CA2154701A1 - Hepatic model - Google Patents
Hepatic modelInfo
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- CA2154701A1 CA2154701A1 CA 2154701 CA2154701A CA2154701A1 CA 2154701 A1 CA2154701 A1 CA 2154701A1 CA 2154701 CA2154701 CA 2154701 CA 2154701 A CA2154701 A CA 2154701A CA 2154701 A1 CA2154701 A1 CA 2154701A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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Abstract
A method for screening a chemical compound for hepatotoxicity or choleostatic potential, hepatic extraction potential, hepatic discharge into the bile, metabolic production or the ability to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile canalicular network, is disclosed.
Description
2 ~ l 5 4 7 01 PCT/US93/10874 TITLE OF THE INVENTION
HEPATIC MODEL
FELO OF THE INVENTION
This invention ye~ ~illS to both a novel and useful method for screening a ch~.mical compound for hepatotoxicity or choleostatic potential, hPp~tic extraction potential, h~.p~tic ~ cl~f~,e into the bile, metabolite production, or ~e ability to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile c~n~licular network. ~e u~e of ~is no~el method Ilesults in ~ess of a ~ep~nce on whole ~nim~l toxicity and drug discovery studies and reduces the total number of ~nim~l.c required for chemical testing. ~ addition, information obtained from this screening procedure is devoid of interference from reactions which may occur with other organs and 5 tissue when whole ~nim~l studies are conducted.
BACKGROUND OF THE INVENTION
Scientific, financial and ethical considerations regarding the use of whole 7nim~1 studies for screening new drug candidates for 20 hepatotoxicity or choleostatic potenti~l, hepatic extraction potential, hepatic discharge, metabolite production and the ability of a chemical compound to alter hepatic cell metabolism have led to a search for an in vitro/ex vivo system which accurately reflects the yivo behaviors of chemical compounds.
The influence of extracellular matrix geometry on hepatocyte function has been reported using a sandwich configuration by Dunn et al. (See J.C.Y. Dunn et al., The FASEB Journal, Vol 3, 174 - 177 (1992); The Journal of Cell Biology, Vol 116, 1043 - 1053 (1992), and Biotechnol. Prog. Vol 7, 237-245 (1991).) In this work, 30 adult rat hepatocytes cultured in a collagen sandwich system m~int~ined normal morphology and a physiologic rate of secretion of albumin, transferrin, fibrinogen, bile acids and urea for at least 42 days. These cells were shown to m~int~in the level of albumin mRNA ~imil~r to that found in the normal liver for this same time period.
-2~ o~
-- 2 -- ~ -The subject matter of this invention has demonstrated that hepatic cell cultures are useful in detennining ~he effect of chemical compounds on hepatic cells and the effect of hepatic cells on the compounds. Rat and porcine hepatic cell cultures were grown on a hard or soft substrate, that is, ungelled or gelled collagen, and subsequently overlaid with a collagen gel. These cells initi~ted uniform formation of bile canaliculi ~roughout the entire culture. Typically, 24 hours after overlaying the collagen gel, the hepatic cell cultures formed are characterized by a nearly complete and continuous bile e~n~
lD network. Morphological analysis of these "sandwiched" cell cultures by electron microscopy co~ ed that c~nnPls had formed between most neighboring cells and that these ch~nnels possessed many of the characteristics of native bile c~n~liculi, such as junctional complexes, microvilli and a terminal actin web subjacent to the apical membrane.
These c~n~liculi are also functional at concel,tl~lillg xenobiotic agents presented to the hepatocyte culture. It has therefore been demonstrated in this invention that hepatic cells cultured in this m~nner can be used to screen a wide variety of chemical compounds with only limited sacrifice of laboratory ~nim~
SUMMARY OF THF INVEN~ION
A method is presented for the in vitro screening of a chemical compound for hepatotoxicity or choleostatic potential, hepatic extraction pot~nti~l, hepatic discharge, metabolite production or the ability of a Ghto.mi~l compound to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile canalicular network, the process col-~.ising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspen~ling ~e cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 --i.--.~es;
(c) adjusting the cell concentration to about 1 to 2 times 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incllb~tin~ the dish co--t~ e cells at about 37C, about g~% ~latiw humidity and an atmosphere of about 5%
C02;
(e) ch~n in~ and repl~r.in~ the hepatocyte culture media after about 2 hours to remove any ~1n~tt~rh~1 cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish c~,-t~i--ing the hepatic cells after ~e cells have spread and established inlelcellular contact, and reincllb~tin~ at the conditions of step (d) to allow ~e added neutralized collagen to gel;
g) ~ in~ to the hepatic cells about an additional 5 ml of hepatocyte culture m~
(h) incubating at the conditions of step (d) until the c~n~licular network is developed as observed by light microscopy;
(i) ~ldin~ from about 1 ~11 to about 5 ml of a solution to the hepatic cell culture, the solution cont~ining from about 1 f~ to about ~0 mM of the chemical compound in a solvent which is comp~tible with the hepatic cell culture, and incubating at the conditions of step (d) for about 1 minute 2s to about 2 weeks;
(j) assessing morphologic or structural changes in the integrity of the canalicular network, cell polarity or cytoskeletal structure; quantifying the amount of the chemical compound colltailled within the hepatic cells or rem~inin~
in the culture solution; quantifying the chemical compound secreted into the bile contained within ~e canalicular network; or measuring the extent of alteration of hepatic cell metabolism.
WO 94tl2662 PCT/US93/10874 DETArLE~ DESCRIPTION OF THE INVENTION
Applicants have found a novel chemical compound screening procedure for the in vitro assessment of hepatotoxicity or choleostatic potential, hepatic extraction potential, hepatic discharge, 5 metabolite production or the ability of a chemical compound to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional 'oile c~n~licular network, the process CO~ ;S~llg ~e steps of:
~a) isola~ing hepatic cells 'oy collagenase ~-fusion;
(b) washing the isolated cells with hepatocyte culture m~
suspending ~e cells in about a ~0:~0 mi~cture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minl~tçs;
(c) adjusting the cell concentration to about 1 to 2 times 106 cells/ml of h~p~tocyte culture media and dispel-~in~ about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incllb~tin~ the dish cont~ining the cells at about 37C, about 95% relative humidity and an atmosphere of about 5%
CO2;
(e) ch~ngin~ and replacing the hepatocyte culture media after about 2 hours to remove any ln~ ched cells;
(f) overlaying about l ml of cold neutralized collagen solution to the dish co.-t~i-,;"g the hepatic cells after ~e cells have spread and established intercellular contact, and reincubating at the conditions of step (d).to allow the added neutralized collagen to gel;
(g) ~ in~ to ~e hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the c~n~licular network is developed as observed by light microscopy;
in~ from about 1 ~l to about ~ ml of a solution to the hepatic cell culture, the solution cont~ining from about l fM to about ~0 mM of the chemical compound in a solvent which is compatible with the hepatic cell culture, and incubating at the conditions of step (d) for about l minute to about 2 weeks;
(j) assessing morphologic or structural changes in the integrity of the c~n~licular network, cell polarity or cytoskeletal structure; quantifying the amount of the chemical compound cu~lA;~ within the hepatic cells or rem7~ining in the ~ulture solution; yu~lliryillg the chPInical ~ o~ d secreted into the l~ile co-~t~ l within the canalicular network; or llleh~u~ g the extent of alteration of hepatic cell metabolism.
