JP2008539787A - Pharmacokinetic-based culture system with biological barrier - Google Patents

Abstract

Systems and methods for microscale pharmacokinetics are disclosed. The interaction of various organs and their drug compounds can be simulated in vitro by using microscale compartments (3722, 3734, 3744) that can be interconnected by microscale channels. Cells and cellular materials associated with the organ can be cultured in such compartments to allow interaction with drug compounds in one or more fluid streams. Such fluid systems may include, for example, digestive fluid flow, blood flow, bile flow, urine flow, and brain fluid flow. Interactions between fluid systems can be simulated by a microscale permeable member (3430).

Description

RELATED APPLICATIONS AND PRIORITY REQUESTS This application is part of US patent application Ser. No. 10 / 133,977 filed Apr. 25, 2002, entitled “DEVICES AND METHODS FOR PHARMOCOKINETIC-BASED CELL CURRENT SYSTEM”. This US patent application claims the benefit of US Provisional Patent Application No. 60 / 286,493, filed April 25, 2001, and this application is also referred to as “MICROSCALE, IN VITRO, CELL COLLTURE. Benefits of US Provisional Patent Application No. 60 / 682,131, filed May 18, 2005, entitled "DEVICE WITH A MICROPOROUS SURFACE THAT MIMICS PHYSIOLOGICAL PARAMETERS" Claims.

Government Rights Statement At least a portion of the disclosure herein is at least partially supported by NASA grant number NAG8-1377. The US government may have certain rights.

BACKGROUND OF THE INVENTION The present disclosure relates to cell culture technology, and more specifically to systems and methods for promoting interactions between fluid systems at the microscale level for pharmacokinetic studies.

Description of Related Art Pharmacokinetics is a study of the fate from the time when drugs and other bioactive compounds are introduced into the body until they are excreted. For example, a series of events related to oral drugs include absorption by various mucosal surfaces, distribution to various tissues through the bloodstream, biotransformation in the liver and other tissues, effects at target sites, and urine or bile It may include excretion of drugs or metabolites into. Pharmacokinetics provides a reasonable means of approaching the metabolism of compounds within biological systems. See, for example, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, and Non-Patent Document 6 for an overview of pharmacokinetic equations and models.

  One of the fundamental challenges faced by researchers in drug, environment, nutrition, consumer product safety, and toxicology research is metabolic data and risk assessment from in vitro cell culture assays to animals. Is an extrapolation. Although some conclusions can be drawn by application of appropriate pharmacokinetic principles, there are still substantial limitations. One concern is that current screening assays utilize cells under conditions that do not replicate their function in the natural environment. Circulating flow, other tissue interactions, and other parameters associated with physiological responses are not found in standard cell culture formats. For example, in a Microscale Cell Culture Analog (CCA) system, cells grow at the bottom of the chamber. These systems have a high non-physiological fluid-to-cell ratio and have an unrealistic ratio of cell types (eg, liver to lung cell ratio). In a variation of the microscale CCA system, cells are grown on microcarrier beads. These systems closely resemble physiological conditions, but are still imperfect because they do not mimic physiological conditions that are accurate enough for predictive studies. Thus, the resulting assay data is not based on the pattern of drug or toxic exposure that can be found in animals.

  Within an organism, concentration, time, and metabolism interact to affect the strength and duration of a pharmacological or toxic response. For example, the presence of liver function in vivo strongly affects drug metabolism and bioavailability. The excretion of the active drug by the liver occurs due to biotransformation and excretion. Biotransformation reactions include reactions catalyzed by cytochrome P450 enzymes that transform many chemically diverse drugs. In the second stage of biotransformation, hydrophilic groups such as glutathione, glucuronic acid, or sulfate can be added to increase water solubility and excretion rate by the kidney.

  Biotransformation can be beneficial but can have undesirable consequences. Toxicity stems from complex interactions between compounds and organisms. During the biotransformation process, the resulting metabolites can be more toxic than the parent compound. The single cell assays used by many people for toxicology screening lack these intercellular and interstitial effects.

  As a result, accurate prediction of human responsiveness to potential drugs is difficult, often unreliable and always expensive. Traditional methods for predicting human response utilize surrogates—usually static homogeneous in vitro cell culture assays or in vivo animal experiments. In vitro cell culture assays are of limited value because they do not accurately mimic the complex environment in which drug candidates are exposed to the human body and cannot accurately predict human risk. Similarly, in vivo animal studies can reveal these complex cell-to-tissue effects that are not observed from in vitro cell-based assays, but in vivo animal experiments are extremely expensive and labor intensive It takes time, and often the results are questionable if they correlate with human risk.

  U.S. Patent No. 6,053,028 published by Shuler et al. Describes a multi-compartment cell culture system. This culture system uses large components such as culture chambers, sensors, and pumps, which require the use of large amounts of media, cells, and test compounds. This system is very expensive to operate and requires a lot of space to operate. Because this system is such a large scale, the physiological parameters are significantly different from those seen in an in vivo situation. It is impossible to accurately generate such a large-scale physiologically realistic state.

The development of microscale screening assays and devices that can provide better, faster, and more efficient predictions of in vivo toxicity and clinical drug performance has been of great interest in many areas. It is something we are working on with the invention. Such a microscale device will accurately produce physiologically realistic parameters and will more closely mimic the desired in vivo system being tested.
US Pat. No. 5,612,188 Paulin and Theil J Pharm Sci. (2000) 89 (1): 16-35 Slob et al. Crit Rev Toxicol. (1997) 27 (3): 261-72 Haddad et al. Toxicol Lett. (1996) 85 (2): 113-26. Hoang Toxicol Lett. (1995) 79 (l-3): 99-106 Knak et al. Toxicol Lett. (1995) 79 (l-3): 87-98 Ball and Schwartz Comput Biol Med. (1994) 24 (4): 269-76.

DISCLOSURE OF THE INVENTION Devices are provided for devices in in vitro cell culture and for microscale cell culture analog (CCA) devices. The device of the present invention allows cells to be maintained in vitro under conditions having pharmacokinetic parameter values similar to those seen in vivo. Desired pharmacokinetic parameters include cell-cell interaction, fluid residence time, fluid-to-cell ratio, relative organ size, cellular metabolism, shear stress, and the like. Providing a pharmacokinetic-based culture system that mimics the natural state of cells enhances the predictive value and in vivo validity of screening and toxicity assays.

  The microscale culture device comprises a fluid network of channels isolated in separate but interconnected chambers. The particular chamber surface shape is designed to provide cell interactions, fluid flow rates, and fluid retention parameters that interact with those found in vivo in the corresponding cells, tissues, or organs. Fluid engineering is designed to accurately represent the essential elements of the circulatory or lymphatic system. In one embodiment, these components are incorporated into a chip format. The design and validation of these surface shapes is based on a Physiological-Based PharmacoKinetic (PBPK) model; a mathematical model that represents the organism as interconnected compartments representing different tissues.

  The device can be seeded with the appropriate cells for each culture chamber. For example, a chamber designed to provide hepatic pharmacokinetic parameters, where hepatocytes are seeded, and adipocytes and fluid seeded in a chamber designed to provide adipocyte pharmacokinetics You may communicate. As a result, pharmacokinetic-based cell culture systems accurately represent, for example, the tissue size ratio, tissue to blood volume ratio, drug residence time of the model animal.

  In one embodiment, the system includes a first microscale culture device and a control instrument. The first microscale culture device has a plurality of microscale chambers with surface shapes that simulate a plurality of in vivo interactions with the medium, each chamber having an inlet and an outlet for the flow of the medium, and Microfluidic channels interconnecting the chambers. The control device is coupled to the first microscale culture device and includes a computer for obtaining data from the first microscale culture device and controlling its pharmacokinetic parameters.

  In another embodiment, the computer includes an input / output interface that includes an interface to a microprocessor, general purpose memory, non-volatile storage elements, and a microscale culture device having one or more sensors, and computer software. The computer software analyzes data from sensors to measure physiological events in multiple chambers of the microscale culture device, adjusts the fluid flow rate of the media in the chambers of the microscale culture device, It can be implemented in a microprocessor to detect a biological or toxic response in the chamber of the scale culture device and to change one or more pharmacokinetic parameters of the microscale culture device upon detection.

  As used herein, the singular includes the plural reference unless the context clearly dictates otherwise. For example, “compound” refers to one or more such compounds, while “cell” refers to other family members and their equivalents in addition to a particular cell, as is known to those of skill in the art. Including.

  One embodiment of the present disclosure maintains a biological material under conditions that provide at least one in vitro pharmacokinetic parameter value that is comparable to the at least one pharmacokinetic parameter value found in vivo. To an apparatus comprising at least one mechanism dimensioned in such a manner. The device further includes a permeable material.

  In one embodiment, the mechanism is a microscale mechanism. In one embodiment, the permeable material is a membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL, cell, cellular material , Tissue, and at least one of the group consisting of tissue pieces.

  In one embodiment, the permeable material further comprises an organic or inorganic material in, on or near the microporous surface.

  In one embodiment, the permeable material is a biological barrier, a passage of a substance through or through the biological barrier, or a through or through the biological barrier. It is configured to simulate at least one of the absorption of the substance. In one embodiment, the biological barrier is a gastrointestinal barrier, blood brain barrier, lung barrier, placental barrier, epidermal barrier, ocular barrier, olfactory barrier, gastroesophageal barrier, mucosa, blood urine barrier, air tissue barrier, blood It is selected from at least one of the group consisting of bile barrier, oral barrier, anorectal barrier, vaginal barrier, and urethral barrier.

  In one embodiment, the at least one pharmacokinetic parameter is: tissue size, tissue size ratio, tissue to blood volume ratio, drug residence time, cell interaction, liquid residence time, liquid to cell ratio, cellular metabolism, shear It is selected from at least one of the group consisting of stress, flow rate, surface shape, circulation transit time, fluid distribution, interaction between tissues and / or organs, and molecular transport by cells.

  In one embodiment, the device measures the absorption, metabolism, or distribution of a substance through or through the permeable material. In one embodiment, the mechanism is a group consisting of at least part of the central nervous system, circulatory system, digestive system, bile duct system, lung system, urinary system, visual system, olfactory system, epidermal system, and lymphatic system. It is configured to represent at least one. In one embodiment, the permeable material is located in or out of the device.

  In one embodiment, the device further comprises at least one microfluidic channel connected to the permeable material.

  In one embodiment, the flow of fluid through or near the permeable material provides at least one pharmacokinetic parameter. In one embodiment, the nature of the fluid flow through the device is based on a mathematical model. In one embodiment, the mathematical model is a Physiologically-Based PharmacoKinetic (“PBPK”) model.

  In one embodiment, the feature or the permeable material is incorporated into a chip format.

  In one embodiment, the permeable material comprises a layer of gastrointestinal cells cultured on the microporous material. In one embodiment, at least a portion of the layer of the gastrointestinal cell is positioned within the device such that fluid can flow along either side of the layer without passing through the layer. In one embodiment, at least a first microscale feature located on the first side of the layer of gastrointestinal cells represents the gastrointestinal tract and at least a second microscale feature located on the second side of the monolayer is Represents the circulatory system. In one embodiment, the device further includes a third microscale mechanism configured to contain the same or different types of biological material.

  In one embodiment, the permeable material comprises a microporous material that is at least partially coated with an organic material.

  In one embodiment, the device further comprises cells located in, on or near both sides of the permeable material. In one embodiment, the device provides absorption properties, metabolic enzyme activity, and / or expression levels. In one embodiment, the cells on either side of the permeable material are of the same type or different types.

  In one embodiment, the device further comprises hepatocytes in, on or near the microporous surface of the permeable material. In one embodiment, at least a portion of the microporous surface comprises a protein that polarizes the hepatocytes.

  In one embodiment, the permeable material comprises a cell line capable of forming a fusible monolayer.

  In one embodiment, the device further comprises a binder that binds hepatocytes to the permeable material. In one embodiment, the binding agent polarizes the hepatocytes. In one embodiment, the binding agent is a protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β-catenin , A cell adhesion molecule, and at least one selected from the group consisting of uvomorulin.

  In one embodiment, the device further comprises a second type of biological material in, on or near the permeable material.

  In one embodiment, the device further comprises fibroblasts in, on or near the permeable material.

  In one embodiment, the device further includes a blood substitute flow proximate to the first side of the permeable material. In one embodiment, the device further includes a bile surrogate flow proximate to the second side of the permeable material.

  One embodiment of the present disclosure maintains a biological material under conditions that provide at least one in vitro pharmacokinetic parameter value that is comparable to the at least one pharmacokinetic parameter value found in vivo. It relates to a method comprising steps. The method further includes passing at least a portion of the permeable material through the substance.

  In one embodiment, the method further comprises maintaining the biological material in or near the microscale mechanism.

  In one embodiment, the permeable material is a membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL, cell, cellular material , Tissue, and at least one of the group consisting of tissue pieces.

  In one embodiment, the permeable material further comprises an organic or inorganic material in, on or near the microporous surface.

  In one embodiment, the permeable material is a biological barrier, a passage of a substance through or through the biological barrier, or a through or through the biological barrier. It is configured to simulate at least one of the absorption of the substance. In one embodiment, the biological barrier is a gastrointestinal barrier, blood brain barrier, blood bile barrier, lung barrier, placental barrier, epidermal barrier, eye barrier, olfactory barrier, gastroesophageal barrier, mucosa, blood urine barrier, air It is selected from at least one of the group consisting of tissue barrier, oral barrier, anorectal barrier, vaginal barrier, and urethral barrier.

  In one embodiment, the at least one pharmacokinetic parameter is: tissue size, tissue size ratio, tissue to blood volume ratio, drug residence time, cell interaction, liquid residence time, liquid to cell ratio, cellular metabolism, shear It is selected from at least one of the group consisting of stress, flow rate, surface shape, circulation transit time, fluid distribution, interaction between tissues and / or organs, and molecular transport by cells.

  In one embodiment, the method further comprises measuring the absorption, metabolism, or distribution of the substance in or through the permeable material. In one embodiment, the mechanism is a group consisting of at least part of the central nervous system, circulatory system, digestive system, bile duct system, lung system, urinary system, visual system, olfactory system, epidermal system, and lymphatic system. It is configured to represent at least one.

  In one embodiment, the method further comprises positioning the permeable material in or out of a microscale device.

  In one embodiment, the method further comprises flowing a fluid through at least one microfluidic channel connected to the permeable material.

  In one embodiment, the flow of fluid through or near the permeable material provides at least one pharmacokinetic parameter. In one embodiment, the nature of the fluid flow through the device is based on a mathematical model. In one embodiment, the mathematical model is a Physiologically-Based PharmacoKinetic (“PBPK”) model.

  In one embodiment, the method further includes incorporating the microscale feature or the permeable material into a chip format.

  In one embodiment, the permeable material comprises a layer of gastrointestinal cells cultured on the microporous material. In one embodiment, the method further comprises positioning at least a portion of the layer of the gastrointestinal cell such that fluid can flow along either side of the layer without passing through the layer. In one embodiment, at least a first microscale feature located on the first side of the layer of gastrointestinal cells represents the gastrointestinal tract and at least a second microscale feature located on the second side of the monolayer is Represents the circulatory system. In one embodiment, the third microscale mechanism is configured to contain the same or different types of biological material.

  In one embodiment, the permeable material comprises a microporous material that is at least partially coated with an organic material.

  In one embodiment, the method further comprises positioning the cell in, on or near both sides of the permeable material. In one embodiment, the method further comprises providing absorption properties, metabolic enzyme activity, and / or expression levels. In one embodiment, the cells on either side of the permeable material are of the same type or different types.

  In one embodiment, the method further comprises locating hepatocytes in, on or near the microporous surface of the permeable material. In one embodiment, at least a portion of the microporous surface comprises a protein that polarizes the hepatocytes.

  In one embodiment, the permeable material comprises a cell line that can form and polarize a confluent monolayer.

  In one embodiment, the method further comprises binding hepatocytes to the permeable material. In one embodiment, the method further comprises polarizing the hepatocyte. In one embodiment, the binding step comprises protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β A binding agent that is at least one selected from the group consisting of catenin, a cell adhesion molecule, and uvomorulin.

  In one embodiment, the method further comprises positioning a second type of biological material in, on or near the permeable material.

  In one embodiment, the method further comprises positioning fibroblasts in, on or near the permeable material.

  In one embodiment, the method further comprises flowing a blood substitute proximate to the first side of the permeable material. In one embodiment, the method further comprises flowing a bile substitute proximate to the second side of the permeable material.

  One embodiment of the present disclosure relates to a method of forming a device. The method is configured to maintain the biological material under conditions that provide at least one in vitro pharmacokinetic parameter value that is comparable to the at least one pharmacokinetic parameter value found in vivo. Forming a mechanism. The method further includes adding, forming, or providing a permeable material. The permeable material is configured such that a substance passes through at least a portion of the permeable material.

  One embodiment of the present disclosure maintains a biological material under conditions that provide at least one in vitro pharmacokinetic parameter value that is comparable to the at least one pharmacokinetic parameter value found in vivo. And a device for providing a permeable barrier.

  One embodiment of the present disclosure relates to a device comprising a microscale permeable material and at least one binder configured to polarize the substance, wherein the substance exhibits at least one property of liver function. .

  In one embodiment, the substance is one or more hepatocytes. In one embodiment, the substance is a genetically modified biological material. In one embodiment, the binding agent binds and polarizes hepatocytes to the microscale permeable material.

  In one embodiment, the device further includes a second material type. In one embodiment, the device further comprises one or more fibroblasts located near at least one surface of the microscale permeable material.

  In one embodiment, the microscale permeable material is organic material, inorganic material, membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL (Matrigel), cells, cellular material, tissue, and at least one selected from the group consisting of tissue pieces. In one embodiment, the microscale permeable material is in, on or near a microporous surface. In one embodiment, the microscale permeable material is a biological barrier, a passage of a substance in or through the biological barrier, or a biological barrier, through, or It is configured to simulate at least one of the absorption of the substance thereby.

  In one embodiment, the device processes the material in, through or through the microscale permeable material. In one embodiment, the treating step further comprises at least one of the group consisting of molecular absorption, extraction, excretion, metabolism, and distribution.

  In one embodiment, the microscale permeable material is located in or out of the device.

  In one embodiment, the device further comprises at least one microfluidic channel connected to the microscale permeable material.

  In one embodiment, the nature of the fluid flow through the device is based on a mathematical model. In one embodiment, the mathematical model is a Physiologically-Based PharmacoKinetic (“PBPK”) model.

  In one embodiment, the feature or the microscale permeable material is incorporated into a chip format. In one embodiment, the device provides absorption properties, metabolic enzyme activity, and / or expression levels.

  In one embodiment, the device further comprises a biological material located in, on or near both sides of the microscale permeable material. In one embodiment, the biological material on either side of the microscale permeable material is of the same type or a different type.

  In one embodiment, the microscale permeable material comprises a cell line capable of forming a confluent monolayer. In one embodiment, the binding agent is a protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β-catenin , A cell adhesion molecule, and at least one selected from the group consisting of uvomorulin.

  In one embodiment, the device further comprises a blood substitute flow proximate to the first side of the microscale permeable material. In one embodiment, the device further includes a flow of bile substitutes proximate to the second side of the microscale permeable material.

  One embodiment of the present disclosure is directed to a method that includes binding a substance that exhibits at least one property of liver function to a microscale permeable material in a manner that polarizes the substance.

  In one embodiment, the substance is one or more hepatocytes. In one embodiment, the substance is a genetically modified biological material.

  In one embodiment, the method further comprises providing a second material type. In one embodiment, the method further comprises positioning one or more fibroblasts in the vicinity of at least one surface of the microscale permeable material.

  In one embodiment, the microscale permeable material is organic material, inorganic material, membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL (Matrigel), cells, cellular material, tissue, and at least one selected from the group consisting of tissue pieces.

  In one embodiment, the method further comprises positioning the microscale permeable material in, on or near a microporous surface.

  In one embodiment, the microscale permeable material is a biological barrier, a passage of a substance in or through the biological barrier, or a biological barrier, through, or Simulate at least one of the absorption of the substance by it.

  In one embodiment, the method further comprises treating the substance in, through or by the microscale permeable material. In one embodiment, the treating step further comprises at least one of the group consisting of molecular absorption, extraction, excretion, metabolism, and distribution.

  In one embodiment, the method further comprises positioning the microscale permeable material in or out of the device.

  In one embodiment, the method further comprises providing at least one microfluidic channel connected to the microscale permeable material.

  In one embodiment, the fluid flow property associated with the at least one property of liver function is based on a mathematical model. In one embodiment, the mathematical model is a Physiologically-Based PharmacoKinetic (“PBPK”) model.

  In one embodiment, the method further includes incorporating the microscale permeable material into a chip format.

  In one embodiment, the method further comprises providing absorption properties, metabolic enzyme activity, and / or expression levels.

  In one embodiment, the method further comprises positioning a biological material in, on or near both sides of the microscale permeable material. In one embodiment, the biological material on either side of the microscale permeable material is of the same type or a different type.

  In one embodiment, the microscale permeable material comprises a cell line capable of forming a confluent monolayer. In one embodiment, the binding step comprises protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β Providing a binding agent selected from at least one of the group consisting of catenin, a cell adhesion molecule, and uvomorulin.

  In one embodiment, the method further includes providing a blood substitute flow proximate to the first side of the microscale permeable material. In one embodiment, the method further comprises providing a bile surrogate flow proximate to the second side of the microscale permeable material.

  One embodiment of the present disclosure relates to a method of forming a device. The method includes forming a microscale permeable material configured to bind and polarize a substance that exhibits at least one property of liver function.

  One embodiment of the present disclosure relates to a microscale device having means for binding a substance that exhibits at least one property of liver function to a microscale permeable material in a manner that polarizes the substance.

  One embodiment of the present disclosure includes a microscale permeable material and at least one substance configured to demonstrate at least one property of liver function, wherein the molecules processed by the substance are the microscale The device is directed to pass through at least a portion of the permeable material.

  One embodiment of the present disclosure includes directing molecules processed by the substance to pass through at least a portion of the microscale permeable material so that the substance exhibits at least one property of liver function. Consists of a method.

  One embodiment of the present disclosure relates to a method of forming a device. The method includes forming the microscale permeable material configured to direct molecules treated by the substance to pass through at least a portion of the microscale permeable material, the substance comprising liver function Is configured to demonstrate at least one property of

  One embodiment of the present disclosure has means for directing molecules treated by the substance to pass through at least a portion of the microscale permeable material, such that the substance exhibits at least one property of liver function. It is related with the device comprised.

  These and other aspects, advantages, and novel features of the present invention will become apparent upon reading the following detailed description and upon reference to the accompanying drawings in which: In the drawings, similar elements may have similar reference numerals.

