Mimicking physiological oxygen levels in a culture medium of at least one organ-on-a-chip module

Abstract

An organ-on-a-chip (OoC) platform can link one or more OoC modules in parallel, mimicking oxygen levels of cultures with biological materials housed therein in each OoC module. Each OoC module can include an oxygen sensor; an oxygen scavenger to decrease an oxygen level within the culture medium surrounding the at least one type of biological material. Each OoC module can be connected to a controller, the controller comprising: a memory storing instructions; and a processor configured to access the memory to execute the instructions to at least: for each of the at least one OoC module, ensure that the oxygen level of the culture medium surrounding the at least one type of biological material is physiological based on control of the oxygen scavenger.

Description

BWH2025-025NONPROVISIONAL APPLICATIONMIMICKING PHYSIOLOGICAL OXYGEN LEVELS IN A CULTURE MEDIUM OF AT LEAST ONE ORGAN-ON-A-CHIP MODULEGOVERNMENT SUPPORT

[0001] This invention was made with government support under 2225698 and 1936105 awarded by the National Science Foundation and 5R01 EB028143-04, 3R01 HL166522-02S1 , 5UG3TR003274-02, 5R00CA201603-05, 5R21 EB025270-03, 5R01 CA282451 -02, 5R01 HL153857-05, 5UH3TR003274-05, and 1 R21 HL168656- 01 A1 awarded by the National Institutes of Health. The government has certain rights in the invention.Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 716,106, filed November 4, 2024, entitled “CLOSED-LOOP MODULAR MULTIORGAN-ON-CHIPS PLATFORM AND USES THEREOF”. The entirety of this provisional application is hereby incorporated by reference for all purposes.Technical Field

[0003] This disclosure relates generally to organ-on-a-chip (OoC) modules, and more specifically to systems and methods that can mimic physiological oxygen levels of biological materials in a culture medium of at least one OoC module.Background

[0004] An organ-on-a-chip (OoC) platform can be used to study disease, test drugs, assess toxicity, and the like. To do so, the OoC platform arranges one or more OoC modules, each which mimics one or more structures and / or functions of microenvironments of one or more biological materials, in parallel such that a single culture medium can flow through each of the one or more OoC modules. Each of the OoC modules can house a unique biological material within the culture medium.

[0005] Current OoC platforms cannot truly mimic the microenvironments of the one or more biological materials within the culture medium at least because current OoC platforms do not provide physiologically appropriate oxygen level(s) to each OoC module. The appropriate oxygen level is critically important to maintainBWH2025-025 functions and allow modulation of behaviors of the one or more biological materials. OoC platforms tend to be housed in incubators that are supplied with an oxygen level of 21%. In such situations, every OoC module in the standard OoC platforms is exposed to the ambient incubator oxygen level of 21 %. However, a 21 % oxygen level is not a physiologically appropriate oxygen level for biological materials within the human body. The physiological oxygen levels in the human body are each greater than 0% and less than 21 % (e.g., alveoli - 13%, artery - 13%, bone - 7%, liver - 13%, kidney - 6%, gut - 8%, vein - 3%). As such, the oxygen level of 21 % cannot accurately model features of one or more biological materials in the human body (e.g., metabolic activities) to study disease, test drugs, assess toxicity, and the like, accurately.Summary

[0006] Described herein are systems and methods that can mimic physiological oxygen levels of one or more biological materials (e.g., cells, tissues, and / or organs) in a culture medium in one or more organ-on-a-chip (OoC) module. The one or more OoC modules can be arranged in parallel in an OoC platform.

[0007] In an aspect, the present disclosure can include a system that can mimic oxygen levels of one or more physiological microenvironments of one or more biological materials (e.g., cells, tissues, and / or organs) in an OoC platform (including one or more OoC module). The system can include the at least one OoC module, each of the at least one OoC module hosting at least one type of biological material within a culture medium. Each of the at least one OoC module can include: an oxygen sensor and at least one oxygen scavenger configured to decrease an oxygen level within the culture medium surrounding the at least one type of biological material. Each of the at least one OoC module cam be connected to a controller. The controller includes a memory storing instructions; and a processor configured to access the memory to execute the instructions to at least: for each of the at least one OoC module, ensure that the oxygen level of the culture medium surrounding the at least one type of biological material is a value from greater than 0% to less than 21 % based on recordings from the oxygen sensor and control of the at least one oxygen scavenger.

