocyte cell line (DYD), exhibits enhanced detoxification activities as characterized by increased GSH content and elevated GST activities, equal to cell strain of immortalized human liver cell and human hepatocyte primary culture separated from the liver tissue, and includes the functional liver cell having the GSH content over 50 nmol/mg protein or 50 nmol/10<6> cells after 24 hr successive cultivation. The set of the functional human liver cell clones is obtained by exposing a normal hepatocyte cell line to toxin-inducing medium condition, and modulating the expression of a related enzyme by the induced material. The extracorporeal liver assistant device is constituted by using a bioreactor 3 housing the set 4 of the functional human liver cell clones.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to the assembly of functional human hepatocyte clones (DYD-) and specificity based on macroporous microcarriers or other biocompatible support matrix systems such as capillaries and ceramics. The present invention relates to an extracorporeal circulation liver assist device (sELAD). The human hepatocyte cell line is established from human regenerative liver tissue and the specific detoxification function in vitro has a higher GSH content than its parental cell, in addition to expressing many of the characteristics of normal human hepatocytes. It is characterized by its amount and GST activity. The present invention also focuses on toxins recognized as potential toxins such as ammonia, phenol or medium molecular weight, as well as toxins from liver necrotic tissue such as electrophilic metabolites and oxides. It also relates to blood detoxification or purification methods.

[0002]

BACKGROUND OF THE INVENTION The functional hepatocytes related to the present invention are distinguished by the ability to detoxify toxins from necrotic liver tissue, which is considered to be one of the most important factors in the development of hepatic encephalopathy in fulminant liver failure. And The hepatocytes are grown on the surface of macroporous microcarriers, resulting in a capacity of 2.5-7.5 × 10 ¹⁰ per bioreactor module.
Individual. Improvements in both the quality and quantity of cultured cells in bioreactors have made sELAD an attractive treatment for the treatment of patients with fulminant liver failure. In the past few years, the study of extracorporeal circulatory liver support for the treatment of fulminant liver failure has shifted its focus from experimental and animal studies to clinical evaluation and application. There are at least four groups in the United States testing bioartificial liver devices under FDA supervision (Roz
ga J et al, J Ann Surg 21
9: 538-46 1994, Sussman NL
et al, Amer J KidDis 18: 3
71-84 1996, Sussman NL, Cli
nInvest Med 19: 393-9 199
6, Dixit V Scan J Gastroe
nterolastroenterol Suppl2
20: 101-14 1996). There are several early reports from Russia (Gerlach J, Transpla
nt Proc 29: 852 1997, Hughe
sR. D et al, Semin Liver
Dis 16: 435-444 1996) and several other European groups have started preclinical studies in Germany, the Netherlands and the UK. Research on bioartificial liver assist devices is also active in Japan.

The immediate purpose of a temporary liver assist device is to maintain patients with acute or fulminant hepatic failure until their own livers have regenerated. According to the available clinical data, the hepatic assist system primarily bridges patients to orthotopic transplantation,
It appears to be used to improve cerebral circulation and hemodynamic indicators. Rozga and his group found that out of 19 patients treated with one to five uses of a bioartificial liver (BAL) device for 7 hours, 13 had an average of 39 patients starting BAL support.
After a time liver transplant, they reported survival. Patients who did not become transplant candidates died (Kamlot A
and Rozga J, Biotech & B
ioeng 50: 382-91 1996). 11 by extracorporeal circulation liver assist device (hereinafter also referred to as ELAD)
Clinical evaluation was performed in 11 patients with acute liver failure who had been treated for up to 4 hours. Nine out of ten showed improved galactose excretion, one recovered without transplant, four received transplant, and the other died (Sussman NL et
al, Amer JKid Dis 18: 371
-84 1996). Complete recovery of the patient with the use of the bioartificial liver assist system was shown only occasionally. Several sporadic clinical reports dealing with the application of hepatocyte bioartificial liver treatment have also been sporadic, with results almost identical to those described above (Sheil AGR et al, Aust.
NZJ Surg 66: 547-52 199
6; Watanabe FD et al, Ann S
urge 225: 484-91 1997).

Although hepatocyte-based hybrid semi-biological extracorporeal liver assist devices have demonstrated significant metabolic effects in a variety of experimental and animal studies, significant metabolic effects have not been fully achieved in human subjects. Could not be confirmed.
In particular, the biochemical measurements and efficacy demonstrated in patients with hepatic failure using the two previously known major lines are not as pronounced as demonstrated in animal studies. The clinical efficacy in these systems is prolonged survival, correction of biochemical abnormalities, and increased toxin clearance. E recently in the UK
An excellent randomized trial conducted to evaluate the value of the LAD (Sussman) and BAL (Rozga) devices showed that enhanced metabolic function in patients was observed in the two systems, The observed effect was not significant (Hughes R. D et al, Semin
Liver Dis16: 435-444 199
6). This suggests that the working capacity of existing devices should at present be limited to clinical use, especially given the number of functions that need to be replaced in fulminant patients.

[0005] In the course of developing extracorporeal liver assist devices, we are now facing both theoretical and technical issues in order to create a transition from a practical point of view to routine clinical use. These disorders are inadequate underlying pathophysiology, reflected in our lack of knowledge in this area, incomplete understanding of hepatic biology and the regulation of hepatocellular proliferation, especially the hepatic nature of patients with fulminant hepatic failure Mechanism of coma or onset of encephalopathy. Technically, either the quality of hepatocytes or the work of hepatocytes in an engineered bioreactor could produce a clinically significant effect in correcting declining liver function in patients with fulminant liver failure Is not enough. Currently, primary cultures, mainly from animals such as pigs or immortalized cells from human hepatocytes, predominately make up the biological components of most biological devices over others.

