RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 63/325,132, filed March 29, 2022, the contents of which are incorporated herein in their entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to a modular microphysiological system including at least one well and tissue-specific niches, each having a separate microenvironment for culturing tissues in the integrated modular microphysiological system.

GOVERNMENT FUNDING

[0003] The present disclosure is made with government support under 75A50121C00017 awarded by Biomedical Advanced Research and Development Authority (BARDA). The government has certain rights in the present disclosure.

BACKGROUND

[0004] Cell cultures are useful for rapid testing of drug candidates at a cellular level, but are not always predictive of their efficacy and/or side effects in human patients. This may be due in part by poor recapitulation of the original pathophysiological milieu and growth conditions, including 2D vs. 3D-growth, lack of extracellular matrix (ECM), lack of surrounding cells (stromal, vascular, immune), as well as lack of molecular signals and physical constraints. For example, tumorigenesis strongly depends on cancer cell interactions with the environment. Animal models provide a better physiological environment, but are laborious, expensive, and do not support fine- grain control of exogenous factors. Over the last decade, bioengineering has entered the field of cancer research, by introducing more physiologically relevant 3-dimensional (3D) models of cancer growth such as tumor spheroids and organoids, vascularization and scaffolds. However, these models fail to recapitulate critical aspects of human pathophysiology, such as the interactions of the tumor compartment with other cells (paracrine interactions) and organs (endocrine interactions). In addition, no currently available bioengineered model is able to model the full complexity of tumor progression, from primary tumor growth to intravasation in the blood stream, and seeding a distal organ site. A human tissue model that could accurately model these aspects of metastatic progression would be transformative to cancer research.

[0005] There is a need to understand human biology and system-wide pathology so we may predict which drugs may work for which patients before clinical trials. While this need is partly addressed using in vitro and animal models for most therapeutic areas, their lack of utility in modeling systemic diseases significantly prohibits the development of drugs for many diseases affecting more than one tissue system. [0006] Cellular cultures that mimic real tissues may extrapolate findings that advance scientific and medical knowledge. However, a culture of cells is not identical to an organ in the body and needs constant attention by researchers to ensure they do not lose viability. Maintenance of cellular cultures and their development may be time-consuming and costly.

[0007] Thus, there is a need for a user-friendly and robot-friendly connected multi-organ system for modeling of systemic disease to allow cell cultures to mimic the systemic environment for cell culture better, while using existing, standardized components, such as a standard pipette, thereby allowing for easier maintenance and scalability of the cultures.

SUMMARY

[0008] According to an aspect of the present disclosure, a modular system configured for culturing biological cells is provided. The modular system includes a pipette configured to exchange media, the pipette including a base and one or more shaft extending from the base, the base being configured to be manipulated by one or more of a hand of a user and a robotic device, the shaft being configured to be transitioned between a disengaged state and an engaged state; one or more transport vessel configured to carry tissue cells, the transport vessel including a body extending between a first end and a second end, the first end of the body defining an opening in communication with a chamber defined by the body, the opening and the chamber being configured to receive at least a portion of the shaft of the pipette, the chamber being configured to frictionally engage the shaft to transition the shaft from the disengaged state toward the engaged state, the second end of the body being configured to allow the transport vessel to be transitioned between a dismounted position and a mounted position; and a platform including a mounting surface, the mounting surface being configured to receive the transport vessel to secure the transport vessel in the mounted position, and wherein the pipette is capable of moving the transport vessel between the dismounted position and the mounted position when the shaft is in the engaged state.

[0009] According to the present disclosure, the chamber of the body of the transport vessel may be substantially cylindrical.

[0010] According to the present disclosure, the chamber of the body of the transport vessel may have a diameter within a range of 1 mm and 9 mm.

[0011] According to the present disclosure, the transport vessel may include a support extending from the body of the transport vessel and the support may be configured to carry a permeable membrane or one or more electrode.

[0012] According to the present disclosure, the second end of the body of the transport vessel may include a first coupling member, the mounting surface of the platform may include a second coupling member, and the transport vessel may be in the mounted position when the first coupling member of the transport vessel is coupled to the second coupling member of the platform.

[0013] According to the present disclosure, the modular system may include an insert including a body extending between a first end and a second end, the first end of the body of the insert may define an opening in communication with a cavity defined by the body of the insert, and the opening and the cavity of the body of the insert may be configured to receive at least a portion of the transport vessel to transition the transport vessel from the dismounted position toward the mounted position.

[0014] According to the present disclosure, the mounting surface of the platform may be configured to receive the insert to secure the insert on the mounting surface.

[0015] According to the present disclosure, the second end of the body of the transport vessel may include a first coupling member, the body of the insert may include a second coupling member in the cavity defined by the body of the insert, and the transport vessel may be in the mounted position when the first coupling member of the body of the transport vessel is coupled to the second coupling member of the body of the insert and the insert is secured on the mounting surface of the platform.

[0016] According to the present disclosure, the platform may include a flow channel, the second end of the body of the insert may define an aperture, and the aperture may be in communication with the flow channel when the insert is secured to the mounting surface of the platform.

[0017] According to the present disclosure, the insert may include a permeable membrane extending across at least a portion of the aperture.

[0018] According to the present disclosure, the modular system may include a plurality of transport vessels and a plurality of shafts may extend from the base of the pipette, and each opening and chamber of each transport vessel of the plurality of transport vessels may be configured to receive a corresponding shaft of the plurality of shafts.

[0019] According to the present disclosure, a space may be defined between each transport vessel of the plurality of transport vessels and an adjacent transport vessel of the plurality of transport vessels when the plurality of transport vessels are in the mounted position and the space may have a length within a range of 4.5 mm to 9 mm.

[0020] According to the present disclosure, the pipette may include an ejector configured to displace the transport vessel to transition the shaft of the pipette from the engaged state toward the disengaged state. [0021] According to the present disclosure, the tissue cells may correspond to an assembly of one or more type of human cells.

[0022] According an aspect of the present disclosure, a method of transporting tissue cells within a system for culturing biological cells is provided. The method includes providing tissue cells within a transport vessel including a body extending between a first end and a second end, the first end of the body defining an opening in communication with a chamber defined by the body, inserting one or more shaft extending from a base of a pipette through the opening and into the chamber of the transport vessel until at least a portion of the shaft is frictionally engaged with the chamber, moving the transport vessel from a dismounted position toward a mounted position on a mounting surface of a platform, and disengaging the shaft of the pipette from the chamber of the transport vessel when the transport vessel is in the mounted position on the mounting surface of the platform.

[0023] According to an aspect of the present disclosure, a transport vessel configured for use within a modular system for culturing tissue cells is provided. The transport vessel is configured to carry tissue cells. The transport vessel includes a body extending between a first end and a second end, the first end of the body defining an opening in communication with a chamber defined by the body, the opening and the chamber being configured to receive at least a portion of a shaft of a pipette, the chamber being configured to frictionally engage the shaft, the second end of the body being configured to allow the transport vessel to be transitioned between a dismounted position and a mounted position within the modular system, and wherein the transport vessel is capable of being moved from the dismounted position toward the mounted position when the chamber of the transport vessel is frictionally engaged with the shaft of the pipette.

[0024] According to the present disclosure, the chamber of the body of the transport vessel may be substantially cylindrical.

[0025] According to the present disclosure, the chamber of the body of the transport vessel may have a diameter within a range of 1 mm and 9 mm.

[0026] According to the present disclosure, the transport vessel may include a support extending from the body of the transport vessel, wherein the support may be configured to carry a permeable membrane or one or more electrode.

[0027] According to an aspect of the present disclosure, a modular system configured for culturing biological cells is provided. The modular system includes a platform including at least one open well for containing culture media and a plurality of tissue-specific modules therein, a fluid circulation loop including an on-board pump and a fluid channel in fluid communication with the plurality of tissue-specific modules configured to circulate vascular fluid through the tissue- specific modules, wherein each of the tissue-specific modules is separated from other tissuespecific modules by a porous membrane in the fluid channel; a lid configured to engage the platform, the lid having at least one opening aligned with the at least one well to allow insertion of a tip of a pipette into the at least one well, wherein the base, the lid, or combinations thereof include at least one sensor, electrical connectivity configured to receive power from an external source, communication of data to remote devices, on-board electronic circuits, and processors or controllers configured to control tissue culture functions, sensing, and communication, wherein each of the plurality of tissue-specific modules includes a transport vessel including a first end configured to releasably engage with a shaft of a pipette and a second end configured to releasably engage with a mounting surface for the transport vessel proximate a bottom of the at least one open well, and a support configured to bear a tissue in the at least one well in contact with the vascular fluid, and wherein each of the plurality of tissue-specific modules is configured to be individually transferred into or out of the at least one well when a pipette is releasably engaged to the first end of the transport vessel.