In accordance with the practice of this invention, it has been found that primary hepatic cells grown on a hard or soft collagen substrate and subsequently overlaid with a collagen gel initi~te~l uniform 5 formation of bile canaliculi throughout the entire culture which can be used in the screening of chemical compounds. Typically, 24 hours after overlay, hepatic cell cultures formed an anastomosing bile c~n~liculi network ~.lercol,llecting a plurality of cells. Morphologic analysis of these "sandwiched" hepatic cell cultures co,.f;....ed that ch~nn~ls had 20 formed between most neighboring cells and that these çh~nnels possessed many of the characteristics of native bile canaliculi, such as, junctional complexes, microvilli, and a t~rmin~l actin web subjacent to the apical membrane.
o~ ; "sandwiched" hepatic cell cultures with 2s antibodies against apical m~mbrane markers (aminopeptidase N, diyc;lJlidyl peptidase IV) showed intense fluorescence ~l~i-.i-.g at the plasma membrane regions associated with the channel stmcture. In addition, double fluorescent labeling ûf hepatic cell cultures fûr actin microfil~ment,s and microtubules before and after channel formation 30 illustrated that an e~treme reorg~ni7~tion of the cellular actin and tubulin occurs during ch~nnel formatiûn. The actin sl~inil~g pattern changed from a diffuse distribution of stress fibers to an intense, peripheral st~inin~ subjacent to the channel membranes. This rearrangement of actin s~ i--g was consistent with the formation of a WO 94/12662 215 47 1 PCT/US93tlO874 peric~n~licular actin web around newly formed channels observed in electron micrographs. Tubulin st~inin~ showed arrays of bundles of microtubules ~tt~ehing to and r~di~tin~ from the leading edges of newly forrning c~n~liculi.
The hepatic cell culture is produced by plating primary hepatocytes on tissue culture dishes coated with gelled or ungelled collagen bas~ llt matrices, allowing the cells to spread ~ut and malce intercellular c~ s, ~en ~verlaying and gelling cAtl~ccllular matrix on top of the cultures. Tissue culture dishes are coated collagen at least 1 day prior to ~ a~ g ~e hcpatocytes. To obtain a hard, ungelled growth substrate, 200-250 ~,11 of 3 mg/ml Vitrogen~E3) (or other collagen solution) is added to each 100 mM tissue culture dish and spread evenly with a teflon policeman. Co~tefl dishes are placed at 37-C overnight then 5 ml fresh tissue culture media is added to neutralize the collagen.
To obtain a gelled b~ement matrix, neutralized Vitrogen@3) is prepared (8:1:1 Vitrogen~), 10X Dulbecco's modiISed Eagles medium, 0.1 N
NaOH) and spread onto petri dishes as described above. The plates are placed at 37c for 30~0 min to allow ~e collagen gel to form, then fresh media is added to the dishes which are stored at 37C. Just before 20 use, the media is aspirated from the precoated culture dishes.
Hepatocytes are isolated using standard collagenase perfusion methods (Selgen, P.O. METHODS BIOL., 1976 13, 29-83.
Livers are perfused with calcium-free buffer for 8-9 min followed by buffer c~llt~ g calcium and collagenase (0.3 mg/ml) for ~10-12 min.
25 The liver is then cut open and released liver cells are separated from undigested tissue with a sterile nylon mesh. The released cells are then divided into two 50-ml ce.,lliruge tubes and washed lX with Dulbecco's modified Eagles medium co~ g 5% fetal calf serum, without horrnonal supplements. The cell pellets are resuspended in a 1:1 30 mixture of 90% isotonic Percoll and media and c~ lifuged for 5 min at 350 X g . The pellets cont~ining viable hepatocytes are resuspended in fresh media, washed one time, and the hepatocytes are added to precoated dishes at a density of 9 x 106 cells/dish and incubated at 37C
in a ~% CO2 incubator. After 2-3 hours, media is replaced wit~ fresh, WO 94/12~i62 PCT/US93/10874 21~701 warm media Col~t~i..i-~g 0.4 ~g/ml dexamethasone, 4 llg/ml insulin, and 20 ng/ml EGF. Media was subsequently replaced on a daily basis with fresh, warm media cont~ining hormonal supplements. Twenty-four to forty-eight hours after plating the cultures are overlaid with a collagen 5 gel by aspirating off the culture media and ~ ling 1 ml of neutralized collagen (8:1:1 Vitrogen: lOX Dulbecco's modified Eagles medium: 0.1 N Na~I). {~ultures are then inc--b~te~l at 37~1C for 3040 min to allow the matrix to gel, after which fresh media is added 'oack to the dish.
The formation of bile canaliculi is then mnnitored with phase contrast o microscopy with an inverted microscope.
In one embodiment of the invention, hepatotoxicity or choleostatic potential of a chçmic~l compound can be ~sessed by adding from about 1 ~l to about ~ ml of a solution cont~ining from about 1 fM
to about 1 mM of the chemical compound in a solvent which is 5 coInp~hble with the hepatic cell culture, and incllb~tin~ the dish cont~inin~ the cells at about 37C, about 95% relative humidity and an atmosphere of about 5% C2 for from about 5 minutes to about 96 hours. The hepatic cell culture is then e~mined to determine the extent of any toxic effect on the hepatic cells produced by the chP!Inical compound.
This method of screening chemical compounds for hepatic cell toxicity is particularly convenient since the formation of well defined c~naliculi allows direct observations to be made of morphological changes to the canaliculi.
In a second embo~ .. t, a method for sc,ee~ g a chemical compound for hepatic extraction potential using this "sandwiched"
hepatic cell culture is acco~ lished by adding from about 1 ~11 to about 5 ml of a solution co~ ining from about 1 pM to about 50 mM of the chemical compound to be screened to the culture and incubating the hepatic cells at about 37C, about 95% relative humidity and an atmosphere of about ~% C2 for from about 1 minllte to about 4 hours.
The amount of the chemical compound contained within the hepatic cells is determined by removing the drug cont~ining buffer and washing the cells with an isotonic salt solution (ie. phosphate buffered salt so~lution ~ ~ 3 ~t 7~
or Hank's balanced salt solution), lysing the cells with detergent solutions (ie. sodium dodecyl sulfate) and detenninin.~ the amount of drug in the lysate and the original incubation solution using analytical procedures such as scintill~tion counting, HPLC, absorbance or fluorescenceq~ tion. Alternativelyin the case of fluorescent ch~ir~l compounds direct q~ tion of the chemical compound can be assessed by direct dele~ tion of cell associated fluorescence.
This method of screenin~ ch~lnical compounds for hepatic cell toxicity is particularly convenient when the compound is o transported into the hepatic cells using active carrier systems such as Type 1 cationic, Type II cationic, anionic, bile salts and asialoglyco~,futeill receptors, or when the compound is passively absorbed into the hepatic cells since they represent the normal for extraction of chPmic~l compounds by the liver.
II1 a third embo-lim~nt of this invention, screenin~ of metabolite production from the interaction of the hepatic cell culture and a chemical compound is accomplished. In this scree.nin~ procedure, from about 1 ~I to about 5 ml of a solution cont~ining from about 1 pM
to about 50 mM of the chemical compound in a solvent which is cornp~tible with the hepatic ceII culture is added to the culture plate.
The culture is then incubated at about 37C, about 95% relative humidity and an atmosphere of about 5% C2 for from about 30 minutes to about 2 weeks. At discrete times, sarnples of the incubation media can be taken and once in~lb~tion is complete, loss of the parent compound and production of new metabolites can be de~e~ ed using cherr~ical analysis techniques such as HPLC, TLC, mass spectroscopy.
Similarly cell associated parent compound and metabolites can be determined from deter~ellt cell Iysates.
In a forth embodiment of this invenhon, ~e potential of a chemical compound to alter hepatic cell metabolism using ~is screening process is also within the scope of Applicants' invention. In this process, from about 1 ,ul to about 5 ml of a solution cont~inin~ from about 1 pM to about 50 mM of the chemical compound in a compatible solvent is added to the hepatic cell culture. Substrate for a metabolic WO 94/12662 PCT/US93tlO874 ~154~01 g pathway is added and the culture is incubated at about 37C, about 95%
relative humidity and an atmosphere of about 5% C2 for from about 30 ~ ,ules to about 2 weeks. The metabolism of the hepatic cells is determined by identifying and quantifying the formation of products 5 from ~e suLsl.ate in the presence and absence of the chemical co~ ound.
