Apparatus and methods for monitoring and controlling biological systems

Abstract

Provided are devices that simulate in vivo conditions of a biological system and that are modular for reliable assembly and disassembly to improve handling of biological systems and subsequent analysis and experimentation therein. A center block having a fluidic network to support a biological material is in fluidic connection with a pair of side channels. With this platform, biological material may be supported and exposed to a plurality of well-defined fluidic characteristics and compositions.

Description

APPARATUS AND METHODS FOR MONITORING AND CONTROLLING BIOLOGICAL SYSTEMSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 538,709, filed on Sept. 15. 2023, which is incorporated by reference herein in its entirety to the extent not inconsistent herewith.BACKGROUND OF INVENTION

[0002] Many in vitro cellular studies are based on cell monolayer cultures and cannot satisfactorily mimic the complex three-dimensional environments and multi- component / constituent structure of biological tissue, including organs, tumors, and soft tissue. The status quo as it pertains to develop new anticancer drugs is to use monolayer (2D) cell culture or animal models in preclinical phase in order to assess efficacy, distribution and toxicity of the compounds. However, 2D cell culture does not recapitulate cell-cell interactions, cellmatrix interactions, nutrient and oxygen gradients, or cell polarity’ and, in addition to all ethical problems, animal models present important differences with human physiology’. Hence, these models appear to be too limited and explain the important rate of failure of drug candidate in clinical phase while rising up the development cost.

[0003] Although some prior art multi-culture systems have been developed, they still suffer from the fundamental disadvantage of not reproducing the complete architecture of the modeled biological system. For example, the tumor microenvironment (TME) has a high impact on cancer cell radiation response and, ultimately, patient survival after radiation treatment. Accordingly, there is a need in the art for cell culture methods and systems that better reflect the actual 3-D properties, both physical and biological, of a biological tissue. In the context of cancer cell treatment, improved cancer cell culture systems that address these issues would be beneficial for a range of therapeutic and treatment applications. One example of such a system is provided in WO 2020 / 264388 (Atty Ref. 338408: 88-19 WO) titled “Method and Apparatus for Interrogating Biological Systems.” Provided herein are devices and related methods having configurations for improved support, monitoring, control and subsequent removal of biological microsystems from the device.SUMMARY OF THE INVENTION

[0004] Provided herein are devices and related methods that address the above problems byutilizing a modular microfluidic device (referred herein as “device”) for simulating in vivo conditions of a biological system. The modular microfluidic device may be configured to facilitate grow th of the one or more 3D structures of cultured biological cells along all three dimensional axes. The methods and systems provided herein are compatible with a range of tissue cells, depending on the application of interest, such as cellular components of an organ, cancer cells of a tumor, cells of the vasculature, such as endothelial cells and smooth muscle cells, and combinations thereof. Any of the methods and systems provided herein may further comprise extracellular matrix, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support of surrounding cells. The devices are characterized as “modular” in that the device is reliably assembled to support a biological system and readily disassembled without damaging the biological system. In this manner, the biological systems may be cultured in vitro but under realistic and controllable fluidic conditions (e.g., flow-rates, fluid composition, with as desired appropriate “challenges” such as a putative therapeutic, radiation challenge, adverse and / or rescue input). The various compartments and components of the biological system may also be removed from the system without undue damage for subsequent assays, applications or testing. In this manner, the “base” test biological material is grown under well-controlled in vitro conditions with a good in vivo model and can be later used in any of a variety of applications. Furthermore, the device modularity are configured so that one or more portions sustaining a biological material, including in another compartment, can be safely and reliably removed without unwanted damage to that another component.

[0005] The devices and related methods are particularly suited for sustaining a multi- cellular culture by a specially -configured multi-compartment layout, generally provided herein as a center block, a first side channel and a second side channel, with each of the compartments independently controllable from the others and thereby precisely tailored to have conditions tailored to the relevant biological material in the component and / or the desired culture conditions. The compartments may also be fluidically and / or diffusively connected, thereby facilitating biological cross-talk between different conditions. As one example, three-dimensionally relevant biological material, such as tumor spheroids, may be supported in the central block compartment,along with lung endothelial cells and normal fibroblasts in the side channels, thereby biologically reproducing vascular and stromal compartments.

[0006] Provided herein are devices useful for sustaining a biological material. As one example, the device can be generally characterized as an in vitro support of a plurality of biological materials. The device is modular, with plug-and-play compartments in the form of a center block, a first side channel, and a second side channel, wherein each side channel is supported by an opposibly positioned center block surface.

[0007] Provided are devices comprising: a center block having: a first surface; a second surface opposibly positioned to the first surface and separated from the first surface by a center block thickness; and a fluidic network in fluidic contact with said fluid inlet port and said fluid outlet port. A first side channel is configured to be positioned adjacent to the center block first surface, the first side channel having: a first fluid inlet port and a first fluid outlet port. The second side channel is similar, expect it is configured to be positioned adjacent to the center block second surface, the second side channel having, a second fluid inlet port; and a second fluid outlet port. In this manner, the first and second channels may facilitate independent fluid flow, including independent fluid compositions and / or shear stress. A first membrane is positioned between the first side channel and the center block first surface and a second membrane positioned between the second side channel and the center block second surface. Each of the membranes are configured to support a biological material, such as a cell culture. The center block fluidic network is configured to support a plurality7of biological materials or organisms, including 3D materials such as organoids, spheroids, tumors or the like. The center block fluidic network is in fluidic contact with the first and second fluid inlet ports and the first and second fluid outlet ports. The first membrane is in fluidic contact with the first inlet port and the first outlet port. The second membrane is in fluidic contact with the second inlet port and the second outlet port.

[0008] The device may further comprise a removable positioning material holder configured to be inserted into the center block fluidic network to support positioning a material, such as a biological material, in at least a well of the center block fluidic network. The positioning material holder may correspond to a gel with the holder shaped to provide a desired indentation in the gel. Alternatively, the material holder may correspond to polymer beads or the like.

[0009] The device may further comprise a removable gasket configured to at least partially surround the center block, wherein the fluidic network of the center block has openingspositioned through the first and second surfaces. A center block enclosure is configured to removably surround the gasket and contain the gasket in a tight configuration for reliable positioning of the material in a plurality of the wells in the center block fluidic network;

[0010] The device may further comprise a connector to connect the center block to each of the first and second side channels, the connector having a central region with a central geometry and a pair of terminal regions with terminal geometries, wherein the central region is between the pair of terminal regions; the center block having a third surface and a fourth surface extending betw een the first and second surfaces, wherein each of the third and fourth surfaces have a recess feature with a recess geometry shaped to contain the connector central geometry; each of the first and second side channels having a pair of edge recess features, each edge recess feature having an edge recess feature geometry shaped to connect to the terminal geometry; wherein the device has: an assembled configuration comprising the connector central region positioned in the center block recess feature and a first of the pair of connector terminal regions positioned in the first side channel edge recess feature and a second of the pair of connector terminal regions positioned in the second side channel edge recess feature; and a disassembled configuration comprising the connector physically separated from the center block and / or the first and second side channels.

[0011] The device may further comprise a first o-ring positioned between the first side channel and the first membrane and a second o-ring positioned betw een the second side channel and the second membrane.

[0012] The device may further comprise a top cap configured to connect to a top surface of the center block to fluidically seal the center block fluidic network from a surrounding environment.

[0013] The device may further comprise a carrier having a plurality of device w ells configured to receive a plurality of devices corresponding to the center block, the first and second membranes, and the first and second side channels. Each of the plurality of device wells may comprise a fastener configured to hold the device in the device wells in a tight-fit configuration; and the number of device w ells may be between 3 and 24.

[0014] The center block fluidic network may comprise betw een 3 and 12 individual wells.

[0015] The device footprint may be configured for positioning an incubator with a plurality of biological fluid reservoirs connected to the inlet ports of the side channels.

[0016] The device may further comprise a gas exchange assembly fluidically connected to the biological fluid reservoirs for introducing a gas composition to the first and second membranes and the center block fluidic network.

[0017] In another example, the device may comprise a center block having a first surface and a second surface opposibly positioned to the first surface. In this manner, the first and second surfaces are separated from each other by a center block thickness. The center block may contain a fluid inlet port and a fluid outlet port in fluidic contact with a fluidic network. The inlet may introduce a fluid to the fluidic network and the outlet may remove fluid from the fluidic network. A first side channel is supported by the center block first surface and a second side channel is supported by the center block second surface. In this context, “supported” may functionally refer to a mo ularity, where the support facilitates reversible connection to the center block for convenient loading / removal of side channels to / from the center block, including in a manner that does not disturb a biological material in the center block and / or biological material in the side channels, while also facilitating desired cross-talk between the compartments.

[0018] Each of the first side channel and second side channels may comprise: a proximal cover having a proximal cover membrane opening and a distal cover, optionally having a distal cover membrane opening. A membrane (first / second) is positioned between the proximal cover and the distal cover and aligned between the proximal cover membrane opening and the distal cover membrane opening. The proximal cover membrane opening and / or the distal cover membrane opening is in fluidic contact with the center block fluidic network. In this configuration, a biological material in the side channel may be supplied by the center block fluidic netw ork, including the inlet and outlet ports by which a fluid is introduced and removed from the device, similar to blood flow- in that feeds a tissue and that is removed from the tissue, such by a blood vessel. The membrane may be used to support a biological material, including a cell culture relevant for the application of interest, such as dependent upon the organoid contained in the center block.

[0019] To ensure a tight and reliable fluid seal, the device may further comprise a first gasket positioned betw een the first side channel proximal cover and the center block first surface and a second gasket positioned between the second side channel proximal cover and the center block second surface.

[0020] The device may further comprise a first double-sided adhesive positioned between the membrane (first) and the distal cover of the first side channel and / or a second double-sided adhesive positioned between the membrane (second) and the distal cover of said second side channel.

[0021] The gasket may comprise a plurality of gasket passages configured to provide a fluid from the center block fluidic network to the membrane.