[0006] According to the basic principles of biology, differentiation and proliferation are inconsistent in most cell types, and these two types of cells are usually located at both ends of the biological lineage. That is why it is very difficult to obtain large quantities of hepatocytes with the desired multifunctionality. The device, containing about 50 g of primary culture from BAL pig liver, has its physiological minimum mass (20
(Range 0-400 g) appears to have fewer cells.
400 g of C3A immortalized cells are housed in ELAD,
With higher cell count devices, major synthetic and metabolic functions were not demonstrated in clinical use (Hughes RD
et al, Liver Transpel Surg
1: 200-6 1995). Even in primary cultures of hepatocytes in certain composite well-designed structures with a higher number of accommodated cells, good effects have been observed only for a limited period of about 7 hours (Galletti PM & J).
auregui HO, in Handbook o
fBioengineering ppl 1952-
66 1995). This is impractical for sustained support of fulminant liver failure patients using fresh pig liver. Currently available methods use hepatocytes in vitro.
It does not seem to be possible to prevent degradation at tro). Significant efforts have been made to improve the work of primary cultures from xenografts and hepatocytes, including established cell lines, but significant differentiation of hepatocyte functions in bioreactors for bioartificial liver assist systems. There were no reports claiming that it had been maintained. Loss of hepatic function in culture was determined in vitro (in v
In vitro), it appears to be related to cellular interactions and cellular responses involving cellular apoptosis (Collins B)
He et al, Transplantation
58: 1162-71 1994). Cells released from the body are placed in a non-physiological environment and
In vivo, it is shut off from a number of regulatory mechanisms that control hepatocyte function. Given that there is no fundamental theoretical breakthrough in harvesting multiple functional hepatocytes, it is no exaggeration to say that hepatocyte bioreactors for bioartificial liver assist are now at their maximum work. .

Another important problem is that bioartificial liver systems do not show significant advantages over artificial liver systems. Some recent artificial liver systems based on membrane technology and sorbents have already been the subject of clinical evaluation (A
sh SR et al, Intl J Artif
Org 15: 151-61 1992). It has been reported that albumin-coated, highly adsorptive dialysis membranes promote removal of potential toxins (Stage J et.
al, Artif Org 17: 809-13
1993). The BAL system still uses traditional activated carbon sorbents in the plasma circuit before the hepatocyte bioreactor (Rozga J & Demetriou).
AA, ASAIO J 41: 831-37 19
95). It is difficult to evaluate what percentage of activated carbon columns are working in the system.

[0008] Despite inadequate knowledge of the pathophysiology of liver failure, biotechnologists and clinicians have invented devices of various designs and simplified the treatment of patients. This is because there is an urgent need for artificial liver assist devices. Both total and bioartificial liver assist systems remove more than 10 potential toxins identified as factors that cause hepatic encephalopathy in patients with hepatic failure by sorbents, dialysis and living hepatocytes However, patient outcomes are not always proportional to these measures of patient circulation. Whether hepatic coma is the result of one or more of these identified metabolic abnormalities is still uncertain.

[0009] The liver cannot clean substances from the portal vein blood by hepatocytes, toxic substances enter the systemic circulation, and toxins accumulated in the patient's circulation come from liver necrotic tissue.
Moreover, the properties and role of necrotic substances in the development of hepatic encephalopathy are less clear. There is indirect evidence that toxins from necrotic tissue have a critical effect on the clinical manifestation of specific stages of acute liver failure. When necrotic tissue is removed from the patient's body before transplantation, that is, when hepatectomy is performed,
Improved systemic hemodynamic indices (systemic vascular resistance and oxygen availability) and reduced intracranial pressure (ICP) can be achieved (Ring)
eB etal, Transplant Proc
20: 552-7 1988; Harrison P
M et al, Gut 32: A837 199
1). Furthermore, if a patient is treated for a period of time with an extracorporeal circulating liver assist device and subsequently undergoes an orthotopic liver transplant, the expectation of the assist device will be more satisfactory than without a transplant because the necrotic liver has not been removed. become. The mechanisms involved in liver injury are complex and interactive. Hepatocytes are damaged by either chemotoxicity or immunocytotoxicity.

In chemically-induced injury, toxic metabolites from necrotic tissue can alter plasma membrane, mitochondria, intracellular ionic homeostasis or denaturing enzyme activity, while
The immune mechanism that involves cell interactions is mediated by cytokines, nitrate and complement components. In both cases, the result is impaired cellular homeostasis. All of these reactions are characterized by the production of active oxides and reactants, which then promote hepatocyte apoptosis and exacerbate their load because the liver is the largest organ to process or remove active oxides It has been confirmed that. Oxidative insult results in mitochondrial dysfunction, membrane damage, and D
Degeneration of NA and other cellular components occurs (Sewell RB
et al, Clin Sci 63: 237 1
982). Excessive production of oxygen groups can lead to changes in enzyme activity, DNA
It leads to poor repair, poor oxygen utilization, glutathione depletion and lipid peroxidation.

Although blood toxins can cause pathological changes such as inflammatory reactions in different parts of the body, brain cells are the most sensitive sites for these toxins, resulting in brain edema characterized by elevated ICP. Occurs. If not treated properly,
The clinical course often involves multiple organ failure (MOF), with severe coagulation abnormalities, renal failure, pulmonary edema or the development of cardiovascular collapse. Therefore, it is inferred that hepatic encephalopathy is an early symptom of multi-organ damage caused by toxin released from necrotic liver tissue. A few reports suggest the involvement of reactive oxygen species toxins from necrotic tissue in the development of hepatic coma, but eradication of necrotic toxins may be an alternative to bioartificial liver assist devices, In future designs of bioreactors for auxiliary systems,
It is inevitable that its role will increase.

[0012] Because of the complexity of the multiple cell interaction and little knowledge of how to culture these differentiated cells, we have established primary cell lines or established cell lines from cells of human origin. One must acknowledge the fact that total replacement by either is difficult, at least for the time being, to obtain a clinically effective device. Numerous experiments have been performed that demonstrate that mimicking liver function with our current technology is impractical. However, previous experience with all-artificial systems suggested that the detoxification effect of toxin removal in the patient's circulation had a beneficial effect on patient survival that could be called a variable. This is because the extracorporeal liver assist system does not necessarily require all multifunctional replacements by hepatocytes in the bioreactor, and the supplementation of any specific functional activity that has been accidentally lost or missing in the patient is an artificial liver assist device. Suggest that it may be another option to obtain a therapeutic effect from.