[0008] In another aspect, the present disclosure can include a method for controlling an oxygen level of a culture medium surrounding at least one type ofBWH2025-025 biological material housed by at least one OoC module. The method can include: receiving, by a system comprising a processor, a recording from an oxygen sensor within the culture medium indicative of the oxygen level of at least a portion of the culture medium; determining, by the system, whether the oxygen level of the culture medium needs to be adjusted to a value from greater than 0% to less than 21 %; and when the oxygen level of the culture medium needs to be adjusted to a value from greater than 0% to less than 21%, and controlling, by the system, at least one oxygen scavenger within the culture medium to ensure that the oxygen level of the culture medium is the value.Brief Description of the Drawings

[0009] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0010] FIG. 1 is a block diagram of an example of a system including an organ- on-a-chip (OOC) module and a controller;

[0011] FIG. 2 is a block diagram of an example of a platform linking a plurality of OoC systems (e.g., the system of FIG. 1 ) in parallel;

[0012] FIG. 3 is a block diagram of a system that can mimic different oxygen levels of physiological microenvironments of biological materials in a culture medium of at least one OoC module of FIG. 1 ;

[0013] FIG. 4 is a block diagram of an example controller of FIG. 3;

[0014] FIG. 5 is a process flow diagram of a method for mimicking a physiological oxygen level of a physiological microenvironment of one or more biological materials in a culture medium of at least one OoC module;

[0015] FIG. 6 shows a customized microfluidic optical setups for real-time oxygen monitoring;

[0016] FIG. 7 shows a module for oxygen-scavenging control;

[0017] FIG. 8 shows a module for oxygen generation;

[0018] FIG. 9 shows a design of a proof-of-concept individual microtissue-on-a- chip platform;BWH2025-025

[0019] FIG. 10 shows evaluations of oxygen effects on physiological activities and toxicity of APAP-poisoning in the closed-loop multi-organ-on-chips platform; and

[0020] FIG. 1 1 shows evaluations of oxygen effects on physiological activities and toxicity of APAP-poisoning in the closed-loop multi-organ-on-chips platform in a conventional hypoxia incubator.Detailed DescriptionI. Definitions

[0021] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0022] As used herein, the singular forms “a,” “an,” and “the” can also include the plural forms, unless the context clearly indicates otherwise.

[0023] As used herein, the terms “comprises” and / or “comprising,” can specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups.

[0024] As used herein, the term “and / or” can include any and all combinations of one or more of the associated listed items.

[0025] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0026] It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.BWH2025-025

[0027] As used herein, the term “organ-on-a-chip module” or “OoC module” refers to a microfluidic or millifluidic device (often miniaturized) that uses living human cells or tissues to replicate the functions of one or more human organs. For example, one or more OoC modules can be used to study disease, test drugs, assess toxicity, and the like. It will be understood that OoC may also be referred to as a different name, often by another biological tissue (cell, tissue, microtissue, etc.)- on-a-chip. OoC is used herein to encompass all biological material-on-a-chip implementations.

[0028] As used herein, the term “platform” can refer to a setup for modulating oxygen levels in microenvironments of each type of microtissue within one or more OoC module. The platform can be closed loop - using at least a controller and one or more oxygen scavengers - to precisely control oxygen levels, mimicking a human’s varying oxygen environments. In some instances, the platform can have a single organ-on-a-chip module. In other instances, the platform can include two or more organ-on-a-chip module (which also may be referred to as a multi-organ-on- chips platform).

[0029] As used herein, the term “culture medium” refers to a fluid within and / or flowing through the microfluidic device of the OoC module. The fluid provides nutrients and / or growth factors for cells and / or tissues to survive and / or grow outside a natural environment.

[0030] As used herein, the term “biological material” refers to any substance that is produced by and / or comes from a living organism. For example, the biological material can include one or more cells, one or more tissues, and / or one or more organs. In some instances, the biological material can be of a micro size and referred to as one or more microtissues.

[0031] As used herein, the term “physiological microenvironment” can refer to the surroundings of biological material. The physiological microenvironment can include a complex and dynamic combination of physical, chemical, and biological signals that influence cellular function, such as mechanical forces, substrate pliability, physical properties of the surroundings, oxygen level, pH, chemical gradients, nearby cells, contents of the extracellular matrix, and the like.