[0028] According to the present disclosure, the at least one well may include a plurality of fluid reservoir compartments, each compartment may include a well configured to be in fluid communication with the circulation loop through the permeable membrane, and the mounting surface for the transport vessel.

[0029] According to the present disclosure, each of the plurality of fluid reservoir compartments may be configured to be individually transferred into or out of the at least one well when a pipette is releasably engaged with the mounting surface for the transport vessel.

[0030] According to the present disclosure, a set of the plurality of the fluid reservoir compartments may be aligned in the platform so that a set of tips of a multi-channel pipette may be capable of transferring fluid into or out of each of the set of fluid reservoir compartments.

[0031] According to the present disclosure, the lid may include a plurality of openings aligned with the plurality of fluid reservoir compartments to provide fluid transfer into or out of each of the plurality of the fluid reservoir compartments.

[0032] According to the present disclosure, the lid may include at least one sensor module configured to dip into at least one of the fluid reservoir compartments.

[0033] According to the present disclosure, the pump may be a piezoelectric pump.

[0034] According to the present disclosure, the platform may have a footprint commensurate to that of a 96-well culture plate.

[0035] According to the present disclosure, two or more platforms may be linked together via a daisy-chained common bus. [0036] According to the present disclosure, the modular system may include an endothelial barrier layer disposed on the permeable membrane between tissue-specific modules.

[0037] According to the present disclosure, the modular system may include at least one tissue in at least one tissue-specific module inserted into the at least one well, wherein the at least one tissue may be selected from a group including bulk tissues, such as one or more of sections of bone matrix, cellular aggregates, cells in hydrogels, and cellular monolayers; tissues needing electromechanical stimulation, such as cardiac tissues; barrier tissues, such as one or more of lung, gut, and skin tissues; or combinations thereof.

[0038] According to the present disclosure, the tissue-specific module may be configured to support a bulk tissue and the transport vessel may include a side extension configured to extend the support to a side of and below the second end of the transport vessel into an open well when the second end of the transport vessel is engaged with the mounting surface, wherein the support may include a tray with a perimeter wall and a porous membrane disposed on a bottom of the perimeter wall to support the bulk tissue.

[0039] According to the present disclosure, the tissue-specific module may be configured to support a tissue needing electromechanical stimulation and the transport vessel may include two flexible pillars configured to support a hydrogel with embedded cells between the pillars, electrodes disposed outboard of the pillars configured to provide cyclic electrical stimulation to the cells, and electrical connectors configured to be in electrical connectivity with electric circuits controlled by microcontrollers to provide power to the electrodes, wherein the flexible pillars and the electrodes may be configured to extend into an open well when the second end of the transport vessel is engaged with the mounting surface.

[0040] According to the present disclosure, the tissue-specific module may be configured to support a barrier tissue and the transport vessel may include an upper well and a lower volumetric compartment bounded by a porous membrane and open on a bottom, wherein a horizontal portion of the porous membrane may be configured to support the barrier tissue in the upper well and the lower volumetric compartment may be configured to extend into an open well when the second end of the transport vessel is engaged with the mounting surface.

[0041] According to an aspect of the present disclosure, a transport vessel configured to be inserted into an open well of a modular system configured for culturing biological cells is provided. The transport vessel includes a body defining a chamber extending between a first end configured to releasably engage with a shaft of a pipette and a second end configured to releasably engage with a mounting surface for the transport vessel proximate the open well, wherein the transport vessel is configured to be transferred into or out of the open well when a pipette is releasably engaged within the chamber.

[0042] According to an aspect of the present disclosure, the transport vessel may be a tissuespecific module configured to support a bulk tissue and the transport vessel may include a side extension configured to extend from the chamber to a support to the side of and below the second end of the chamber into the open well when the second end is engaged with the mounting surface, wherein the support may include a tray with a perimeter wall and a porous membrane disposed on a bottom of the perimeter wall to support the bulk tissue.

[0043] According to an aspect of the present disclosure, the transport vessel may be a tissuespecific module configured to support a tissue needing electromechanical stimulation and the transport vessel may include two flexible pillars configured to support a hydrogel with embedded cells between the pillars, electrodes disposed outboard of the pillars configured to provide cyclic electrical stimulation to the cells, and electrical connectors configured to be in electrical connectivity with electric circuits controlled by microcontrollers to provide power to the electrodes, wherein the flexible pillars and the electrodes may be configured to extend into the open well when the second end of the transport vessel is engaged with the mounting surface.

[0044] According to an aspect of the present disclosure, the transport vessel may be a tissuespecific module configured to support a barrier tissue and the transport vessel may include an upper well and a lower volumetric compartment bounded by a porous membrane and open on a bottom, wherein a horizontal portion of the porous membrane may be configured to support the barrier tissue in the upper well and the lower volumetric compartment is configured to extend into the open well when the second end of the transport vessel is engaged with the mounting surface. [0045] According to an aspect of the present disclosure, the transport vessel may be a stimulation module configured to provide electromechanical stimulation to tissues and the transport vessel may include electrodes configured to provide cyclic electrical stimulation to the cells and electrical connectors configured to be in electrical connectivity with electric circuits controlled by microcontrollers to provide power to the electrodes, wherein the electrodes may be configured to extend into the open well when the second end of the transport vessel is engaged with the mounting surface.

[0046] According to an aspect of the present disclosure, the transport vessel may be a sensor module and the transport vessel may include a sensor disposed below the transport vessel configured to extend into the open well when the second end of the transport vessel is engaged with the mounting surface and electrical connectors configured to be in electrical connectivity with electric circuits controlled by microcontrollers to control the sensors and receive information therefrom.

[0047] According to an aspect of the present disclosure, a method for culturing at least one tissue is provided. The method includes providing a modular system according to any aspect of the present disclosure provided herein, disposing a first tissue into a first tissue-specific transport vessel for supporting the first tissue and optionally disposing additional tissues into additional tissue-specific transport vessels for supporting the additional tissues, inserting the first tissuespecific transport vessel and optional additional tissue-specific transport vessels into at least one well of the platform of the system, loading a vascular fluid or culture medium into the fluid circulation loop of the system, circulating the vascular fluid or culture medium through the first tissue-specific transport vessel and optional additional tissue-specific transport vessels.

[0048] According to the present disclosure, the method may include loading endothelial cells into the vascular fluid or culture medium, thereby forming an endothelial barrier layer on the permeable membrane.

[0049] In the manner described and according to aspects illustrated herein, the structures and/or relationships referred to above are capable of providing a user-friendly and robot-friendly connected multi-organ system for modeling of systemic disease to allow cell cultures to mimic the systemic environment for cell culture better, while using existing, standardized components, such as a standard pipette, thereby allowing for easier maintenance and scalability of the cultures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] A detailed description of various aspects, features and embodiments of the subject matter described herein is provide with reference to the accompanying drawings, which are briefly described below. The drawings are illustrative and not necessarily drawn to scale, with some components being exaggerated for clarity. The drawings illustrate various aspects and features of the present subject matter and may illustrate one or more embodiment(s) or example(s) of the present subject matter in whole or in part. Together with the description, the drawings serve to explain the principles of the disclosed subject matter.

[0051] Figs. 1A-1 F show aspects of a platform of a system that enables maintenance of tissue-specific niches while allowing for tissue cross-talk, in accordance with aspects of the disclosure.

[0052] Figs. 2A-2B show aspects of a smart lid of the system of Figs. 1 A-1 F.

[0053] Figs. 3A-3B show aspects of fluid transfer into/out of the system of Figs. 1 A-1 F.

[0054] Figs. 4A-4B show a schematic depiction of the operation of an on-board piezoelectric pump of the system of Figs. 1A-1F. [0055] Figs. 5A-5B show aspects of electrical connectors for use in the system of Figs. 1A- 1 F.

[0056] Figs. 6A-6D show aspects of tissue-specific tissue transport vessels of the system of Figs. 1A-1 F.

[0057] Fig. 7 shows aspects of a tissue-specific tissue transport vessel for supporting bulk tissues in the system of Figs. 1A-1 F.

[0058] Figs. 8A-8B show aspects of a tissue-specific tissue transport vessel for supporting tissues needing electromechanical stimulation in the system of Figs. 1A-1 F.

[0059] Figs. 9A-9D show aspects of a tissue-specific tissue transport vessel for supporting barrier tissues in the system of Figs. 1A-1 F.

