WO1993011498A1 - Systeme de culture cellulaire automatique en compartiments multiples - Google Patents

Systeme de culture cellulaire automatique en compartiments multiples Download PDF

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Publication number
WO1993011498A1
WO1993011498A1 PCT/US1992/010130 US9210130W WO9311498A1 WO 1993011498 A1 WO1993011498 A1 WO 1993011498A1 US 9210130 W US9210130 W US 9210130W WO 9311498 A1 WO9311498 A1 WO 9311498A1
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cell
compartments
test
naphthalene
compartment
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PCT/US1992/010130
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English (en)
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Michael L. Shuler
John G. Babish
Lisa M. Sweeney
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Cornell Research Foundation, Inc.
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Priority to EP92925396A priority Critical patent/EP0620939A4/en
Priority to JP5510211A priority patent/JPH07501224A/ja
Publication of WO1993011498A1 publication Critical patent/WO1993011498A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/44—Multiple separable units; Modules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

Definitions

  • SUBSTITUTE SHEET test chemical Pharmacological changes or damage induced by a chemical at peak concentrations may be reversed as the chemical concentration falls and the cell may return to pre-exposure status before a subsequent exposure to the chemical.
  • Glutathione is a tripeptide that is metabolically coupled to reactive (harmful) chemicals by an enzyme known as glutathione transferase.
  • the cell utilizes GSH to protect itself against chemical damage.
  • GSH reserves can be depleted. When subsequent exposures occur before repletion of GSH stores, the cell will manifest a toxic response.
  • the multicompartmental cell culture analog device described herein is capable of modeling the chemical absorption, distribution, metabolism and elimination capacity of any species. The device will allow drug or toxicant and
  • This species specificity can be examined in the multicompartmental cell mammalian culture system by using combinations of mouse x rat cell types to determine whether the mouse pulmonary cells are as sensitive as rat pulmonary cells when mouse or rat hepatocytes are present.
  • the specific isozymes responsible for the generation of reactive metabolites of naphthalene can be identified by using cells containing only single isozymes of cytochrome P-450 (e.g. cytochrome P-450IA1) in the hepatocyte compartment. These cells are created by transvection of the gene coding for the cytochrome of interest.
  • a physiologically based pharmacokinetic model of naphtha gene was constructed from previous experiments describing the biotransformation of the kinetics of the chemical in several species. This model was used to predict the behavior of naphthalene in mice, rats and humans.
  • the mathematical determinants of compartmental volume and flow rates between and among compartments are used to hand set the mechanical pumping devices of the multicompartmental cell mammalian culture system. Additionally, the modeling provides the information relevant to compartmental volumes of distribution for the individual cellular compartments.
  • the present invention is directed to a device that allows the exchange of metabolites or test chemicals among compartments containing cells of differing origin at controlled flow rates, providing a method and means to study the metabolic interactions of several cell types in a multiple compartment cell culturing device.
  • the invention provides for more accurate physiologic and kinetic modeling of the distribution of chemicals among cell types.
  • One or more pumping devices can be incorporated into the system to allow for controlled flow rates among and between cellular compartments.
  • shunts and reservoirs can be added to further control the kinetics of metabolite or test chemical distribution to individual compartments.
  • a model for the DNA component uridine bromodeoxyuridine
  • neoplastic cells H4II-E
  • normal cells lymphocytes
  • Vd volume of distribution
  • Dose CO A Vd where CO is the concentration at time zero in the system and Dose is in g/kg.
  • CO the concentration at time zero in the system and Dose is in g/kg.
  • the invention described allows the determination of dose directly in mg/kg as well as an estimate of the difference between effects on H4II-E cells (target cells) and normal lymphocytes (nontarget cells) .
  • the model using uridine can be adapted for the in vitro screening of anti-neoplastic drugs for efficacy against cancer cells.
  • the system described herein permits tumor cells and normal cells to be exposed to candidate anti-cancer drugs simultaneously, which will permit the determination of a drug's ability to discriminate between the two cell types, prior to human or animal testing. Drugs which do not discriminate between cancer cells and normal cells can be eliminated immediately.
  • the culture system of the invention can determine the efficacy and toxicity simultaneously of a potential pharmaceutical drug, so that the therapeutic index of the drug will be immediately known. Accordingly, a drug that may initially appear to be promising, but whose development would eventually be terminated due to toxicity discovered following administration to humans or animals, can be eliminated immediately using the system of the invention.
  • multicompartmental cell culture system Another use for the multicompartmental cell culture system is testing pro-drugs for both efficacy and their ability to be metabolically transformed into the active moiety.
  • the system would contain hepatocytes with human biotransforming enzymes, as well as target cell types. In this manner, the multicompartmental cell culture system of the invention will mimic the actual metabolism of a drug in a human or animal system.
  • the system is also designed to permit determination of multiple dose regimens, since drugs are administered on the same mg/kg basis used for human drug dosages and clearance rates are physiologically based. Such multiple dose
  • the multicompartmental cell culture system of the invention besides use for screening and studying the metabolic profile of a drug, can also be used to identify interactions with other drugs or foods and food additives that effectively halt development of a drug or a biologically active compound in the final stages of clinical testing.
  • Another use for the multicompartmental cell culture system of the above invention is the determination of the effects of cellular metabolites of test chemicals on secondary cells, which would prove useful for the agrichemical and chemical industry.
  • the multicompartmental cell culture system of the invention would have at least two cultures in separate chambers, namely hepatocytes (for example, human, rat or mouse) and pulmonary Clara cells (for example, human, rat or mouse) .
  • Reactive metabolites produced by the hepatocytes in the first culture may circulate and affect the pulmonary cells in the second culture, provide an in vitro, interactive model which mimics the intact mammalian system.
  • PBBK physiologically-based pharmokinetic model
  • Pharmacological changes or damage induced by a chemical at peak concentrations may be reversed as the chemical concentration falls and the cell may return to preexposure status before the subsequent exposure to the chemical.
  • the frequency of exposure is as critical a determinant of cellular toxicity as the amount of the exposure to a chemical.
