Integrated microfluidic well plate

Abstract

An integrated microfluidic well plate (IMWP) system disclosed herein includes a well plate frame including a plurality of wells. At least one first well (or well type) of the plurality of wells includes a reservoir. At least one second well (or well type) includes a scaffold disposed within the second well. Such an IMWP system can also comprise a microfluidic layer disposed beneath the well plate frame. The microfluidic layer fluidly couples the reservoir to the scaffold via one or more microfluidic channels disposed within the microfluidic layer.

Description

Attorney Docket No.: PCT.1313INTEGRATED MICROFLUIDIC WELL PLATECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority pursuant to 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 740,666, filed December 31, 2024, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Drug and therapeutics development are generally time-consuming and costly endeavors. In vivo testing of new drugs involves a phased, highly-regulated approach that helps to ensure the efficacy of the underlying drugs and / or therapies, and helps to ensure the safety of patients upon whom they are being tested, but also often entails a multi-billion dollar (and multi-year) regulatory approval processes. In silico modeling of drugs and therapies can also be time-consuming and costly, and can lead to results that are inherently less accurate than those derived from in vivo testing. The success of in silico (i.e., computer) modeling and testing is also dependent on the underlying data upon which those computer models are based. When in vivo testing and in silico modeling result in counter-indications and / or inconclusive results, years of development time and millions (or even billions) of dollars of research and development investments are often lost.SUMMARY

[0003] The present disclosure presents systems, methodologies, and apparatuses that enable quick prototyping and modeling of organs, tissues, cells, biological processes, and other mechanisms, via a flexible platform that allows for adaptive and / or iterative build and refinements of models. In one aspect, a platform is provided for generating an organ-on- a-chip (OoC) and / or other biological models. In some cases, such an OoC or "organ chip" includes a 3D-printed scaffold that includes a vascular structure and optionally an infill surrounding the vascular structure, wherein the scaffold is capable of being seeded with active cells (e.g., endothelial cells for the vascular structure and / or hepatocytes for the infill) such that the functioning of organs (for example, human organs such as a liver) may be replicated or mimicked accurately. Additionally, organ chips, as described herein, in some embodiments, may be defined by one or more such scaffolds disposed in a microfluidic well plate or portion thereof. Moreover, in some cases, an organ chip described herein may further include microfluidic controls and / or sensors. In someAttorney Docket No.: PCT.1313preferred embodiments, an organ chip described herein comprises or is defined by an integrated microfluidic well plate (IMWP) including the foregoing components, all within a standardized footprint.

[0004] As stated above, the present embodiments include an integrated microfluidic well plate (IMWP), associated assemblies and systems, and associated methods of use. In some embodiments, the IMWP includes integrated microfluidic pumps and valves, as described herein, thereby enabling enhanced control and sensing of in vitro models (for example, of organs on a chip, synthetic tissues, live cells, etc.), all within a footprint that is compatible with SLAS-ANS1 microwell plate standards and as such is compatible with automated liquid handling systems and automated readers for detection and monitoring processes.

[0005] In one aspect, the present embodiments are directed to an integrated microfluidic well plate (IMWP) system including a well plate frame including a plurality of wells (e.g., including first, second, third, and nth wells, where n may be an integer ranging from 4 to 96, 4 to 384, or 4 to 1536). At least one first well (or well type) of the plurality of wells includes a reservoir. In some cases, an IMWP system described herein comprises multiple reservoirs disposed in or defined by multiple wells of the well plate frame (e.g.. n wells of the plurality of wells can comprise n reservoirs, with one reservoir per well). Additionally, in some implementations of an IMWP system described herein, at least one scaffold is disposed within at least one well (e.g., a second well or well ty pe) of the plurality of wells. It is also possible for n scaffolds to be disposed within n wells of the plurality of wells (e.g., in a 1:1 manner), where n is an integer as described herein. Further, in some cases, an IMWP system described herein also comprises a microfluidic layer disposed beneath the well plate frame. Moreover, the microfluidic layer can fluidly couple the reservoir to at least one neighboring reservoir and / or scaffold via one or more microfluidic channels disposed within the microfluidic layer.

[0006] In some embodiments, a system described herein includes a bottom seal layer attached to the bottom of the microfluidic layer for sealing microfluidic layer to the well plate frame.

[0007] In some embodiments, the bottom seal layer transmits therethrough 90% or greater of light within the visible and / or near-infrared wavelengths, and has a thickness (e.g., an average thickness) in a range from about 25 microns (pm) to 200 pm. Other transparencies and thicknesses are also possible.

[0008] In some embodiments, the bottom seal layer is formed from or composed of at least one of a cyclic olefin copolymer (COC) such as a norbomene-ethylene copolymer or aAttorney Docket No.: PCT.1313tetracyclododecene-ethylene copolymer; a cyclic olefin polymer (COP) such a polynorbomene; a fluorinated ethylene propylene (FEP) or copolymer of hexafluoropropylene and tetrafluoroethylene; and a polyethylene terephthalate (PET). Other materials may also be used in some instances.

[0009] In some embodiments, a system described herein further includes at least one pump disposed within the microfluidic layer. The pump can be fluidly coupled to the microfluidic channels. Moreover, in some embodiments comprising a pump, a circular hole within the microfluidic layer houses the pump. Other configurations are also possible.

[0010] In some embodiments, the pump is or includes a rotational pump including at least one impeller blade. Additionally, in some embodiments, the pump includes a magnet coupled thereto. In some embodiments, a system described herein includes at least one pump disposed within the microfluidic layer, wherein the pump includes a piezoelectric pump.

[0011] In some embodiments, a system described herein includes a non-circular hole (e.g.„ square, rectangular, oval-shaped, etc.) within the microfluidic layer, and the hole houses a scaffold described herein.

[0012] In some embodiments, a system described herein includes a magnetic driving base disposed beneath the microfluidic layer, wherein the magnetic driving base includes one or more sets of driving coils, and wherein each of the sets of driving coils is disposed beneath a corresponding well of the pump, and the sets of driving coils are configured to actuate the corresponding pump.

[0013] In some embodiments, a system described herein includes a bottom seal layer attached to the bottom of the microfluidic layer for sealing the microfluidic layer to the well plate frame, the bottom seal layer being disposed between the magnetic driving base and the microfluidic layer, wherein, in operation, at least one magnetic field from the sets of driving coils traverses the bottom seal layer to actuate the pump.

[0014] In some embodiments, a pump described herein has or includes an outer diameter that is 3-20% smaller than that of each well (or of the average well diameter) of the plurality of wells.

[0015] In some embodiments, a pump described herein operates at a frequency within a range from about 40 Hz to about 400 Hz. In some embodiments, a pump describe herein includes a piezoelectric actuator configured to operate such that it does not exceed a strain of greater than 5%.Attorney Docket No.: PCT.1313

[0016] In some embodiments, a pump described herein includes a piezoelectric actuator configured to operate such that it does not exceed a total travel distance (or total strain) greater than about 50 pm.

[0017] In some embodiments, the microfluidic layer of a system described herein includes at least one micro check valve fluidly coupled to the microchannel. In some such embodiments, the micro check valve includes a Tesla valve with a width in a range from about 0.1 millimeters (mm) to about 0.3 mm. In some embodiments, the micro check valve includes at least one of a thin membrane valve, duckbill valve, and a passive micro check valve.

[0018] In some embodiments, a microfluidic layer described herein includes a width of about 1 mm to about 3 mm.

[0019] In some embodiments, the microfluidic layer of a system described herein is formed from or composed of at least one of the following: a cyclic olefin copolymer (COC), a cyclic olefin polymer (COP), and a fluorinated ethylene propylene (FEP).

[0020] In some embodiments, a microfluidic layer described herein is formed by at least one of injection molding and hot embossing.

[0021] In some embodiments, microfluidic channels of a system described herein include an inner diameter within a range from about 50 pm to about 500 pm.

[0022] In some embodiments, microfluidic channels of a system described herein are formed via voids within the microfluidic layer.

[0023] In some embodiments, the plurality of wells of a system described herein are spaced out relative to each other to meet ANSI / SLAS Microplate Standards.

[0024] In some embodiments, a system described herein includes at least one sensor operably coupled thereto for sensing at least one parameter while the system is in use. In some such embodiments, the sensor includes embedded platinum, carbon-based, indium tin oxide, gold, and / or iridium oxide electrodes. In some embodiments, the sensor of a system described herein includes a trans-endothelial electrical resistance (TEER) sensor, a barrier integrity sensor, a MEMS-based flow meter, a biophotonic sensor, a surface plasmon resonance (SPR) sensor, and / or a sensor formed from or composed at least partially of poly vinylidene fluoride (PVDF).

[0025] In some embodiments, a system described herein includes a sealing lid configured to interface with the well plate frame such that a fluidic seal is maintained within the reservoir. In some such embodiments, the sealing lid includes at least one pneumatic channel disposed therein, wherein the pneumatic channel fluidly couples the reserv oir to an external pressure source. In someAttorney Docket No.: PCT.1313such embodiments, the pneumatic channel is coupled to the external pressure source via a pneumatic interface including a microfilter. In some such embodiments, the microfilter includes a pore size of about 0.22 pm.

[0026] In some embodiments, the pneumatic channel of a system described herein includes multiple pneumatic channels, and the pneumatic interface includes multiple pneumatic interfaces, and the external pressure source includes multiple external pressure sources. In some such instances, each pressure source is coupled to one of the multiple pneumatic channels via one of the multiple pneumatic interfaces (e.g., in a 1:1 manner). In some embodiments, the multiple external pressure sources of a system described herein comprise multiple pumps.

[0027] In some embodiments, a system described herein includes at least one sampling port disposed within a top surface of the sealing lid, wherein the sampling port enables access to the reservoir. In some such embodiments, the sampling port includes a cap-based sampling port, such as a sampling port cap disposed within a top surface of the cap-based sampling port. In some embodiments, the port is a septum-based sampling port including a septum layer disposed within a top surface of the septum-based sampling port. In some embodiments, the system includes at least one septum disposed within a top surface of the sealing lid.

[0028] In some embodiments, a system described herein includes at least one pneumatic channel disposed within the well plate, the pneumatic channel fluidly coupling the reservoir to an external pressure source. In some such embodiments, the pneumatic channel is coupled to the external pressure source via a pneumatic interface disposed within a side surface of the well plate.

[0029] In some embodiments, the microfluidic layer of a system described herein is coupled to the scaffold via a hosebarb coupling attached to the microfluidic layer, and the hosebarb coupling interfaces with a hosebarb disposed within a void disposed within a bottom surface of an overhang portion of a scaffold described herein. In some embodiments, the microfluidic layer of a system described herein is coupled to a scaffold via an adhesive applied to the microfluidic layer that interfaces with a bottom surface of an overhang portion of the scaffold.

[0030] In some embodiments, a system described herein includes a seal clip disposed at least partially between a scaffold and the well plate frame, the seal clip being configured to place the scaffold within the well plate frame, and to subsequently remove the scaffold from the well plate frame. In some such embodiments, the seal clip includes two upper prongs, a mid portion, and two lower prongs, and each of the two upper prongs includes a hole disposed therethrough to facilitate removal of the seal clip and scaffold from the well plate frame.Attorney Docket No.: PCT.1313

[0031] In some embodiments, a scaffold of a system described herein is at least partially formed from or composed of a hydrogel material. In some embodiments, a scaffold of a system described herein includes at least one bio-printed passage disposed therethrough, and the bio-printed passage simulates a vasculature. In some such instances, the scaffold is seeded with at least one live cell.

[0032] In some embodiments, the bio-printed passage of a system described herein includes a network of bioprinted passages arranged such that they define an interstitial space between the network of bioprinted passages and adjacent to at least a portion of the bioprinted passages. In some such cases, one or more live cells is seeded within the interstitial space.

[0033] In some embodiments, the well plate of a system described herein is or includes a 24-well plate.

[0034] In some embodiments, the well plate of a system described herein includes up to 12 units, at least 12 units, or exactly 12 units, wherein each unit includes a first well used as the reservoir (or a first, reservoir-type well, which may also be referred to as a ‘'reservoir well’’) and a second well with a scaffold disposed within the second well (or a second, scaffold-type well, which may also be referred to as a “scaffold well’’). In some such instances, the first well (that is, the reservoir well or reservoir-type well) and the second well (that is, the scaffold well or scaffold-type well) are fluidly coupled to one another.

[0035] Moreover, in some embodiments, the well plate of a system described herein includes up to 8 units, at least 8 units, or exactly 8 units, wherein each unit includes the following: a first well used as a primary reservoir (which may also be referred to as a "primary reservoir well”); a second well with a scaffold disposed within the second well (which may also be referred to as a “scaffold well”); and a third well used as an auxiliary reservoir (which may also be referred to as an “auxiliary reservoir well”). In some such cases, the second well (the scaffold well) is selectively fluidly coupled to both the first well (the primary reservoir well) and the third well (the auxiliary reservoir well). Further, in some instances, the first well (the primary reservoir well) is also selectively fluidly coupled to the third w ell (the auxiliary reservoir well).

[0036] In some embodiments, the bottom seal layer of a system described herein includes a semi-permeable film that enables oxygen transport thereacross.

[0037] Associated methods of using a system or IMWP described herein are also contemplated. For example, in some cases, a method described herein comprises disposing one or more live cells in a scaffold described herein and flowing a biological, biologic, or pharmaceutical medium (e.g., comprising a therapeutic agent) through the scaffold (and in contact with the live cells) using theAttorney Docket No.: PCT.1313IMWP, such as by disposing the medium in a reservoir or reservoir well described herein and flowing the medium through a vascular structure of the scaffold.

[0038] These and other embodiments are described in more detail in the detailed description below.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 illustrates a perspective view of an organ chip, according to some embodiments described herein.

[0040] Figure 2 illustrates a perspective view of an organ chip, according to some embodiments described herein.

[0041] Figure 3 illustrates a cutaway perspective view of an organ chip, according to some embodiments described herein.

[0042] Figure 4A illustrates a view of a vasculature configuration, according to one embodiment described herein.

[0043] Figure 4B illustrates a view of a vasculature configuration, according to one embodiment described herein.

[0044] Figure 4C illustrates a view of a vasculature configuration, according to one embodiment described herein.

[0045] Figure 5A illustrates a top view of a vasculature configuration, according to one embodiment described herein.

[0046] Figure 5B illustrates a side view of a vasculature configuration, according to one embodiment described herein.

[0047] Figure 6 illustrates a cutaway perspective view of a vasculature configuration, according to one embodiment described herein.

[0048] Figure 7A illustrates a top view of a vasculature configuration, according to one embodiment described herein.

[0049] Figure 7B illustrates a side view of a vasculature configuration, according to one embodiment described herein.

[0050] Figure 8 illustrates a cutaway perspective view of a vasculature configuration, according to one embodiment described herein.

[0051] Figure 9 illustrates a perspective view of a liver-on-a-chip model, according to one embodiment described herein.Attorney Docket No.: PCT.1313

[0052] Figure 10 illustrates atop view of a liver-on-a-chip model, according to one embodiment described herein.

[0053] Figure 11 illustrates a side view of a liver-on-a-chip model, according to one embodiment described herein.

[0054] Figure 12 illustrates atop view of a liver-on-a-chip model, according to one embodiment described herein.

[0055] Figure 13 illustrates a perspective view of an integrated microfluidic well plate, according to one embodiment described herein.

[0056] Figure 14A illustrates an exemplary top view of an integrated microfluidic well plate, according to one embodiment described herein.

[0057] Figure 14B illustrates an exemplary top close-up view of a unit of an integrated microfluidic well plate, according to one embodiment described herein.

[0058] Figure 15A illustrates an exploded perspective view of an integrated microfluidic well plate, including a pump, according to one embodiment described herein.

[0059] Figure 15B illustrates a perspective view of an integrated microfluidic well plate, including a pump, according to one embodiment described herein.

[0060] Figure 15C schematically illustrates an example of a rotational pump architecture, according to one embodiment described herein.

[0061] Figure 16 illustrates an exploded perspective view of an integrated microfluidic well plate, including a pump, according to one embodiment described herein.

[0062] Figure 17A illustrates a cutaway perspective view of an integrated microfluidic well plate, including a pump, according to one embodiment described herein.