[0060] Figs. 10A-10F show aspects of a platform of an alternative configuration of the system of 1A-1F, which enables maintenance of tissue-specific niches while allowing for tissue crosstalk, in accordance with aspects of the disclosure.

[0061] Figs. 11A-11D show aspects of a vascular insert for use in the system of Figs. 10A- 10F.

[0062] Figs. 12A-12E show aspects of transport vessels for use in the system of Figs. 10A- 10F.

[0063] Figs. 13A-13B shows aspects of storage of the systems of 1A-1 F and/or 10A-10F in automated microwell equipment, in accordance with aspects of the disclosure.

DETAILED DESCRIPTION

[0046] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the as aspects disclosed and claimed herein. In this description, the use of the singular includes the plural, the words “a” or “an” mean “at least one,” and the use of “or” means “and/or,” unless specifically stated otherwise. Furthermore, the use of the term “including,” as well as other forms, such as “includes” and “included” is not limiting. Also, terms such as “element” or “component” encompass both elements or components including one unit and elements or components that include more than one unit unless specifically stated otherwise. The use of the term “or” in the claims and the present disclosure is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0047] Use of the term “about,” when used with a numerical value, is intended to include +/- 10%. For example, if a number of amino acids is identified as about 200, this would include 180 to 220 (plus or minus 10%). [0048] The terms “patient,” “individual,” and “subject” are used interchangeably herein, and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

[0049] Bioengineered tissue systems offer a new paradigm for modeling human pathophysiology and testing drug efficacy and safety. However, establishing physiological communication between multiple tissues, while also preserving their individual phenotypes, is a major challenge due to the conflicting requirements for maintaining each tissue-specific regulatory niche. Herein, a biomimetic bioreactor system, in which each tissue may be cultured in its own specific optimized environment within the system, is described. Each environment is a specific tissue niche that is separated by a selectively permeable endothelial barrier from recirculating flow containing circulating monocytes. The specific tissues, however, are linked by vascular perfusion in the system. The tissues maintain their molecular, structural, and functional phenotypes over four weeks of culture. The system, thus, defines a plurality of bioengineered human tissue niches that are linked by vascular perfusion to enable recapitulated effects and biomarkers of multi-organ drug toxicities. In the system described herein, multiple tissues may be physiologically connected without sacrificing their individual biological fidelity. Overall, the bioreactor system allows systemic level tissue communication, maintains the engineered tissue phenotypes, and may thereby facilitate clinical translation.

[0050] Other approaches are unable to maintain individual tissue health and functionality, gene expression, proteomics, and/or drug responses when connecting multiple tissues together. For example, current methods rely on either transferring supernatant between tissues or the use of “common media” containing the factors required by all tissues. In contrast, the organs in our body maintain their own environments while being linked by vasculature lined with selectively permeable endothelium.

[0051 ] Notably, the system is configured according to established standards, allowing it to be used with standardized equipment for cell culture, facilitating automated tissue culture that more closely mimics in vivo tissue growth and maintenance.

[0052] Generally, aspects of the disclosed subject matter provide a highly advanced “patient on a chip” model that uses vascular perfusion to physiologically integrate target tissues. In aspects, the subject matter provides a system with bioengineered human tissue niches linked by vascular perfusion that may recapitulate effects and biomarkers of multi-organ drug toxicities. The system allows for systemic level tissue communication, maintains the engineered tissue phenotypes, and may thereby facilitate clinical translation. The system provides a human, systemic model of patient specific disease “on-a-chip” or “in-a-dish,” benefiting drug developers, patients, clinicians, and the healthcare economy.

[0053] In aspects, a microwell tissue culture system for engineered or patient-sourced tissues configured to established standards to allow for integration within manual and automated applications is provided. The system includes a platform having footprint similar to that of a microwell cellular culture plate. Conditions offered by the system allow for development of tissue cultures to simulate organs and in vivo conditions. The system incorporates standardized dimensions and innovative design for culture media flow and loading of cellular samples. Components within the system may be loaded and maintained by a human or robot, allowing for rapid introduction of cultures to be grown and maintained in a relevant environment. Once they are mature, the system may be used to test the effects of a variety of stimuli, including potential therapeutics.

[0054] The system’s footprint may conform to microwell plate standards (ANSI/SUKS) to allow the system to be readily integrated with a wide array of robotic and analytical equipment. Usability and adherence to standards provides compatibility with a wide range of equipment and does not require capital expenditure to use. Electrical connections and on-board signal routing may enable connection to a wide array of sensors and actuators that may be located within the system. Specific features for loading cells, priming flow paths, and transferring tissues robotically may also be incorporated. The specific configuration and placement of tissue reservoirs enables rapid sampling or media exchange with standard multi-channel pipettes. Specialized tissue-specific reservoirs support a variety of cellular types and their environments. The system may be used manually for research and investigative work but is also automation-friendly for use in higher throughput quality controlled environments.

[0055] The system may include “smart features,” such as on-board electronics, sensing, and communication of data to remote devices. A “smart lid” may provide a means to make electrical connections to the platform and additional space to locate various integrated circuits or microcontrollers.

[0056] On-board pumping may reduce burden on the user and faciliate robotic automation by eliminating a need for an external pump. In aspects, on-board piezoelectric diaphragm pumps may provide for circulation of fluids among a plurality tissue-specific compartments.

[0057] The platform is configured to allow for integration of an endothelial barrier between tissue-specific compartments. It provides increased automation compared to previous systems by the ability for simultaneous media exchange to/from multiple compartments using multi- channel (8- or 16-channel) multi-channel pipettes. These and other configurations described herein allow for rapid testing and development of therapeutics.

[0058] In aspects, a modular system (also referred to herein as a “bioreactor system’’) is provided. The system may be configured according to ANSI/SLAS standards so that it may be adaptable to and operated using existing cell culture infrastructure. The system may have dimensions and configurations similar to standard microwell culture plates. The system includes a platform with at least one open well for containing culture media and tissue-specific modules therein and a porous membrane integrated into the at least one well by, for example thermal bonding/welding using heat, ultrasound, or laser, and fluid circulation loop(s) for circulating vascular fluid through the tissue-specific modules. A lid may be configured with at least one opening aligned with the at least one well to allow insertion of a standard pipette tip into the at least one well. The open-well configuration, as opposed to an enclosed microfluidic network, allows for a user or robot to interface with the system easily for handling of liquid media, transfer of cells or tissues, insertion of physical sensors, easy cleaning, etc. For example, the base may include a plurality of compartments for tissue-specific cell culture and a plurality of openings in the lid to allow for loading/seeding of a vascular barrier by pipette and rapid sampling using multichannel pipettes. The platform, the lid, or a combination thereof may include electrical connectors, circuits, controllers, sensors, etc. for “smart” functions for culturing tissues. “Cordless” connections of electrical power and signals using magnetic connectors may be incorporated into the base and/or lid. Sensors for optical sensing (fluorescence, colorimetric) or other properties may be integrated in the base and/or lid or inserted into the compartments using pipette-adapted sensor modules. Microcontroller(s) for control of pump, stimulation of tissues, and integration with on-board sensing may be integrated into the platform, the lid or a combination thereof. For example, a “smart lid” provides for making electrical connections to the platform, and additional space to locate various integrated circuits or microcontrollers. The smart lid may also provide a modular structure for locating sensors that may be dipped into the well(s) in the platform. Through-holes may be placed above desired sampling areas so medium may be exchanged with the lid installed using multi-channel pipettes.

[0059] Individual systems may be linked for multi-system control, power, and communication via adaisy-chained common bus enabling data logging, control of embedded programs, user interface, etc.

[0060] The system may be configured to provide on-board pumping of fluids using diaphragm pumps, such as piezoelectric pumps, through the fluid circulation loop. [0061 ] A set of modular tissue transport components enables handling of tissues with unique requirements. The transport components may be installed/removed on demand with human or robotic interface. Tissues may be transferred manually or robotically. The tissue-specific modules feature automation-friendly transfer of the tissue-specific modules into and out of the wells, including transport vessels configured to engage a standard pipette. The body of the transport vessel is modeled after a pipette shaft so that a standard pipette may engage, transfer (and eject) a tissue from one location to another. For example, different tissue may follow different timelines for maturation, but need to be connected and/or integrated for an experiment in a multi-tissue chip in the system. The tissue compartments in the platform of the system may be designed for different tissue types, such as electrically excitable tissues (any), barrier tissues (lung, gut, skin, etc.), bulk aggregates (liver, bone), and/or bulk tissues (sections of bone matrix, cellular aggregates, cells in hydrogels, and/or monolayers).