[0032] As used herein, the term “sample” refers to at least a portion of the biological material within a culture housed within the OoC module.BWH2025-025

[0033] As used herein, the term “controller” refers to one or more devices (which may run software via a processor) that that regulate function(s) of at least a portion of a system. For example, the controller can allow a closed loop of one or more OoC module to mimic oxygen levels of one or more physiological microenvironments of one or more biological materials.II. Overview

[0034] An organ-on-a-chip (OoC) platform (linking one or more OoC modules in parallel) can be used to study disease, test drugs, assess toxicity, and the like. To accurately mimic these structures and / or functions, physiologically relevant parameters are required to be added to a culture medium flowing through each of the one or more OoC modules supporting one or more biological materials therein to mimic physiological microenvironments of the one or more biological materials. It has been well-known that oxygen level is a critical microenvironmental parameter to maintain cellular and / or tissue functions and modulate cell and / or tissue behaviors. However, standard OoC platforms tend to be housed in incubators that are supplied with an ambient oxygen level of 21 %, which is not a physiologically appropriate oxygen level for biological materials within the human body. In fact, the oxygen level of 21% is higher than most, if not all, physiological oxygen concentrations (different in each type of cell and / or tissue, ranging from greater than 0% to less than 21 %). As such, the ambient oxygen level of 21% cannot accurately represent the metabolic activities of one or more biological materials in the human body to study disease, test drugs, assess toxicity, and the like. For the OoC platform to use correct models, oxygen levels within the culture medium of traveling through each of the one or more OoC modules should be modulated to physiological oxygen levels (for the biological material(s) therein). In cases where hypoxia incubators are used, each hypoxia incubator can only be set at a certain oxygen level (e.g., the whole incubator can be set to a certain static oxygen level). If only a single organ is cultured, this might work fine, but if multiple organs are cultured, each of the multiple organs prefers a specific oxygen level, all of the different organs are at less than ideal conditions.

[0035] Described herein are systems and methods that can employ an OoC platform to enable real-time monitoring and control of oxygen levels of a culture medium within one or more OoC modules. Each of the one or more OoC modules can include one or more oxygen sensors and one or more oxygen scavengers. OneBWH2025-025 or more controllers can allow oxygen levels of the culture medium within the OoC module to be monitored in real time and tightly controlled such that the oxygen levels match the physiological microenvironment of the type of cells and / or tissue therein. The tight control allows for independent, precise up-regulation and down-regulation of dissolved oxygen in the perfused culture medium to meet the physiological oxygen level of the specific biological material. The one or more oxygen scavengers can include one or more chemicals to perform oxygen scavenging functions.

[0036] Examples of oxygen scavengers are shown in Table 1 . It will be understood that these examples are meant to provide context and are not meant to be limiting and other chemicals can be used to perform oxygen scavenging functions according to this disclosure.Table 1 - Example Chemicals that can be used to Perform Oxygen ScavengingFunctionsBWH2025-025BWH2025-025BWH2025-025III. Systems

[0037] Organ-on-a-chip (OoC) modules (an example OoC module 102 is shown in FIG. 1) can be used to mimic structure and / or function of one or more biological materials housed in a culture medium. Each OoC module can create and / or simulate a 3D microenvironment (e.g., a physiological microenvironment that is specific for a certain one or more biological material, such as cells, tissue, organ, etc.) that is engineered so to culture the biological materials in such a way that provides a more physiologically relevant environment compared to traditional 2D culture. Each OoC module can include a microfluidic or millifluidic module (including the culture medium) and an integrated bioreactor (including the biologicalBWH2025-025 material(s)). The OoC module (e.g., length of the module less than 500 cm, 100 cm, 50 cm, 10 cm, or the like) can include channels of one centimeter, 1 millimeter, 100 micrometers, 50 micrometers, 10 micrometers, etc. or less in diameter. In some instances, the biological materials can be sized at the microscale level (e.g., to fit within the channels of the microfluidic module). Examples of biological materials that can be held by each OoC module can include, but are not limited to alveoli microtissue, arterial microtissue, bone microtissue, liver microtissue, kidney microtissue, gut microtissue, and / or vein microtissue.

[0038] An OoC platform can group one or more OoC modules, often with different biological materials, in parallel with a common culture medium (an example of an OoC platform 200 in shown in FIG. 2). The OoC platform aims to mimic functions and interactions of at least a portion of a human body (e.g., to provide robust and accurate predictions of drug / toxin effects in the human body). OoC platforms have been developed with a goal of achieving animal-free tests and personalized precision medicine. However, current OoC platforms are not able to recreate the at least the portion of the human body at least because biological parameters, such as oxygen level, are not met for the individual biological materials within the OoC platform.