Detailed Description of Embodiments The inventors have developed a Microscale Cell Culture Analog (CCA) system. Such a microscale CCA system has many advantages over previous microscale systems. The microscale system uses fewer reagents, fewer cells (which allows the use of true primary cells rather than cultured cells) and is more physiologically realistic (eg, residence time , Organ ratio, shear stress), lower device cost and smaller size (available for multiple tests and statistical analysis). Furthermore, multiple biosensors can be incorporated on the same chip.

  In its simplest terms, the chip of the present invention provides an accurate in vitro surrogate for whole animals or humans. To achieve this, the initial design was generated using a Physiological-Based PharmacoKinetic (PBPK) model—a mathematical model that represents the organism as an interconnected compartment specific to a particular organ. It has been done. From the PBPK model and published empirical data, a long-term extensive development program allows known tissue size ratios, tissue-to-blood volume ratios, drug residence times, and other important animal or human physiological parameters Resulting in a micro-scale device that imitates exactly. In short, the chip technology of the present invention is a whole animal or human microscale model (˜1 / 100,000 for humans).

  In operation, the device replicates the recirculating multi-organ system by sequestering living cells into separate interconnected “organ” compartments (see, eg, FIG. 15). Fluid engineering is designed to accurately mimic the interactions between the intrinsic elements of the circulatory system and the organ system. Each organ compartment contains a specific cell type that has been carefully selected or modified to mimic the intrinsic function of the entire corresponding organ (eg, xenobiotic metabolism by the liver). The cell type may be adherent or non-adherent and may be derived from standard cell culture lines or primary tissues. Human cells are used for human surrogate or other species of cells as appropriate.

  The organ compartments are connected by a recirculating medium that acts as a “blood substitute”. The test agent in the medium is distributed and interacts with cells in the organ compartment, just as it is in the human body or whole animal. The effects of these compounds and / or their metabolites on various cell types measure or monitor key physiological events such as cell death, cell proliferation, differentiation, immune response, perturbations in metabolic or signal transduction pathways To be detected. Pharmacokinetic data can also be measured by collecting and analyzing aliquots of media for drug metabolites.

  The microscale chip device of the present invention also presents both the cost and throughput advantages of conventional cell culture assays, as well as the high information content of whole animal models. However, unlike whole animal testing, the chips are cheap and mostly disposable. Low liquid volume (˜5 μl) devices provide the high sensitivity and throughput characteristics of microfluidic devices. Furthermore, the device readout is flexible and assay independent—almost all cell types and assays can be used without modification. Real time monitoring is feasible because multiple biological assays based on optical interrogation and readout (eg, fluorescence, luminescence) are available. Alternatively, standard pathology, biochemistry, genomic, or proteomic assays can be directly utilized as a system that can be designed to be fully compatible with conventional coverslips (22 mm x 22 mm) or 96-well formats. . Furthermore, genetically modified cells can be used for special end-user applications. Additional compartments and modules can also be included to analyze absorption of the gastrointestinal tract or blood brain barrier using a “3D” chip.

  Unlike traditional in vitro assays, the chip of the present invention more closely mimics the complex multi-tissue (liver, lung, fat, circulatory system, etc.) ecology of whole organisms. Because drug candidates are exposed to more realistic animal or human physiological environments, information content (eg, absorption, distribution, bioaccumulation, metabolism, excretion, efficacy, and more) than general in vitro assays Toxic). These benefits directly affect the safety and efficacy prediction of drug lead compounds, especially their prioritization prior to entering expensive and time-consuming nonclinical or clinical trials. This prioritization increases drug development throughput, reduces the number of animals required for toxicological screening, reduces the cost of nonclinical research, and lacks potential toxicity or efficacy before entering these trials Increase the efficiency of clinical trials by allowing rapid and direct assessment of

  These demonstrate some of the advantages of the chip technology of the present invention. In summary, data acquisition is rapid when compared to traditional in vitro cell culture assays, animal experiments, or clinical trials. The data is also robust and provides highly predictive content that is not available from conventional in vitro assays. The chip platform is designed to be perfectly compatible with existing assays-those performed in standard coverslips or 96-well formats. The device itself can be configured for any animal species or combination of multiple organ compartments. Individual chips are priced cost-effectively as consumables. Moreover, the low volume device further reduces reagent costs for screening potential compounds.

  Unlike currently available technologies, the chip system significantly increases not only the clinical phase, but also the success rate in reducing the number of compounds required to undergo preclinical testing. As a result, pharmaceutical companies have determined (1) which drug candidates are likely to be toxic to humans early in the development process; (2) the species of animal that best predicts human response. Make a good choice; (3) Determine which drug candidates have the potential to be effective. Thus, the chip of the present invention greatly increases the success rate and reduces the development time of marketable drugs.

Pharmacokinetic-based microscale culture devices Devices in in vitro cell culture and methods for CCA devices are provided. The subject methods and devices provide a means by which cells are maintained in vitro in a physiologically representative environment, thereby improving the predictive value and in vivo validity of screening and toxicity assays. The microscale pharmacokinetic culture device of the present invention is prepared by seeding appropriate cells in each culture chamber, and then using the culture system for compound screening, toxicity assays, models for the development of desired cells, models for infection kinetics, etc. Can be used for An input variable is added to the established culture system, which may be, for example, a compound, sample, gene sequence, pathogen, cell (such as a stem cell or progenitor cell). To measure the response of cells to input variables, including the pH of the medium, the concentration of O ₂ and CO _{2 in} the medium, the expression of proteins and other cell markers, the survival of cells, or the release of cell products into the medium In addition, various cell outputs can be evaluated.

  Culture substrate or device designs and surface shapes are provided for the unique growth conditions of the present invention. Each device comprises one or more chambers that are interconnected by fluid channels. Each chamber may have a different surface shape configuration than the other chambers present on the device. For example, one embodiment of the device consists of chambers representing lungs, liver, and other tissues (FIG. 18A). The lung chamber of this embodiment contains a high ridge of 5 μm to achieve realistic cell-to-liquid volume ratio and liquid residence time (FIG. 18B). The liver chamber of this embodiment contains 20 μm high pillars to achieve realistic cell-to-liquid volume ratios and liquid residence times (FIG. 18C). The device also includes inlet and outlet ports that allow the medium to circulate.

  In one embodiment, the culture device is in a chip format, i.e., chambers and fluid channels are created or molded from the created master, so that the device is one as a single unit or on a separation unit. Formed as a modular system with more than one chamber. In general, the chip format is provided on a small scale, and usually a piece does not exceed about 10 cm and even a piece does not exceed about 5 cm. A piece may be no more than about 2 cm. In another example, the chips may be stored in a 96 well format, where individual chips are less than 0.9 cm x 0.9 cm. The chambers and fluid channels are correspondingly micro-scale sizes.

  In another embodiment, the culture device is in the form of an integrated device consisting of a tabletop device that houses a plurality of microscale chips made as disposable plastic polymer-based components. The instrument consists of a base with a recess to accommodate individual cell chips, or a single “chip” in standard 96 well format (ie, 96 individual chips are in an 8 × 12 format). It may be. The top of the instrument, when closed, seals the chip and provides a fluid interconnect. The instrument contains a low volume pump to recirculate fluid to the chip and a small 3 way valve with an injection loop to provide for the introduction of test compounds or directly from a 96 or 384 well plate Pull out the compound. This instrument can be used to assess efficacy, toxicity, and / or metabolite production for multiple compounds. The instrument can also incorporate on-chip fluorescence detection for real-time physiological function monitoring utilizing well-characterized biomarkers.

  The device may include a mechanism for obtaining signals from cells and media. Signals from different chambers and channels can be monitored in real time. For example, the biosensor may be incorporated into the device or external to the device, allowing real-time readout of the physiological state of cells that are in the system.

  The present invention includes, for example, a series of pharmaceutical compositions, vaccine formulations, cytotoxic chemicals, mutagens, cytokines, chemokines, growth factors, hormones, inhibitory compounds, chemotherapeutic agents, and other compounds or host of factors, etc. It provides an ideal system for high-throughput screening to identify positive or negative responses to other substances. The substance to be tested can be naturally occurring or synthetic, organic or inorganic.

  For example, the activity of a cytotoxic compound can be measured by its ability to injure or kill cells in culture. This can be easily assessed by vital staining techniques. The effects of growth / restriction factors can be assessed by analyzing the intracellular content of the matrix, for example by total cell number and differential cell number. This can be accomplished using standard cytological and / or histological techniques, including the use of immunocytochemistry with antibodies that define type-specific cellular antigens. The effects of various drugs on normal cells cultured in the device can be evaluated. For example, drugs that increase erythropoiesis can be tested in bone marrow culture. For example, drugs that affect cholesterol metabolism by reducing cholesterol production can be tested in the liver system. Tumor cell cultures can be used, for example, as a model system to test the efficacy of anti-tumor agents.

  The device of the present invention can be used as a model system for the study of physiological or pathological conditions. For example, in certain embodiments of the invention, the device may be used as a model for the blood brain barrier, and such a model system may be used to study the penetration of substances through the blood brain barrier. In further embodiments, and without limitation, a device containing a mucosal epithelium may be used as a model system for studying herpesvirus or papillomavirus infection, such model system comprising an antiviral drug Can be used to test efficacy.

  The devices of the present invention can also be used to assist in the diagnosis and treatment of malignant tumors and diseases. For example, a biopsy of any tissue (eg, bone marrow, skin, liver) can be taken from a patient suspected of having a malignant tumor. The patient's culture is used in vitro to identify the most effective ones, i.e. those that kill malignant or diseased cells and do not harm normal cells. Compounds can be screened. Subsequently, these agents can be used to treat patients.

  In yet another embodiment of the invention, the device can be used in vitro to produce biological products in high yield. For example, cells that naturally produce large quantities of specific biological products (eg, growth factors, restriction factors, peptide hormones, antibodies) or host cells that have been genetically modified to produce foreign gene products Can be expanded clonally using a vitro device. When the transformed cells excrete the gene product into the culture medium, the product can be used using standard separation techniques (eg, a few but, for example, HPLC, column chromatography, electrophoresis techniques) or It can be easily isolated from the conditioned medium. A “bioreactor” can be devised that utilizes a continuous flow method for feeding cultures in vitro. In principle, as fresh media passes through the culture of the device, the gene product will be washed away from the culture along with the cells released from the culture. The gene product can be isolated from the effluent of spent or conditioned media (eg, by HPLC column chromatography, electrophoresis).

  The present invention also provides a system for screening or measuring the effects of various environmental conditions or compounds in biological systems. For example, it is possible to mimic or vary the state of air or water in the device. It is possible to test the influence of different known or suspected toxic substances. The present invention further provides a system for screening consumer products such as cosmetics, cleaning agents, or liquids. A system for measuring the safety and / or efficacy of a dietary supplement, dietary supplement, or food additive is also provided. The present invention can also be used as a miniature bioreactor or cell production platform to produce large quantities of cell products.

  A typical efficacy or toxicity experiment using the chip format microscale culture device of the present invention is completed within 24 to 48 hours, depending on the experimental design. However, extended experiments may be performed to test the effects of chronic exposure (eg, genotoxicity, carcinogenicity, or potential disease).

  The present invention provides novel devices, systems, and methods as described herein. Unless defined otherwise, generally, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For clarity, definitions of some terms used herein to describe the present invention are set forth below. Unless explicitly indicated otherwise, these definitions apply to terms as they are used throughout this specification.

Definition of Terms Pharmacokinetic-based culture system: An in vitro cell culture system in which cells are maintained under conditions that provide pharmacokinetic parameter values that mimic those found in vivo. A pharmacokinetic culture device comprises a fluid network of channels isolated in separate but interconnected chambers, with specific chamber surface shapes being found in vivo in the corresponding cell, tissue, or organ system Designed to provide interacting cell interactions, liquid flow rates, and liquid retention parameters. The device is seeded with cells suitable for the conditions being modeled, such as liver cells in a liver-based culture chamber, lung cells in a lung-based culture chamber, etc. to provide a culture system. .

  The culture system of the present invention provides at least one pharmacokinetic parameter value, preferably at least two parameter values, comparable to those obtained in vivo for a desired cell, tissue or organ system, and more than two Can provide comparable parameter values. Desired pharmacokinetic parameters include, for example, cell-cell interaction, liquid residence time, liquid-to-cell ratio, metabolism by cells, or shear stress.

  A comparable value means that the actual value does not deviate from the theoretical value by more than 25%. For example, the calculated or theoretical value for fluid residence time in the rat lung compartment is 2 seconds, and the actual value measured in the lung cell culture chamber of the rat CCA device is 2.5 ± 0.7 seconds. Met.

  Pharmacokinetic parameter values are obtained by using the equations of the PBPK model. Such equations have been described in the art, for example, see Paulin and Theil (2000) J Pharm Sci., Incorporated by reference. 89 (l): 16-35; Slob et al. (1997) Crit Rev Toxicol. 27 (3): 261-72; Haddad et al. (1996) Toxicol Lett. 85 (2): 113-26; Hoang (1995) Toxicol Lett. 79 (l-3): 99-106; Knak et al. (1995) Toxicol Lett. 79 (l-3): 87-98; and Ball and Schwartz (1994) Compute Biol Med. 24 (4): 269-76. Pharmacokinetic parameters can also be obtained from published literature, for example, see Buckpitt et al. (1984) J. MoI. Pharmacol. Exp. Ther. 231: 291-300; DelRaso (1993) Toxicol. Lett. 68: 91-99; Haies et al. (1981) Am. Rev. Respir. Dis. 123: 533-541.

  Specific physiological parameters desired include tissue or organ fluid residence time, tissue or organ mass, fluid to cell volume ratio, cell shear stress, and the like. Physiologically relevant parameter values can be obtained experimentally by conventional methods, or can be obtained from values known in the art and publicly available. Desired pharmacokinetic parameter values are obtained for animals, usually mammals, but other animal models such as insects, fish, reptiles, or birds can also find use. Mammals are laboratory animals such as mice, rats, rabbits, or guinea pig mammals of economic value, such as horses, sheep, goats, cows, dogs, or cats; primates including monkeys, apes, or humans; Etc. Animals of different ages, such as fetuses, newborns, infants, children, adults, or older people; and different physiological conditions, eg, when affected, after contact with a pharmaceutically active agent, after infection, or Under different atmospheric pressure conditions, different values can be obtained and used.

  Similar to the mass balance equations that can be applied to various substances modeled in the system, information related to pharmacokinetic parameter values can be obtained from data processing components of the culture system, such as a look-up in a general purpose memory provided for storage Optionally provided on an uptable or the like. These equations represent physiological pharmacokinetic models of various biological / chemical substances in the system.

  Pharmacokinetic culture device: The culture device of the present invention provides a substrate for cell growth. Each device comprises at least one chamber, usually at least two chambers, and may comprise more than two chambers, which are interconnected by fluid channels. The chambers can be on a single substrate or on different substrates. Preferably, each chamber has a different surface shape configuration than the other chambers present on the device. The device contains a cover to seal the chamber and the channel and comprises at least one inlet and outlet port that allows for media recirculation. The device includes a mechanism for pumping media through the system. The medium is designed to maintain the survival of the cultured cells. The device includes a mechanism that allows a test compound to be introduced into the system.

  In one embodiment of the present invention, the device is made on a microscale as a single unit that does not exceed about 2.5 cm on a side and preferably comprises at least two interconnected chambers. The two organ compartments are connected by a channel approximately 50-150 μm wide and 15-25 μm deep. For example, one chamber can represent a lung comprising an interconnected array of parallel channels, usually at least about 10 channels, preferably at least about 20 channels. Such channels may have common microfluidic dimensions, for example, about 30-50 μm wide, 5-15 μm deep, and 3-7 mm long. Another compartment can represent a liver, typically comprising two or more parallel channels in a serpentine shape, about 50-150 μm wide, 15-25 μm deep, and 5-15 cm long.

  The device will usually include a mechanism for obtaining signals from cells and media. Signals from different chambers and channels can be monitored in real time. For example, the biosensor may be integrated into the device or external to the device, allowing real-time readout of the physiological state that is in the system.

  The pharmacokinetic culture device of the present invention can be provided as a chip or a substrate. Such microsystems, in addition to enhancing fluid dynamics, save space and can be produced inexpensively, especially when used in highly parallel systems. The culture device is formed from a polymer such as but not limited to polystyrene and can be discarded after use without the need for sterilization. As a result, in vitro subsystems can be produced inexpensively and used extensively. Also, cells can be grown in a three-dimensional manner, for example to form a tube, thereby more closely replicating the iv vivo environment.

  To mimic an animal's metabolic response to any particular agent, a set of parallel or multiple sequences comprises multiple (ie, at least two) cell culture systems, where each system may be identical. However, it may be varied according to a predetermined parameter value or input agent and concentration. The array may comprise at least about 10, or as many as 100 or more systems. Advantageously, the cell culture system on the microchip can be stored in a single chamber so that all underlying cell culture systems are exposed to the same conditions during the assay.

  Alternatively, multiple chips may be incorporated to form a single device, for example to mimic the gastrointestinal barrier or blood brain barrier.

  Cells: The cells used in the assays of the present invention may be an organism, a single cell type derived from an organism, or a mixture of cell types, as is common in in vivo situations. The culture conditions may include, for example, temperature, pH, presence of factors, presence of other cell types, and the like. As described above, a variety of animal cells can be used, including any animal that can obtain pharmacokinetic parameter values.

  The present invention is suitable for use with any cell type including primary cells, stem cells, progenitor cells, normal, genetically engineered, genetically modified, immortalized and transformed cell lines. Yes. The present invention is suitable for use with a single cell type or cell line, or a combination of different cell types. Preferably, the cultured cells maintain the ability to respond to stimuli, leading to a response in their naturally occurring counterpart. These can be derived from any source, such as eukaryotic or prokaryotic cells. The eukaryotic cell may in fact be a plant or an animal such as a human, monkey or rodent. The cells can be of any tissue type (eg, heart, stomach, kidney, intestine, lung, liver, fat, bone, cartilage, skeletal muscle, smooth muscle, heart muscle, bone marrow, muscle, brain, pancreas) and cell type ( For example, epithelium, endothelium, mesenchyme, adipocyte, hematopoiesis). In addition, cross sections of tissues or organs may be used. For example, arterial, venous, gastrointestinal tract, esophagus, or colon cross sections may be used.

  Cells that have been genetically modified or engineered to contain non-natural “recombinant” (also called “exogenous”) nucleic acid sequences, or modified by antisense technology to provide gain or loss of genetic function Cells may be utilized in the present invention. Methods for generating genetically engineered cells are known in the art, see, for example, “Current Protocols in Molecular Biology” (Ausubel et al., John Wiley & Sons, New York, NY, 2000). The cells can be terminally differentiated or undifferentiated, such as stem cells. The cells of the present invention may be cultured cells from various genetically diverse individuals that can react differently to biological agents and pharmacological substances. Genetic diversity can have indirect and direct effects on susceptibility. In the direct case, even a single nucleotide change that causes a single nucleotide polymorphism (SNP) alters the amino acid sequence of the protein and contributes directly to the disease or susceptibility Can do. For example, several APO-lipoprotein E genotypes have been associated with the onset and progression of Alzheimer's disease in some individuals.

  If several polymorphisms are associated with a particular disease phenotype, the cells of individuals identified as carriers of the polymorphism are studied for developmental abnormalities using non-carrier carriers as controls. obtain. Since several different cell types can be studied simultaneously and conjugated to related cells, the present invention provides an experimental system for studying developmental abnormalities associated with specific genetic disease symptoms. For example, neural progenitor cells, glial cells, or other cells of neural origin can be used in the device to characterize the cellular effects of compounds on the nervous system. The system also allows cells to identify genetic elements that affect not only responses to changes in receptor expression and / or function, but also drug sensitivity, chemokine and cytokine responses, responses to growth factors, hormones, and inhibitors. Can also be set up to study. This information can be invaluable in designing therapeutic methodologies for diseases of genetic origin or genetic predisposition.

  In one embodiment of the invention, the cell is involved in detoxification and metabolism of a pharmaceutically active compound, eg, liver cells including hepatocytes; kidney cells including tubule cells; Adipocytes including adipocytes that can be produced. These cells can be integrated with a culture system having cells such as lung cells involved in the respiration and oxidation processes. These cells are cells that are particularly sensitive to damage from the desired agent, such as intestinal epithelial cells, bone marrow cells, and other normally rapidly dividing cells for agents that affect cell division You may combine. Nervous cells may be present to monitor the effect of the agent on neurotoxicity and the like.

  There is also interest in the growth characteristics of tumors and the response of surrounding tissues and the immune system to tumor growth. Degenerative diseases, including the affected tissue and surrounding areas, can be exploited to measure both the response of the affected tissue and the interaction with other parts of the body.

The term “environment” or “culture conditions” encompasses cells, media, factors, time, and temperature. The environment can also include drugs and other compounds, certain atmospheric conditions, pH, salt composition, minerals, and the like. Cell culture is generally performed in an aseptic environment that mimics physiological conditions, eg, in an incubator containing 37 ° C., 92-95% humidified air / 5-5% CO ₂ atmosphere. Cell culture can be performed in a nutrient mixture containing undefined biological fluids such as fetal bovine serum or in a medium that does not contain fully defined serum. Various media are known in the art and are commercially available.

  As used herein, the term “physiological state” is used to monitor cell culture conditions very specifically in order to mimic as closely as possible the natural tissue conditions of a particular cell type in vivo. It is defined as meaning. These conditions include parameters such as fluid residence time (ie, the time that fluid remains in the organ); cell-to-blood volume ratio, normal stress on the cells, and compartment size comparable to that of the natural organ.

Screening assay: Drugs, toxins, cells, pathogens, samples, etc., collectively referred to herein as “input variables”, are added to a pharmacokinetic-based culture system and then the desired output variable change For example, biological activity is screened by evaluating cultured cells for consumption of O ₂ , production of CO ₂ , cell survival, or expression of the desired protein. Input variables are generally added to the cell medium in culture, either in solution or in a readily soluble form. Input variables can be added using a flow-through system or by adding boluses to other static solutions. In the flow-through system, two fluids are used, one is a physiologically neutral solution and the other is the same solution with the addition of a test compound. The first fluid passes through the cell, followed by the second fluid. In the single solution method, a bolus of test input variables is added to the volume of media surrounding the cells. The overall composition of the medium should not change significantly with the addition of boluses or between the two solutions in the flow-through method.

  Preferred input variable formulations do not contain additional ingredients that have a significant effect on the overall formulation, such as preservatives. Accordingly, preferred formulations comprise a bioactive agent and a physiologically acceptable carrier such as water, ethanol, or DMSO. However, if the agent is a liquid without an excipient, the formulation may be only the compound itself.

  Preferred input variables are viruses, virus particles, liposomes, nanoparticles, biodegradable polymers, radiolabeled particles, radiolabeled biomolecules, toxin conjugated particles, toxin conjugated biomolecules, and particles or bios conjugated with stabilizers. Including but not limited to molecules. A “stabilizer” is an agent used to stabilize a drug and provide controlled release. Such agents include albumin, polyethylene glycol, poly (ethylene-co-vinyl acetate), and poly (lactide-co-glycolide).