  • This characteristic waxing and waning pattern of chemical exposure and resultant cellular responses cannot be simulated by the application of mathematical functions to results of a cell culture experiment performed with static exposure concentrations, but this characteristic waxing and waning pattern can be produced in the multicompartmental cell culture system of the invention.
  • the monitoring and adjustments made to the model system of the invention permit a close resemblance to animal or human models without resorting to the use of living organisms.
  • a bank of four to five of the multicompartmental cell culture system units can be set up, each system representing a particular percentile of the population. Due to the genetic variation of humans with respect to xenobiotic metabolism, each of the units is set up to model the metabolism of a particular population segment. Segments can include the elderly, neonates, and pediatric population, as well as account for difference due to gender, ethnicity, or physical condition.
  • Cells from any species of organism, which can be adapted to tissue culture can be used in the system. These organisms can include human beings, laboratory animals such as rats, mice, and hamster, domestic farm laboratory animals, fish, insects, plants, unicellular organisms, and viruses. The choice of organisms is not meant to be limited by the preceding list. Particular embodiments can mix complete organisms in one or more compartments with cell, tissue, or organ cultures in one or more of the other compartments.
  • Cells from an organism with a particular condition can also be selected for inclusion within the system.
  • Age, ethnicity, gender are other aspects that can be considered in cell selection.
  • the system can also include cells that have been engineered in various ways.
  • the cells used could be chimeric species (for example, human x mouse combinations) or can be cells transvected with foreign or altered genes.
  • This invention provides methods and means that allow cells of different species to be exposed simultaneously to chemicals or metabolites under conditions of exchange that may be altered to model one or more than one species.
  • the multicompartmental cell culture system described above can be automated by attaching a microprocessor to control the circulation of media, metabolites and drugs within the system.
  • various containers, sensors, probes and measuring devices can be included as part of the overall system to control or vary the cellular environment.
  • Means are included to provide computer control and monitoring of the cell interactions in the multiple compartment cell culturing device.
  • the computer-based system provides substantial flexibility in testing, as by enabling programmably varying the number and types of cell culturing compartments, the configuration of fluid paths, fluid flow rates, parameters to be monitored, and the manner of recording and displaying test results.
  • Fig. la A simplified schematic of the multiple cell compartments of the apparatus of Fig. lc.
  • Fig. lb A simplified schematic of the multiple cell compartments of the appratus of Fig. lc, with the addition of a compartment for kidney cells and provision for both arterial and venous blood flow.
  • FIG. lc A schematic diagram of an apparatus applying the principles of this invention, showing the multiple cell compartments connected to a reservoir.
  • Fig. 2 Bar graph depicting the viability of human lymphocytes and H4II-E cells following 24 hr exposure to BrdU in the device described in Figure 1 and 30 sec exposure to UV light;
  • FIG. 10 GSH levels in mouse liver four hours after ip dosing with naphthalene. Simulation. *Data of Warren et al., 1982;
  • Fig. 11 Measured NPSH levels and simulated GSH levels in the lung four hours after ip injection of naphthalene into a rat. Simulation. Data of O'Brien et al., 1985; Fig. 12. Measured NPSH levels and simulated GSH levels in the liver four hours after ip injection of naphthalene into.a rat. Simulation. *Data of O'Brien et al., 1985;
  • Fig. 13 Sensitivity of naphthalene disposition to initial GSH level in the mouse liver. 4.1 mM 6.6 mM 8.6 mM; Figs. 14 and 15. Sensitivity of covalent binding levels (four hours after ip injection of naphthalene) to initial GSH level in mouse liver. 4.1 mM 6.6mM 8.6 mM.
  • FIG. 16 A schematic block diagram of the system of the present invention.
  • FIG. 17 A diagram illustrating a large number of cell compartments interconnected by a conduit system with reconfigurable fluid paths
  • Fig. 18 A flow chart of major components of a software program for a system of the present invention.
  • Figs. 19a, 19b, 19c, 19d and 19e Flow charts of subroutines associated with the software of the present invention.
  • lymphocytes collected from the 14 mL blood sample were seeded into 100 mL of RPMI 1640 (Gibco, Grand Island, NY) and placed in a Forma Scientific waterjacketed incubator (Fisher Scientific, Philadelphia, PA) set at 370C with 5 percent C02 overnight.
  • MCCS multicompartmental cell culture system
  • SUBSTITUTE SHEET normal and transformed cells to an antineoplastic compound simultaneously.
  • the volume of distribution of the system was 500 mL.
  • Each compartment (T-flask, Becton Dickinson, Lincoln Park, NJ) contained 75 mL of cell culture medium (RPMI 1640 as previously described) .
  • the flow rate was set at 5 mL per min and maintained by means of a peristaltic pump (Millipore, Bedford, MA) .
  • the entire system was placed inside a Forma Scientific water-jacketed incubator (Fisher Scientific, Philadelphia, PA) set at 370C with 5 percent C02.
  • the MCCS was treated with 5-bromo-2'-deoxyuridine (BrdU) by injecting 1 mL of dimethylsulfoxide containing 750 mg of BrdU into the reservoir (Fig. lc) .
  • the system was allowed to run for 24 hr. Separate T-f lasks containing untreated lymphocytes and hepatoma cells were incubated concurrently as total viability controls.
  • both human lymphocytes and rat hepatoma cells were removed from their compartments, placed on a hemocytometer and exposed to a UV light source for 30 sec. Viable cells were determined by trypan blue dye exclusion immediately following UV exposure.
  • MCCS to allow different cell types to exchange metabolites.
  • MCCS MCCS
  • methods such as the following can be employed to identify potential antineoplastic agents that are effective against transformed cells, but have low toxicity to normal.
  • MCCS Physiologically Based Pharmacokinetic Model for Naphthalene
  • the MCCS system can be modified to account for the expected metabolic functions. In order to do this, it is necessary to thoroughly understand the metabolism of certain cell types and compounds.
  • the following example using naphthalene describes how to work out the equations necessary to set up a physiologically based pharmokinetic (PBPK) model. Once these equations have been derived and testing, then the MCCS system would be set up with the required measuring devices such as probes and spectrophotometer to monitor the metabolism of the system.
  • PBPK physiologically based pharmokinetic
  • additional reservoirs would be added in order to maintain or adjust the gas mixture, acidity, and to add various compounds or metabolites as required to completely model a physiologically-based system.
