21 January 2010 (21.01.2010)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`
`(10) International Publication Number
`WO 2010/009307 A2
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA. CH,CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU,ID,IL, IN,IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG,PH,PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT,
`TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(51) International Patent Classification:
`C12M 3/06 (2006.01)
`BOLL 3/00 (2006.01)
`Int
`ti
`1 Application Number:
`International
`Application Number:
`PCT/US2009/050830
`
`21)
`(21)
`
`(22)
`
`InternationalFiling Date:
`
`16 July 2009 (16.07.2009)
`.
`English
`English
`
`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`61/08 1.080
`
`16 July 2008 (16.07.2008)
`
`(71) Applicant (for all designated States except US): CHIL-
`DREN'S MEDICAL CENTER CORPORATION[US/
`US]; 55 Shattuck Street, Boston, MA 02115 (US).
`Inventors; and
`Inventors/Applicants (for US only): INGBER, Donald,
`E. [US/US]; 71 Montgomery Street, Boston, MA 02116
`(US). HUH, Dongeun [KR/US];
`115 Peterborough
`Street, #44, Boston, MA 02215 (US).
`
`(72)
`(75)
`
`us (84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW,GIL,
`GM,KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, 7M,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HR, HU,IE,IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SL, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR,NE,SN, TD, TG).
`
`Published:
`
`(74) Agents: RESNICK, David et al.; Nixon Peabody Llp, — witheut international search report and to be republished
`100 SummerStreet, Boston, MA 02110 (US).
`upon receipt of that report (Rule 48.2(g))
`
`(54) Title: ORGAN MIMIC DEVICE WITH MICROCHANNELS AND METHODS OF USE AND MANUFACTURING
`THEREOF
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`
`wo2010/009307A2IMITINMINIIMNITMNINYTATUIAAUAA
`
`112
`
`114
`
`Display
`
`Fluid
`Source
`
`118
`
`116
`
`
` inter|104n 104 406 no
`
`
`
`
`
`
`Pressure
`
`Source
`
` TaninterfaceTanDevice
`
`
`100
`Collector
`
`
`
`FIG. 1
`
`(57) Abstract: System and method includes a body having a central microchannel separated by one or more porous membranes.
`The membranes are configured to divide the central microchannel into a two or more parallel central microchannels, wherein one
`or more first fluids are applied through the first central microchannel and one or more second fluids are applied through the sec-
`ond or more central microchannels. The surfaces of each porous membrane can be coated with cell adhesive molecules to support
`the attachment of cells and promote their organization into tissues on the upper and lower surface of the membrane. The pores
`may be large enough to only permit exchange of gases and small chemicals, or to permit migration and transchannel passage of
`large proteins and whole living cells. Fluid pressure, flow and channel geometry also may be varied to apply a desired mechanical
`force to one or both tissue layers.
`
`

`

`WO 2010/009307
`
`PCT/US2009/0350830
`
`ORGAN MIMIC DEVICE WITH MICROCHANNELS AND METHODS OF USE
`AND MANUFACTURING THEREOF
`
`CROSS REFERENCE TO RELATED APPLICATION
`
`[0001]
`
`This application claims benefit under 35 U.S.C. § 119(e) of the U.S. provisional
`
`application No. 61/081,080 filed July 16, 2008, the contents of which are incorporated herein
`
`by reference in its entirety.
`
`GOVERNMENT SUPPORT
`
`[0002]
`
`This invention was made with Government support under Grant No.: NIH RO1
`
`ES016665-01A1 awarded by the National Institutes of Health. The Government has certain
`
`rights in the invention.
`
`TECHNICAL FIELD
`
`[0003]
`
`The present disclosure relates generally to an organ mimic device with
`
`microchannels and methods of use and manufacturing thereof.
`
`BACKGROUND
`
`[0004]
`
`Mechanical forces - pushes, pulls, tensions, compressions - are important
`
`regulators of cell development and behavior. Tensegrity provides the structure that
`
`determines how these physical forces are distributed inside a cell or tissue, and how and
`
`where they exert their influence.
