Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • 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
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/04—Screening or testing on artificial tissues
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/54—Collagen; Gelatin
• • 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

Definitions

• This invention pertains to both a novel and useful method for screening a chemical compound for hepatotoxicity or choleostatic potential, hepatic extraction potential, hepatic discharge into the bile, metabolite production, or the ability to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile canalicular network.
• the use of mis novel method results in less of a dependence on whole animal toxicity and drug discovery studies and reduces the total number of animals required for chemical testing.
• information obtained from this screening procedure is devoid of interference from reactions which may occur with other organs and tissue when whole animal studies are conducted.
• hepatic cell cultures are useful in determining the 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 initiated uniform formation of bile canaliculi throughout 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 canaliculi network.
• step (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;
• step (h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
• step (i) adding from about 1 ⁇ l to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 fM to about 50 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 1 minute to about 2 weeks;
• Applicants have found a novel chemical compound screening procedure for the in vitro assessment of hepatotoxicity or choleostatic potential, hepatic extraction potential, hepatic discharge, metabolite production or the ability of a chemical compound to alter hepatic cell metabolism, using a primary hepatic cell culture with a functional bile canalicular network, the process comprising the steps of:
• step (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;
• step (h) incubating at the conditions of step (d) until the canalicular network is developed as observed by light microscopy;
• step (i) adding from about 1 ⁇ l to about 5 ml of a solution to the hepatic cell culture, the solution containing from about 1 fM to about 50 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 1 minute to about 2 weeks; (j) assessing mo ⁇ hologic or structural changes in the integrity of the canalicular network, cell polarity or cytoskeletal structure; quantifying the amount of the chemical compound contained within the hepatic cells or remaining in the culture solution; quantifying the chemical compound secreted into the bile contained within the canalicular network; or measuring the extent of alteration of hepatic cell metabolism.
• hepatic cell cultures formed an anastomosing bile canaliculi network interconnecting a plurality of cells.
• Mo ⁇ hologic analysis of these "sandwiched" hepatic cell cultures confirmed that channels had formed between most neighboring cells and that these channels possessed many of the characteristics of native bile canaliculi, such as, junctional complexes, microvilli, and a terminal actin web subjacent to the apical membrane.
• Immunostaining "sandwiched" hepatic cell cultures with antibodies against apical membrane markers showed intense fluorescence staining at the plasma membrane regions associated with the channel structure.
• double fluorescent labeling of hepatic cell cultures for actin microfilaments and microtubules before and after channel formation illustrated that an extreme reorganization of the cellular actin and tubulin occurs during channel formation.
• the actin staining pattern changed from a diffuse distribution of stress fibers to an intense, peripheral staining subjacent to the channel membranes.
• the hepatic cell culture is produced by plating primary hepatocytes on tissue culture dishes coated with gelled or ungelled collagen basement matrices, allowing the cells to spread out and make intercellular contacts, then overlaying and gelling extracellular matrix on top of the cultures.
• Tissue culture dishes are coated collagen at least 1 day prior to preparing the hepatocytes.
• 200-250 ⁇ l of 3 mg/ml Vitrogen® (or other collagen solution) is added to each 100 mM tissue culture dish and spread evenly with a teflon policeman. Coated dishes are placed at 37°C overnight then 5 ml fresh tissue culture media is added to neutralize the collagen.
• neutralized Vitrogen® is prepared (8:1:1 Vitrogen®, 10X Dulbecco's modified Eagles medium, 0.1 N NaOH) and spread onto petri dishes as described above. The plates are placed at 37°c for 30-40 min to allow the collagen gel to form, then fresh media is added to the dishes which are stored at 37 °C. Just before 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 containing calcium and collagenase (0.3 mg/ml) for ⁇ 10-12 min.
• 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 centrifuge tubes and washed IX with Dulbecco's modified Eagles medium containing 5% fetal calf serum, without hormonal supplements.
• the cell pellets are resuspended in a 1:1 mixture of 90% isotonic Percoll and media and centrifuged for 5 min at
• the pellets containing viable hepatocytes are resuspended in fresh media, washed one time, and the hepatocytes are added to precoated dishes at a density of 9 x 10 ⁇ cells/dish and incubated at 37°C in a 5% C0 2 incubator. After 2-3 hours, media is replaced with fresh, warm media containing 0.4 ⁇ g/ml dexamethasone, 4 ⁇ g/ml insulin, and
• bile canaliculi The formation of bile canaliculi is then monitored with phase contrast microscopy with an inverted microscope.