A fifth em~od-nlel-l of this invention relies on the fact that hc;~alocyles grown in ~is collagen sandwich confi~,uldli~l. have been shown to be competent at xenobiotic excretion into the canaliculi. The o potential for hepatic discharge of a chemical compound into bile may be determined using this screerlin~ process. From about 1 ~11 to about 5 ml of a solution col,t;1i"i"~ from about 1 pM to about 50 mM of a ch~mic~l compound to be screened, in a solvent which is co~ &lible with the hepatic cell culture, is added to the hepatic cell culture. The culture is 15 then incubated at about 37C, about 95% relative hllmi~lity and an atmosphere of about 5% C2 for from about 1 ~ te to about 4 hours.
At this time the cells are washed using phosphate l)urrered saline and then the c~n~liculi are disrupted with EDTA. That is, EDTA disrupts the tight junctions which seal the canaliculi without bre~kin~ cell 20 membranes. This technique allowed for the measurement of material which was secreted into the c~n~licl l~r network without i..telrelence from the material which had been incorporated into the cell. When 5 ~1 of 1 mg/ml ca.l,oxyfluorescein diacetate or ~ ~1 of 1 mg/ml rho~mine B were introduced into the medium, these compounds were 25 observed by fluorescence microscopy to be selectively secreted and conce..ll~ted into c~n~ ular network of ~'sandwiched" hepatocytes.
Carboxyfluorescein fluorescence was released by treatment with ethylene~ min~o-tetraacetic acid (EDTA) which is consistent with the formation of macrodomains bounded by tight junctions.
This method is also applicable to the screening of many chemical co~ ,ounds siml-lt~neously. Any number of compounds which can be solubilized by the co~ tihle solvent may be delivered to the hepatic cell culture simultaneously. In the event that hepatotoxicity is observed, each of these compounds may then be individually scrèened to 7~1 determine which compound or groups of compounds produced the effect.
By "screening" is meant the determination of potential for specified activity for a series or mixture of compounds.
By "chemical compound" is meant any chemical agent or mi~tllre of chPmir~ l agents with potential toxic or therapeutic properties.
~y "co~ uous" is meant that ~n~licllli form a multicellular network of channels throughout the cell culture which o integrate a plurality of cells within the culture and are contiguous to many cells as distinct from isolated cell couplets.
By "hepatotoxicity" is meant an activity which results in deleterious effects on normal liver function or rlimini~hes the viability of liver cells.
By "choleostatic potential" is meant the propensity to decrease normal biliary output.
By "primary hepatic cell culture" is meant normal liver cells m~i,.t~ d in vitro.
By "functional bile c~n~licular network" is meant anastomosing .l.ler~oll-lected tubular canals between hepatocytes which m~int~in the capability for directional secretion of chemical compounds.
By "collagenase perfusion" is meant a techni-lue for se~a.d~ g and isolating hepatocytes wherein in situ perfusion of the liver with a collagenase solution results in enzymatic disruption of 25 intercellular contacts and cell/basement membrane contacts.
By "hepatocyte culture media" is meant a m~inten~n~e solution col.t~ nutrients and growth factors necessary for supporting hepatocyte viability, for example, Dulbecco's modified medium with 5% fe~al calf serum, nonessential amino acids, gl~-t~mine, 30 antibiotics, antimycotics supplemented with 0.4 ,ug/ml dexamethasone, 4 ug/ml in~-llin, and 2 ng/ml epideImal growth factor.
By "Percoll solution" is meant a mixture of colloidal polyvinylpyrrolidone coated silica used for centrifugal separation of cells and cellular components on the basis of density.
wo 94/12662 2 ~ 5 4 7 Q I PCT/US93/10874 By "culture dish precoated with collagen" is meant a plastic well or plate in which a collagen gel has been adsorbed onto the surface or ungelled collagen has been dried onto the surface.
By "overlaying about 1 ml of cold neutralized collagen" is 5 meant the process of depositing a solution of collagen on the top of cells previously adhered to a culture dish or well.
By " i~ r~!~ r contact" is meant ~h~sions between neighboring cells.
By "canalicular network is developed" is meant a meshwork of continuous c~n~liculi has formed which illlelcol-llects multiple cells.
By "solvent which is compatible with the hepatic cell culture" or "coInp~hble solvent" is meant a solution which, over the nor nal course of experimental procedures and hepatic cell m~int~nance, 5 does not elicit adverse biochemical or morphologic changes in cultured hepatic cells.
By "morphologic or structural changes in the integrity of the c~n~licular network" is meant alterations to the physical appearance of the canaliculi and/or redistribution of membrane cGnl~ollents or cytoskeletal compon~ntc, i.e. actin or tubulin.
By "cell polarity" is meant non-uniform distribution of cellular and membrane components which confer a unique sidedness to a cell, i.e. a side which faces the canaliculi and a side which faces sinusoidal spaces.
By "hepatic extraction potential" is meant the tendency for a chemical compound to be absorbed into the liver.
By "~e potential for hepatic discharge of a chemical compound into bile" is meant the tendency for a chemical compound to be secreted into bile canaliculi.
-~ 30 By "screening of metabolite production from a chemicalcompound" is meant the determin~tion of the potential of a chemical compound to be chemically modified by er~ymatic pathways in the liver.
~ ~ 3 47 ~ i By "the potential of a chemical compound to alter hepatic cell metabolism" is meant the ability of a chemical co~ ,ound to interact with a biochemical pathway in the liver producing qll~ntit~tive or qualitative changes in a product.
By "measuring ~e e~ctent of alteration of hepatic cell metabolism" is meant using analytical technillues, such as radiolabeled-or fluorescent ~ubsl~tes or chemical analysis, to .l~tc~ e 4..~..t;~;1tj~e or ql~lit~tive changes in product formation from bioçhP-mic~l pathways.
The chemical compound used in this screen can be quantified using any standard analytical technique. Standard analytical techniques include but are not limited to the use of high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy (MS), electrophoresis, optical absorption, fluorescence qualllilation, quantitation of radioactive isotopes.
lS Assessment of morphologic or structural changes in the integrity of the c~n~licular network, cell polarity or cytoskeletal st~cture is accomplished using light microscopy, fluorescence microscopy, electron microscopy, chemical modification of proteins, antibody labelling.
FXAMPLES
F.XAMP~.F. 1 ~ffect of colla.~en overlay on rat hepatocytes Experiments were perforrned in which neutralized bovine derrnal collagen (Vitrogen(~)) was allowed to gel on the top surface of primary rat hepatocytes grown on tissue culture plates coated with dried ungelled collagen or with gelled collagen. After the overlaid collagen matrix gelled, tissue culture media was added to the dish and the cells 30 were incubated at 37C in a 95% air 5% C02 hllmiclified incubator.
Phase contrast microscopy of the cells 1 day later showed that the cells had developed an elaborate network of canalicular ch~nnels which extended across a plurality of cells which appeared as refractile channels which ci~ scribed the cells. These channels were confirrned to be bile 2 1 ~ 4 7 ~ 1 c~n~liculi by electron microscopy and fluorescence microscopy.
- Electron microscopy of the overlaid hepatocytes showed microvilli containing ch~nnels between neighboring cells which are bound on each side by tight junctions and have a dense actin network subjacent to the 5 canalicular membranes. Using fluorescent-labeled antibodies, fluorescence microscopy co~ med the presence of the apical markers aminopeptidase N and Dipeptidyl peptidase in the G~n~licular ch~nn~ls similar to observations in intact liver. Similarly, fluorescent-conjugates of phalloidin lemon~trated that actin in the hepatocytes is highly o localized to the peric~n~licular region simil~r to in intact liver.
The morphology of collagen overlaid cells grown on gelled collagen b~em~nt m~.~nbrane was found to be better than cells grown on dried collagen as assessed by electron microscopy. Phase contrast microscopy showed that rat tail collagen, bovine tendon collagen, and 5 bovine dermal collagen were all effective matrixes for the basement membrane and for the overlaid matrix, and that extracellular matrix from Engelbreth-Holm-Swarm mouse tumors (MatrigellM) was also effective as an overlay matrix.