[0062] Applications of cell culture using the system disclosed herein include various tissue models (barrier tissues, monolayer, multi-layer, organoid or air/liquid, parenchymal/bulk, uniqueneeds tissues (e.g. stimulation)). Specific cell or tissue types include cardiac, lung, bone, liver, skin, gut, etc. Cell sources may be universal or patient-specific and may be primary or induced pluripotent stem (IPS) cells. Cell culture may be conducted for raising multiple tissue samples of a common type under identical conditions, or studying mixed tissue samples having a common vasculature while maintaining individual tissue integrity. For example, the system may be used for rapid, high volume screening, dosing or early clinical trials suing multiple tissues for “organ- on-a-chip” (OOC) concepts. The system provides for human-tissue culture enabling studies of systemic disease including aspects of metastasis, immune conditions, biologies, fibrosis, infectious diseases, aging, potency assays (cell therapy, exosomes, antibodies, vaccines), etc.

[0063] The system enables: 1) maintaining biological fidelity of tissues (engineered or patient derived) over long and short term culture times, 2) maintaining tissues in their niche while allowing communication via secreted factors, 3) allow cells and biologies (secreted factors, exosomes) to move between compartments, 4) allow circulating cells to preferentially extravasate from the vasculature into tissues as biologically appropriate (metastasis to expected tissue site, immune infiltration into damaged tissues, cell therapy), 5) drug studies in the integrated platform that has more clinical relevance as an integrated system versus the sum of its parts, qualifying use as a clinical human correlate for disease modeling and drug testing.

[0064] The system allows for versatility in connecting millimeter-sized engineered tissues via vascular perfusion, with each tissue in an individual, optimized microenvironment, while still allowing for natural, selectively permeable cytokine and cellular cross-talk across a vascular endothelium separating the tissue and perfusion spaces. In aspects, the system is modular and various different tissues, organoids, or patient biopsies may be combined as desired. In aspects, the system enables multiple tissues to be plugged directly into the platform of the system, which is configured to receive the tissues, to enable multi-tissue studies in a way that tissues are connected through a vascular network, while maintaining tissue-specific niches for enhanced functionality, transcriptomics, and clinically relevant drug responses. Additionally, the ability to include human immune interactions and cellular movement from one compartment to another, as within the human body, is a major innovation facilitating a translational impact of the system.

[0065] Referring to Fig. 1A-1 D, the bioreactor system 1 is a configurable, “plug-and-play” modular system including a main platform 10 configured to hold a plurality of tissue-specific niches (not shown) in individual compartments connected by a common vasculatory circulation system. Fig. 1A shows a perspective view of the platform 10, which includes a base 11 and a perimeter wall 12 extending above the base 11. The platform 10 may have a footprint that conforms to microwell plate standards (ANSI/SLAS) to allow the system 1 to be readily integrated with a wide array of robotic and analytical equipment. Following these standards (size, footprint, location of wells, distance of key surfaces, and associated tolerances) enables the system 1 to have compatibility with a wide range of analytical and robotic equipment (e.g. microscopy, plate readers, plate handlers, liquid handlers, robotic incubators, etc.) that are configured according to the ANSI/SLAS standards. Providing the platform 10 according the ANSI/SLAS standards allows for use of the platform 10 by humans and robots using existing and readily available equipment. The system 1 shown in Figs. 1A-1 F includes four “organ-on-a-chip” modules in a footprint of a standard 96-well plate.

[0066] The platform 10 may have a rectangular shaped base 11 including a plurality of compartments (may also be referred to herein as “wells”) 13 defined by sidewalls. Each compartment 13 has a bottom surface and sidewalls that separate the tissue-specific niche environments from each other. The compartments 13 are configured to receive a “plug” or insertable tissue-specific devices (tissue-specific transport vessels 60, described further below) and thereby define separated, tissue-specific niches of the system 1. Bottom surfaces of the compartments 13 include an opening to allow selective flow of vascular media into the tissuespecific niche environments. In aspects, the compartments 13 may be configured in four groups of four compartments 13. Fourfluid circulation loops 14 including tubing segments 15 and 16 and pumps 17 may be included in the system 1 , each of which may be configured to circulate fluids (e.g. vascular media) through a set or group of four compartments 13. Each circulation loop 14 continues on the bottom of the base 11, as described in relation to Figs. 1 B-1 E. Each group of compartments 13 and associated fluid circulation loop 14 includes an “organ-on-a-chip” module. Fig. 1 B shows a close-up cross-section of an individual “organ-on-a-chip” module. A tube 15A and channel 18A below the compartments 13 include a portion of a circulation loop 14 for a first module. Tube 16B and pump 17B include a portion of a circulation loop 14 for a second module. [0067] Fig. 1C shows a top view of the platform 10. Channel 18 runs below a group of tissue compartments 13 to complete the circulation loop 14. The bottom surfaces of the compartments 13 include projections 20 for receiving the insertable tissue-specific devices. To this end, the bottom surface of each compartments 13 and, thus, the platform 10, is also referred to herein as a “mounting surface.” Apertures 21 at the bottom of the compartments 13 are in fluid communication with channel 18. A porous membrane 29 is disposed between the compartments 13 and channel 18. In aspects, the porous membrane 29 is fabricated from a durable porous material such as track-etched polycarbonate or poly(ethylene terephthalate) (PET) having a pore size of about 5 to 30 pm, such as 20 pm. A second set of openings 22 at the bottom of the compartments 13 provide a viewing window into the compartments 13 for observing the tissue cultures in the individual tissue compartments. Two types of tissue compartments 13 are shown. A first type 13A has larger dimensions than type 13B and does not include an aperture 21 to provide for inclusion of a port 23 for delivering fluid and cells into the vascular fluid circulation loop 14. Port 23 is configured to compressively seal with a tip 39 of a pipette 38, 68. The pipette 38, 68 may deliver cells (e.g. endothelial cells) into the channel 18 so the cells may attach to the porous membrane 29 and form a confluent layer as the cells attach. Optionally, a second port 24 may be provided for the pipette 38, 68 to deliver medium into the circulation loop 14 in order to "prime" a pump (i.e., push air out of the circulation loop 14). Fig. 1 D shows a close-up view of a portion of the base 11 to show details of the features of an upper surface of the base 11 .

[0068] Fig. 1 E shows a bottom view of the platform 10. Porous membrane 29 may be thermally welded (by heat, ultrasonic, or laser welding) onto the bottom of the thermoplastic platform 10 to provide a manifold below each of the apertures 21 including a common medium flow channel that shares a porous boundary with multiple tissue-specific wells. A thin, transparent (e.g. glass or plastic) cover plate 26 may be attached to the bottom of platform 10 by an adhesive layer 25 to seal the bottom of the vascular flow channel 18, with glass viewing windows for observation of an endothelial barrier layer established on the bottom of porous membrane 29. Optionally, heat and pressure may be applied during the assembly of the transparent surface so that a raised thermoplastic border on apertures 21 melts and forms an impermeable barrier, preventing fluid contact with any adhesive. In embodiments, plate 26 is melt- sealed to the bottom of platform 10 to provide a barrier to contain the fluids in the apertures 21 of the platform 10. Fig. 1 F shows a close-up view of a portion of the platform 10 to show details of the features of the bottom surface of the platform 10.

[0069] Fig. 2A shows an exploded view of the system 1 including the platform 10 and a lid 30. Lid 30 includes a planar top portion 31 and a perimeter sidewall including a first portion 32 and second portion 33 configured to releasably engage with a sidewall 12 of the platform 10. A plurality of openings 34 in planar top portion 31 are configured to allow access and fluid connectivity to the compartments 13 in the platform 10. In aspects, the lid 30 may be a “smart lid” including “smart features,” such as on-board electronics, sensing, and communication of data to remote devices. The lid 30 may also be configured to provide electrical connections to the platform 10 and additional space to locate various integrated circuits or microcontrollers. Referring to Fig. 2A, the lid 30 may include inserts 35 configured to reach down into the compartments 13 and provide sensors, etc. for observation of tissues in the compartments 13. A zone 36, shown schematically, on a planar portion 31 , provides a space for installing integrated circuits, microcontrollers, communication modules, etc. to enable monitoring and control of conditions. Electrical circuits (not shown) may connect sensors, controllers, etc. together and to internal power sources (batteries) or external sources (AC power) to power operation and provide communication among the components. In aspects, circuits may also connect to tissue-specific structures in the compartments 13 in the platform 10. Fig. 2B shows a view of system 1 in which the lid 30 is engaged to the platform 10 to allow for a contained culture system for culturing tissues therein.