[0063] Figure 17B illustrates a close-up perspective view of a portion of an integrated microfluidic well plate, including a pump, according to one embodiment described herein.

[0064] Figure 18A illustrates a perspective view of a rotational pump, including a rotating disk, according to one embodiment described herein.

[0065] Figure 18B illustrates a perspective view of an example of a rotational pump, according to one embodiment described herein.

[0066] Figure 19 illustrates a partially exploded perspective view of an integrated microfluidic well plate, including a piezoelectric pump, according to one embodiment described herein.

[0067] Figure 20 illustrates a partially exploded perspective view of an integrated microfluidic well plate, including a piezoelectric pump, according to one embodiment described herein.Attorney Docket No.: PCT.1313

[0068] Figure 21A illustrates a perspective view of an integrated microfluidic well plate, including a piezoelectric pump, according to one embodiment described herein.

[0069] Figure 21B illustrates a cutaway perspective view of an integrated microfluidic well plate, including a piezoelectric pump, according to one embodiment described herein.

[0070] Figure 22 illustrates a perspective view of an integrated microfluidic well plate, including a pressure-driven pump, according to one embodiment described herein.

[0071] Figure 23 A illustrates a perspective view of an integrated microfluidic well plate, including a pressure-driven pump, according to one embodiment described herein.

[0072] Figure 23B illustrates a perspective view of an integrated microfluidic well plate, including a pressure-driven pump, according to one embodiment described herein.

[0073] Figure 24A illustrates a perspective view of an integrated microfluidic well plate, including a pressure-driven pump, according to one embodiment described herein.

[0074] Figure 24B illustrates a cutaway perspective view of an integrated microfluidic well plate, including a pressure-driven pump, according to one embodiment described herein.

[0075] Figure 25A illustrates a perspective view of an integrated microfluidic well plate, including a pressure-driven pump, pneumatic interface and microfilter. with an expanded portion, according to one embodiment described herein.

[0076] Figure 25B illustrates a cutaway perspective view of an integrated microfluidic well plate, including a pressure-driven pump, septum-based sampling port, according to one embodiment described herein.

[0077] Figure 26A illustrates a top view of a sealed lid and septum-based sampling port, according to one embodiment described herein.

[0078] Figure 26B illustrates a perspective view of a sealed lid and septum-based sampling port, according to one embodiment described herein.

[0079] Figure 27 illustrates a perspective view of an individual unit of a system, according to one embodiment described herein.

[0080] Figure 28A illustrates an exploded perspective view of a handling method of an individual scaffold, according to one embodiment described herein.

[0081] Figure 28B illustrates an exemplary front view of a seal clip, according to one embodiment described herein.

[0082] Figure 29 illustrates a perspective view of a mechanical coupling mechanism, according to one embodiment described herein.Attorney Docket No.: PCT.1313

[0083] Figure 30A illustrates a side view of an adhesive coupling mechanism, according to one embodiment described herein.

[0084] Figure 30B illustrates a perspective cross section view of an adhesive coupling mechanism, according to one embodiment described herein.DETAILED DESCRIPTIONDefinitions

[0085] In order for the present disclosure to be more readily understood, certain terms are defined below. Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[0086] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0087] A, an, at least one, one or more: It is to be noted that the term ‘“a"' or ’an" entity refers to one or more of that entity. For example, "a cell” is understood to represent one or more cells. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein, unless the context of a particular use requires otherwise.

[0088] Additive manufacturing: The terms “additive manufacturing,” “three-dimensional printing system,” “three-dimensional printer,” “printing,” and the like generally describe various solid freeform fabrication techniques for making three-dimensional articles or objects by selective laser sintering (SLS), stereolithography (SLA), dynamic light projection (DLP), selective deposition, jetting, fused deposition modeling (FDM), multijet modeling (MJM), and other additive manufacturing techniques now known in the art or that may be known in the future that use a build material or ink to fabricate three-dimensional objects

[0089] ANSI / SLAS Microplate Standards: As used herein, the term “ANSI / SLAS Microplate Standards” refers to standards set for microplate development by the Society' for Laboratory Automation and Screening (SLAS) and the American National Standards Institute (ANSI), particularly those approved as of December 31, 2024. Five ANSI / SLAS standards include ANSI / SLAS 1-2004: Microplates - Footprint Dimensions; ANSI / SLAS 2-2004: Microplates -Height Dimensions; ANSI / SLAS 3-2004: Microplates - Bottom Outside Flange Dimensions;Attorney Docket No.: PCT.1313ANSI-SLAS 4-2004: Microplates - Well Positions; and ANSI / SLAS 6-2012: Microplates - Well Bottom Elevation. The latter of these specific standards (Well Bottom Elevation) generally specifies definitions and a test method for each of Microplate Well Bottom Elevation (WBE). Well Bottom Elevation Variation (WBEV), or Intra-Well Bottom Elevation Variation (IWBEV).Well Botom Elevation (WBE) is the distance from the resting plane to the inside bottom surface of any well at a well position; it can be reported as a nominal value with a tolerance.Well Botom Elevation Variation (WBEV) is the maximum allowable spread between the highest and lowest WBE points on an individual plate; it can be reported as a maximum value.Intra-Well Botom Elevation Variation (IWBEV) is the range (maximum-minimum) of the distance from the resting plane to anywhere on the inside bottom surface of any individual well; it can be reported as a maximum value.Well Depth is the distance from the maximum projection of any individual well to anywhere on the inside bottom surface of the well; it can be reported as a nominal value with a tolerance.Botom Thickness is the mean thickness of all the well bottoms on any individual plate; it is reported as a nominal value.Well Botom Width is the internal dimension (width or diameter) of any individual flat bottom well, measured at the inside bottom surface. It is measured to the theoretical sharp comer (to the tangency between the well’s inside bottom surface and the sidewalls) and is reported as a nominal value.

[0090] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%. 6%, 5%, 4%, 3%, 2%. 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0091] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility' to, severity' of, stage of, etc. the disease,Attorney Docket No.: PCT.1313disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated'’ with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, electrostatic, magnetism, and combinations thereof.

[0092] Bioprinting: The term “bioprinting,” as used herein, refers to 3D printing or additive manufacturing with biocompatible materials such as hydrogels onto which live cells may be stably adhered and / or otherwise functionalized. It is to be understood that a scaffold described herein, in some cases, may be bioprinted. That is, in some embodiments, 3D printing or additive manufacturing (e.g., using a hydrogel as a “build material” or “ink”) may be used to form a scaffold described herein.

[0093] Comprising: A composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are required or essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any system, device, composition, or method described as “comprising” (or which “comprises”) one or more named elements or steps also describes the corresponding, more limited system, device, composition, or method “consisting essentially of (or which “consists essentially of’) the same named elements or steps, meaning that the system, device, composition, or method includes the named required or essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the system, device, composition, or method. It is also understood that any system, device, composition, or method described herein as “comprising” or “consisting essentially of’ one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method “consisting of’ (or “consists of’) the named elements or steps to the exclusion of any other unnamed element or step. In any system, device, composition, or method disclosed herein, known or disclosed equivalents of any named required or essential element or step may be substituted for that element or step.

[0094] Continuous: A “continuous” process (such as a continuous build process or manufacturing process) refers to a single process that proceeds in a single manner or mode (e.g.,Attorney Docket No.: PCT.1313additive manufacturing carried out by DLP, in a layer-by-layer manner), as opposed to a “batch” process or “discontinuous” or “multi-stage” process in which the manner or mode changes. For instance, an exemplary non-continuous process would be injection molding to form one structure, followed by surface treatment of the structure. These two steps together would not constitute a “continuous” process as used herein. Similarly, another example of a non-continuous process is a process that first forms a structure using additive manufacturing, and then forms a different structure using a different additive manufacturing process or “print job.” and then joins the two structures together in a separate step (e.g., using an adhesive or mechanical fastener). The term “continuous with,” when used in reference to a structure, is synonymous with “integral with” as described herein.

[0095] Digital light processing (DLP): As used herein and as understood by a person of ordinary skill in the art, the term, “digital light processing” (DLP) refers to a 3D printing technology used to rapidly produce photopolymer parts using a projected light source to cure an entire layer at once. In some cases, DLP uses a range of wavelengths from 360 nm to 405 nm, typically as the light source in connection with a photocurable polymer resin.

[0096] Directions or orientations: As one example, it is to be understood that a “vertical” orientation or a “vertical” direction is an “up and down” orientation or direction (e.g., based on the orientation of the Tong axis’ of the relevant structure, as compared to the ‘short axis’ of the relevant structure). Moreover, such an “up and down" or “vertical” orientation or direction can be relative to the force of gravity exerted by the earth, such that “down” is tow ard the ground, and “up” is toward the sky, when the relevant platform, component, device, or system is used as intended and as described herein. However, it is further to be understood that terms such as “vertical” and “horizontal” as used herein are to be understood as terms relative to one another to indicate different directions or orientations, analogous to the standard use of terms such as “x-direction” and “y-direction” and “z-direction” in a Cartesian coordinate system, or “length” and “width” and “height” in geometry. Thus, for instance, unless the context clearly indicates otherwise, a “vertical” direction or orientation described herein is orthogonal or perpendicular to a “horizontal” direction or orientation described herein. These terms may also be replaced, in general, with Cartesian directions or orientations, such as “z” (e.g., for “height” or the “vertical” direction or orientation) and “x” or “y” (e.g., for “length” or “width” or the “horizontal” direction or orientation).

[0097] Hydrogel: As used herein, the term, “hydrogel” refers to a three-dimensional network composed of or formed from polymers (in some cases, hydrophilic polymers), such as may beAttorney Docket No.: PCT.1313synthesized by crosslinking water-soluble polymers. Hydrogels can retain a large quantity of water within their network (e.g., such that water constitutes most or all of the fluid phase of the biphasic gel), including without destroying the original structure (e.g., of the non-fluid phase). Hydrogels can have flexibility' and swelling or non-swelling properties.

[0098] Improve, increase, inhibit, and reduce: As used herein, the terms ‘improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in absence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0099] Integral with: As used herein, the term “integral with” refers to two entities or components that are seamlessly j oined to one another, without use of an attachment mechanism or means such as an adhesive or mechanical fastener. Such “integral” entities or components, for example, may be formed by producing both entities in the same additive manufacturing “print job” or other continuous, single process. That is, in some cases, a component, structure, or portion that is “integral with” another component, structure, or portion (or that has an “integral structure”) may be formed from the same material as the other component, structure, or portion (including as may occur if both are formed from the same build material in the same additive manufacturing print job or process).

[0100] Interstitial space and infill: As used herein, the term “interstitial space” refers to a space, void, or volume in between other structures described herein. An “interstitial infill” refers to a structure or material (e.g., a hydrogel or hydrogel structure) disposed within an interstitial space. As one example, an “interstitial” space may be a volume or void wherein a cell in a fluid(s) and / or a polymer (e.g., a hydrogel) is seeded to create a tissue or tissue-like structure. The tissue or tissuelike structure, in this example, may be described as an “infill.” Infills are further described below in the context of “scaffolds.”

[0101] Lattice or Lattice Structure: As used herein, the term, “lattice” or “lattice structure” refers to a repeating two-dimensional or three-dimensional pattern of structural members formed of 3D-printed hydrogels. The pattern may occur on a nanoscale (10 nm or greater), microscale (e.g., ones of microns or tens of microns), millimeter scale, and / or macroscale (i.e., as distinguishedAtorney Docket No.: PCT.1313from co stal latice structures, which may be formed and / or repeating on atomic or molecular level, on the order of ones to tens of Angstroms).

[0102] Ranges: All ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of ‘’1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9. All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10” should generally be considered to include the end points 5 and 10. Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0103] Scaffold: As used herein and as understood by the skilled person, the term, “scaffold” refers to a structural framework or matrix, sometimes used within physical models of living tissues (e.g., synthetic tissues), that may also be used for drug discovery and for assessing the effectiveness of various therapies. In some cases, a scaffold supports internal passages that approximate the geometries of human vasculatures, tissues, and other structures, and may be operatively coupled to cells seeded in an interstitial space within the scaffold. A scaffold can thus enable delivery of biologically active materials to the cells and / or tissue(s) via fluid(s) flowing through the vasculature, and transport through vasculature walls. More particularly, a scaffold described herein, in some cases, may be formed from a hydrogel, such as a bioprinted hydrogel. Moreover, in some preferred embodiments, a scaffold described herein has a continuous or integral structure, or is printed all at once (as one “print job” in an additive manufacturing process). Further, in some preferred embodiments, a scaffold described herein has two primary components, substructures, or portions, both of which can be essentially defined by printing an overall structure that has voids arranged in a desired manner: one primary component can be a vascular structure, and the other primary component can be an infill that surrounds the vascular structure and is “fed” by the vascular structure. An infill can be “fed” by a vascular structure in the sense that the vascular structure transports media (e.g.. nutrients, pharmaceutically active agents, etc.) to and from the infill (e.g., due to diffusion of the media through walls of the vascular structure). Moreover, in some cases, as described above, the infill is organ-specific or has an organ-specific structure,Attorney Docket No.: PCT.1313meaning a structure that is intended to mimic or simulate a specific organ. For example, a liverspecific infill may comprise microspheres.

[0104] Units, prefixes, and symbols: Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, unless expressly stated otherwise.

[0105] Vasculature, Vascularized, and Vascular: As used herein, the terms “vasculature”, “vascularized”, and “vascular” refer to a single vessel or a network (i.e., a two-dimensional network, a three-dimensional network) of vessels that enable distribution of drugs, biologies, biological fluids, and / or other substances to a cell. As understood by a person of ordinary skill in the art, the term “vasculature” refers to a perfusable 2D or 3D interconnected tubular transport system. More particularly, a “vasculature” or “vascular structure” can convey biological fluids, nutrients, drugs, biologies, and / or gases or other substances to a cell, cellular aggregate, or tissue, including to maintain viability of the cell / tissue. “Vasculature” or a “vascular structure” may also convey waste away from a cell, cellular aggregate, or tissue. In some cases, “vasculature” or a “vascular structure” described herein interpenetrates or is embedded within another network or within a tissue or a tissue mimic or substitute or extracellular matrix, such as a hydrogel structure described herein. For instance, in some instances, a vasculature or vascular structure described herein comprises one or more channels layered with, partially or completely overlapping, surrounding, embedded within, and / or interwoven with one or more channels of a tissue or other network, or embedded within a hydrogel structure. Further, in some embodiments, the “vasculature” or “vascular structure” of a device, system, or method described herein does not consist of or comprise regular cylinders or other regular geometric shapes (e.g., such regular geometric shapes joined together to form a network), but instead has an irregular geometric structure, shape, or cross-section. Additionally, in some cases, the “vasculature” or “vascular structure” of a device, system, or method described herein has a perfusable architecture or structure mimicking a mammalian (e.g., human) vascular system that includes an artery, arteriole, capillary bed, venule, vein, or a combination of two or more of the foregoing. In some cases, a vasculature or vascular structure described herein obeys or corresponds to Murray’s Law, as further described below. It is further to be understood that an entity that is “vascularized” has a vasculature or vascular structure as described above.Attorney Docket No.: PCT.1313Organ Systems and Models

[0106] The present disclosure provides technologies to generate a model organ and / or tissue. In some aspects, the present disclosure provides methods to generate a model organ and / or tissue that includes a bioprinted entity, comprising a polymer (e.g., a hydrogel), and a cell associated with the bioprinted entity and / or seeded therein. In some embodiments, the model organ and / or tissue is one aspect of a system or ecosystem (e.g., organ-on-a-chip) which may be used for drug disco very, drug delivery’, and / or for assessing the effectiveness (e.g., therapeutic result, safety, toxicity, etc.) of various therapies. In some aspects, the model organ and / or tissue is optionally vascularized.

[0107] Fig. 1 illustrates a view of an organ chip 10, according to some embodiments. As shown in Fig- 1, the vasculature 14 disposed within a hydrogel scaffold 12 of the chip 10 may have a larger diameter at each of the inlets and outlets, and may include a plurality or network of smaller vasculature passages 14A connecting therebetween. A large part of the branching vasculature of the mammalian circulatory and respiratory systems obeys or is consistent with Murray’s Law, which states that the cube of the radius of a parent vessel equals the sum of the cubes of the radii of the daughters. Where this law is obeyed, a functional relationship exists between vessel radius and volumetric flow, average linear velocity of flow, velocity profile, vessel-wall shear stress, Reynolds number, and pressure gradient in individual vessels. Accordingly, in some embodiments according to the present disclosure, the internal diameter of the vasculature decreases from the vasculature inlet to the center of the organ chip 10, and then increases from the center of the organ chip 10 to the vasculature outlet.