F~xAMpLE 2 F.ffect of col!~en overlay on porcine hepatocytes Experiments were per~ormed in which neutralized collagen (Vitrogen~)) was allowed to gel on the top surface of primary porcine 25 hepatocytes grown on tissue culture plates coated with dried ungelled collagen or wi~ gelled collagen. After ~e overlaid collagen matrix gelled, tissue culture media was added to ~e dish and the cells were incubated at 37C in a 95% air 5% C2 humidified incubator. Phase contrast microscopy of the cells, 1 day later, showed that the cells had 30 developed an elaborate network of canalicular channels which extended across a plurality of cells similar to observations with rat liver hepatocytes.
WO 94/12662 2~5 47 01 PCT/US93/10874 F.XAMPLE 3 Collagen overlaid hepatocytes concentrate xenobiotics into ~eir bile canaliculi Experiments were performed to demonstrate the ability of collagen sandwiched hepatocytes to transport dicarboxyfluorescein from the interior of cells to ~e c~n~lir.uli and concelll.ate the compoun in the c-~n~liculi. Five days after collagen overlaying, ~e cul~res were incubated at 37 C in Hanks balanced salt solution cont~ining 1-10 ~g/ml dicarboxyfluorescein di~ret~te to allow uptake of dicarboxyfluorescein tli~ret~te and conversion of the probe to dicarboxyfluorescein. After five to fifteen minutes resi~ l dicarboxyfluorescein (li~r,et~te was removed and the cells were washed 3-4 times with Hank's balanced salt solution. Uptake of the dicarboxyfluorescein was confirmed by 15 fluorescence microscopy and was localized to the cell interior as well as some accllm~ tion in the bile canaliculi. After 15-30 minntes of further incubation at 37C comparisons of phase contrast and fluorescence microscopy showed the dicarboxyfluorescein to be localized almost exclusively in the bile canaliculi. Using 2mM EDTA
20 the diGarboxyfluorescein concentrated in the canaliculi is released after disruption of the tight junctions which seal the canaliculi.
F,XAMPLE 4 2s Passive absolption and secretion into bile canaliculi by collagen sandwiched hepatocyte,s E~yeliments were performed to demonstrate ~at Rho-l~mine B which is passively absorbed by the liver and secreted into the bile is taken up and secreted into the bile canaliculi by collagen 30 sandwiched hepatocytes. Pive days after collagen overlaying, the cultures were incllb~te-l at 37C in Hanks balanced salt solution cont~ininp; 1 ~g/ml rho~l~min~, B. After five minl-tes residual rhodarnine B was removed and the cells were washed 3-4 times with Hank's balanced salt solution. After ~-20 minlltes further incubation at 37C
WO 94/12662 Z~ 7 Q I PCT/US93/10874 comparisons of phase contrast and fluorescence microscopy showed the rho-l~min~ B to be highly concentrated in the canaliculi and less concentrated diffusely distributed in the cell interior.
EXAMP~ F ~S
Active ~ptake of bile salts by col!~pen sandwiched l~ alocytes Experim~s were performed to rlemQn.ctrate active uptake of bile acids by Collagen sandwiched hepatocytes. Twenty-four hours after overlaying collagen, hepatocytes were rinsed with Hanks balanced salt solution and incubated with lmM taurocholate with 0.1 ~Ci 140C-taurocholate at 37C or at 4DC. At set time points the taurocholate cont~ining buffer was removed and the culture plates were washed with Hank's b~l~n~ed salt solution. The hepatocytes were then lysed with 5 detergent and ~e amount of taurocholate taken up by the hepatocytes was determined based on the amount of radioactive taurocholate present in the lysate. The active uptake of taurocholate was distinguished from the passive uptake and adsorption based on the dirr~lcllce between uptake at 37C and 4C. Uptake experiments were also performed on 20 unsandwiched hepatocytes at comparable cell density and showed that the collagen overlaid hepatocytes were better at taurocholate uptake (see Table 1).
2s 21~47Ql TABLE 1. TAUROCHOLATE UPTAKE
Taurocholate Uptake (nMoles/ dish) Time: 0 min 2 min 15 min Collagen Sandwiched 0 27 40 Unsandwiched 0 12 25
HEPATIC MODEL
FELO OF THE INVENTION
This invention ye~ ~illS to both a novel and useful method for screening a ch~.mical compound for hepatotoxicity or choleostatic potential, hPp~tic extraction potential, h~.p~tic ~ cl~f~,e into the bile, metabolite production, or ~e ability to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile c~n~licular network. ~e u~e of ~is no~el method Ilesults in ~ess of a ~ep~nce on whole ~nim~l toxicity and drug discovery studies and reduces the total number of ~nim~l.c required for chemical testing. ~ addition, information obtained from this screening procedure is devoid of interference from reactions which may occur with other organs and 5 tissue when whole ~nim~l studies are conducted.
BACKGROUND OF THE INVENTION
Scientific, financial and ethical considerations regarding the use of whole 7nim~1 studies for screening new drug candidates for 20 hepatotoxicity or choleostatic potenti~l, hepatic extraction potential, hepatic discharge, metabolite production and the ability of a chemical compound to alter hepatic cell metabolism have led to a search for an in vitro/ex vivo system which accurately reflects the yivo behaviors of chemical compounds.
The influence of extracellular matrix geometry on hepatocyte function has been reported using a sandwich configuration by Dunn et al. (See J.C.Y. Dunn et al., The FASEB Journal, Vol 3, 174 - 177 (1992); The Journal of Cell Biology, Vol 116, 1043 - 1053 (1992), and Biotechnol. Prog. Vol 7, 237-245 (1991).) In this work, 30 adult rat hepatocytes cultured in a collagen sandwich system m~int~ined normal morphology and a physiologic rate of secretion of albumin, transferrin, fibrinogen, bile acids and urea for at least 42 days. These cells were shown to m~int~in the level of albumin mRNA ~imil~r to that found in the normal liver for this same time period.
-2~ o~
-- 2 -- ~ -The subject matter of this invention has demonstrated that hepatic cell cultures are useful in detennining ~he effect of chemical compounds on hepatic cells and the effect of hepatic cells on the compounds. Rat and porcine hepatic cell cultures were grown on a hard or soft substrate, that is, ungelled or gelled collagen, and subsequently overlaid with a collagen gel. These cells initi~ted uniform formation of bile canaliculi ~roughout the entire culture. Typically, 24 hours after overlaying the collagen gel, the hepatic cell cultures formed are characterized by a nearly complete and continuous bile e~n~
lD network. Morphological analysis of these "sandwiched" cell cultures by electron microscopy co~ ed that c~nnPls had formed between most neighboring cells and that these ch~nnels possessed many of the characteristics of native bile c~n~liculi, such as junctional complexes, microvilli and a terminal actin web subjacent to the apical membrane.
These c~n~liculi are also functional at concel,tl~lillg xenobiotic agents presented to the hepatocyte culture. It has therefore been demonstrated in this invention that hepatic cells cultured in this m~nner can be used to screen a wide variety of chemical compounds with only limited sacrifice of laboratory ~nim~
SUMMARY OF THF INVEN~ION
A method is presented for the in vitro screening of a chemical compound for hepatotoxicity or choleostatic potential, hepatic extraction pot~nti~l, hepatic discharge, metabolite production or the ability of a Ghto.mi~l compound to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile canalicular network, the process col-~.ising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspen~ling ~e cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 --i.--.~es;
(c) adjusting the cell concentration to about 1 to 2 times 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incllb~tin~ the dish co--t~ e cells at about 37C, about g~% ~latiw humidity and an atmosphere of about 5%
C02;
(e) ch~n in~ and repl~r.in~ the hepatocyte culture media after about 2 hours to remove any ~1n~tt~rh~1 cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish c~,-t~i--ing the hepatic cells after ~e cells have spread and established inlelcellular contact, and reincllb~tin~ at the conditions of step (d) to allow ~e added neutralized collagen to gel;
g) ~ in~ to the hepatic cells about an additional 5 ml of hepatocyte culture m~
(h) incubating at the conditions of step (d) until the c~n~licular network is developed as observed by light microscopy;
(i) ~ldin~ from about 1 ~11 to about 5 ml of a solution to the hepatic cell culture, the solution cont~ining from about 1 f~ to about ~0 mM of the chemical compound in a solvent which is comp~tible with the hepatic cell culture, and incubating at the conditions of step (d) for about 1 minute 2s to about 2 weeks;
(j) assessing morphologic or structural changes in the integrity of the canalicular network, cell polarity or cytoskeletal structure; quantifying the amount of the chemical compound colltailled within the hepatic cells or rem~inin~
in the culture solution; quantifying the chemical compound secreted into the bile contained within ~e canalicular network; or measuring the extent of alteration of hepatic cell metabolism.