  Multiple assays can be run in parallel with different input variable concentrations to obtain response differences for different concentrations. As is known in the art, a range of concentrations derived from 1:10 or other log scale dilutions is generally used to determine the effective concentration of an agent. The concentration can be further purified by secondary dilution if necessary. In general, one of these concentrations serves as a negative control, i.e. is zero concentration or below the level of detection.

  The input variables of interest are often organic molecules, but include multiple chemical classifications. A preferred embodiment is the use of the method of the invention for screening the toxicity of a sample, for example an environmental sample or a drug. Candidate agents may contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonds, and generally will include at least amine, carbonyl, hydroxyl, or carboxyl groups, preferably at least 2 of these chemical functional groups. Including one. Candidate agents often include cyclic carbon or heterocyclic structures, and / or aromatic or polycyclic aromatic structures, replacing one or more of the above functional groups. Candidate agents are also found in biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

  Pharmacologically active drugs and genetically active molecules are included. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. A typical pharmaceutical suitable for the present invention is “The Pharmacological Basis of Therapeutics” Goodman and Gilman, McGraw-Hill, New York, New York (1996), 9th edition, Drugs Acting at Synapit at Sect. the Central Nervous System; Autocoids: Drug Therapy of Information; Water, Salts and Ions; Drugs Affecting Rental Function and Electrolyte Metaltology; scular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; And described in the Toxicology section It is shall, all of which is incorporated by reference herein. Toxins and biological and chemical warfare agents are also included, see, for example, Somani, S .; M.M. (Eds.), “Chemical Warfare Agents” (Academic Press, New York, 1992).

  Test compounds include all the classes of molecules described above and may further include samples of unknown content. Many samples will contain compounds in solution, but solid samples that can be dissolved in a suitable solvent may be assayed. Samples of interest are environmental samples such as groundwater, seawater, or mining waste; biological samples such as lysates or tissue samples prepared from crops; production samples such as time during pharmaceutical preparation A library of compounds prepared for analysis; and the like. Samples of interest include compounds that are being evaluated for potential therapeutic value, such as drug candidates from plants or fungal cells.

  The term “sample” also includes the fluids described above to which additional components have been added, such as components that affect ionic strength, pH, or total protein concentration. The sample can also be processed to achieve at least partial fractionation or concentration. Biological samples may be stored, for example, under nitrogen, frozen, or combinations thereof if care is taken to reduce degradation of the compound. The sample volume used is sufficient to allow measurable detection, and usually about 0.1 μl to 1 ml of biological sample is sufficient.

  Compounds and candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Several means are available for random or designated synthesis of a wide variety of organic compounds and biomolecules, including, for example, expression of randomly chosen oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds are available or readily produced in the form of bacterial, fungal, plant and animal extracts. Furthermore, natural or synthetically produced libraries and compounds can be easily modified by conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. it can. Known pharmacological agents can be subjected to designated or random chemical modifications such as acylation, alkylation, esterification, amidation, etc. to produce structural analogs.

  Output variable: An output variable is a quantifiable element of a cell, particularly an element that can be accurately measured in a high-throughput system. Output is derived from, for example, survival, respiration, metabolism, cell surface determinants, receptors, proteins or conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, mRNA, DNA, or such cellular components Can be any cellular component or cellular product. Most outputs will provide a quantitative readout, but in some cases semi-quantitative or qualitative results will be obtained. The read information may include a single measurement or may include an average, median value, or variance. As a feature, the range of read values is obtained from each output. Variations are expected and a range of values representing a set of test outputs can be established using standard statistical methods.

  Various methods can be utilized to quantify the presence of the selected marker. A convenient way to measure the amount of molecule present is to label the molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, or enzymatically active. Fluorescent and luminescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins, but also to specific conformations, cleavage products, or site modifications such as phosphorylation reactions. Individual peptides and proteins can be modified to be autofluorescent, for example, by expressing them as green fluorescent protein chimeras in cells (reviewed Jones et al. (1999) Trends Biotechnol. 17 (12): 477. ~ 81).

  Output variables can be measured by immunoassays, such as immunohistochemistry, radioimmunoassay (RadioImmunoAssay; RIA), or enzyme linked immunosorbent assay (ELISA) and related non-enzymatic techniques. These techniques utilize specific antibodies as particularly useful reporter molecules due to their high specificity for attaching to single molecule targets. Cell surface ELISA or related non-enzymatic or fluorescence based methods allow measurement of cell surface parameters. Information read from such an assay can be the average fluorescence associated with the cell surface molecule or cytokine in which the individual fluorescent antibody was detected, or the average fluorescence intensity, median fluorescence intensity, dispersion of fluorescence intensity, or in between There can be some relationship.

  Data analysis: Results of screening assays can be compared to results obtained from reference compounds, concentration curves, controls, and the like. Comparison of results is achieved through the use of appropriate inference protocols, Al systems, statistical comparisons, and the like.

  A database of reference output data can be compiled. These databases contain results from known agents or combinations of agents, as well as references from analyzes of cells treated under environmental conditions where single or multiple environmental conditions or parameters have been removed or specifically modified. May also be included. A data matrix can be generated, each point of the data matrix corresponding to information read from the output variable, and the data representing each output is sourced from replicate measurement results, eg, multiple individual cells of the same type. May be emitted.

  The read information may be an average, average, median, or variance, or other statistically or mathematically derived value associated with the measurement result. The output readout information can be further purified by direct comparison with the corresponding reference readout information. The absolute value obtained for each output under the same conditions will display the variation inherent in the living biological system and reflect not only the variation inherent between individuals but also individual cell variations. .

Cell Culture and Cell Culture Devices The culture devices of the present invention comprise a fluid network of channels that are separate but interconnected and preferably isolated within one or more chambers incorporated in a chip format. Specific chamber surface shapes are designed to provide cell interactions, fluid flow rates, and fluid retention parameters that interact with those found in vivo in the corresponding cell, tissue, or organ system.

  An optimized chamber surface shape can be developed by repeating the procedure of testing parameter values in response to changes in fluid flow and dimensions until a selected value is obtained. Substrate optimization involves selecting the number of chambers, selecting the chamber surface shape that provides the correct cell-to-volume ratio, selecting the chamber size that provides the correct tissue or organ size ratio, and the correct liquid Selecting an optimal fluid flow rate that provides a residence time, and then calculating a cell shear stress based on these values. If the cell shear stress exceeds the maximum allowable value, a new parameter value is selected and the process is repeated. Another embodiment of a CCA device includes one in which cells grow in hollow tubes rather than on the bottom and sides of the channel or chamber. Cells growing in such three-dimensional tissue constructs have been demonstrated to be more authentic for some in vivo tissues (Griffith (1998) PhARMA Biol. Biotech. Conf., Coronado, CA). March 15-18).

  In creating a 3D culture device, three intrinsic design parameters are considered. The first is the residence time where the fluid is in contact with a particular tissue or is in a well. The residence time is chosen to reflect the amount of time that blood is in contact with the organ tissue represented by the well within one passage of the circulatory system. The second is the radius of the tube in which the cells grow. For example, the radius of the tube for replicating the liver is in the range of 200-400 μm. It should be noted that if the radius of the tube becomes too large, the cells are essentially flat and form a monolayer on the tube.

  The third parameter is the rate of flow that reaches each module. The flow from the chamber is segmented by adjusting the surface shape of the flow channel. The channels or tubes to each module or chamber are generally of different lengths to balance the pressure drop and balance the flow. The fluid can be recirculated by the pump after leaving other tissues. The flow rate through the tube was calculated from the tube size and residence time. In view of the flow rate, the shear stress on the cells was calculated to ensure that the value did not exceed the cell stress limit. Due to the very short residence time required in lung tissue, it is not possible to use the well and tube approach for this organ. Because the shear stress is too high, the lung tissue section remains unaffected by the lung tissue monolayer.

  Since the system of the present invention is bi-directional (ie, the computer not only senses but also controls conditions within the test range), corrections are dynamically added to the system, appropriately noted and implemented. Can be documented to inform researchers of the kinetics of the study within.

  The data collected by the computer consists of a collection of data necessary for continuous in-line monitoring of the test chemical effluent from each compartment. As described above, sensors, preferably flow-through types, are placed in-line with the effluent from each compartment to detect, analyze, and provide quantitative data regarding the test chemical effluent from each compartment. The

  The microprocessor is also useful in calculating a Physiologically-Based PharmacoKinetic (PBPK) model for a particular test chemical. These calculations can serve as a basis for setting flow rates between compartments and the rate of elimination of test chemicals from the system. However, it may also serve as a theoretical estimate for the test chemical. At the end of the experiment, predictions regarding the concentrations of test compounds and metabolites made by the PBPK measurements can be compared to sensor data. The hard copy output compares the PBPK model with the experimental results.

  Several prototype CCA systems were built and tested. FIG. 17A depicts a “first generation” three-compartment device. The dimensions are as follows: the wafer is 2 cm × 2 cm; the lung chamber has 20 channels (length 5 mm) 40 μm × 20 μm (width × depth); the liver chamber has 2 channels (length 100 mm) It shall have 100 micrometers x 20 micrometers (width x depth). The first step in using this device is to inject fluid using a syringe pump until all chambers are full. Second, the fluid is recirculated using a peristaltic pump. FIG. 17B shows a cross-sectional view of the device demonstrating the fluid optics of the system. A 400 μm thick elastomer showed good sealability and plexiglass and gel loading tips were found to be much less brittle than other materials. This device had the problem of high pressure drop and leakage when bent at 90 °.

  Cell adhesion studies were performed using this “first generation” device. L2 cells were placed in the lung chamber and H4IIE cells were placed in the liver chamber. Poly-D-lysine was adsorbed on the surface of the chamber to promote cell attachment in the channel. Unfortunately, because the cells were attached to the outside of the trench, different substrates were tested and the surface was modified.

  FIG. 18A depicts a “second generation” device. The dimensions were as follows: chip 2 cm × 2 cm; etching 20 μm deep; lung chamber 2 mm × 2 mm (width × length); liver chamber 7.5 mm × 10 mm (width × length). The lung chamber contained a high ridge of 5 μm to increase cell adhesion (FIG. 18B), and the liver chamber contained a high column of 20 μm to simulate leaching (FIG. 18C).

  FIG. 19 depicts a “third generation” device. Dimensions are as follows: tip 2 cm x 2 cm; lung chamber 2 mm x 2 mm (width x length); liver chamber 3.7 mm x 3.8 mm (width x length); "other tissue" chamber 7 mm x 7 mm (width x length). Fluid was split from the lung chamber, 20% to the liver and 80% to the other tissue chambers. Part of the chamber (dashed line part) is 100 μm deep to reduce pressure drop and the other part (solid line part) is 20 μm deep to give a realistic liquid cell ratio.

  FIG. 20 is a flow diagram representing a five compartment PBPK model CCA. In this device, a chamber representing fat cells, a chamber representing slowly perfused fluid and rapidly perfused fluid are added. Such devices can be used for bioaccumulation studies, cytotoxicity studies, and metabolic activities. Other devices with various permutations can also be developed. For example, a diaphragm pump with gas exchange, or an online biosensor, or a micro electromechanical (MEM) pump, or a biosensor and an electronic interface may be added. Devices can be developed to mimic oral drug delivery. Alternatively, devices can be developed to mimic the blood brain barrier.

Production Cell culture devices generally comprise a collection of separation elements, such as chambers, channels, inlets, or outlets, that when properly mated or engaged, form the culture devices of the present invention. Preferably, the element is provided in an integrated “chip-based” format.

  Device fluidics are scaled appropriately to fit the size of the device. In a chip-based format, the fluid connection is a “microfluidic” and such a system has a passage, chamber, or having at least one internal cross-sectional dimension, eg, about 0.1 μm to 500 μm in depth or width. Contains fluidic elements such as conduits. In the device of the present invention, the channel between the chambers generally comprises at least one microscale channel.

  In general, a microfluidic device comprises a top portion, a bottom portion, and an inner portion, the inner portion substantially defining the channel and chamber of the device. In a preferred embodiment, the bottom portion will comprise a solid substrate that is substantially planar in structure, the substrate having at least one substantially flat top surface. Various substrate materials may be used as the bottom portion. In general, since devices are microfabricated, substrate materials are generally known microfabrication techniques such as photolithography, thin film deposition, wet chemical etching, reactive ion etching, inductively coupled plasma deep silicon etching, laser cutting, air wear. The choice will be based on technology, injection molding, embossing, and compatibility with other technologies.

  The substrate material of the present invention is a polymer material such as plastic such as polystyrene, polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON (trademark)), polyvinylchloride (PolyvinylChloride; PVC), polydimethyl. Siloxane (PolyDiMethyl Siloxane; PDMS), polysulfone, etc. are provided. Such substrates are easily manufactured from microfabricated masters using well-known molding techniques such as injection molding, embossing, or stamping, or by polymerizing the polymer precursor material in the mold. The Such polymer substrate materials are preferred because of their general inertness to the most extreme reaction conditions, as well as their ease of manufacture, low cost, and disposal. These polymeric materials are treated surfaces such as derivatized or coated to enhance their usefulness within the system, for example, to provide enhanced fluid orientation, cell attachment, or cell separation. Surface may be included.

  The channels and / or chambers of the microfluidic device are generally fabricated in the top or bottom portion of the substrate using the microfabrication techniques described above, such as microscale grooves or press fit. The lower surface of the upper portion of the microfluidic device is generally provided with a second planar substrate, which is then overlaid and bonded to the surface of the bottom substrate, the device channel at the interface of these two components And / or seal the chamber (inner part). The bonding of the top portion to the bottom portion can be done by using various known methods, depending on the nature of the substrate material. For example, in the case of a glass substrate, a thermal bonding technique using high temperature and pressure can be used to bond the top portion of the device to the bottom portion. In order to prevent over-melting of the substrate material, polymer substrates can be bonded using similar techniques except that the temperatures used are generally low. Alternative methods may be used to bond the polymer parts of the device together, including acoustic welding techniques or the use of adhesives such as UV curable adhesives.

  The device will generally comprise a pump, such as a low flow peristaltic pump. A small bore flexible tube will be attached to the outlet of the device and will pass through the peristaltic pump and will be attached to the inlet of the device, thereby forming a closed loop system. The pump generally operates at a flow rate of about 1 μL / min. The pump system may be any fluid pump device such as a diaphragm, may be integral to the CCA device (chip-based system), or may be a separate component as described above.

  The device may be connected or interfaced to a processor that stores and / or analyzes signals from each biosensor. The processor sequentially transfers the data to computer memory (hard disk or RAM), from which the data can be used by a software program to further analyze, print, and / or display the results.

DESCRIPTION OF EXEMPLARY EMBODIMENTS In the following detailed description of specific embodiments, reference is made to the accompanying drawings that form a part hereof, and which are shown for purposes of illustrating specific embodiments in which the invention may be practiced. refer. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

  FIG. 1 is a block diagram of an in vitro system according to the present invention. The lung cell simulation chamber 102 receives the oxidation medium from the gas exchange device 103. Such an oxidizing medium is obtained by contacting the medium with an oxygen-containing gas such that the medium absorbs the oxygen-containing gas and desorbs the carbon dioxide-containing gas. The medium exiting the lung cell simulation chamber 102 is similar to mammalian arterial blood 106. Subsequently, the oxygen-containing medium constituting the arterial blood 106 is supplied to the liver simulation chamber 108, the other tissue simulation chamber 110, the fat simulation chamber 112, and the kidney simulation chamber 114. The medium starting from the liver simulation chamber 108, the other tissue simulation chamber 110, the fat simulation chamber 112, and the kidney simulation chamber 114 is similar to the mammalian venous blood 104. As shown in FIG. 1, the medium corresponding to venous blood 104 is returned to the lung cell simulation chamber 102. The system of the present invention also includes an intestinal simulation chamber 116 and an abdominal cavity simulation chamber 118, both of which constitute a site for test compound introduction. As with mammals, waste fluid 115 is withdrawn from the kidney simulated chamber 114.

  FIG. 2 is a simplified schematic diagram of one embodiment of the system 200 of the present invention. System 200 includes a lung cell culture chamber 210, a hepatocyte culture chamber 212, an adipocyte culture chamber 213, another tissue chamber 214, and a gas exchange chamber 250. Chambers 210, 212, 213, 214, and 250 are formed on a silicon substrate, commonly referred to as chip 230. It should be noted that more than four cell culture chambers can be stored or formed on a single chip 230. A fluid path 240 connects the chambers 210, 212, 213, 214, and 250.

  The chamber has an inlet 211 and an outlet 215. The inlet 211 is located at one end of the gas exchange chamber 250. The outlet 215 is located at one end of the hepatocyte culture chamber 212. Chambers 210, 212, 213, 214, and 250 and fluid path 240 are located substantially between inlet 211 and outlet 215. The system includes a pump 260 for circulating fluid within the system 200. A microtubule 270 connects the outlet 215 of the pump 260 and the inlet side. A microtubule 271 connects the outlet side of the pump 260 to the inlet 211. Cell culture chambers 210, 212, 213, 214, gas exchange chamber 250, fluid path 240, and pump 260 form system 200. The system may include an additional cell culture chamber. One common cell culture chamber that is added is a simulated kidney.

  FIG. 3 is a schematic illustrating another embodiment of the present invention. In FIG. 3, a first signal path 310, a second signal path 320, and a third signal path 330 are provided on the chip 230. Signals for monitoring various aspects of each cell culture system 200 can be taken from the chip 230 and at specific locations on the chip 230 and transferred to an output outside the chip 230. As an example, the signal paths 310, 320, 330 on the chip 230 are integrated embedded waveguides. In such embodiments, the chip 230 may be made of silicon, glass, or polymer. Waveguides 310, 320, 330 will have transducers 312, 322, 332 located here to transmit light to the edge of the chip and convert the optical signal into an electrical signal. Subsequently, the cells in system 200 may be monitored for fluorescence, luminescence, or absorption, or all these characteristics, to investigate and monitor the cells in system 200. A light source is required to confirm fluorescence. The light source is used to probe molecules such as waveguides 310, 320, 330 or optical fibers and signal carriers that capture the signal and send it to the chip 230. The signal carriers 310, 320, 330 will direct the light towards a photodetector near the end of the signal carrying portion of the chip 310, 320, 330.

  FIG. 4 is a schematic diagram illustrating another embodiment of the system 200 of the present invention. In this embodiment, biosensors 410, 420, 430, 440, 450, and 460 are positioned upstream and downstream of each cell culture chamber of chip 230. Biosensors 410, 420, 430, 440, 450, 460 monitor the oxygen, carbon dioxide, and / or pH of the medium. These sensors allow the system 200 to be monitored and the gas concentration adjusted as needed to maintain a healthy environment. Biosensors also provide useful information about cell metabolism and survival when located just upstream and downstream of each cell compartment.

  5A-5G illustrate the steps used to make a polymer-based disposable tip 230. The silicon wafer 20 is spin coated with a thin layer of photoresist 21 (FIG. 5A). Photoresist 21 is exposed to ultraviolet light 22 through a photomask 23 that includes the desired features (FIG. 5B). The UV-exposed photoresist 21 is developed in a suitable solvent, thereby exposing the silicon 20 (FIG. 5C). Silicon 20 is etched to the desired depth using an inductively coupled plasma etching system (FIG. 5D). The remaining photoresist is removed with a suitable solvent (FIG. 5E). As shown in FIG. 5E, an ultrathin gold (or Ti) plating base 24 is deposited on the silicon substrate 20 to create a template for an electroplating process. A sample is immersed in a nickel sulfamate bath, and nickel 25 is electroplated onto the silicon template 20 using gold acting as a conductive film until the nickel thickness is sufficient. The nickel master is generated from the gold layer, and gold becomes part of the nickel master. This forms the Ni mechanism 25 shown in FIG. 5F. Calibrate the plating speed, template diameter, and template thickness as a function of plating current to about 45 nm / min. After fabrication, the mechanism 25 is inspected using a microscope to verify the dimensions of the mechanism. The remaining nickel features 25 should be uniform and have the desired shape. Nickel master 25 and polymer substrate 26 are heated to slightly above the glass transition temperature of the polymer. The nickel master 25 and the polymer 26 are brought into contact, and the mechanism of the nickel master 25 is embossed into the polymer substrate 26. Removal of the nickel master 25 produces a polymer 26 that includes the same mechanism as the original silicon wafer 20 (FIG. 5G).

  FIG. 6 is a schematic diagram of a third embodiment of the system 200 of the present invention. In this embodiment, biosensors 600, 602, 604 are positioned around the periphery of chip 230. The biosensors 600, 602, 604 are used to further monitor the state of the cells of the system 200 created on the chip 230. Advantageously, the chip 230 can be made disposable at a minimal cost by positioning the biosensors 600, 602, 604 around the periphery of the chip 230. In other words, it is not necessary to discard the biosensors 600, 602, and 604 together with the chip 230. It should be noted that the biosensors 600, 602, 604 may be provided on the disposable chip 230. This particular option would not be cost effective because the biosensor 600, 602, 604 that disposes of the chip 230 results in discarding the biosensor 600, 602, 604 as well. If the biosensor 600, 602, 604 is positioned off the chip 230, the biosensor 600, 602, 604 is most cost effective because it is reused rather than discarded after each use. Each of biosensors 600, 602, 604 is connected to an input of computer 620.

  FIG. 7 is a schematic detailing the computer 620 in further detail. Computer 620 monitors and adjusts the operation of system 200 for each chip 230. The computer 620 includes a microprocessor provided with an input / output interface 700 and an internal register / cache memory 702. As shown, the microprocessor 798 includes a keyboard 704 via a connection 716, a non-volatile storage memory 706, a general purpose memory 708, and a lookup table 710 via a connector 718, and a printer / printer via a connector 720. The plotter recorder 712 and the display 714 are connected by an interface.

  The non-volatile storage memory 706 may be in the form of a CD writable memory, a magnetic tape memory, a disk drive, or the like. The look-up table 710 may physically comprise a portion of a general purpose memory 708 that is provided for storage of a set of mass balance equations that can be applied to various materials being modeled in the system. These equations represent physiological pharmacokinetic models of various biological / chemical substances in the system. The internal register / cache memory 702 and the general purpose memory 708 contain a system program in the form of a plurality of program instructions and special data for automatically controlling virtually all functions within the system 200 of each chip 230. The computer can also control and regulate the pump 260 associated with the system 200.