[0022] The device may further comprise a holder operably connected to the center block configured to reversibly and slideably engage the first side channel with the center block for removal of the first side channel without adversely impacting a biological organism supported by the membrane. The device may further comprise a second holder operably connected to the center block configured to reversibly and slideably engage the second side channel with the center block for removal of the second side channel without adversely impacting a biological organism supported by the membrane of the second side channel. In this context, without adversely impacting refers to minimal disruption such that the organism remains living and capable of being reintroduced to the device without substantial impact. Alternatively, a functional definition is that an assay outcome is substantially similar compared to an equivalent biological organism where there has not been a removal.

[0023] The biological organism may comprise a cell culture or a biological tissue.

[0024] The device may further comprise a plurality of mounting protrusions extending from each of the center block first surface and the second surface. A plurality of orifices may extend through each of said first side channel and said second side channel configured to receive the plurality7of mounting protrusions. Such mounting protrusions are another example of facilitating a reliable support of the side channels by the center block, without sacrificing the modularity and plug-and-play type configuration of the device, while avoiding unwanted leakage.

[0025] The central block may further comprise a gas network configured to provide a controlled flow of gas to the membranes. This is particularly relevant to ensure the biological material associated with each of the membranes are exposed and maintained at biologically - appropriate levels of gas (e.g., oxygen and carbon dioxide within a desired range) and / or a desired gas composition is introduced to the membrane, including as a challenge and / or a therapeutic for the biological material. In this manner, precise gas and liquid compositions are independently controllable to each of the compartments.

[0026] The device may further comprise: a carrier having a plurality' of device passages, wherein each of the carrier device passages is configured to receive and maintain one of the devices in a tight-fit configuration. This aspect is relevant for providing a highly parallel set-up, useful for massively parallel experiments, including ranging up to any number of devices, such as between 2 and 100, including between 10 and 100, depending on the application of interest, where different biological materials can be exposed to different biological conditions in a simultaneous (e.g.. parallel) manner. This provides a significant decrease in experimental time compared to conventional in vitro culture systems. To facilitate ease of loading and unloading, the carrier may further comprise a cam system configured to secure the device in the device passage. The device may further comprise a manifold, wherein the manifold secures the carrier with the plurality of devices positioned in the plurality of device passages of the carrier.

[0027] The device may further comprise a programmable microincubator connected to the manifold to control a temperature and / or gas composition of fluid in the center block fluid network. Such a programmable microincubator allows for electronic control of any number of biologically-relevant parameters, including by electronic control of a heater, a pump, fluidic control components such as valves, concentration of various components of a gas and / or liquid, etc. Optionally, automated feedback loops may be utilized wherein a sensor of a biological parameter in the device is used to input a control signal to a controller, including a controller of the microincubator, so that the biological parameter is maintained within a user-desired range.

[0028] The plurality of side channels and / or the fluidic network may be configured in a standard footprint of a multi-well format having a number of wells ranging from between 6 wells and 384 wells.

[0029] Any of the devices may further comprise a heater to control a temperature, including a thermoelectric device and / or combination with peltier cooler and resistive heater.

[0030] Any of the devices may further comprise a pump operably connected to the fluid inlet and / or fluid outlet to provide a controllable flow rate of a culture media in the center block fluidic network. A pump operably connected to the fluid inlet refers to the driving of fluid into the inlet from an upstream higher pressure region, and subsequently along the fluidic network and any fluid passages connected thereto, out of the device at the fluid outlet. A pump operably connected to the fluid outlet refers to pulling of the fluid under a lesser pressure, from a feed reserv oir that is upstream of the fluid inlet.

[0031] Any of the devices may further comprise a pair of stability rails, wherein each of the center and side blocks comprise stability rail orifices through which said stability rail passes. This is another configuration that facilitates reliable support and removal of side channels to the respective center block first and second surfaces.

[0032] Any of the devices may further comprise a pump, a sensor and a fluidic controller configured to control one or more fluidic parameters of fluid flow to the center block fluidic network.

[0033] Any of the devices may further comprise fluidic reservoirs operably connected to the pumps and a valve for introducing one or more fluids to the fluid inlet port.

[0034] Any of the devices may further comprise a holder configured to force the side channels toward the center block. The holder may be connected to the center block in which the side channels are positioned. The holder may comprise stability rails to help align and guide the side channels to the center block surfaces. The holder may comprise a clamp that provides a force to force the side channels against the center block.

[0035] Any of the devices may further comprise an incubator having an optically -transparent surface configured for optical imaging a plurality of center block and side channels without removal from the incubator. In this manner, an optically-based assessment of the biological material associated with the center block and / or side channels may be made without removing the biological material from the device.

[0036] Any of the devices may further comprise a manifold operably connected to the fluid inlet and / or outlet ports, configured to control one or more fluid parameters, including flow-rate and / or fluid composition.

[0037] To facilitate support of a biological material in the center block, the center block may further comprise a well and a gel positioned in the well configured to support the biological material in the well. The biological material may be a three-dimensional organoid, such as a sphere having a diameter, including an effective diameter ranging from between 10 pm to 10 mm.

[0038] Also provided herein are methods of using any of the disclosed devices to sustain a biological material. “Sustain7’ is used broadly herein and can refer to relatively long-term cellculture all the way to acute pharmaceutical testing, and any intervening time. For example, the biological material may be grown in the device via cell culture. Alternatively, the biological material may be provided to the membrane of the side channel and the side channel plugged into the device (e.g., supported by a center block first or second surface).

[0039] Accordingly, provided herein are methods of sustaining a biological material, such as by providing the device, introducing a biological material to the membrane, flowing a biological fluid through the center block fluid network, and controlling one or more biological parameters of the flowing biological fluid. In this manner the biological material is sustained, particularly because the biological fluid is able to flow from the center block fluid network to the membrane.

[0040] The biological material may be selected from the group consisting of: a cell culture; a biological tissue; and a tumor.

[0041] The biological parameter may be one or more of a chemical composition (t pe and / or concentration or dose), mechano-transduction, fluid composition, flow rate, shear stress, fluid pH, fluid temperature and the like.

[0042] Also provided herein are methods of making any of the disclosed devices. In an example, the method may comprise the steps of providing a center block surrounded by a gasket and supported in the center block enclosure. A heated gel in liquid form (e.g., agarose) is dispensed into the wells in the fluidic network of the center block. For example, each well maybe configured to hold any desired amount of gel, including between about 50 pL - 250 pL volume of gel, including about 65 pL. A removable positioning material holder is inserted into the wells to provide a desired shape and volume in the well and well bottom, depending on the biological material or organism to be positioned in the well. The temperature is cooled to solidify the gel into a solid. The removable positioning material holder is removed from the center block and the center block is removed from the removable gasket and the center block enclosure. At this point, the center block has the desired biological holding volume in each of the fluidic network of wells and can be assembled with the side channels and first and second membranes. As described herein, the various components between the side channels and the center block (e.g., membrane, o-ring, adhesive, etc.) can be reliably positioned and secured by connecting the center block with the side channels with a pair of connectors that provides a force in a direction from each of the side channels toward the center block, such as by a shaped connector havingterminal ends that fit into ends of the side block and a central region that fits into the ends of the center block, such as within correspondingly shaped recess features.

[0043] Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the devices and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is an illustration of the assembly of the center block and side channels that together form a device, also referred herein as a modular microfluidic device. The side channel is formed of PC cover (0.1 mm thick) 110, membrane 300, adhesive 300 (double sided) and PC cover (1 mm thick) 500, with gasket 400 to reliably contact side channel to a center block surface.

[0045] FIG. 2 illustrates a center block design, with inlet ports, including for gas and / or liquid.

[0046] FIG. 3A and FIG. 3B are an assembled and disassembled device, respectively, including made by 3D printing, with laser cut side channels.

[0047] FIG. 4A illustrates a side channel that is configured to support different functionality, including culture and analysis in a single side channel. The side channel(s) can be removed from the center block without adversely impacting (e.g., breaking or disturbing) cells. FIG. 4B illustrates a carrier configured to hold multiple devices.

[0048] FIG. 5A illustrates a programmable microincubator having a multi-well (e.g.. 96) footprint with temperature and gas control. The device can interface with a microscope for realtime cell viewing. The low profile design is compatible with, for example, an inverted microscope. FIG. 5B illustrates various components in a disassembled configuration, including with fluid inlet and outlet ports positioned in the side channels.

[0049] FIG. 6 shows a microincubator with a heat source and transparent window for real-time viewing.

[0050] FIGs. 7A-7B illustrate various tubing connection configurations, such as flanged tubing, tubing connection mount, quick connect to manifold.

[0051] FIG. 8 shows another device embodiment, with a 6-well configuration in the center block.

[0052] FIGs. 9A-9B illustrate gasket-membrane joining configurations.

[0053] FIGs. 10A-10B and 11 are a gasket-membrane fabrication summary.

[0054] FIG. 12 is a schematic illustrating a wall enclosure around the channel. The side channel may be fully enclosed by a wall and once assembled, the wall can pinch the gasket to reliably seal the channel.

[0055] FIG. 13 is a schematic of without wall (left panel) and with wall (right panel).

[0056] FIG. 14A is a schematic illustration of a FLASH device with the unassembled device (left side of arrow) and assembled and connected device (right side of arrow). FIG. 14B is a side view illustration of the assembled device showing inlet and outlet ports and a plurality of reserv oirs for supporting a biological material with a fluid.

[0057] FIG. 15A is another embodiment of a FLASH device illustrating fluidic input and output through center block with one pair of fasteners that connect side channels to center block. FIG. 15B is a cut-away illustration of the assembled device showing inlet and outlet ports and a plurality of reservoirs for supporting a biological material with a fluid with the dashed box noting a surface that is desirably flat.

[0058] FIG. 16A-16B is a schematic defining various components of the microfluidic device of FIG. 15A-15B

[0059] FIG. 17 summarizes the flow of a fluid (e.g., blood) to the center block and associated channels and returned out of the center block for the device of FIGs 15A-16.