  • the entire setup would be controlled by a microprocessor programmed to account for both the metabolic equations and to adjust the various parameters in order to closely maintain a metabolic model of a human or animal system.
  • Naphthalene is a commercial ly important compound produced from coal tar and petroleum.
  • the toxicology of naphthalene shows unusual species and tissue specificity.
  • the rodent LD50s are 380 mg/kp ip in male Swiss-Webster mice
  • Tissue binding is not necessarily indicative of toxicity or metabolism at that site, with similar binding levels occurring in lung and liver, but toxicity is limited to the Clara cells of the lung. For the lung, increases in binding are associated with increasing severity of tissue damage. Sensitivity of the target cell or circulation of reactive metabolites from the liver to the lung have been proposed as
  • PBPK physiologically based pharmacokinetic model
  • Our model allows the reactive metabolites, the two naphthalene oxide enantiomers, to circulate throughout the body, unlike previous PBPKs where metabolites have been restricted to the tissues in which they are generated.
  • Our model is essentially a system of parallel PBPKs which are bridged by the biotransformation of naphthalene to naphthalene oxide in the lung and liver.
  • Circulating metabolites have been offered as explanations for the toxicity of 3-methylindole (Yost et al., 1990), dichlorethylene (Okine and Gram, 1986), and bromobenzene (Casini et al., 1986).
  • SUBSTITUTE SHEET Livermore Solver for Ordinary Differential Equations, with Automatic Switching Method for Stiff and Non-stiff Problems, Linda R. Petzold and Alan C. Hindmarch, Lawrence Livermore National Laboratory, Livermore, CA
  • a model based on a 22-g mouse was developed.
  • the blood volume was calculated from the allometric relation in Kaplan et al. (1983) and divided between venous and arterial blood 2:1 (Gearhart et al., 1990).
  • the weight of the lung, liver, kidney and fat were also calculated from allometric relations (Calder, 1984) .
  • Andersen et al. (1987) calculated the volume of rapidly and slowly perfused tissues as totaling 83 percent of the body weight.
  • the "other tissues" in our model are assumed to weigh 83 percent of the body weight less the weight of the blood and kidney.
  • the cardiac output was calculated as in Kaplan et al. (1983) and the renal blood flow as in Calder (1984). The liver and fat are assumed to receive 24 and 5 percent of the cardiac output (Andersen et al., 1987). The remainder of the cardiac output flows to the other perfused tissues.
  • Anatomical parameters for a 220-g rat are the same as those used in Gearhart et al. (1990) with the volumes and flows for rapidly and slowly perfused tissues, brain, and diaphragm summed for our lumped "other tissues" compartment.
  • the parameters used in simulation are summarized in Table 1.
  • Tissue Blood Partition Coefficients From the known solubility characteristics of naphthalene in air, water (Vargaftik, 1975) and octanol-water (Hansch and Leo, 1979) systems, it is possible to preduct tissue:blood partition coefficients were calculated for lung (0.627), liver (5.41), kidney (3.87) fat (796) and muscle (4.13). The partition coefficient for muscle was used for the other tissues compartment of our model. The same partition coefficients were used for naphthalene and napthalene oxide. The partition coefficient for lung was found to be inconsistent with levels of covalent binding occurring in this
  • the lung value is an average of two literature values (O'Brien et al., 1985 and Warren et al., 1982).
  • the liver value is an average of several appearing in the literature ranging from 4.1 mM (Buonarati et al., 1989) and 8.6 mM (Richieri and Buckpitt,
  • naphthalene was modeled as the sum of two separate reactions, each producing a different enantiomer, with the Vmax's summing to the value calculated as described above.
  • the 1R, 2S- naphthalene oxide/lS, 2R- naphthalene oxide ratio (RS/SR) is 30:1 and 10:1 for naphthalene concentrations of 0.015 and 1.0 mM (Buckpitt and Frankliin, 1989, and Buckpitt et al., 1987). 5 Vmax's and Km's were fit to the reaction rate and ratio data by trial and error.
  • Vmax's for conversion of each enantiomer in the lung and liver were determined by trial and
  • Vmax's for conjugation with each enantiomer are assumed equal, and the Km for reaction with each enantiomer is assumed to be one-half of the 5 value reported for racemic naphthalene oxide.
  • the naphthalene oxide-GSH conjugation Vmax's were determined by fitting data from incubations of lung and liver microsomes and cytosolic protein with GSH or nepatocyte cultures with concentrations of naphthalene ranging from 0.005 to 1.5 mM (Buckpitt et al., 1987, Buckpitt et al. , 1984, and Richieri and Buckpitt, 1987) .
  • SUBSTIT scaled as if there were 1.8 and 16.4 mg/g of cytosolic protein in the lung and liver.
  • the amounts of total protein are 13.8 mg/mouse lung (Kanekal et al., 1990) and 112 mg/mouse liver (Cha and Bueding, 1979) .
  • these values are multiplied by the ratio of the organ weights in rat and mouse.
  • Uptake from an oral dose was modeled as first order with respect to the amount yet to be absorbed.
  • D'Souza and Andersen (1988) report that oral absorption rates of 1.0 hr are typical for halogenated hydrocarbons administered po in a corn oil vehicle, and used 0.5- 1. 0/hr (0.008-0. 017/min) for vinylidene chloride. An initial estimate in this range was used for naphthalene.
  • mice retreated with buthionine sulfoximine (BSO) hepatic and pulmonary GSH are depleted.
  • BSO buthionine sulfoximine
  • the proposed mechanism is that BSO blocks GSH synthesis Griffith and Meister, 1979)
  • the GSH synthesis rate is set equal to zero, and the initial levels of GSH set to the experimentally measured values.
  • the natural turnover of GSH by degradation is allowed to continue, in addition to depletion by conjugation with naphthalene oxide.
  • test cases were simulated and compared to the base model to test sensitivity of the model to various parameters. These cases were disposition of a lOOmg/kg ip dose of naphthalene and the levels of covalent binding in lung and. liver resulting from 200 and 400 mg/kg ip doses of naphthalene.