  Fluid flow may also be provided from the flow meter via the input / output interface 700 as input to the microprocessor 798. This makes it possible to strictly control the fluid flow rate in the system by adjusting the program commands sent to the pumps 260 via the pump control lines. For example, the flow rates are 9.5 μL / min in conduit 58, 2.5 μL / min through flow meter 66, 7 μL / min through flow meter 78, and 2.5 μL / min in conduit 70. Can be set to The temperature of the medium in the container 50 can also be adjusted by the microprocessor 798, which receives temperature measurement results from the temperature probe 792 via the input / output interface 700 and the temperature indicator line 728. In response to these signals, microprocessor 798 turns heating coil 790 on and off via input / output interface 700 and heating coil control line 730.

  Biological and toxic reactions / changes in the cell culture chambers 210 and 212 are detected by sensors 600, 602, and 604, respectively, and communicated to the microprocessor 798 via the control line as well as the input / output interface 700. The sensor can be designed to represent test results in terms of a particular value or range of wavelengths to represent the test results.

  Microprocessor 798 can also be very easily adapted to include programs for providing researchers with interactive control via keyboard 704. This makes it possible, for example, to direct the computer to always specifically check the state of any of the culture compartments.

  A further option provided by the present invention is the ability to recall pre-stored test results for similar experiments by recalling information from CD / tape memory 706. Accordingly, the memory 706 holds historical data collected from publicly available information, data collected from prior performance tests conducted using the system of the present invention, or data derived from theoretical calculations. Can be pre-programmed. The provision of the CD / tape memory also makes it possible to use the system as an information retrieval tool. The system can obtain study data associated with a particular test compound or with a particular cell line, for example, based on selection information input to the microprocessor 798 via the keyboard 704. Inclusion or development of a large library of information in memory 706 enables researchers to more intelligently configure and plan test runs.

  FIG. 8 is a schematic showing more than one chip 230 housed in a single housing 800. The housing 800 may be an environmental chamber that maintains the same conditions for each of the chips 230 in the housing. The housing 800 includes a plurality of chip positions 810, 812, 814, 816. The output from each chip 230 or chip location 810, 812, 814, 816 is input to a computer 620. Subsequently, the computer 620 can monitor the system 200 from a plurality of chips 230 in real time.

  FIG. 9 is a schematic showing that the test can include a set of chips 230 in different housings 800, 900. The output of each of the chips 230 can be monitored for changes in the environment, such as when the temperature is slightly higher. Each of the chips within one housing can have the same cell culture therein, that is, the chip 230 within the housing 800 forms different parts of mammals or interdependent organs within the housing. It is further envisaged to have chips interconnected to each other.

  Consider FIGS. 2-4 and 6-9 using a two-dimensional cell culture chamber 210, 212, 213, 214. Since three-dimensional tissue culture constructs may be closer to the real in their metabolism, yet another chip 1000 addresses the inclusion of the three-dimensional construct. The following describes the creation of a microscale cell culture analog (“CCA”) device that incorporates three-dimensional tissue in a modular format. The CCA device or chip 1000 incorporates a flow-over approach for lung cell chambers and a flow-through approach for other organs. The flow-through approach to CCA design is discussed further below.

  FIG. 10 shows the outline of the chip 1000 and the flow method. The chip 1000 includes four wells or tissue modules. Chip 1000 includes lung well 1010, liver well 1020, fat well 1030, slowly perfused well 1040, and rapidly perfused well 1050. A tube is used to circulate fluid through the tip 1000. The pump 1060 moves the fluid through the pipe. Lung well 1010 first receives all the flow. After the lung 1010, the fluid will be segmented into four tissue modules. The liver module will get 25% of the flow, the fat module will get 9%, the slowly perfused module will get 15%, and the rapidly perfused module will get 51%. The flow from the lung well 1010 is segmented by adjusting the surface shape of the flow channel. The channels to each module are of different lengths to balance the pressure drop and balance the flow. After leaving the other tissue, the fluid circulates through pump 1060 and returns to the lung compartment. Each of the well or tissue modules 1020, 1030, 1040, 1050 holds tissue. The tissue is retained in microscale tubes 1022, 1032, 1042, 1052 in wells 1020, 1030, 1040, 1050. As shown in FIG. 10, there is only one microscale tube 1022, 1032, 1042, 1052 per well 1020, 1030, 1040, 1050, respectively. It should be noted that multiple microtubules may be placed in the well.

  In operation, there are two methods that allow 3D tissue to be incorporated into a CCA device or chip 1000. Both methods involve the flow of inoculum media through polystyrene or glass microscale tubes. The cells under test adhere to the inside of the tube and collect within the three-dimensional tissue. The tubes are collected, bundled and placed in wells on the chip 1000. Each well becomes an organ module through which the aqueous drug passes and contacts the tissue.

  The first method for enabling the incorporation of 3D tissue involves a flow-through reactor strategy. An opening is formed in the silicon wafer, and then the channeled medium passes through the opening. Silicon on the inner surface of the opening provided a scaffold for cells and concentrated in the three-dimensional tissue. To apply this technique to the polymer CCA1000, the polymer tube may be treated with adhesion protein or the cells may be cultured in serum-supplemented medium. Both serum and adhesion protein allow cells to stick to the inner surface of the tube.

  The second method involves culturing cells in a HARV microgravity reactor. By building a scaffold with a tube in the center of the rotary reactor, or by introducing a floating tube into the medium, the cells form three-dimensional aggregates within some of the tube. Due to the enhanced activity of cells grown in microgravity, these tissue contractions have superior function compared to two-dimensional tissues or tissues formed by the methods described above. Tissue with a tube inside can be separated according to weight or density and placed on the device.

  FIG. 11 is a partially exploded isometric view of a cell culture analog device 1100 that incorporates a chip 1000. The chip 1000 includes a lung cell culture region 1010 and a plurality of wells connected to the lung cell culture region 1010. The wells include a liver tissue well 1020, an adipose tissue well 1030, a slowly perfused well 1040, and a rapidly perfused well 1050. Microscale tubes containing various tissues are contained in wells 1020, 1030, 1040, and 1050. Each well includes an output to the elastomer bottom 1110 attached to the chip 1000. Elastomer 1110 is part of the pump. The actuator 1120 compresses the elastomer to produce a pumping action that moves the fluid of the system 1100 or to circulate the fluid of the system 1100 back from the well to the lung tissue module 1010 via the return line 1130. A glass layer is placed on top of the chip to cover lung tissue module 1010 and various wells 1020, 1030, 1040, and 1050. It should be noted that channels 1021, 1031, 1041, and 1051 are sized to produce several flow rates through the various wells 1020, 1030, 1040, and 1050. Rather than adjusting the length and width of the various channels 1021, 1031, 1041, 1051, other along the channel to provide variation in flow rates to the various wells 1020, 1030, 1040, and 1050 It is thought that a flow restrictor can be placed. The glass top 1140 can be replaced with a flexible membrane, and a plunger ball type valve can be added so that the flow in the channels 1021, 1031, 1041, and 1051 can be adjusted depending on other than the dimensions of the channel. .

  The chip 1100 may be made of silicon, but it is more cost effective to make the chip 1000 from polystyrene or some other suitable plastic. Each chip is first formed in silicon by conventional means. Subsequently, a nickel master is formed from silicon. In other words, the chip 1000 is manufactured by replicating polystyrene and silicone elastomer on a silicon and nickel master. Of course, the first step in the production of polymer chips is to produce the chips on a silicon wafer. First, a layer of photoresist 1210 is placed on a silicon wafer 1200. A mask is put on the photoresist 1210. The mask contains a pattern of lung tissue culture region 1010. The mask allows ultraviolet light to pass through the photoresist and expose only the portion corresponding to the lung region 1010. The photoresist is then developed to produce openings 1211 corresponding to the lung tissue culture region 1010. Next, the silicon wafer with the photoresist is etched to produce lung openings 1010 in the silicon wafer 1200. Subsequently, the photoresist 1210 is removed from the silicon wafer 1200, leaving the lung well 1010 on the silicon wafer. Subsequently, another layer of photoresist 1220 is placed on the wafer 1200. A mask is placed on the wafer. The mask allows exposure of various wells or fluid channels including 1021, 1031, 1041, and 1051, which are used to connect lung well 1010 with various wells 1020, 1030, 1040, and 1050. . The mask exposes the photoresist in the region of the fluid channel. The photoresist is then developed to remove the exposed photoresist corresponding to the fluid flow channel. Subsequently, the exposed area is etched to a desired depth. Thereafter, the remaining photoresist 1220 is removed, leaving the lung well 1010 and the other wells 1020, 1030, 1040, and 1050 on the silicon wafer 1200. The next step is to apply a third layer of photoresist 1230. A mask is applied over the photoresist, and the mask has openings corresponding to the various wells 1020, 1030, 1040, and 1050. The photoresist is masked and exposed to ultraviolet light to produce openings corresponding to various wells. The photoresist is developed, leaving exposed silicon areas for wells 1020, 1030, 1040, and 1050. The chip and photoresist 1230 are then etched to produce wells 1020, 1030, 1040, and 1050. The openings corresponding to the tissue modules 1020, 1030, 1040, 1050 are etched with plasma to a depth of about 750 micrometers. The opening is further wet etched with KOH at 250 micrometers to form a tapered end. KOH will etch silicon at an angle of 54.7 degrees along its crystal plane. Subsequently, the photoresist is removed and a silicon wafer is formed from which a nickel master can be made.

  Nickel master 1250 is created by electroplating nickel on the silicon chip. Next, the polymer substrate 1000 is cast or embossed using the nickel master. For replication molding, the polymer is melted or solubilized in a suitable solvent and poured onto the nickel master 1250 and solidified into the same shape as the original silicon chip. See FIG. 5 for embossing. Subsequently, the polymer chip 1000 is attached to the silicone elastomer trough 1110. Since the polymer and silicone are self-sealing, the layers will form a single unit. A pneumatic actuator 1120 is placed under the chip to pump fluid collected from the various tissue modules 1020, 1030, 1040, 1050. Every second, the trough is filled with 0.032 microliters of fluid. The actuator then pushes up the silicone, allowing fluid to escape through the microtubules and back to the lung compartment 1010. Elastomeric trough 1110 and actuator 1120 form pump 260 (shown in FIG. 12). Subsequently, an elastomer-coated polymethyl methacrylate (PLEXIGLAS ™) 1140 seals the top of the wafer or chip 1000.

  To balance the tensile pressure created when the silicone is filled with liquid, the polymethyl methacrylate (PLEXIGLAS ™) on the lung cell compartment 1010 is removed and replaced with a silicone membrane. This membrane moves up and down under the action of a silicone pump to keep the pressure in the device balanced. Prior to applying elastomer-coated polymethylmethacrylate (PLEXIGLAS ™) over the chip 1000, various microscale tubes are placed in the wells. A machine for handling microtubules includes an adhesive arm that lowers to retrieve a specific number of tissue-loaded tubes. The machine transports the tube to the device and tightly packages the tube within each module well 1020, 1030, 1040, 1050. Tight packaging allows frictional forces to keep the tube in place despite any agitation on the cell culture analog device. This minimizes fluid flow leakage around the tube in each well 1020, 1030, 1040, 1050. Even when tight, approximately 5-10% of the fluid flow bypasses the tube and flows directly to the silicone-based or elastomeric trough 1110.

  FIG. 13 shows an elastomeric trough. An elastomeric trough is a piece of silicone elastomer with an essentially rectangular opening in it. The rectangular opening acts as a fluid container for fluid originating from wells 1020, 1030, 1040, and 1050. Elastomeric trough 1110 has an opening on one side denoted by reference numeral 1300. The return line 1130 has one end attached to the opening 1300 in the elastomer trough 1110 and the other end attached to the lung well 1010 of the chip 1000.

  In yet another embodiment, the elastomeric trough 1110 is replaced with a silicone elastomer pump 1400, which is shown in FIG. The silicone elastomer pump 1400 is designed to more accurately reproduce the circulatory system flow on the chip 1000 and throughout the system depicted by reference number 1100. Pump 1400 includes a first lung chamber 1410 and a second system chamber 1412 that are actuated by isolation actuators 1420 and 1422. Multiple chambers 1410 and 1412 create a physiologically more realistic pump pattern with a multi-trough elastomer base at the bottom of the chip 1000. By creating a plurality of chambers 1410 and 1412 in the silicone elastomer trough 1400 by having an actuator that pushes up the base section at specific time intervals, the pumping action of the heart is replicated.

  FIG. 28A is a block diagram diagram illustrating a system for controlling a microscale culture device, according to one embodiment of the present invention. In this embodiment, system 2800 includes a first microscale culture device 2806 coupled to controller 2802. The first microscale culture device 2806 includes a plurality of microscale chambers (2808, 2810, 2812, and 2814) having a surface shape that simulates a plurality of in vivo interactions with the culture medium, each chamber comprising: Includes inlets and outlets for media flow and microfluidic channels interconnecting the chambers. The controller 2802 includes a computer 2804 for obtaining data from the first microscale culture device 2806 and controlling its pharmacokinetic parameters.

  In another embodiment, the first microscale culture device 2806 is formed on a computerized chip. The first microscale culture device 2806 further includes one or more sensors coupled to a controller 2802 for measuring physiological events in the chamber. The sensor includes one or more biosensors that monitor medium oxygen, carbon dioxide, or pH. Controller 2802 holds first microscale culture device 2806 and seals the top of first microscale culture device 2806 to establish a microfluidic channel. The control equipment 2802 provides microfluidic interconnections whereby the microfluidic flows to / from the device. In another implementation, the computer 2804 controls a pharmacokinetic parameter of the first microscale culture device 2806 selected from the group consisting of pump group speed, temperature, length of experiment, and frequency of data acquisition. In one implementation, the computer 2804 provides a setup screen so that the operator can also manually specify the pump speed, device temperature, length of experiment, and frequency of data acquisition (eg, every 15 minutes). To do. In another implementation, the computer 2804 controls a pharmacokinetic parameter in the first microscale culture device 2806 selected from the group consisting of flow rate, chamber surface shape, and number of cells. In this implementation, the system 2800 provides a more rapid and sensitive response compared to whole animal studies and traditional tissue culture studies. By controlling the parameters, the system 2800 is no longer physiological based. In another implementation, the computer 2804 may generate a first microscale culture device to create a medium residence time in the chamber (2808, 2810, 2812, and 2814) that is comparable to that encountered in vivo. One or more pumps in 2806 are further controlled. In another implementation, the computer 2804 further controls one or more valves distributed along the microfluidic channel in a manner consistent with pharmacokinetic parameter values associated with the simulated part of the organism.

  In another embodiment, the system 2800 further includes a second microscale culture device having a plurality of microscale chambers having a surface shape that simulates a plurality of in vivo interactions with the media, each chamber comprising a media And inlets and outlets for the flow of gas and microfluidic channels interconnecting the chambers. The control device 2802 is connected to the second microscale culture device.

  FIG. 28B is a block diagram illustrating another embodiment of a system for controlling a microscale culture device. In this embodiment, the system 2816 includes a first microscale culture device 2806 coupled to a control device 2818. The control device 2818 is a computer 2804, a pump 2820 for controlling microfluidic circulation in the microfluidic channel of the first microscale culture device 2806, and a temperature for controlling the first microscale culture device 2806. A heating element 2822, a light source 2824, and a photodetector 2826 for detecting fluorescence emission from the cell compartment in the first microscale culture device 2806. In one implementation, the computer 2804 records data representing fluorescence intensity using a type of measuring instrument selected from the group consisting of colorimetric, fluorescent, luminescent, and radiometric. In another implementation, the heating element 2822 maintains the first microscale culture device 2806 at a temperature of 37 degrees Celsius.

  FIG. 29 is a flow diagram illustrating a computerized method for dynamically controlling a microscale culture device, according to one embodiment of the present invention. In this embodiment, computerized method 2900 includes blocks 2902, 2904, 2906, and 2908. Block 2902 includes analyzing data from a plurality of sensors for measuring physiological events in the plurality of chambers of the microscale culture device. Block 2904 includes adjusting the fluid flow rate of the media in the chamber of the microscale culture device. Block 2906 includes detecting a biological or toxic response in the chamber of the microscale culture device. Block 2908 includes changing one or more pharmacokinetic parameters of the microscale culture device upon such detection.

  In one embodiment, block 2906 (ie, detecting) includes detecting a change in the size of the cell compartment of the microscale culture device. In one implementation, block 2908 (i.e., changing) comprises selecting a drug selected from the group consisting of cell-cell interaction, liquid residence time, liquid-to-cell ratio, cellular metabolism, and shear stress in a microscale culture device. Changing the kinetic parameters. In another implementation, block 2908 includes changing a pharmacokinetic parameter selected from the group consisting of flow rate, chamber surface shape, and number of cells in the microscale culture device.

  In another embodiment, the computerized method 2900 further includes optimizing the chamber surface shape within the microscale culture device, the optimizing step including selecting a chamber quantity and an appropriate Selecting a chamber surface shape that provides a tissue or organ size ratio; selecting an optimal fluid flow rate that provides an adequate liquid residence time; and calculating a cell shear stress.

  In another embodiment, the computerized method 2900 further includes adjusting the temperature of the medium. In yet another embodiment, the computerized method 2900 further comprises detecting fluorescence emission from the cell compartment of the microscale culture device.

  In another embodiment, a computer-readable medium includes computer-executable instructions stored thereon for performing the various embodiments of the computerized method described above. In one implementation, the computer-readable medium includes a memory or a storage device. In another implementation, a computer readable medium includes a computer data signal embodied in a carrier wave.

  FIG. 30 is a block diagram illustrating a computer for controlling a microscale culture device according to one embodiment of the invention. In this embodiment, the computer 3000 includes an input / output interface 3008 that includes an interface with a microprocessor 3002, a general purpose memory 3004, a non-volatile storage element 3006, and a microscale culture device having one or more sensors, and computer software. Including. The computer software adjusts the fluid flow rate of the medium in the multiple chambers of the microscale culture device to detect biological or toxic reactions in the chambers of the microscale culture device, and upon detection, Executable in microprocessor 3002 to change one or more pharmacokinetic parameters.

  In one embodiment, non-volatile storage element 3006 includes historical data taken from publicly available information, data collected from prior performance tests, or data derived from theoretical calculations. The computer software adjusts the fluid flow rate by sending commands to one or more pumps of the microscale culture device via the pump control line. In one implementation, the computer software is further executable in the microprocessor 3002 to adjust the temperature of the medium. The computer software adjusts the temperature by sending commands to the heating coil of the microscale culture device via the heating coil control line.

  In another embodiment, the computer 3000 is a look-up table coupled to a general purpose memory 3004 for storing a set of mass balance equations representing physiological pharmacokinetic models of various biological or chemical substances in the system. Further included is a memory and a cache memory coupled to the microprocessor 3002 for storing computer software.

  In another embodiment, the input / output interface 3008 further includes a keyboard interface, a display interface, and a printer / plotter recorder interface. In one implementation, the computer 3000 uses these input / output interfaces to connect to keyboards, displays, and printer / plotter recorder peripheral devices.

The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention and are not intended to limit the scope considered as the invention. .

  Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, concentration), but some experimental error and deviation have occurred. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Method In the experimental process, the following method was used:
Cell culture. The cells were obtained from the American Type Culture Collection (Manassas, VA)
Propagated in recommended complete growth medium in a tissue culture incubator (95% O ₂ /5% CO ₂ ). For HepG2 and HepG2 / C3A cells, the recommended medium is Eagle's minimum essential medium (Earle's balanced salt solution, 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino supplement, 1.5 g / L sodium bicarbonate, and 10% fetal bovine serum) (EMEM). McCoy's 5A medium with 1.5 mM L-glutamine, 1.5 g / L sodium bicarbonate, and 10% fetal calf serum is recommended for HCT-116.

  Growth curve. Growth curves were measured by plating the cells at an initial low density in a 35 mm dish. Every day, cell numbers were determined by detaching the cells with trypsin-EDTA and visually counting the cells using a hemocytometer. The measurement was performed in three ways.

  Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Cells were cultured on glass coverslips treated with collagen, MATRIGEL ™, or polylysine as needed. HepG2 / C3A grown to ~ 90% confluent monolayer was desorbed with trypsin-EDTA and precipitated for ~ 500g for 5 minutes. RNA was isolated and purified with the RNEASY ™ kit (Qiagen) according to the manufacturer's protocol. Adult human liver total RNA was purchased from Ambion. The amount and purity of the isolated RNA (260/280 nm ratio) was measured with a BIOPHOTOMETER ™ spectrophotometer (Eppendorf). The isolated RNA was then incubated with 2 U Dnase I for 25 minutes at 37 ° C. and then inactivated with DNase Inactivation Reagent (Ambion).

  The RT reaction was performed using a mixture of 5 μg RNA, 10 μM oligo dT primer that was heated to 72 ° C. for 2 minutes and then placed on ice for 2 minutes. Next, 5 mM DTT, 600 μM dNTP mix, 40 U rRNasin, 200 U SUPERSCRIPT II ™ were combined in reverse transcriptase buffer and incubated at 42 ° C. for 1 hour.

  Primers specific for the cytochrome P450 isoform were used and 2.0 μl of first strand cDNA was used in a 50 μl PCR reaction (Rodriguez-Antona, C., Jober, R., Gomez-Lechon, M.-J. , And Castell, JV (2000), Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies, Arch. Biochem. Biophys. The PCR conditions were 28 cycles of 94 ° C for 4 minutes followed by 94 ° C for 40 seconds, 60 ° C for 45 seconds, 72 ° C for 50 seconds, and 72 ° C for the last 4 minutes extension.

  PCR products were separated by electrophoresis on a 1.2% agarose gel and visualized by staining with SYBR Gold to compare authenticity with appropriate molecular weight standards. To quantify the amplified cDNA, 15 μl of each PCR reaction was diluted with 0.1 × Tris-EDTA buffer and stained with PICOGREEN ™ (Molecular Probes) at a final concentration of 1: 400. When fluorescence was measured, it was 480 nm excitation and 520 nm emission. Results were normalized to β-actin and performed in triplicate from at least two separate experiments.

  Cell survival, death, and apoptosis assays. Cell viability and cell death were measured using trypan blue dye exclusion or LIVE / DEAD staining (Molecular Probes). Trypan blue (GIBCO) is usually excluded from the cytoplasm and distinguishes cells with damaged membranes by visually staining dead or dying cells blue. At the end of the experiment, a 1: 1 dilution of 0.4% (w / v) trypan blue solution is added to the recirculation medium of the chip device. This solution was pumped through the tip for 30 minutes at room temperature and discarded. The housing was removed from the pump and visualized under a reflection microscope (Micromaster, Fisher).

  LIVE / DEAD staining is a two-component staining consisting of calcein AM and ethidium homodimer. Living cells actively hydrolyze the acetoxymethyl ester (AM) portion of calcein AM to produce the bright green fluorescence of calcein. In contrast, cells with damaged membrane integrity usually allow membrane-impermeable ethidium homodimers to stain the nuclei of dead or dying cells in a fluorescent red color. The cell permeable nuclear stain Hoechst 33342 acts as a general stain for all cells. With the appropriate filter set, live cells are fluoresceed green, dying or dead cells are red, and all cells are quantified by blue nuclear fluorescence. In the experiments described herein, trypan blue was 0.2% (w / v), calcein AM was 1: 20,000, propidium iodide was 1: 5,000, and Hoechst 33342 was 10 μg / ml. used. Cells were visualized by M2Bio stereofluorescence microscope (Zeiss). The AU experiment was repeated at least three times to produce three measurement results.