[0060] FIG. 18 summarizes relevant aspects of the center block (fluidic network with six wells for receiving biological material) and side channels, including preference for a 90° orientation, imaging of bottom of device, membrane in PDMS not necessarily, uniform (not shown), and no hole in the membrane.

[0061] FIGs. 19-21 illustrate an embodiment where a plurality of devices are combined to facilitate a more high-throughput configuration, including in a CAM configuration.

[0062] FIG. 22A illustrates a carrier in an open position (bottom image) ready to receive a plurality of Asteroid (Apparatus to Simulate Tumor Environment and Reproduce Organs) devices (top panel). FIG. 22B illustrates four Asteroid devices locked into the carrier via a CAM system that forces together toward the central block the side channels.

[0063] FIG. 23A is an exploded view of one embodiment of the carrier. FIG. 23B provides further details of the cam system and switch embodiment for reliable receipt and fluidic connection to a plurality of the devices.

[0064] FIGs. 24A-24B illustrate gasket / membrane patterns, where silicone gaskets with adhesive worked better than non-adhesive gaskets; Gaskets with holes worked better than long channel version. FIG. 24B is preferred, with the long channel of FIG. 24A having membrane wrinkling.

[0065] FIGs. 25-26 illustrate different fluidic network geometries.

[0066] FIG. 27 is an exploded view of a device (top panel) and the adhesive film (bottom panel).

[0067] FIGs. 28-36 illustrate various components.

[0068] FIGs. 37-39 illustrate use of gel in the fluidic network wells to facilitate support of the biological material within the wells of the center block.

[0069] FIGs. 40-50 illustrate various embodiments.

[0070] FIGs. 51-60 illustrate a rmcrocontrollable incubator that is optically transparent for viewing a plurality of the devices, along with fluidic loading and valve control and various device embodiments.

[0071] FIG. 61 illustrates an assembled device with inlay key lock (top panel) and related fluid flow test in an injection molded device (bottom panel).

[0072] FIG. 62 illustrates a lock and key configuration between the center block and the side channels.

[0073] FIG. 63 illustrates a gasket cover for agarose and to facilitate gel dispensing into the wells, including to facilitate automatable cell loading to the wells.

[0074] FIG. 64 ASTEROIDS device. Top panel is an exploded view of an ASTEROIDS embodiment. Middle panel is a schema representing the cellular interaction between the different ASTEROIDS compartments. Bottom panels are tumor spheroids observed directly into the ASTEROIDS under bright field (left image) and fluorescence (right image, Cy5-F-actin (red)) microscopy.

[0075] FIG. 65A: Assembled view of the microincubator. FIG. 65B, Exploded view diagram of the programmable microincubator supporting operational function of 5 ASTEROIDS devices FIG. 65C, 96-well plate holder earn ing 5 mock ASTEROIDS devices in a plate reader.

[0076] FIG. 66A-66F illustrates Co-culture of lung TME cells within the ASTEROIDS for 7 days. FIG. 66A, Photograph of the assembled ASTEROIDS prototype used for this data acquisition. FIG. 66B, Fluorescent picture of the membrane seeded with HULECs stained with F-actin (green) and DAPI. FIG. 66C, Fluorescent pictures of HULECs (i) and IMR-90 (ii) stained with live / dead assay. FIG. 66D, Fluorescent picture of HULECs stained with VE- cadherin (red). FIG. 66E, Calculated permeability coefficients for 5 (LMW) and 70 (HMW) kDa dextran across the porous membrane in presence or not of HULECs. FIG. 66F, Volume grow th of A549 spheroids with a fluorescent picture of a spheroid stained with live / dead assay at day 7.

[0077] FIG. 67 is a graph of Immune cells migration efficiency through 3 and 8 pm membranes in presence of chemoattractant and HULECs.

[0078] FIG. 68 illustrate the HULECs form a functional endothelial barrier.

[0079] FIG. 69 illustrate the PBMCs can attach to activated endothelial cells under physiological blood-flow- velocity.

[0080] FIG. 70A illustrates a removable positioning material holder, such as a gel material, center block, gasket, and a center block enclosure useful for shaping a material to reliably position a biological organism in the center block fluidic netw ork w ell position. FIG. 70B illustrates the components of FIG. 70A assembled.

[0081] FIG. 71 illustrates the gel holder and center block, with the gel holder removed after the liquid gel has polymerized. The center block and gel holder are removed, leaving a shaped impression in the solidified gel, ready to receive a biological organism, such as a tumor, organoid or spheroid, at a desired position that can interact with fluid flow from the side channel(s).

[0082] FIG. 72A illustrates a media cap assembly useful for containing a fluid introduced to or removed from the device. FIG. 72B is the assembled components of FIG. 72A.

[0083] FIG. 73A are the various separate components (disassembled configuration) of a device, illustrating relative positions. FIG. 73B illustrates the components of FIG. 73A in an assembled configuration. FIG. 73C is an open view illustrating the fluidic network.

[0084] FIG. 74 illustrates a gas exchanger that is a cartridge assembly that can sen e as bioreactor control of pH, CO2, useful for support of biological organisms and materials.

[0085] FIGs. 75A-75C illustrates a carrier and components thereof useful for securing a plurality of the devices.

[0086] FIG. 76 illustrates a membrane holder useful for receiving and storing membranes from the devices and / or deploying membranes to the devices.

[0087] FIG. 77 illustrates a workstation, including incubator for temperature control, fluid reservoir, gas exchanges and pumps useful for controlling parameters that impact biological organisms and materials in the device.DETAILED DESCRIPTION OF THE INVENTION

[0088] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. Where applicable in the following description, definitions of specific terms and phrases are provided to clarify their specific use in the context of the invention.

[0089] In the following description, numerous specific details of the devices, device components and methods of the present invention are set forth in order to provide a thorough explanation of the precise nature of the invention. It will be apparent, however, to those of skill in the art that the invention can be practiced without these specific details.

[0090] As used herein, “biological organism” is used broadly to refer to an in vitro biological unit of biological system that is useful in a variety7of applications, including modelling and testing systems. A biological organism may correspond to an organ, or a portion thereof, including cellular components thereof. For example, a biological organism may be a lung, liver, intestine, heart, brain, etc., or cells thereof, including cells associated with blood vessels (e.g., endothelial cells, smooth muscle cells, and the like). A biological organism may be a collectionof one or more organs, tissue and / or biological fluid (e.g., blood, plasma, interstitial fluid, etc.) organized to perform one or more biological functions. For example, a biological organism may be the respirator}' system, digestive system, cardiovascular system, endocrine system, immune system, etc. A biological organism may include a biological abnormality such as cancer. Accordingly, a “vascularized” biological organism refers to a network of conduits within the biological organism that is capable, at least in part, of transporting required oxygen and nutrients to the biological cells of the biological organism and removing waste product from the biological cell. The exchange may be similar to how exchange occurs in blood vessels, such as by diffusion. The biological organisms are, of course, compatible with bulk media control and / or biomechanical conditions, where the cultured cells are immersed in a cell culture fluid that can be replaced or supplemented as desired. “Biological microorganism” is a type of biological system that has at least a portion with a characteristic dimension that is less than about 1 mm to 5 mm and, more specifically, a chamber within the device that received the biological material with a dimension less than 5 mm or less than f mm. “Biological material” may be used interchangeably with “biological organism”.

[0091] “Three dimensional structures” or “3D structures” of cultured biological cells refers to a collection of cultured cells joined together via cell adhesion wherein the collection of cells is many cells across in all three spatial dimensions. A 3D structure of cultured biological cells may be at least 10 cells across in all three spatial dimensions. A 3D structure of cultured biological cells may be at least 100 cells across in all three spatial dimensions. Accordingly, as used herein a monolayer of cultured cells is not considered a 3D structure of cultured biological cells, fn some embodiments, a 3D structure of cultured biological cells is a tissue, an organ, or a cancer mass. A 3D structure of cultured biological cells can include any eukaryotic cells that are able to self-organize in order to form a 3D structure that influences the spatial organization of the cell surface receptors engaged in interactions with surrounding cells, and may also induce physical constraints on the cells. These spatial and physical aspects in 3D cellular structures affect the signal transduction from the outside to the inside of cells, and ultimately influence gene expression and cellular behavior in a manner closer to that of the in vivo environment than a standard cellular monolayer culture. The systems and methods provided herein are compatible with a range of biological cell sources, including cells that have not been cultured, so long as the seeded cells are capable of subsequent cell culture after being introduced to the microfluidic device.

[0092] ‘‘CAM system” refers to a rotatable or slideable linkage used to efficiently and reliable mount multiple devices into a carrier.

[0093] “Tight-fit” refers to components that may fit together under force that can be generated by a typical human hand / finger, but that cannot be removed without an application of force beyond a simple gravitational force.

[0094] The devices disclosed herein provide a platform technology mimicking a range of biological systems, including human ovarian TME, including culture of 3D cancer structures in close contact with several tissue compartments and extracellular matrix, in dynamic flow condition, while having high-throughput capacity and a ready-to-use configuration for subsequent biological analysis. Indeed, the device is highly customizable for any cell type. For example, the different chambers that support a biological material may be controlled independently allowing cell culture in different medium, flow condition (shear stress, flow rate, etc.), gas environment, etc. Moreover, the device may be directly compatible with most of the common experimental methods and thus ready-to-use for microscopy imaging or plate reader analysis.

[0095] In one embodiment, the tumor-on-chip devices disclosed herein combine the advantages of standard cell culture (human cells, easy to handle, low cost) and animals models (complex spatial organization, interaction of cells) in order to mimic in vitro the human in vivo tumor microenvironment (TME). This approach is especially beneficial for the study of cancer with a specific TME, such as ovarian cancer of which TME includes not only vascular and stroma system but also the unique presence of peritoneal cavity which drives many factors responsible of toxicity, metastasis dissemination, etc.