[0108] It is to be understood that a scaffold described herein may be formed from any hydrogel not inconsistent with the technical objectives of the present disclosure. For example, in some cases, the hydrogel is formed from the polymerization or gelation of a composition comprising an acrylate component (e.g., one or more hydroxyalkylacrylates present in the hydrogel or hydrogel-forming composition in an amount of 10-90 weight percent, based on the total weight of the hydrogel or hydrogel -forming composition), a photoinitiator component (e.g., a monoacylphosphine oxide (MAPO) salt or bisacylphosphine oxide (BAPO) salt, which may in some instances be a sodium or lithium MAPO or BAPO salt, and which may be present in an amount of 0.1 -5 weight percent, based on the total weight of the hydrogel or hydrogel-forming composition), an optional non-curable absorber component (e.g., sulfonated quinoline yellow, which may be present in an amount of 0.1-5 weight percent, based on the total weight of the hydrogel or hydrogel-forming composition), and water (e.g., as the “balance” of the hydrogel or hydrogel-forming composition,Attorney Docket No.: PCT.1313or in an amount of 20-80 weight percent). Non-limiting examples of hydrogels which may be suitable for bioprinting a scaffold in some embodiments described herein include those disclosed in U.S. Patent 12,384,935 or U.S. Patent Application Publication 2024 / 0091412 Al, the entireties of which are hereby incorporated by reference.

[0109] Fig.2 illustrates a view of an organ chip, according to aspects of some embodiments. Fig.3 illustrates a view of another organ chip, according to some embodiments.

[0110] Figs. 4A. 4B, and 4C illustrate views of vasculature configurations, according to some embodiments. In the configuration shown in Fig. 4A, the vasculature 14 includes a single passageway or channel through the interstitial infill 16 (the reference character 14 may also be used to indicate this single passageway or channel). The vasculature 14 may take a serpentine path making several turns (for example 90-degree, 180-degree, 270-degree, and / or other number of degree turns) through the interstitial infill 16 (see the discussion of Figs. 5-9 and other figures below for a further description of the interstitial infill 16) and in between a vasculature inlet and a vasculature outlet (not shown in Fig. 4A). In the configuration shown in Fig. 4B, the scaffold 12 may include multiple vasculatures 14 (or channels, or passageways), each vasculature 14 including an internal network of connections or passageways disposed within the interstitial infill 16 between a vasculature inlet and a vasculature outlet (not shown in Fig. 4B). In the configuration shown in Fig. 4B, the multiple vasculatures 14 may not connect to each other. In the configuration shown in Fig. 4C, the scaffold 12 may include an open vasculature 14 where passageways connect to adjacent passageways within the interstitial infill 16 forming a two dimensional and / or three dimensional interconnected vasculature 14. These interconnected adjacent passageways are represented in Fig. 4C by the white channels or network generally identified using reference character 14.

[0111] Figs.5A, 5B, and 6 illustrate views of still other vasculature configurations 14, according to some embodiments. In the embodiments illustrated in Figs. 5A, 5B. and 6, the interstitial infill 16 includes a repeating lattice structure or framework, wherein some lattice elements are represented by the structures 16a in Fig. 6.

[0112] Figs.7A, 7B, and 8 illustrate view s of vasculature configurations 14, according to aspects of the present embodiments. In the embodiments illustrated in Figs. 7A, 7B, and 8, the interstitial infill 16 includes a plurality of packed and interconnected microspheres, denoted using reference character 16b in Fig.8.Attorney Docket No.: PCT.1313

[0113] In some embodiments, the model organ is a liver. In some aspects, a model liver comprises a cell chamber (to metabolize a biologically active material e g., a drug), an access point (to enable delivery or sampling of a fluid(s) comprising a cell and / or a biologically active material), an inlet, and an outlet. In some embodiments, the model liver is vascularized. In some embodiments, the model liver is not vascularized.

[0114] Figs. 9-12 illustrate a model liver, according to aspects of some embodiments described herein. The model liver may include or be defined by a scaffold, which may comprise or be a bioprinted entity. The model liver may also be described as a “chip / ’ See reference character 10 in Figs. 9-12. As noted herein, such a chip can comprise a bile duct, a chamber, an inlet, an outlet, and optionally vasculature. In some such embodiments, the model liver may comprise a cell associated with the scaffold and / or seeded therein, wherein the cell is optionally an endothelial cell. In some embodiments, the model liver optionally comprises a cell associated with the vasculature and / or seeded therein, wherein the cell is optionally an LSEC (liver sinusoidal endothelial cell). In some embodiments, the model liver optionally comprises a cell associated with the bile duct and / or seeded therein, wherein the cell is optionally a cholangiocyte. It will be appreciated by one of skill in the art that the association of a cell may occur with a combination of one or more aspects of the scaffold (e.g., bioprinted entity, bile duct, chamber, inlet, outlet, vasculature), and furthermore that a plurality of cell types may be associated with said scaffold.

[0115] Referring still to Figs.9-12, the chip 10 may include a first fluid network (i. e. , vasculature 14) and a second fluid network (i.e., bile duct 18). The first and second fluid networks f4, f8 may be intertwined and or interconnected (i.e., with interlinking fluid passageways) without actually being fluidly coupled (that is, the fluid within one network does not mix with the fluid within the other network, particularly because the fluid channels / passageways of the two networks are distinct and not in direct fluid communication with one another at points of intertwining or contact, other than may occur via diffusion through the walls of the networks). Both the first and second fluid networks 14, 18 may be operatively coupled (for example, functionally coupled) to the interstitial space and interstitial infill 16 disposed therein such that drugs and / or other biological substances that flow through either the first and / or second fluid networks 14, 18 may pass through the vasculature walls or bile duct walls and into the interstitial infill (i.e.. to the active cells seeded therein). In Figs.9-12, the interstitial infill is denoted using reference character 16, consistent with other figures herein.Attorney Docket No.: PCT.1313Integrated Microfluidic Well Plate (IMWP)

[0116] The present disclosure, in some embodiments, presents a custom-designed well plate to redefine organ-on-a-chip technology’ by consolidating scaffold, housing, and microfluidic interfaces with an integrated pumping system within a single ANSI / SLAS format micro well plate. An integrated microfluidic well plate (IMWP) may achieve a complete integration of pumps within the plate, restricting all liquid handling to the plate itself, and therefore eliminating the need for tubing or fluidic and / or gas interfaces and interconnects with external control systems.

[0117] The design of some implementations of an IMWP described herein, in some embodiments, addresses several challenges related to organ-on-a-chip designs. Traditional systems require bulky external components that take up valuable lab space and are difficult to scale. An IMWP described herein, in some cases, may integrate all components - pumps, scaffolds, interconnects, and fluidics - within a modular ANSI / SLAS format well plate, drastically reducing its footprint. That is, in some cases, an IMWP described herein includes no external components, where an “external” component is a component that is outside an ANSI / SLAS format or footprint. More particularly, in some embodiments, none of the pumps, scaffolds, interconnects, or fluidics of the well plate or system is an external component. The ANSI / SLAS format of an IMWP allows easy scalability with multiple plates in high-throughput arrays, and ensures compatibility with standard lab automation tools, enabling high-throughput workflows without custom adaptations and modifications. Conventional systems rely on complex external tubing and connections, increasing setup complexity and increasing contamination risks. An IMWP may embed pumps and fluidics within the plate itself, with low dead volume interconnects, making operation as simple as placing the plate on a base (for example, a magnetic base). By eliminating external connections, an IMWP may reduce setup time, enhance reliability, and / or minimize excessive handling, while also reducing contamination risks. In traditional systems, ensuring reliable seals and consistent media flow can be challenging. Design of an IMWP may secure fluid paths internally within the plate, reducing leak risks without needing O-rings or external tubing, or interconnection systems.

[0118] Additionally, each channel ithin an IMWP described herein, in some embodiments, may include an individually controlled pump, allowing precise, scaffold-specific flow' adjustments for efficient nutrient and waste exchange, which can enhance the physiological relevance of experiments. Several conventional systems require custom setups for high-content imaging, further complicating workflows. SLAS-ANSI / SLAS standard format of an IMWP described herein may be compatible with standard microscopes, enabling high-content imaging withoutAttorney Docket No.: PCT.1313modifications, as well as standard format readers (such as fluorescence, luminescence, infrared (IR) and ultraviolet (UV) microscopes, imagers, or readers).

[0119] Turning in more detail to the microplate footprint of an IMWP described herein, in some embodiments, the outside dimension of the base footprint, measured at any point along the side, are Length (at 127.76 mm ± 0.25 mm) x Width (at 85.48 mm ± 0.25 mm). In some embodiments, the outside dimension of the base footprint is measured within 12.7 mm of the outside comers. In some embodiments, the footprint is continuous and uninterrupted around the base of the plate. Turning to the comer radius of the plate’s bottom flange, in some embodiments, each comer has a comer radius to the outside of 3.18 mm ± 1.6 mm.

[0120] Turning in more detail to the plate height of some embodiments of the microplate of an IMWP described herein, the plate height, with or without external clearance to the plate bottom, when measured from the resting plane to the maximum protmsion of the perimeter wells, is 14.35 mm ± 0.25 mm. In some embodiments, the overall plate height, measured from the resting plane to the maximum protrusion of the plate, is 14.35 mm ± 0.76 mm. In some embodiments, the maximum projection above the top-stacking surface is 0.76 mm, where the top-stacking surface is the surface on which another plate would rest when stacked one on another. In some embodiments, when resting on a flat surface, the top surface of the plate is parallel to the resting surface within 0.76 mm. In some embodiments, the minimum clearance from the resting plane to the plane of the bottom external surface of the wells, that is the external clearance to the plate bottom, is 1 mm (0.0394 inches). In some variations, this clearance is limited to the area of the wells.

[0121] Turning to flange height of the microplate of some embodiments of an IMWP described herein, the flange height typically is measured from the bottom-resting plane to the top of the flange. In some embodiments, all four sides have the same flange height. In some embodiments, the height of the bottom outside flange is short, that is 2.41 mm± 0.38 mm. In some embodiments, the height of the bottom outside flange is medium, that is 6.10 mm ± 0.38 mm. In other embodiments, the height of the bottom outside flange is tall, that is 7.62 mm ± 0.38 mm. For any flange height, in one variation the flange width of the bottom outside flange as measured at the top of the flange is a minimum of 1.27 mm. In one embodiment, the quantity and location of chamfer(s) or comer notches is optional. In one embodiment, the chamfer does not include the flange. In some embodiments, the flange height includes intermptions. In some variations, all four sides have the same flange height except for a defined interruption centered along one side, such as the long side. In some embodiments, each long side of the plate has a single interruption or projection on center.Attorney Docket No.: PCT.1313In some embodiments, such as a short flange with interruptions, each edge of the interruption is a minimum of 47.8 mm from the nearest edge of the part; in some variations, the height of the flange at the interruption does not exceed 6.85 mm. In another embodiment, dual flange heights may be used, wherein the height of the bottom outside flange is 2.41 mm ± 0.38 mm along the short sides of the plate; in one variation the height of the bottom outside flange is 7.62 mm ± 0.38 mm along the long sides of the plate.

[0122] Turning to the well positions of the microplate of some embodiments of an IMWP described herein, typically, the left outside edge of the part is defined as the two 12.7 mm areas (as measured from the comers) and the top edge of the part is defined as the two 12.7 mm areas (as measured from the comers) as described above with respect to footprint dimensions. In some embodiments, the microplate is a 96 well microplate. In some embodiments, the wells in a 96 well microplate are arranged as eight rows by twelve columns. With regard to the well column position, in some embodiments, the distance between the left outside edge of the plate and the center of the first column of wells is 14.38 mm. In some embodiments, each following column is an additional 9 mm in distance from the left outside edge of the plate. With regard to the well row position, in some embodiments, the distance between the top outside edge of the plate and the center of the first row of wells is 11.24 mm. In some embodiments, each following row is an additional 9 mm in distance from the top outside edge of the plate. With regard to positional tolerance, in some embodiments, the positional tolerance of the well centers is specified using true position. In some embodiments, the center of each well is within a 0.70 mm diameter of the specified location; such a tolerance applies regardless of feature size. In other embodiments, the microplate is a 384 well microplate. In some embodiments, the wells in a 384 well microplate are arranged as sixteen rows by twenty-four columns. With regard to well column position, in some embodiments, the distance between the left outside edge of the plate and the center of the first column of wells is 12.13 mm. In some embodiments, each following column is an additional 4.5 mm in distance from the left outside edge of the plate. With regard to well row position, in some embodiments, the distance between the top outside edge of the plate and the center of the first row of wells is 8.99 mm. In some embodiments, each following row is an additional 4.5 mm in distance from the top outside edge of the plate. With regard to positional tolerance, in some embodiments, the positional tolerance of the well centers is specified using True Position. In some embodiments, the center of each well is within a 0.70 mm diameter of the specified location. In some embodiments, the tolerance applies regardless of feature size. In other embodiments, the microplate is a 1536 wellAttorney Docket No.: PCT.1313microplate. In some embodiments, the wells in a 1536 well microplate are arranged as thirty7-two rows by forty-eight columns. With regard to well column position, in some embodiments, the distance between the left outside edge of the plate and the center of the first column of wells is 11.005 mm. In some embodiments, each following column is an additional 2.25 mm in distance from the left outside edge of the plate. With regard to well row position, in some embodiments, the distance betw een the top outside edge of the plate and the center of the first row of wells is 7.865 mm. In some embodiments, each following row is an additional 2.25 mm in distance from the top outside edge of the plate. With regard to positional tolerance, in some embodiments, the center of each well is within a 0.50 mm diameter of the specified location. In some embodiments, the tolerance applies regardless of feature size. With regard to well markings, in some variations of any aspect or embodiments disclosed herein, the top left well of the plate is marked in a distinguishing manner, including but not limited to the letter A or numeral 1 located on the lefthand side of the well; in another variation, the top left well of the plate is marked on the upper side of the well.

[0123] Further, design of an IMWP described herein, in some instances, may allow easy handling and sampling, as with any standard well plate, simplifying workflows and reducing transition time for imaging and analysis. The design features of an IMWP described herein, in some embodiments, may collectively enhance the functionality, reliability, and scalability of organ-on-a-chip systems, rendering them more practical for research, biotechnology and pharma applications.

[0124] Fig. 13 illustrates an example of an IMWP (denoted using reference character 20), according to some embodiments described herein. In some embodiments, the IMWP 20 includes a well plate frame 22, individual wells 24, a microfluidic layer 34, a bottom seal 38, one or more scaffolds 32, and one or more pumps 30. In some embodiments, the microfluidic layer 34 includes one or more individual microfluidic channels 36. In some embodiments, a pump 30 is disposed at the base or bottom of an individual well 24 that is used as a fluidic reservoir 28 (that is, a "‘reservoir well” that can provide fluid media such as biological media or reagent media for use in an assay or other method described herein), and a scaffold 32 is disposed inside a neighboring individual well 24 (such a neighboring well comprising a scaffold may be referred to herein as a “scaffold well”). In this example, the pump 30 is a rotational pump. However, other pumps may also be used. A microfluidic channel 36 may be used to link the pump 30 to the corresponding scaffold 32, creating an independent unit 26 that can operate separately without fluidic connections to other units. For clarity, reference character 26 is disposed on the opposite side of Fig. 13, and it will be readilyAttorney Docket No.: PCT.1313understood by the skilled person that any “set” of “reservoir well” and “scaffold well” may be linked microfluidically as described herein to form an independent unit 26. It is also to be understood that a given independent unit 26 need not be limited to only one reservoir well and one scaffold well. Instead, a single of reservoir well can be combined with a plurality of scaffold wells to form an independent unit, or a plurality of reservoir wells can be combined a single scaffold well to form an independent unit, or a plurality of reser oir wells can be combined a plurality of scaffold wells to form an independent unit, and the specific number or combination is not particularly limited.