WO 94tl2662 PCT/US93/10874 DETArLE~ DESCRIPTION OF THE INVENTION
Applicants have found a novel chemical compound screening procedure for the in vitro assessment of hepatotoxicity or choleostatic potential, hepatic extraction potential, hepatic discharge, 5 metabolite production or the ability of a chemical compound to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional 'oile c~n~licular network, the process CO~ ;S~llg ~e steps of:
~a) isola~ing hepatic cells 'oy collagenase ~-fusion;
(b) washing the isolated cells with hepatocyte culture m~
suspending ~e cells in about a ~0:~0 mi~cture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minl~tçs;
(c) adjusting the cell concentration to about 1 to 2 times 106 cells/ml of h~p~tocyte culture media and dispel-~in~ about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incllb~tin~ the dish cont~ining the cells at about 37C, about 95% relative humidity and an atmosphere of about 5%
CO2;
(e) ch~ngin~ and replacing the hepatocyte culture media after about 2 hours to remove any ln~ ched cells;
(f) overlaying about l ml of cold neutralized collagen solution to the dish co.-t~i-,;"g the hepatic cells after ~e cells have spread and established intercellular contact, and reincubating at the conditions of step (d).to allow the added neutralized collagen to gel;
(g) ~ in~ to ~e hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the c~n~licular network is developed as observed by light microscopy;
in~ from about 1 ~l to about ~ ml of a solution to the hepatic cell culture, the solution cont~ining from about l fM to about ~0 mM of the chemical compound in a solvent which is compatible with the hepatic cell culture, and incubating at the conditions of step (d) for about l minute to about 2 weeks;
(j) assessing morphologic or structural changes in the integrity of the c~n~licular network, cell polarity or cytoskeletal structure; quantifying the amount of the chemical compound cu~lA;~ within the hepatic cells or rem7~ining in the ~ulture solution; yu~lliryillg the chPInical ~ o~ d secreted into the l~ile co-~t~ l within the canalicular network; or llleh~u~ g the extent of alteration of hepatic cell metabolism.
In accordance with the practice of this invention, it has been found that primary hepatic cells grown on a hard or soft collagen substrate and subsequently overlaid with a collagen gel initi~te~l uniform 5 formation of bile canaliculi throughout the entire culture which can be used in the screening of chemical compounds. Typically, 24 hours after overlay, hepatic cell cultures formed an anastomosing bile c~n~liculi network ~.lercol,llecting a plurality of cells. Morphologic analysis of these "sandwiched" hepatic cell cultures co,.f;....ed that ch~nn~ls had 20 formed between most neighboring cells and that these çh~nnels possessed many of the characteristics of native bile canaliculi, such as, junctional complexes, microvilli, and a t~rmin~l actin web subjacent to the apical membrane.
o~ ; "sandwiched" hepatic cell cultures with 2s antibodies against apical m~mbrane markers (aminopeptidase N, diyc;lJlidyl peptidase IV) showed intense fluorescence ~l~i-.i-.g at the plasma membrane regions associated with the channel stmcture. In addition, double fluorescent labeling ûf hepatic cell cultures fûr actin microfil~ment,s and microtubules before and after channel formation 30 illustrated that an e~treme reorg~ni7~tion of the cellular actin and tubulin occurs during ch~nnel formatiûn. The actin sl~inil~g pattern changed from a diffuse distribution of stress fibers to an intense, peripheral st~inin~ subjacent to the channel membranes. This rearrangement of actin s~ i--g was consistent with the formation of a WO 94/12662 215 47 1 PCT/US93tlO874 peric~n~licular actin web around newly formed channels observed in electron micrographs. Tubulin st~inin~ showed arrays of bundles of microtubules ~tt~ehing to and r~di~tin~ from the leading edges of newly forrning c~n~liculi.
The hepatic cell culture is produced by plating primary hepatocytes on tissue culture dishes coated with gelled or ungelled collagen bas~ llt matrices, allowing the cells to spread ~ut and malce intercellular c~ s, ~en ~verlaying and gelling cAtl~ccllular matrix on top of the cultures. Tissue culture dishes are coated collagen at least 1 day prior to ~ a~ g ~e hcpatocytes. To obtain a hard, ungelled growth substrate, 200-250 ~,11 of 3 mg/ml Vitrogen~E3) (or other collagen solution) is added to each 100 mM tissue culture dish and spread evenly with a teflon policeman. Co~tefl dishes are placed at 37-C overnight then 5 ml fresh tissue culture media is added to neutralize the collagen.
To obtain a gelled b~ement matrix, neutralized Vitrogen@3) is prepared (8:1:1 Vitrogen~), 10X Dulbecco's modiISed Eagles medium, 0.1 N
NaOH) and spread onto petri dishes as described above. The plates are placed at 37c for 30~0 min to allow ~e collagen gel to form, then fresh media is added to the dishes which are stored at 37C. Just before 20 use, the media is aspirated from the precoated culture dishes.
Hepatocytes are isolated using standard collagenase perfusion methods (Selgen, P.O. METHODS BIOL., 1976 13, 29-83.
Livers are perfused with calcium-free buffer for 8-9 min followed by buffer c~llt~ g calcium and collagenase (0.3 mg/ml) for ~10-12 min.
25 The liver is then cut open and released liver cells are separated from undigested tissue with a sterile nylon mesh. The released cells are then divided into two 50-ml ce.,lliruge tubes and washed lX with Dulbecco's modified Eagles medium co~ g 5% fetal calf serum, without horrnonal supplements. The cell pellets are resuspended in a 1:1 30 mixture of 90% isotonic Percoll and media and c~ lifuged for 5 min at 350 X g . The pellets cont~ining viable hepatocytes are resuspended in fresh media, washed one time, and the hepatocytes are added to precoated dishes at a density of 9 x 106 cells/dish and incubated at 37C
in a ~% CO2 incubator. After 2-3 hours, media is replaced wit~ fresh, WO 94/12~i62 PCT/US93/10874 21~701 warm media Col~t~i..i-~g 0.4 ~g/ml dexamethasone, 4 llg/ml insulin, and 20 ng/ml EGF. Media was subsequently replaced on a daily basis with fresh, warm media cont~ining hormonal supplements. Twenty-four to forty-eight hours after plating the cultures are overlaid with a collagen 5 gel by aspirating off the culture media and ~ ling 1 ml of neutralized collagen (8:1:1 Vitrogen: lOX Dulbecco's modified Eagles medium: 0.1 N Na~I). {~ultures are then inc--b~te~l at 37~1C for 3040 min to allow the matrix to gel, after which fresh media is added 'oack to the dish.
The formation of bile canaliculi is then mnnitored with phase contrast o microscopy with an inverted microscope.
In one embodiment of the invention, hepatotoxicity or choleostatic potential of a chçmic~l compound can be ~sessed by adding from about 1 ~l to about ~ ml of a solution cont~ining from about 1 fM
to about 1 mM of the chemical compound in a solvent which is 5 coInp~hble with the hepatic cell culture, and incllb~tin~ the dish cont~inin~ the cells at about 37C, about 95% relative humidity and an atmosphere of about 5% C2 for from about 5 minutes to about 96 hours. The hepatic cell culture is then e~mined to determine the extent of any toxic effect on the hepatic cells produced by the chP!Inical compound.