[0096] In one example, the different perfused chambers of the fluidic network, including in the center block and / or membrane of the side channels, provide functionality to mimic and control independently both the flow condition of vascular system and peritoneal cavity. In some embodiments, the device allows for flexible design w orkflow (numbers of replicates, conditions, high-throughput, etc.) and analysis with diverse bottom / top optical readers.

[0097] In one embodiment, a tumor-on-chip may grow ovarian cancer cells spheroids, surrounded by functional monolayer of endothelial cells and fibroblasts cells seeded on membranes and cultured in representative dynamic condition. The device is user friendly by being directly compatible with microscopes and microplate readers and allowing investigation ofseveral biological samples at the same time. If desired, fasteners such as clips may be used to ensure a tight fluid seal while facilitating convenience as to accessing the different cell compartments. The devices and methods are compatible with a range of membranes, including membranes with different pores, such as different pore density and / or sizes. Any type of cell can be used with the membranes, so long as the cell is capable of adhering to the membrane and is compatible with the instant cell culture techniques.

[0098] The invention can be further understood by the following non-limiting examples.

[0099] Example 1: Exemplary modular microfluidic device embodiments

[0100] FIGs 1-65C are schematic illustrations of various devices having modular microfluidic components for simulating in vivo conditions of a biological system while configured to support biological organisms. For example, the devices and methods disclosed herein facilitate the culture of 3D cancer structures in close contact with non-cancerous dynamic compartments by combining the 3D architecture and heterogeneity of a tumor with its multi-cellular biochemical and mechanical environment. Exemplary embodiments illustrated in FIGs. 1-65C, provide a modular platform that is compatible with lab settings and are advantageously configured for automation, mass production, and increased high-throughput.

[0101] FIGs. 1-2 schematically illustrate the various components of one embodiment of the modular microfluidic device for simulating in vivo conditions of a biological system. Exemplary illustrated components include a center block 10, also referred to as a T block, with a first surface 20 and second surface 30 with a center block thickness separating the surfaces indicated by double sided arrow 40. Various ports, including a fluid inlet port 50 and a fluid outlet port 60, are provided for fluid (e.g., gas and / or liquid). The fluid inlets and outlets can be positioned in any of a variety of locations, including in the center block and / or the side channels. The devices are versatile in that they can accommodate any of a variety of fluidic inlets and outlets, so long as there is a reliable fluidic contact between the wells where the biological material is located, the side channels, and the fluid inlet 50 and outlet ports 60.

[0102] Also illustrated are a first side channel 100 and a second side channel 500 supported by center block 10 first surface 20 and second surface 30, respectively. Each side channel is formed from a proximal cover 110 with a proximal cover membrane opening 600, a distal cover 130, and a membrane 300. Optionally, there is a first adhesive, which may be a first double-sided adhesive 140, and a second double sided adhesive 150 which may facilitate support and attachment ofmembranes to the side chamber (FIG. 33). The adhesive may be, for example, a low-tack biocompatible surgical grade adhesive that prevents leaking while allowing easy disassembly of the modular microfluidic device. Preferably, this allows for subsequent analysis of the cells seeded on the membrane. The adhesive can also be, for example, a double-sided medical pressure sensitive adhesive. Each side channel (100 and 500) is preferably separated from the center block by, for example, microns-size porous membrane optionally facilitating: (1) cell attachment to reproduce a physiological barrier as well as tumor-normal interface; and (2) cellular and molecular exchanges between the side channel and well (FIG. 64 middle panel illustrating a biological material that is an organoid 900 with other biological material corresponding to endothelial cells and fibroblasts, with attendant flows labeled as Vin and Vout in the respective side channels).

[0103] In an embodiment, orifices 190 extend through the first and second side channels and are configured for receiving center block mounting protrusions 180 (FIGs. 3A-3B) from each of either the center block first surface or the center block second surface. Optionally, the modular microfluidic device includes a first gasket 400, and a second gasket 800, including gasket passages 120 (see, e.g., FIGs 9A-9B) for fluid communication from center block fluidic network and first and second side channels. Preferably, the first gasket 400 and the second gasket 800 are positioned between the first side channel 100 proximal cover and the center block first surface 20 and the second side channel 500 and the center block second surface 30, respectively. The first gasket and the second gasket may contain one or more passages configured to facilitate fluid exchange from the center block fluidic network 70 to the membrane 300. The gaskets may optionally be fabricated from, for example, polydimethylsiloxane or silicone and may optionally contain an adhesive side.

[0104] Also illustrated is a first holder 160, and a second holder 170 (FIG. 62) operably connected to the center block and configured to reversibly and slideably engage the first side channel or the second side channel with the center block to remove the first side channel or the second side channel without adversely impacting, for example, a biological organism being supported by the membrane. Various carrier 200 designs (FIGs. 4A-4B, 22A-23B) may be configured with one or more device passages wherein each device passage is configured to carry a modular microfluidic device in a tight fit configuration. Further, a manifold 240 (FIG. 5B) may optionally be provided to secure a carrier with a plurality of modular microfluidic devices. The manifold may further have or be connected to a programmable microincubator 260 (FIG. 5A).Optionally, the microincubator further comprises heater, such as a peltier heater 320 and may optionally provide temperature and gas control. Further pumps 340 operably connected to the fluid inlet or fluid outlet may transport fluid from fluidic reservoirs 420 with the required flow velocity and appropriate shear stress for each compartment (center block and side channels), including through the use of valves (FIG. 45). For example, the programmable microincubator may provide a complete independent platform (FIG. 65A-65C), including with software-based control of one or more fluid parameters, including through the use of a graphical user interface. In this configuration, the devices can be locked up and aligned in a multiwall format that will fit in a controlled environmental chamber (FIG. 65B). The microincubator may be assembled with an optically -transparent surface 460 (FIGs. 6-7 A) configured, for example, for optical imaging of a plurality of center block and side channels without removal from the incubator 440. In this configuration, the devices can be locked up and aligned in a multi-well format consisting of, for example, 8 wells, 96 wells, or 256 wells 280.

[0105] In another embodiment, modular microfluidic devices may be locked into the carrier via a CAM system 220 that forces together toward the central block the side channels. FIG. 23B illustrates the CAM system in a switch embodiment for reliable receipt and fluidic connection to a plurality of the modular microfluidic devices.

[0106] In a further embodiment, the modular microfluidic device may optionally be stabilized by a pair of stability rails 360 which may be secured through stability’ rail orifices 380 located, for example, on the center blocks and the side channels (FIG. 43).

[0107] In an embodiment, the modular microfluidic device may be utilized to study biological organisms 900 in a modular microfluidic device for simulating in vivo conditions. For example, the biological organism may be microvascular endothelial cell (HULECs), human fetal lung cells (IMR-90), adenocarcinomic human alveolar basal epithelial cells (A549), immune cells, cancer cells, fibroblasts, or endothelial cells. The biological organisms may be embedded in a gel 480 within the chamber (FIG. 37), or seeded on a membrane that separates the chambers.

[0108] Example 2: Biological characterization

[0109] FIGs 66A-69 are biological characterization of a biological organism incorporated into a device of the instant invention, illustrating the devices are biologically relevant. In one example, the device was used to co-culture different ty pes of cells. Live / Dead assays for HULECs, IMR-90, and A549 cells were conducted staining with F-actin and DAPI and confirmthat the different types of cells, e.g., endothelial cells (HULEC-5a) and fibroblasts (IMR-90) seeded in the perfused chamber of the device remain living after 7 days of incubation in the device (FIG. 66C). In addition spheroid volume increased over the course of the 7 day incubation indicating the devices support 3D organoid growth, including spheroid growth (FIG. 66F). FIG. 68 shows results of a Dextran assay for a device seeded w ith HULECs cells. The permeability for both low molecular weight (LMW) and high molecular weight (HMW) decreased when the device was seeded with HULECs cells suggesting the establishment of a functional endothelial barrier.

[0110] In one embodiment, data indicate that different types of cells within the device communicate between each other. Overexpression of the Myc target gene pathway in tumor endothelial cells has been shown in human lung biopsies. When HULECs cells were co-cultured with A549 spheroids, HULECs cells overexpressed Myc target gene pathway while HULECs cells cultured alone did not as assessed by qPCR. These data indicate that different types of cells co-cultured in the device can communicate and reproduce an in vivo response. In addition, genes involved in ECM organization, ECM-cell and cell-cell junctions were overexpressed in A549 cells co-cultured with HULEC-5a cells compared to A549 cells cultured alone further indicated the existence of cellular communication within the device of the instant application.

[0111] In another experiment using the device, immune cells were perfused in the device seeded with endothelial cells. (FIG. 69). FIG. 69 shows a fluorescent picture of a leukocyte (CD45) interacting with an endothelial cells (HULEC-5a cell) stained with F-actin. The data show that injection of TNF-a activates HULEC-5a and increases the attachment of CD45- positive cells (peripheral blood mononuclear cells (PBMCs)). In addition, the presence A549 spheroids increases PBMCs attachment suggesting that PBMCs can sense the presence of A549 cells. qPCR analysis show ed that expression of pro-inflammatory genes in A549 cells increased in the presence of PBMCs.

[0112] In another experiment, HULEC-5a cells were seeded in the device and exposed to ionizing radiation to determine whether the cells respond to ionizing radiation and display in vivo features. Fluorescent pictures of HULEC-5a cells stained with VE-cadherin and phalloidin indicated that the adherens junction of the cells was disrupted after radiation. A Dextran assay w as conducted after radiation and showed that permeability of the HULEC-5a membrane increased after radiation further confirming the disruption of the endothelial barrier. In addition, fluorescent images indicated that the HULEC-5a cell nucleus increased after irradiation withionizing radiation. HULEC-5a and A549 cells were irradiated with increasing doses of 8 Gy and 24 Gy. The number of 53BP1 foci in HULEC-5a and A549 nuclei increased after irradiation in a dose-dependent manner demonstrating that cells respond to radiation induced DNA damage. Additionally, p53-dependent DNA damage response genes were assessed using qPCR. DDB2 and CDKN1A gene expression increased after irradiation confirming that cells respond to irradiation damages. A549 spheroids were stained with Ki67 after irradiation and fluorescent pictures showed that proliferation activity of A549 spheroids decreased after irradiation. Quantification of Ki67 at protein (based on fluorescent pictures) and transcript (using qPCR) level confirmed the decrease in Ki67 expression after irradiation. qPCR showed that the expression of ferroptosis gene biomarkers in A549 cells increased after irradiation suggesting that the decrease in cell proliferation may be due to ferroptosis-related cell death. Taken together, these data indicate that cells within the device respond to ionizing radiation and display in vivo features of irradiated tissue.