`
`[0005]
`
`In the human body, most cells are constantly subjected to mechanical forces, such
`
`as tension or compression. Application of mechanical strain to cells in culture simulates the
`
`in vivo environment, causing dramatic morphologic changes and biomechanical responsesin
`
`the cells. There are both long and short term changes that occur whencells are mechanically
`
`loaded in culture, such as alterations in the rate and amount of DNA or RNAsynthesis or
`
`degradation, protein expression and secretion, the rate of cell division and alignment, changes
`
`in energy metabolism, changesin rates of macromolecular synthesis or degradation, and other
`
`changes in biochemistry and bicenergetics.
`
`

`

`WO 2010/009307
`
`PCT/US2009/0350830
`
`[0006]
`
`Every cell has an internal scaffolding, or cytoskeleton, a lattice formed from
`
`molecular "struts and wires". The "wires" are a crisscrossing network of fine cables, known
`
`as microfilaments, that stretch from the cell membrane to the nucleus, exerting an inward
`
`pull. Opposing the pull are microtubules, the thicker compression-bearing "struts” of the
`
`cytoskeleton, and specialized receptor molecules on the cell's outer membrane that anchor the
`
`cell to the extracellular matrix, the fibrous substance that holds groups of cells together. This
`
`balance of forces is the hallmark of tensegrity.
`
`[0007]
`
`Tissues are built from groups of cells, like eggs sitting on the "egg carton” of the
`
`extracellular matrix. The receptor molecules anchoring cells to the matrix, known as
`
`integrins, connect the cells to the wider world. Mechanical force on a tissueis felt first by
`
`integrins at these anchoring points, and then is carried by the cytoskeleton to regions deep
`
`inside each cell. Inside the cell, the force might vibrate or change the shape of a protein
`
`molecule, triggering a biochemical reaction, or tug on a chromosomein the nucleus,
`
`activating a gene.
`
`[0008]
`
`Cells also can be said to have "tone," just like muscles, because of the constant
`
`pull of the cytoskeletal filaments. Much like a stretched violin string produces different
`
`sounds when force is applied at different points along its length, the cell processes chemical
`
`signals differently depending on how muchit is distorted.
`
`[0009]
`
`A growth factor will have different effects depending on how muchthecell is
`
`stretched. Cells that are stretched and flattened, like those in the surfaces of wounds, tend to
`
`grow and multiply, whereas rounded cells, cramped by overly crowded conditions, switch on
`
`a "suicide" program and die. In contrast, cells that are neither stretched norretracted carry on
`
`with their intended functions.
`
`[0010]
`
`Another tenet of cellular tensegrity is that physical location matters. When
`
`regulatory molecules float around loose insidethe cell, their activities are little affected by
`
`mechanical forces that act on the cell as a whole. But when they're attached to the
`
`cytoskeleton, they becomepart of the larger network, and are in a position to influence
`
`cellular decision-making. Many regulatory and signaling molecules are anchored on the
`
`cytoskeletonat the cell's surface membrane, in spots known as adhesion sites, where integrins
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`cluster. These prime locations are key signal-processing centers, like nodes on a computer
`
`network, where neighboring molecules can receive mechanical information from the outside
`
`world and exchangesignals.
`
`[0011]
`
`Thus, assessing the full effects of drugs, drug delivery vehicles, nanodiagnostics
`
`or therapies or environmental stressors, such as particles, gases, and toxins, in a physiological
`
`environment requires not only a study of the cell-cell and cell-chemical interactions, but also
`
`a study of how these interactions are affected by physiological mechanical forces in both
`
`healthy tissues and tissues affected with diseases.
`
`[0012]
`
`Methodsof altering the mechanical environmentor response ofcells in culture
`
`have included wounding cells by scraping a monolayer, applying magnetic orelectric fields,
`
`or by applyingstatic or cyclic tension or compression with a screw device, hydraulic
`
`pressure, or weights directly to the cultured cells. Shear stress has also been induced by
`
`subjecting the cells to fluid flow. However, few of these procedures have allowed for
`
`quantitation of the applied strains or provided regulation to achieve a broad reproducible
`
`range of cyclic deformations within a culture microenvironment that maintains
`
`physiologically relevant tissue-tissue interactions.