Nevertheless, which of many different functions needs to be replaced in fulminant hepatic failure is probably the most fundamental and important issue to be considered. Synthetic, biotransformation or secretory functions are crucial for the recovery of hepatic coma, but we do not know exactly which is the major category. It is well known that acute liver failure is usually associated with hepatic encephalopathy, resulting in high mortality. The main purpose of the use of the artificial liver assist system is to prevent or treat fulminant hepatic failure patients from developing hepatic encephalopathy. The reversible and systemic properties of hepatic encephalopathy strongly suggest that metabolic abnormalities, rather than direct toxic damage, are involved in the pathogenesis of encephalopathy. Moreover, there is a great deal of evidence that many toxins accumulating in the patient's circulation during the development of liver failure should be responsible for the onset of encephalopathy. Therefore, detoxification or toxin removal in patients with fulminant hepatic failure is considered to be a major task assigned to extracorporeal circulation devices. However, the fact that toxin removal from patients by sorbents or dialysis does not significantly improve survival in patients with severe encephalopathy strongly suggests that blood purification may be more specific and selective. And it would be less effective without the involvement of the enzymatic conversion in living hepatocytes.

The detoxification effect of hepatocytes is that metabolites, toxins and other harmful substances are cytochrome P-45.
It means that the enzyme is converted into a readily soluble complex by an oxidizing action of 0 or various conjugations. It is the most important aspect of hepatocyte differentiation. It disappears easily in cell culture and decreases in converted cell lines. Drug metabolizable cytochrome P
-450 enzyme activity is a key activity and may be the most difficult activity to replace. Conjugation ability is important in the detoxification of bilirubin, bile acids, phenol and a wide range of metabolites. However, some researchers have suggested that these activities may be induced to some extent when the cells meet in different culture conditions.

[0015]

SUMMARY OF THE INVENTION In vivo (in v
Since it is difficult to obtain an extracorporeal circulatory assist device with all of the hepatocellular biochemical potentials of iv), we maintain some detoxification functions in ELAD or are important for patient recovery. An attempt was made to enhance beneficial specific functional activity. In the present invention, the specific functional cells are in vivo (in vivo).
A method and apparatus or device is described that enhances detoxification in immortalized human hepatocytes by a cellular approach so that it is readily available in large quantities, equivalent to its equivalent in vivo.

[0016]

When the liver detoxification action is examined, glutathione and the glutathione S-transferase system (GSH / GST) play an important role in removing toxic substances accumulated in the circulatory system of patients with liver failure. I have. GSH is the most commonly used low molecular weight peptide with numerous physiological and metabolic effects. GSH
Participates in reactions that destroy hydrogen peroxide, organic peroxides, free radicals and foreign substances or drugs. GSH is also involved in the metabolism of various endogenous substances by a number of chemical mechanisms. The GSH / GST pathway represents part of the adaptive response mechanism to chemical insults triggered by electrophiles, the activated form of most metabolites. GSH /
Induction of the GST pathway appears to detoxify some of the toxic carbonyl, peroxide, and epoxide-containing metabolites produced inside cells by oxidative insult (Meister
A. in The river: Biology a
nd PATHHOLOGY, 1988 by Rav
en Press, Ltd. , New Yor
k). Because GSH acts to protect cells from reactive oxygen intermediates, free radicals and toxic compounds, cells G
Increasing SH levels and GST activity may be effective under certain conditions. Several clinical studies have shown that liver damage can be prevented and minimized by the application of glutathione precursors (Prescott LF et al., Lanc.
et 2: 109-13 1976; Rumac
BHet al, Arch Intern Med
141: 380-5 1981; Milkstei
n MJ et al, N Engl J Med
319: 1557-62 1988). By supplementing the enzyme with a substrate in GSH synthesis, cellular GSH levels can be partially increased. However, GSH synthesis and GS
Constitutive activation (enhancement of activity) with T activity modulates cells under incremental toxin-induced culture.
ing) (Hamilt
on TC, in Glutathione ST
transferase Structure, Func
Tion and Clinical Implica
ions, pp 173-85 1996). The same applies to the induction of the cell defense system against oxides.

Modulation of intracellular GSH content and GST activity is about to be performed in an immortalized hepatocyte cell line DYD, a human hepatocyte clone established from normal liver tissue. It is a highly differentiated cell line with a high density of growth potential. By conferring them a specific detoxification function, cells have the ability to convert toxins more specifically, quickly and efficiently, thereby increasing the efficiency of bioartificial liver assisted bioreactors. The amount of hepatocytes inoculated into a bioreactor is another important factor affecting the efficiency of existing bioreactors. Although no one can calculate the exact number of hepatocytes in a bioreactor, a generally accepted value for cell mass is 100-300 g with an extracorporeal circulation assist device. In the present invention,
The amount of cells in a single bioreactor is aimed at reaching 2.5-7.5 billion cells. To accommodate such a vast number of cells, special bioreactor structures have been constructed. Cells can grow and confluent on macroporous microcarriers, and then the microcarriers with attached cells are removed into the bioreactor, where nutrients and nutrients are maintained to maintain the stable functional action of hepatocytes. A continuous supply of oxygen is guaranteed.

To activate the detoxification function, highly differentiated human hepatocytes are a prerequisite for an ideal liver assist system. Immortalized cell lines with highly differentiated functions are an attractive approach. In addition to unlimited cell division capacity, immortalized human hepatocytes obviate concerns about species-specific metabolic differences, and infusion of human proteins reduces immune-mediated responses, especially after prolonged or repeated use. The establishment of such cell lines is possible with advanced techniques of cell and tissue culture. Nevertheless, in order to ensure clonal expansion of hepatocytes from normal liver tissue, for example, a new matrix,
Specific manipulations in cell line development are required, such as the use of growth factors or conditioned media, and induction of clonal expansion.