[0125] In the configuration of Fig. 13, a plurality of pumps 30 are disposed at the base of a plurality of center wells 24 of the IMWP 20 (where “center” wells are those not on a long edge of the IMWP, or that have neighboring wells on both sides, left and right), while the scaffolds 32 are disposed inside outer wells 24 (where “outer” wells are those on a long edge of the IMWP, or that have only one neighboring well, on the left or right but not both). In this example, the IMWP 20 corresponds to a 24-well plate format, accommodating 12 units (of 2 wells each — one “reservoir well” and one “scaffold well”) and therefore supporting 12 scaffolds. In some embodiments, 48 units may be supported by using 48 pumps 30 (which may be smaller than the pumps 30 used in a 24-well plate format) and 48 scaffolds 32 in a similar manner within a 96-well plate format, achieving a higher throughput.

[0126] Still referring to Fig. 13, in some embodiments, the bottom seal 38 has specifications comparable to those of ANSI / SLAS standards glass-bottom plates. Additionally, in some cases, the bottom seal 38 is fully compatible with high content imaging (HCI), and readers such as fluorescence, luminescence, IR, and UV imagers and readers. In some embodiments, for example, the bottom seal 38 has greater than 90% light transmission or transparency across visible and nearinfrared wavelengths, where the transparency is calculated based on the percent of incident photons within the relevant wavelength range. For example, in some cases, the bottom seal 38 has a light transmission or transparency of at least 90% or greater than 90% within a spectral window of SOO-SOO nm, 380-750 nm, 780 nm to 3 pm, or 780 nm to 1.4 pm. Further, in some embodiments, the bottom seal 38 has an optically flat surface to minimize distortion, and a thickness selected or optimized for optical microscopy to ensure compatibility with dry and immersion objectives. Moreover, in some embodiments, the bottom seal 38 includes a thickness or average thickness in a range of about 100-300 pm, such as 150-200 pm, 140-210 pm, 130-220 pm, or about 120-230 pm. Additionally, in some embodiments, the bottom seal 38 includes a thickness or averageAttorney Docket No.: PCT.1313thickness in a range of about 25 gm to about 200 gm. In some embodiments, the bottom seal 38 offers clarity and precision comparable to traditional glass-bottom plates, enabling seamless integration of an IMWP described herein with automated HCI platforms for applications such as cell viability, morphology, and phenotypic analysis .

[0127] According to aspects of the present embodiments, various types of pumps may be integrated into or used with an IMWP described herein including but not necessarily limited to rotational pumps, piezoelectric pumps, pressure driven pumps, and other types of pumps, as described herein. In some embodiments, the primary requirements for the bottom seal of the well plate (for example, in the rotational pump embodiment) are optical transparency and a thickness suitable for HCI. Several commercially available fdms fulfill the necessary' sealing criteria and exhibit good sorption properties, making them suitable for use as the bottom seal material. Examples of potential materials include cyclic olefin copolymer (COC). cyclic olefin polymer (COP), fluorinated ethylene propylene (FEP), and polyethylene terephthalate (PET). Other types of materials may also be used for the bottom seal 38, as long as the relevant transparency and sealing requirements are met. In some preferred embodiments, the material used for the bottom seal 38 provides high optical transparency or clarity with minimal autofluorescence or scattering (e.g., less than 5%, less than 1%, less than 0.5%, or less than 0.1% of incident photons within the imaging spectrum cause or experience autofluorescence or scattering when interacting with the bottom seal material). In this manner, compatibility' with high-content imaging systems can be obtained in some cases. Smoothness and transparency, in some preferred embodiments, are prioritized for optimal imaging, particularly in fluorescence or brightfield microscopy. In addition, in some preferred embodiments, the material used for the bottom seal 38 exhibits low sorption rates to prevent absorption or leaching of reagents, thereby helping to ensure assay reliability and minimizing compound loss or contamination. The membrane thickness for the bottom seal 38 can range from 25 pm to 200 pm, with the exact value chosen to balance mechanical integrity with imaging performance. Further, in some embodiments, the bottom seal 38 or membrane may include a semi-permeable film that allows oxygen to transport thereacross, but that simultaneously sen es as a fluid barrier.

[0128] Turning again to the figures, Fig. 14A illustrates an exemplary top view of an IMWP plate 20 (e.g., the IMWP plate 20 illustrated in Fig. 13), according to some embodiments described herein. In some embodiments, IMWP 20 includes 24 independent units 26. In some embodiments, IMWP 20 includes 12 independent units 26, as shown in Fig. 14A. In some embodiments, IMWPAttorney Docket No.: PCT.131320 includes 48 independent units 26. A close-up of a unit 26 is shown in Fig. 14B. In some embodiments, a unit 26 includes a pump 30, a scaffold 32, a microfluid channel 34, and a microfluidic layer 34. As shown in Fig. 14B, in some embodiments, the pump 30 occupies approximately the same area or footprint 25 as an individual well 24. For example, in some embodiments, the pump 30 (e.g.„ a rotational pump) has an outer diameter or largest dimension (e.g., in an xy-plane) that is slightly smaller than the that of an individual well 24. For example, in some embodiments, the pump has an outer diameter or largest dimension that is 5%-20%, or 5%-15%, or 5%-10%, or 3%-8% smaller than an outer diameter of each individual circular well 24.

[0129] An IMWP as described herein, in some embodiments, offers an exceptional combination of precision, flexibility, and scalability, thereby providing capabilities with respect to real-time monitoring and functional assessment in organ-on-a-chip systems. Versatility of the IMWP as described herein enables a wide range of applications. In some embodiments, an IMWP may be used for drug development and toxicology. For example, comprehensive metabolic and barrier integrity data may ensure detailed insights into drug efficacy and safety. In some embodiments, an IMWP may be used for neurobiological studies. For example, high-density microelectrode arrays may provide precise measurements for research on neurological diseases and treatments. In some embodiments, an IMWP may be used for mechanobiology. For example, piezoelectric and strain sensors may enhance understanding of tissue mechanics in cardiovascular, musculoskeletal, and respiratory models. In some embodiments, an IMWP may be used for dynamic environmental simulations. For example, integrated gas and / or chemical sensors may support studies of hypoxia, nutrient gradients, and other physiologically relevant conditions.

[0130] Other impellers or configurations of rotational pumps, such as disc pumps or similar mechanisms, may also be used in connection with an IMWP configuration described herein. In each case, the impeller or pump configuration may be selected or optimized for the specific flow rates and application requirements to ensure efficient and reliable operation, and the particular parameters used for a given use case are not especially limited. Instead, as will be appreciated by the skilled person, the system’s flexibility allow s for customization of the rotational pump design to meet various experimental and / or operational needs.Attorney Docket No.: PCT.1313Pumping Mechanisms

[0131] In some embodiments, an IMWP described herein may include a rotational pump, utilizing magnetic coupling to drive fluid flow without external tubing or mechanical interfaces. In some such embodiments, the rotational pump includes an impeller. In some embodiments, the rotational pump includes a disc pumps. A rotational pump configuration may be selected or optimized for specific flow rates and / or application requirements to ensure an efficient and reliable operation, as indicated above. The flexibility of the IMWP as described herein enables customization of a rotational pump design to meet various experimental and / or operational needs.

[0132] Fig. 15A illustrates an example of an integrated microfluidic well plate 20, including a rotational pump, according to some embodiments described herein. In some embodiments, the IMWP 20 includes a well plate frame 22, a reservoir 28, a scaffold 32, a microfluidic layer 34, a bottom seal 38, a pump impeller 40, a magnet 42, and a membrane 44. The well plate frame 22 may incorporate the pump impeller 40, which is securely attached to the magnet 42, disposed within a cavity inside the microfluidic layer 34, and sealed by the thin membrane 44. In some embodiments, the membrane 44 separates the impeller 40 and the magnet 42 from a magnetic driver. When the IMWP 20 is placed on a magnetic driver, the driver’s alternating magnetic field engages with the magnet 42, inducing rotation of the impeller 40 (which is rigidly coupled to the magnet 42) and therefore, directing the fluid within microfluidic layer 34.

[0133] Fig. 15B illustrates an example of an integrated microfluidic well plate 20, including a rotational pump, according to aspects of the present embodiments. In some embodiments, the IMWP 20 includes a reserv oir 28, a scaffold 32, a microfluidic layer 34, a microfluidic channel 36, and a pump impeller 40. The pump impeller 40 may be disposed within a cavity inside the microfluidic layer 34. In some embodiments, the impeller 40 includes a hub 41 and a plurality of blades 43. In this example, the hub 41 (e.g., shaft) is the most inner part of the impeller 40 and longitudinal axis of hub 41 is aligned with the longitudinal axis of the reservoir 28 (and is also concentric with the magnet 42). The blades 43 are disposed radially and circumferentially about the longitudinal axis of the bearing 41. The bearing 41 (or hub 43) may only allow the rotation of the blades 43 about its longitudinal axis and prevent movements in other directions (i.e., the impeller 40 is free to rotate about the hub 41, but is prevented from translating (for example, laterally or vertically) within the IMWP). In some embodiments, the IMWP 20 includes a magnet 42 (shown in Fig. 15A) secured to the impeller 40. In some embodiments, the IMWP 20 is placedAttorney Docket No.: PCT.1313on a driver, whose alternating magnetic field engages with the magnet, and induces rotation of the impeller 40. The rotational motion of the impeller 40 results in generation of shear forces on the fluid within reservoir 28, creating a pressure that drives the fluid into the microfluidic channel 36. The media may then flow through the scaffold 32 and cycle back to the top of the reservoir 28, completing a circulation process 46. The direction of the circulation 46, for example, corresponds to a clockwise rotation of impeller 40. In some embodiments, the magnet 42 may sit on top of the impeller 40. In some embodiments, the impeller 40 may sit on top of the magnet 42. In some embodiments, as shown in Fig. 15C, the magnet 42 may be integrated into the pump 30 such that the magnet 42 is disposed radially around the hub 41, with the impeller 40 being disposed radially, around the magnet 42. In the embodiment of Fig. 15C, for example, an outer wall of the magnet 42 may form an inner wall of the impeller 40. In some embodiments, the hub 41, magnet 42, and impeller 40 are all disposed concentrically about a center of the hub 41.

[0134] Fig. 16 illustrates an example of an IMWP 20, including a rotational pump, according to aspects of the present embodiments. In some embodiments, the IMWP 20 includes a well plate frame 22, one or more scaffolds 32, a microfluidic layer 34, a bottom seal 38, one or more pump impellers 40. one or more magnets 42, a magnetic driving base 48, and a plurality of driving coils 50. In some embodiments, the magnetic driving base 48 is a planar, electrified base including a plurality of driving coils 50, each set of driving coils 50 being disposed beneath a corresponding pump magnetically coupled thereto. Accordingly, in operation, the well plate assembly 20 shown in Fig. 16 and according to the present embodiments includes touchless power transfer between each set of driving coils 50 and the corresponding pump. In operation, magnetic fields forming the magnetic coupling between each magnet 42 and the corresponding set of driving coils 50 are oriented such that they traverse the bottom seal 38.

[0135] Fig. 17A illustrates an example of an integrated microfluidic well plate 20, including a rotational pump, according to aspects of the present embodiments. In some embodiments, the IMWP 20 includes a well plate frame 22, one or more reservoirs (or “reservoir wells”) 28, one or more scaffolds (or “scaffold wells”) 32, one or more pump impellers 40, a magnetic driving base 48, and a plurality of driving coils 50. A selected area 21 of one impeller 40 and the magnetic driving base 48 is shown in Fig. 17B. In some embodiments, the impeller 40 includes a hub 41, a plurality of blades 43, a magnet 42, and a membrane 44. In some embodiments, the magnetic driving base 48 includes a plurality of coils 50. The driving coils 50 generate localized magnetic fields that transfer energy through the membrane 44 to the magnet 42. In some embodiments, theAttorney Docket No.: PCT.1313membrane 44 is configured or optimized to minimize resistance to a magnetic field while preserving the structural integrity necessary for efficient system operation. The coupling between the magnetic field and the magnet 42 inside the impeller 40 may provide sufficient rotational force to drive the impeller 40, enabling fluid movement 51 through the microfluidic channels. In some embodiments, the alternating magnetic fields may be precisely controlled, allowing for independent operation of each pump and facilitating customizable flow rates for each channel. This mechanism eliminates the need for direct mechanical connections, enhancing reliability, maintaining sterility, and simplifying the overall system design.

[0136] Fig. ISA illustrates an example of a rotational pump 30, including a rotating disk 52, according to aspects of the present embodiments. In some embodiments, the rotating disc 52 offers an alternative to an impeller 40 while retaining the same architecture, layout, and magnetic driving mechanism.

[0137] Fig. 18B illustrates an example of a rotational pump 30, according to aspects of the present embodiments. In some embodiments, a rotational pump 30 includes a rotating disk 52, a channel 54, and inlet / outlet 55. The channel 54 may include a width 56 and a height 57. In operation, the rotating disk 52 engages with an alternating magnetic field of a driver, resulting in a rotation 51 of the rotating disk 52. By replacing an impeller 40 with the rotating disc 52, an IMWP described herein, in some cases, can achieve similar fluid movement capabilities without altering the underlying design or operational principles, ensuring flexibility and adaptability to various application requirements.

[0138] Rotational pumps are well-suited for experiments requiring recirculating flow within a steady flow rate regime, typically in applications that include flow variation not greater than 10-fold, (for example, where the ratio of maximum (or “max”) flow to minimum (or “min”) flow ranges from lx to lOx, etc.). The compact design associated with the rotational pump requires only one reservoir per scaffold and eliminates the need for valves, simplifying both the well plate design and the pumping mechanism. Accordingly, the present embodiments are well-suited for high-throughput applications, due to the small footprint of the rotational pump, which allows for a greater number of wells. Additionally, the absence of valves minimizes potential flow disruptions or dead zones, which is particularly beneficial for shear-sensitive biological systems such as organoids or fragile cell cultures. In some embodiments, other types of pumps may be used in connection with applications requiring unidirectional flow, flow variations of greater than lOx, and / or in connection with the introduction of cells that could be affected by shear stress. WhileAttorney Docket No.: PCT.1313compact, integrating these pumps within an SLAS / ANSI-format plate may require additional space for housing and powering, potentially affecting the well plate’s density. Fine-tuning shear stress may also require additional mechanisms to replicate in vivo-like conditions accurately.

[0139] In some embodiments, an IMWP described herein may include a piezoelectric (PZT) pump, utilizing PZT actuation to achieve precise fluid flow without external tubing or mechanical interfaces. Fig. 19 illustrates an example of an integrated microfluidic well plate 60, including a PZT pump, according to aspects of the present embodiments. In some embodiments, the IMWP 60 includes a well plate frame 22, a reservoir 28, a scaffold 32, a hosebarb coupling 114, a microfluidic layer 34, a piezoelectric (PZT) transducer 62, a pump chamber 64, and a flexible membrane 66. In this example, as the PZT transducer 62 vibrates, it induces compression and relaxation of the flexible membrane 66 inside the pump chamber 64. In some embodiments, during the relaxation phase of the flexible membrane 66, media from the reservoir 28 flows into the chamber, and during the compression phase, the flexible membrane 66 displaces the fluid from the reservoir 28 to the microfluidic channel 36. This creates a controlled circulation of media through the scaffold 32 and back into the reservoir 28, ensuring continuous nutrient and waste exchange to support physiological conditions for organ-on-a-chip applications. In some embodiments, the integrated microfluidic well plate 60 includes micro check valves (for example, disposed within the microfluidic channel 36) used in connection with the piezoelectric transducer 62 to ensure fluid flow only in a single direction.