  Numerous methods can be used to monitor apoptosis, or programmed cell death (Smyth, PG, Berman, SA, and Bursztajn, S. (2000), Markers of apoptosis. : Methods for elucidating the mechanical of apoptotic cell death from the nervous system, Biotechniques, 32: 648-665). To distinguish apoptosis from necrosis, at least two separate indicators of apoptosis are required (Wronsla, R., Golob, N., and Gryger, E. (2002), Two-color, fluorescence-based microplate. assay for apoptosis detection, Biotechniques, 32: 666-668). One method, annexin V-FITC binding, relies on the observation that annexin V is tightly bound to phosphatidylserine in the presence of divalent calcium (Williamson, P., Eijnde, Svd., And Schlegel). R.A. (2001), Phosphatidylserine exposure and phagocytosis of apoptotic cells, In Apoptosis, L. M. Schwartz and J. D. Ashwell, Ed. Normally, phosphatidylserine is present on the inner leaflet of the cell membrane, but translocates to the cell membrane early in apoptosis. Cells that have been apoptotic when exposed to annexin labeled with fluorophosphate exhibit distinct membrane staining. On a microscale chip, the annexin V-FITC label is placed on the jaw by first flushing the system with PBS and then recirculating Annexin V-FITC (10 μg / ml in Clontech in Annexin V binding buffer) for 30 minutes. Direct visualization. Subsequently, the cells were directly visualized using a FITC filter set.

  In contrast to Annexin V labeling, the APOPTAG ™ kit (Intergen Co., MA) exposed free 3'-OH DNA ends exposed during visualization using DNA degradation and immunofluorescence methods associated with apoptosis. Terminal deoxynucleotide transferase (Li, X., Traganos, F., Melamed, MR, and Darzynewicz, Z. (1995), Single-step procedure for labeling DNA strand breaks with flourescein-or BODPY-conjugated deoxynucleotides: detection of apoptosis and bromodex yuridine information, Cytometry 20, 172-180). Although this method is highly specific for apoptosis, the procedure cannot be performed on-chip due to the solidification and incubation steps. Briefly, microscale chips are run under certain experimental conditions, cell chips are removed from their housing units, fixed in 1% paraformaldehyde, and APOPTAG ™ using manufacturer's protocol. Treated with kit.

  Microscale chip fabrication and experimental methods. A microscale chip was fabricated as follows: a pattern was designed using Computer Assisted Design (CAD) software (Cadence), and a chrome photomask was created using GCA / Mann3600F Optical Pattern Generator. did. Subsequently, the wafer is exposed to ultraviolet rays through a photomask using a Karl Suss MA6 Contact Aligner, and this high-resolution pattern is converted into silicon containing a thin coating (˜1 μm) of a positive photoresist (Shipley 1813) Transferred to a wafer (3 inches in diameter). After exposure, the photoresist was developed to expose the silicon through the photoresist layer in the defined pattern. The exposed silicon was etched to a specific depth (20-100 μm) using a PlasmaTherm SLR 770 ICP Deep Silicon Etch System. The photoresist was stripped from the wafer using acetone. Individual 22 mm square microscale chips were diced from the wafer, washed in Nanostrip (Cyantek), rinsed in distilled water, and dried in a 170 ° C. drying oven.

The surface of silicon in the organ compartment was treated with collagen to promote cell attachment. About 10 μl of a 1 mg / ml solution of type I collagen was deposited on the surface of the microscale chip and incubated at room temperature for 30 minutes. The collagen solution was removed and the organ compartment was rinsed with cell culture medium. Cells were dissociated from the cell culture dish, the number of cells was measured, and the concentration was adjusted so that there was a confluent monolayer of cells within each cell compartment. For example, in the microchip described above in FIG. 2 (above), 10 μl of a 2,400 cell / μl suspension of L2 cells is deposited in the lung chamber of the cell chip and 15 μl of H4IIE cells are suspended at 3,400 cells / μl. The turbid liquid was deposited in the liver chamber. Cells were allowed to attach overnight in a CO ₂ incubator. When the cells attached, the chip was assembled in an acrylic chip housing. The top of the housing contains fluid interconnects to provide cell culture fluid to the chip. A stainless steel tube is connected to a micro-diameter pump tube and inserted into a small hole at the top of the microcentrifuge tube containing the medium with or without the test compound. Connect the pump tube to the peristaltic pump, prime with this solution and connect to the inlet port of the chip housing. The recirculating fluid circuit is completed by connecting a small section of a pump tube with a stainless steel tube connected to the end to the outlet port and inserting the tube into a small hole at the top of the microtube. Place the entire instrument in a 37 ° C. CO ₂ incubator. A schematic diagram of this setting is presented in FIG.

Example 1
Circulation of a system for replicating rats In the design of the chip 1000, all necessary chambers were housed in a silicon chip not exceeding 2 cm x 2 cm. This size tip is easy to manufacture and adapts to the size of the connecting tube and pumping device intended to be used to direct fluid flow. There are several other important factors that constrain device design, listed below along with the acceptable values for each variable. This one embodiment of the device consists of two compartment systems, one compartment representing the rat liver and one compartment representing the rat lung. The total size of the chip is 2 cm × 2 cm, 20 parallel channels 40 μm wide, 10 μm deep, 5 mm long to serve as a “lung” chamber, and a width to serve as a “liver” chamber It consists of an interconnected array of two parallel channels in a serpentine shape, 100 μm, 20 μm deep and 10 cm long. The two organ compartments are connected by a channel 100 μm wide and 20 μm deep. There are many other possible surface shapes, dimensions, number of chambers, etc. This design was chosen as an example.

計算例
チャネルまたはチャンバ計算：
これらの計算では、システム１１００のチップ１０００に関し、上述した方法によって、前回の反復から流速を得たものと仮定する。

Calculation example Channel or chamber calculation:
These calculations assume that the flow rate has been obtained from the previous iteration for the chip 1000 of the system 1100 in the manner described above.

Thus, Q = 8.05 × 10 ⁵ μm ³ / trench seconds.

  Subsequently, the liquid residence time in the trench was calculated as follows:

次に、「細胞長さ」中の細胞数を計算した。

Next, the number of cells in the “cell length” was calculated.

続いて、チャネル／チャンバの細胞長さ容積を計算した。

Subsequently, the cell length volume of the channel / chamber was calculated.

細胞長さ容積も測定した。

Cell length volume was also measured.

液体の細胞長さ容積は、チャネル／チャンバの細胞長さ容積から単純に細胞の細胞長さ容積を減じたものである。細胞の細胞長さ容積と液体の細胞長さ容積の比は、システムの液体対細胞容積比を求めるものである。

The cell length volume of the liquid is simply the cell length volume of the cell minus the cell length volume of the channel / chamber. The ratio of the cell length volume of the cell to the cell length volume of the liquid determines the liquid to cell volume ratio of the system.

所与の流速と関連付けられた個々の細胞にかかるせん断力を測定した。液体の細胞長さ容積および細胞の直径に基づいて、液体を通過させるために利用可能なアベレージ表面積を計算した。

The shear force on individual cells associated with a given flow rate was measured. Based on the cell length volume of the liquid and the cell diameter, the average surface area available to pass the liquid was calculated.

続いて、チャネル内のアベレージ線速度を計算した。

Subsequently, the average linear velocity in the channel was calculated.

層流を前提として、球体にかかる抵抗を計算するためにストークスの法則を使用し、個々の細胞が受ける全せん断力を推定した。

Assuming laminar flow, Stokes' law was used to calculate the drag on the sphere, and the total shear force experienced by individual cells was estimated.

次に、チャネル／チャンバ内における液体の実際の滞留時間を検証し、チャネル／チャンバ内にある全細胞数まで計算した。

Next, the actual residence time of the liquid in the channel / chamber was verified and calculated to the total number of cells in the channel / chamber.

Ｉ．Ｂ．膜酸化計算：
シリコーン膜の酸化する面積を以下のようにして測定した：
まず、細胞の酸素摂取速度（Ｏｘｙｇｅｎ Ｕｐｔａｋｅ Ｒａｔｅ；ＯＵＲ）の近似値を求める：

I. B. Membrane oxidation calculation:
The oxidized area of the silicone membrane was measured as follows:
First, an approximate value of the oxygen uptake rate (OUR) of a cell is obtained:

続いて、液体媒質を再酸化するのに十分か否かを判断するために、膜の内側にかかる酸素の分圧を計算する。これは、多孔質膜を通るガスの流束の方程式を使用して為され、ここでＱは膜透過性である。Ｊは細胞へのガスの流束を表し、ｚは膜の厚さである：

Subsequently, to determine whether the liquid medium is sufficient to reoxidize, the partial pressure of oxygen applied to the inside of the membrane is calculated. This is done using the equation of gas flux through the porous membrane, where Q is membrane permeable. J represents the gas flux into the cell and z is the membrane thickness:

この圧力は、膜と接触している液体媒質に２００秒で酸素を飽和させるのに十分である。内側の酸素分圧を最大化するように、反復様式で膜の面積を測定した。

This pressure is sufficient to saturate oxygen in the liquid medium in contact with the membrane in 200 seconds. Membrane area was measured in an iterative fashion to maximize the inner oxygen partial pressure.

（実施例２）
４つの臓器コンパートメントチップ
チップは、４つの臓器コンパートメント―異物代謝に関与する臓器を表すための「肝臓」コンパートメント、標的組織を表す「肺」コンパートメント、部位に疎水性化合物の生物蓄積を提供するための「脂肪」コンパートメント、および、非代謝、非蓄積の組織において循環パターンを模倣するのを補佐するための「その他の組織」コンパートメント―から成るように設計した（図１５）。これらの、およびその他の臓器コンパートメント（例えば、腎臓、心臓、結腸、または筋肉）は、ＣＡＤファイルとして完全にモジュール化され、任意の構成または組み合わせで作製されることができる。デバイス自体を任意の数の基板（例えば、シリコン、ガラス、またはプラスチック）内に産生することができる。

(Example 2)
4 organ compartment chips The chip is composed of 4 organ compartments—the “liver” compartment to represent the organs involved in xenobiotic metabolism, the “lung” compartment to represent the target tissue, and the site to provide bioaccumulation of hydrophobic compounds It was designed to consist of a “fat” compartment and an “other tissue” compartment to assist in mimicking the circulatory pattern in non-metabolizing, non-accumulating tissues (FIG. 15). These and other organ compartments (eg, kidney, heart, colon, or muscle) are fully modularized as CAD files and can be created in any configuration or combination. The device itself can be produced in any number of substrates (eg, silicon, glass, or plastic).

  Once the cells were seeded in the appropriate compartment, the chip was assembled in the Lucite manifold. This manifold holds 4 tips and contains a transparent top so cells could be observed in situ. The upper part containing the fluid is interconnected to provide the cell culture medium to the chip. Using a peristaltic pump, the medium was pumped through the chip at a flow rate of 0.5 μl / min. The media was recirculated in a closed loop consisting of a fluid container (total volume ˜15 to 50 μl), micro-diameter tube, and chip compartments and channels.

  Using a three-compartment system with human HepG2-C3A cells in the liver compartment and HT29 colon cancer cells in the target tissue compartment, the cells remain viable for over 144 hours under continuous operation I understood. HepG2-C3A cells are a well-characterized human hepatocyte cell line that is known to express various hepatic metabolic enzymes at levels comparable to fresh primary human hepatocytes. In these experiments, cells were seeded in the appropriate compartments and the specially formulated cell culture medium was recirculated through the system for up to 144 hours. At various time points within 30 minutes, the medium was switched to PBS-containing LIVE / DEAD fluorescent reagent (Dual Fluorescent Stain (Molecular Probes, Inc., Eugene, Oreg., USA)). Cells were visualized under a fluorescence microscope and fluorescent images of the same field were obtained using an appropriate filter set. Live cells emitted green fluorescence, while dead cells were red (data not shown).

(Example 3)
Drug metabolism within the chip A microscale chip with three compartments, liver, target tissue, and other tissues was used to study the metabolism of two widely used prodrugs, tegafur and sulindac sulfoxide. Both prodrugs require conversion to active metabolism by enzymes present in the liver and have a cytotoxic effect on the target organ. For the prodrug sulindac sulfoxide, its anti-inflammatory and cancer chemopreventive properties are derived from its sulfide and sulfone metabolites catalyzed by the liver enzyme sulfoxide reductase. The sulfone metabolite (and the second sulfone metabolite) has been demonstrated to induce apoptosis in some cancer cells (eg, colon cancer).

  With proper treatment planning, 5-FU itself is extremely toxic to normal cells, so its prodrug, tegafur [5-fluoro-1- (2-tetrahydrofuryl) -2,4 (1H, 3H)- Administration of [pyrimidinedione]. However, unlike sulindac, tegafur is first converted to 5-FU in the liver by cytochrome P450 2A6.

  To test the efficacy of sulindac, HepG2-C3A cells were seeded in the liver compartment and HT29 human colon cancer cells in the target tissue compartment of the microscale chip. 100 micromoles of sulindac (requires manufacturer) was added to the recirculation medium for 24 hours and the chip was treated as described above—live cells fluoresced green while dead cells fluoresced red. (Data not shown). In the absence of HepG2-C3A hepatocytes, a minimal level of cell death (similar to vehicle control) was observed. These results indicate that the drug can be metabolized within the liver compartment and thus circulate to the target and induce its biological effects as much as in live animals or humans. It has been demonstrated.

  A prodrug for cancer treatment, tegafur, was tested in a microscale chip system. For efficacy, tegafur requires metabolic activation to its active form 5-fluorouracil (5-FU) by cytochrome P450 enzymes present in the liver (Ikeda, K., Yoshisue, K., Matsushima, E., Nagayama, S., Kobayashi, K., Tyson, CA, Chiba, K., and Kawaguchi, Y. (2000), Bioactivation of terafur toyrocyl to fluoriscyl to 5-fluoric 450 2A6 in human river microsomes in vitro, Clin. Cancer Res., 6, 4409-4415; Komatsu, T., Ya mazaki, H., Shimada, N., Nakajima, M., and Yokoi, T. (2000), Roles of cytochromes P450 1A2, 2A6, and 2C8 in 5-fluorouraformation. Micromes, Drug Met.Disp., 28, 1457-1463; Yamazaki, H., Komatsu, T., Takemoto, K., Shimada, N., Nakajima, M., and Yokoi, T. (2001). cytochrome P450 IA and 3A enzymes in evolved in bio ctivation of tegafur to 5-fluorouracil and autoinduced by tegafur liver microsomes, Drug Met.Disp., 29,794~797). Proper treatment regulations require the administration of its prodrug, tegafur, because 5-FU itself is extremely toxic to normal cells. 5-FU is currently the most effective adjuvant therapy for patients with colon cancer (Hwang, PM, Bunz, F., Yu, J., Rago, C, Chan, TA, Murphy, M.) P., Kelso, GF, Smith, R.A.J., Kinzler, K.W., and Vogelstein, B. (2001), Ferredoxin reductase effects p53-dependent, 5-fluorourcidal-inducto-purecapped in collective cancer cells, Nat. Med., 7, 1111-1117). Like most chemotherapeutic agents, 5-FU induces significant apoptosis in susceptible cells by the generation of reactive oxygen species (Hwang, PM, Bunz, F., Yu, J., Rago, C , Chan, TA, Murphy, MP, Kelso, GF, Smith, RAJ, Kinzler, KW, and Vogelstein, B. (2001), Ferredoxin reductase. effects p53-dependent, 5-fluorouracil-induced in collective cancer cells, Nat. Med., 7, 1111-1117).

  To measure the cytotoxic effect of tegafur on colon cancer cells, microscale chips were prepared with HepG2-C3A cells in the liver compartment and HCT-116 human colon cancer cells in the target tissue compartment. Tegafur was added to the recirculation medium at various concentrations for 24 hours and the cells were labeled with Hoechst 33342, a membrane-permeable DNA dye, and ethidium homodimer, a membrane-impermeable DNA dye (see methods section). All cells emitted blue fluorescence while dead cells were marked with a fluorescent red ethidium homodimer (data not shown). Tegafur was cytotoxic to HCT-116 cells in a dose-dependent manner within this microscale chip system, but was ineffective in conventional cell culture assays (FIGS. 16A and 16B). 5-FU also triggered cell death in a conventional cell culture assay, but no cytotoxicity was observed until 48 hours after exposure compared to 24 hours after exposure to tegafur with a microscale chip.

  To demonstrate that the liver compartment was involved in the bioactivation of tegafur, only HCT-116 was plated on a microscale chip. There were no cells in the liver compartment. Tegafur or 5-FU was added to the recirculation medium for 24 hours and the chips were processed as described above (data not shown). Tegafur did not cause significant cell death of HCT-116 in the absence of the liver compartment, while the active metabolite 5-FU caused substantial cell death. Furthermore, tegafur had no effect when HCT-116 was replaced with HT-29 colon cancer cells (data not shown). This was likely due to the mutant p53 present in HT-29 cells, which is required for 5-FU cytotoxicity. Together, these experiments demonstrated that tegafur like sulindac is metabolized to the active drug within the liver compartment and circulated to another organ compartment to drain cancer cells. These effects were mechanically distinguishable by chip-sulindac was effective even in the absence of active p53, tegafur was not.

Example 4
Multiple cell cultures in a single organ compartment It is also possible to use a mixture of multiple cell types within a single organ compartment. In one study, the hepatocyte cell line HepG2 / C3A (from ATCC) is used in the liver compartment. Cells are propagated in McCoy's 5A medium supplemented with 1.5 mM L-glutamine, 1.5 g / L sodium bicarbonate, and 10% fetal calf serum. To more closely mimic in vivo organs, a mixture of primary hepatocytes and fibroblasts may be used with macrophages (Kupffer cells) in a 1 to 2 ratio.

  In another example, a mixture of cells or cell lines derived from lung epithelial cells is used to more closely mimic lung tissue. This includes a mixture of type I epithelial cells, type II epithelial cells (large alveolar cells), fibroblasts, macrophages, and mast cells.

(Example 5)
Optimization of tissue culture conditions in a chip-based system A tissue culture medium was developed that is compatible with two different rat cell culture lines, H4IIE (rat liver cell line) and L2 (rat lung cell line). Preliminary experiments show that a 1: 1 mixture of DMEM and Hams F12K medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS) is able to survive both H4IIE and L2 cells on a microscale chip. Was maintained for up to 20 hours of continuous operation. This media formulation was used for all rat-based microscale chip studies.

  Appropriate human cell lines that realistically mimic human liver function were selected. Furthermore, the optimal cell culture medium formulation for maintaining the human cell line on the microscale chip was determined. The basal expression levels of the three major cytochrome P450 (CYP) isoforms (1A2, 3A4, and 2D6) in HepG2 and HepG2 / C3A (HepG2 subclone) cell lines were examined. CYP-1A2, 2D6, and 3A4 were examined because they are responsible for 80-90% metabolism of known drugs (Hodgson, J. (2001), ADMET-turning chemicals into drugs, Nat. Biotech., 19, 722-726). This cell line was tested because the C3A subclone of the HepG2 hepatocyte cell line was reported to be a more carefully selected cell line that exhibited more “liver-like” properties, particularly significantly higher CYP expression compared to the parent cell line. (Kelly, JH (1994), Permanent human hepatocyte cell line and its use in a liver assist device (LAD), US Pat. No. 5,290,684). RT-PCR analysis confirmed that the amount of basal CYP in HepG2 / C3A cells was considerably larger than that of parental HepG2 and comparable to that of adult human liver (FIG. 23).

  In all subsequent experiments, HepG2 / C3A cells were used as liver substitutes. A number of media components were compared to select common media for use during microscale chip experiments (DMEM, McCoy 5A, RPMI 1640, MEM, F12, F12K, Wayout's, CMRL, MEM , And Iscove's modified Dulbecco medium). Analysis of inorganic salts, glucose, amino acid composition, and vitamin content suggested that EMEM, DMEM, McCoy's 5A, and RPMI were the most appropriate “generic” media among the media examined. After several passages, the cells were divided and subcultured in the following medium.

  Earle's minimum essential medium supplemented with Earle's balanced salt solution, 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino supplement, 1.5 g / L sodium bicarbonate, and 10% fetal calf serum (EMEM).

  Dulbecco's modified Eagle medium (DMEM) with 4 mM L-glutamine, 4.5 g / L glucose, 1.5 g / L sodium bicarbonate, and 10% fetal calf serum.

  McCoy's 5A medium (McCoy's) with 1.5 mM L-glutamine, 1.5 g / L sodium bicarbonate, and 10% fetal calf serum.

  RPMI 1640 medium (RPMI) supplemented with 2 mM L-glutamine, 4.5 g / L glucose, 1.0 mM sodium pyruvate, 1.5 g / L sodium bicarbonate.

  Subsequently, growth curves representing both cell lines in each medium were measured as described in the method section (FIG. 24). DMEM was found to be unsuitable for HepG2 / C3A cells since significant changes in cell morphology were observed and adhesion was observed after passage 5 (not shown). Similarly, after 3 days in RPMI, a significant decrease in HepG2 / C3A and survival and growth of HCT-116 were noted. Both cell lines grew well in McCoy's and EMEM compared to their preferred medium.

  RT-PCR was then used to examine the expression levels of these CYP isoforms in HepG2 / C3A cells growing in EMEM or McCoy's (see Methods section) (Figure 25) . The results showed that EMEM is superior to McCoy's in maintaining CYP expression and is a preferred medium for HepG2 / C3A. The effect of different growth substrates on CYP expression was studied (Figure 26). A comparison of silicon treated with poly-D-lysine or collagen as an attachment substrate and cells grown on polystyrene with standard tissue culture treatment was performed. The results also showed that EMEM supports the growth of both HepG2 / C3A and HCT-116 cells and that collagen is a preferred substrate based on RT-PCR CYP expression analysis.

  Using these conditions, long-term cell survival of these cells, namely HepG2 / C3A and HCT-116, was studied under continuous operation in a microscale chip system. Using a three-compartment system with human HepG2 / C3A cells in the liver compartment and HCT-116 colon cancer cells in the target tissue compartment, the cells remain viable for more than 144 hours under continuous operation Prove that there is. In these experiments, cells were seeded in the appropriate compartment and EMEM was recirculated through the system for up to 144 hours. At various time points (6, 24, 48, 72, 96, 120, and 144 hours), live or dead cells were visualized using LIVE / DEAD staining (data not shown). Cells were visualized under a fluorescence microscope and fluorescent images of the same field were obtained using an appropriate filter set. Live cells emitted green fluorescence, while dead cells were red (data not shown).