[0039] Generally traditional OoC platforms are supplied with an ambient oxygen level (e.g., set to a single value, such as 21%), which is not a physiologically appropriate oxygen level for biological materials within the human body.Alternatively, the whole incubator can be set to a certain static oxygen level (e.g., a single value greater than 0% and less than 21%). However, the physiological oxygen levels for different biological materials within the human body are each greater than 0% and less than 21% and each have their own value. Examples of oxygen levels within the human body include but are not limited to alveoli - 13%, artery - 13%, bone - 7%, liver - 13%, kidney - 6%, gut - 8%, vein - 3%. As such, the oxygen level of 21% cannot accurately represent functions of one or more biological materials (e.g., metabolic activity) in the human body to correctly study disease, test drugs, assess toxicity, and the like. This can be corrected such that each type of biological tissue can be housed in an OoC module with a culture medium fixed to the appropriate oxygen level.

[0040] A system 100 of FIG. 1 can include an OoC module 102 linked to a controller 104. As noted, the OoC module 102 can include a microfluidic moduleBWH2025-025(including the culture medium) and an integrated bioreactor (including one or more biological material). The system 100 can be an example of a platform with a single OoC module 102. It will be understood that the OoC module 102 can have and / or be associated with one or more different / alternative components therewithin (e.g., an oxygen sensor, at least one oxygen scavenger, etc.). The OoC module 102 can receive microfluidic / milifluidic inputs (IN) and provide microfluidic / milifluidic outputs (OUT). The microfluidic / milifluidic inputs and outputs refer to inputs and outputs of the culture medium.

[0041] The controller 104 can fix the oxygen level of the culture medium for the specific biological material within the OoC module 102 (e.g., based on at least input to the controller from the oxygen sensor and output from the controller to the one or more oxygen scavenger). In some instances, the controller 104 can be tied to the one or more oxygen scavenger so that the sensing and controlling are paired. The controller 104 may be a portion (e.g., a partition) of a larger controller. Although not illustrated, it will be understood that the controller 104 can have the capabilities of a memory and a processor (which may be implemented as a microprocessor).

[0042] The controller 104 can ensure that the oxygen level of the culture medium surrounding the at least one type of biological material matches or substantially matches (e.g., is within a threshold from) a stored value required by the at least one type of biological material. In some instances, the threshold can define a set of values away from the stored value (e.g., + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1 %, + / - 0.5%, + / - 0.25%, or the like). The stored values each can be any value from greater than 0% to less than 21%. As an example, the oxygen level of the culture medium can be determined by an oxygen sensor and any corrective action can be taken by at least one oxygen scavenger when instructed by the controller 104 (based on a comparison between recordings from the oxygen sensor and a stored ideal oxygen saturation stored in memory accessible by the processor of the controller).

[0043] FIG. 2 shows an example of a platform 200 linking a plurality of OoC systems 100-(1-N) in parallel. It should be understood that N can be equal to any integer greater than or equal to two. The platform 200 can be used to study both the reaction of the cells in each OoC module but also the many effects the different OoC systems 100-(1-N) have on one other. As an example, when the platform 200 is used for drug screening, each of the OoC systems 100-(1-N) can be affected by theBWH2025-025 drug, and each of the OoC systems can have an effect on one another (negative, positive, or neutral) during drug screening.

[0044] As an example, each of the OoC systems 100-( 1 -N) can be similar to the system 100 of FIG. 1 (in which each OoC system 100 can include an OoC module 102 in bidirectional communication with a controller 104). In platform 200 the inputs (IN in FIG. 1) into each of the plurality of OoC systems 100-(1 -N) can be connected and the outputs (OUT in FIG. 1 ) of each of the plurality of OoC systems can also be connected (such that the OoC systems 100-(1 -N) are in parallel to one another). Although each of the OoC systems 100-(1-N) houses a unique biological material, the OoC systems share a culture medium.