`
`[0013]
`
`Living organsare three-dimensional vascularized structures composed of two or
`
`more closely apposed tissues that function collectively and transport materials, cells and
`
`information across tissue-tissue interfaces in the presence of dynamic mechanical forces, such
`
`as fluid shear and mechanicalstrain. Creation of microdevices containing living cells that
`
`recreate these physiologicaltissue-tissue interfaces and permit fluid flow and dynamic
`
`mechanical distortion would have great value for study of complex organ functions, e.g.,
`
`immunecell trafficking, nutrient absorption, infection, oxygen and carbon dioxide exchange,
`
`etc., and for drug screening, toxicology, diagnostics and therapeutics.
`
`[0014]
`
`The alveolar-capillary unit plays a vital role in the maintenance of normal
`
`physiological function of the lung as well as in the pathogenesis and progression of various
`
`pulmonary diseases. Because of the complex architecture of the lung, the small size of lung
`
`alveoli and their surrounding microvessels, and the dynamic mechanical motionsof this
`
`organ, it is difficult to study this structure at the microscale.
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`[0015]
`
`The lung has an anatomically unique structure having a hierarchical branching
`
`network of conducting tubes that enable convective gas transport to and from the microscopic
`
`alveolar compartments where gas exchange occurs. The alveolus is the most important
`
`functional unit of the lung for normal respiration, and it is most clinically relevantin thatit is
`
`the blood-gas barrier or interface, as well as the site where surfactants act to permit air entry
`
`and where immunecells, pathogens and fluids accumulate in patients with acute respiratory
`
`distress syndrome (ARDS) or infections, such as pneumonia.
`
`[0016]
`
`The blood-gas barrier or tissue-tissue interface between the pulmonary capillaries
`
`and the alveolar lumen is composed of a single layer of alveolar epithelium closely
`
`juxtaposed to a single layer of capillary endothelium separated by a thin extracellular matrix
`
`(ECM), which forms through cellular and molecular self-assembly in the embryo. Virtually
`
`all analysis of the function of the alveolar-capillary unit has been carried out in whole animal
`
`studies because it has not been possible to regenerate this organ-level structure in vitro.
`
`[0017]
`
`A major challenge lies in the lack of experimental tools that can promote
`
`assembly of multi-cellular and multi-tissue organ-like structures that exhibit the key
`
`structural organization, physiological functions, and physiological or pathological mechanical
`
`activity of the lung alveolar-capillary unit, which normally undergoes repeated expansion and
`
`contraction during each respiratory cycle. This limitation could be overcomeif it were
`
`possible to regenerate this organ-level structure and recreate its physiological mechanical
`
`microenvironmentin vitro. However, this has not been fully accomplished.
`
`[0018]
`
`Whatis needed is a organ mimic device capable of being used in vitro or in vivo
`
`which performstissue-tissue related functions and which also allowscells to naturally
`
`organize in the device in response to not only chemical but also mechanical forces and allows
`
`the study of cell behavior through a membrane which mimics tissue-tissue physiology.
`
`OVERVIEW
`
`[0019]
`
`System and method comprises a body having a central microchannel separated by
`
`one or more porous membranes. The membranes are configured to divide the central
`
`microchannel into a two or more closely apposed parallel central microchannels, wherein one
`
`or morefirst fluids are applied through the first central microchannel and one or more second
`
`fluids are applied through the second or more central microchannels. The surfaces of each
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`porous membrane can be coated with cell adhesive molecules to support the attachment of
`
`cells and promote their organization into tissues on the upper and lower surface of each
`
`membrane, thereby creating one or more tissue-tissue interfaces separated by porous
`
`membranes between the adjacent parallel fluid channels. The membrane may be porous,
`
`flexible, elastic, or a combination thereof with pores large enough to only permit exchange of
`
`gases and small chemicals, or large enough to permit migration and transchannel passage of
`
`large proteins, as well as whole living cells. Fluid pressure, flow characteristics and channel
`
`geometry also may be varied to apply a desired fluid shear stress to one or both tissue layers.
`
`[0020]
`
`In an embodiment, operating channels adjacent to the central channel are applied a
`
`positive or negative pressure which creates a pressure differential that causes the membrane
`
`to selectively expand and retract in response to the pressure, thereby further physiologically
`
`simulating mechanical force of a living tissue-tissue interface.