[0113] Example 3: Controllers

[0114] The instant devices and related methods may be controlled, at least in part, with software. For example, a graphical user interface provides ease of control of one or more flow parameters corresponding to fluid flow rate. For example, a controller may be used to control the incubator temperature, gas exchanger, pump and humidity as shown in FIG. 77.

[0115] Example 4: Device applications

[0116] Emergence of organoids and organ-on-chip technologies have recently provided two advanced cancer models by their capacity to recapitulate the three-dimensional (3D) architecture and heterogeneity' of the tumor, and its multi-cellular biochemical and mechanical environment, respectively. Although complementary’, these technologies have not been combined yet to create an even more physiologically relevant human model. Therefore, provided herein are devices, specifically microphysiological systems described by the acronym: ASTEROIDS (Apparatus to Simulate Tumor Environment and Reproduce Organs) in an Interactive and Dynamic System. ASTEROIDS devices facilitate the culture of 3D biological materials, including cancer structures in close contact yvith non-cancerous dynamic compartments. In addition, it has been specifically engineered to overcome some well-known limitations of the current advanced models and thus provide a versatile and modular platform that is compatible yvith most lab settings and adapted for automation, mass production and increased throughput. We perform, as proof of concept, multi-cellular culture by seeding lung tumor spheroids along with lung endothelial cells and normal fibroblasts to reproduce vascular and stromal compartments. The preliminary data (including other data provided in Example 2), demonstrate that the ASTEROIDS platform can sustain the co-culture for at least 7 days and that the cells even demonstrate establishment and maintenance of key tissue features such as endothelial barrier function or tumor pathophysiological gradients. We demonstrate that the ASTEROIDS device can reproduce some major in vivo hallmarks of the biological environment, including in vivo lung TME. First, we assess the capacity of the ASTEROIDS to promote intercellular communication between tumor spheroids and non- cancerous cells. Results are compared with recent in vivo data obtained from lung biopsies that showed the stromal phenotype molding induced by cancer cells. Second, the tumor immunology is investigated by injecting immune cells in the perfused vascular compartment and by assessing their interaction with endothelial cells by measuring adhesion, extravasation and colonization of lung cancer spheroids. In addition, throughout this whole characterization process, the platform interface is subjected to regular evaluation testing in order to define whether the ASTEROIDS technology can provide a robust platform adapted for a large range of bioassays performed by any biomedical scientists without engineering background. The ASTEROIDS devices have the capacity to reproduce key characteristics of tumor biology, while providing a user-friendly and versatile platform accessible to all. This helps address major current challenges in cancer research by offering opportunity to most biomedical labs to easily work at low cost with a physiologically relevant model, including a model of any system, such as a human model.

[0117] Current in vitro human cancer models lack the combination of major features of the tumor environment, limiting their physiological relevance and efficiency. This project utilizes a new bioengineered approach combining the three-dimensional structure of the tumor with its multi-cellular and dynamic habitat in order to reproduce more accurately key in vivo cancer hallmarks. Such technology can be applied to obtain fundamental findings relevant to clinical settings, thus improving applications including drug screening, biomarkers discovery and our cancer biology knowledge that will ultimately benefit cancer patients.

[0118] Major advances in fundamental, translational or clinical oncology research rely on the need of non-animal models that can recapitulate the complex human tumor microenvironment (TME) more accurately. The devices provide the ability to mimic the unique biological landscape, including multiples properties through its three-dimensional (3D) cellular morphology, the biochemical signaling governing the interaction of its multi-cellular components and themechanical forces that occur during tumor progression. Approaches include: (1) the culture of 3D cellular structures (i.e. organoids) and (2) the use of microfluidics devices reproducing a specific tissue function also known as organ-on-chip (OOC) [1], First organoids form a complex structure that is self-organizing and self-assembling, involving differentiation of cells in response to physical and chemical cues [2], The structure is organized with different layers of heterogeneous cells proliferating, quiescent and necrotic cells to provide a 3D cellular organization. Different cell lineages can even appear reflecting a specific structure and function of an organ. Nevertheless, such structures are still limited by their static cell culture environment that implies uncontrolled delivery of compounds, non-physiological cell- to-media ratio, uncontrolled shear forces during media exchanges, as well as highly variable conditions between media exchanges [3], In addition, these structures are usually cultured alone and thus lack of cellular microenvironment and accurate organ representation [4], On the other side, OOC devices contain small chambers for cell culture, enabling control over local gradients, fluid flow, tissue mechanics, and composition of the local environment [5] . Therefore, they allow the presence and integration of multiple cell types to reflect a more physiological balance of cells and the presence of biomechanical forces relevant to the tissue being modelled |6J. However, even with these features, high cellular fidelity is usually not reproduced mainly due to the lack of 3D structure. Moreover, OOC technologies are often created at the expense of throughput, industry'- standard form factors, and compatibility' with state-of-the-art data collection tools [7], Therefore, there is a need to integrate organoids in OOC platform, through synergistic engineering, in order to create a relevant in vitro model mimicking the human in vivo TME. In the absence of such platform technology, the lack of the integration of physiologically relevant cellular architecture, biochemical signaling and physical forces in biomedical human research systems will continue to provide false-positive results from inadequate models [8,9] and delay key discoveries in human cancer biology’.

[0119] The devices provided herein build on our previous gut-on-chip that successfully reproduced the intestine human microbial interaction [10-12] (see, e.g., WO 2020 / 264388. which is specifically incorporated by reference herein). The instant devices are configured to allow culture of 3D cellular structures in close contact with two perfused chambers (‘’side channels”) reproducing vascular and stromal compartments, thus recapitulating both organoids and OOC properties on a single platform. In addition, the overall device platform is configurable to facilitate biological high- throughput analysis by matching a standard format of 96 well plate to provide a versatile platform with features suitable for automation addressing the main limitations of current models. We demonstrate that the ASTEROIDS platform can sustain multi-cells culturefor up to 7 days suggesting the feasibility of the concept. Usefulness of the devices provided herein are demonstrated by:

[0120] 1 : Reproducing the cellular interactions, biochemical signaling, and transcriptomics changes observed in in vivo systems, including lung TME. ASTEROIDS devices are run with either endothelial or fibroblasts cells in presence or absence of lung cancer spheroids. Secreted cytokines and transcriptomics profile of stromal cells will be measured for each condition and compared with in vivo data obtained from lung biopsies.

[0121] 2: Assessing the immune-endothelial-tumor interaction in the dynamic ASTEROIDS environment. Immune cells are injected in the vascular compartment perfused at physiological blood velocity and their interaction with endothelial cells are assessed by measuring adhesion, extravasation and colonization of lung cancer spheroids.

[0122] With this, the devices are demonstrated as having the ability to effectively reproduce key biological features, including for TME, such as tumor-induced phenotypical changes of normal cells and immune cell tumor invasion. These data collectively demonstrate applicability of this platform to address maj or biological cancer challenges, help the discovery' of new biomarkers or the screening of new therapeutic compounds and delivery modalities.

[0123] To date, tumor-on-chip can be mostly classified in two categories. First, the spheroid- on-chips are the simplest devices where a tumor spheroid, embedded or not in extracellular matrix (ECM), is directly placed under flow in the microfluidics system [13-19], In these platforms, nutrients and oxygen permeate by diffusion into tumor organoids from channels that emulate capillary circuits, and the 3D nature of real tumors is present. However, their main limitations lie in the lack of healthy tissue, vascularization, and the interactions between the different cells present in the TME. Thus, the other type of cancer-on-chip rely on the culture of cancer tissues in contact with non-cancerous tissues. These platforms consist of several chambers that share the same media and where the cells are either embedded in gel within the chamber, or seeded on membrane that separate the chambers [20-24], Although they reconstitute the multicellular environment, often integrate the vascular component and model the physical forces, these platforms do not integrate tumor 3D structure. Consequently, they reproduce with difficulty the cell-cell and cell-matrix interactions between tumor cells and the TME, as well as the oxygen and nutrient gradients and, therefore, can lead to phenotypes that widely differ from in vivo environment [25,26],

[0124] Very few devices tried to integrate 3D cell culture in a dynamic multi-cellular environment and remained in a simplistic configuration with limited capacities. For instance, Aung et al.