• hepatotoxicity or choleostatic potential of a chemical compound can be assessed by adding from about 1 ⁇ l to about 5 ml of a solution containing from about 1 f to about 1 mM of the chemical compound in a solvent which is compatible with the hepatic cell culture, and incubating the dish containing the cells at about 37°C, about 95% relative humidity and an atmosphere of about 5% C0 2 for from about 5 minutes to about 96 hours. The hepatic cell culture is then examined to determine the extent of any toxic effect on the hepatic cells produced by the chemical compound.
• This method of screening chemical compounds for hepatic cell toxicity is particularly convenient since the formation of well defined canaliculi allows direct observations to be made of mo ⁇ hological changes to the canaliculi.
• a method for screening a chemical compound for hepatic extraction potential using this "sandwiched" hepatic cell culture is accomplished by adding from about 1 ⁇ l to about
• the amount of the chemical compound contained within the hepatic cells is determined by removing the drug containing buffer and washing the cells with an isotonic salt solution (ie. phosphate buffered salt solution or Hank's balanced salt solution), lysing the cells with detergent solutions (ie. sodium dodecyl sulfate) and determining the amount of drug in the lysate and the original incubation solution using analytical procedures such as scintillation counting, HPLC, absorbance or fluorescence quantitation. Alternatively in the case of fluorescent chemical compounds direct quantitation of the chemical compound can be assessed by direct determination of cell associated fluorescence.
• an isotonic salt solution ie. phosphate buffered salt solution or Hank's balanced salt solution
• detergent solutions ie. sodium dodecyl sulfate
• This method of screening chemical compounds for hepatic cell toxicity is particularly convenient when the compound is transported into the hepatic cells using active carrier systems such as Type I cationic, Type II cationic, anionic, bile salts and asialoglycoprotein receptors, or when the compound is passively absorbed into the hepatic cells since they represent the normal for extraction of chemical compounds by the liver.
• active carrier systems such as Type I cationic, Type II cationic, anionic, bile salts and asialoglycoprotein receptors
• screening of metabolite production from the interaction of the hepatic cell culture and a chemical compound is accomplished.
• this screening procedure from about 1 ⁇ l to about 5 ml of a solution containing from about 1 pM to about 50 mM of the chemical compound in a solvent which is compatible with the hepatic cell culture is added to the culture plate.
• the culture is then incubated at about 37°C, about 95% relative humidity and an atmosphere of about 5% C0 2 for from about 30 minutes to about 2 weeks.
• samples of the incubation media can be taken and once incubation is complete, loss of the parent compound and production of new metabolites can be determined using chemical analysis techniques such as HPLC, TLC, mass spectroscopy.
• cell associated parent compound and metabolites can be determined from detergent cell lysates.
• the potential of a chemical compound to alter hepatic cell metabolism using this screening process is also within the scope of Applicants' invention.
• this process from about 1 ⁇ l to about 5 ml of a solution containing 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 pathway is added and the culture is incubated at about 37 ⁇ C, about 95% relative humidity and an atmosphere of about 5% C0 2 for from about
• the metabolism of the hepatic cells is determined by identifying and quantifying the formation of products from the substrate in the presence and absence of the chemical compound.
• a fifth embodiment of this invention relies on the fact that hepatocytes grown in this collagen sandwich configuration have been shown to be competent at xenobiotic excretion into the canaliculi.
• the potential for hepatic discharge of a chemical compound into bile may be determined using this screening process. From about 1 ⁇ l to about 5 ml of a solution containing from about 1 pM to about 50 mM of a chemical compound to be screened, in a solvent which is compatible with the hepatic cell culture, is added to the hepatic cell culture. The culture is then incubated at about 37°C, about 95% relative humidity and an atmosphere of about 5% C0 2 for from about 1 minute to about 4 hours.
• This method is also applicable to the screening of many chemical compounds simultaneously. Any number of compounds which can be solubilized by the compatible 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 screened to determine which compound or groups of compounds produced the effect.
• screening is meant the determination of potential for specified activity for a series or mixture of compounds.
• chemical compound is meant any chemical agent or mixture of chemical agents with potential toxic or therapeutic properties.
• canaliculi form a multicellular network of channels throughout the cell culture which integrate a plurality of cells within the culture and are contiguous to many cells as distinct from isolated cell couplets.
• hepatotoxicity is meant an activity which results in deleterious effects on normal liver function or diminishes the viability of liver cells.