`
`[0021]
`
`In another embodiment, three or more parallel microchannels are separated by a
`
`plurality of parallel porous membranes whichare lined by a commontissue type in the
`
`central channel and two different tissue types on the opposite sides of the membranes in the
`
`two outer channels. An example would be a cancer mimic device in which cancercells are
`
`grown in the central microchannel and on the inner surfaces of both porous membranes,
`
`while capillary endothelium is grown on the opposite surface of one porous membrane and
`
`lymphatic endothelium is grown on the opposite surface of the second porous membrane.
`
`This recreates the tumor microarchitecture and permits study of delivery of oxygen, nutrients,
`
`drugs and immunecells via the vascular conduit as well as tumor cell egress and metastasis
`
`via the lymphatic microchannel.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0022]
`
`The accompanying drawings, which are incorporated into and constitute a part of
`
`this specification, illustrate one or more examples of embodiments and, together with the
`
`description of example embodiments, serve to explain the principles and implementations of
`
`the embodiments. In the drawings:
`
`[0023]
`
`Figure 1 illustrates a block diagram of a system employing an example organ
`
`mimic device in accordance with an embodiment.
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`[0024]
`
`Figure 2A illustrates a perspective view of a organ mimic device in accordance
`
`with an embodiment.
`
`[0025]
`
`Figure 2B illustrates an exploded view of the organ mimic device in accordance
`
`with an embodiment.
`
`[0026]
`
`Figures 2C-2Dillustrate perspective viewsoftissue-tissue interface regions of the
`
`device in accordance with an embodiment.
`
`[0027]
`
`Figures 2E-2G illustrate top down cross sectional views of the tissue-tissue
`
`interface regions of the device in accordance with one or more embodiments.
`
`[0028]
`
`Figures 3A-3B illustrate perspective viewsof tissue-tissue interface regions of the
`
`device in accordance with an embodiment.
`
`[0029]
`
`Figures 3C-3Eillustrate perspective views of the membrane in accordance with
`
`one or more embodiments.
`
`[0030]
`
`Figures 4A-4Cillustrate perspective views of the formation of the membraneof a
`
`two channel device in accordance with an embodiment.
`
`[0031]
`
`Figure 4D illustrates a side view of the membrane of the tissue-tissue interface
`
`device in accordance with an embodiment.
`
`[0032]
`
`Figures 5A-5E illustrate perspective views of the formation of the organ mimic
`
`device in accordance with an embodiment.
`
`[0033]
`
`Figure 6 illustrates a system diagram employing an organ mimic device with
`
`multiple channels in accordance with an embodiment.
`
`[0034]
`
`Figures 7A-7B illustrate perspective views of the organ mimic device in
`
`accordance with an embodiment.
`
`[0035]
`
`Figure 7C illustrates a side view of the membrane of the organ mimic device in
`
`accordance with an embodiment.
`
`[0036]
`
`Figures 8 and 9 illustrate ROS generation over time in accordance with an
`
`experiment conducting with the present device.
`
`DESCRIPTION OF EXAMPLE EMBODIMENTS
`
`[0037]
`
`Example embodiments are described herein in the context of an organ simulating
`
`device and methods of use and manufacturing thereof. Those of ordinary skill in the art will
`
`realize that the following description is illustrative only and is not intended to be in any way
`
`limiting. Other embodiments will readily suggest themselves to such skilled persons having
`
`the benefit of this disclosure. Reference will now be madein detail to implementations of the
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`example embodimentsas illustrated in the accompanying drawings. The same reference
`
`indicators will be used throughout the drawings and the following description to refer to the
`
`same orlike items. It is understood that the phrase “an embodiment” encompasses more than
`
`one embodimentandis thus not limited to only one embodimentfor brevity’s sake.
`
`[0038]
`
`In accordance with this disclosure, the organ mimic device (also referred to as
`
`“present device’’) is preferably utilized in an overall system incorporating sensors, computers,
`
`displays and other computing equipmentutilizing software, data components, process steps
`
`and/or data structures. The components, process steps, and/or data structures described
`
`herein with respect to the computer system with which the organ mimic device is employed
`
`may be implemented using various types of operating systems, computing platforms,
`
`computer programs, and/or general purpose machines. In addition, those of ordinary skill in
`
`the art will recognize that devices of a less general purpose nature, such as hardwired devices,
`
`field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), or
`
`the like, may also be used without departing from the scope andspirit of the inventive
`
`concepts disclosed herein.