(Example 6)
Assay for detection of toxicity on microscale chips Trypan blue is the most common stain used to distinguish viable cells from non-viable cells, and only non-viable cells absorb the dye and become blue Looks like. Conversely, living healthy cells appear to be circular and refracted without absorbing the blue dye. To determine cell survival on the microscale chip, experiments were performed using trypan blue. Trypan blue (see method section) is easy to use and only requires a light microscope to visualize, but over time, viable cells will absorb trypan blue, which can affect the results. Also, trypan blue has a higher affinity for serum proteins than for cellular proteins, so the background is dark when using serum-containing media. Therefore, alternative methods for distinguishing viable cells from dead cells were studied.

  Cells grown on glass coverslips were used to optimize the LIVE / DEAD assay (see Methods section). Briefly, HepG2 / C3A cells were seeded on glass coverslips treated with poly-D-lysine and treated with and without 1 μM staurosporine for 24 hours. Staurosporine is a broad-spectrum protein kinase inhibitor and is known to induce apoptosis in various cell types (Smyth, PG, Bemian, SA, and Bursztajn, S. (2002). ), Markers of apoptosis: methods for elucidating the mechanism of an apoptotic cell death from the nervous system, Biotechniques, 32, 648-66. The coverslips were washed with Phosphate Buffered Saline (PBS), LIVE / DEAD reagent was added and incubated for 30 minutes at room temperature. Cover slips were removed and visualized (data not shown). Staurosporine was clearly found to cause cell death of HepG2 / C3A cells (data not shown).

  Subsequently, the assay for detection of cytotoxicity in the microscale chip system was optimized. As described in the methods section, the microscale cell chip was seeded with HepG2 / C3A cells in the liver compartment and HCT-116 cells in the target tissue compartment. Cell chips were loaded into a microscale chip system and processed with and without 1 μM staurosporine as described above. After 24 hours of incubation, the recirculation medium was switched to PBS and allowed to flow through the system for 30 minutes and then discarded, followed by switching to PBS containing LIVE / DEAD reagent and allowed to flow through the system for an additional 30 minutes. The acrylic housing containing the cell chip was removed from the system, placed under a stereofluorescence microscope, and the cell chip was visualized through the transparent top of the housing (data not shown). Cells were visualized under a fluorescence microscope and fluorescent images of the same field were obtained using an appropriate filter set. Live cells emitted green fluorescence, while dead cells were red (data not shown). 1 μM staurosporine caused significant HCT-116 cell death after treatment for 24 hours compared to untreated control cell chips (data not shown).

(Example 7)
Two chip-based assays to detect the occurrence of cell death and distinguish between apoptosis and necrosis Two different assays to detect apoptosis were examined. The first assay was the immunofluorescence-based terminal deoxynucleotide transferase BrdU nick end labeling method available as a kit form as APOPTAG (Intergen Co., Mass.); TUNEL (See method section). Initially, the assay was optimized using cells grown on glass coverslips. Briefly, HepG2 / C3A cells were seeded on glass coverslips treated with poly-D-lysine and treated with and without staurosporine. Cover slips were processed as described above (see method section). Various staurosporine concentrations and treatment times were tested and the results showed that 1 μM staurosporine caused significant apoptosis after 24 hours incubation compared to untreated controls (data not shown) ). Next, the assay for detecting apoptosis in a microscale chip system was optimized and a comparison of the APOPTAG method with the LIVE / DEAD staining method was performed. As described in the methods section, the microscale cell chip was seeded with HepG2 / C3A cells in the liver compartment and HCT-116 cells in the target tissue compartment. Cell chips were loaded into a microscale chip system and processed with and without 1 μM staurosporine as described above. After 24 hours of incubation, the recirculation medium was switched to PBS for 30 minutes. Half of the cell chip was removed from the housing and the APOPTAG ™ assay was performed as described above. Other cell chips were left in the microscale chip system and subjected to the LIVE / DEAD staining method as described above. Cells were visualized under a fluorescence microscope and fluorescent images of the same field were obtained using an appropriate filter set. Live cells emitted green fluorescence, while dead cells were red (data not shown). Both techniques produced very similar results, ie 24 hours exposure to 1 μM staurosporine showed significant apoptosis (or cytotoxicity) for HCT-116 cells compared to untreated controls. (Data not shown).

  Annexin V-FITC was used to detect apoptosis in the microscale chip system as described in the methods section. Briefly, HepG2 / C3A cells were seeded in the liver compartment and HCT-116 cells in the target tissue compartment of the microscale cell chip. Cell chips were loaded into a microscale chip system and processed with and without 1 μM staurosporine as described above. After 6 hours of incubation, the recirculation medium was switched to PBS containing Annexin V-FITC and Hoechst 33342 and allowed to flow through the system for 30 minutes. Cell chips were removed from the acrylic housing and visualized under a fluorescence microscope. Cells were visualized under a fluorescence microscope and fluorescent images of the same field were obtained using an appropriate filter set. Live cells emitted green fluorescence, while dead cells were red (data not shown). 1 μM staurosporine caused significant apoptosis after 6 hours of treatment compared to untreated control cell chips (data not shown).

(Example 8)
Use of naphthalene as a model toxicant Naphthalene was used to study toxicology because pulmonary toxicity requires enzymatic conversion in the liver. Therefore, the effect of naphthalene on rat lung cell lines was studied. In these experiments, a three-compartment (liver, lung, and other tissue) rat-based microscale chip with H4IIE cells in the liver compartment and rat L2 cells in the lung compartment was used. Microscale chips were made and prepared for experiments as described in the methods section.

  The microscale chip system was operated for 20 hours in the presence or absence of 250 μg / ml naphthalene before switching to trypan blue with PBS. The solution was recirculated through the cell chip for 30 minutes and the chip visualized under a light microscope (see method section). Naphthalene caused significant cell death of rat L2 cells in the lung compartment of the cell chip, but no cell death was observed in the absence of naphthalene (data not shown). No cell death was observed in the H4IIE cell compartment with or without naphthalene or in the L2 cell compartment in the absence of H4IIE cells (data not shown).

  These results demonstrate that naphthalene is activated in the “liver” compartment and that toxic metabolites circulate in the “lung” causing cell death. These results were obtained with a benchtop CCA device and are consistent with the data expected from the PBPK model (Sweeney, LM, Shuler, ML, Babish, JG, and Ghanem, A (1995), A cell culture of rodent physology: application of napthalene toxiology, Toxicol, in Vitro, 9, 307-316).

Example 9
Human microscale chip prototype A human biochip prototype was prepared containing compartments representing lung, target tissue, and other tissues. The compartment and channel dimensions were as follows:

Entrance: 1mm x 1mm
Liver: width 3.2 mm x length 4 mm
Target tissue: 2mm x 2mm
Other tissues: width 340 μm x length 110 mm
Outlet: 1mm x 1mm
Channel connecting the liver to the Y connection: width 440 μm
Channel from Y connection to target tissue: width 100 μm
Create a human biochip prototype as described above. The arrangement of the organ compartment is intended to simulate exposure to a compound (drug) taken orally. When a compound is taken orally, it is absorbed into the blood from the small or large intestine. From there, it circulates directly to the liver via the portal vein and is distributed throughout the body (FIG. 27). Thus, in this design, the liver is the first organ compartment and is then divided into other tissues, target tissue compartments and chambers. The other tissue compartment represents the distribution and stagnation of blood in the body, and the target tissue compartment represents the desired therapeutic target (eg, colon cancer cells representing a colon tumor).

CONCLUSION The present invention typically includes a first cell culture chamber having a receiving end and an outlet end, a second cell culture chamber having a receiving end and an outlet end, and an outlet end of the first cell culture chamber. A pharmacokinetic-based culture device and system comprising a conduit connecting a portion to a receiving end of a second cell culture chamber. Preferably, the device is chip-based, i.e., microscale size. The medium can circulate through the first cell culture chamber, through the conduit, and through the second culture chamber. The medium can also be oxidized at one or more points in the recirculation loop.

  The device may include a mechanism for transmitting the signal from the device part to the off-chip position, for example with a waveguide for transmitting the signal from the device part to the off-chip position. . There may be a plurality of waveguides, for example, the first waveguide transmits signals from the first chamber, the second waveguide transmits signals from the second chamber, and so on. Become.

  In one embodiment, at least one of the first cell culture chamber and the second cell culture chamber is three-dimensional. In another embodiment, both the first cell culture chamber and the second cell culture chamber are three-dimensional.

  A device for maintaining cells in a viable state also includes a fluid circulation mechanism, and may be a flow-through fluid circulation mechanism or a fluid circulation mechanism that recirculates fluid. A device for maintaining cells in a viable state also includes a fluid pathway connecting at least the first and second compartments. In one embodiment, bubbles in the fluid path are removed with a bubble remover. The device can further include a pump mechanism. The pump mechanism may be located on the substrate.

  A method is provided for sizing the substrate to maintain at least two cell types viable in the at least two cell chambers. The method includes measuring the cell type retained on the substrate and applying physiological pharmacokinetic model constraints to measure the physical properties of the substrate. Applying the constraints of the physiological pharmacokinetic model comprises measuring the type of chamber formed on the substrate, the step comprising measuring the surface shape of at least one of the cell chambers; Measuring the surface shape of the flow path interconnecting the two cell chambers. Applying the constraints of the physiological pharmacokinetic model may include measuring the flow medium composition of the flow path.

  All publications and patent applications cited herein are hereby incorporated by reference as if each individual publication or patent application were instructed to be specifically and individually incorporated by reference. Incorporated.

  It should be understood that the above description is intended to be illustrative and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

  One embodiment of the invention relates to a microscale permeable material. Although some embodiments of the present invention describe permeable materials as biological barriers associated with microscale devices, microscale permeable materials can exist in a wide variety of contexts and devices. Should be understood.

  As an example of a suitable microscale device, a biological material can be obtained under conditions that provide a value for one or more pharmacokinetic parameters in vitro that is comparable to the value of at least one pharmacokinetic parameter found in vivo. There may be one or more microscale features dimensioned to maintain. Details regarding the formation and operation of various embodiments of the microscale mechanism are disclosed above. For purposes of the following description, “microscale” may mean dimensions in the range of about 0.1 μm to about 500 μm. Thus, the microscale feature can be sized such that at least one of its dimensions falls within the microscale range. It will also be appreciated that various embodiments of the present disclosure may be implemented on a scale greater than the microscale level defined above. For purposes of the following description, “millimeter scale” may mean a dimension in the range of about 0.1 mm to about 100 mm. Accordingly, one or more features of the present disclosure may be a millimeter scale feature, wherein at least one of its dimensions falls within the millimeter scale range. It will be appreciated that some mechanisms may have a combination of dimensions, one on the microscale and the other on the millimeter scale. Such a mechanism can be characterized as either of two scales. Furthermore, the various features of the present disclosure may be implemented with dimensions outside the ranges defined above. For example, in one embodiment, the microscale feature may have a dimension of less than 0.1 μm or greater than 500 μm. Similarly, in one embodiment, the millimeter scale feature may have a dimension that is less than 0.1 mm or greater than 100 mm.

  In other embodiments, the microscale permeable material facilitates interactions between different fluid systems. For example, drugs taken orally enter the gastrointestinal (GI) system. One or more compounds associated with the drug can pass from the GI system to the circulatory blood through the inner wall of the small intestine. Drug compounds in the blood can reach and affect various organs and / or systems. For example, drug compounds can pass from the blood to the cerebral fluid system, thereby affecting the brain.

  In another example, the drug compound can pass from the blood to the bile duct system in the liver and enter the enterohepatic recirculation cycle. Drug compounds can remain in the enterohepatic circulation for a long time, resulting in high concentrations in the liver, which can lead to unexpected hepatotoxicity.

  Thus, it can be seen that by revealing the passage of drug compounds or their metabolites between different systems, a better understanding of the pharmacokinetics of the drugs involved can be obtained.

  FIG. 31 illustrates that, in one embodiment, an interaction 3100 between the first and second fluid systems 3102, 3140 is provided and under in vitro conditions under conditions with physiological parameter values similar to those seen in vivo. It can be maintained. For illustrative purposes, the first fluid system 3102 includes one or more microscale features, and the second fluid system 3104 also includes one or more microscale features.

  As further shown in FIG. 31, the interaction 3100 between the first and second systems 3102, 3104 is the passage of one or more compounds from the first system 3102 to the second system 3104 (depicted by arrows 3106. And / or the passage of one or more compounds from the second system 3104 to the first system 3102 (depicted by arrows 3108).

  FIG. 32 shows a block diagram of an example biological system 3110 having several example fluid systems that can be formed using microscale features. Vascular system 3112, GI system 3114, bile duct system 3116, and cerebral fluid system 3118 are some non-limiting examples that can be simulated using microscale mechanisms.

  In one embodiment, at least one intersystem interaction is provided between microscale mechanism-based systems. Various intersystem interactions are described in further detail below.

  Figures 33A-33D show non-limiting examples of various interaction configurations that may be provided for two or more fluid systems. In one embodiment, as shown in FIG. 33A, a two-system configuration 3120 may include an interaction 3172 between the two systems “A” and “B” (3162 and 3164). FIG. 33B illustrates that, in one embodiment, triplet 3130 is not only an interaction 3176 between B and “C” (3164 and 3166), but also an interaction 3174 between A and B (3162 and 3164). Indicates that it can be included. FIG. 33C illustrates that in one embodiment, a four-system configuration 3140 is an interaction between B and “D” (3164 and 3168) in addition to interactions 3178 and 3180 similar to interactions 3174 and 3176 of FIG. 33B. 3182 can be included.

  In one embodiment, the pharmacokinetics associated with interactions 3178 and 3180 (FIG. 33C) may be substantially the same as that of interactions 3174 and 3176 (FIG. 33B). In another embodiment, the presence of additional interaction 3182 (FIG. 33C) can significantly alter the pharmacokinetics associated with interactions 3178 and 3180 from that of interactions 3174 and 3176 (FIG. 33B).

  FIG. 33D illustrates that in one embodiment 3150, multiple systems (eg, three) can be configured to provide and simulate recirculation functionality. In the illustrated example, systems A and B (3162 and 3164) are via interaction 3184; systems B and C (3164 and 3166) are via interaction 3186; and systems C and A (3166 and 3162) Are shown to interact via interaction 3188.

  Specific examples of the configuration shown in FIGS. 33A to 33D will be described in detail below. Other configurations are possible.

  34A-34C show various views of one embodiment of a two fluid system configuration 3200. FIG. 34A shows a partially exploded view of the assembly view of FIG. 34B, and FIG. 34C shows a top view. The first system is shown to include a layer 3220 that defines one or more compartments (depicted as compartments 3222). As shown, compartment 3222 is supplied with fluid for pharmacokinetic studies via inflow path 3212 (defined by cover layer 3210) and inflow through the inflow channel 3260 (shown as arrow 3250). obtain. Fluid from the compartment 3222 can exit the outflow channel 3262, through the outflow path 3214 (defined by the cover layer 3210) and out as an outflow flow (shown as an arrow 3252).

  The second system is shown to include a layer 3230 that defines one or more compartments (depicted as compartments 3232, 3234, 3236). As shown, the compartments 3232, 3234, and 3236 have input flow (shown as arrows 3254) through the inflow path 3242 (defined by the cover layer 3240) and the inflow channel 3270 connected to the compartment 3232. Through which fluids for pharmacokinetic studies can be supplied. Fluid from compartment 3232 may be supplied to other compartments 3234 and 3236 via channels 3272, 3274, and 3278. Fluid from compartments 3234 and 3236 can exit through outlet channels 3276 and 3280 through outlet path 3244 (defined by cover layer 3240) as an output flow (shown as arrow 3256).

  In one embodiment, the formation of compartments, inflow and outflow paths, and various channels of the first and second systems can be formed by the various techniques disclosed above. Also, fluid circulation for the two fluid systems can be accomplished by the various techniques disclosed above.

  As shown in FIGS. 34A-34C, a two-fluid system configuration 3200 is positioned between at least one of the first system 3220 compartments and at least one of the second system 3230 compartments. Material 3224 is included. In the illustrated example, the permeable material 3224 is depicted as being positioned between the compartments 3222 and 3232, and thus provides fluid interaction between the first and second systems 3220 and 3230. Making it possible. The permeable material 3224 will be described in further detail below.

  In FIGS. 34A-34C, compartments 3222 and 3232 and permeable material 3224 are depicted as having different dimensions. This is merely for the purpose of clarity of illustration. The permeable material 3224 may be sized to be smaller, larger or substantially identical to either or both of the compartments 3222 and 3232. In one embodiment, the permeable material 3224 may be placed either partially or substantially inside the compartment, or between the compartments 3222 and 3232.

  FIG. 34D shows a partially exploded view of an embodiment 3200 that is a variation of the example configuration shown in FIG. 34A. As shown, the two-fluid system configuration 3200 includes one or more cell culture compartments (depicted as compartments 3914) and / or one or more biological barriers (depicted as barriers 3916). A first module 3902 having a first culture system may be included.

  As shown, the two-fluid system configuration 3200 may include a second module 3904 having a second culture system that includes one or more cell culture compartments (depicted as compartments 3918 and 3920). In one embodiment, the second module 3904 may include one or more biological barriers (not shown).

  In one embodiment, as shown, the two-fluid system configuration 3200 can include a fluid interconnect 3910 that facilitates fluid flow for the first culture system 3902. In one embodiment, a housing top 3900 can be positioned over the first module 3902 to define a fluid path for the fluid interconnect 3910.

  Similarly, fluid interconnect 3922 facilitates fluid flow for second culture system 3904. In one embodiment, a housing bottom 3906 can be positioned under the second module 3904 to define a fluid path for the fluid interconnect 3922.

  For the purposes of the description herein, a “permeable” material is any biological material or material that allows passage of one or more materials in a selective manner as found in or simulates a biological system. Contains non-biological materials. Thus, a permeable material as used herein can include a semi-permeable material.

  The aforementioned two-system configuration 3200 provides an in vitro environment for pharmacokinetic studies of combinations such as, but not limited to, GI-blood, blood-bile, blood-brain, blood-tissue, and blood-urine can do.

  35A and 35B show a partially exploded view and an assembled view of one embodiment of a three fluid system configuration 3290. The first system is shown to include a layer 3300 that defines one or more compartments (depicted as compartment 3304). The second system is shown to include a layer 3320 that defines one or more compartments (depicted as compartments 3322, 3324, and 3328). The third system is shown to include a layer 3340 that defines one or more compartments (depicted as compartments 3342).

  In one embodiment, the first system 3300 may be supplied with fluid flow (arrows 3350 and 3352) through paths 3302a and 3302b. Third system 3340 may be supplied with fluid flow (arrows 3354 and 3356) through paths 3344a and 3344b. The second system 3320 may have a circulation that provides a connection between the first and second systems 3300 and 3340. The compartment 3322 that interacts with the first system 3300 can be interconnected with the compartment 3328 that interacts with the third system 3340 via channels (not shown) and paths 3326a and 3326b.

  As shown in FIGS. 35A and 35B, the three fluid system configuration 3290 can include two permeable material assemblies 3310 and 3330. First permeable material assembly 3310 is shown configured such that permeable material 3312 is positioned between compartments 3304 and 3322 of first and second systems 3300 and 3320. The second permeable material assembly 3330 is shown configured such that the permeable material 3332 is positioned between the compartments 3328 and 3342 of the second and third systems 3320 and 3340.

  In configuration example 3290 shown in FIGS. 35A and 35B, permeable materials 3312 and 3332 are depicted as being part of separate layers 3310 and 3330. In one embodiment, the permeable materials 3312 and 3332 can be formed to be part of one of their adjacent layers. For example, the permeable material 3312 can be formed as part of any of the layers 3300 and 3320 such that the permeable material 3312 separates the compartments 3304 and 3322. Similarly, permeable material 3322 can be formed as part of either of layers 3320 and 3340 such that permeable material 3322 separates compartments 3328 and 3342.

  In one embodiment, the permeable materials 3312 and 3322 can be configured to promote their respective intersystem interactions. The permeable materials 3312 and 3322 are described in further detail below.

  In one embodiment, the triple system configuration may be implemented in the manner described above with reference to FIGS. 35A and 35B. FIG. 36 shows a block diagram of an example 3360 of such a three fluid system. The drug delivery system 3362 can be represented by the first system 3300 (FIGS. 35A and 35B); the organ system 3364 can be represented by the second system 3320; the brain 3366 can be represented by the third system 3340. Can be represented. Interaction 3370 between drug delivery system 3362 and organ system 3364 can be represented by permeable material assembly 3310; interaction 3372 between organ system 3364 and brain 3366 is represented by permeable material assembly 3330. Can be done.

  In a three-system application 3360, drug delivery system 3362 may include a GI system, and the organ system may include various organs (other than the brain) and vascular system. Thus, interaction 3370 may involve the passage of one or more compounds associated with the drug from the GI system to the blood; interaction 3372 may involve the passage of one or more compounds associated with the drug from the blood. It may include passage through the brain fluid system.

  It will be appreciated that other three-system configurations are possible.

  FIG. 37 shows a block diagram of a configuration example 3380 including the liver 3384. Liver 3384 is shown to interact with GI tract 3382 via enterohepatic circulation (shown as arrows 3390 and 3392). Liver 3384 is shown to also interact with urinary system 3388 (depicted by arrow 3396) and tissue 3386 (depicted by arrow 3394). The interaction 3396 between the liver 3384 and the urinary system 3388 can be facilitated by the blood circulatory system acting as a mediator. Similarly, the blood circulatory system can promote the interaction 3394 between the liver 3384 and the tissue 3386.

  FIG. 38 shows that vasculature 3406 can also promote enterohepatic circulation processes including liver 3384 and GI tract 3382. FIG. As shown, the bile duct system 3402 (of the liver 3384) interacts with the GI system 3404 (arrow 3410), which in turn interacts with the circulatory system 3406 (arrow 3412). The circulatory system 3406 interacts with the bile duct system 3402 (arrow 3414), thereby forming a recirculation process.

  As is generally known, the liver produces bile acids that are delivered to the small intestine to aid digestion. Within the gastrointestinal tract, bile acids are converted to conjugated bile salts (primary or secondary), which are absorbed either actively or passively into the hepatic portal circulation for recycling by the liver. Is done. In general, each bile salt molecule is reused about 20 times within the enterohepatic cycle.

  One of the consequences of the recycling process described above is that the drug or its components can remain in the enterohepatic circulation for an extended period of time. Thus, some molecules that would otherwise be toxic can accumulate in the liver and become toxic. Thus, pharmacokinetics associated with the enterohepatic recirculation process can provide an important understanding of the toxicity (or non-toxicity) of the drug being tested.

  As described above, various features of the interaction between the different fluid systems described above can be facilitated by one or more permeable materials. In some embodiments, such permeable material may be part of a microscale permeable device.