[0140] Still referring to Fig. 19, in some embodiments the piezoelectric actuator operates such that an amplitude does not exceed 5% strain on the attached membrane, corresponding to a travel distance of approximately 50 pm or less. The driving frequency of the pump depends on the desired flow rates and pressure, as well as the characteristics of the microfluidic system. For instance, in a Tesla valve configuration, the fluid within the valve must achieve a Reynolds number between 100 and 1000 to function effectively. Consequently, the driving frequency can be tailored to the system’s resistance and valve dimensions. In some embodiments, the system may include Tesla valves with a width of approximately 0.2 mm (for example, from about 0.1 mm to about 0.3 mm, or from about 0.15 mm to about 0.25 mm), and a driving frequency within a range of 40-400 Hz. In some embodiments, other valve designs may operate under different microfluidic regimes, requiring adjustments to the corresponding driving frequency. In operation, depending on the specific use case, a driving frequency of 40-400 Hz may be significantly faster than the pulsation frequencies of most biological mechanical signaling processes, such as cardiac or musculoskeletalAttorney Docket No.: PCT.1313systems. However, according to aspects of the present embodiments, it is possible to superimpose a physiologically relevant wave through amplitude modulation of the piezoelectric pump pulsations. By modulating the actuator's amplitude to mimic biological rhythms, such as a heartbeat or muscle contraction, the system could achieve pulsation patterns relevant to specific biological processes. This capability provides an additional layer of flexibility, allowing the piezoelectric configuration to simulate physiologically meaningful mechanical environments while maintaining its precision for microfluidic applications. According to the present disclosure, the system / assemblies described herein may incorporate selective PZT pump amplitude modulation to align with experimental goals.

[0141] Referring still to Fig. 19, in some embodiments, the PZT pump operates in coordination with micro check valves to ensure a unidirectional flow. In some embodiments, the micro check valve may include various types of valves including Tesla valves, thin membrane valves, duckbill valves, and other suitable types of micro check valves. In some embodiments, the micro check valve may be passive, meaning that it does not require active control to function, thereby simplifying the control module and reducing the complexify of the system. Depending on the specific application requirements, such as flow rates, operating pressures, and material compatibility, one or more of the various types of micro check valves may be particularly well-suited for the given application.

[0142] Fig. 20 illustrates an example of an IMWP 60, including a piezoelectric pump, according to some embodiments described herein. As depicted in Fig.20, the IMWP 60 includes a well plate frame 22, one or more reservoirs (or reservoir wells) 28. one or more scaffolds 32. one or more seal clips 70, holes 76 disposed within the one or more seal clips 70, a microfluidic layer 34, and a flexible membrane 66. The seal clips 70 may be used to place the scaffolds into each scaffold compartment (or “scaffold well’') 73, and then to subsequently remove each scaffold 32 from each scaffold compartment 73. In addition, each of the seal clips 70 may be used to apply downward pressure on the scaffold 32 such that the hosebarb couplings 114 (shown in Fig. 19) are fluidly coupled into the scaffold 32, as described herein. Accordingly, each of the seal clips 70 includes a center span (see element 74 in Fig. 21B, Fig. 28A, or Fig. 28B) that includes an area that approximately matches (e.g., within 10% or less, within 5% or less, or within 1% or less) the top surface of the scaffold 32 such that the downward force may be evenly distributed across the scaffold 32, thereby reducing the likelihood of causing damage to the scaffold 32. As shown in Fig.20, the well plate frame 22 may include one or more slots 71 or spaces into which the seal clipAttorney Docket No.: PCT.131370 may fit, thereby providing a scaffold compartment with an area (e.g., a bottom area or footprint) corresponding exactly or nearly exactly to (e.g., within 3% or within 1%) the amount of area required by the scaffold 32 and the seal clip 70. Stated otherwise, in operation, the seal clip remains within the well plate frame 22, and seals the gap between the well plate frame 22 and the scaffold 32. Each of the holes 76 may be used to remove seal clips 70 (and scaffold 32) from the well plate frame 22 (for example, using fish wire, thread, other cord or wire, a prong, and / or other tool). The scaffold 32 may fit snugly within the seal clips 70 such that a slight compression fit is sufficient to allow the seal clips 70 to hold the scaffold 32. As shown in Fig.20, the microfluidic layer 34 may include alternating cutouts 75, 77 to enable space for the reservoir / wells and scaffolds 32 respectively to fit. The cutouts 75 for the wells may be substantially circular while the cutouts 77 for the scaffolds may be substantially rectangular or square. More generally, the shape of the cutouts can correspond, respectively, to the shape of the compartment, well, or object disposed above the cutouts.

[0143] Fig. 21A illustrates an example of an IMWP 60, including a PZT pump 62, according to some embodiments. In some embodiments, the IMWP 60 includes a well plate frame 22, one or more reservoirs 28, a scaffold 32, and a seal clip 70.

[0144] Fig. 21B illustrates an example of an IMWP 60, including a PZT pump, according to some embodiments. In some embodiments, the IMWP 60 includes a well plate frame 22, a reservoir 28, a scaffold 32, a seal clip 70, recesses 79, and a microfluidic layer 34. Recesses 79 may be disposed within the well plate frame 22 near the top of the scaffold compartment 73 such that two prongs 72 of the seal clip 70 may be grasped or squeezed together in order to remove the scaffold 32 from the well plate frame 22 (e.g., when the seal clip 70 is formed from a flexible, deformable, or elastic material that can be ‘flexed’ in this manner). According to some embodiments, the piezoelectric pump 62 can provide a balance of simplicity and precision, providing highly accurate flow control desired for experiments involving microphysiological systems (MPS) that require fine-tuned fluid delivery. Piezoelectric pumps 62 are compact enough to be integrated directly into individual wells, enabling localized flow control and reducing interwell variability. In some cases, piezoelectric pumps 62 may be easier to drive and require simpler control systems compared to pressure-driven configurations. Vibrations introduced by the pump can be managed such that they do not impact sensitive biological assays, such as organoids or primary cell cultures. For example, in some embodiments, vibration-dampening materials or isolation techniques may be used in the vicinity of the piezoelectric pump 62. Additionally, PZTAttorney Docket No.: PCT.1313pumps 62 may be used in connection with valves (for example, micro check valves) to control flow direction. According to some embodiments, piezoelectric pumps 62 can integrate well with automated liquid handling systems, making them suitable for high-throughput workflows.

[0145] In some embodiments, an IMWP may include a pressure-driven pump, utilizing controlled pressure to drive fluid flow between reservoirs and microfluidic channels without external tubing or mechanical interfaces. Fig. 22 illustrates an example of an integrated microfluidic well plate 80, including a pressure-driven pump, according to some embodiments. In some embodiments, the IMWP 80 includes a plurality of independent units 81, each unit including a well plate frame 22 (or portion of the overall well plate frame 22, including to define the relevant wells of the unit), a primary reservoir (or "primary reservoir well’’) 28A, an auxiliary reservoir (or “auxiliary' reservoir well”) 28B, and a scaffold 32 (which may be disposed in a “scaffold well,” as described herein).

[0146] Fig. 23A illustrates an example of a unit 81 of an IMWP 80, including a pressure-driven pump (or system), according to some embodiments. In some embodiments, the unit 81 includes a well plate frame 22 (or portion thereof), a primary reservoir 28A, an auxiliary reservoir 28B, a scaffold 32, a microfluidic layer 34, a bottom seal 38, a sealed lid 82, cap-based sampling ports 84 (which, e.g., may be threaded), and sampling port caps 86 (which, e.g., may be threaded). The specialized sealed lid 82 may enable seamless coupling of the well plate frame 22 to the reservoirs 28A, 28B, facilitating aseptic connection, disconnection, and operation.

[0147] Fig. 23B illustrates an example of a unit 81 of an IMWP plate 80, including a pressure-driven pump (or system), according to some embodiments. In some embodiments, the unit 81 includes a well plate frame 22 (or portion thereof), a primary reservoir 28A, an auxiliary reservoir 28B, a scaffold 32, a microfluidic layer 34, a bottom seal 38, a sealed lid 82, cap-based sampling ports 84, sampling port caps 86, cap sealing O-rings 88, and lid sealing O-rings 90. As depicted in Fig. 23B, portions of the microchannels 36 of the microfluidic layer 34 are shown, particularly connecting the scaffold 32 to the reservoirs 28A, 28B. However, it is to be understood, with reference to the entirety of the present disclosure, that specific microchannels of a microfluidic layer described herein are not necessarily shown in detail, for the sake of simplicity. As understood by the skilled person, the precise arrangement and configuration of the microchannels of a microfluidic layer described herein are not particularly limited and may be selected as needed or desired to enable fluid flow from between the various components of a given IMWP describedAttorney Docket No.: PCT.1313herein (e.g., to enable fluid flow between a reservoir and a scaffold, between scaffolds, between reservoirs, etc.).

[0148] Referring again to Figs. 23A and 23B, the system 80 may employ a hermetically sealed lid 82 that integrates with pneumatic connectors 87 that fluidly connect to each pressure vessel (where a “pressure vessel” can refer to a sealed compartment such as a well). For example, a plurality7of pneumatic channels 98 (shown in Figs. 26A and 26B) disposed within the sealed lid 82 are each fluidly connected to the pressure vessels (i.e., reservoirs 28A, 28B, etc.) via the pneumatic connectors 87, which may be vertically oriented and are also disposed within the sealed lid 82. In this embodiment, filtered air is introduced through these pneumatic connectors 87 and channels 98 to ensure contamination-free operation. A low-pressure, high-sensitivity regulated pressure source, combined with a controlled feedback loop, is used to pressurize the vessels. The pressure source(s) (not shown) may be connected to each pneumatic channel 98 and connector 87 via pneumatic interfaces 92 (shown in Fig. 24A) disposed within a sidewall of the sealed lid 82. In some embodiments, the advanced feedback system as described herein continuously monitors and adjusts both the pressure and airflow7delivered to the reservoirs, ensuring precise control over media movement. The regulated pressure drives the media flow selectively from one reservoir 28A, through the scaffold 32, and into the second reservoir 28B, maintaining consistent and reliable operation throughout. The sealed lid 82 may include a plurality of sampling ports 84 (e.g., capbased sampling ports) disposed therein, each sampling port 84 located centrally above (i.e., concentric with) a centerline (i.e., center axis) of a corresponding pressure vessel or reser oir 28 A, 28B. The system 80 may also include a knob 86 or sampling port cap 86 at the top of each pressure vessel to allow7for sampling from the top of each reservoir 28A, 28B when necessary or desired. In some embodiments, the system 80 include septa (e.g., a septum associated with each sampling port 84) to be used in connection with a needle / syringe when sampling, i.e., rather than the knobs 86 / sampling port caps 86.

[0149] Still referring to Figs. 23A and 23B, each of the sampling port caps 86 may include external threading corresponding to internal threads disposed within each of the sampling ports 84, thereby enabling each of the sampling port caps 86 to be twisted or securely screwed into a corresponding sampling port 84. This design approach comprising pressure vessels and pressure driven flow, as descnbed herein, facilitates top-access sampling, thereby enabling or improving compatibility7with automation systems. The system 80 may include caps 86 or septa, as well as alternative ways to seal the well plate. In some embodiments, the IMWP (or system) 80 mayAttorney Docket No.: PCT.1313include a bonded fdm used as a seal, which can be peeled off and replaced again when needed. In some embodiments, the system 80 may include all pressure channels 98 being integrated directly into or with the plate itself (i. e. , well plate frame 22), thereby eliminating the need for a sealed lid 82. In embodiments in which the well plate frame 22 includes pressure channels 98 integrated therein, the pneumatic interfaces may be disposed within the side of the well plate frame 22, thereby providing an alternative means of maintaining a pressurized environment while accommodating different operational requirements.

[0150] Referring still to Figs. 23A and 23B, in some embodiments, flow directionality may be managed through a predefined flow path and a system of sampling port caps 86 to allow multiple flow configurations. In general, a flow path operates by directing fluid from a more pressurized reservoir or container to a less pressurized reservoir or container. In some embodiments, the IMWP 80 includes a bidirectional flow. In this configuration, the primary reservoir 28A is pressurized, pushing the media through the scaffold 32 to the auxiliary reservoir 28B. Once the media has been fully transferred, the primary reservoir 28A is depressurized, and the auxiliary reservoir 28B is pressurized. This reverses the flow, driving the media back through the scaffold 32 and into the primary reservoir 28A. In some embodiments, the IMWP 80 includes a unidirectional, non-recirculatory flow. In this configuration, the primary reservoir 28A is pressurized, pushing the media through the scaffold 32 and into the auxiliary reservoir 28B. Once the transfer is complete, the primary reservoir 28A is depressurized, and both reservoirs are vented. The reservoirs may be vented by opening the sealed lid 82 and / or the sampling port caps 86. Subsequently, the waste is retrieved, and the primary reservoir 28A is refilled to restart the process. In some embodiments, the IMWP 80 includes a recirculatory flow. In this configuration, a series of sampling port caps 86 redirect the flow between the reservoirs 28A, 28B through a secondary flow path that bypasses the scaffold 32. The media is then recirculated through the scaffold 32 in one consistent direction, maintaining uniform perfusion and nutrient exchange within the scaffold 32. This configuration allows for continuous, controlled unidirectional flow through the scaffold 32 while preserving media recirculation efficiency. In some embodiments, the IMWP 80 (or system) includes advanced automation with additional reservoirs and flow paths. Additional reservoirs, valves, and flow paths may be incorporated to enable automated reagent addition, real-time sampling, and / or waste removal. These features may enhance system functionality, allowing for activities such as dynamic media replenishment, gradient creation, and automated endpoint sampling, and providing greaterAttorney Docket No.: PCT.1313flexibility for complex experimental workflows. Each flow configuration may be designed to accommodate specific experimental needs, offering flexibility and precision in fluid handling.

[0151] Fig. 24A illustrates an example of an IMWP 80, including a pressure-driven pump, according to some embodiments. In some embodiments, the IMWP 80 includes a well plate frame 22 (or portion thereof), a sealed lid 82, cap-based sampling ports 84, sampling port caps 86, and a plurality7of pneumatic interfaces 92.

[0152] Fig. 24B illustrates an example of a unit 81 of an IMWP 80, including a pressure-driven pump, according to some embodiments. In some embodiments, the unit 81 includes a well plate frame 22 (or portion thereof), a primary reservoir 28A, an auxiliary reservoir 28B, a scaffold 32, a microfluidic layer 34, a sealed lid 82, cap-based sampling ports 84, sampling port caps 86, cap sealing O-rings 88, and lid sealing O-rings 90.

[0153] Fig. 25A illustrates an example of an IMWP 80, including a pressure-driven pump (or system), according to some embodiments. In some embodiments, the IMWP 80 includes a well plate frame 22 (or portion thereof), a sealed lid 82, septum-based sampling ports 94, and a plurality of pneumatic interfaces 92. In some embodiments, a pneumatic interface 92 includes a microfilter 96. According to aspects of the present disclosure, embodiments that include the sampling port caps 86 rather than septum-based sampling ports 94 may also include microfilters 96 disposed within the pneumatic interfaces 92.

[0154] Fig. 25B illustrates an example of an IMWP 80, including a pressure-driven pump (or system), according to some embodiments. In some embodiments, the IMWP 80 includes a well plate frame 22 (or portion thereof), a primary reservoir 28A, an auxiliary reservoir 28B, a scaffold 32, a sealed lid 82, septum-based sampling ports 94 (each with a septum layer 95 disposed at the top of each septum-based sampling port 94 such that each septum layer 95 is co-planar with the top surface of the sealed lid 82), and lid sealing O-rings 90. Each of the lid sealing O-rings 90 may be centered about a corresponding septum-based sampling port 94 disposed within an inner circumference of one of the reservoirs, and around a corresponding outer circumference of an interfacing downwardly protruding cylindrical feature 97 of the sealed lid 82, thereby forming a tight seal (e.g., leak proof seal under normal operating temperature and pressure, such as 0.5-1.5 atmosphere (atm) and 22-37°C) between each reservoir 28A, 28B and the sealed lid 82.

[0155] Fig. 26A illustrates an example of a sealed lid 82, according to some embodiments. In some embodiments, the sealed lid 82 includes a plurality of pneumatic interfaces 92 and a plurality of pneumatic channels 98. Each of the pneumatic channels 98 may be configured such that theyAttorney Docket No.: PCT.1313extend both lengthwise and widthwise (longitudinally and laterally) within the sealed lid such that they fluidly connect each pneumatic interface 92 with a corresponding reservoir 28A, 28B.