`
`[0039]
`
`Where a method comprising a series of process steps is implemented by a
`
`computer or a machine with use with the organ mimic device described below and those
`
`process steps can bestored as a series of instructions readable by the machine, they may be
`
`stored on a tangible medium such as a computer memorydevice (e.g., ROM (Read Only
`
`Memory), PROM (Programmable Read Only Memory), EEPROM (Electrically Eraseable
`
`Programmable Read Only Memory), FLASH Memory, Jump Drive, and the like), magnetic
`
`storage medium (e.g., tape, magnetic disk drive, and the like), optical storage medium (e.g.,
`
`CD-ROM, DVD-ROM, papercard, paper tape and the like) and other types of program
`
`memory.
`
`[0040]
`
`Embodiments of the present device can be applied in numerousfields including
`
`basic biological science, life science research, drug discovery and development, drug safety
`
`testing, chemical and biological assays, as well as tissue and organ engineering.
`
`In an
`
`embodiment, the organ mimic device can be used as microvascular network structures for
`
`basic research in cardiovascular, cancer, and organ-specific disease biology. Furthermore,
`
`one or more embodiments of the device find application in organ assist devices for liver,
`
`kidney, lung, intestine, bone marrow, and other organs and tissues, as well as in organ
`
`reeplacement structures.
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`[0041]
`
`The cellular responses to the various environmental cues can be monitored using
`
`various systems that can be combined with the present device. One can monitor changes in
`
`pH using well knownsensors. One can also sample cells, continuously or periodically for
`
`measurement of changes in gene transcription or changesin cellular biochemistry or
`
`structural organization. For example, one can measure reactive oxygen species (ROIs) that
`
`are a sign of cellular stress. One can also subject the “tissue” grown on the porous membrane
`
`to microscopic analysis, immunohistochemical analysis, in situ hybridization analysis, or
`
`typical pathological analysis using staining, such as hematoxylin and eosin staining. Samples
`
`for these analysis can be carried out in real-time, or taken after an experimentor by taking
`
`small biopsies at different stages during a study or an experiment.
`
`[0042]
`
`Onecan subject the cells grown on the membrane to other cells, such as immune
`
`system cells or bacterial cells, to antibodies or antibody-directed cells, for example to target
`
`specific cellular receptors. One can expose the cells to viruses or other particles. To assist in
`
`detection of movement of externally supplied substances, such as cells, viruses, particles or
`
`proteins, one can naturally label them using typical meanssuch as radioactive or fluorescent
`
`labels.
`
`[0043]
`
`Cells can be grown, cultured and analyzed using the present device for 1, 2, 3, 4,
`
`5, 6, or 7 days, between at least 1-2 weeks, and even over 2 weeks. For example, as
`
`discussed below,it has been shown that co-culture of alveolar epithelial cells with pulmonary
`
`microvascular endothelial cells on a thin porous membrane in an embodimentof the
`
`described device could be grown for over two weeks without loss of viability of the cells.
`
`[0044]
`
`The organ mimic device described herein has manydifferent applications
`
`including, but not limited to, identification of markers of disease; assessing efficacy of anti-
`
`cancer therapeutics; testing gene therapy vectors; drug development; screening; studies of
`
`cells, especially stem cells and bone marrow cells; studies on biotransformation, absorption,
`
`clearance, metabolism, and activation of xenobiotics; studies on bioavailability and transport
`
`of chemical or biological agents across epithelial or endothelial layers; studies on transport of
`
`biological or chemical agents across the blood-brain barrier; studies on transport of biological
`
`or chemical agents across the intestinal epithelial barrier; studies on acute basal toxicity of
`
`chemical agents; studies on acute local or acute organ-specific toxicity of chemical agents;
`
`studies on chronic basal toxicity of chemical agents; studies on chronic local or chronic
`
`organ-specific toxicity of chemical agents; studies on teratogenicity of chemical agents;
`
`studies on genotoxicity, carcinogenicity, and mutagenicity of chemical agents; detection of
`
`infectious biological agents and biological weapons; detection of harmful chemical agents
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`and chemical weapons; studies on infectious diseases; studies on the efficacy of chemical or
`
`biological agents to treat disease; studies on the optimal dose range of agents to treat disease;
`
`prediction of the response of organsin vivo to biological agents; prediction of the
`
`pharmacokinetics of chemical or biological agents; prediction of the pharmacodynamics of
`
`chemical or biological agents; studies concerning the impact of genetic content on response
`
`to agents; studies on genetranscription in response to chemicalor biological agents; studies
`
`on protein expression in response to chemical or biological agents; studies on changes in
`
`metabolism in response to chemical or biological agents. The organ mimic device can also
`
`be used to screen on the cells, for an effect of the cells on the materials (for example, in a
`
`manner equivalent to tissue metabolism of a drug).