[0027] and Ko et al

[0028] developed microfluidic devices in which they cultured spheroids surrounded by endothelial cells. Although these devices have shown promises to study cancer cells-endothelium interaction and tumor angiogenesis, their configuration design and the lack of real perfusion system prevent the control of shear stress and fluid velocity, and thus the formation of a hollow blood vessels in which immune cells could be perfused to study immune cell invasion. In addition, they remain limited to endothelial cells only and do not integrate other stromal cells. Similarly, Jeong et al. presented a microfluidic poly dimethylsiloxane (PDMS) device that integrated tumor spheroids with cancer-associated fibroblasts

[0029] , That device demonstrated ability to mimic the reciprocal interaction between these cells but fail to implement additional cellular component as well as a physiologically relevant, efficient and controlled perfusion. Another PDMS device showed capacity to co-culture mammary epithelial cells in close contact to fibroblast embedded in ECM

[0030] , Interestingly, authors managed to seed ductal carcinoma in situ spheroids on the epithelial layer thus reproducing the microarchitecture of normal human mammary duct and its interaction with early stage tumor. However, besides the lack of vascularization and immune component, these chips relied on soft lithography fabrication process which is labor-intensive, limits mass production and high throughput analysis, and used PDMS. a compound known to absorb small hydrophobic molecules that can affect cellular communication or the outcome of drug screening

[0031] ,

[0125] In order to provide a platform that can combine 3D cell culture in close contact to multiple non-cancerous tissues in a physiologically relevant physical environment, we designed an Apparatus to Simulate Tumor Environment and Reproduce Organ in an Interactive and Dynamic System (ASTEROIDS) (FIG. 64). The ASTEROIDS comprises a center block (exemplified by a central piece (also referred herein as a T-block) with six round-bottom well), with the wells also referred herein as a “fluidic network” (e.g., receive biological material in the wells and in fluidic contact with the side channels), surrounded by two side channels (independent chambers on each side of center block), allowing cell culture with different growth conditions at the same time, and perfused by peristaltic pumps that can provide the required flow velocity and appropriate shear stress for each compartment (FIG. 64). The fluid inlets and outlets can be positioned in any of a variety of locations, including in the center block and / or the side channels. The devices are flexible in that they can accommodate any of a variety of fluidic inlets andoutlets, so long as there is a reliable fluidic contact between the wells where the biological material is located, the side channels, and the fluid inlet and outlet ports.

[0126] Each side channel is separated from the center block by microns-size porous membrane allowing (1) cell attachment to reproduce physiological barrier as well as tumor-normal interface and (2) cellular and molecular exchanges between chamber and well (FIG. 64 middle panel). The membranes are attached to the side chamber by a low-tack biocompatible surgical grade adhesive that prevents leaking while allowing easy disassembly at the end of the experiment to make the cells seeded on the membrane accessible for subsequent analysis (e.g. imaging, protein or RNA extraction, etc.). The central wells in the central block have been optimized to allow 3D cell culture

[0032] and are optically clear for a direct real-time visualization of biological material under bright or fluorescent microscopy (FIG. 64 bottom panel). In order to provide a platform highly compatible with most of laboratory' setting and equipment, the device can further comprise a programmable microincubator to provide a complete independent platform (FIG. 65A), including with software-based control of one or more fluid parameters. In this configuration, the devices can be locked up and aligned in a 96-well plate format holder that will fit in a controlled environmental chamber (FIG. 65B). In addition, the 96-well plate format holder is removable to allow direct analysis of the biological material (including the spheroids) in a plate reader as of a standard multi-well plate (FIG. 65C). With the “plug and play” configuration, the device and platform provided herein provides an elegant and fast connection of several devices, linked by their vascular compartments to form a multi-organ system. This configuration is beneficial, for example, for screening and identification of new cancer drugs as a 3D liver-on-chip device could be linked with a 3D tumor-on-chip and kidne -on-chip devices to assess drug metabolization, tumor control efficiency and clearance. This is an elegant solution for reliably, efficiently and conveniently providing multiple independent devices, with each individual device having in turn independently controllable or modular compartments (central and first and second side channels), with attendant savings in time due to the potential for massively parallel individual experiments.

[0127] Interestingly, the data reflects that the device is amenable with co-culturing of different cell types for up to 7 days or more. The device has been assembled (FIG. 66A) and seeded with human lung endothelial cells (HULECs) in one chamber and pulmonary' fibroblasts in the other chamber (IMR-90). Pre-formed lung cancer (A549) spheroids are then introduced in the central well of the center block fluidic network and embedded in Matrigel®. After 7 days of perfusion, imaging of the entire membrane revealed a high confluent cell monolayer demonstrating thatcells were able to attach along the entire length of the membrane (FIG. 66B). Live / dead assay further showed that both HULECs and IMR-90 cells were alive (FIG. 66C). Additional analysis on HULECs cells revealed that endothelial cells form VE-cadherin cell junctions suggesting the formation of an endothelial barrier (FIG. 66D). To confirm this observation, the results reflect that, after injection of fluorescent dextran inside the vascular chamber, apparent permeability (Papp) was significantly higher when HULECs were absent from the membrane compared to the empty membrane (FIG. 66E). Finally, spheroids grow th was followed over the 7-days period and results showed that spheroid volume increased with spheroids keeping their 3D structure and displaying a standard pattern of necrotic inner core and proliferating cells at the periphery (FIG. 66F). All together, these results demonstrate the potential of the ASTEROIDS platform to sustain in close contact the co-culture of two major non- cancerous tissue of the TME vascular and stromal compartments) with tumor spheroids for 7 days. The cells even demonstrated establishment and maintenance of key tissue features such as endothelial barrier function or tumor pathophysiological gradients.

[0128] As previously described, the current tumor-on-chips suffer from the lack of integration of either 3D cellular structures to accurately recapitulate tumor biology' or the numerous non- cancerous tissues to reproduce the complex biochemical interaction present in the TME which is vital for tumor development or drug response. Moreover, those bioengineered platforms have often been developed without taking in consideration the need of the end-user and thus are not well suited for a routine use in biological labs. Therefore, we provide a newly designed tumoron-chip platform that satisfy many or all of these requirements, and tailorable to the application of interest. The platform is highly adaptable to the desired design workflow and experimental outcomes and can uniquely combine key features on both the biological and engineering side, such as:

[0129] The ASTEROIDS integrate in close contact a 3D cell culture with up to three non- cancerous tissue; The ASTEROIDS provide efficient perfusion that is compatible with physiological fluid stress, such as shear stress over a biological organism or material; The ASTEROIDS parts can be readily separated to collect both spheroids and membranes and allow the end- user to perform a large panel of diverse biological analysis. Accordingly, any of the devices and methods may relate to a removable biological organism or biological material from each of the central block and side channels (first and second), with the side channels in the form of a membrane-supported biological organism or material and the central block a 3D biologicalorganism or material, such as an organoid. The device can be formed from thermoplastics which confers (i) biocompatibility, (ii) low absorption, (iii) optical transparency, (iv) mass production (i.e. injection molding) and (v) low cost. The 96-well format of the platform is adapted to most lab settings and equipment, opening the perspective of an automated and independent platform for increased throughput and satisfying the need of replicability. The extreme modularity of the platform allows a multi-device configuration where the ASTEROIDS can be linked together to form a multi-organ system (i.e. body-on-chip).

[0130] Cell communication is required for proper cellular activities or movements, and both failure and excess of this crosstalk can lead to tissue pathology. Through the secretion of soluble factors or membrane vesicles, normal and cancerous cells dynamically transmit reciprocal information that is vital in tumor fate, highlighting the importance of non-cancerous tissue in tumor biology and the importance of integrating them in cancer models

[0033] , Another way of cell interaction is through adhesion molecules and gap junctions to provide a direct communication (i.e. intracrine, autocrine and juxtacrine) underlining the need of recreating a 3D tumor structure that can also form these critical cell junctions

[0034] , Because the ASTEROIDS integrate both of these characteristics in a dynamically relevant environment, we hypothesize that our device is able to reproduce such complex interaction. This platform facilitates intercellular communication within the ASTEROIDS device as demonstrated by the non-cancerous cells seeded on the membrane and the tumor spheroids and this reciprocal interaction can lead to phenotypical changes of normal cells as showed by recent human in vivo data. Indeed, by comparing gene expression of cells from human lung TME with matching non-malignant lung samples, profiling approach revealed a highly complex TME that profoundly molds stromal cells [35,36], Results showed that tumor endothelial cells are remodeled to downregulate their antigen presentation and immune cell homing activities, thus contributing to tumor immunotolerance, while lung tumor fibroblasts displayed a low- level of elastin and high level of collagen compared to matching normal fibroblasts, thus participating to the stiffening of the TME. We seed lung endothelial cells and fibroblasts in the side chambers, in presence or not of lung tumor spheroids, and assess if such phenotypical and transcriptional changes occur within the ASTEROIDS device.

[0131] The ASTEROIDS is made of multiple materials. The outer manifolds can be 3D printed using a Form2 SLA printer while the center block can be machined from polycarbonate plastic sheets. They are pressed together with double-sided medical pressure sensitive adhesive. Track- etched Polycarbonate membranes (8 pm pores) separate the chambers. Once the device isassembled, it is cleaned with 70% ethanol and left to dry. Then, it is irradiated with X-Rays and kept in sterile condition until cell culture.

[0132] Before proceeding to cell seeding, porous membranes are coated with collagen I (50 pg / mL) for 4 hours. After membrane coating, central wells are pre-loaded with Matrigel Matrix in order to prepare for tumor spheroid loading. Matrigel recapitulates cell-ECM interaction while preventing the spheroids to attach the bottom of the well and the loss of its 3D structure. HULECs will be first seeded on membrane into one of the chambers and allow to attach for 2 hours. Device is flipped at 180° and IMR-90 will be seeded on the membrane of the other chamber and allow to attach for 2 hours. After cell seeding on the membrane, A549 spheroids, previously pre-formed into a 96 round-bottom well ultra-low affinity plate, are loaded in the well and embedded in Matrigel. Control devices comprise (1) ASTEROIDS with HULECs and IMR- 90 cells on membrane, Matrigel in central wells but no A549 spheroids and (2) ASTEROIDS with collagen-coated membranes but no seeded HULECs and IMR-90 but with A549 Matrigel- embedded spheroids. After seeding, cells are allowed to attach overnight in their respective medium. The next day, endothelial chamber is perfused at a flow rate of 31 pL / min to reach a fluid velocity of 0.286 mm / s similar to blood velocity encountered in tumor blood vessels [37,38], Fibroblast is perfused at a flow rate of 0.5 pL / min to reach similar velocity (~ 1-40 pm / s) as interstitial fluid displacement [39-41], Based on the data, devices are cultured for 7 days to let the cells acclimate and form endothelial barrier for HULECs.