  • the parameters tested for sensitivity included steady state levels of GSH, cardiac output, blood flow to the fat, tissue volumes, and partition coefficients.
  • v nanomoles of product/mg microsomal protein/minute ratio: ratio of conjugates of 1R, 2S- and IS, 2R- napthalene oxide
  • Table 7 presents observed levels of mercapturates with predicted amounts of glutathione conjugates.
  • An initial GSH concentration of 6.8 mM was used for rat liver to match the values measured experimentally.
  • Six and one-half hours after administration of a 200 mg/kg oral dose the liver GSH was 17 percent of the initial concentration (Summer et al. , 1979) .
  • simulation predicts GSH levels 70, 36, and 30 percent of the initial level 6 1/2 hours after administration of naphthalene.
  • Figs. 7 and 8 Simulated and experimental values for covalent binding following ip dosing of a mouse are shown in Figs. 7 and 8. GSH values for these simulations and experiments are shown in Figs. 9 and 10.
  • Figs. 11 and 12 show experimentally determined depletion of nonprotein sulfhydryls (NPSH) and predicted GSH levels following ip administration of naphthalene to a rat.
  • NPSH nonprotein sulfhydryls
  • mice with BSO depletes GSH in the liver more than in the lung
  • GSH levels in lung and liver were 86 and 35 percent of the control values before administration of a 200 mg/kg dose of naphthalene (Buckpitt and Warren, 1983) .
  • a change in lung weight does not significantly affect covalent binding, but a shift in the RS/SR ratio for the overall disposition of a 100 mmg/kg dose of naphthalene does occur, if the lung weight is decreased to 0.125 gl the ratio of conjugate 2 to conjugates 1 and 3 is 1.03, but if it is increased to 0.5 g the ratio increases to 1.11.
  • the simulations of covalent binding over a range of doses are also quite accurate.
  • the sharp increases in covalent binding occur between 200 and 400 mg/kg for both the experiments and simulations (Figs. 7 and 8) and the simulation predicts the binding levels in the liver quite well.
  • the model underpredicts covalent binding and over predicts GSH levels in the lung at higher doses (Figs. 7 and 9) . This suggests that the model assumption of equilibrium between blood and lung for naphthalene oxide concentrations may not be entirely accurate, or that the partition coefficient is too low.
  • the simulation of pretreatments also matches the available in vivo data. Similar increases in binding after depletion of GSH were observed in the lung for simulation and experiment, in terms of percent increase, the liver binding was way off, but the nature of the increase was the same (binding levels of less than 300 pmole/mg without BSO, over 1000 pmole/mg with BSO) . Detailed whole animal and in vitro data for the rat is not as readily available as for the mouse, but the model does make a fairly accurate prediction of GSH levels in the rat after ip dosing (Figs. 11 and 12) .
  • Table 8 summarizes binding levels measured and predicted for doses at or near the LD50s. Since all the simulations have the same weaknesses, it is probably more reliable to compare the simulations to each other than each to the experimentally determined values. All the simulations for male rodents show similar levels of binding in the lung. This suggests that the differences in rodent sensitivity to naphthalene are due to the pharmacokinetics, not to differences between mouse and rat target cells (Clara cells) .
  • PBPK PBPK which accurately models the toxicology of naphthalene, providing a way to study species and route differences. This was accomplished by incorporating the circulation of reactive metabolites with existing GSH synthesis model. This model can be a paradigm for other compounds which exhibit similar mechanisms of toxicity.
  • Fig. la pictorially illustrates a housing 10 containing various cell culturing compartments including compartments 12, 14, 16 and 18 which simulate, respectively, the lungs, liver, tissue and fat of a species
  • the cell culture compartments may contain cell cultures from more than one species of organism, for example, human lymphocytes in one compartment and rat hepatoma cells in another compartment.
  • the choice of cell type, such as kidney versus pancreas can also be varied as needed.
  • the displays 20 enable internal circuitry (not shown) to output and display various test results as shown.
  • Reference numeral 22 designates pushbuttons, levers or the like through which an operator may control the system in housing 10.
  • Fig. lb is a schematic illustrating the circulatory system of the present invention that is designed to simulate the flow of arterial blood from a chamber 24, via an arterial blood supply network 26, to the aforementioned cell
  • Figs. 2 was previously described in Example 1 and Figure 3 and 5-15 were previously described in Example 2.
  • Fig. 16 is a simplified block diagram showing a system with two cell culturing compartments, and a computer controlled circulation system. This system essentially constitutes a tissue culture incubator with, compartments, optionally plug-in, for various cell cultures utilized for any particular experiment. Flow rates between and among the tissues are established under control of the microprocessor 102 and various settings for the system which are inputted through the keyboard 108 for the species or physiological conditions being modeled. System software (Fig. 18) operating in conjunction with the microprocessor 102 allows for the real time determination of physiologically based flow rates to and from the various compartments.
  • the media circulating through the various compartments consist of standard tissue culture media with horse serum and various surfactants.
  • the media allows the different cell cultures to not only survive but also communicate with each other.
  • Cell cultures may be added by means of convenient snap-in units.
  • Biological containment and sterile conditions are maintained as needed, depending on the particular test system.
  • Test materials may be administered through an injection port.
  • In-line monitors of cell viability such as albumin production or drug (metabolite) concentration, can provide real time indication of a desired physiological effect or cell specific drug metabolism profile.
  • These variables are designed to be determined by means of a flow-through multi-channel spectrophotometer.
  • a display may be provided to indicate thereon results pertaining to the various compartments.
  • the system of Fig. 16 may be used as a single stand- alone unit or as a bank of four to five systems as desired, for example, to simulate different population segments. This may be beneficial in order to test for the genetic variability of humans with respect to xenobiotic metabolism.
  • units of the present invention may be used for representing various well- defined segments of the population with known differences with respect to drug metabolism such as neonates or the elderly.
  • the system of the present invention can be used to determine the biological and toxicological effects of chemicals and pharmaceuticals in human populations.
  • the system of the present invention does not have the attendant uncertainties concerning differences in metabolic profiles between the testing species (such as rats) and humans, because biotransformation reactions are model after human metabolism.