• choleostatic potential is meant the propensity to decrease normal biliary output.
• primary hepatic cell culture normal liver cells maintained in vitro.
• bile canalicular network anastomosing interconnected tubular canals between hepatocytes which maintain the capability for directional secretion of chemical compounds.
• collagenase perfusion is meant a technique for separating and isolating hepatocytes wherein in situ perfusion of the liver with a collagenase solution results in enzymatic disruption of intercellular contacts and cell/basement membrane contacts.
• hepatocyte culture media is meant a maintenance solution containing nutrients and growth factors necessary for supporting hepatocyte viability, for example, Dulbecco's modified medium with 5% fetal calf serum, nonessential amino acids, glutamine, antibiotics, antimycotics supplemented with 0.4 ⁇ g/ml dexamethasone, 4 ug/ml insulin, and 2 ng/ml epidermal growth factor.
• 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.
• 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.
• laying about 1 ml of cold neutralized collagen is meant the process of depositing a solution of collagen on the top of cells previously adhered to a culture dish or well.
• intercellular contact is meant adhesions between neighboring cells.
• canalicular network is developed. a meshwork of continuous canaliculi has formed which interconnects multiple cells.
• solvent which is compatible with the hepatic cell culture or “compatible solvent” is meant a solution which, over the normal course of experimental procedures and hepatic cell maintenance, does not elicit adverse biochemical or mo ⁇ hologic changes in cultured hepatic cells.
• ⁇ hologic or structural changes in the integrity of the canalicular network is meant alterations to the physical appearance of the canaliculi and/or redistribution of membrane components or cytoskeletal components, i.e. actin or tubulin.
• 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.
• hepatic extraction potential is meant the tendency for a chemical compound to be absorbed into the liver.
• the potential for hepatic discharge of a chemical compound into bile is meant the tendency for a chemical compound to be secreted into bile canaliculi.
• screening of metabolite production from a chemical compound is meant the determination of the potential of a chemical compound to be chemically modified by enzymatic pathways in the liver.
• the potential of a chemical compound to alter hepatic cell metabolism is meant the ability of a chemical compound to interact with a biochemical pathway in the liver producing quantitative or qualitative changes in a product.
• measuring the extent of alteration of hepatic cell metabolism is meant using analytical techniques, such as radiolabeled- or fluorescent substrates or chemical analysis, to determine quantitative or qualitative changes in product formation from biochemical 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 abso ⁇ tion, fluorescence quantitation, quantitation of radioactive isotopes.
• Phase contrast microscopy of the cells 1 day later showed that the cells had developed an elaborate network of canalicular channels which extended across a plurality of cells which appeared as refractile channels which circumscribed the cells. These channels were confirmed to be bile canaliculi by electron microscopy and fluorescence microscopy. Electron microscopy of the overlaid hepatocytes showed microvilli containing channels between neighboring cells which are bound on each side by tight junctions and have a dense actin network subjacent to the canalicular membranes. Using fluorescent-labeled antibodies, fluorescence microscopy confirmed the presence of the apical markers aminopeptidase N and Dipeptidyl peptidase in the canalicular channels similar to observations in intact liver. Similarly, fluorescent-conjugates of phalloidin demonstrated that actin in the hepatocytes is highly localized to the pericanalicular region similar to in intact liver.
• Collagen overlaid hepatocytes concentrate xenobiotics into their bile canaliculi
• Rhodamine B which is passively absorbed by the liver and secreted into the bile is taken up and secreted into the bile canaliculi by collagen sandwiched hepatocytes.
• the cultures were incubated at 37°C in Hanks balanced salt solution containing 1 ⁇ g/ml rhodamine B. After five minutes residual rhodamine B was removed and the cells were washed 3-4 times with Hank's balanced salt solution. After 5-20 minutes further incubation at 37 °C comparisons of phase contrast and fluorescence microscopy showed the rhodamine B to be highly concentrated in the canaliculi and less concentrated diffusely distributed in the cell interior.
• the active uptake of taurocholate was distinguished from the passive uptake and adso ⁇ tion based on the difference between uptake at 37 ⁇ C and 4 ⁇ C.
• Uptake experiments were also performed on unsandwiched hepatocytes at comparable cell density and showed that the collagen overlaid hepatocytes were better at taurocholate uptake (see Table 1).

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  • 1993-11-12 AU AU55527/94A patent/AU5552794A/en not_active Abandoned
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