[0045] The OoC systems 100-(1 -N) can each be associated with a controller. The controller(s) can ensure an oxygen level of the culture media in each of the OoC systems 100-(1 -N) (each housing a biological material) is physiologically appropriate for the biological material housed therein. In some examples, the OoC systems 100- (1 -N) each can have and / or be associated with a unique controller (e.g., controller 104 of system 100). In other instances, the OoC systems 100-(1 -N) can each be associated with separate lines of code and / or partitions within one or more common controllers. For each of the OoC systems 100-(1-N), the controller can ensure that the oxygen level of a portion of the culture medium surrounding the at least one type of biological material housed therein matches or substantially matches (within a threshold from) a stored value required by the at least one type of biological material. In some instances, the threshold can be the same for the different types of biological materials. However, each of the OoC systems 100-(1 -N) can have different thresholds and / or thresholds set uniquely (e.g., set based on the biological material). For example, the thresholds each can be + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, + / - 0.5%, + / - 0.25%, or the like. The controller can store in memory an ideal value for each biomaterial, which can be any value from greater than 0% to less than 21%.

[0046] Each OoC system 100-(1 -N) can have at least an oxygen sensor and an oxygen scavenger. As an example, each OoC system 100-(1 -N) can be a fluidic module where oxygen scavenging chemicals used for oxygen scavenging functions are flown into the bottom of the fludic module while the culture medium is flown through the top of the fluidic module and the top flow and bottom flow are separated by a membrane (e.g., a PDMS membrane) that is only permeable to oxygen (or other gasses) but not oxygen scavenging species of the chemicals and the cultureBWH2025-025 medium. The controller can determine the oxygen level near and / or within each OoC system 100-(1 - N) based on recordings by one or more oxygen sensors within each OoC system. Based on the oxygen level, the controller can determine whether any corrective action is needed for each OoC system 100-(1-N). Each corrective action can be taken independently for each OoC system 100-(1 -N) by at least one oxygen scavenger (within the OoC system when instructed by the controller). A decision regarding the corrective action can be based on a comparison between recordings from the oxygen sensor and a stored ideal oxygen saturation stored in memory accessible by the processor of the controller.

[0047] FIG.3 illustrates an example of a system 300 that can mimic different oxygen levels of physiological microenvironments of biological materials in a culture medium of at least one OoC module (e.g., a single OoC module of FIG. 1 and / or a platform 200 of FIG. 2). The system 300 can include at least a part of the OoC module 102 (which may be within the platform 200). Although not illustrated, it will be understood that the OoC module can include and / or host at least one type of biological material within the culture medium (which may be shared with other OoC modules when arranged as a platform). Each OoC module can include one or more oxygen sensor 302, at least one oxygen scavenger 304 (one is illustrated), and may include an oxygen generator 304 (this may be common for the entire platform and / or specific to modules within the platform). As an example, when the medium is circulated, the oxygen generator would be necessary, but in cases of unidirectional flow without circulation, the oxygen generator may not be needed, The one or more oxygen sensor 302 can detect the oxygen level within the OoC module (e.g., oxygen level of culture media surrounding the biological material). For example, the one or more oxygen sensor 302 can detect an amount of dissolved oxygen in the culture medium within the OoC module. The at least one oxygen scavenger 304 can decrease the decrease the oxygen level within the culture medium surrounding the at least one type of biological material. The oxygen generator 306 can be used increase the oxygen level within the culture medium (e.g., of the entire system, including surrounding the at least one type of biological material in the OoC module) to a standard level (e.g., 21%). For example, the at least one oxygen scavenger 304 can be configured to down-regulate the amount of the dissolved oxygen in the culture medium and the at least one oxygen generator 306 can be configured to up- regulate the amount of the dissolved oxygen in the culture medium. For example,BWH2025-025 the at least one oxygen generator 306 can deliver an ambient level of 21% (e.g., to the entire platform 200) and the at least one oxygen scavenger 304 (which can be within a single OoC system 100(1 -N) of the platform) can downregulate the oxygen level in the single OoC system.

[0048] The at least one oxygen sensor 302 can send a signal comprising the detected oxygen level (e.g., level of dissolved oxygen within the culture medium in the OoC module housing the biological material) to a controller 310 (e.g., in some instances similar to controller 104). The at least one oxygen sensor 302 can send the signal to the controller according to a wireless and / or wired connection. It will be understood that each of the OoC modules is connected to the controller 310 (which, as described above, can be a common controller that may be separately partitioned or may be a separate controller for each OoC module). The controller can include a memory storing instructions and a processor configured to access the memory to execute the instructions to determine whether a corrective action is needed. It will be understood that the memory and processor can be any type of device known to execute such functionality. In some instances, the memory and processor can be at least partially embedded within the same device (e.g., a microprocessor). When the processor 310 deems that the corrective action is needed, the controller 310 can send instructions (across a wireless connection and / or wired connection) to the one or more oxygen scavenger 304 when a corrective action is to be taken.