[0070] Fig. 3A shows a top view of the system 1 to show how it the system may be configured to interact with standard cell culturing equipment to provide a microwell tissue culture system for both engineered or patient-sourced tissues, while also being configured to established standards to allow for integration within manual and automated applications. The system 1 includes a microwell cellular culture platform 10 that incorporates standardized dimensions and innovative design for culture media flow and loading of cellular samples. Each platform 10 may be loaded and maintained by a human or robot, allowing for rapid introduction of cultures to be grown and maintained in a relevant environment. Once they are mature, the platform 10 may be used to test the effects of a variety of stimuli, including potential therapeutics. A lid 30A is disposed over the platform 10. In this view, the lid 30A is a standard culture plate lid having 96 openings 34A in a 12 x 8 array. It is contemplated that other lids, such as lid 30 shown in Figs. 2A and 2B, may be configured to cover a variety of configurations of tissue culture compartments 13 in the platform 10. A set of eight openings 34 are aligned in a row defined by rectangle 37 to provide access to eight tissue compartments 13 below openings 34. In this view, four openings 34 access compartments 13 of a first culture module and four other openings 34 access compartments 13 of a second culture module. Additional openings access compartments 13 in two more culture modules. It may be appreciated that lids 30, 30A may not include 96 openings, but may be configured with openings only where needed to access the compartments 13 in the platform 10. Fig. 3B shows use of an automated cell culture apparatus including an automated multi-channel pipette 38. As shown, the multi-channel pipette 38 may access eight compartments simultaneously using eight pipette tips 39. An 8-channel pipette 38 may simultaneously exchange media to/from multiple compartments 13 (by hand or robotically). While Figs. 3A and 3B show an 8-channel version (based on a standard 96-well pattern), other configurations maybe able to reduce size and increase the number of compartments 13 for compatibility with a 384-well pattern (24 x 16 array), which would utilize a 16-channel pipette 38.

[0071 ] The platform 10 includes piezoelectric pump(s) 17 to circulate fluid through the circulation loop 14 to simulate a vascular system connecting the individual tissues in their compartments 13. Piezoelectric driven micropumps rely on an electromechanical property of a piezo ceramic to deform in response to applied voltage. A piezoelectric disk attached to the membrane causes diaphragm deflection driven by an external axial electric field, expanding and contracting a chamber of the micropump. Figs. 4A and 4B show the operating principle for two piezoelectric pumps in series to drive fluid entering at the left of flow channel 40 and exiting at the right. In a first state shown in Fig. 4A, the applied field expands piezoelectric disk 41 A, deflecting a diaphragm 42A upward and expanding a chamber 43A, drawing fluid through a gate 44A. Flaps 45A and 46A act as one-way valves to keep fluid moving in a rightward direction. The applied field also contracts a piezoelectric disk 41 B, deflecting a diaphragm 42B downward and contracting a chamber 43B, pushing fluid out of the chamber 43B through gate 44A. Flaps 45B and 46B act as one-way valves to keep fluid moving in the rightward direction. As shown in Fig. 4B, reversing the applied field reverses the positions of all components 41-46A and 42-46B. Depending on an amount of fluid to be pumped and the dimensions allowed for the pump 17, a plurality of such chambers 43A, 43B may be configured in series and/or parallel to move fluid through the circulation loop 14. As shown in Figs. 1A-1 F, 2B, and 3A, pumps 17 may be disposed in open volumes between the platform 10 and lid 30. Additionally or alternatively, pumps 17 and/or fluid channels 15 and 16 may be disposed within the platform 10 to conserve space. Commercially available piezoelectric pumps that are user-programmable up to 6 mL/min (e.g. Bartels mp6, Bartels Mikrotechnik, Dortmund, DE) may be suitable for use in the system 1.

[0072] Figs. 5A and 5B show aspects of electrical connectors that may be used in the system 1. In Fig. 5A, a magnetic pogo connector 50 is disposed in the platform 10 for connection to an external power source for powering pumps 17. Additionally or alternatively, a similar pogo connector may be included in lid 30 to power electrical components in the lid 30 and/or provide communication connectivity for data transmission, control signals, etc. Fig. 5B shows enlarged views of pogo connectors 50 and 55. Pogo connector 50 includes magnetic connectors 51 to quickly connect to similar connectors 56 on pogo connector 55. Connectors 51 and 55 may each include magnets or ferromagnetic metal plates where magnetic attraction between connectors 51 and 56 holds connectors 50 and 55 together. Magnetic connectors 51 and 55 may include magnets disposed so that magnetic attraction/repulsion between connectors 51 and 55 aligns connectors 50 and 55 in the proper configuration to allow for circuit matching. Connector 50 includes female sockets 52 to receive male pin connectors 57 on connector 55. Connector 55 also includes a cord 58 to electrically connect to external devices and/or power sources.

[0073] The compartments 13 in the platform 10 may use specialized mini-bioreactors depending on intended use. Figs. 6A-6D show aspects of transport vessels (also may be referred to herein as “tissue-specific insertable devices”) 60 configured for placement in the compartments 13 for culturing tissues in the platform 10. A variety of modular transport vessels 60 enables handling of tissues with unique requirements. The transport vessels 60 may be installed/removed on demand with human or robotic interface. Three different transport vessels 60 are shown in Fig. 6A: a first transport vessel 61, a second transport vessel 62, and a third transport vessel 63 (may be collectively referred to herein as the “transport vessels 60”). Each of the transport vessels 60 includes a body 70 extending between an upper end 64A (also referred to herein as a “first end”) and a lower end (also referred to herein as a “second end”) 64B and defining chamber 64 between the upper end 64A and the lower end 64B. The body 70 defines an opening 74 at the upper end 64A that is in communication with the chamber 64. The body 70 and, thus, the chamber 64 and the opening 70, are configured according to ANSI/SLAS standards so the transport vessel 60 may be used with a standard single-channel pipette 68 and/or a multi-channel pipette 38. The overall shape and dimensions of the body 70 and, thus, the chamber 64 and the opening 74, are substantially similar to a shaft 68A of a standard pipette 38, 68. In examples, the shaft 68A of the pipette 68 extends from a base 69 of the pipette, which is configured to be manipulated by a hand of a user and/or a robotic device. In examples, the body 70 and, thus, the chamber 64 and the opening 74, may have a diameter within a range of 4.5 mm and 9 mm, or a diameter of 4.5 mm or 9 mm. Additionally or alternatively, the upper end 64A is configured with an inner diameter configured to releasably engage with the shaft 68A of the pipette 38, 68. The lower end 64B may be configured to releasbly engage projections 20 on the bottom of the compartments 13 (see Figs. 1 B-1D). To this end, the lower end 64B may be understood as including a first coupling member and the compartments 13 may be understood as including a second coupling member. It is contemplated that any coupling member capable of forming an interference-fit engagement between the lower end 64B and the compartments 13 may be suitable for use with the system 1. The transport vessels 60 facilitate pick-and-place operations to load or unload the transport vessels 60 in the platform 10 using manual or robotic pipettes. The transport vessles 61, 62 and 63 are configured for use with tissue-specific supports 65, 66, and 67, respectively. Although three tissue-specific transport vessels 60 are shown, it is contemplated that other transport vessels 60 may be envisioned and designed to support a given tissue sample.

[0074] Fig. 6B shows a standard manual single-channel pipette 68 that may be used to pick and place transport vessels 60, such as transport vessel 62, for example. Insertion of the pipette shaft 68A into upper end 64A of the transport vessel 62 allows the pipette 68 to engage the transport vessel 62 and move the transport vessel 62 into position in a corresponding compartment 13 and engage the lower end 64B with projections 20. T o this end, it may be understood that the pipette shaft 68A is configured to be transitioned between a disengaged state and an engaged state, in which the pipette shaft is 68A is either disengaged from the transport vessel 62 or engaged with the transport vessel 62, respectively. Additionally, it may be understood that the transport vessel 62 is configured to be transitioned between a dismounted position and a mounted position, in which the transport vessel 62 is dismounted from the platform 10 and, thus, the compartment 13, and in which the transport vessel 62 is mounted on the platform 10 and, thus, within the compartment 13, respectively. Once the transport vessel 62 is firmly engaged to projections 20, an ejector 75 of the pipette 38, 68 may be activated to displace the transport vessel 62 to release pipette shaft 68A from the transport vessel 62, thereby leaving the transport vessel 62 within the compartment 13.