  • the system can be adjusted as needed: to model the metabolism of other organisms, for example, the horse; to model host/parasite relation, for example, malarial parasites and cell cultures from host organisms; or to model an ecosystem, for example, screening pesticides against insects (whole or cell cultures) , plants (whole or cell cultures) and mammalian cell cultures.
  • the system of the present invention is also superior to existing in-vitro systems because of its capacity for intra-cellular communication.
  • the present invention can provide a superior method of screening early stage drugs for efficacy and toxicity simultaneously. With such information, research efforts can be focused on those pharmaceuticals most likely to be useful
  • Example 1 A physiologically-based system of the present invention has been constructed that models the 15 kinetics of the occupationally significant chemical naphthalene in a mouse or a rat, as described in Example 2.
  • a media reservoir 50 holds a test chemical for being supplied via conduit 52 to a pump 54.
  • the pump 54 conducts the test chemical to a first cell 20 culturing compartment 56 holding a first cell culture, for example, mouse pulmonary LL/2 cells obtained from the American Type Culture Collection (Rockville, Md.) .
  • the effluent carrying the test chemical interacts with the LL/2 cells and continues via the conduit 58 to the second cell compartment 25 60, holding a second cell culture, for example, H415-E, urged along by the further pump 62 and passing by needle valve 64 and flow meter 66, as shown.
  • the circulatory path to the compartment 56 is completed by the conduit 68 in which a pump 70 is connected in series.
  • the effluent in media reservoir 50 may be pressurized to a predetermined pressure exerted by gas supplied to the media reservoir 50 from a gas reservoir 78 via the flow meter 80.
  • Biological, toxological and other effects induced by 5 the test chemical on the cell cultures in the compartments 56 and 60 can be monitored or discerned by means of in-line connected spectrophotometer 82 and 84 to monitor the effluent in the conduits 68 and 58, respectively.
  • the effects of the test chemical on the culture cells in the compartment 60 can be ascertained with the spectrophotometer 82 and those in the compartment 56 by means of the spectrophotometer 84.
  • the computer system 100 may comprise a microprocessor 102 having an input/output interface 104 and internal register or cache memory 106. in a typical setup, the microprocessor 102 interfaces to: a keyboard 108 through which operator instructions and test definitions and other information may be entered to the microprocessor 102; a non-volatile storage memory 110, which may comprise a CD writable memory, a magnetic tape memory, or the like; a general purpose memory 112; and look-up tables 114.
  • the look-up tables 114 may physically comprise a portion of the general purpose memory 112 which has been set aside for the storage therein of a set of mass balanced equations applicable to various substances to be modeled in the system of the present invention, in essence, the mass balance equations represent physiologically based pharmacokinetic models for various biological/chemical substances and systems.
  • the microprocessor 102 may further be interfaced to a display 116 and to a printer/plotter recorder 118 which may provide hard copy of various test parameters and results in printed or graphical format.
  • the memories 106 and/or 112 contain a system program in the form of a plurality of program instructions and special data for
  • the temperature of the media in the reservoir 50 is regulated by the microprocessor 102 outputting computer generated commands, via its input/output line 120, to turn the heater coil 122 on and off, responsive to temperature measurements taken with the temperature probe 124 and communicated to the microprocessor 102 via line 126.
  • Fluid flow monitoring lines 128a, 128b and 128c provide inputs to the microprocessor 102 from flow meters 66, 80 and 76, respectively, enabling precise control over the fluid flow between the various compartments.
  • the fluid flow can be adjusted by program commands transmitted to the pumps 70, 54 and 62 via control lines 130a, 130b and 130c, respectively.
  • the flow rates may be set to 9.5 mL/min in conduit 58, 2.5 mL/min through the flowmeter 66, 7 mL/min through the flowmeter 76, and 2.5 mL/min in the conduit 68.
  • spectrophotometer 82 and 84 Software commands to the spectrophotometer 82 and 84 to output discrete or ranges of wavelengths and outputs of the spectrophotometer representing test results are communicated via microprocessor input/output lines 132a and 132b. It is presently contemplated that the spectrophotometer will be control led to output electromagnetic radiation in the range from about 260 to 700 nanometers, as a single, multiple, or a sequentially outputted range of wavelengths.
  • vent 134 extending from the media reservoir 50 conducts gaseous by-products to a transducer or biological sensor 136 which then provides a suitable electrical output to the microprocessor 102, enabling further analysis/control over the chemical/biological activity in the system of the present invention.
  • the computer software may initially enter decision block 140 to determine whether the system is configured to proceed in an automatic mode, applicable when the cell compartments 60 and 56 are constructed as electrically "intelligent', compartments as described further on, or in accordance with a semiautomatic mode, in the semiautomatic mode, the program proceeds via blocks 142, 144, 146 and 148 to receive operational instructions that are entered via the keyboard 108 to define the species which the system is about to model (block 142) , the cell type in each compartment (block 144) , the number and volume of cellular compartments (block 146) , and the temperature and / or wavelength or range of wave lengths at which the experiment is to be carried out.
  • step 150 one or more balance equations needed to properly model the experiment being run are fetched from the look-up table memory 114, based on the species model, cell type, number and volume of cellular compartments, etc. Thereafter the program proceeds to software block 152 to calculate a physiological based pharmacokinetic model for the chemical being tested.
  • Software instructions associated with block 154 cause the input/output interface 104 of microprocessor 102 to output physiologically based pump settings determining the fluid flow rates for various cell compartments. Thereafter, upon turning the heater 122 "ON" in step 156, the software proceeds to decision block 158 to monitor the output of the temperature probe 124 to ensure that fluid flow shall begin only after the effluent in media reservoir 50 has reached the correct temperature.
  • the program proceeds to the block 164 where general housekeeping tasks, including test data gathering and storing and, optionally, displaying intermediate as well as final data reflecting on the progress of the experiment, are attend to.
  • general housekeeping tasks including test data gathering and storing and, optionally, displaying intermediate as well as final data reflecting on the progress of the experiment.
  • the program proceeds to the decision block 176 to determine whether an end-of-test flag (to be described) has been set. if so, the program proceeds to its defined "test end" at block 177. Otherwise, the program sequentially proceed through the subroutines 166, 168, 170, 172 and 174.