[0049] FIG. 4 illustrates an example of the controller 310 that can determine whether corrective action is needed to bring the oxygen level surrounding a biological material within a single OoC module at a time (however, the action can be repeated for each OoC system by the controller 310 and / or by other controllers). In some instances, this action can be performed by controller 310 in parallel (and does not need to be in serial) if there is enough memory capacity. In other instances, this action can be performed in serial by the controller 310. In still other instances, this action can be performed parallel in part and serial in another part by the controller 310.

[0050] As noted, the controller 310 has a memory 312 that stores instructions and a processor 314 configured to access the memory and execute the instructions to determine whether a corrective action is needed. Upon execution of the instructions, the controller 314 can receive 402 data (within a signal from at least one oxygen sensor) regarding an oxygen level of the culture medium around theBWH2025-025 biological material at a time. The controller 310 can analyze the data and determine the oxygen level of the culture medium surrounding the at least one type of biological material at the time based on the recording. The oxygen level of the culture at the time can be compared to an ideal oxygen level (for the biological material) stored in memory 312. The comparison can allow the controller 310 to determine whether the oxygen level of the culture at the time is in range 406 of the ideal oxygen level (for the biological material). The determination of whether oxygen level of the culture at the time can be in a “target oxygen level” may be equal or within range for the biological material (depending on the biological material). The “target oxygen level range” for the biological material can be stored in the memory 410. For example, the target oxygen level range related to different types of biological materials can include alveoli microtissue -12.5-13.5%, arterial microtissue - 12.5-13.5%, bone microtissue - 6.5-7.5%, liver microtissue - 12.5-13.5%, kidney microtissue - 5.5-6.5%, gut microtissue - 7.5-8.5%, and / or vein microtissue - 2.5-3.5% (however, the values may be different and the list is not exclusive).

[0051] When the oxygen level of the culture medium surrounding the at least one type of biological material at the time matches the target oxygen level for the at least one type of biological material (Yes 408), another recording can be received at another time (back to receive 402). When the oxygen level of the culture medium surrounding the at least one type of biological material at the time does not match the target oxygen levels for the at least one type of biological material (No 410) a corrective action can be taken by regulating at least one oxygen scavenger (within the culture medium near the biological material) to ensure that the oxygen level of the culture medium surrounding the at least one type of biological material matches the target oxygen level for the at least one type of biological material. After the corrective action 412 is taken, another recording can be received at another time (back to receive 402). As an example, the corrective action can be adjusting the flow rate of the scavenging solution in the bottom layer of the scavenger.IV. Methods

[0052] Another aspect of the present disclosure can include a method 500 for mimicking different oxygen levels of physiological microenvironments of biological materials (e.g., cells, tissues, and / or organs) in a culture medium of at least one organ-on-a-chip (OoC) module or platform of modules (e.g., shown in FIGS. 1 and 2). It should be noted that the OoC module can create and / or simulate a 3D orBWH2025-025 compartmentalized microenvironment (e.g., a physiological microenvironment that is specific for a certain one or more biological material) that is engineered so to culture the biological materials in such a way that provides a more physiologically relevant environment compared to traditional 2D culture. The OoC modules can include a microfluidic module (including the culture medium) and an integrated bioreactor (including the biological materials). Examples of biological materials include, but are not limited to alveoli microtissue, arterial microtissue, bone microtissue, liver microtissue, kidney microtissue, gut microtissue, and / or vein microtissue. The OoC platform can group one or more OoC modules, often with different biological materials, in parallel and / or in serial in a common culture medium.

[0053] In other words, the method 500 can control the oxygen level of one or more portions of a culture medium surrounding one or more types of biological materials (but each segment of the platform is controlled independently). The system 300 of FIG. 3, and the example controller shown in FIG. 4, for example, can be used to execute the method 500. In some instances, at least a portion of the method 500 can be executed using and / or aided by a processor (e.g., one or more steps of the method 500 can be executed by a system including a processor).