Enhancement of differentiated hepatocyte function specific to hepatocyte detoxification can be brought about by the induction and regulation of glutathione S-transferase, which is considered to represent a major group of detoxifying enzymes. All eukaryotic species have a number of cytosolic and membrane-bound GST isotopes, each of which exhibit distinct catalytic and non-catalytic binding characteristics (Hayes JD et al, Crit Re
v Biochemmol Biol 30: 445-
600 1995). Due to the involvement of several isotopic enzymes, the GST supergenes provide several layers of protection against harmful chemicals. Evidence suggests that GST expression levels are determinants of the sensitivity of cells to a wide range of hazardous chemicals.

In many organisms, GST activity increases after exposure to foreign substances. Induction of GST has been most thoroughly studied in rodents, and at least 100 different chemicals have been used as inducers of GST in rats and mice. The range of non-biological substances used as inducers is G
It suggests that ST induction is part of the adaptive response mechanism to chemical invasion that is widely distributed in nature. According to rodent studies, the adaptive response to chemical invasion is clearly pluripotent, implicating the induction of many drug metabolizing enzymes. Collectively, these detoxifying enzymes provide protection against a wide range of harmful substances. In addition to these effects, evidence also suggests that GST is involved in protection against oxidative insults (Lenehan PF).
et al, Cancer Chemother P
Pharmacol 35: 377-86 1995).

Several enhancers have been found to enhance GST in animal cells.
It is well known that expression can be regulated. Some enhancers interact with GST genetic elements that respond to non-living substances. They transcriptionally activate the GST gene by different mechanisms according to their genetic element structure (Hayes JD et al, Critical
Reviews in Biochemistry
and Molecular Biology 30:
445-600, 1995). Constitutive expression of GST activation is dependent upon its effective modulation (modulatio
n).

The immediate purpose of the extracorporeal circulatory assist system is to maintain patients with fulminant hepatic failure until the patient's own liver has regenerated, or to bridge the patient to orthotopic transplantation. Blood detoxification is considered a prerequisite for extracorporeal hepatic support,
Most liver assist systems used rely on it.
Metabolism of toxic substances by living cells in hepatocyte bioreactors will reduce toxicity. This has a beneficial effect on patient recovery. The present invention describes a novel detoxification system based on a special functional hepatocyte cell line called a specific extracorporeal circulation liver assist device. The term "specific extracorporeal liver assist device" (sELAD) means a unique extracorporeal liver assist system. The term `` specific '' is used to describe a number of specific, but important, declining, but very important, components of the extracorporeal circulatory assist system in the recovery of hepatic coma in patients with fulminant hepatic failure It means having extraordinary functional activity. Since it is difficult to obtain an extracorporeal circulatory assist system with all of the biochemical capabilities of in vivo hepatocytes, the present invention provides a system having some of the major liver functions, especially Detoxification, which is a characteristic of the system, is adopted with priority.

As used in this claim, the term "specific extracorporeal liver assist device" refers to a hepatocyte inoculated with this system having a content several times that of a freshly isolated or transformed hepatocyte. Having the detoxifying enzyme and the detoxifying substance also means that it is an extracorporeally circulating liver assist system with enhanced or increased detoxification. A bioartificial liver assist system utilizing the principles of the present invention is another effective option for treating patients with fulminant liver failure. The devices and methods of the present invention can be used to bridge a patient to orthotopic transplantation as well as to prevent the patient from developing encephalopathy. The pathway for enhancing the specific detoxification function of hepatocytes described in the present invention is based on the immortalized human hepatocyte cell line (DY
D) modulates the values of glutathione (GSH) and glutathione S-transferase (GST) in D, so that a sufficient amount of specific functional liver equivalent to its in vivo counterpart Cells are readily available.

[0024]

[Effects] Examination of the liver detoxification effect revealed that glutathione and glutathione S-transferase (GSH / G
ST) plays an important role in removing toxic substances accumulated in the circulation of patients with liver failure. Eukaryotic cells have evolved several mechanisms that protect cellular components from highly reactive molecules entering or being biotransformed in the cells. The high affinity of electrophiles for thiol groups over hydroxyl or amino groups is a chemical-physical basis, especially that high concentrations of thiols can be more protective than others. GSH is the most predominant low molecular weight peptide with numerous physiological and metabolic effects. GSH is involved in reactions that destroy hydrogen peroxide, organic peroxides, free radicals, and foreign substances or drugs. Generally, the activity of GSH results in a detoxification because the formation of a thioether bond between the electrophile and GSH almost always results in a conjugate that is less reactive than its parent compound. Conjugation of GSH with noxious electrophilic components of cells increases the solubility of hydrophobic non-biological materials, and these hydrophobic non-biological materials are easily extracellularly released by the ATP-dependent glutathione S-conjugate efflux pump (GSH-Px). Can be transported to

The conjugation reaction between GSH and non-living substances represents the first step in the synthesis of mercapturic acid, an important group of excretion products. After conjugation with GSH, the next step in mercapturic acid biosynthesis requires the continuous action of glutamyltranspeptide, cysteinylglycinase and N-acetyltransferase, leading to end products in urine. Since GSH acts to protect cells from reactive oxygen intermediates, free radicals and toxic compounds, increased cellular GSH levels and GST activity are:
Numerous toxins from necrotic tissue can be beneficial under certain clinical conditions, such as hepatic coma flowing into the bloodstream.