This method of screening chemical compounds for hepatic cell toxicity is particularly convenient since the formation of well defined c~naliculi allows direct observations to be made of morphological changes to the canaliculi.
In a second embo~ .. t, a method for sc,ee~ g a chemical compound for hepatic extraction potential using this "sandwiched"
hepatic cell culture is acco~ lished by adding from about 1 ~11 to about 5 ml of a solution co~ ining from about 1 pM to about 50 mM of the chemical compound to be screened to the culture and incubating the hepatic cells at about 37C, about 95% relative humidity and an atmosphere of about ~% C2 for from about 1 minllte to about 4 hours.
The amount of the chemical compound contained within the hepatic cells is determined by removing the drug cont~ining buffer and washing the cells with an isotonic salt solution (ie. phosphate buffered salt so~lution ~ ~ 3 ~t 7~
or Hank's balanced salt solution), lysing the cells with detergent solutions (ie. sodium dodecyl sulfate) and detenninin.~ the amount of drug in the lysate and the original incubation solution using analytical procedures such as scintill~tion counting, HPLC, absorbance or fluorescenceq~ tion. Alternativelyin the case of fluorescent ch~ir~l compounds direct q~ tion of the chemical compound can be assessed by direct dele~ tion of cell associated fluorescence.
This method of screenin~ ch~lnical compounds for hepatic cell toxicity is particularly convenient when the compound is o transported into the hepatic cells using active carrier systems such as Type 1 cationic, Type II cationic, anionic, bile salts and asialoglyco~,futeill receptors, or when the compound is passively absorbed into the hepatic cells since they represent the normal for extraction of chPmic~l compounds by the liver.
II1 a third embo-lim~nt of this invention, screenin~ of metabolite production from the interaction of the hepatic cell culture and a chemical compound is accomplished. In this scree.nin~ procedure, from about 1 ~I to about 5 ml of a solution cont~ining from about 1 pM
to about 50 mM of the chemical compound in a solvent which is cornp~tible with the hepatic ceII culture is added to the culture plate.
The culture is then incubated at about 37C, about 95% relative humidity and an atmosphere of about 5% C2 for from about 30 minutes to about 2 weeks. At discrete times, sarnples of the incubation media can be taken and once in~lb~tion is complete, loss of the parent compound and production of new metabolites can be de~e~ ed using cherr~ical analysis techniques such as HPLC, TLC, mass spectroscopy.
Similarly cell associated parent compound and metabolites can be determined from deter~ellt cell Iysates.
In a forth embodiment of this invenhon, ~e potential of a chemical compound to alter hepatic cell metabolism using ~is screening process is also within the scope of Applicants' invention. In this process, from about 1 ,ul to about 5 ml of a solution cont~inin~ from about 1 pM to about 50 mM of the chemical compound in a compatible solvent is added to the hepatic cell culture. Substrate for a metabolic WO 94/12662 PCT/US93tlO874 ~154~01 g pathway is added and the culture is incubated at about 37C, about 95%
relative humidity and an atmosphere of about 5% C2 for from about 30 ~ ,ules to about 2 weeks. The metabolism of the hepatic cells is determined by identifying and quantifying the formation of products 5 from ~e suLsl.ate in the presence and absence of the chemical co~ ound.
A fifth em~od-nlel-l of this invention relies on the fact that hc;~alocyles grown in ~is collagen sandwich confi~,uldli~l. have been shown to be competent at xenobiotic excretion into the canaliculi. The o potential for hepatic discharge of a chemical compound into bile may be determined using this screerlin~ process. From about 1 ~11 to about 5 ml of a solution col,t;1i"i"~ from about 1 pM to about 50 mM of a ch~mic~l compound to be screened, in a solvent which is co~ &lible with the hepatic cell culture, is added to the hepatic cell culture. The culture is 15 then incubated at about 37C, about 95% relative hllmi~lity and an atmosphere of about 5% C2 for from about 1 ~ te to about 4 hours.
At this time the cells are washed using phosphate l)urrered saline and then the c~n~liculi are disrupted with EDTA. That is, EDTA disrupts the tight junctions which seal the canaliculi without bre~kin~ cell 20 membranes. This technique allowed for the measurement of material which was secreted into the c~n~licl l~r network without i..telrelence from the material which had been incorporated into the cell. When 5 ~1 of 1 mg/ml ca.l,oxyfluorescein diacetate or ~ ~1 of 1 mg/ml rho~mine B were introduced into the medium, these compounds were 25 observed by fluorescence microscopy to be selectively secreted and conce..ll~ted into c~n~ ular network of ~'sandwiched" hepatocytes.
Carboxyfluorescein fluorescence was released by treatment with ethylene~ min~o-tetraacetic acid (EDTA) which is consistent with the formation of macrodomains bounded by tight junctions.
This method is also applicable to the screening of many chemical co~ ,ounds siml-lt~neously. Any number of compounds which can be solubilized by the co~ tihle solvent may be delivered to the hepatic cell culture simultaneously. In the event that hepatotoxicity is observed, each of these compounds may then be individually scrèened to 7~1 determine which compound or groups of compounds produced the effect.
By "screening" is meant the determination of potential for specified activity for a series or mixture of compounds.
By "chemical compound" is meant any chemical agent or mi~tllre of chPmir~ l agents with potential toxic or therapeutic properties.
~y "co~ uous" is meant that ~n~licllli form a multicellular network of channels throughout the cell culture which o integrate a plurality of cells within the culture and are contiguous to many cells as distinct from isolated cell couplets.
By "hepatotoxicity" is meant an activity which results in deleterious effects on normal liver function or rlimini~hes the viability of liver cells.
By "choleostatic potential" is meant the propensity to decrease normal biliary output.
By "primary hepatic cell culture" is meant normal liver cells m~i,.t~ d in vitro.
By "functional bile c~n~licular network" is meant anastomosing .l.ler~oll-lected tubular canals between hepatocytes which m~int~in the capability for directional secretion of chemical compounds.
By "collagenase perfusion" is meant a techni-lue for se~a.d~ g and isolating hepatocytes wherein in situ perfusion of the liver with a collagenase solution results in enzymatic disruption of 25 intercellular contacts and cell/basement membrane contacts.
By "hepatocyte culture media" is meant a m~inten~n~e solution col.t~ nutrients and growth factors necessary for supporting hepatocyte viability, for example, Dulbecco's modified medium with 5% fe~al calf serum, nonessential amino acids, gl~-t~mine, 30 antibiotics, antimycotics supplemented with 0.4 ,ug/ml dexamethasone, 4 ug/ml in~-llin, and 2 ng/ml epideImal growth factor.
By "Percoll solution" is meant a mixture of colloidal polyvinylpyrrolidone coated silica used for centrifugal separation of cells and cellular components on the basis of density.
wo 94/12662 2 ~ 5 4 7 Q I PCT/US93/10874 By "culture dish precoated with collagen" is meant a plastic well or plate in which a collagen gel has been adsorbed onto the surface or ungelled collagen has been dried onto the surface.
By "overlaying about 1 ml of cold neutralized collagen" is 5 meant the process of depositing a solution of collagen on the top of cells previously adhered to a culture dish or well.
By " i~ r~!~ r contact" is meant ~h~sions between neighboring cells.
By "canalicular network is developed" is meant a meshwork of continuous c~n~liculi has formed which illlelcol-llects multiple cells.
By "solvent which is compatible with the hepatic cell culture" or "coInp~hble solvent" is meant a solution which, over the nor nal course of experimental procedures and hepatic cell m~int~nance, 5 does not elicit adverse biochemical or morphologic changes in cultured hepatic cells.
By "morphologic or structural changes in the integrity of the c~n~licular network" is meant alterations to the physical appearance of the canaliculi and/or redistribution of membrane cGnl~ollents or cytoskeletal compon~ntc, i.e. actin or tubulin.
By "cell polarity" is meant non-uniform distribution of cellular and membrane components which confer a unique sidedness to a cell, i.e. a side which faces the canaliculi and a side which faces sinusoidal spaces.
By "hepatic extraction potential" is meant the tendency for a chemical compound to be absorbed into the liver.