`
`[0045]
`
`The present device may be used by one to simulate the mechanical load
`
`environment of walking, running, breathing, peristalsis, flow of flow orurine, orthe beat of a
`
`heart, to cells cultured from mechanically active tissues, such as heart, lung, skeletal muscle,
`
`bone, ligament, tendon, cartilage, smooth musclecells, intestine, kidney, endothelial cells and
`
`cells from other tissues. Rather than test the biological or biochemical responses of a cell ina
`
`static environment, the investigator can apply a range of frequencies, amplitudes and duration
`
`of mechanical stresses, including tension, compression and shear, to cultured cells.
`
`[0046]
`
`A skilled artisan can implant various types of cells on the surfaces of the
`
`membrane. Cells include any cell type from a multicellular structure, including nematodes,
`
`amoebas, up to mammals such as humans. Cell types implanted on the device depend on the
`
`type of organ or organ function one wishes to mimic, and the tissues that comprise those
`
`organs. More details of the various types of cells implantable on the membraneof the present
`
`device are discussed below.
`
`[0047]
`
`One can also co-culture various stem cells, such as bone marrow cells, induced
`
`adult stem cells, embryonal stem cells or stem cells isolated from adult tissues on either or
`
`both sides of the porous membrane. Using different culture media in the chambers feeding
`
`each layer of cells, one can allow different differentiation cues to reach the stem cell layers
`
`thereby differentiating the cells to different cell types. One can also mix cell types on the
`
`same side of the membrane to create co-cultures of different cells without membrane
`
`separation.
`
`[0048]
`
`Using the organ mimic device described herein, one can study biotransformation,
`
`absorption, clearance, metabolism, and activation of xenobiotics, as well as drug delivery.
`
`The bioavailability and transport of chemical and biological agents across epithelial layers as
`
`in the intestine, endothelial layers as in blood vessels, and across the blood-brain barrier can
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`also be studied. The acute basal toxicity, acute local toxicity or acute organ-specific toxicity,
`
`teratogenicity, genotoxicity, carcinogenicity, and mutagenicity, of chemical agents can also
`
`be studied. Effects of infectious biological agents, biological weapons, harmful chemical
`
`agents and chemical weaponscan also be detected and studied. Infectious diseases and the
`
`efficacy of chemical and biological agents to treat these diseases, as well as optimal dosage
`
`ranges for these agents, can be studied. The response of organs in vivo to chemical and
`
`biological agents, and the pharmacokinetics and pharmacodynamicsof these agents can be
`
`detected and studied using the present device. The impact of genetic content on response to
`
`the agents can be studied. The amount of protein and gene expression in response to chemical
`
`or biological agents can be determined. Changes in metabolism in response to chemical or
`
`biological agents can be studied as well using the present device.
`
`[0049]
`
`The advantages of the organ mimic device, as opposed to conventional cell
`
`cultures or tissue cultures, are numerous. For instance, when cells are placed in the organ
`
`mimic device, fibroblast, SMC (smooth muscle cell) and EC (endothelial cell) differentiation
`
`can occur that reestablishes a defined three-dimensional architectural tissue-tissue
`
`relationships that are close to the iv vivo situation, and cell functions and responses to
`
`pharmacological agents or active substances or products can be investigated at the tissue and
`
`organ levels.
`
`[0050]
`
`In addition, many cellular or tissue activities are amenable to detection in the
`
`organ mimic device, including, but not limited to, diffusion rate of the drugs into and through
`
`the layered tissues in transported flow channel; cell morphology, differentiation and secretion
`
`changesat different layers; cell locomotion, growth, apoptosis, and the like. Further, the
`
`effect of various drugs on different types of cells located at different layers of the system may
`
`be assessed easily.