[0054] Unless otherwise stated, the methods described herein generally follow the usual practices widely known in related fields. Moreover, examples of how elements of the method can operate and / or what the elements can be are described in the Systems section above. Moreover, additional steps may be required to perform the method and these steps will be obvious to a person having ordinary skill in the art.

[0055] The method 500 can be executed by a controller to work on a single OoC module. The single OoC module can have at least an oxygen sensor configured to detect an amount of oxygen (e.g., dissolved within the culture medium) for a surrounding distance for a time and at least one oxygen scavenger configured to decrease an oxygen level within the culture medium surrounding the at least one type of biological material (at the time). In some instances, the OoC module can have and / or be associated with at least one oxygen generator configured to increase the oxygen level within the culture medium surrounding the at least one type of biological material (however, the oxygen generator may be associated with the entire platform). Generally, the at least one oxygen scavenger is configured to down- regulate the amount of the dissolved oxygen in the culture medium and the at leastBWH2025-025 one oxygen generator configured to up-regulate the amount of the dissolved oxygen in the culture medium.

[0056] At 502, a recording can be received (by the controller) from the oxygen sensor within a culture medium indicative of the oxygen level of the culture medium (the level of dissolved oxygen within the culture medium inside the OoC module) at a time. At 504, a determination can be made (by the controller based on consulting the memory) of whether the oxygen level at the time (within the culture medium inside the OoC module) needs to be adjusted. For example, the controller can determine whether the oxygen level is at a value greater than 0% and less than 21% that is preselected for the biological material (at the time). As another example, the controller can determine whether the oxygen level within a predefined range for the biological material (e.g., within a target oxygen level range related to different types of biological materials, such as (may be variable ranges with different tissue types) alveoli microtissue -12.5-13.5%, arterial microtissue - 12.5-13.5%, bone microtissue - 6.5-7.5%, liver microtissue - 12.5-13.5%, kidney microtissue - 5.5-6.5%, gut microtissue - 7.5-8.5%, and / or vein microtissue - 2.5-3.5%) (at the time)? As a further example, the controller can determine whether the oxygen level equal to a stored oxygen level for the biological material (at the time). If the answer is no, the method 500 can revert back to step 502. If the answer is yes, at 506, at least one oxygen scavenger can be controlled to set the oxygen level to a value. Controlling the at least one oxygen scavenger can ensure that the oxygen level of the culture medium (e.g., surrounding the biological tissue) in the value. Then, once the value is set, the method 500 can revert back to step 502. Steps 502, 504, and 506 can be repeated at another time. However, it should be understood that the method 500 can be conducted sequentially and / or done in tandem for another area (e.g., another OoC module with a different biological sample) with numbers stored for the different biological sample.V. Experimental

[0057] This experiment illustrates a microtissue-on-a-chip platform that can accommodate different physiological oxygen levels in different microenvironments of different microtissue. Not only can the microtissue-on-a-chip platform enable realtime monitoring of physiological oxygen levels, but more importantly, the microtissue- on-a-chip platform can provide tight control of the physiological oxygen levels in the range of 4-20% in each microenvironment. Moreover, the scavenger describedBWH2025-025 herein refers to the entire microfluidic module with the culture medium flowing through the top and the chemical (scavenger chemical) flowing through the bottom separated by a membrane (e.g., PMMA membrane).Methods

[0058] Materials

[0059] NaaSOs, CoSO4, tris(4,7-diphenyl-1 ,10-phenanthroline) ruthenium (II) dichloride (Ru(ddp), Alfa Aesar), dichloromethane, isopropanol, phosphate-buffered saline (PBS), APAP, sodium dodecyl sulfate (SDS), toluene, gelatin from porcine skin (Type A, -300 g Bloom, average Mw =90,000 Da), albumin, chloroform, methacrylic anhydride (MA) were purchased from Sigma-Aldrich. Lithium phenyl- (2,4,6-trimethylbenzoyl) phosphinate (LAP, photoinitiator, PI) was purchased from Allevi. Dulbecco’s modified Eagle’s medium (DMEM), live / dead kit, fetal bovine serum (FBS), and penicillin-streptomycin was purchased from Life Technologies. LIVE / DEAD Viability / Cytotoxicity Kit, PrestoBlue cell proliferation reagent, TRIzol reagent, and human albumin enzyme-linked immunosorbent assay (ELISA) kit were purchased from Thermofisher. Human KIM-1 DuoSet ELISA kit was purchased from Bio-Techne, and ET-1 ELISA kit was purchased from Abeam. iScript Reverse Transcription Kit (1708841 ) was purchased from Bio-Rad. Syringe filters (0.22 pm in pore size) were purchased from VWR International, polydimethylsiloxane (PDMS) was purchased from Dow Corning Sylgard 184. Black and transparent poly(methyl methacrylate) (PMMA) were purchased from McMaster-carr. Epoxy glue was purchased from Amazon. All of the optics were purchased from Thorlabs, Inc.