By "~e potential for hepatic discharge of a chemical compound into bile" is meant the tendency for a chemical compound to be secreted into bile canaliculi.
-~ 30 By "screening of metabolite production from a chemicalcompound" is meant the determin~tion of the potential of a chemical compound to be chemically modified by er~ymatic pathways in the liver.
~ ~ 3 47 ~ i By "the potential of a chemical compound to alter hepatic cell metabolism" is meant the ability of a chemical co~ ,ound to interact with a biochemical pathway in the liver producing qll~ntit~tive or qualitative changes in a product.
By "measuring ~e e~ctent of alteration of hepatic cell metabolism" is meant using analytical technillues, such as radiolabeled-or fluorescent ~ubsl~tes or chemical analysis, to .l~tc~ e 4..~..t;~;1tj~e or ql~lit~tive changes in product formation from bioçhP-mic~l pathways.
The chemical compound used in this screen can be quantified using any standard analytical technique. Standard analytical techniques include but are not limited to the use of high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy (MS), electrophoresis, optical absorption, fluorescence qualllilation, quantitation of radioactive isotopes.
lS Assessment of morphologic or structural changes in the integrity of the c~n~licular network, cell polarity or cytoskeletal st~cture is accomplished using light microscopy, fluorescence microscopy, electron microscopy, chemical modification of proteins, antibody labelling.
FXAMPLES
F.XAMP~.F. 1 ~ffect of colla.~en overlay on rat hepatocytes Experiments were perforrned in which neutralized bovine derrnal collagen (Vitrogen(~)) was allowed to gel on the top surface of primary rat hepatocytes grown on tissue culture plates coated with dried ungelled collagen or with gelled collagen. After the overlaid collagen matrix gelled, tissue culture media was added to the dish and the cells 30 were incubated at 37C in a 95% air 5% C02 hllmiclified incubator.
Phase contrast microscopy of the cells 1 day later showed that the cells had developed an elaborate network of canalicular ch~nnels which extended across a plurality of cells which appeared as refractile channels which ci~ scribed the cells. These channels were confirrned to be bile 2 1 ~ 4 7 ~ 1 c~n~liculi by electron microscopy and fluorescence microscopy.
- Electron microscopy of the overlaid hepatocytes showed microvilli containing ch~nnels between neighboring cells which are bound on each side by tight junctions and have a dense actin network subjacent to the 5 canalicular membranes. Using fluorescent-labeled antibodies, fluorescence microscopy co~ med the presence of the apical markers aminopeptidase N and Dipeptidyl peptidase in the G~n~licular ch~nn~ls similar to observations in intact liver. Similarly, fluorescent-conjugates of phalloidin lemon~trated that actin in the hepatocytes is highly o localized to the peric~n~licular region simil~r to in intact liver.
The morphology of collagen overlaid cells grown on gelled collagen b~em~nt m~.~nbrane was found to be better than cells grown on dried collagen as assessed by electron microscopy. Phase contrast microscopy showed that rat tail collagen, bovine tendon collagen, and 5 bovine dermal collagen were all effective matrixes for the basement membrane and for the overlaid matrix, and that extracellular matrix from Engelbreth-Holm-Swarm mouse tumors (MatrigellM) was also effective as an overlay matrix.
F~xAMpLE 2 F.ffect of col!~en overlay on porcine hepatocytes Experiments were per~ormed in which neutralized collagen (Vitrogen~)) was allowed to gel on the top surface of primary porcine 25 hepatocytes grown on tissue culture plates coated with dried ungelled collagen or wi~ gelled collagen. After ~e overlaid collagen matrix gelled, tissue culture media was added to ~e dish and the cells were incubated at 37C in a 95% air 5% C2 humidified incubator. Phase contrast microscopy of the cells, 1 day later, showed that the cells had 30 developed an elaborate network of canalicular channels which extended across a plurality of cells similar to observations with rat liver hepatocytes.
WO 94/12662 2~5 47 01 PCT/US93/10874 F.XAMPLE 3 Collagen overlaid hepatocytes concentrate xenobiotics into ~eir bile canaliculi Experiments were performed to demonstrate the ability of collagen sandwiched hepatocytes to transport dicarboxyfluorescein from the interior of cells to ~e c~n~lir.uli and concelll.ate the compoun in the c-~n~liculi. Five days after collagen overlaying, ~e cul~res were incubated at 37 C in Hanks balanced salt solution cont~ining 1-10 ~g/ml dicarboxyfluorescein di~ret~te to allow uptake of dicarboxyfluorescein tli~ret~te and conversion of the probe to dicarboxyfluorescein. After five to fifteen minutes resi~ l dicarboxyfluorescein (li~r,et~te was removed and the cells were washed 3-4 times with Hank's balanced salt solution. Uptake of the dicarboxyfluorescein was confirmed by 15 fluorescence microscopy and was localized to the cell interior as well as some accllm~ tion in the bile canaliculi. After 15-30 minntes of further incubation at 37C comparisons of phase contrast and fluorescence microscopy showed the dicarboxyfluorescein to be localized almost exclusively in the bile canaliculi. Using 2mM EDTA
20 the diGarboxyfluorescein concentrated in the canaliculi is released after disruption of the tight junctions which seal the canaliculi.
F,XAMPLE 4 2s Passive absolption and secretion into bile canaliculi by collagen sandwiched hepatocyte,s E~yeliments were performed to demonstrate ~at Rho-l~mine B which is passively absorbed by the liver and secreted into the bile is taken up and secreted into the bile canaliculi by collagen 30 sandwiched hepatocytes. Pive days after collagen overlaying, the cultures were incllb~te-l at 37C in Hanks balanced salt solution cont~ininp; 1 ~g/ml rho~l~min~, B. After five minl-tes residual rhodarnine B was removed and the cells were washed 3-4 times with Hank's balanced salt solution. After ~-20 minlltes further incubation at 37C
WO 94/12662 Z~ 7 Q I PCT/US93/10874 comparisons of phase contrast and fluorescence microscopy showed the rho-l~min~ B to be highly concentrated in the canaliculi and less concentrated diffusely distributed in the cell interior.
EXAMP~ F ~S
Active ~ptake of bile salts by col!~pen sandwiched l~ alocytes Experim~s were performed to rlemQn.ctrate active uptake of bile acids by Collagen sandwiched hepatocytes. Twenty-four hours after overlaying collagen, hepatocytes were rinsed with Hanks balanced salt solution and incubated with lmM taurocholate with 0.1 ~Ci 140C-taurocholate at 37C or at 4DC. At set time points the taurocholate cont~ining buffer was removed and the culture plates were washed with Hank's b~l~n~ed salt solution. The hepatocytes were then lysed with 5 detergent and ~e amount of taurocholate taken up by the hepatocytes was determined based on the amount of radioactive taurocholate present in the lysate. The active uptake of taurocholate was distinguished from the passive uptake and adsorption based on the dirr~lcllce between uptake at 37C and 4C. Uptake experiments were also performed on 20 unsandwiched hepatocytes at comparable cell density and showed that the collagen overlaid hepatocytes were better at taurocholate uptake (see Table 1).
2s 21~47Ql TABLE 1. TAUROCHOLATE UPTAKE
Taurocholate Uptake (nMoles/ dish) Time: 0 min 2 min 15 min Collagen Sandwiched 0 27 40 Unsandwiched 0 12 25
Claims (13)
1. A method for screening a chemical compound for hepatotoxicity or choleostatic potential using a primary hepatic cell culture with a functional bile canalicular network, the cell culture grown on ungelled or gelled collagen and subsequently overlaid with a collagen gel, the process comprising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 fM to about 1 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 5 minutes to about 96 hours;
(j) assessing morphologic or structural changes in the integrity of the canalicular network, cell polarity or cytoskeletal structure.
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 fM to about 1 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 5 minutes to about 96 hours;
(j) assessing morphologic or structural changes in the integrity of the canalicular network, cell polarity or cytoskeletal structure.