[0156] Fig. 26B illustrates an example of a sealed lid 82, according to some embodiments. In some embodiments, the sealed lid 82 includes a plurality of pneumatic interfaces 92 and a plurality of pneumatic channels 98. As shown in Fig. 26B, each of the pneumatic channels 98 connects to a vertically-oriented pneumatic connector 87 which fluidly connects to one of the reservoirs 28A, 28B. According to aspects of the present disclosure, aseptic conditions may be maintained by ensuring that all liquid handling processes are confined within the well plate frame 22. The air used to drive fluid movement can be pre-filtered within the pump system to eliminate contaminants. Additionally, 0.22 pm filters (i.e., microfilters 96) integrated into the pneumatic interfaces 92 of the plate lid 82 provide an additional barrier, preventing the ingress of any contaminants that could be present in the gas source. This multi-layered approach helps maintain the sterility of the system throughout its operation. In addition, the air used to drive the fluid movement enables the liquids to remain within the well-plate frame 22. The pressure-driven pump configurations according to the present disclosure are highly versatile and well-suited for applications requiring unidirectional or bidirectional flow, as well as a wide range of flow rates, such as those with max-min flow ratios of lx to lOOOx. This flexibility makes the present embodiments preferred for replicating dynamic biological environments, including vascularized tissues or perfused organ systems. The pressure-driven embodiments described herein, in some cases, also accommodate recirculating flow by adding valves. Pressure-driven systems described herein in some instances, may include at least two reservoirs per scaffold. The pneumatic interfaces 92, channels 98, and connectors 87 described herein may also be used to enable 1) gas exchange for oxygenation in long-term cell cultures, 2) robust sterility maintenance through embedded 0.22 pm filters, and 3) strategies to mitigate air bubble formation, such as degassing systems (for example, in connection with bubble traps). Furthermore, in some embodiments, the IMWP / system 80 of the present disclosure may include integrated real-time monitoring sensors for parameters such as flow rate, pressure, pH, and oxygen levels, which can enhance experimental precision and control.Microfluidic Layer

[0157] As described herein, in some cases, a microfluidic layer is integrated or included at the bottom of an IMWP and may be customized for specific applications. The microfluidic layer mayAttorney Docket No.: PCT.1313have any configuration and be formed in any manner not inconsistent with the technical objectives of the present disclosure. In some embodiments, for example, a microfluidic layer may be manufactured via injection molding. In some applications, a microfluidic layer may direct fluid from a pump outlet to a scaffold inlet and subsequently, direct the fluid back to a reservoir. In some applications, one or more valves may be incorporated into an IMWP to enable flow direction changes and / or automated sampling. Sampling may also be efficiently achieved using standard automation setups, performed from the top of a well plate (i.e., standard method) in connection with automated liquid handling systems. Fig. 27 illustrates an example of an individual unit 26, according to some embodiments described herein. In some embodiments, the unit 26 includes a well plate frame 22 (or a portion thereof), a reservoir 28, a pump 30, a scaffold 32, a microfluidic layer 34, and one or more microfluidic channel(s) 36. In some embodiments, the pump 30 is a rotational pump. In the example illustrated in Fig. 27, the microfluidic layer 34 directs the fluid from the pump 30 to the scaffold 32 and subsequently, from the scaffold 32 to the reservoir 28.

[0158] Referring still to Fig. 27, the thickness of the microfluidic layer 34 may vary depending on the specific design requirements. In some embodiments, the thickness of the microfluidic layer ranges from 1 mm to 3 mm. In some embodiments, thinner layers may be preferred as they can improve performance and efficiency, provided they still meet the structural and functional needs of the system. In some embodiments, the microfluidic layer 34 may be composed of materials that offer high optical clarity, chemical resistance, and manufacturability. Suitable options include a cyclic olefin copolymer (COC) such as a norbornene-ethylene copolymer or a tetracyclododecene-ethylene copolymer; a cyclic olefin polymer (COP) such a polynorbomene; a fluorinated ethylene propylene (FEP) or copolymer of hexafluoropropylene and tetrafluoroethylene; and a polyethylene terephthalate (PET). Other materials may also be used in some instances, such as other materials that are well-suited for applications that require optical transparency and low sorption (such as described herein). In some embodiments, the microfluidic layer may be manufactured via injection molding and / or hot embossing, each of which are compatible with the potential microfluidic layer 34 materials described herein, and each of which allow for high-precision fabrication of microfluidic features while enabling scalability and cost efficiency for high-throughput applications. In some embodiments, the inner diameters of microfluidic channels 36 (i.e., flow passages within the microfluidic plate / layer 34) may range from about 50 pm to about 500 pm, depending on the application and the desired flow dynamics. Smaller diameters (closer to 50 pm) are ty pically used for precise control of microfluidic flows, such as mimicking capillary-level fluidAttorney Docket No.: PCT.1313dynamics. Larger diameters (up to 500 pm) are more suitable for higher flow rates or when reduced pressure drop is critical. The specific dimensions of the microfluidic flow channels are not particularly limited and may be selected based on factors such as one or more of the following: (1) the required or desired flow rate and pressure, (2) the type of media being circulated, (3) compatibility with biological and / or chemical application, and / or (4) the overall design constraints of the Microphysiological Systems (MPS) system. Accordingly, embodiments and configurations according to the present disclosure balance precision, manufacturability, and compatibility with typical microfluidic requirements in an SLAS / ANSI-format well plate.

[0159] Still referring to Fig. 27, and also to previous figures, the IMWP 20, 60, 80 according to the present disclosure may include key external dimensions that conform to the SBS-format standards (127.76 mm x 85.48 mm footprint). This standardization helps provide compatibility with existing high-throughput systems and automation equipment. The thickness of the microfluidic layer 34 is particularly constrained by these requirements, in some cases. If the microfluidic layer 34 is too thick, it reduces the available height for other components, such as the well plate frame 22. This reduction may ultimately impact the liquid volume capacity within the wells, which is an important parameter for many applications. Therefore, balance is preferred to ensure the microfluidic layer 34 is functional while maintaining compatibility with SBS-format dimensions and preserving sufficient liquid volume.

[0160] Additionally, in some cases, the interface between the pump inlet / outlet 30 and the microfluidic layer 34 is designed to provide a seamless and leak-proof connection while maintaining sterility. In this system 20, 60, 80, the pump 30 assembly is integrated directly within the well plate, and the inlet and outlet align precisely with the microfluidic channels 36 within the microfluidic layer 34 (e.g., within 1%). In some embodiments, to eliminate the need for external tubing or cylindrical couplings, the layers are welded or fused together using methods such as thermal, ultrasonic, and / or laser welding. This process creates a permanent bond between the pump assembly and the microfluidic layer, ensuring a secure and precise interface. The welded connection(s) can largely eliminate the risk of fluid leakage, reduce dead volumes, and enhance the overall robustness of the system 20, 60, 80. By directly coupling the pump to the microfluidic channels, the system can in some cases achieve reliable and consistent fluid transfer, which is preferred for maintaining precise control over media flow and providing uniform perfusion in MPS. This integration supports physiological conditions and streamlines the architecture, making the system efficient and reliable for advanced tissue modeling and experimental applications.Attorney Docket No.: PCT.1313According to the present disclosure, the microfluidic layer 34 can be a solid layer wi th microfluidic channels 36 (i.e., flow passages) fornied by voids. In some embodiments, microfluidic features may be embossed directly onto a bottom surface of the well plate frame 22. and sealed with a bottom film.

[0161] Additionally, in some embodiments, an IMWP described herein (e.g., IMWP 20, 60, or 80) utilizes a coupling mechanism to allow a hydrogel scaffold to form a functional interface with the microfluidic layer 34. A coupling mechanism can help ensure a scaffold 32 remains stable while maintaining preferred contact with the microfluidic layer 34, supporting efficient fluid exchange and consistent experimental conditions. In some embodiments, a coupling mechanism includes mechanical means, such as clamps or clips. In some embodiments, a coupling mechanism includes specialized adhesives. In some embodiments, other coupling mechanisms may be used. The flexibility of coupling mechanisms may also allow for easy assembly, disassembly, and scalability, rendering an IMWP 20, 60, 80 adaptable to a wide range of experimental setups and research needs.

[0162] Fig. 28A illustrates an example of a handling method 100 of a scaffold 32, according to some embodiments. In some embodiments, the handling method includes a well plate frame 22 (or portion thereof), a scaffold 32, and a seal clip 70. In some embodiments, the seal clip 70 includes two top prongs 72, a mid-portion 74, two bottom prongs 81, and two holes 76 (i.e., a hole 76 disposed within each of the top prongs 72). In use, the top prongs 72 may be pinched or squeezed together enabling the bottom prongs 81 to be disposed on either side of the scaffold 32, thereby enabling the scaffold to be grasped and picked up (and subsequently placed within the well plate frame 22). The mid-portion 74 may include a surface that approximately matches that of a top surface 35 of the scaffold 32, thereby enabling the mid portion 74 to be used to gently push the scaffold 32 dow n into place in a distributed fashion (i.e., with the force being distributed across the top surface 35 of the scaffold 32), avoiding the application of too large of a downward force in any one area (which could damage the scaffold 32).

[0163] Fig.28B illustrates an exemplary front view' of a seal clip 70, according to aspects of the present embodiments. In some embodiments, the seal clip 70 includes two top prongs 72, a midportion 74, and two bottom prongs 81. In some embodiments, the seal clip 70 is pre-formed so as to have elasticity such that a slight compressive spring force may be applied by each of the bottom prongs 81 onto the sides of the scaffold 32, thereby allowing the scaffold 32 to be picked up and moved. For example, Fig. 28B shows an embodiment of a seal clip 70 in a resting state. As theAttorney Docket No.: PCT.1313two top prongs 72 are pressed together, the two bottom prongs 81 move farther apart, thereby allowing them to be placed around the scaffold 32. In some embodiments, the seal clip 70 does not include a pre-formed spring force, and instead fits precisely around and over the scaffold, thereby enabling friction and / or a slight compression fit to allow the seal clip 70 to grasp the scaffold 32.

[0164] Fig. 29 illustrates an example of a mechanical coupling mechanism 110, according to some embodiments. In some embodiments, the mechanical coupling mechanism 110 includes a well plate frame 22 (or portion thereof), a scaffold 32, a scaffold overhang 33, a microfluidic channel 36, a seal clip 70, a hosebarb connector 112, and a hosebarb coupling 114. The hosebarb connector 112, for example, may be created by including a void in the overhang 33 that corresponds to the geometry of the hosebarb coupling 114, which is coupled to and / or integrated with the microfluidic layer 34). Such a void may be created as part of an additive manufacturing process used to form the overall scaffold, as further described herein. An additional view of the hosebarb coupling 114 integrated onto the microfluidic layer 34 is shown in Fig. 19. In some embodiments, the scaffold 32 is securely positioned within its slot by the seal clip 70 and precisely aligned with the microfluidic channel 36. A microfluidic seal is achieved through the mechanical engagement of the mating connectors 112, 114 on the scaffold 32 and microfluidic layer 34, while the seal clip 70 applies a controlled force through mid-portion 74 to compress the scaffold 32 against the bottom of the well. The mechanical coupling mechanism 110 may ensure a secure, reversible attachment that provides a robust and reliable connection, enabling efficient and consistent fluid transfer between the scaffold 32 and the microfluidic layer 34.

[0165] Fig. 30A illustrates an example of an adhesive coupling mechanism 120, according to some embodiments. In some embodiments, the adhesive coupling mechanism 120 includes a scaffold 32, a microfluidic channel 36, and an adhesive 122. Fig. 30B illustrates an exemplary cross section of an adhesive coupling mechanism 120, according to some embodiments. In some embodiments, the adhesive coupling mechanism 120 includes a well plate frame 22 (or portion thereof), a scaffold 32, a scaffold overhang 33, a microfluidic channel 36, a seal clip 70, and an adhesive 122. In this example, an interface is achieved by precisely aligning the scaffold 32 with the microfluidic channel 36, further applying a versatile bonding solution. This interface is secured using an adhesive for a permanent seal, ensuring durability and leak-proof operation. In some embodiments, the adhesive coupling mechanism accommodates scaffold inlets located at various positions, such as the top, the bottom, or the sides of a scaffold 32, thereby providing designAttorney Docket No.: PCT.1313flexibility while maintaining a reliable and robust seal for efficient fluid transfer between the scaffold 32 and a microfluidic layer 34.Sensor Integration

[0166] In some embodiments, an integrated microfluidic well plate (IMWP) described herein may be designed to integrate a variety of sensors tailored to specific applications, enabling realtime, multi-modal monitoring of tissue performance and therapeutic insights within an organ-on-a-chip system. Sensors may be embedded into a cartridge or a well plate in any manner not inconsistent with the technical objectives of the present disclosure, such as by using advanced microfabrication techniques, which may provide modularity and scalability. By leveraging advanced technologies, the platform may offer precise, scalable, and customizable functionality. In some embodiments, a sensor includes an electrochemical sensor. For example, embedded platinum, carbon-based, or iridium oxide electrodes may be used to monitor critical metabolic parameters such as oxygen consumption, pH, glucose, and lactate levels, and therefore provide real-time data on cellular metabolism and tissue health. In some embodiments, a sensor includes an electrophysiological sensor. For example, arrays of high-density microelectrodes, fabricated from gold or indium tin oxide (ITO), may be used to record neuronal activity in neurobiological constructs, thereby offering high-resolution insights into electrical signaling and tissue excitability.

[0167] In some embodiments, a sensor includes a barrier integrity' sensor. For example, trans-epithelial or trans-endothelial electrical resistance (TEER) sensors, composed of planar electrodes with biocompatible coatings such as polydimethylsiloxane (PDMS). may be used to assess tight junction integrity’ and barrier function in epithelial or endothelial tissues. In some embodiments, a sensor includes an optical sensor. For example, fluorescence and absorbance systems, coupled with embedded optical fibers, may be used to monitor key parameters such as oxygenation, calcium signaling, and pH using biocompatible fluorescent probes, and therefore provide non-invasive, high-sensitivity measurements. In some embodiments, a sensor includes a mechanical sensor. For example, piezoelectric sensors, made from materials such as polyvinylidene fluoride (PVDF), may be used to measure tissue contractility and scaffold deformation, thereby providing data on mechanical properties in cardiac, musculoskeletal, and lung models. In some embodiments, strain and stress sensors may be used to monitor forces exerted by or on tissues, particularly in systems modeling dynamic physiological processes such as breathing or blood flow.Attorney Docket No.: PCT.1313

[0168] In some embodiments, a sensor incorporated into the present system 20, 60, 80 includes an environmental sensor. For example, thin-film thermistors may be used to track temperature to ensure physiological stability. In some embodiments, MEMS-based flow meters may be used to monitor microfluidic flow rates, ensuring precise nutrient delivery and waste removal. In some embodiments, dissolved oxygen and carbon dioxide sensors may be used to provide data on gas exchange, which is critical for cell viability. In some embodiments, a sensor includes a chemical and biomolecular sensor. For example, enzyme-based biosensors may be used to detect specific metabolic byproducts or nutrients, such as urea, ammo acids, or lipids, thereby expanding metabolic monitoring that is possible with the present disclosed systems 20, 60, 80. In some embodiments, antibody-based sensors may be used to measure secreted biomarkers, cytokines, or hormones for functional tissue assessment. In some embodiments, a DNA or RNA sensor may be used to track gene expression or contamination, enabling advanced diagnostics and quality control. In some embodiments, a sensor includes a gas sensor. For example, volatile organic compounds (VOC) and ammonia sensors may be used to detect byproducts of cellular metabolism and / or environmental contamination, thereby enhancing overall system reliability.

[0169] In some embodiments, a sensor incorporated into the present disclosed systems 20, 60, 80 may include an advanced biophotonic sensor. For example, label-free biosensors, such as surface plasmon resonance (SPR) sensors, may be used to provide real-time measurements of cellular adhesion, morphology7, and molecular interactions without the need for additional markers. In some embodiments, a sensor includes a flow sensor. A flow sensor may be capable of measuring microfluidic flow rates with high precision using optical, differential pressure, and / or thermal detection methods. The thin, transparent bottom of an IMWP 20 may enable non-invasive optical flow measurements, enhancing accuracy and compatibility with dynamic monitoring systems. In some embodiments, a sensor includes a pressure sensor. For example, MEMS-based pressure sensors may be used to provide real-time data on microfluidic pressure gradients within the system. As another example, optical methods may be employed for pressure monitoring, leveraging the transparent bottom for precise and localized measurements. In some embodiments, a sensor is a wireless sensor. Wireless sensors may enable real-time data acquisition and transmission, simplifying monitoring in high-throughput or long-term studies, and facilitating remote control and data analysis. The particular sensor used in a system described herein is not particularly limited, as understood by the skilled person. The foregoing example sensors are provided for illustrationAttorney Docket No.: PCT.1313purposes in the context of specific embodiments. Other sensors and sensor configurations are also possible and contemplated by the present disclosure.EXAMPLES

[0170] The following examples are intended to illustrate but not limit the disclosed embodiments. The following examples are useful to confirm aspects of the disclosure described above and to exemplify certain embodiments of the disclosure.