Constitutive activation of GSH synthesis and GST activity can be achieved by modulating cells under toxin-induced culture conditions. Chemicals that induce GST and GSH are very diverse. These include carcinogens, cytotoxins, chemotherapeutics,
Also includes heavy metals and metal-containing chemicals. GST induction is also made by reactive oxygen species. Examples of these chemicals include N-acetoxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine, aflatoxin B1-8,9-epoxide, benzo [a] pyrene-4. , 5-oxide, ben {[a] anthracene-
5,6-oxide, ben {[a] anthracene-8,9
-Diol-10,11-oxide, butadiene monoepoxide, + antichrysene-1,2-diol-3,4-
Oxide, 5-hydroxymethylchrysene-sulfate, 7,12-dihydroxymethylbenz [[a] anthracene-sulfate, 7-hydroxymethyl-12-
Methylbenzo [a] anthracene-sulfate, 1-
Methyl-2-nitro-1-nitrosoguanidine, 1-nitropyrene-4,5-oxide, 1-nitropyrene-
9,10-oxide, 4-nitroquinoline 1-oxide, benzo [a] pyrene, dimethylaminoazobenzene, 7,12-dimethylbenzo [a] anthracene,
5,9-dimethyl-7H-dibenzo [c, g] carbazole, 3-methylcholanthrene, acetochlor, acifluorfen, arachlor, aldrin, atrazine, azinefosmethyl, 1,4-benzoquinone, chlorimuron-ethyl , Cumene hydroperoxide, DDT,
N, N-diallyl-2-chloroacetamide, diazinon, diclobenyl, diclofluanide, 2,4-dichlorophenoxy-acetic acid, S-ethyl N, N-dipropylthiocarbamate sulfoxide, ethylene oxide, ethyl parathion, phenoxy Aprop-ethyl, fluorodiphene, lindane, malathion, methyl bromide, methyl chloride, methyl parathion, metolachlor, trans, trans-muconaldehyde, naphthalene 1,2-oxide, parathion, propachlor, propetanphos,
Styrene oxide, tetrachlorvinphos, trans-stilbene oxide, tridiphene, vinyl chloride,
Ampicillin, fosfomycin, penicillin, 1,3-
Bis (2-chloroethyl) -1-nitrosourea, chlorambucil, cyclophosphamide, ethacrynic acid, mechlorethamine, melphalan, mitozantrone, nitrogen mustard, thiotepa, acrolein, adenine propenal, cholesterol α-oxide, cytosine propenal, Dilinoleoylphosphatidylchlorin hydroperoxide, dilinoleoylphosphatidylethanolamine hydroperoxide, dilinoleoylphosphatidylglycerol hydroperoxide, epoxyeicosatrienoic acid, 4-hydroxynonenal, linoleic acid hydroperoxide, methyl linoleate ozonized, thymine Propenal, uracil propenal, chlorotrifluoroethane, 1,4-dibromo-2,3-epoxybutane, Romometan, 1,3-dichloroacetone, dichloroacetylene, dichloromethane, 1,2,
3,4-diepoxybutane, 1,2-epoxy-4-bromobutane, dibromide ethylene, hexachlorobutadiene, allobarbital, 1-β-D-arabinofuranosylcytosine, barbital, bisethylxanthogen,
Butylhydroxyanisole, butylhydroxytoluene, 3,5-di-tert-butylcatechol, ter
t-butylhydroquinone, 2-n-butylthiophene,
Cisplatin, clonazepam, cyclophosphamide, dexamethasone, diethyldithiocarbamic acid, diethyl maleate, diethylnitrosamine, 5,6-dihydro-2H-pyran-2-one, dimethyl fumarate, dimethyl maleate, dimethyl itaconate, disulfiram, 1,2-dithiol-3-thione, elsine, erythroline, ethanol, ethoxyquin, 5-ethyl-5-phenylhydantoin, 2-n-heptylfuran, α-hexachlorocyclohexane, γ-hexachlorocyclohexane, hexachlorobenzene, 3,4 , 5,3 ',
4 ', 5'-hexachlorobiphenyl, 2,4,5
2 ', 4', 5'-hexachlorobiphenyl, 2,3
5,2 ′, 3 ′, 5′-hexachlorobiphenyl, interferon-α / β, iproplatin, isosafrole, lead acetate, P-methoxyphenol, methyl acrylate, 3-methylcholanthrene, 2-methylene-4-butyrolactone , Methyl selenocyanate, jasmine xylene, β-naphthoflavon, ortibraz, phenobarbital, polychlorinated biphenyl, propylthiouracil, rifampicin, trans-stilbene oxide,
Steptozotocin, sudan, 1,4-bis- [2-
(3,5-dichloropyridyloxy)] benzene, tetrachlorodibenzo-P-dioxin, 2,3,5,6-
Tetrafluorophenol, 1- (2-thiazolylazo) -2-naphthol, vinylidene chloride, adriamycin, arsenic acid, bleomycin, 2- [3- (chloroethyl) -3-nitrosoureido] -D-dioxyglucopyranose, ethacrynic acid , Etoposide, hepsulfam, mitomycin C, mitoxantrone, novantrone, oxazaphosphorin, TGF-β1, and vincristine. To our knowledge, most GST substrates are either non-biological materials or products of oxidative invasion in nature. A common feature of these substances is that they are involved in the development of metabolic processes or metabolic cascades.
And some endogenous substances are also derivatives of GST.

Modulation of intracellular GSH content and GST activity was determined by replicating hepatocyte cell line D, a human hepatocyte clone established from normal liver tissue.
Performed in YD. Modulation of cells (modu
The rating procedure is described in detail in Example 1. By conferring on them an enhanced specific detoxification function, hepatocytes convert toxins more specifically, quickly and efficiently, thus increasing the efficiency of bioartificial liver assisted bioreactors. The amount of hepatocytes inoculated into a bioreactor is another important factor affecting the efficiency of current bioreactors. Although no one can calculate the exact number of hepatocytes in a bioreactor, a generally accepted value for cell mass is 100-300 g for extracorporeal circulation assist devices. The cell amount of the present invention is 2.5 to 7.5 billion cells per module, about 2
5-75 grams. Special bioreactor structures are constructed to accommodate such a vast number of cells. Functional hepatocytes can grow and confluent on macroporous microcarriers, and then withdraw the microcarriers with attached cells into the bioreactor, where they maintain a stable functional effect of the hepatocytes. A continuous supply of nutrients and oxygen is guaranteed.