  • Fig. 19a depicts the temperature monitoring subroutine which includes the software block 180 responsible for reading a temperature measurement off the probe 124, comparing the measured temperature to an internally stored desired temperature (block 182) , and determining whether the appropriate temperature has been reached (block 184) . if the temperature is above the desired value, the heater 122 is turned off in block 186. Otherwise, the heater 122 is either turned or kept turned oh in block 188. The software thereafter proceeds, via return block 190, to the main software code.
  • the program enters the block 192 wherein an internally kept display request table is consulted (block 192) to check for predetermined or operator requested quantities/ parameters/test results that are to be displayed on the display 116. Based on the information selection criteria read in block 192, the program selects (block 194) the appropriate quantities and parameters to be displayed and organizes the same into a form that is suitable for being transmitted to the display 116. After data transmission to the display 116 in block 196, the program returns to the main code via the subroutine return block 198.
  • Fig. 19c generally follows Fig. 19b, the functions in the blocks 200, 202, 204 and 206 corresponding, respectively, to the functions of blocks 192, 194, 196, 198 in Fig. 19b, enabling producing a hard copy report on the recorder 118.
  • the end-of-test subroutine includes a first block 208 for testing, if applicable, whether the program has already been running for a predetermined time period.
  • the end-of-test condition is determined on the basis of having achieved certain test results, e.g. quantity of cancerous cells, toxicity level, etc.
  • the subroutine in Fig. I9e is responsible for outputting, if desired, certain visual or audible alarms in
  • the system of the present invention is accordingly designed to allow cell forms, of humans or other species, to be exposed to a test chemical in a manner consistent with in vivo exposure.
  • the system incorporates several novel features.
  • the compartments in which the biological cells reside are constructed to be mathematically equivalent to the volume of distribution (physical size) of the organ or tissue being modeled.
  • the compartment for the hepatocyte culture would represent the volume of distribution for the liver.
  • the flow rates between the various compartments are biologically based to correctly represent the flow rates between and among the corresponding biological organs, tissue, etc. In this way, cells residing in the compartments are exposed to concentration of test materials in a manner consistent with human or animal exposure to the test material.
  • the system of Fig. 16 constitutes a microprocessor controlled instrument that performs the functions of exposing the biological cells of different tissue origin to test material at a rate modeling a selected species. Species modeling and flow control are performed by the microprocessor which serves as the overall controller so that it carefully controls flow rates, temperature, and other conditions within the system to mimic, as closely as possible, conditions within the human or other species being modeled.
  • the invention permits with appropriate inputs to model, among other things, the numbers of compartments, the volume of compartments, cell type, etc.
  • the microprocessor is able to configure the correct model for a given problem on the basis of a database of physiological organ flow rates obtained from general reference information for each species.
  • Data gathering by the computer consists of the collection of data required for continuous in-Line monitoring of test chemical eflux from each compartment.
  • the spectrophotometer preferably of the flow-through type, are disposed in-line with the outflow from each compartment, to thus detect, analyze and provide quantitative data regarding the test chemical eflux from each compartment.
  • the microprocessor may also serve to compute a physiologically-based pharmacokinetic (PBPK) model for a particular test chemical. These calculations may serve as the basis for setting the flow rates among compartments and excretions rates for the test chemical from the system. However, they may also serve as a theoretical estimate for the test chemical.
  • PBPK physiologically-based pharmacokinetic
  • FIG. 16 has been drawn to show only a pair of cell compartments 60 arid 56.
  • Fig. 17 illustrates a system comprising a complex of conduits interconnecting a larger number of cell compartments, namely cell compartments 250-268.
  • Each of the compartments 250-268 constitutes, in accordance with the embodiment of Fig. 17, an "intelligent" modularly constructed device that can be plug-in fitted into predesignated slots in the housing 10 shown in Fig. la.
  • Uniformly configured pneumatic and electrical connections may be provided for the compartments 250-268 to enable automatic engagement of electrical and pneumatic connections (not shown) provided in the housing 10.
  • the pneumatic connections 259a, 259b and 259c have been drawn only for compartment 258, for which the electrical connection 259d to the electrical bus 270 is also shown.
  • compartments can be designed as no more than a piece of tubing to provide flow through to the next compartment, for increased flexibility.
  • An electronic circuit or module (see module 250' for compartment 250, 252' for the compartment 252, etc.) has been designed to hold characterizing information, readable by the microprocessor 102 via the data bus 270.
  • the electronic module 250', etc. define for the given compartment its cell type, the species to be modeled, etc.
  • the information may be stored in an on-board, non-volatile memory, in a bank of switches, or in any other form used for data storage.
  • the modules 250', 252', etc. may optionally comprise intelligence as by including an onboard microprocessor (not shown) and circuitry for communicating with the system microprocessor 102 via serial data transmission, e.g. an RS 232 serial data bus.
  • the microprocessor 102 upon plugging-in of any of the compartments into one of the slots in the housing 10, the microprocessor 102 is enabled to automatically ascertain many aspects pertaining to the test to be run.
  • the network of fluid carrying conduits including the conduits 272, 274, 276, etc. in Fig. 17 provides direct and indirect fluid connection paths between the various compartments 258-268, each conduit branch including a respective valve (see, for example, the valves 280, 282, 284, etc.) .
  • the system of this invention can be configured to provide a very large number of different test setups.
  • the media reservoir 150 in Fig. 17 and the various control valves 280, 282, etc. can be configured so that a test chemical injected into the first compartment 250 flows through a circulation path including only the compartments 250, 252, 254, 256 and 258.
  • valves 280, 282, etc. may be so set up that the fluid circulation path is through the compartments 250, 252, 266 and 268. It is also possible to establish two parallel and independent circulation paths, which may prove to be helpful for simultaneously analyzing the efficacy of a given test chemical on different cell cultures. For example, one circulation path may involve the compartments 250, 252, 254, 256 and 258 and another compartments 268, 266, 264, 262 and 260. More conduits than shown in Fig. 17 may be provided.
  • each or several of the control valves 280, 282, etc. may be configured
  • SUBSTITUTE SHEET as pairs of anti-parallel connected one-way valves, to provide control over the direction of fluid flow.