[0133] After 7 days of culture, membranes are cut in half. One half is fixed with 4% paraformaldehyde for immunofluorescence (IF) staining while the other half is lysed to extract RNA fortranscriptomics analysis. For HULECs, cells will be stained with ethidium-homodimer III, annexin V and phospho-MLKL to investigate if cells entered in programmed necrosis (necroptosis) as in vivo study showed that tumor cells induce necroptosis of endothelial cells, thus promoting tumor cell extravasation and metastasis

[0042] , For IMR-90, cells will be stained for cancer-associated fibroblasts biomarkers (e.g. a-SMA, ROCK2, DLG1, FAP, FSP1, etc.) identified from human lung biopsies

[0043] , Staining intensity’ is measured by ImageJ and compared between stromal cells from ASTEROIDS with and without spheroids to determine expression changes. RNA is sent to a commercial company to perform RNA-sequencing following high-quality standard. Raw data is analyzed in consultation with biostatisticians in the Biostatistics and Study Design Services. In addition, studies have shown that cytokines (i.e. GM- CSF, IFN-y, IL-lb, IL- 2, IL- 4, IL-8, IL-12b, IL-17a, IL-23, and TNF-a) expression levels aresignificantly increased in the serum of lung cancer patients compared to healthy controls [44,45], Consequently, supernatant from the vascular chamber is collected to quantify and compare cytokine levels by ELISA between ASTEROIDS with and without spheroids. Finally, the shape and growth of spheroids is followed between ASTEROIDS with and without HULECs / IMR-90 cells on the membrane in order to assess if the presence of stromal cells induced an invasive phenotype as previously documented

[0035] , Spheroids are imaged using bright field microscopy and images will be analyzed using ReViSP software to measure volume from 2D projection and invadopodia. Moreover, by incorporating a degradation marker (DQ-Green BSA) into the Matrigel, we quantitate cancer cell-induced matrix degradation by measuring fluorescence intensity with ImageJ

[0046] .

[0134] Infiltrating cells of the immune system are increasingly accepted to be generic constituents of tumors and the tumor-host immunological interactions are a key regulator of the TME and cancer progression

[0047] , These inflammatory cells operate in conflicting ways: both tumor-antagonizing and tumor-promoting leukocytes can be found, in various proportions, in most if not all neoplastic lesions. The ASTEROIDS has been designed to anticipate immune cell infiltration from the vascular compartment to the central well. Our preliminary data showed that, using Transwell inserts, 8 pm pore size membrane allowed lymphocytes seeded in upper compartment to reach bottom compartment in presence of the chemoattractant SDF-la, while 3 pm pore size membrane prevented any migration (FIG. 67). When HULECs are presents on the membrane, lymphocytes could still cross the membrane although their migration efficiency was less efficient compared to the empty membrane. Using the appropriate membrane, ASTEROIDS simulate immune cells tumor invasion. Therefore, immune cells injected in the ASTEROIDS can be recognized and captured by the endothelial cells, migrate through the endothelial barrier to infiltrate the central wells with the tumor spheroids. Our approach includes the perfusion of human peripheral blood mononuclear cells (PBMCs) at physiological flow-rates (e.g., shear stress, Reynolds number) in the ASTEROIDS vascular chamber where endothelial cells are previously activated to initiate inflammatory state. Immune cells are tracked on the membrane and within the central wells using IF microscopy.

[0135] The ASTEROIDS platform is assembled, and the cells are seeded following the protocol described previously. One of the hallmark of cancer is the avoidance of immune recognition by alteration of tumor-cell characteristics, as well as by creation of a local immunosuppressive network that inactivates innate and adaptive cytolytic effectors

[0048] , To avoid such situation, priorto the injection of PBMCs, an inflammatory state is induced by perfusing the devices with proinflammatory cytokine tumor necrosis factor alpha (TNF-a) to activate HULECs. Activation will be assessed by measuring the expression of cell surface proteins and adhesion receptors such as ICAM-1, VCAM-1 and E-selectin using western blot or IF. Twenty-four hours after activation, PBMCs are injected at a physiological concentration of 2 million cells / mL and at a flow rate of 31 pL / min mimicking tumor blood velocity. PBMCs are first isolated from healthy and lung cancer donors using Ficoll-Hypaque density gradient centrifugation from EDTA-collected whole blood and resuspended in RP MI-1640 supplemented 10% FBS. Adhesion of immune cells to activated endothelial cells are assessed shortly (~1 h) after injection by IF using CD45 antibody, as well as CD4 and CD8 to determine which subpopulation preferentially attached. Prolonged perfusion is used to allow attached cells to extravasate and colonize tumor spheroids.Extravasation and infiltration is assessed by visualizing tumor-infiltrating cells directly within the central wells using IF. In order to assess if the presence of immune cells modified the TME reproduced in the ASTEROIDS, cytokine level will also be quantified after the PBMCs perfusion and compared with samples collected from specific aim #1. For all experiments, control devices comprise ASTEROIDS without TNF-a or without tumor spheroids (with or without TNF-a). Immune cell profiling in cancer has shown that tumor-infiltrating immune cells displayed specific gene and protein profile that ultimately guide their response against tumors [49,50], Therefore, we also assess the efficiency in adhesion and infiltration between PBMCs from normal and cancer donors after co-culture. Number of PBMCs present both on membrane and in the wells will be the primary outcome to determine efficiency of immune response.

[0136] ASTEROIDS can reproduce the concept of immunosurveillance because the perfused immune cells can attach endothelial cells and colonize the tumor spheroids. Such data support evidence that the ASTEROIDS is compatible with investigation of tumor immunology and as a screening model for assessing efficiency of new immunotherapeutic approaches. PBMCs perfusion have been employed in many organ-on-chips [51,52], To promote immune cell adhesion, we use TNF-a, but other compounds, such as IL-6, IL- ip or LPS, can be used in case of non- activation of the HULECs. In addition, studies have shown the importance of shear stress (i.e. 0.5 to 2 dynes / cm2) for optimal lymphocyte adhesion to vessel walls during trafficking [53- 55], In this study, we decided to use a flow rate matching average blood velocity7in tumor blood vessel, leading to a shear of 0.4 dynes / cm2. If adhesion is not successful, different flow rates will be tested to reach shear stress around 1 dynes / cm2. If the PBMCs adhere but do not cross the membrane, there should be assessed if this lack of infiltration is due to the configuration of theASTEROIDS or the immune escape from the tumor spheroid. In this this case, chemoattractant can be injected in the wells to ensure PBMCs attraction.

[0137] EXAMPLE 5: Devices to simulate tumor environment and reproduce organs.

[0138] FIGs 70-77 illustrate another device embodiment, particularly suited as an apparatus to simulate tumor environment and reproduce organs or '‘ASTEROID’’. The device has similar core features to that of the previous figures and Examples, with FIG. 70A-70B illustrating an embodiment to facilitate positioning of a gel in the fluidic network 70 of center block 10, specifically wells 71 of fluidic network. A gasket 401 can surround the center block first and second surfaces (20 30) and to ensure a gel does not leak out of the fluidic network. A removable gel holder 481 is configured to be positioned in the center block and facilitate placement of a gel in the center block fluidic network 70, including the portions of the fluidic network described as wells 71 where a biological organism, such as a tumor or tumor model having 3-D geometry (e.g., organoid). Center block enclosure 12 is configured to hold and contain the gasket 401 and center block 30 within a central enclosure volume 13 formed in center block enclosure. The top panel of FIG. 70 is an exploded view and the bottom panel an assembled view.

[0139] FIG. 71 focusses on use of a removable gel holder 481 to apply gels 482 in a controlled manner to the fluidic network 70 of center block 10. In particular, gel 482 is positioned in a well 71 of the fluidic network 70. In this manner, the gel can support a biological organism, including a 3-D tumor or organoid, and ensure appropriate positioning, including to fluidic contact with the fluid introduced via the side channel inlet ports.

[0140] The Gel Clamp Assembly illustrated in FIGs 70A-71 is exemplified as a platform designed for the molding of agarose gel cups. The open sides of the center block 10 are sealed using a silicone gasket’ and secured within the center block enclosure, which effectively functions as a holder. This holder ensures the proper alignment of the center block. Heated agarose gel in liquid form is dispensed into the wells of the center block fluidic network, with each w ell accommodating 65 pL of gel. After the gel is dispensed, the removable gel holder is inserted into the wells to shape the bottom of the cups within the gel. The assembly is then allowed to rest at room temperature for approximately 15 minutes. Once the gel has solidified, the center block and removable gel holder are removed from the enclosure and gasket. At this point, the gel is fully molded, and the removable gel holder is removed from the center block, leaving a cup-shaped impression in the solidified agarose. In this manner, any geometry7impression can be made by providing a corresponding shaped mold. The geometry may be selected based on the biological material to be provided to the center block.

[0141] FIGs. 72A-72B illustrate a tubing cap assembly useful as a fluid reservoir of biologically-relevant liquids that are to be introduced and / or removed from the device. A media cap 720 formed from a gasket top 721 enclosure and a gasket bottom 722 enclosure connects to reservoir 723, such as a test tube or vial. Fluidic connection is facilitated by connectors formed from a barbed fitting 724, passages 725 though a gasket, and tubing 726. The assembly utilizes a cylindrical locking mechanism to secure the top and bottom caps when rotated into place. A soft 40-durometer silicone gasket is fitted between the caps to ensure a tight seal. The bottom cap features a threaded undercut that attaches to a standard ’5 mL Tube' vial. The "Gasket’ contains three holes with double-barbed fittings for tubing connections. One tubing extension reaches into the ‘5 mL Tube’ for media siphoning via a peristaltic pump. The second tubing is designed for external liquid dispensing, and the third barbed fitting provides access for atmospheric pressure regulation and filter attachment.