Referring to the embodiment depicted in FIG. 1, it comprises a cartridge 1 for plasma separation for patient circulation, an adsorption column 2, a hepatocyte bioreactor 3, and a dialysis cartridge 6 for nutrient / waste circulation and exchange. FIG. 1 shows a schematic view of a system of the living-artificial liver assist device. The hepatocyte bioreactor contains said functional hepatocytes 4 grown at very high density on a container 43 and a macroporous microcarrier 5. The principle of the liver assist of the present invention is that the dialysate circulates through the bioreactor 3 at a constant rate outside the capillary of the plasma separation system 1 while the patient's blood / plasma passes through the plasma separation system 1 for plasma separation. It flows inside the capillary of system 1. The dialysate carries the toxin or substance from the patient into the bioreactor 3 where the functional hepatocytes 4 are growing on the macroporous microcarrier 5.
Functional hepatocytes 4 grown on macroporous microcarriers 5 will biotransform or metabolize these toxins or substances into less toxic or non-toxic metabolites.
To maintain hepatocyte viability and functionality in culture, a nutrient supply and metabolite removal system is connected to hepatocyte bioreactor 3 by use of dialysis cartridge 6 connecting bioreactor 3 and media reservoir 34. You. Adsorption column 2 circulates patient circulation 11 in bioreactor 3
Is connected to Activated carbon is preferred as the adsorbent. Some immunosorbents can also be used.

With respect to the plasma filtration cartridge 1, the blood /
A preferred cartridge membrane is selected that allows the plasma circulation to communicate with the hepatocyte bioreactor 3 and allows efficient exchange of a wide range of molecular weight compounds, such as toxins and nutrients, accumulated in the patient with fulminant hepatic failure. The membrane selected as the plasma filtration cartridge 1 includes, for example, ammonia, aromatic amino acids, fatty acids, mercaptan, endogenous benzodiazepine, bile acid, bilirubin, medium molecular weight, cytokine,
It must be permeable to toxins that are potentially important for encephalopathy development in patients with fulminant liver failure, such as endotoxins and the like. However, because many known toxins are bound to albumin as a carrier in the patient's blood, free passage of albumin can be achieved by further limiting the molecular weight of the membrane to efficiently purify the blood. Will. Therefore, a suitable plasma filtration membrane designed for such purpose was installed. Such membranes are commercially available. The membrane is made of cellulose, cellulose acetate, polyacrylonitrile or polysulfone.

The hepatocyte bioreactor 3 includes a container 43 and a centrifugal pump 40 in which the hepatocytes are grown at high density on the macroporous microcarrier 5. The container is made of a plastic material that is non-toxic to cells and has high mechanical strength. The centrifugal pump 40 is connected to the bottom of the container 43.
At the top of the container 43 there is an opening 30 as a hepatocyte inlet, and on both sides of the container 43 there are four openings designed as dialysate inlets 24, 31 and dialysate outlets 25, 32.
The upper opening 30 is used as an injection port for the macroporous microcarrier into the container 43. Filters are fixed at each inlet and outlet to prevent cells from entering the dialysate circulation. On the other hand, a pressure sensor 39 in the dialysis circuit monitors pressure changes indicative of circuit coagulation.

The composition of the dialysate circulation is a William E medium supplemented with 10-30% normal human serum or plasma. The purpose of replenishing normal human serum or plasma is to compensate for declining liver synthesis by injecting human natural blood components, increase colloid permeability in dialysate, and patient and dialysate circulation Is to facilitate mass exchange between the two. At the bottom of the container 43, a centrifugal pump for suspending the macroporous microcarrier is fixed. Macroporous microcarriers serve as a matrix to fix hepatocytes. In the selected embodiment, the container 4
3 is made of polypropylene, polycarbonate or polysulfone. Macroporous microcarrier is Ph
armasia LKB Biotechnology
Cytodex ^™ , Cytopor by the company
supplied as e ^TM and Cytoline ^TM. The diameter of the macroporous microcarrier is preferably 30-500 μm, most preferably 100-300 μm. The microcarrier is made of cellulose, cross-linked dextran, polyethylene and / or silica.

Hepatocyte viability and functionality in the bioreactor is maintained by a nutrient supply and metabolic waste removal system. This system comprises a dialysis cartridge 6 for performing a large-scale exchange between the hepatocyte bioreactor 3 and the medium reservoir 34, a pH sensor 35 and a dissolved oxygen sensor 37, p.
An injection cylinder 36 for injecting the H adjusting liquid, and an oxygen generator system 38 for adjusting the pH value and the dissolved oxygen concentration in the circulating liquid in the capillary are provided. The dialysate flows outside the capillary of the dialysis cartridge 6. This part of the dialysate is not supplemented with plasma proteins.
It is an illiamE cell culture medium. Hepatocyte culture in macroporous microcarriers allows for practical high-yield culture of anchorage-dependent cells. In macroporous microcarriers, cultured hepatocytes proliferate not only as a monolayer on the surface of the microcarrier, but also inside the pores of the microcarrier. By using microcarriers in suspension or perfusion culture systems, it is possible to achieve yields of 10 ^{7 to} 10 ⁸ cells / ml.

The hepatocyte bioreactor is the central part of the bioartificial liver assist system. The hepatocytes are sealed in the container 43 and suspended by the vortex pump 40. Container 4
The material 3 is preferably polypropylene, polycarbonate, polystyrene or polysulfone. The advantages of this type of material for the container structure are: It has been found to be non-toxic to cell viability and metabolism. 2. Can be easily sterilized. 3. When the centrifugal pump 40 operates, its mechanical strength withstands vibration. On both sides of the container 43 there are dialysate inlets 24, 26 and outlets 25, 27. They are specially manufactured to match the standard plastic parts of commonly used dialysis cartridges. In addition to the dialysis inlet and outlet, filters (not shown) are attached to each inlet and outlet to prevent cells and their debris or even larger particles from entering the blood circulation. In order to successfully prevent leakage of cellular components into the dialysate circulation and to minimize interference therewith, the pore size of the filter is less than 10 μm, preferably 1-5 μm. If the particles solidify the inlet or outlet, the filter can be replaced. The filter material is made of Millpore cellulose membrane.