  • a test sample withdrawing conduit network 292 might be set up for withdrawing samples of the effluent from desired compartments and supplying the same to a master spectrophotometer 294 providing a single output 296 to the microprocessor 102.
  • the microprocessor 102 may control a network of valves 296 to sequentially select for testing the effluent from the different compartments 250-268.
  • the conduit network 292 may be constructed as a waveguide network for directing electromagnetic radiation to the different locations in the fluid conduit system of Fig. 17 and for receiving a resultant spectra at the spectrophotometer 294.
  • the system may include an additional fluid reservoir 151' for holding a flushing solution for being pumped through the entire system to remove any residues from prior tests.
  • the standardized compartments will enable providing to test laboratories pre-prepared cell culture compartments with prepared and pre-identified species, cell type, etc.
  • the software of the computer system of Fig. 16 is designed to model different physiological flow rates, and to reflect different pathological conditions, including a person at rest, during exercise, or at sleep.
  • the versatility of the microprocessor 102 further extends to the display function which might provide on the display 116 continuously updated readings of spectrophotometric results in the form of a number between 1 and 2 or as an anti-log ' number ranging between 0 to 100 or as alphanumeric text providing quantitative and qualitative information.
  • the microprocessor 102 is also quite easily adaptable to include a program to provide the researcher with interactive control via keyboard 108 enabling, for example, directing the computer to specifically check on the conditions of any of the culture compartments at any given time.
  • the system of the present invention may also be deployed for the purposes of taking measurements for determining whether healthy cells are in the midst of being transformed to cancerous cells and data relevant thereto.
  • the diversity and flexibility of the present invention also permits carrying out tests using multiple culture compartments of parallel cell types to test the effects of an agent on different cell types simultaneously.
  • the compartments may be filled with cell lines of different species, e.g., mouse, rat, human, of different organs, e.g., lung, heart, and kidneys, or of different populations, e. g. , neonates, middle-aged, and elderly. Many other combinations of parallel cell cultures are feasible and useful.
  • a further option provided by the present invention is the ability to recall previously stored test results for similar experiments by recalling information from the CD/tape memory 110.
  • the memory 110 may be preprogrammed to hold historical data taken from published information, data gathered from previously run tests conducted with the system of the present invention or data derived from theoretical calculations.
  • CD/tape memory also affords the possibility of the system being used as an information researching tool to obtain, for example, research data pertaining to a particular test chemical, or to a particular
  • Fig. lc is a schematic of another embodiment applying the principles of this invention, and shows multiple cell compartments including compartments 21, 23 and 25 connected to a reservoir 27.
  • a single pump 29 maintains a 10 constant flow rate through the entire device.
  • a shunt 3, is incorporated to provide a finer control over the effective flow rate to the compartments 21, 23 and 25 and to decrease foaming. Additional pumps and shunts (not shown) could be incorporated to maintain differential flows among the 15 compartments 21, 23 and 25.
  • the circulation path includes conduit 33 feeding effluent to compartment 21, intercompartment conduits 35 and 37, conduit 39 from compartment 25 to reservoir 27, and conduit 41 through which effluent is drawn from the reservoir 20 27 by the pump 29.
  • Fig. lc may incorporate the flow meters, valves, computer system, spectrophotometers and other elements and features which have been described above in relation to Fig. 2516.
  • Buckpitt, A.R. and Franklin, R.B. (1989) Relationship of naphtha lene and 2-methyl-naphthalene metabolism to pulmonary bronchiolar epithelial cell necrosis. Pharmac.Ther. 41, 45393-410.
  • Buckpitt, A.R. and Warren, D.L. (1983) Evidence for the formation, export, and covalent binding of reactive metabolites in extrahepatic tissues in vivo. 50 J.Pharmacol.Ex .Ther. 225, 8-16.
  • Yost, G.S., Buckpitt, A.R. , Roth, R.A. , and McLemore, T.L. (1989) Mechanisms of lung injury by systemically administered chemicals.
  • the parameter is not tissue specific.
  • VLURSD maximum velocity (v) for reaction in the lung (LU) of II?, 2S-napthalene oxide (RS) to dihydrodiol (D) .
  • KLIGGS rate (K) in the liver (LI) of glutathione (G) at steady state (SS) .
  • TD Time Delay for glutathione synthetase synthesis
  • CLIN/dt QLI*(CPVN-CVLIN)/WLI- VLINRS*CLIN/ (CLIN+KMLINRS)
  • CVN ((2VLIN*QLI+CCVKN*QK+CVFN*QF+CVON*QO/QC For CVRS and CVSR, replace N in the above equation with RS or SR.
  • CPVN CAN+KIP/QLI (ip dose)
  • CPVN CAN+KUP*AOR*EXP(-KUP*T)/QLI (oral dose)
  • CARS 2 CARS ! ( l+KNOH*WBL*2 / 3 / QC)
  • CVRS 2 CVRS 1 (l+KNOH*WBL/3/QC)
  • KLUGS*CLUG+CLUSR/(CLUSR+KMNOC) )*UNITS d KLUG/dt (KLUGSS+KMGSS)/(CLIGPT+KMGSS)-KGSD*KLUG
  • VLIRSD*CLIRSD/ (CLIRSD+KMRSDI) +VLISRD*CLISRD/(CLISRD+KMSRDI) )*UNIT2*WLI d CONJUGATE2/dt VLUNOC*(CLUG/ (CLUG+KMGC) ) *(CLURS/ (CLURS+KMNOC) *UNIT5*WLU

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Abstract

Procédé et appareil permettant l'interaction métabolique de certains types de cellules dans des compartiments multiples séparés. Plusieurs chambres (21, 23, 25), contenant chacune un type de cellule différent, sont reliées pour permettre une exposition simultanée à des agents chimiques ajoutés ou un échange de métabolites dans les divers compartiments cellulaires. L'écoulement dans les compartiments est régulé par une ou plusieurs pompes (29), le ou les débit(s) peut ou peuvent être régulés pour obtenir des débits physiologiques. Par conséquent le dispositif peut servir de système de reproduction d'un modèle physiologique permettant l'interaction entre au moins deux types de cellules ou de tissus.