[0142] FIGs. 73A-73C illustrates device with center block 10, first 100 and second 500 side channels, o-ring 731. first 300 and second 301 membranes, connector 732, and top cap 733, in a disassembled (top panel) and an assembled configuration (bottom panels). First fluid inlet port 50 and first fluid outlet port 60 are provided in first side channel. Similar ports are provided in the second side channel. To facilitate reliable assembly and disassembly, connector 732 is configured to reversibly connect the center block to the first and second side channels, with membranes disposed therein. Connector 732 has central region with central region geometry 733 and a pair of terminal regions with terminal geometries 734. The connector can mate with the center block at third 735 and fourth 736 surfaces having recess feature geometry 737 and with the side channels at recess features 777. In this manner, the device can have an assembled configuration 778 and a disassembled configuration 779 with the center block separated from the side channels. O-rings 780 may be used to facilitate fluidic seal of the device. Top cap 733 connected to top surface 16 of center block 10 provides further fluidic seal of the center block fluidic network.

[0143] A gas exchanger 740 is illustrated in FIG. 74. Components of gas exchanger may include, as labeled by 1-7, corresponding to cartridge laminate gas layer, membrane, adhesive layer, cartridge laminate liquid layer, adhesive gas layer, membrane, and cartridge laminate liquid layer, respectively. The Gas Exchanger is a cartridge assembly comprising polycarbonate ‘Laminate’ layers, which are machined with channels for liquid and gas flow. These sections areseparated by an air-permeable ‘Membrane’ that allows gas exchange while restricting liquid flow. The layers are bonded using double-sided adhesive and include barbed connectors for flexible tubing. The gas exchanger serves as a bioreactor, enabling precise pH control of cell culture media. CO2 is introduced through the gas line and permeates the membrane, dissolving in the liquid channel to optimize conditions for biological growth. Additionally, a secondary vacuum channel above the liquid channel, separated by another air-permeable membrane, removes air bubbles from the liquid pathway.

[0144] FIGs 75A-75C illustrate a plurality of devices in an assembled configuration 778 incorporated into a carrier 200 at carrier wells 280 to facilitate high-throughput culture and experiments. Fasteners 750 can help secure the devices to the carrier wells, exemplified in this example as linear wave springs. The carrier is engineered to securely hold a plurality of assembled ‘ASTEROIDS Devices’, illustrated in this case as three devices. It incorporates a ‘Linear Wave Spring’ that applies compression, ensuring a tight fit and preventing leaks in the side channels of the devices. The carrier is designed to follow a 96-well plate footprint, with openings aligned to match standard well locations, facilitating its use with plate readers for the detection of biological, chemical, or physical phenomena.

[0145] It can be useful to have a special holder configured to hole the membranes, either removed from the device or to be inserted into the device, such as between side channels and center block, as described herein. FIG. 76 illustrates a membrane holder configured to accommodate membranes after cell culture, taking advantage of the ability to quickly and without damage remove membranes from the device. The ‘Spacer’ and ‘Upper Enclosure’ lid feature openings and dividers that enable the dispensing of two distinct reagents onto the cells on the membrane. A ‘Silicone Sheet’ positioned between the ‘Spacer’ and the membrane creates a leak-proof seal, preventing cross-contamination between wells. The holder is capable of securing a plurality of membranes, in this example up to six membranes. Components are numbered 1-6 as: spacer membrane holder (compression shim); silicone sheet; upper enclosure, membrane sheet, lower enclosure, and hook latch. The upper panel is an exploded view, with the bottom panels the assembled holder from different perspectives.

[0146] FIG. 77 illustrates a workstation that comprehensively controls the cell culture parameters to any number of devices provided in carriers. The ASTEROIDS Workstation is designed to house the carrier with multiple assembled devices. These devices are connected via barbed fittings to flexible silicone tubing, which links to the gas exchanger’, peristaltic pump,and media tubing cap bottles. The workstation maintains optimal incubation temperatures for cell growth, with the entire system functioning to precisely control environmental conditions such as media perfusion, pH, CO2 levels, humidity, and temperature for cell and tissue culture applications. The numbered 1-5 components correspond to incubator, gas exchanger, pump, carrier, and media tubing cap assembly, respectively.STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

[0147] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0148] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.

[0149] When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.

[0150] Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated.

[0151] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0152] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.

[0153] As used herein, '‘comprising’’ is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0154] One of ordinary' skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

CLAIMSWe claim:

1. A device comprising: a center block having: a first surface; a second surface opposibly positioned to the first surface and separated from the first surface by a center block thickness; and a fluidic network in fluidic contact with said fluid inlet port and said fluid outlet port; a first side channel configured to be positioned adjacent to the center block first surface, the first side channel having: a first fluid inlet port; a first fluid outlet port; a second side channel configured to be positioned adjacent to the center block second surface, the second side channel having: a second fluid inlet port; and a second fluid outlet port; a first membrane positioned between the first side channel and the center block first surface; a second membrane positioned between the second side channel and the center block second surface; wherein: the center block fluidic network is in fluidic contact with the first and second fluid inlet ports and the first and second fluid outlet ports; the first membrane is in fluidic contact with the first inlet port and the first outlet port; the second membrane is in fluidic contact with the second inlet port and the second outlet port.

2. The device of claim 1, further comprising a removable positioning material holder configured to be inserted into the center block fluidic network to support positioning a material in at least a well of the center block fluidic network.

3. The device of claim 2, further comprising: a removable gasket configured to at least partially surround the center block, wherein the fluidic network of the center block has openings positioned through the first and second surfaces;a center block enclosure configured to removably surround the gasket and contain the gasket in a tight configuration for reliable positioning of the material in a plurality of the wells in the center block fluidic network;4. The device of any one of claims 1-3, further comprising: a connector to connect the center block to each of the first and second side channels, the connector having a central region with a central geometry’ and a pair of terminal regions with terminal geometries, wherein the central region is between the pair of terminal regions; the center block having a third surface and a fourth surface extending between the first and second surfaces, wherein each of the third and fourth surfaces have a recess feature with a recess geometry shaped to contain the connector central geometry; each of the first and second side channels having a pair of edge recess features, each edge recess feature having an edge recess feature geometry shaped to connect to the terminal geometry; wherein the device has: an assembled configuration comprising the connector central region positioned in the center block recess feature and a first of the pair of connector terminal regions positioned in the first side channel edge recess feature and a second of the pair of connector terminal regions positioned in the second side channel edge recess feature; and a disassembled configuration comprising the connector physically separated from the center block and / or the first and second side channels.

5. The device of any one of claims 1-4, further comprising a first o-ring positioned between the first side channel and the first membrane and a second o-ring positioned between the second side channel the second membrane.

6. The device of any one of claims 1-5, further comprising atop cap configured to connect to a top surface of the center block to fluidically seal the center block fluidic network from a surrounding environment.

7. The device of any one of claims 1-6. further comprising a carrier having a plurality of device wells configured to receive a plurality of devices corresponding to the center block, the first and second membranes, and the first and second side channels.

8. The device of claim 7, wherein each of the plurality of device wells comprises: a fastener configured to hold the device in the device wells in a tight-fit configuration; and the number of device wells is between 3 and 24.

9. The device of any one of claims 1-8. wherein the center block fluidic network comprises between 3 and 12 individual wells.

10. The device of any one of claims 1-9, having a device footprint configured for positioning an incubator with a plurality of biological fluid reservoirs connected to the inlet ports of the side channels.

11. The device of claim 10 further comprising a gas exchange assembly fluidically connected to the biological fluid reservoirs for introducing a gas composition to the first and second membranes and the center block fluidic network.

12. A device comprising: a center block having: a first surface; a second surface opposibly positioned to the first surface and separated from the first surface a center block thickness; a fluid inlet port; a fluid outlet port; a fluidic network in fluidic contact with said fluid inlet port and said fluid outlet port; a first side channel supported by said center block first surface; a second side channel supported by said center block second surface, wherein each of said first side channel and said second side channel comprise: a proximal cover having a proximal cover membrane opening; a distal cover; a membrane positioned between said proximal cover and said distal cover and aligned between said proximal cover membrane opening and said distal cover membrane opening; wherein said proximal cover membrane opening and / or said distal cover membrane opening is in fluidic contact with said center block fluidic network.

13. The device of claim 12, further comprising a holder operably connected to said center block configured to reversibly and slideably engage said first side channel with said center block for removal of said first side channel without adversely impacting a biological organism supported by said membrane.

14. The device of claim 13, further comprising a second holder operably connected to said center block configured to reversibly and slideably engage said second side channel with said center block for removal of said second side channel without adversely impacting a biological organism supported by said membrane of said second side channel.

15. The device of any one of claims 1-14, wherein the membranes and / or fluidic network are configured to support a biological organism, such as a biological organism comprising a cell culture or an isolated biological tissue.

16. The device of any one of claims 1-15, further comprising: a plurality of mounting protrusions extending from each of said center block first surface and said second surface; and a plurality of orifices extending through each of said first side channel and said second side channel configured to receive said plurality of mounting protrusions.

17. The device of any one of claims 1-6, 9-17, further comprising: a carrier having a plurality of device passages, wherein each of said carrier device passage is configured to receive and maintain one of said devices in a tight-fit configuration.

18. The device of claim 17, wherein said carrier further comprises: a cam system configured to secure the device in the device passage.

19. The device of any of claims 1-17, further comprising a pump and fluidic controller configured to control one or more fluidic parameters of fluid flow to the center block fluidic network.

20. The device of any of claims 19, further comprising fluidic reservoirs operably connected to the pumps for introducing one or more fluids to the fluid inlet ports.

21. The device of any one of claims 1-20, further comprising a gel positioned in a well of the center block configured to provide support to a biological material in the well.

22. A method of sustaining a plurality of unique biological materials, the method comprising the steps of: providing the device of any one of claims 1-21 ; introducing a first biological material to the first membrane; introducing a second biological material to the second membrane; introducing a third biological material to the center block fluidic network; flowing a biological fluid through said first and second side channels; and controlling one or more biological parameters of said flowing biological fluid; thereby sustaining said plurality of unique biological materials.

23. The method of claim 22, wherein said biological material is selected from the group consisting of: a cell culture; a biological tissue; a tumor.

24. A method of making any of the devices of claims 1-21.

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