The tubing connecting the components of the overall system is a silicone tubing, having similar criteria to those routinely used in dialysis treatment, and has the characteristics of being manufactured in the same manner. The macroporous microcarrier has an outer surface and an inner space in which hepatocytes growing in suspension can be fixed. This macroporous microcarrier increases productivity by increasing cell density per reactor volume. Hepatocytes fully grown on macroporous microcarriers were -10
^{It can be as high as 8} cells / ml. Macroporous microcarrier culture begins with inoculation of the hepatocytes 4 into microcarriers 5. Hepatocytes 4 that are in a good nutritional state during the logarithmic growth phase are harvested by digestion with proteolytic enzymes and transferred to a flask so that cells can be transferred to microcarriers 5 even if sorted. After 5 hours, add the medium to the final volume. In order to obtain a sufficient number of cells, a fluidized bed culture system is used and productivity is scaled up. The high cell density created by the macroporous microcarrier 5 allows any metabolites to be rapidly separated. Therefore, a steady state is always maintained at the set value by the medium perfusion and oxygen supply system. Cell growth can be monitored by biochemical measurements and cell counts. When the cells 4 are confluent on the macroporous microcarrier 5, the cells are harvested.

The macroporous microcarrier 5 to which the hepatocytes 4 that have been sufficiently grown are attached is placed in the bioreactor 3 under aseptic conditions.
Is transferred from the upper opening 30 to the container 43. The amount of cells transferred is in the range of 5-7.5 × 10 ¹⁰ cells, usually 5 × 10
There are ^ten . The amount of cells in a bioreactor will largely depend on the clinical need depending on the severity of the patient's condition. After transferring the macroporous microcarrier, bioreactor 3 is first filled with fresh medium containing 10-30% human serum or plasma WillamEE medium. As the dialysate flows through the capillary outer space of the plasma filtration system 1, the medium must fill the circuit connected to the bioreactor 3. The dialysate is circulated by a peristaltic pump 23.

The functional hepatocytes are maintained by the continuous flow of nutrient medium through the dialysis membrane 6 and the toxic cell waste products diffuse through the dialysis membrane 6 into the medium reservoir 34. The selective membrane allows exclusive free passage of small molecules such as amino acids and mercapturic acids between the intracapillary and extracapillary spaces. However, the dialysis membrane 6 is not permeable to high molecular weight compounds such as plasma proteins. 12 membranes suitable for the purpose
It has a molecular weight cut-off in the range of 000-14,000 kd. Cellulose membranes are the best candidates for such membranes. Other membranes that can be used in the present invention include polysulfone,
Polypropylene is included. The composition of the dialysate is William E without serum. The medium reservoir is replaced after a period of time while patient circulation is being initiated. The nutrient medium from the medium reservoir 34 passes through the semi-permeable membrane of the dialysis cartridge 6 to feed the hepatocytes 4 on the microcarriers 5. Cell waste products and toxin conjugates are removed by the dialysate, which is subsequently changed periodically. In this way, the system substitutes for the excretory function of the liver.

Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following examples.

Example 1 Establishment of a Functional Human Hepatocyte Line and Modulation of Its GSH Content and GST Activity Cisplatin (cis-diaminedichlor)
oplatinum (cis-DDP) is a chemotherapeutic agent for a wide range of tumors. The mechanism of this drug kills cells in a way that causes cytotoxic effects. In order to obtain particularly functional hepatocytes that are characterized by a higher detoxification capacity than their parental cells, the established human hepatocyte cell line DYD was
Modulates content and GST activity (modulati
ng). All cell culture reagents, including William E medium, fetal calf serum (FCS), penicillin-streptomycin, sodium pyruvate and L-glutamine, are from Gibco Life T
technologies Ltd. Purchased from. All other chemicals are Sigma (St. Lou) unless otherwise noted.
is, USA). Established human liver DYD cells
10% (v / v) in an atmosphere containing 5% CO ₂ gas in air
v) Growed as a monophasic culture in William E medium supplemented with FCS. Cells are trypsin and ED
Subcultured weekly using TA at a ratio of 1: 3. Cisplatin is available from Sigma (St. Louis, USA)
Purchased from. It was dissolved in Hans solution to prepare a stock solution. Stock solutions stored at 20 ° C. were diluted according to the concentration required for the medium. In a medium supplemented with serum, 0.5 μg of DYD cells were added once a week for 24 hours.
/ Ml of cisplatin. During this time, a medium without cisplatin was used as a conventional medium for culturing cells. This procedure was repeated, increasing the concentration of cisplatin from 0.5 μg / ml to 5.05 μg / ml during the 10-month dosing period. Cells were cloned and the cloned cells were stored in liquid nitrogen.
A population of clones was selected using primordial DYD-, followed by several weeks of drug exposure time. DYD cells and their clones were grown in 24-well plates [Constar, USA] for measurement of GSH and GST. Glutathione and glutathione S-transferase were quantified in each batch of cloned cells. The measurement of the total intracellular glutathione value was performed by a fluorimetric method. GST activity in cell homogenate is 50 μm
Direct spectrophotometry was performed using 1-chloro-2,4-dinitrobenzene (CDNB).

Results: Table 1 shows the GSH content and GST activity in three different DYD cell clones. As shown in Table 1, G in these three clones
SH content is compared to that of parental cells. GSH levels are elevated in all three clones and correlate well with the duration of drug exposure. GST activity was enhanced with different values in these clones. Compared to GSH, increased GST activity may play an important role in conjugating toxins to GSH.