[0075] Fig. 6C shows a top view of several compartments 13 of a culture module in the platform 10. As shown, each of the transport vessels 61 , 62, and 63 are inserted into corresponding compartments 13B so that tissue supports 65, 66, and 67 are disposed in apertures 21 in compartments 13B. In a compartment 13A, which does not have an aperture 21 , transport vessels 60, with a tissue-specific support 65, 66, and 67, as further described below, are not normally placed therein. Compartment 13A may serve as a reservoir for culture medium or vascular fluid. Compartment 13A may include projections 20 to allow transport vessels 60 without a tissue-specific support 65, 66, and 67 to be placed therein. For example, transport vessels 60 with sensors may be disposed in compartment 13A. In aspects, sensors may include optical sensors for light detection (e.g. fluorescence, colorimetric, etc.) and/or for measurement. Additionally or alternatively, sensors for detecting conditions of the vascular fluid (e.g. tempearture, pH, etc.) and/or compounds within the fluid. Transport vessels 60 including sensors may include an insert 35 disposed on the underside of the lid 30 as shown in Figs. 2A-2B. Alternatively, transport vessels 60 including sensors, as shown in Fig. 12E, may be used. Transport vessels 60 with a reporter tissue may be placed in compartment 13A for culturing and observing tissue in a shared-reservour format.

[0076] Fig. 6D shows a bottom view of the culture module, where the tissue-specific supports 65, 66 and 67 are visible in the apertures 21. Figs. 6C-6D show a suite of three different tissue-specific transport vessels 60 in compartments 13B to provide an organ-on-a-chip module that allows for different tissue types to interact with each other while maintaining tissue-specific integrity. However, other applications may include a variety of alternate configurations of modules and tissue-specific transport vessels 60 depending on the desired needs. For example, a tissue-culturing application may include a plurality of identical tissue-specific transport vessels 60 in a single platform 10 to culture a plurality of similar tissue samples under common conditions to provide tissues that may transferred into organ-on-a-chip modules in different platforms 10 for further culturing and investigation. Transfer of tissues between platfroms 10 may be accomplished using pick-and-place operations manually or robotically. The chambers 64 of the transport vessels 60 are configured to allow the pipette 38, 68 to engage the chambers 64 to lift the tissue-specific transport vessels 60 from a first compartment and transfer it to a second compartment with minimal disturbance to a tissue supported by the tissue support 65, 66, and 67 on the transport vessels 60.

[0077] Fig. 7 shows a close-up perspective view of a tissue-specific transport vessel 61. Transport vessel 61 may be used to culture bulk tissues such as sections of bone matrix, cellular aggregates, cells in hydrogels, and cellular monolayers. Transport vessel 61 includes the body 70 and chamber 64 as described above. Transport vessel 61 includes a side extension 71 on the body 70 and, thus, the chamber 64, that extends tissue support 65 to the side of and below the lower end 64B so that the tissue support 65 is configured to extend into an aperture 21 when the lower end 64B is engaged to the projections 20 at the bottom of a corresponding compartment 13B. Tissue support 65 includes a tray with a perimeter wall 72 and a porous membrane 73 disposed on a bottom of the perimeter wall to support the bulk tissue. It is contemplated that an endothelial barrier may be included and/or deposited on only the porous membrane 73 of the tissue support 65, and not the porous membrane 29 of the platform 10. Additionally or alternatively, track-etched PETE may be used for the porous membrane 73, so as to allow for sufficient light transmission to image tissues.

[0078] Figs. 8A-8B show aspects of a tissue-specific tissue transport vessel 62 for supporting and culturing tissues needing electromechanical stimulation for proper development, such as cardiac cells. Fig. 8A shows a close-up perspective view of the transport vessel 62. Transport vessel 62 includes the body 70 and chamber 64 as described above and the tissue-specific support 66. Tissue-specific support 66 includes two flexible pillars 81 configured to support a hydrogel 82 with embedded cells between the pillars 81, which are configured to extend into an aperture 21. Electrodes 83 disposed outboard of the pillars 81 are configured to provide cyclic electrical stimulation to the cells, which react by contracting/extending in response to the stimulation. Electrical connectors 84 are configured to be in electrical connectivity with electric circuits controlled by microcontrollers and provide power to the electrodes to provide the electrical stimulation to the cells in the hydrogel 82. In aspects, connectors 84 are arranged at the upper end 64A of the transport vessel 62 to connect with circuits and controllers in the lid 30.

[0079] In asepcts, it is contemplated that the pillars 81 may be formed and/or manufactured out of any elastomer (e.g. thermoplastic elastomer (TPE), thermoplastic polyurethane (TPU), etc.) and/or the like that is sufficient for injection molding and/or thermal processing. Additionally or alternatively, the pillars 81 may be overmolded via reaction molding or room-temperature vulcanization of liquid silicone rubber. Additionally or alternatively, the pillars 81 may be formed by centrifugal casting of polydimethylsiloxane (PDMS, Dow Corning Sylgard 184) molded onto a polycarbonate support frame. In such a process, the tissue-specific support 66 is first inserted into Delrin (polyoxymethylene) molds fabricated by CNC machining. PDMS with a 10:1 ratio of silicone elastomer base to curing agent is centrifugally cast at the relative centrifugal force (RCF) of 400 for 5 min, and cured in an oven at 60 °C for 1 hour. The resulting support 66 includes the pair of pillars 81 to support the formation of one tissue.

[0080] In aspects, the pillars 81 may have a diameter within a range of 100 urn to 1 mm, a length within a range of 2 mm and 10 mm, and be spaced apart within a range of 2 mm to 6 mm in an axis-to-axis distance, and designed to subject the tissues to mechanical loading, mimicking the forces human myocardium are exposed in the heart. Hydrogel compaction causes passive tension in the tissues stretched between the two pillars 81 , inducing elongation and alignment. Tissues are formed on the pillars 81 by inserting the pillars 81 into a reservoir (e.g. compartment 13B that surrounds the pillars filled with 100 pL of cell suspension in hydrogel. Pillars 81 may also include a SEBS-based thermoplastic elastomer on a polypropylene core (see Fig. 12C).

[0081 ] For example, human iPS cells may be differentiated into cardiomyocytes with high efficacy. Fibrin hydrogel 82 encapsulating human iPS cell-derived cardiomyocytes and dermal human fibroblasts is compacted around flexible pillars 81. The pillars 81 subject the hydrogel to mechanical loading designed to mimic that in the native heart. The hydrogel compaction causes passive tension in the tissues as they are stretched and aligned between the pillars 81. Synchronous contractions force the tissues to work against the pillars 81. The tissues may matured over 4 weeks of culture and may be exposed to various test substances. For example, the tissues may be exposed to drug candidates to look for potential cardiac side effects.

[0082] Figs. 9A-9D show aspects of a tissue-specific transport vessel 63 for supporting barrier tissues, such as lung, gut, skin, etc. Fig. 9A shows a close-up perspective view of the tissue-specific transport vessel 63. Transport vessel 63 includes the body 70 and the chamber 64 as described above and the tissue-specific support 67. Tissue-specific support 67 includes an upper well 91 and a lower volumetric compartment 92 bounded by porous membrane(s) 93 and open on a bottom configured to fit in an aperture 21 of a corresponding compartment 13B. Tissue may be disposed in the upper well 91 and housed above a horizontal portion of membrane 93. Upper well 91 may provide a reservoir for fluid that the barrier tissue is pysiol og ical ly suited to block. In aspects (i.e. with respect to lung and skin tissues), the fluid may be air. Vertical portions of membrane 93 allow transport from the surrounding medium through the horizontal membrane to the tissue. Lower compartment 92 may contain a hydrogel. Membrane 93 may include a unitary membrane that provides both horizontal and vertical portions, or the horizontal and vertical membranes may be separate portions.

[0083] In aspects, manufacturing approaches to integration of the membrane 93 may include injection overmolding (i.e. insertion of the membrane 93 into a cavity of mold tooling for the tissuespecific support 67 prior to injection molding), thermal bonding (e.g., heat staking, ultrasonic welding, laser welding, and IR welding), and adhesive bonding. Additionally or alternatively, the membrane 93 may be be supported and sealed to the tissue-specific support 67 by a second injection molding step in which the tissue-specific support 67 and the membrane 93 are inserted into the cavity of the mold tooling cavity for a subsequent injection). In aspects, a material of the membrane 93 may be the same or substantially similar to the tissue-specific support 67, such that the membrane 93 is also elastomeric (e.g. any suitable thermoplastic elastomer or thermoplastic urethane). Additionally or alternatively, membrane 93 may be attached to the tissue-specific support 67 by melt welding. Fig. 9B shows the transport vessel 63 in an inverted configuration to show the open bottom of lower compartment 92. Fig. 9C shows a cross-section and Fig. 9D shows a top view of the transport vessel 63. As with the tissue support 65, it is contemplated that an endothelial barrier may be included and/or deposited on only the porous membrane 93 of the tissue support 67, and not the porous membrane 29 of the platform 10. Additionally or alternatively, track-etched hydrophilic polyester (PETE) may be used for the porous membrane 93, so as to allow for sufficient light transmission to image tissues. It is contempalted that the above manufacturing processes for membrane 93 may also apply to other membranes 29, 73, and 135 as discussed herein, with respect to their respective structural relationships. [0084] An alternative configuration of the system 1 is shown in Figs. 10A-12E. In aspects of the alternative configuration of the system 1 , the vascularized membranes for each tissue have been modularized and made compatible for manual or robotic handling via standard multi-channel pipettes 38. Tissues may be inserted directly into vasculature compartments, or with specialized modules that plug into the compartments. These compartments may incorporate any of the tissue-specific modules described herein and enable tissue-specific vasculature, for example, for later assembly into an organ-on-a-chip circuit.