[0171] These non-limiting examples demonstrate particular features, use cases and advantages of provided technologies - e.g., of provided integrated microfluidic well plates, associated systems, and methods of use.Example 1: Drug Development and Toxicology

[0172] The present example demonstrates that a system provided herein, in some embodiments, achieves detailed insights into drug efficacy and safety. In some embodiments, detailed insight into drug efficacy and safety is achieved through comprehensive metabolic and barrier integrity measurements.

[0173] In one example, epithelial or endothelial cells are seeded on a surface of a system described herein (for example, on a surface of scaffold 32) at 37 degrees Celsius and 5% CO2. The surface is comprised of trans-epithelial or trans-endothelial electrical resistance (TEER) sensors, comprised of planar electrodes with biocompatible coatings such as polydimethylsiloxane (PDMS). Cells are grown to confluency. forming a monolayer. The cells (e.g., endothelial, epithelial, etc.) are exposed to an antibody-drug-conjugate (ADC) (e.g., Trastuzumab deruxtecan (T-Dxd)) while electrical resistance is measured via the TEER sensors. If tight junctions and / or plasma membrane of the cells are damaged upon exposure to an ADC, the TEER will detect a corresponding disruption in cellular electrical resistance.

[0174] In some embodiments, a system provided herein further comprises enzyme-based biosensors (for example, disposed within or around scaffold 32) that detect metabolic byproducts or nutrients, such as urea and / or amino acids. Hepatocytes are seeded on a surface of the system 20, 60, 80 described herein (for example, on a surface of scaffold 32) as previously described for endothelial cells, and are then exposed to a therapeutic (e.g., fialuridine, entecavir, troglitazone, clozapine, olanzapine, acetaminophen, metacetamol, tolcapone, entacapone, nefazodone, buspirone, trovafloxacin, levofloxacin, diclofenac, or amiodarone). In some embodiments, pumpAttorney Docket No.: PCT.131330 and / or microvalves (e.g., micro check valves) as described herein may be used to regulate the exposure of the endothelial cells to the therapeutic. Urea production by the hepatocyte culture, in response to each therapeutic, is then measured. Therapeutics that cause hepatocytes to release urea (above a pre-determined threshold) are assessed as toxic (or relatively toxic) to hepatocytes.

[0175] The achievements of the system 20, 60, 80 provided herein are not limited to the particular cells and / or compounds described in this example. A skilled artisan would appreciate, upon reading this disclosure, that the system 20, 60, 80 provided herein can achieve detailed insights into drug efficacy and safety (e.g., toxicology) of various compounds against various cell types.Example 2: Neurobiological Studies

[0176] The present example demonstrates that a system described herein (e.g., IMWP 20, 60, 80) can achieve detailed insights into aspects of central nervous system functionality in response to treatment (e.g., drugs, biologies, therapeutics, compounds). In some embodiments, a system 20, 60, 80 provided herein records neuronal activity' to achieve high-resolution insights into electrical signaling and tissue excitability. In some embodiments, a system provided herein provides insights into efficacy and safety of drugs (e.g., therapeutics, compounds) for the amelioration of neurological diseases.

[0177] Human iPSC-derived cortical neurons are seeded on a surface of the system described herein (for example, on a surface of scaffold 32) and cultured at 37 degrees Celsius and 5% CO2.Electrophysiological activity is recorded using arrays of high-density microelectrodes fabricated from gold or indium tin oxide (ITO) to assess neuronal long-term potentiation (LTP), indicative of neuronal learning and memory formation.

[0178] To assess physiological function (e.g., electrical excitability) in response to a pathogenic stimuli, neurons are exposed to intracellular solution (140 mM K-gluconate, 4 mM NaCl, 0.5 mM CaCh, 1 mM MgCh, 1 mM EGTA, 5 mM HEPES acid, 5 mM HEPES base, and / or 5 mM Na2ATP) and baseline electrical activity and LTP of the neurons are recorded. In some embodiments, pump 30 and / or microvalves (e.g., micro check valves) as described herein may be used to regulate the exposure of the neurons to the intracellular solution. LTP may subsequently be induced, and the neuronal response may be recorded.

[0179] Neurons are treated with approximately 5uM pathogenic Afty for 1 hour and neuronal activity is recorded. Neurons are subsequently co-treated with Apty and an agent (e.g., aAttorney Docket No.: PCT.1313therapeutic, memantine, donepezil, saracatinib, rolipram) for 72 hours to mimic therapeutic intervention for Alzheimer’s disease. Controls include untreated conditions. Recordings of neuronal activity are recorded at 1, 24, 48, and 72 hours. Analysis of neuronal action potentials indicate that A 42 serves to dampen neuronal activity. Restoration of neuronal activity (e.g., action potential) for the duration of the 72-hour recording, upon agent (e.g., memantine) exposure, indicates rescue of neuronal electrical activity.

[0180] A skilled artisan will appreciate, upon reading the present disclosure, additional drugs and / or cell types may be tested to investigate their mechanism in modulating action potential propagation. In some embodiments, changes in inward sodium currents of a tested cell may also or instead be recorded.Example 3: Mechanobiology

[0181] The present example demonstrates that a system described herein (e.g., a system 20, 60, 80) can achieve detailed insights into mechanical properties (e.g., contractility, scaffold deformation, strain) of various models, tissues, and / or cells (e.g., cardiac, musculoskeletal, and lung models). In some embodiments, a system 20, 60, 80 provided herein comprises piezoelectric sensors that achieve measurements of one or more mechanical properties in various models and cell types. In some embodiments, the piezoelectric sensor(s) is made from materials such as polyvinylidene fluoride (PVDF).

[0182] H9-derived cardiomyocytes (H9-CM) are seeded on a surface (e.g., a scaffold 32 surface) of a system 20, 60, 80 described herein. In some embodiments, the surface is pre-coated with a substance (e.g., gelatin, MatrigeL Synthemax) that promotes the adherence of H9-CM to the surface. H9-CM are differentiated into contracting cardiomyocytes as described in Lyra-Leite et al. STAR Protoc. 2022 Aug 18;3(3):101560, the entire contents of which are incorporated herein by reference.

[0183] The beating rate, represented in beats per 10 seconds, of cardiomyocytes are subsequently evaluated to establish a baseline. To test the effects of a therapeutic on beating, cardiomyocytes are incubated with an agent (e.g., isoproterenol, propranolol, or blebbistatin) for approximately 10-30 minutes. In some embodiments, pump 30 and / or microvalves (e.g., micro check valves) as described herein may be used in connection with incubation of the cardiomyocytes with the agent. In addition, in some embodiments, a piezoelectric pump (e.g., including a piezoelectric transducer 62 as described herein) may be used to vary the incubation rate (e.g., by varying the operationalAttorney Docket No.: PCT.1313frequency (for example, in a range from about 40 Hz to about 400 Hz) of the piezoelectric transducer 62) to further assess the effect of beating. Beating rate is measured during this incubation. After incubation, the agent containing media is replaced with fresh media and beating is measured for an additional 60 minutes to determine if the restoration of beating frequency to baseline occurs. Cytotoxicity of various agents is also assessed by measuring the cessation of beating in response to an agent. As a positive control for cytotoxicity', cardiomyocytes are incubated with doxorubicin (DOX) and beating frequency is measured for 72 hours.

[0184] In some embodiments, a system 20, 60, 80 provided herein achieves measurements of cardiomyocyte fractional shortening (i.e., the amount a heart muscle cell contracts and shortens). Fractional shortening may be measured in response to various mechanical, chemical (e.g., drugs, therapeutic), and / or environmental (e.g.. oxygen) stimuli. A system 20, 60, 80 provided herein, in some cases, can achieve assessment of cardiomyocyte contractile function through measurement and calculation of the change in length of a cardiomyocyte during a contraction cycle (e.g., fractional shortening).

[0185] A skilled artisan will appreciate, upon reading the present disclosure, that additional cell types (e.g., lung, skeletal) may be used to interrogate tissue mechanics of each respective cell type. A skilled artisan will appreciate, upon reading the present disclosure, that various cell types can be used in combination with various agents (e.g., drugs, compounds, therapeutics) and / or environmental conditions to investigate the role of each drug and / or condition in a disease associated with that cell type.

[0186] In some embodiments, a system described herein (e.g.. a system 20, 60, or 80) can achieve the identification of therapeutics for treatment of various cardiovascular diseases. In some embodiments, a system described herein achieves the measurement of the physiological consequence of genetic manipulation that identifies potential therapeutic drug targets.Example 4: Dynamic Environmental Simulations

[0187] The present example demonstrates that a system described herein (such as a system 20, 60, or 80) can achieve detailed insights into the role environmental conditions (e.g., nutrient, gas, ionic) play on various cell types and models. In some embodiments, a system described herein comprises optical sensors (e.g., fibers) that detect, for example, fluorescence and / or absorbance, to achieve measurements of changes in, for example, oxygenation, calcium signaling, and pH.Attorney Docket No.: PCT.1313

[0188] In some embodiments, lung endothelial cells are plated on a surface (e.g., a surface of scaffold 32) of a system 20, 60, 80 described herein and grown to confluency. Cells are loaded with a membrane permeable fluorescent calcium indicator (e.g., FURA-2-AM) for approximately 30 minutes at 37 degrees Celsius. Cells are subsequently washed with fresh mediate remove any fluorescent indicator that has not entered the cells. To calibrate fluorescent measurements in order to correlate fluorescent signal with calcium concentration, cells are exposed to the ionophore ionomycin, to release calcium globally in each cell, establishing a fluorescent signal that is representative of the maximum amount of calcium within a cell. Subsequently, according to aspects of the present disclosure, cells may be exposed to calcium chelating EGTA and a detergent (e.g., Triton X-100) to lyse cells and establish a minimum fluorescent signal representing the absence of free calcium.

[0189] Optical sensors of a system 20. 60, 80 described herein measure the changes in fluorescence, optionally ratiometrically (e.g., using the ratio of two different fluorescent wavelengths, one wavelength representing calcium bound to the fluorescent indicator and the other wavelength representing fluorescent indicator unbound to calcium). Cells are then exposed to a range of environmental conditions (e.g., changes in pH, oxygen concentration) that represent disease forming conditions (e.g., hypoxia). In some embodiments, pump 30 and / or microvalves (e.g., micro check valves) as described herein may be used to regulate the exposure of the cells to the range of environmental conditions. For example, the pump 30 may be used to vary the pH of solutions that flow through the vasculature of the scaffold 32. In addition, the pressure driven system 80 of the present embodiments (e.g., in connection with the sealed lid 82, and the pneumatic passageways and connections 87, 92, 98) may be used to regulate the amount of oxygen present in the system 80. Changes in cytosolic calcium are measured in response to such stimuli, providing insight into the role environmental conditions play in cellular stress and the mechanisms cell types employ to mitigate such stress. A significant increase in calcium, above baseline, indicates a potential homeostatic response to stress. When cytosolic calcium is restored to baseline levels, the cell type is determined to maintain homeostasis in response to the environmental stimuli. If calcium levels continue to rise and do not restore to baseline levels, the environmental stimuli is determined to produce an irreversible disruption in cellular homeostasis.

[0190] Cells can also be loaded with a membrane permeable fluorescent pH probe (e.g., BCECF-AM, i.e., (2',7'-bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein, Acetoxymethyl Ester)) toAttorney Docket No.: PCT.1313perform the previously described experiment, but measuring changes in cytosolic pH in response to environmental stimuli.

[0191] According to the present disclosure, additional measurements may be made in response to environmental stimuli using gas sensors of a system described herein. In some embodiments, a system described herein achieves detection of volatile organic compounds and / or byproducts of cellular metabolism and / or environmental contamination.

[0192] In some embodiments, a system described herein comprises advanced biophotonic sensors. In some embodiments, a system 20, 60, 80 described herein achieves real-time measurements of cellular adhesion (e.g., cell to substrate interaction, cell to cell interaction), morphology (e.g., migration, motility), and molecular interactions (e.g., antibody interactions) using a biophotonic sensor (e.g., without the need for additional chemical probes and / or markers).

[0193] In some embodiments, a system 20. 60, 80 described herein achieves real-time measurements using surface plasmon resonance (SPR) sensors. A cancer cell (e.g., breast cancer cell, tumor cell) is plated on a surface described herein. A CAR T-cell, engineered to bind to the cancer cell is added to the system. The binding of the CAR T-cell to its target (e.g., a cancer cell) is assessed through the measurements of the refractive index, near the surface the cancel cells are adhered to (e.g., a surface of scaffold 32), which is directly proportional to the amount of mass bound to that surface. Such measurements, using a system provided herein, can provide insights into the efficacy of the CAR T-cell under examination, and whether it can achieve therapeutic effect (e.g., cytotoxicity of target cancer cell).ADDITIONAL EMBODIMENTS

[0194] Some additional non-limiting example embodiments are described below. It is to be understood that features of the various components, platforms, scaffolds, systems, and methods described herein can be used with one another. Various other combinations are contemplated in the present disclosure, as will be appreciated by the person of ordinary skill in the art, in view of the teachings of the present disclosure.

[0195] Embodiment 1. An integrated microfluidic well plate (IMWP) system comprising: a well plate frame comprising a plurality of wells, at least one first well of the plurality of wells comprising a reservoir;at least one scaffold disposed within at least one second well of the plurality of wells; andAttorney Docket No.: PCT.1313a microfluidic layer disposed beneath the well plate frame, the microfluidic layer fluidly coupling the reservoir to at least one neighboring reservoir and / or to the at least one scaffold via one or more microfluidic channels disposed within the microfluidic layer.

[0196] Embodiment 2. The system of Embodiment 1, further comprising a bottom seal layer attached to the bottom of the microfluidic layer for sealing microfluidic layer to the well plate frame.

[0197] Embodiment 3. The system of Embodiment 2, wherein the bottom seal layer transmits 90% or greater of light within the visible and / or near-infrared wavelengths therethrough, and wherein the bottom seal layer comprises a thickness in a range from about 25 pm to about 200 pm.

[0198] Embodiment 4. The system of Embodiment 2, wherein the bottom seal layer is formed from at least one of a cyclic olefin copolymer (COC), a cyclic olefin polymer (COP), a fluorinated ethylene propylene (FEP), and a polyethylene terephthalate (PET).

[0199] Embodiment 5. The system of any one of Embodiments 1-4, further comprising at least one pump, wherein the at least one pump is fluidly coupled to the one or more microfluidic channels.

[0200] Embodiment 6. The system of Embodiment 5, wherein the at least one pump comprises a rotational pump comprising at least one impeller blade.

[0201] Embodiment 7. The system of Embodiment 5, wherein the at least one pump comprises a magnet.

[0202] Embodiment 8. The system of Embodiment 5. wherein the at least one pump is disposed within the microfluidic layer, and the at least one pump comprises a piezoelectric pump.

[0203] Embodiment 9. The system of Embodiment 8, further comprising a non-circular hole within the microfluidic layer housing the at least one scaffold.

[0204] Embodiment 10. The system of Embodiment 7, further comprising a magnetic driving base disposed beneath the microfluidic layer, the magnetic driving base comprising one or more sets of driving coils,wherein each of the one or more sets of driving coils is disposed beneath a corresponding well of the at least one pump, the one or more sets of driving coils configured to actuate the corresponding pump.Attorney Docket No.: PCT.1313

[0205] Embodiment 11. The system of Embodiment 10, further comprising a bottom seal layer attached to the bottom of the microfluidic layer for sealing the microfluidic layer to the well plate frame, the bottom seal layer being disposed between the magnetic driving base and the microfluidic layer,wherein, in operation, at least one magnetic field from the one or more sets of driving coils traverses the bottom seal layer to actuate the at least one pump.

[0206] Embodiment 12. The system of any one of Embodiments 5-11, wherein the at least one pump comprises an outer diameter that is about 3-20% smaller than that of each well of the plurality of wells.