[0040]

[Table 1]

Example 2 Characterization of Established Functional Cell Lines and Comparison of the Primary Culture of Human Hepatocytes with Immortalized Cells The functional hepatocyte clone of the present invention was established from human liver regenerating tissues but was enhanced. Has GSH content and GST activity. Table 2 shows the GSH content and GST activity of this cell line, an immortalized human hepatocyte cell line (using Hep G2 cells as an example), the cell line in the primary culture of hepatocytes. Primary cultures of hepatocytes are fresh, freshly isolated from liver tissue. Hepatocytes were separated by 0.25% collagenase and then dispersed in buffered saline. After washing and filtering through a cell mesh, a 10 cm dish was seeded with cells at a concentration of 5 × 10 ^{6 in} a Petri dish. For cell culture, 10% FCS in DMEM medium
Is used. Hep G2 cells and DYD-
The culture of 50 cells is the same as in Example 1.

[0042]

[Table 2] * Grant M. H, et al: Biochemical Pharmacology, 37 (17): 3365-68 1988 Doostar H, et al: Xenobiotica, 20 (4): 435-41 1990 ** Grant M. H, et al: Biochemical Pharmacology, 36 (14): 2311-16 1987.

Example 3 Scale-Up of Established Functional Cells on Macroporous Microcarriers Macroporous microcarriers are purchased from Pharmacia under the trade names Cytopore ^™ and Cytoline ^™ . Cytopore ^™ is cellulose and C
ytoline ^™ is made of polyethylene and silica. Prior to use of both microcarriers, hydrophilize the dried microcarriers in fresh distilled water or PBS,
It is necessary to swell. Replace the distilled water with the medium and equilibrate the microcarriers overnight under culture conditions.
In order to prepare a uniform culture environment and reduce the shearing force that is applied, a perfusion culture system is used so that high-density cell culture can be supplied. In this experiment, a fluidized bed or packed bed reactor is the optimal reactor for Cytoline microcarriers. This type of reactor combines high density culture and low shear in the perfusion method. A cell suspension was prepared by trypsin separation of cells grown in a nutrient state in the exponential growth phase in a flask. Microcarrier loading 2 ×
It was added into a large flask at a rate of 10 ⁶ / ml. During the attachment phase, the flask was placed in an incubator and gently agitated every 30 minutes to obtain a uniform cell distribution on the microcarrier. After 5 hours, sufficient medium was added and the microcarrier was placed in the bioreactor. In fluid bed culture,
The maximum volume of packed microcarriers that could be used was 50% of the reactor volume. In a packed bed culture, the maximum volume of packed microcarriers that could be used was 75% of the reactor volume. To maintain a high cell density in the bioreactor, no toxic metabolites should accumulate and the pH value should be maintained at a set value. Desirable oxygen concentration values should also be maintained. The perfusion medium was changed periodically. It usually takes 1-2 weeks for DYD hepatocytes to confluence under perfusion conditions. During cell harvesting, its density reaches 10 ⁷ -10 ⁸ / ml in a fluidized bed bioreactor.

[Brief description of the drawings]

FIG. 1 is an overall structure of a specific extracorporeal circulation assisted liver device (sELAD) using a special functional hepatocyte having enhanced detoxification action proposed in the present invention.

FIG. 2 is a diagram showing an adsorption column.

FIG. 3 is a diagram showing a hepatocyte bioreactor.

[Explanation of symbols]

 1 Plasma Filtration Cartridge for Patient Circulation 2 Adsorbent Perfusion Tank 3 Hepatocyte Bioreactor 4 Functional Hepatocytes 5 Microcarrier 6 Nutrition / Waste Circulation Dialysis Cartridge 11 Patient Subject 12 Pump 13 Blood Inlet 14 Blood Outlet 20 Sorbent 21 Dialysis Liquid outlet 22 Dialysate inlet 23 Pump 24 Dialysate inlet 25 Dialysate outlet 26 Dialysate inlet 27 Dialysate outlet 30 Bioreactor opening 31 Bioreactor circulation inlet 32 Bioreactor circulation outlet 33 Pump 34 Medium reservoir 35 pH sensor 36 pH adjustment Syringe for liquid 37 Dissolved oxygen sensor 38 Artificial lung 39 Pressure sensor 40 Centrifugal pump 41 Dialysis circulation outlet 42 Dialysis circulation inlet 43 Container

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Kazuo Izumi 12-17 Kakocho, Naka-ku, Hiroshima-shi, Hiroshima, Japan

Claims (10)

[Claims] 1. A functional human hepatocyte clone (DYD-) which is derived from a normal hepatocyte cell line (DYD) and which exhibits enhanced detoxification, characterized by increased GSH content and increased GST activity. set. 2. The functional human of claim 1, wherein said increase in GSH content and increase in GST activity are comparable to primary cultures of human hepatocytes released from immortalized human hepatocyte cell lines and liver tissue. A collection of hepatocyte clones. 3. The method of claim 1 wherein said functional hepatocytes exceed 50 nmol / mg protein or 50 nm after 24 hours of subculture.
2. The population of human hepatocyte clones of claim 1 having a GSH content greater than ol / 10 ⁶ cells. Wherein said functional hepatocytes has GST activity exceeding min 15 nmol / mg exceeding protein or per minute 80 nmol / 10 ⁶ cells by substrate CDNB after 24 hours subculture, human claim 1, wherein A collection of hepatocyte clones. 5. The functional human of claim 1, comprising the step of exposing a normal hepatocyte cell line to toxin-inducing medium conditions and modulating the expression of a related enzyme by an inducer in said cell line. A method for obtaining a set of hepatocyte clones. 6. The method of obtaining a population of functional human hepatocyte clones according to claim 5, wherein the inducer has either carcinogenic or cytotoxic properties. 7. An extracorporeal circulation liver assist device comprising a bioreactor containing a collection of functional human hepatocyte clones according to claim 1. 8. The extracorporeal circulation of claim 7, wherein said functional human hepatocyte clones are selectively propagated on macroporous microcarriers or other biocompatible support matrix systems such as capillaries and ceramics. Liver assist device. 9. The extracorporeal circulation liver assist device according to claim 7, further comprising a plasma filtration circulation system and a dialysis cartridge circulation system. 10. The extracorporeal circulation liver assist device according to claim 9, wherein the plasma filtration circulation system includes an adsorption column.