[0085] Physiologically mature organ-on-a-chip circuits that contain multiple tissue types may follow different timelines for maturation for each tissue type. The culture of these tissues may be staggered, maintained, and quality controlled such that integration of multiple tissue types into an organ-on-a-chip circuit may happen simultaneously and with better control over tissue properties. Specific tissues may be combined/matured with vasculature at the time of, or prior to, integration with other tissues.

[0086] The modularization of the vasculature reservoir enables tissue-specific vasculature, which increases physiologic relevance, enables quality control, and allows for a tissue/vasculature period of co-maturation prior to multi-tissue integration. In aspects, a tissue plus its associated vasculature is treated as a single tissue unit.

[0087] Engineered (or primary) tissues have different form factors and only a subset are directly transferrable by standard liquid handling (e.g. small aggregates, spheroids, organoids). The alternative configuration of the system 1 enables custom modules adapted for specific tissue structures to be handled with tools that do not adversely disturb the tissues. In aspects, the reservoir may be large enough to receive and culture tissue samples larger than possible in other systems.

[0088] The pipette shaft 68A interface or adapter for handling the tissue-specific modules is chosen to enable the tissue modules to be used manually with hand-held pipettes 38, 68, or robotically, with automated liquid handling. This is advantageous for ease of use, familiarity, standardization, and adoption since such tools are prevalent and allows users to tap into capabilities of proven systems.

[0089] As described above, on-board pumping and self-coupling magnetic electrical ports enable a perfusion-based chip to be robotically transferred into and out of an incubator.

[0090] Figs. 10A-10F show aspects of a platform 100 according to the alternative configuration of the system 1. Two isolated organ circuits per platform 100 configured to have dimensions of a standardized microculture plate are illustrated in Figs. 10A-10F. Each organ circuit may include up to three tissues each, although a main use may include two different tissues for each circuit. However, other configurations of modules may be envisioned. The platform 100 is configured to allow rapid media exchange by use of a manual or automated (robotic) multichannel pipette 38. For example, compartments in the entire platform 100 may be sampled or refreshed simultaneously, or selected compartments may be sampled. The platform 100 may include a transparent bottom (e.g. glass or clear polymer) for clear visible microscopy.

[0091 ] Figs. 10A, 10D, and 10E show top views of the platform 100. Platform 10, 100 may be CNC-machined or injection molded from plastics such as polysulfone. Additionally or alternatively, the platform 10, 100 may be formed via additive manufacturing (e.g. 3D printing). It is contemplated that the platform 10, 100 may be manufactured out of any material that may be sufficient for the purposes of the system 1 , such as polystyrene (PS), polyetheretherketone (PEEK), polyetherimide (PEI), polycarbonate (PC), and/or any rigid plastic compatible with cell culture. The platform 100 has a rectangular shaped base 110 including one or more well (also referred to herein as a “mounting surface”) 113. In aspects, the one or more well may be defined by sidewalls 111. To this end, each well 113 may include a bottom surface and sidewalls 111 for receiving one or more vasculature insert 119 to provide specific niche environments separate from each other. In aspects, three inserts 119 are disposed in each well 113. The vasculature inserts 119 are configured to receive a “plug” or insertable tissue-specific transport vessel 160, as described above and further below, and thereby define separated, tissue-specific niches of the system 1. The vasculature insert 119 includes impermeable sidewalls and a bottom including a porous membrane 135 and an aperture 121 to allow selective flow of vascular media into the tissue-specific niche environments. Fluid channels 115 and 116 on the top of base 110 are in fluid connectivity with a pump 117 to form a portion of a circulation loop for circulating culture medium (vascular fluid) through the module. Port 123 is configured to receive and compressively seal with a pipette tip 39 for delivering fluid and cells into the vascular fluid circulation loop. The pipette 38, 68 may deliver cells (e.g. endothelial cells) into a channel 118 at the bottom of wells 113 so they may attach to a porous membrane 135 (see Fig. 11 B) in the vasculature insert 119 and form a confluent layer as the cells attach. Optionally, a second port (not shown) may be provided for a pipette 38, 68 to deliver medium into the pumping loop in order to "prime" the pump 117 (that is, push air out of the circulation loop). The platform 100 may also include through-holes 130 configured to receive screws to connect the platform 100 to a pump housing (not shown).

[0092] Fig. 10B shows a bottom view of platform 100. An elongate opening 118A in the bottom of wells 113 is shown. A thin, transparent (e.g. glass or plastic) cover plate 126 may be attached to the bottom of platform 100 by an adhesive layer 125 to seal the bottom of a vascular flow channel 118, with glass viewing windows formed by contacting the bottoms of apertures 121 in the vasculature inserts 119 with the transparent cover plate 126. Optionally, heat and pressure may be applied during the assembly of the cover plate 126 so that a raised thermoplastic border on apertures 121 melts and forms an impermeable barrier, preventing fluid contact with any adhesive. In aspects, cover plate 126 is melt-sealed to the bottom of platform 100 to provide a barrier to contain the fluids in the wells 113 of the platform 100. The porous membranes 135 incorporated into each of the vasculature inserts 119 allow fluid to pass from channel 118 into the bottoms of the vasculature inserts 119. Channel 118 and porous membranes 135 combine to form a manifold below each of the vasculature inserts 119 including a common medium flow a channel that shares a porous boundary with multiple tissue-specific apertures 121 in the inserts 119. Additionally or alternatively, the porous membrane 135 may be provided as part of the platform 100, such that the porous membrane 135 is included across at least a portion of the channel 118. Fig. 10B shows that the bottom of platform 100 includes an essentially smooth planar surface, but it is contemplated that niches in the bottom may provide space for pumps and/or electronics, electrical circuits, controllers, etc. to provide “smart” functionality to the platform 100.

[0093] Figs. 10C, 10D, and 10F show a perspective top view of the platform 100. The base 110 includes the perimeter sidewall 111 and a flange 112 on base 110 to engage with a lid (not shown). In aspects, the lid may be similar to the lid 30, 30A shown in Figs. 2A and 2B. Fig. 10C shows how an 8-channel multi-channel pipette 138 may insert pipette tip array 139 into the vasculature inserts 119 to place or remove tissue-specific transport vessels 160 into the vasculature inserts 119. The multi-channel pipette 138 may also transfer fluids into/out of the vasculature inserts 119 via standard pipette tips 39 (see Fig. 3B).

[0094] Fig. 10D shows a perspective top view of a tray 140 including the platform 100. Tray 140 is sized to match that of a standard 96-well plate. Platform 100 occupies half of tray 140 and the other half provides room for piezoelectric pumps 117, integrated circuits, controllers, etc. Tubing connecting fluid channels 115 and 116 to pumps 117 to complete a circulation loop is not shown. Tray 140 includes perimeter sidewall 141 and flange 142 configured to engage a lid, such as similar to the lid 30, 30A. An electronics package 144 included on the tray 140 may include integrated circuits, controllers, processors, sensor linkages, communications modules, etc. to provide smart capabilities to the platform 100. On-board routing for electrical stimulation of certain tissue types (e.g. cardiac tissue) may be enabled. Power connectors 145 supply power to drive the piezoelectric pumps 117. An electrical connector 150, similar to connector 50, is disposed in the sidewall 141 to receive power from an external source via a connector similar to connector 55 and is connected to electronics package 144 and power connectors 145 to deliver power to operate the pumps 117 and electronics.

[0095] In aspects, the platform 100 has two organ-on-a-chip circuits arranged lengthwise in the tray 140, with vasculature reservoirs arranged side by side. However, other arrangements may be envisioned. For example, the platform 100 may be configured so that the two organ-on-a-chip circuits are arranged side-by-side in tray 140 and the ends of vasculature inserts 119 of one organ-on-a-chip circuit are adjacent to the ends of vasculature inserts 119 of the second organ-on-a-chip circuit. This arrangement would allow all eight channels of an 8-channel multi-channel pipette 138 to access the interior of the two organ-on-a-chip circuits (four for each organ-on-a-chip circuit).