[0207] Embodiment 13. The system of any one of Embodiments 5-12, wherein the at least one pump operates at a frequency within a range from about 40 Hz to about 400 Hz.

[0208] Embodiment 14. The system of Embodiment 5, wherein the at least one pump comprises a piezoelectric actuator configured to operate such that it does not exceed a strain of greater than about 5%.

[0209] Embodiment 15. The system of Embodiment 5, wherein the at least one pump comprises a piezoelectric actuator configured to operate such that it does not exceed a total travel distance (or total strain) greater than about 50 pm.

[0210] Embodiment 16. The system of any one of the preceding Embodiments, wherein the microfluidic layer comprises at least one micro check valve fluidly coupled to the one or more microchannels.

[0211] Embodiment 17. The system of Embodiment 16, wherein the at least one micro check valve comprises a Tesla valve with a width in a range from about 0.1 mm to about 0.3 mm.

[0212] Embodiment 18. The system of Embodiment 16, wherein the at least one micro check valve comprises at least one of a thin membrane valve, a duckbill valve, and a passive micro check valve.

[0213] Embodiment 19. The system of any one of the preceding Embodiments, wherein the microfluidic layer has a width of about 1 mm to about 3 mm.

[0214] Embodiment 20. The system of any one of the preceding Embodiments, wherein the microfluidic layer is formed from at least one of a cyclic olefin copolymer (COC), a cyclic olefin polymer (COP), and a fluorinated ethylene propylene (FEP).

[0215] Embodiment 21. The system of any one of the preceding Embodiments, wherein the microfluidic layer is formed by injection molding and / or hot embossing.Attorney Docket No.: PCT.1313

[0216] Embodiment 22. The system of any one of the preceding Embodiments, wherein the one or more microfluidic channels comprise an inner diameter (or average inner diameter) within a range from about 50 pm to about 500 pm.

[0217] Embodiment 23. The system of any one of the preceding Embodiments, wherein the microfluidic channels are formed via voids within the microfluidic layer.

[0218] Embodiment 24. The system of any one of the preceding Embodiments, wherein the system has one or more features (e.g., spacing of the plurality of wells relative to each other) to meet, satisfy, or comply with one or more ANSI / SLAS Microplate Standards.

[0219] Embodiment 25. The system of any one of the preceding Embodiments, further comprising at least one sensor operably coupled thereto for sensing at least one parameter while the system is in use.

[0220] Embodiment 26. The system of Embodiment 25, wherein the at least one sensor comprises embedded platinum, carbon-based, indium tin oxide, gold, and / or iridium oxide electrodes.

[0221] Embodiment 27. The system of Embodiment 25, wherein the at least one sensor comprises a trans-endothelial electrical resistance (TEER) sensor, a barrier integrity sensor, a MEMS-based flow meter, a biophotonic sensor, a surface plasmon resonance (SPR) sensor, and / or a sensor composed at least partially of polyvinylidene fluoride (PVDF).

[0222] Embodiment 28. The system of any one of the preceding Embodiments, further comprising a sealing lid configured to interface with the well plate frame such that a fluidic seal is maintained within the reservoir.

[0223] Embodiment 29. The system of Embodiment 28, wherein the sealing lid comprises at least one pneumatic channel disposed therein, the at least one pneumatic channel fluidly coupling the reservoir to an external pressure source.

[0224] Embodiment 30. The system of Embodiment 29, wherein the at least one pneumatic channel is coupled to the external pressure source via a pneumatic interface comprising a microfilter.

[0225] Embodiment 31. The system of Embodiment 30, wherein the microfilter comprises a pore size of about 0.22 pm.

[0226] Embodiment 32. The system of Embodiment 30, wherein the at least one pneumatic channel comprises multiple pneumatic channels,wherein the pneumatic interface comprises multiple pneumatic interfaces, andAttorney Docket No.: PCT.1313wherein the external pressure source comprises multiple external pressure sources, each pressure source coupled to one of the multiple pneumatic channels via one of the multiple pneumatic interfaces.

[0227] Embodiment 33. The system of Embodiment 28, further comprising at least one sampling port disposed within a top surface of the sealing lid, wherein the at least one sampling port enables access to the reservoir.

[0228] Embodiment 34. The system of Embodiment 33, wherein the at least one sampling port comprises a cap-based sampling port comprising a sampling port cap disposed within a top surface of the cap-based sampling port.

[0229] Embodiment 35. The system of Embodiment 33, wherein the at least one sampling port is a septum-based sampling port comprising a septum layer disposed within a top surface of the septum-based sampling port.

[0230] Embodiment 36. The system of Embodiment 28, comprising at least one septum disposed within a top surface of the sealing lid.

[0231] Embodiment 37. The system of Embodiment 32, wherein the multiple external pressure sources comprise multiple pumps.

[0232] Embodiment 38. The system of Embodiment 28, further comprising at least one pneumatic channel disposed within the well plate, the at least one pneumatic channel fluidly coupling the reservoir to an external pressure source.

[0233] Embodiment 39. The system of Embodiment 38, wherein the at least one pneumatic channel is coupled to the external pressure source via a pneumatic interface disposed within a side surface of the well plate.

[0234] Embodiment 40. The system of any one of the preceding Embodiments, wherein the microfluidic layer is coupled to the at least one scaffold via a hosebarb coupling attached to the microfluidic layer that interfaces with a hosebarb disposed within a void disposed within a bottom surface of an overhang portion of the scaffold.

[0235] Embodiment 41. The system of any one of the preceding Embodiments, wherein the microfluidic layer is coupled to the at least one scaffold via an adhesive applied to the microfluidic layer that interfaces with a bottom surface of an overhang portion of the scaffold.

[0236] Embodiment 42. The system of any one of the preceding Embodiments, further comprising a seal clip disposed at least partially between the scaffold and the well plateAttorney Docket No.: PCT.1313frame, the seal clip being configured to place the scaffold within the well plate frame, and to subsequently remove the scaffold from the well plate frame.

[0237] Embodiment 43. The system of Embodiment 42, wherein the seal clip comprises two upper prongs, a mid portion, and two lower prongs, andwherein each of the two upper prongs comprises a hole disposed therethrough to facilitate removal of the seal clip and scaffold from the well plate frame.

[0238] Embodiment 44. The system of any one of the preceding Embodiments, wherein the scaffold is fully or at least partially formed from a hydrogel material, and wherein the scaffold is optionally formed via additive manufacturing.

[0239] Embodiment 45. The system of Embodiment 44, wherein the scaffold comprises at least one bio-printed passage disposed therethrough, the at least one bio-printed passage simulating a vasculature, andwherein the scaffold is seeded with at least one live cell.

[0240] Embodiment 46. The system of Embodiment 45, wherein the at least one bio-printed passage comprises a network of bioprinted passages arranged such that they define an interstitial space between the network of bioprinted passages and adjacent to at least a portion of the bioprinted passages, andwherein the at least one live cell is seeded within the interstitial space.

[0241] Embodiment 47. The system of any one of the preceding Embodiments, wherein the well plate comprises a 24-well plate.

[0242] Embodiment 48. The system of Embodiment 47, wherein the well plate comprises 12 units, wherein each unit comprises a first well used as the reservoir and a scaffold disposed within a second well, wherein the first well and the second well are fluidly coupled to one another.

[0243] Embodiment 49. The system of Embodiment 47, wherein the well plate comprises 8 units, wherein each unit comprises:a first well used as a primary reservoir;a scaffold disposed within a second well; anda third well used as an auxiliary reservoir,wherein the second well is selectively fluidly coupled to both the first well and the third well, andwherein the first well is selectively fluidly coupled to the third well.Attorney Docket No.: PCT.1313

[0244] Embodiment 50. The system of Embodiment 2, wherein the bottom seal layer comprises a semi-permeable film that enables oxygen transport thereacross.

[0245] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosures described herein. The scope of the present disclosures are not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

Attorney Docket No.: PCT.1313CLAIMS1. An integrated microfluidic well plate (IMWP) system comprising:a well plate frame comprising a plurality of wells, at least one first well of the plurality of wells comprising a reservoir;at least one scaffold disposed within at least one second well of the plurality of wells; anda microfluidic layer disposed beneath the well plate frame, the microfluidic layer fluidly coupling the reservoir to at least one neighboring reservoir and / or to the at least one scaffold via one or more microfluidic channels disposed within the microfluidic layer.

2. The system of claim 1, comprising a bottom seal layer attached to the bottom of the microfluidic layer for sealing microfluidic layer to the well plate frame.

3. The system of claim 2, wherein the bottom seal layer transmits 90% or greater of light within the visible and / or near-infrared wavelengths therethrough, andwherein the bottom seal layer comprises a thickness in a range from 25 pm to 200 pm.

4. The system of claim 2, wherein the bottom seal layer is formed from at least one of a cyclic olefin copolymer (COC), a cyclic olefin polymer (COP), a fluorinated ethylene propylene (FEP), and a polyethylene terephthalate (PET).

5. The system of claim 1, further comprising at least one pump, wherein the at least one pump is fluidly coupled to the one or more microfluidic channels.

6. The system of claim 5, wherein the at least one pump comprises a rotational pump comprising at least one impeller blade.

7. The system of claim 5, wherein the at least one pump comprises a magnet.

8. The system of claim 5, wherein the at least one pump is disposed within the microfluidic layer, and the at least one pump comprises a piezoelectric pump.Attorney Docket No.: PCT.13139. The system of claim 8. further comprising a non-circular hole within the microfluidic layer housing the at least one scaffold.

10. The system of claim 7, further comprising a magnetic driving base disposed beneath the microfluidic layer, the magnetic driving base comprising one or more sets of driving coils,wherein each of the one or more sets of driving coils is disposed beneath a corresponding well of the at least one pump, the one or more sets of driving coils configured to actuate the corresponding pump.

11. The system of claim 10, comprising a bottom seal layer attached to the bottom of the microfluidic layer for sealing the microfluidic layer to the well plate frame, the bottom seal layer being disposed between the magnetic driving base and the microfluidic layer, wherein, in operation, at least one magnetic field from the one or more sets of driving coils traverses the bottom seal layer to actuate the at least one pump.

12. The system of claim 5, wherein the at least one pump comprises an outer diameter that is 3-20% smaller than that of each well of the plurality of wells.

13. The system of claim 5, wherein the at least one pump operates at a frequency within a range from about 40 Hz to about 400 Hz.

14. The system of claim 5, wherein the at least one pump comprises a piezoelectric actuator configured to operate such that it does not exceed a strain of greater than 5%.

15. The system of claim 5, wherein the at least one pump comprises a piezoelectric actuator configured to operate such that it does not exceed a total travel distance (or total strain) greater than 50 pm.

16. The system of claim 1, wherein the microfluidic layer comprises at least one micro check valve fluidly coupled to the one or more microchannels.Attorney Docket No.: PCT.131317. The system of claim 16, wherein the at least one micro check valve comprises a Tesla valve with a width in a range from 0.1 mm to 0.3 mm.

18. The system of claim 16, wherein the at least one micro check valve comprises at least one of a thin membrane valve, a duckbill valve, and a passive micro check valve.

19. The system of claim 1, wherein the microfluidic layer has a width of 1 mm to 3 mm.

20. The system of claim 1, wherein the microfluidic layer is formed from at least one of a cyclic olefin copolymer (COC), a cyclic olefin polymer (COP), and a fluorinated ethylene propylene (FEP).

21. The system of claim 1, wherein the microfluidic layer is formed by injection molding and / or hot embossing.

22. The system of claim 1, wherein the one or more microfluidic channels comprise an inner diameter within a range from 50 pm to 500 pm.

23. The system of claim 1, wherein the microfluidic channels are formed via voids within the microfluidic layer.

24. The system of claim 1, wherein the plurality of wells are spaced out relative to each other to meet ANSI / SLAS Microplate Standards.

25. The system of claim 1, further comprising at least one sensor operably coupled thereto for sensing at least one parameter while the system is in use.

26. The system of claim 25, wherein the at least one sensor comprises embedded platinum, carbon-based, indium tin oxide, gold, and / or iridium oxide electrodes.

27. The system of claim 25, wherein the at least one sensor comprises a trans-endothelial electrical resistance (TEER) sensor, a barrier integrity sensor, a MEMS-based flow meter, a biophotonic sensor, a surface plasmon resonance (SPR) sensor, and / or a sensor composed at least partially of polyvinylidene fluoride (PVDF).Attorney Docket No.: PCT.131328. The system of claim 1, comprising a sealing lid configured to interface with the well plate frame such that a fluidic seal is maintained within the reservoir.

29. The system of claim 28, wherein the sealing lid comprises at least one pneumatic channel disposed therein, the at least one pneumatic channel fluidly coupling the reservoir to an external pressure source.

30. The system of claim 29, wherein the at least one pneumatic channel is coupled to the external pressure source via a pneumatic interface comprising a microfilter.

31. The system of claim 30, wherein the microfilter comprises a pore size of 0.22 pm.

32. The system of claim 30, wherein the at least one pneumatic channel comprises multiple pneumatic channels,wherein the pneumatic interface comprises multiple pneumatic interfaces, and wherein the external pressure source comprises multiple external pressure sources, each pressure source coupled to one of the multiple pneumatic channels via one of the multiple pneumatic interfaces.

33. The system of claim 28, comprising at least one sampling port disposed within a top surface of the sealing lid, wherein the at least one sampling port enables access to the reservoir.

34. The system of claim 33, wherein the at least one sampling port comprises a cap-based sampling port comprising a sampling port cap disposed within a top surface of the cap-based sampling port.

35. The system of claim 33, wherein the at least one sampling port is a septum-based sampling port comprising a septum layer disposed within a top surface of the septum-based sampling port.

36. The system of claim 28, comprising at least one septum disposed within a top surface of the sealing lid.Attorney Docket No.: PCT.131337. The system of claim 32, wherein the multiple external pressure sources comprise multiple pumps.

38. The system of claim 28, further comprising at least one pneumatic channel disposed within the well plate, the at least one pneumatic channel fluidly coupling the reservoir to an external pressure source.

39. The system of claim 38, wherein the at least one pneumatic channel is coupled to the external pressure source via a pneumatic interface disposed within a side surface of the well plate.

40. The system of claim 1, wherein the microfluidic layer is coupled to the at least one scaffold via a hosebarb coupling attached to the microfluidic layer that interfaces with a hosebarb disposed within a void disposed within a bottom surface of an overhang portion of the scaffold.

41. The system of claim 1, wherein the microfluidic layer is coupled to the at least one scaffold via an adhesive applied to the microfluidic layer that interfaces with a bottom surface of an overhang portion of the scaffold.

42. The system of claim 1, further comprising a seal clip disposed at least partially between the scaffold and the well plate frame, the seal clip being configured to place the scaffold within the well plate frame, and to subsequently remove the scaffold from the well plate frame.

43. The system of claim 42, wherein the seal clip comprises two upper prongs, a mid portion, and two lower prongs, andwherein each of the two upper prongs comprises a hole disposed therethrough to facilitate removal of the seal clip and scaffold from the well plate frame.

44. The system of claim 1, wherein the scaffold is at least partially formed from a hydrogel material.Attorney Docket No.: PCT.131345. The system of claim 44, wherein the scaffold comprises at least one bio-printed passage disposed therethrough, the at least one bio-printed passage simulating a vasculature, andwherein the scaffold is seeded with at least one live cell.

46. The system of claim 45, wherein the at least one bio-printed passage comprises a network of bioprinted passages arranged such that they define an interstitial space between the network of bioprinted passages and adjacent to at least a portion of the bioprinted passages, andwherein the at least one live cell is seeded within the interstitial space.

47. The system of claim 1, wherein the well plate comprises a 24-well plate.

48. The system of claim 47, wherein the well plate comprises 12 units, wherein each unit comprises a first well used as the reservoir and a scaffold disposed within a second well, wherein the first well and the second well are fluidly coupled to one another.

49. The system of claim 47, wherein the well plate comprises 8 units, wherein each unit comprises:a first well used as a primary reservoir;a scaffold disposed within a second well; anda third well used as an auxiliary reservoir,wherein the second well is selectively fluidly coupled to both the first well and the third well, andwherein the first well is selectively fluidly coupled to the third well.

50. The system of claim 2, wherein the bottom seal layer comprises a semi-permeable film that enables oxygen transport thereacross.

Images