2 A method for screening a chemical compound for hepatic extraction potential using a primary hepatic cell culture with a functional bile canalicular network, the cell culture grown on ungelled or gelled collagen and subsequently overlaid with a collagen gel, the process comprising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 1 minute to about 4 hours;
(j) quantifying the amount of the chemical compound contained within the hepatic cells or remaining in the solution of step (i).
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 1 minute to about 4 hours;
(j) quantifying the amount of the chemical compound contained within the hepatic cells or remaining in the solution of step (i).
3. A method for screening the potential for hepatic discharge of a chemical compound into bile using a primary hepatic cell culture with a functional bile canalicular network, the cell culture grown on ungelled or gelled collagen and subsequently overlaid with a collagen gel, the process comprising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 1 minute to about 4 hours;
(j) quantifying the chemical compound secreted into the bile contained within the canalicular network.
(a) isolating hepatic cells by collagenase perfusion;
(b) washing the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 1 minute to about 4 hours;
(j) quantifying the chemical compound secreted into the bile contained within the canalicular network.
4. A method for screening of metabolite production from a chemical compound using a primary hepatic cell culture with a functional bile canalicular network, the cell culture grown on ungelled or gelled collagen and subsequently overlaid with a collagen gel, the process comprising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washig the isolated cells with hepatocyte culture media, suspendingthe cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (4) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 30 minutes to about 2 weeks;
(j) quantifying the loss of chemical compound or production of metabolites.
(a) isolating hepatic cells by collagenase perfusion;
(b) washig the isolated cells with hepatocyte culture media, suspendingthe cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (4) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 30 minutes to about 2 weeks;
(j) quantifying the loss of chemical compound or production of metabolites.
5. A method for screening the potential of a chemical compound to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile canalicular network, the cell culture grown on ungelled or gelled collagen and subsequently overlaid with a collagen gel, the process comprising the steps of:
(a) isolating hepatic cells by collagenase perfusion;
(b) washig the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;.
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 30 minutes to about 2 weeks;
(j) measuring the extent of alteration of hepatic cell metabolism.
(a) isolating hepatic cells by collagenase perfusion;
(b) washig the isolated cells with hepatocyte culture media, suspending the cells in about a 50:50 mixture of the hepatocyte culture media and about 90% isotonic Percoll solution, and centrifuging at about 350 times gravity for about 5 minutes;
(c) adjusting the cell concentration to about 1 - 2 x 106 cells/ml of hepatocyte culture media and dispensing about 6 ml of cell suspension to a culture dish precoated with collagen;
(d) incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% CO2;.
(e) changing and replacing the hepatocyte culture media after about 2 hours to remove any unattached cells;
(f) overlaying about 1 ml of cold neutralized collagen solution to the dish containing the hepatic cells after the cells have spread and established intercellular contact, and reincubating at the conditions of step (d) to allow the added neutralized collagen to gel;
(g) adding to the hepatic cells about an additional 5 ml of hepatocyte culture media;
(h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
(i) adding from about 1 µl to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with hepatic cell culture, and incubating at the conditions of step (d) for about 30 minutes to about 2 weeks;
(j) measuring the extent of alteration of hepatic cell metabolism.
6. The method of screening a chemical compound as in claims 1,2,3,4 or 5 wherein the chemical compounds is tgransported into hepatic cells using active carrier systems selected from the group concisting of Type I cationic, Type II cationic, anionic,m bile salts, and asialoglycoprotein receptors.
7. The method of screening a chemical compound as in claims 1,2,3,4 or 5 wherein the chemical compound is passively absorbed into the hepatic cells.
8. The method of screening a chemical compound as in claims 1,2,3,4 or 5 wherein the hepatic cell culture is maintained between a basement and overlaid gelled extracellular matrix.
9. The method of screen a chemical compound as in claims 1,2,3,4 or 5 wherein the functional bile canaliculi form an anastomosing network interconnecting a plurality of cells.
10. The method of screening a chemical compound as in claims 1, 2, 3, 4 or 5 wherein the hepatic cells are obtained from rat and porcine liver.
11. The method of screening a chemical compound as in claims 1, 2, 3, 4 or 5 wherein the collagen is gelled on the culture dish prior to the addition of the cell suspension.
12. The method of screening a chemical compound as in claims 1, 2, 3, 4 or 5 wherein the collagen stock solution is equal to or greater than 3 mg/ml.
13. The method of screening a chemical compound as in claim 1, 2, 3, 4 or 5 wherein a plurality of chemical compounds are screened simultaneously using the same culture dish.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US98196492A | 1992-11-25 | 1992-11-25 | |
| US981,964 | 1992-11-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2154701A1 true CA2154701A1 (en) | 1994-06-09 |
Family
ID=25528763
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2154701 Abandoned CA2154701A1 (en) | 1992-11-25 | 1993-11-12 | Hepatic model |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0673433A1 (en) |
| JP (1) | JPH08503610A (en) |
| AU (1) | AU5552794A (en) |
| CA (1) | CA2154701A1 (en) |
| WO (1) | WO1994012662A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7601494B2 (en) | 1999-03-17 | 2009-10-13 | The University Of North Carolina At Chapel Hill | Method of screening candidate compounds for susceptibility to biliary excretion |
| ATE437360T1 (en) * | 1999-03-17 | 2009-08-15 | Univ North Carolina | SCREENING PROCEDURES FOR CANDIDATE COMPOUNDS FOR SUSPENSIBILITY TO BALE EXCRETION |
| WO2001059062A2 (en) * | 2000-02-11 | 2001-08-16 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for obtaining in vitro polarized hepatoma cell lines |
| US7034553B2 (en) | 2003-12-05 | 2006-04-25 | Prodont, Inc. | Direct resistance measurement corrosion probe |
| JP6087264B2 (en) | 2013-11-29 | 2017-03-01 | 株式会社日立ハイテクノロジーズ | Component analyzer, medicinal effect analyzer, and analysis method |
| US10444228B2 (en) | 2014-08-22 | 2019-10-15 | Biopredic International | Method of screening a candidate compound for inducing bile canalicular function disorders |
| WO2016036785A1 (en) * | 2014-09-02 | 2016-03-10 | Hurel Corporation | In vitro biliary excretion assay |
| JP6758026B2 (en) | 2015-04-17 | 2020-09-23 | 株式会社日立ハイテク | Component analyzer, drug component analyzer, component analysis method and drug component analysis method |
| WO2017085119A1 (en) * | 2015-11-16 | 2017-05-26 | Insphero Ag | Method and assay for the assessment of a cholestatic risk of a compound |
| KR101864410B1 (en) * | 2016-01-21 | 2018-06-05 | 한국화학연구원 | Three dimensional hepatocyte culturing unit, evaluating system for hepatotoxicity and method using the same |
| JP6756493B2 (en) * | 2016-02-29 | 2020-09-16 | 米満 吉和 | High-performance hepatocytes and their utilization |
| JP7113132B2 (en) | 2019-02-26 | 2022-08-04 | 株式会社日立ハイテク | Intracellular dynamics analysis method for compounds |
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| US4914032A (en) * | 1985-06-06 | 1990-04-03 | Centro De Investigation Y Estudios Avanzados Del Instituto Politecnico Nacional | Process for the long-term surviving culture of hepatocytes |
| US5032508A (en) * | 1988-09-08 | 1991-07-16 | Marrow-Tech, Inc. | Three-dimensional cell and tissue culture system |
-
1993
- 1993-11-12 AU AU55527/94A patent/AU5552794A/en not_active Abandoned
- 1993-11-12 JP JP6513179A patent/JPH08503610A/en active Pending
- 1993-11-12 CA CA 2154701 patent/CA2154701A1/en not_active Abandoned
- 1993-11-12 EP EP94900612A patent/EP0673433A1/en not_active Withdrawn
- 1993-11-12 WO PCT/US1993/010874 patent/WO1994012662A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU5552794A (en) | 1994-06-22 |
| JPH08503610A (en) | 1996-04-23 |
| EP0673433A1 (en) | 1995-09-27 |
| WO1994012662A1 (en) | 1994-06-09 |
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