  As described in more detail below, one or more features of the present disclosure can provide various systems and methods, alone or in combination. For example, the device is dimensioned to maintain the biological material under conditions that provide at least one in vitro pharmacokinetic parameter value that is comparable to the at least one pharmacokinetic parameter value found in vivo. It can have at least one determined mechanism and a permeable material. The permeable material is described in further detail below. In one embodiment, the at least one mechanism includes a microscale mechanism.

  In one embodiment, the at least one mechanism comprises the following non-limiting system examples: central nervous system, circulatory system, digestive system, bile duct system, lung system, urinary system, visual system, olfactory system, epidermal system, and It may be configured to represent at least part of one or more of the lymphatic systems.

  In one embodiment, as described herein, the device further comprises at least one microfluidic channel connected to the permeable material. Such channels can facilitate fluid flow in, through, or close to the permeable material so as to provide at least one pharmacokinetic parameter. In one embodiment, such fluid flow properties may be based on a mathematical model, such as a Physiologically-Based PharmacoKinetic (“PBPK”) model.

  In one embodiment, at least one feature and / or permeable material may be incorporated into the chip format.

  In one embodiment, the permeable material can be located in or out of the device. In one embodiment, the permeable material may comprise a microporous material that is at least partially coated with an organic material.

  In one embodiment, the cells may be located in, on or near both sides of the permeable material. In one embodiment, a device having such cells can facilitate the measurement or estimation of parameters such as absorption properties, metabolic enzyme activity, and / or expression levels. In one embodiment, the cells on either side of the permeable material may be of the same type or different types.

  FIG. 39 illustrates one embodiment of a microscale permeable device 3420 having a permeable material 3430 that can facilitate one or more interactions between two fluid systems. Some non-limiting examples of permeable material 3430 include the following: membrane, porous membrane, porous silicon, microporous silicon, semipermeable membrane, microporous polymer, porous polycarbonate membrane, alginate, It may include collagen, MATRIGEL, cells, cellular material, tissue, and tissue pieces.

  In one embodiment, the permeable material 3430 can include an organic or inorganic material in, on or near the microporous surface of the permeable material 3430.

  In one embodiment, the permeable material 3430 includes a microporous material. Non-limiting examples of such microporous materials include: organic or inorganic materials cultured, vapor deposited, or inserted in, on or near the microporous surface of the microporous material. obtain.

  In one embodiment, the permeable material 3430 may be a biological barrier, a passage of a substance in or through the biological barrier, or a through or through the biological barrier. It may be configured to simulate at least one of the absorption of the substance. In one embodiment, the biological barrier is: gastrointestinal barrier, blood brain barrier, lung barrier, placental barrier, epidermal barrier, eye barrier, olfactory barrier, gastroesophageal barrier, mucosa, blood urine barrier, air tissue It may include at least one of a barrier, blood bile barrier, oral barrier, anorectal barrier, vaginal barrier, and urethral barrier.

  In one embodiment, the permeable material 3430 can facilitate measurement of various pharmacokinetic parameters while taking part in one or more intersystem interactions. These pharmacokinetic parameters include: tissue size, tissue size ratio, tissue to blood volume ratio, drug residence time, cell interaction, liquid residence time, fluid to cell ratio, cellular metabolism, shear stress, flow rate , Surface shape, circulation transit time, fluid distribution, interaction between tissues and / or organs, and molecular transport by cells.

  In one embodiment, the permeable material 3430 can facilitate measurement of absorption, metabolism, or distribution of a substance through or through the permeable material.

  In one embodiment, the permeable material 3430 is formed, contained, inserted, assembled, made in a device that includes a plurality of microscale features representing two or more fluid systems, or Can be configured.

  In one embodiment, either or both sides of the permeable material 3430 can be configured to allow culturing, attachment, or positioning of the cell or cellular material. Such a configuration allows measurement of parameters such as absorption characteristics, metabolic enzyme activity, and / or expression levels.

  In one embodiment, the permeable material 3430 can include a cell line that can form and polarize a confluent monolayer.

  In one embodiment, the permeable material 3430 can include a microscale permeable material 3432. In one embodiment, the microscale permeable material 3432 can include a microporous substrate having a plurality of pores. In some embodiments, the pores generally have a dimension less than about 10 μm. In some embodiments, the microporous substrate inhibits the passage of particles having a dimension greater than about 10 μm.

  In one embodiment, the microporous substrate can be formed from porous silicon having pores with dimensions in the range of about 0.1 to 10 μm. The thickness “T” of such a substrate may range from about 5 to 100 μm. In one embodiment, the microporous substrate can be formed from a porous polycarbonate membrane having pores with dimensions in the range of about 0.4 μm. The thickness “T” of such a substrate may be in the range of about 100 μm. In one embodiment, the microporous substrate can be formed from a porous low stress silicon nitride material having pores with dimensions ranging from about 0.2 to 1 μm. The thickness “T” of such a substrate may range from about 2 to 5 μm.

  In one embodiment, the lateral dimension “L” (perpendicular to the direction defining the thickness) may have any value relative to the lateral dimension of the compartment 438. For a given thickness, the larger the surface area (and hence the greater the lateral dimension), the more likely it is to provide a greater amount of interaction between the two fluid systems. Thus, the amount of material passage between the two systems can be controlled by providing surface areas with different lateral sizes. Accordingly, the lateral dimension L may be less than, substantially equal to, or greater than the corresponding lateral dimension of the compartment 3438 (as shown in the example of FIG. 39).

  In one embodiment, the microporous substrate has a lateral dimension in the range of about 0.1 to 10 mm. In an embodiment, the lateral dimension is about 3.4 mm × 4 mm.

  As further shown in FIG. 39, the permeable assembly 3430 may further include one or more specific function cells 3434 positioned on, in and / or around the microscale permeable material 3432. The specific function cell 3434 will be described in more detail below, taking the interaction between the blood system and the bile duct system as an example. However, it will be appreciated that cells of different specific functions can be positioned relative to the microscale permeable material 3432 to provide the desired functionality for different intersystem interactions.

  In one embodiment, the permeable assembly 3430 can include one or more binders 3445 that facilitate binding of the cells 3434 to the surface of the microscale permeable material 3432. Examples of binder 3445 are described in further detail below.

  In one embodiment, the permeable assembly 3430 can further include one or more features 3436 that provide functionality similar to fibroblasts. In one embodiment, the specific function cells 3434 can be distributed on the surface of the microscale permeable material 3432 and the fibroblasts 3436 are not occupied by the cells 3434 on the surface of the microscale permeable material 3432. Can fill the area. In such a configuration, fibroblasts can provide sealing functionality such that the passage of material through the permeable assembly 3430 occurs almost exclusively through the cells 3434.

  In one embodiment, fibroblasts 3436 can provide a favorable environment for cell 3434 growth and maintenance. In one embodiment, fibroblast 3436 can provide both functionalities—not only sealing of permeable material 3430 but also cell growth and maintenance.

  In one embodiment, the permeable assembly 3430 can be formed as part of the layer 3422 to define a compartment 3438 on at least one side of the permeable assembly 3430. In other embodiments, both sides of the permeable assembly 3430 can define their respective compartments. In such a configuration, the permeable assembly 3430 can have cells 3434 and a binder 3434 on either or both sides of the permeable assembly 3430.

  Figures 40A and 40B illustrate various example situations where a permeable assembly may be provided to allow interaction between different fluid systems. This intersystem interaction example is based on the enterohepatic recirculation process. However, other intersystem interactions can be made in a similar manner.

  FIG. 40A shows one embodiment of an interaction configuration 3440 between the blood flow system and the bile flow system. In one embodiment, the permeable assembly 3442 may be interposed between blood flow and bile flow and may include a microscale permeable material 3444. In some embodiments, the microscale permeable material 3444 can be formed and dimensioned in a manner similar to that described above with reference to FIG.

  In one embodiment, the permeable assembly 3442 may further include one or more specific function cells 3446. For blood-bile interactions, cells 3446 can include hepatocytes.

  Liver hepatocytes may be polar cells, and different surfaces of differentiated hepatocytes may have intrinsic functions. In one embodiment, the basolateral sinus-like membrane and the distal bile duct membrane in the liver can be simulated in the following manner. Isolated hepatocytes are generally not polarized. Hepatocytes are generally polarized when in physical contact with neighboring hepatocytes. A capillary bile duct can be formed between two or more of such parallel cells.

  External cues can be important for epithelial cell polarization, and the physical contact between two neighboring hepatocytes appears to be a signal representing such hepatocyte polarization. Hepatocytes can form connections with neighboring hepatocytes through binding of binding proteins or adhesion proteins, and the interaction of these proteins appears to be an important signal representing capillary bile morphogenesis .

  As shown in FIG. 40A, these proteins can act as binding agents 3445 that promote hepatocyte 3446 binding to the microscale substrate 3444 and hepatocyte polarization. In some embodiments, these proteins are gap junction proteins (eg, connexin 32), tight junction proteins (eg, Occludin, Claudin-1, ZO-1, ZO-2), It may include adhesion-binding proteins (eg, E-cadherin and β-catenin), and cell adhesion molecules (eg, uvomorulin).

  In one embodiment, one or more of these proteins attached to the microscale permeable substrate 3444 can actually mimic the plasma membrane surface of neighboring hepatocytes. When isolated hepatocytes bind to this surface, the hepatocytes can be induced to polarize, resulting in a tip or capillary (3449b) on the surface of the microscale permeable substrate 3444. The opposite side may be formed with a basolateral or sinusoidal surface (3449a).

  In one embodiment, hepatocytes 3446 can be seeded at a suitable density to inhibit cell-cell interactions. Once hepatocytes 3446 are attached to the microscale permeable substrate 3444, to form a “blood bile” barrier by substantially sealing the microscale permeable surface in areas not occupied by the hepatocytes 3446. And / or fibroblasts 3448 or other suitable cells can be cultured on the surface to provide a favorable environment for hepatocyte growth.

  In one embodiment, one or more selected compounds desired may be introduced in order not to affect hepatocytes 3446. Such compounds can be transported through the hepatocytes 3446 across the microscale permeable surface into the bile surrogate flow of the device 3440. The presence of the compound and its metabolites can be measured in the flow of bile surrogates to measure biliary excretion.

  When bile is transferred to the GI system and reabsorbed into the blood system, “bile” in the GI system is composed of the following compounds: bile salts (chenodeoxycholic acid, hyodeoxycholic acid, cholic acid, α-mulicholic acid, and phospholipids (phosphatidylcholine (˜82%), trace amounts of phosphatidylinositol, phosphatidylserine, and sphingomyelin); bile alcohols (5β-cholestane-3α, 7α, 12α, 26-tetrol); and amino acids Can be included.

  In one embodiment, bile flow can be coupled to GI flow to further mimic enterohepatic recirculation. In one embodiment, bile can be mixed with GI fluid. Such mixing can be accomplished, for example, in a manner as described in more detail below.

  In one embodiment 3460 as shown in FIG. 40B, GI flow can be coupled to the blood flow to further mimic enterohepatic recirculation. The interaction 3460 may include a permeable assembly 3462 having a microscale permeable substrate 3464 and a surface defined by one or more specific function cells 3466. In one embodiment, the specific function cell 3466 may comprise an intestinal epithelial cell. In one embodiment, Caco-2 cells 3468 can be provided in the vicinity of cell 3466 to facilitate in vitro absorption of the compound from the GI flow into the bloodstream.

  In one embodiment, the permeable material 3462 can include a layer of gastrointestinal cells cultured on a microscale permeable substrate 3464. In one embodiment, at least a portion of the layer of gastrointestinal cells is positioned within device 3460 such that fluid can flow along either side of the layer without passing through the layer. In one embodiment, at least a first microscale feature located on the first side of the layer of gastrointestinal cells can represent the gastrointestinal tract and at least a second microscale located on the second side of the monolayer. The mechanism can represent a circulatory system. In one embodiment, a third microscale mechanism may be provided and configured to contain the same or different types of biological material.

  41A and 41B show partially exploded and assembled views of an example embodiment of a device 3700 that can provide a pharmacokinetic simulation of the enterohepatic recirculation process described above. Device 3700 may include a GI substitution module 3720 that can provide GI-blood interaction functionality similar to that described above with reference to FIG. 40B. Device 3700 can also include an organ system module 3730 that can provide blood-bile interaction functionality similar to that described above with reference to FIG. 40A. Housing caps 3710 and 3760 can provide a housing for device 3700 and can also provide paths for various fluid flows.

  As shown, the GI flow to and from the GI substitution module 3720 (arrow 3770) and from there (arrow 3772) may be provided by respective paths 3712 and 3714. Similarly, blood flow to and from the blood side of organ system module 3730 (arrow 3774) and from there (arrow 3776) may be provided by respective pathways 3762, 3750 and 3752, 3768. Similarly, biliary flow to the bile side of organ system module 3730 (arrow 3778) and from (3780) therefrom may be provided by respective pathways 3764 and 3766.

  As shown, the GI substitute module 3720 can include a compartment 3722 that includes a permeable assembly having a microscale permeable substrate 3724. The microscale permeable substrate 3724 may be formed from any one or combination of the materials described above with reference to FIG. The permeable assembly may include intestinal epithelial cells 3726 formed on a microscale permeable substrate 3724. In one embodiment, the GI side of compartment 3722 may include Caco-2 cell 3728 in the vicinity of cell 3726. As is generally known, Caco-2 cells can promote in vitro absorption of compounds from the gut to the blood.

  Compounds absorbed through the permeable assembly of the GI surrogate module 3720 can enter the blood system in the compartment 3732 of the organ system module 3730. Blood can circulate between compartment 3732 and one or more other compartments. For purposes of illustration, a compartment 3734 having a permeable assembly for blood-bile interaction and a compartment 3744 simulating a target organ (via a target cell 3746) are shown. In one embodiment, target organ 3744 may include an organ or tissue that may be affected by drug activity. For example, when testing heart drugs, the target organ 3744 may be the heart. In another example, when testing drug toxicity, the target organ may be the pancreas.

  The permeable assembly of compartment 3734 is shown to include a microscale permeable substrate 3736. The microscale permeable substrate 3736 can be formed from any one or combination of the materials described above with reference to FIG. The permeable assembly may include hepatocytes 3738 formed on a microscale permeable substrate 3736. In one embodiment, hepatocytes 3738 can be bound to the microscale permeable substrate 3736 via a binder in the manner described above with reference to FIG. 40A. In one embodiment, the permeable assembly may further include fibroblasts 3740 to provide functionality as described above with reference to FIG. 40A.

  The permeable assembly of the organ system module 3730 can promote blood-bile interactions between blood flow (in the space 3742 of the compartment 3734) and bile flow (on the other side of the permeable assembly). it can. Subsequently, the bile flow can be circulated via paths 3764 and 3766, and the bile can be reintroduced (not shown) into the GI flow.

  FIG. 41C shows another partially exploded view of an organ system module 3700 similar to that shown in FIG. 41A. In FIG. 41C, compartment 3734 having a permeable assembly for blood-bile interaction is shown in more detail by the appendix. In one embodiment, the permeable assembly of compartment 3734 may be similar to that described above with reference to FIG. Thus, the permeable assembly 3430 can include a permeable material 3432 and cells or cellular material 3434 formed on either or both sides of the permeable material 3432. In one embodiment, cells 3434 may be hepatocytes that can be combined as described herein. In an embodiment where hepatocytes are used, the permeable assembly 3430 may further include fibroblasts 3436.

  FIG. 42 depicts various example fluid flow schematics 3800 that may be implemented in the example enterohepatic recirculation simulation device of FIGS. 41A and 41B. In one embodiment, a GI fluid flow 3802 (depicted as a dotted line) may be provided to flow through a GI tract compartment 3810 having a GI blood barrier 3812 as described herein. The GI fluid stream 3802 may be caused to flow from the GI fluid container 3850 to another container (not shown). In one embodiment, the GI fluid stream 3802 does not recirculate.

  As shown in FIG. 42, GI-bile interaction can be facilitated by the GI blood barrier 3812. Blood flow 3804 is depicted as a solid line. Blood flow, shown as 3804a, interacts with the GI flow 3802 in the GI tract compartment 3810 via the barrier 3812 and is directed to the liver compartment 3820. Blood bile barrier 3822 (as described herein) can facilitate the interaction of blood flow 3804a with bile flow 3806. In one embodiment, bile flow 3806 from liver compartment 3820 can be provided from bile fluid container 3860. In one embodiment, the bile stream 3806 from the liver compartment 3820 is mixed with the GI stream 3802 at a location upstream of the GI tract compartment 3810, thereby providing bile recirculation functionality from the liver compartment 3820. it can.

  In one embodiment, blood flow 3804a from liver compartment 3820 can be directed to one or more other compartments. For example, blood flow 3804c (via 3804b) is shown to provide blood to target tissue compartment 3830, and blood flow 3804e (via 3804b) provides blood to other tissue compartments 3840. It is shown. Blood flows 3804d and 3804f from compartments 3830 and 3840 can be recombined with blood flow 3804g, which can become part of blood flow 3804a at a location upstream of GI tract compartment 3810.

  43A through 43E illustrate various stages of fabrication of one embodiment of the microscale permeable device described above. FIG. 44 illustrates one embodiment of a process 3520 that can perform the fabrication of the devices of FIGS. 43A-43E.

  As shown in FIG. 43A, an opening 3502 may be formed on the substrate 3500. Such opening formation may be accomplished within process block 3522.

  As shown in FIG. 43B, a microscale transparent substrate 3504 can be formed in the opening 3502. Formation of such a microscale transmissive substrate 3504 can be accomplished within process block 3524.

  One or more binders 3506 may be positioned on the microscale permeable substrate 3504, as shown in FIG. 43C. Providing such a binder 3506 may be accomplished within process block 3526.

  As shown in FIG. 43D, one or more specific function cells 3508 can be bound to the microscale permeable substrate 3504 via a binder 3506. Such specific function cell 3508 binding may be accomplished within process block 3528.

  As shown in FIG. 43E, one or more fibroblasts 3510 may be introduced between cells 3508 of a specific function to provide a seal and / or promote cell 3508 growth and maintenance. . Such introduction of fibroblasts 3510 may be accomplished within process block 3530.

  In one embodiment, the microscale transmissive substrate 3504 can be formed by the following non-limiting example. The microporous surface can be formed from silicon by etching with HF (Hydrofluoric Acid) under application of a bias. Microporous surfaces can be produced using standard photolithography and etching techniques for pore sizes greater than about 0.4 microns in diameter, or electron beams for pore sizes less than about 0.4 microns in diameter. It can also be formed from low stress silicon nitride thin films by using lithography and etching.

  In one non-limiting example embodiment, the binding protein can be micropatterned onto a microporous surface by utilizing microcontact printing techniques. Replication molding techniques can be used to produce a silicone elastomer “rubber stamp”. By immersing the rubber stamp in a solution of binding protein and subsequently depositing these binding proteins on the surface of the microporous material, a micropattern of binding protein can be produced. This process is commonly known as microcontact printing.

  In one non-limiting example embodiment, hepatocytes can be attached to the binding protein, and after attachment, fibroblasts are introduced to the surface and substantially all of the microporous surface not occupied by hepatocytes. Can be attached to the area.

  Other manufacturing techniques may be used.

  In one embodiment, the microscale permeable material (such as 3432 in FIG. 39) and at least one binder (such as 3506 in FIG. 43C) can define the device. The at least one binding agent can be configured to polarize the substance, wherein the substance exhibits at least one property of liver function.

  In one embodiment, the substance may be one or more hepatocytes. In one embodiment, the substance may be a genetically modified biological material.

  In one embodiment, the binding agent can bind and polarize hepatocytes to the microscale permeable material.

  In one embodiment, the device may include a microscale permeable material (such as 3432 in FIG. 39) and at least one substance configured to demonstrate at least one property of liver function. The treated molecules can be directed to pass through at least a portion of the microscale permeable material.

  FIG. 45 illustrates non-limiting examples of various combinations of systems that can be coupled using one or more techniques of this disclosure. The microscale permeable device 3540 can allow interaction between the blood system and the bile duct system. The microscale permeable device 3542 can allow interaction between the blood system and the GI system. The selected connection (depicted as arrow 3544) can allow interaction between the bile duct system and the GI system (eg, by mixing at a selected location). The microscale permeable device 3546 can allow interaction between the blood system and the brain system. The microscale permeable device 3548 can allow interaction between the blood system and the urinary system.

  It will be appreciated that other intersystem interactions are possible through microscale permeable devices. Accordingly, as shown in FIG. 46, in general, the microscale permeable device 3550 can allow interaction between the first fluid system and the second fluid system.

  In the above description, various embodiments of microscale permeable devices are depicted as being part of a layer that is part of either the system layer or the separation layer. In such a configuration, compartments associated with different systems are depicted as being formed on different layers.

  In some embodiments, this is not necessarily a requirement. For example, in one embodiment, an organ system module (3730 in FIGS. 41A and 41B) can be formed on one side of the layer and a GI substitute module (3720 in FIGS. 41A and 41B) can be formed on the other side of the same layer. It can be formed on the side.

  In another example embodiment, a microscale permeable device may be formed on a given layer such that it defines two compartments, with each compartment representing a separate system. Thus, as shown in the example embodiment 3560 of FIG. 47, a microscale permeable device 3562 can be formed on the layer to define and separate the two compartments 3564 and 3566. Thus, the first compartment 3564 can represent a first fluid system and the second compartment 3566 can represent a second fluid system. The microscale permeable device 3562 can provide interaction between the first and second fluid systems. If appropriate, more complex systems such as those shown in FIGS. 41A and 41B can be formed.

  Although the embodiments disclosed above illustrate and point out the basic novel features of the present invention when applied to the above-disclosed embodiments, the detailed forms of the devices, systems, and / or methods illustrated It should be understood that various omissions, substitutions, and changes in may be made by those skilled in the art without departing from the scope of the invention. Accordingly, the scope of the invention should not be limited to the foregoing description, but should be defined by the appended claims.