`
`[0051]
`
`For drug discovery, for example, there can be two advantages for using the organ
`
`mimic device described herein: (1) the organ mimic deviceis better able to mimic in vivo
`
`layered architecture of tissues and therefore allow one to study drug effect at the organ level
`
`in addition to at the cellular and tissue levels; and (2) the organ mimic device decreases the
`
`use of in vivo tissue models andthe use of animals for drug selection and toxicology studies.
`
`[0052]
`
`In addition to drug discovery and development, the organ mimic device described
`
`herein may be also useful in basic and clinical research. For example, the organ mimic
`
`device can be used to research the mechanism of tumorigenesis. It is well established that in
`
`vivo cancer progression is modulated by the host and the tumor micro-environment, including
`
`10
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`the stromal cells and extracellular matrix (ECM). For example, stromal cells were found
`
`being able to convert benign epithelial cells to malignant cells, thereby ECM was found to
`
`affect the tumor formation. There is growing evidence that cells growing in defined
`
`architecture are more resistant to cytotoxic agents than cells in mono layers. Therefore, a
`
`organ mimic device is a better means for simulating the original growth characteristics of
`
`cancer cells and thereby better reflects the real drug’s sensitivity of in vivo tumors.
`
`[0053]
`
`The organ mimic device can be employed in engineering a variety of tissues
`
`including, but not limited to, the cardiovascular system, lung, intestine, kidney, brain, bone
`
`marrow, bones, teeth, and skin. If the device is fabricated with a suitable biocompatible
`
`and/or biodegradable material, such as poly-lactide-co-glycolide acid (PLGA), the organ
`
`mimic device maybe used for transplantation or implantation in vivo. Moreover,the ability to
`
`spatially localize and control interactions of several cell types presents an opportunity to
`
`engineerhierarchically, and to create more physiologically correct tissue and organ analogs.
`
`The arrangement of multiple cell types in defined arrangement has beneficial effects on cell
`
`differentiation, maintenance, and functional longevity.
`
`[0054]
`
`The organ mimic device can also allow different growth factors, chemicals, gases
`
`and nutrients to be addedto different cell types according to the needs of cells and their
`
`existence im vivo. Controlling the location of those factors or proteins may direct the process
`
`of specific cell remodeling and functioning, and also may provide the molecular cues to the
`
`whole system, resulting in such beneficial developments as neotissue, cell remodeling,
`
`enhanced secretion, and the like.
`
`[0055]
`
`In yet another aspect, the organ mimic device can be utilized as multi cell type
`
`cellular microarrays, such as microfluidic devices. Using the organ mimic device, pattern
`
`integrity of cellular arrays can be maintained. Thesecellular microarrays may constitute the
`
`future "lab-on-a-chip”, particularly when multiplexed and automated. These miniaturized
`
`multi cell type cultures will facilitate the observation of cell dynamics with faster, less noisy
`
`assays, having built-in complexity that will allow cells to exhibit im vivo-like responses to the
`
`array.
`
`[0056]
`
`In yet anotheraspect, the organ mimic device can be utilized as biological sensors.
`
`Cell-based biosensors can provide more information than other biosensors because cells often
`
`have multifaceted physiological responsesto stimuli, as well as novel mechanismsto amplify
`
`these responses. All cell types in the organ mimic device can be used to monitor different
`
`aspects of an analyte at the same time; different cell type in the organ mimic device can be
`
`used to monitor different analytes at the same time; or a mixture of both types of monitoring.
`
`11
`
`

`

`WO 2010/009307
`
`PCT/US2009/050830
`
`Cells ranging from E. coli to cells of mammalian lines have been used as sensors for
`
`applications in environmental monitoring, toxin detection, and physiological monitoring.
`
`[0057]
`
`In yet anotheraspect, the organ mimic device can be used in understanding
`
`fundamental processes in cell biology and cell-ECM interactions. The in vivo remodeling
`
`process is a complicated, dynamic, reciprocal process between cells and ECMs. The organ
`
`mimic device would be able to capture the complexity of these biological systems, rendering
`
`these systems amenable to investigation and beneficial manipulation. Furthermore, coupled
`
`with imaging too