PCT/US1992/010130 1991-11-25 1992-11-23 Systeme de culture cellulaire automatique en compartiments multiples WO1993011498A1 (fr)

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EP92925396A EP0620939A4 (en) 1991-11-25 1992-11-23 Automated multicompartmental cell culture system.
JP5510211A JPH07501224A (ja) 1991-11-25 1992-11-23 自動化多区画細胞培養システム

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WO1996040858A1 (fr) * 1995-06-07 1996-12-19 Aastrom Biosciences, Inc. Appareil et procede destines a la conservation et a la culture de cellules biologiques
US5985653A (en) * 1995-06-07 1999-11-16 Aastrom Biosciences, Inc. Incubator apparatus for use in a system for maintaining and growing biological cells
US5994129A (en) * 1995-06-07 1999-11-30 Aastrom Biosciences, Inc. Portable cassette for use in maintaining and growing biological cells
US6096532A (en) * 1995-06-07 2000-08-01 Aastrom Biosciences, Inc. Processor apparatus for use in a system for maintaining and growing biological cells
US6228635B1 (en) 1995-06-07 2001-05-08 Aastrom Bioscience, Inc. Portable cell growth cassette for use in maintaining and growing biological cells
WO2003027223A2 (fr) * 2001-04-25 2003-04-03 Cornell Research Foundation, Inc. Dispositifs et procedes pour systeme de culture cellulaire pharmacocinetique
EP1820846A1 (fr) * 2001-04-25 2007-08-22 Cornell Research Foundation, Inc. Système de culture de cellules
WO2008095165A2 (fr) * 2007-02-01 2008-08-07 Dana-Farber Cancer Institute, Inc. Systèmes de coculture cellulaire et utilisations de ceux-ci
US7919307B2 (en) 2005-05-09 2011-04-05 Alpha Plan Gmbh Supply system for cell culture module
US8647861B2 (en) 2008-07-16 2014-02-11 Children's Medical Center Corporation Organ mimic device with microchannels and methods of use and manufacturing thereof
US9725687B2 (en) 2011-12-09 2017-08-08 President And Fellows Of Harvard College Integrated human organ-on-chip microphysiological systems
GB2553074A (en) * 2016-02-05 2018-02-28 Revivocell Ltd A cell culture device
EP3572498A4 (fr) * 2017-01-18 2020-11-18 Shinko Chemical Co., Ltd. Dispositif d'évaluation de substances chimiques et méthode d'évaluation de substances chimiques

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JP2019521715A (ja) * 2016-07-21 2019-08-08 セリアドＣｅｌｙａｄ 細胞を自動的に独立並列してバッチ処理するための方法および装置

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040858A1 (fr) * 1995-06-07 1996-12-19 Aastrom Biosciences, Inc. Appareil et procede destines a la conservation et a la culture de cellules biologiques
US5985653A (en) * 1995-06-07 1999-11-16 Aastrom Biosciences, Inc. Incubator apparatus for use in a system for maintaining and growing biological cells
US5994129A (en) * 1995-06-07 1999-11-30 Aastrom Biosciences, Inc. Portable cassette for use in maintaining and growing biological cells
US6096532A (en) * 1995-06-07 2000-08-01 Aastrom Biosciences, Inc. Processor apparatus for use in a system for maintaining and growing biological cells
US6228635B1 (en) 1995-06-07 2001-05-08 Aastrom Bioscience, Inc. Portable cell growth cassette for use in maintaining and growing biological cells
US6238908B1 (en) 1995-06-07 2001-05-29 Aastrom Biosciences, Inc. Apparatus and method for maintaining and growth biological cells
AU2002359234B2 (en) * 2001-04-25 2007-11-15 Cornell Research Foundation, Inc. Devices and methods for pharmacokinetic-based cell culture system
WO2003027223A3 (fr) * 2001-04-25 2003-12-18 Cornell Res Foundation Inc Dispositifs et procedes pour systeme de culture cellulaire pharmacocinetique
EP1820846A1 (fr) * 2001-04-25 2007-08-22 Cornell Research Foundation, Inc. Système de culture de cellules
US7288405B2 (en) 2001-04-25 2007-10-30 Cornell Research Foundation, Inc. Devices and methods for pharmacokinetic-based cell culture system
WO2003027223A2 (fr) * 2001-04-25 2003-04-03 Cornell Research Foundation, Inc. Dispositifs et procedes pour systeme de culture cellulaire pharmacocinetique
US8030061B2 (en) 2001-04-25 2011-10-04 Cornell Research Foundation, Inc. Devices and methods for pharmacokinetic-based cell culture system
US7919307B2 (en) 2005-05-09 2011-04-05 Alpha Plan Gmbh Supply system for cell culture module
WO2008095165A3 (fr) * 2007-02-01 2009-05-07 Dana Farber Cancer Inst Inc Systèmes de coculture cellulaire et utilisations de ceux-ci
WO2008095165A2 (fr) * 2007-02-01 2008-08-07 Dana-Farber Cancer Institute, Inc. Systèmes de coculture cellulaire et utilisations de ceux-ci
US8647861B2 (en) 2008-07-16 2014-02-11 Children's Medical Center Corporation Organ mimic device with microchannels and methods of use and manufacturing thereof
US9725687B2 (en) 2011-12-09 2017-08-08 President And Fellows Of Harvard College Integrated human organ-on-chip microphysiological systems
US10954482B2 (en) 2011-12-09 2021-03-23 President And Fellows Of Harvard College Integrated human organ-on-chip microphysiological systems
US11773359B2 (en) 2011-12-09 2023-10-03 President And Fellows Of Harvard College Integrated human organ-on-chip microphysiological systems
GB2553074A (en) * 2016-02-05 2018-02-28 Revivocell Ltd A cell culture device
GB2553074B (en) * 2016-02-05 2020-11-18 Revivocell Ltd A cell culture device
EP3572498A4 (fr) * 2017-01-18 2020-11-18 Shinko Chemical Co., Ltd. Dispositif d'évaluation de substances chimiques et méthode d'évaluation de substances chimiques

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JPH07501224A (ja) 1995-02-09
EP0620939A1 (fr) 1994-10-26

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