FIG. 1 is a block diagram of a system according to the present invention. FIG. 2 is a simplified perspective view of one embodiment of the appearance of the system of the present invention. FIG. 3 is a detailed schematic diagram of another embodiment of the system of the present invention. FIG. 4 is a schematic diagram of yet another embodiment of the system of the present invention. 5A-5G show the steps used to make a chip from plastic. FIG. 5A illustrates the step of coating a silicon wafer with a positive photoresist material. FIG. 5B shows the step of exposing the resist-coated silicon wafer to UV light with a photo material. FIG. 5C shows the step of developing the photoresist material. FIG. 5D shows the step of etching silicon. FIG. 5E shows the steps of stripping the photoresist material and evaporating the gold. FIG. 5F shows the step of electroplating nickel. FIG. 5G shows the steps of removing the silicon and embossing the polymer. FIG. 6 is a schematic diagram of yet another embodiment of the system of the present invention. FIG. 7 is a schematic detailing the computer associated with the chip. FIG. 8 is a schematic showing more than one chip located within the housing. FIG. 9 is a schematic of a system that includes a set of chips in different housings. FIG. 10 is a schematic of yet another embodiment of a chip. FIG. 11 is an isometric partial exploded view of the system. FIG. 12 is an isometric view of the steps of creating a jaw associated with the system shown in FIG. FIG. 13 is an isometric view of the single trough elastic portion of the pump associated with the system shown in FIG. FIG. 14 is an isometric view of the multi-trough elastic portion of the pump. FIG. 15 is a schematic diagram of a four compartment chip. FIG. 16 shows tegafur dose response. The chip was seeded with HepG2-C3A cells in the liver compartment and HCT-116 colon cancer cells in the target tissue compartment. The chips were treated with the indicated concentration of tegafur for 24 hours. The first graph (FIG. 16A) is a plot of dead cells versus percentage of tegafur or 5-FU concentration 24 hours after recirculation on the chip. The second graph (FIG. 16B) is a similar dose response using a conventional in vitro cell culture assay, with HCT-116 cells using 48 hours exposure. HCT-116 cells were plated on polylysine treated glass coverslips and exposed to the indicated concentrations of tegafur or 5-FU. After 48 hours incubation, the coverslips were processed as described above and the percentage of cell death was measured. FIG. 16 shows tegafur dose response. The chip was seeded with HepG2-C3A cells in the liver compartment and HCT-116 colon cancer cells in the target tissue compartment. The chips were treated with the indicated concentration of tegafur for 24 hours. The first graph (FIG. 16A) is a plot of dead cells versus percentage of tegafur or 5-FU concentration 24 hours after recirculation on the chip. The second graph (FIG. 16B) is a similar dose response using a conventional in vitro cell culture assay, with HCT-116 cells using 48 hours exposure. HCT-116 cells were plated on polylysine treated glass coverslips and exposed to the indicated concentrations of tegafur or 5-FU. After 48 hours incubation, the coverslips were processed as described above and the percentage of cell death was measured. FIG. 17A depicts a “first generation” three-compartment device. FIG. 17B shows a cross-sectional view of the device. FIG. 17A depicts a “first generation” three-compartment device. FIG. 17B shows a cross-sectional view of the device. FIG. 18A depicts a “second generation” device. FIG. 18B depicts a 5 μm high ridge in the chamber and FIG. 18C depicts a 20 μm high pillar in the chamber. FIG. 18A depicts a “second generation” device. FIG. 18B depicts a 5 μm high ridge in the chamber and FIG. 18C depicts a 20 μm high pillar in the chamber. FIG. 18A depicts a “second generation” device. FIG. 18B depicts a 5 μm high ridge in the chamber and FIG. 18C depicts a 20 μm high pillar in the chamber. FIG. 19 depicts a “third generation” device. FIG. 20 is a flow diagram representing a five compartment PBPK model CCA. FIG. 21 depicts a human biochip prototype containing compartments representing lungs, target tissues, and other tissues. The dimensions of the compartment and channel are as follows: Inlet: 1 mm × 1 mm Liver: width 3.2 mm × length 4 mm Target tissue: width 2 mm × length 2 mm Other tissue: width 340 μm × length 110 mm Exit: 1 mm × 1 mm Channel connecting the liver to the Y connection: width 440 μm Channel from Y connection to the target tissue: width 100 μm FIG. 22 depicts a schematic diagram of a microscale chip system. FIG. 23 depicts HepG2, HepG2 / C3A, and human liver basal CYP expression levels. Standard error of 3 separate measurements. FIG. 24A depicts HepG2 / C3A growth curves in EMEM, DMEM, McCoy's, and RPMI. FIG. 24B depicts HCT-116 growth curves in EMEM, DMEM, McCoy's, and RPMI. Standard error of 3 separate measurements. FIG. 24A depicts HepG2 / C3A growth curves in EMEM, DMEM, McCoy's, and RPMI. FIG. 24B depicts HCT-116 growth curves in EMEM, DMEM, McCoy's, and RPMI. Standard error of 3 separate measurements. FIG. 25 depicts RT-PCR measurements of CYP isoform expression in HepG2 / C3A under different growth medium conditions. FIG. 26 depicts RT-PCR measurements of CYP isoform expression in HepG2 / C3A grown on different substrates. FIG. 27 depicts a human biochip prototype. FIG. 28A is a block diagram diagram illustrating a system for controlling a microscale culture device, according to one embodiment of the present invention. FIG. 28B is a block diagram diagram illustrating a system for controlling a microscale culture device according to another embodiment of the present invention. FIG. 28A is a block diagram diagram illustrating a system for controlling a microscale culture device, according to one embodiment of the present invention. FIG. 28B is a block diagram diagram illustrating a system for controlling a microscale culture device according to another embodiment of the present invention. FIG. 29 is a flow diagram illustrating a computerized method for dynamically controlling a microscale culture device, according to one embodiment of the present invention. FIG. 30 is a block diagram illustrating a computer for controlling a microscale culture device according to one embodiment of the invention. FIG. 31 illustrates that in one embodiment, the interaction between the first and second fluid systems can be provided and maintained in vitro under conditions having physiological parameters similar to those found in vivo. Indicates. FIG. 32 shows a block diagram of several example fluid systems in which various intersystem interactions can be simulated in vitro. FIG. 33A shows an example interaction between two fluid systems. FIG. 33B illustrates that in one embodiment, a given fluid system can interact with more than one fluid system. FIG. 33C illustrates that in one embodiment, a given fluid system can interact with more than two fluid systems. FIG. 33D illustrates that, in one embodiment, fluid system interactions can provide recirculation functionality. FIG. 34A shows a partially exploded view of an example embodiment of a two-fluid system configuration where intersystem interaction can be facilitated by a permeable material. FIG. 34B shows an assembly view of the two fluid system of FIG. 34A. FIG. 34C shows a top view of the two fluid system of FIG. 34A. FIG. 34D illustrates one embodiment of a variation of the system of FIG. 34A. FIG. 35A shows a partially exploded view of an example embodiment of a three fluid system configuration, where the interaction between the two systems can be facilitated by one or more permeable materials. FIG. 35B shows an assembly view of the three fluid system of FIG. 35A. FIG. 36 shows a block diagram of a three-fluid system example where the organ system is depicted as interacting with a drug delivery system such as the gastrointestinal (GI) system and a target system such as the brain system. . FIG. 37 shows a block diagram of an example configuration that includes various intersystem interactions involving the liver, such interactions may be part of a recirculation process such as enterohepatic circulation. FIG. 38 shows a block diagram depicting the enterohepatic circulation of FIG. FIG. 39 illustrates one embodiment of a microscale permeable device having a permeable material that can facilitate one or more interactions between two fluid systems. FIG. 40A illustrates one embodiment of a microscale permeable device configured to promote interaction between the blood system and the bile duct system. FIG. 40B illustrates one embodiment of a microscale permeable device configured to facilitate the interaction between the GI system and the blood system. 41A and 41B show a partially exploded view and an assembled view of one embodiment of the enterohepatic circulation simulation device. And 41B show a partially exploded view and an assembled view of one embodiment of the enterohepatic circulation simulation device. FIG. 41C shows another partially exploded view of FIG. 41A, showing one embodiment of a microscale permeable device in more detail. FIG. 42 shows schematic depictions illustrating various fluid flows that may be implemented in the example enterohepatic circulation simulation device of FIGS. 41A and 41B. 43A through 43E illustrate various stages of fabrication of one embodiment of the microscale permeable device of FIG. FIG. 44 illustrates one embodiment of a process for making the microscale permeable device of FIGS. 43A-43D. FIG. 45 shows a non-limiting example of an intersystem interaction that can be facilitated by a microscale permeable device. FIG. 46 shows an overall depiction of the intersystem interaction between the first and second systems facilitated by the microscale permeable material. FIG. 47 shows that, in one embodiment, a microscale permeable device can be configured to promote intersystem interaction between two compartments formed on the same layer, the two compartments being 2 Indicates that it is part of two different systems.

Claims (118)

at least sized to maintain the biological material under conditions that provide at least one in vitro pharmacokinetic parameter value comparable to that of at least one pharmacokinetic parameter found in vivo. One microscale mechanism,
A permeable material;
A device comprising:   The permeable material is a membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL, cell, cellular material, tissue, and tissue The device of claim 1, wherein the device is selected from at least one of the group consisting of strips.   The device of claim 1, wherein the permeable material further comprises an organic or inorganic material in, on or near the microporous surface.   The permeable material may be a biological barrier, a passage of a substance in or through the biological barrier, or an absorption of a substance through or through the biological barrier. The device of claim 1, wherein the device is configured to simulate at least one of them.   The biological barrier includes: gastrointestinal barrier, blood brain barrier, lung barrier, placental barrier, epidermal barrier, eye barrier, olfactory barrier, gastroesophageal barrier, mucosa, blood urine barrier, air tissue barrier, blood bile barrier, oral barrier 5. The device of claim 4, wherein the device is selected from at least one of the group consisting of: anorectal barrier, vaginal barrier, and urethral barrier.   The at least one pharmacokinetic parameter includes tissue size, tissue size ratio, tissue to blood volume ratio, drug residence time, cell-to-cell interaction, liquid residence time, liquid to cell ratio, metabolism by cells, shear stress, flow rate, surface The device of claim 1 selected from at least one of the group consisting of shape, circulation transit time, fluid distribution, interaction between tissues and / or organs, and molecular transport by cells.   The device of claim 1, wherein the device measures the absorption, metabolism, excretion, or distribution of substances in or through the permeable material.   The mechanism represents at least one of the group consisting of at least part of the central nervous system, circulatory system, digestive system, bile duct system, lung system, urinary system, visual system, olfactory system, epidermal system, and lymphatic system The device of claim 1, configured as follows.   The device of claim 1, wherein the permeable material is located in or out of the device.   The device of claim 1, further comprising at least one microfluidic channel connected to the permeable material.   The device of claim 1, wherein a flow of fluid through or in proximity to the permeable material provides at least one pharmacokinetic parameter.   The device of claim 11, wherein the nature of the fluid flow through the device is based on a mathematical model.   13. The device of claim 12, wherein the mathematical model is a Physiologically-Based PharmacoKinetic ("PBPK") model.   The device of claim 1, wherein the feature or the permeable material is incorporated into a chip format.   The device of claim 1, wherein the permeable material comprises a layer of gastrointestinal cells cultured on a microporous material.   16. The device of claim 15, wherein at least a portion of the layer of the gastrointestinal cells is positioned within the device such that fluid can flow along either side of the layer without passing through the layer.   At least a first microscale mechanism located on the first side of the layer of gastrointestinal cells represents the gastrointestinal tract, and at least a second microscale mechanism located on the second side of the monolayer represents the circulatory system. The device of claim 16.   18. The device of claim 17, further comprising a third microscale feature configured to contain the same or different types of biological material.   The device of claim 1, wherein the permeable material comprises a microporous material that is at least partially coated with an organic material.   The device of claim 1, further comprising cells located in, on or near both sides of the permeable material.   21. The device of claim 20, wherein the device provides absorption properties, metabolic enzyme activity, and / or expression levels.   21. The device of claim 20, wherein the cells on either side of the permeable material are of the same type or different types.   The device of claim 1, further comprising hepatocytes in, on or near a microporous surface of the permeable material.   24. The device of claim 23, wherein at least a portion of the microporous surface comprises a protein that polarizes the hepatocytes.   The device of claim 1, wherein the permeable material comprises a cell line capable of forming a fusible monolayer.   The device of claim 1, further comprising a binder that binds hepatocytes to the permeable material.   27. The device of claim 26, wherein the binding agent polarizes the hepatocytes.   The binding agent includes protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β-catenin, cell adhesion molecule, 27. The device of claim 26, comprising at least one selected from the group consisting of and uvomorulin.   The device of claim 1, further comprising a second type of biological material in, on or near the permeable material.   The device of claim 1, further comprising fibroblasts in, on or near the permeable material.   The device of claim 1, further comprising a blood substitute flow proximate to the first side of the permeable material.   32. The device of claim 31, further comprising a bile surrogate flow proximate to the second side of the permeable material. maintaining the biological material under conditions that provide at least one in vitro pharmacokinetic parameter value comparable to the at least one pharmacokinetic parameter value found in vivo;
Passing a substance through at least a portion of a permeable material;
Including methods.   34. The method of claim 33, further comprising maintaining the biological material in or near a microscale mechanism.   The permeable material is a membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL, cell, cellular material, tissue, and tissue 34. The method of claim 33, wherein the method is selected from at least one of the group consisting of strips.   34. The method of claim 33, wherein the permeable material further comprises an organic or inorganic material in, on or near a microporous surface.   The permeable material may be a biological barrier, a passage of a substance in or through the biological barrier, or an absorption of a substance through or through the biological barrier. 34. The method of claim 33, configured to simulate at least one of them.   The biological barrier includes gastrointestinal barrier, blood brain barrier, blood bile barrier, lung barrier, placental barrier, epidermal barrier, eye barrier, olfactory barrier, gastroesophageal barrier, mucosa, blood urine barrier, and air tissue barrier, oral cavity 38. The method of claim 37, wherein the method is selected from at least one of the group consisting of a barrier, an anorectal barrier, a vaginal barrier, and a urethral barrier.   The at least one pharmacokinetic parameter includes tissue size, tissue size ratio, tissue to blood volume ratio, drug residence time, cell-to-cell interaction, liquid residence time, liquid to cell ratio, metabolism by cells, shear stress, flow rate, surface 34. The method of claim 33, selected from at least one of the group consisting of shape, circulation transit time, fluid distribution, interaction between tissues and / or organs, and molecular transport by cells.   34. The method of claim 33, further comprising the step of measuring the absorption, metabolism, or distribution of substances in or through the permeable material.   The mechanism represents at least one of the group consisting of at least part of the central nervous system, circulatory system, digestive system, bile duct system, lung system, urinary system, visual system, olfactory system, epidermal system, and lymphatic system 35. The method of claim 34, configured as follows.   34. The method of claim 33, further comprising positioning the permeable material in or out of a microscale device.   34. The method of claim 33, further comprising flowing a fluid through at least one microfluidic channel connected to the permeable material.   34. The method of claim 33, wherein a flow of fluid through or through the permeable material provides at least one pharmacokinetic parameter.   45. The method of claim 44, wherein the nature of the fluid flow through the device is based on a mathematical model.   46. The method of claim 45, wherein the mathematical model is a Physiologically-Based PharmacoKinetic ("PBPK") model.   34. The method of claim 33, further comprising incorporating the microscale feature or the permeable material into a chip format.   34. The method of claim 33, wherein the permeable material comprises a layer of gastrointestinal cells cultured on a microporous material.   49. The method of claim 48, further comprising positioning at least a portion of the layer of gastrointestinal cells such that fluid can flow along either side of the layer without passing through the layer.   At least a first microscale mechanism located on the first side of the layer of gastrointestinal cells represents the gastrointestinal tract, and at least a second microscale mechanism located on the second side of the monolayer represents the circulatory system. 50. The method of claim 49.   51. The method of claim 50, further comprising a third microscale feature configured to contain the same or different types of biological material.   34. The method of claim 33, wherein the permeable material comprises a microporous material that is at least partially coated with an organic material.   34. The method of claim 33, further comprising positioning the cell in, on or near both sides of the permeable material.   54. The method of claim 53, further comprising providing absorption characteristics, metabolic enzyme activity, and / or expression levels.   54. The method of claim 53, wherein the cells on either side of the permeable material are of the same type or different types.   34. The method of claim 33, further comprising positioning hepatocytes in, on or near the microporous surface of the permeable material.   57. The method of claim 56, wherein at least a portion of the microporous surface comprises a protein that polarizes the hepatocytes.   34. The method of claim 33, wherein the permeable material comprises a cell line capable of forming and polarizing a confluent monolayer.   34. The method of claim 33, further comprising binding hepatocytes to the permeable material.   60. The method of claim 59, further comprising polarizing the hepatocyte.   The binding step includes protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β-catenin, cell adhesion molecule 60. The method of claim 59, comprising a binding agent that is at least one selected from the group consisting of:   34. The method of claim 33, further comprising positioning a second type of biological material in, on or near the permeable material.   34. The method of claim 33, further comprising positioning fibroblasts in, on or near the permeable material.   34. The method of claim 33, further comprising flowing a blood substitute proximate to the first side of the permeable material.   65. The method of claim 64, further comprising flowing a bile substitute proximate to the second side of the permeable material. A method of forming a device comprising:
Form a mechanism configured to maintain the biological material under conditions that provide at least one in vitro pharmacokinetic parameter value comparable to that of at least one pharmacokinetic parameter found in vivo And steps to
Adding, forming, or providing a permeable material, wherein the material is configured to pass a substance through at least a portion of the permeable material;
Including methods. means for maintaining the biological material under conditions that provide at least one in vitro pharmacokinetic parameter value comparable to that of at least one pharmacokinetic parameter found in vivo;
Means for providing a permeable barrier;
A device comprising: A microscale permeable material;
At least one binder configured to polarize the substance, the substance exhibiting at least one property of liver function;
A device comprising:   69. The device of claim 68, wherein the substance is one or more hepatocytes.   69. The device of claim 68, wherein the substance is a genetically modified biological material.   69. The device of claim 68, wherein the binding agent binds and polarizes hepatocytes to the microscale permeable material.   69. The device of claim 68, further comprising a second material type.   The device of claim 1, further comprising one or more fibroblasts located near at least one surface of the microscale permeable material.   The microscale permeable material is organic material, inorganic material, membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL (matrigel), cell 69. The device of claim 68, wherein the device is selected from at least one of the group consisting of: cell material, tissue, and tissue piece.   69. The device of claim 68, wherein the microscale permeable material is in, on or near a microporous surface.   The microscale permeable material may be a biological barrier, a passage of a substance in or through the biological barrier, or an absorption of a substance in, or through the biological barrier. 69. The device of claim 68, wherein the device is configured to simulate at least one of.   69. The device of claim 68, wherein the device treats the substance in or through the microscale permeable material.   78. The device of claim 77, wherein the processing step further comprises at least one of the group consisting of molecular absorption, extraction, excretion, metabolism, and distribution.   69. The device of claim 68, wherein the microscale permeable material is located in or out of the device.   69. The device of claim 68, further comprising at least one microfluidic channel connected to the microscale permeable material.   69. The device of claim 68, wherein the nature of the fluid flow through the device is based on a mathematical model.   82. The device of claim 81, wherein the mathematical model is a Physiologically-Based PharmacoKinetic ("PBPK") model.   69. The device of claim 68, wherein the feature or the microscale permeable material is incorporated into a chip format.   69. The device of claim 68, wherein the device provides absorption properties, metabolic enzyme activity, and / or expression levels.   69. The device of claim 68, further comprising positioning a biological material in, on or near both sides of the microscale permeable material.   86. The device of claim 85, wherein the biological material on either side of the microscale permeable material is of the same type or a different type.   69. The device of claim 68, wherein the microscale permeable material comprises a cell line capable of forming a fusible monolayer.   The binding agent includes protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β-catenin, cell adhesion molecule, 69. The device of claim 68, comprising at least one selected from the group consisting of and uvomorulin.   69. The device of claim 68, further comprising a blood substitute flow proximate to the first side of the microscale permeable material.   90. The device of claim 89, further comprising a bile surrogate flow proximate to a second side of the microscale permeable material.   Binding a substance manifesting at least one property of liver function to the microscale permeable material in a manner that polarizes the substance.   92. The method of claim 91, wherein the substance is one or more hepatocytes.   92. The method of claim 91, wherein the substance is a genetically modified biological material.   92. The method of claim 91, further comprising providing a second material type.   92. The method of claim 91, further comprising positioning one or more fibroblasts in the vicinity of at least one surface of the microscale permeable material.   The microscale permeable material is organic material, inorganic material, membrane, porous membrane, microporous silicon, semipermeable membrane, microporous material, microporous polymer, alginate, collagen, MATRIGEL (matrigel), cell 94. The method of claim 91, wherein the method is selected from at least one of the group consisting of: a cell material, a tissue, and a tissue piece.   92. The method of claim 91, further comprising positioning the microscale permeable material in, on or near a microporous surface.   The microscale permeable material may be a biological barrier, the passage of a substance in or through the biological barrier, or the absorption of a substance in, or through the biological barrier. 92. The method of claim 91, wherein at least one of   92. The method of claim 91, further comprising the step of processing the substance through or through the microscale permeable material.   100. The method of claim 99, wherein the treating step further comprises at least one of the group consisting of molecular absorption, extraction, excretion, metabolism, and distribution.   92. The method of claim 91, further comprising positioning the microscale permeable material in or out of the device.   92. The method of claim 91, further comprising providing at least one microfluidic channel connected to the microscale permeable material.   92. The method of claim 91, wherein the fluid flow property associated with the at least one property of liver function is based on a mathematical model.   104. The method of claim 103, wherein the mathematical model is a Physiologically-Based PharmacoKinetic ("PBPK") model.   92. The method of claim 91, further comprising incorporating the microscale permeable material into a chip format.   92. The method of claim 91, further comprising providing an absorption characteristic, metabolic enzyme activity, and / or expression level.   92. The method of claim 91, further comprising positioning a biological material in, on or near both sides of the microscale permeable material.   108. The method of claim 107, wherein the biological material on either side of the microscale permeable material is of the same type or of a different type.   92. The method of claim 91, wherein the microscale permeable material comprises a cell line capable of forming a fusible monolayer.   The binding step includes protein, connexin 32, tight junction protein, Occludin, Claudin-1, ZO-1, ZO-2, adhesion binding protein, E-cadherin, β-catenin, cell adhesion molecule 92. The method of claim 91, comprising providing a binding agent selected from at least one of the group consisting of:   92. The method of claim 91, further comprising providing a blood substitute flow proximate to the first side of the microscale permeable material.   92. The method of claim 91, further comprising providing a bile surrogate flow proximate to a second side of the microscale permeable material.   A method of forming a device comprising forming a microscale permeable material configured to bind and polarize a substance that exhibits at least one property of liver function.   A microscale device comprising means for binding a substance manifesting at least one property of liver function to a microscale permeable material in a manner that polarizes the substance. A microscale permeable material;
And at least one substance configured to manifest at least one property of liver function, wherein molecules processed by the substance are directed to pass through at least a portion of the microscale permeable material. ,
device.   A method comprising directing molecules treated with a substance through at least a portion of a microscale permeable material, wherein the substance is configured to demonstrate at least one property of liver function.   A method of forming a device comprising forming a microscale permeable material configured to direct molecules treated with a substance through at least a portion of the microscale permeable material; The method wherein the substance is configured to demonstrate at least one property of liver function.   A device comprising means for directing molecules treated by a substance to pass through at least a portion of a microscale permeable material, the substance configured to demonstrate at least one property of liver function.

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