[0096] Figs. 11A-11 D show aspects of a vasculature insert 119 for use with platform 100. Fig. 11A shows a top view of vasculature insert 119 including a perimeter sidewall 128 and a bottom 129. Projections 120 on the upper side of bottom 129 of the vascular insert 119 define positions 122 (may also be referred to herein as a “mounting surface”) for receiving tissue-specific transport vessels 160 or transport vessels 160 including sensors. Four positions 122 are shown, but it is contemplated that more or less positions may be included.

[0097] Fig. 11 B shows a bottom perspective view of vasculature insert 119. Projections 131 on the underside of bottom 129 are configured to be received within complimentary recesses 114 defined on a surface of each well 113 (may also be referred to herein as a “mounting surface”) to secure each vasculature insert 119 within the corresponding well 113 (also see Fig. 10F). Alternatively, the projections 131 on the underside of the bottom 129 may be configured to hold the vasculature insert 119 above the bottom of each well 113 to provide space for a common reservoir of vascular fluid that may circulate into the vasculature inserts 119 via the porous membrane 135. A transparent window 136 at the bottom of the aperture 121 allows for viewing of the tissue in the vasculature insert 119. As discussed, the porous membrane 135 is included across at least a portion of the bottom of the aperture 121. Additionally or alternatively, a seal 127 may be provided on the aperture 121 to seal the interface between the aperture 121 and the channel 118.

[0098] Fig. 11C shows a photograph of a vasculature insert 119, which is fabricated from transparent polymeric materials. It may be prepared by two-stage injection molding having a polypropylene rigid core structure, over-molded with a Styrene ethylene butylene styrene SEBS- based thermoplastic elastomer with an integral track-etched PET membrane filter (8um). [0099] Fig. 11 D shows a schematic representation of how an 8-channel multi-channel pipette 138 may be used to transfer tissue-specific transport vessels 160 into or out of the vasculature inserts 119. Tissue-specific transport vessels 160 may be configured substantially similar to the transport vessels 61 , 62 and 63 (see Figs. 6A-6D, 7, 8A, 8B and 9A-9D).

[00100] Figs 12A-12D show aspects of a cardiac cell-specific transport vessel 162 for use with the vasculature insert 119. Fig. 12A shows a cut-away view of the transport vessel 162 disposed in the vasculature insert 119. Similar to insert 62, insert 162 has a body 190 defining a chamber 164 having an upper end 164A configured to engage a the shaft 68A of a standard pipette 38, 68, so as to allow for moving and/or placing the transport vessel 162 into or out of the vasculature insert 119. The lower end 164B is configured to be received by and/or engaged with projections 120 at a position 122C (may also be referred to herein as a “mounting surface”) adjacent to the aperture 121. To this end, the lower end 164B may be understood as including a first coupling member and the inserts may be understood as including a second coupling member, as discussed above. A tissue-specific support 165 includes a pair of flexible pillars 166 configured to support a hydrogel (see 82 in Fig. 8A) with embedded cells between the pillars 166. The pillars 166 extend downward into the aperture 121. Electrodes disposed outboard of the pillars 166 are configured to provide cyclic electrical stimulation to the cells, which react by contracting/extending in response to the stimulation. In aspects, the electrodes are disposed in a separate stimulation module and/or transport vessel 170 (see Fig. 12E) disposed at an adjacent position 122B (may also be referred to herein as a “mounting surface”) to provide the electrical stimulation to the cells in the hydrogel.

[00101 ] Fig. 12B shows a top view of the vascular insert 119 with cardiac cell-specific transport vessel 162 disposed therein, with the pillars 166 extending into the aperture 121. Positions of electrodes for electromechanical stimulation are indicated by dots 171 and the position of the separate stimulation module 170 is shown by a dashed circle 172 at the position 122B. It is contemplated that a transport vessel similar to the transport vessel 162 incorporating the flexible pillars 166 and the electrodes in a unitary transport vessel may be used instead of separate tissue and stimulation transport vessels 60, 160. Use of separate transport vessels 60, 160 may be advantageous for easier loading of the hydrogel onto the pillars 166 and incorporation of cardiac cells or cardiac cells precursors, e.g. induced pluripotent stem cells (iPSCs), into the hydrogel prior to electromechanical stimulation. Further, the tissue-specific transport vessel 162 may be disposed of after use, while the stimulation module 170 may be sterilized and reused. [00102] Fig. 12C shows photographs of the tissue support 165 and flexible pillars 166. The pillars 166 may be formed from a SEBS-based thermoplastic elastomer (TPE) overmolded onto a polypropylene rigid core.

[00103] The stimulation module 170 is shown in Fig. 12D. The stimulation module 170 is similar to the transport vessel 62, 162, except it does not include the pillars 66, 166. The stimulation module 170 includes the base 190 and, thus, the chamber 164, as described above. Electrodes 173 are disposed below the base 190 and, thus, the chamber 164, and spaced apart so that the electrodes are disposed outboard of the pillars 166 at positions 171 shown in Fig. 12B. The electrodes 173 are configured to provide cyclic electrical stimulation to the cells, which react by contracting/extending in response to the stimulation. Electrical connectors 174 are configured to be in electrical connectivity with electric circuits controlled by microcontrollers to provide the electrical stimulation to the cells in the hydrogel. In the embodiment shown, connectors 174 are located at the top of the stimulation module to connect with circuits and controllers in the lid 30.

[00104] Although Figs. 12A-D relate to the cardiac-specific tissue transport vessel 162, transport vessels 160 configured for culturing tissues other than cardiac tissues may be used in the vascular inserts 119. For example, transport vessels 160 similar to tissue-specific transport vessels 61 and 63 may also be used in a similar manner.

[00105] A sensor module 180 is shown in Fig. 12E. The sensor module 180 is similar to the stimulation module 170, except sensors replace the electrodes 173. The sensor module 180 includes the body 190 and, thus, the chamber 164, as described above. Sensor(s) at positions 183 and/or 184 may be disposed below the body 190 and, thus, the chamber 164, so that the sensors may reach into apertures 21 or 121. Electrical connectors 185 are configured to be in electrical connectivity with electric circuits controlled by microcontrollers to control the sensors and receive information therefrom. In aspects, connectors 185 are located at the top of the sensor module 180 to connect with circuits and controllers in the lid 30.

[00106] Referring to Figures 13A-13B, the system 1 including the cell culture platforms 10, 100 and transport vessels 60, 160 described herein provide “smart,” robotic automation-friendly plates for culturing tissues. The plates are capable of electrification, with low release-force electrical connectors for connecting to external power, control and communication functions. Electronics functions may be included in a smart microwell platform 10, 100 and/or smart lid 30. Multi-system control, power, and communication (e.g. data logging, control of embedded programs, etc.) is accomplished via a daisy-chained common bus connecting a plurality of systems 1 described herein to external electronic modules including servers, processors, controllers, memory, data stacks, user interface, etc. and power supplies. The systems 1 may be configured according to ANSI/SLAS standards for ready integration into existing cell culture infrastructure, wherein the platforms 10, 100 and/or lid 30 may be connected to power/communications buses in the infrastructure.

[00107] The platforms 10, 100 are stackable into vertical cell culture racks with a power/communications bus running along vertical structure of microplate stack uisng existing robotic systems for handling cell culture platforms. Fig. 13A shows an automated robotic handler 200 stacking a single system 1 according to aspects herein into a vertical stacking rack 210 capable of holding a stack of a plurality of culture platforms 10, 100. A power/communications bus is not visible in Fig. 13A. Fig. 13B shows a representative robotic platform hotel 220 enabling robotic storage of several platforms 10, 100, with a front cover of the platform hotell 220 being removed to show an inner configuration. The platform hotel 220 includes two carousels 221, each including a plurality of platform racks 210. Armature 222 is configured to rotate the carousels 221 to position a specific platform rack 210 to a front of the platform hotel 220 for loading or unloading culture platforms 10, 100 inlcuding the systems 1. A robotic handler 200 may be used to manipulate the platfroms 10, 100. Replacement of the front cover provides a climate controlled incubator for culturing tissues.

[00108] While the disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are considered illustrative or exemplary and not restrictive. The disclosure is not limited to the disclosed aspects. It is contemplated that various omissions, substitutions, and changes in the form and details of the devices or process illustrated may be made by those skilled in the art without departing from the disclosure. This description is in no way meant to be limiting, but rather should be taken as illustrative of the general principles of the disclosure. The scope of the disclosure should be determined with reference to the following claims.