[0060] Chip-fabrication

[0061] Raw-cast transparent and black PMMA sheets (0.95 cm in thickness) were used to fabricate microfluidic chips. The PMMA chip had a low gas permeability coefficient (2.5x10-12m2s-1). A commercial CO2 laser-cutting machine (25 W, wavelength 10.6 pm, Universal Laser Systems) with an adjustable X-Y-Z for laser micromachining of the PMMA sheets. The laser-cutting process was operated in software VLS.2.30 for adjusting ablation power, speed, and distance. CorelDRAW Graphics Suite X5 was used to design the geometry of the chip. The PMMA chip was cleaned with chloroform vapor treatment. The PMMA chips engraved with microchannels were exposed to chloroform vapor at room temperature for 5 min and then 70 °C for 30 min. The surface of the microchannels became smooth and clean after treatment. The bioreactor was assembled in a resealable manner to allow forBWH2025-025 the opening and closing of the system for subsequent biological assays when necessary. For fabrication of the oxygen-scavenger, open microchannels were ablated on the two pieces of PMMA chips. Subsequently, a PDMS membrane was fabricated by spin-coating at 5,000 rpm with a ratio of 8:1 , 10:1 , or 12:1 and incubated at 80 °C for 2 h. The PDMS membrane was then sandwiched between two pieces of PMMA chips and sealed underwent thermal treatment in a vacuum oven at 50 °C and -30 psi for 2 h. The inlet and outlet were connected to the main microchannel, while the inlet of the bottom microchannel was connected with Na2SC>3 solution integrated with the syringe pump. For fabrication of the mixer, microchannels in the PMMA chips were created by CO2 laser-ablation and were sealed by thermal treatment. For fabrication of the oxygen-generator, an open- channeled PDMS microfluidic chip was fabricated by a photolithography technique. The microchannels were sealed by a PDMS membrane, which were sandwiched between PMMA chips. A closed-loop modular organ-on-a-chip platform was integrated by a Teflon tubing or a Tygon microbore tubing (inner diameter: 2 mm; Cole-Parmer). All the connections were sealed by epoxy.

[0062] Biosensor-fabrication

[0063] PDMS beads were generated using a 3-to- 1 converging microfluidic flowfocusing devices made of PDMS with 100-pm dimensions. A surfactant solution (0.5% SDS) was flown through the side channels (50 pL / min), to form the continuous phase, and a 1 :1 mixture of toluene and 1 :10 PDMS pre-polymer solution (Sylgard 184) was flowed through the middle channel (1 pL / min), to form the dispersive phase. The collected PDMS microdroplets along with the continuous phase were maintained at room temperature overnight, then put into a 60QC oven overnight. The continuous phase solution was then removed, the cured PDMS beads washed with DI water, and incubated at room temperature in the dark in a 5 mg / mL dichloromethane solution of Ru(ddp) (tris(4,7-diphenyl-1 ,10-phenanthroline) ruthenium(ll) dichloride (Alfa Asar) for 24 h. The dichloromethane solution was then removed, the beads washed with 5-mg mL-1Ru(ddp) isopropyl alcohol solution, then DI water. The beads were stored in the dark until use.

[0064] The monodispersed oxygen-sensing beads were gently brushed onto the microchannel and fixed by the PDMS curing agent. The oxygen-sensor was integrated with a customizable mini-microscope, which was fabricated from a blackBWH2025-025PMMA sheet. The top layer was assembled with LED light as an excitation source, while a web camera was assembled at the bottom of the microscope. Band-pass optical filters were mounted above and below the sensing chips respectively. Micrographs were captured from the software, and the emission intensity was measured by MATLAB programs.

[0065] Integration

[0066] The whole system was controlled by a custom-coded MATLAB program, which served as the central control hub for real-time oxygen-monitoring and controlling. The program controlled an Arduino Mega 2560 microcontroller, which was connected as a master to a syringe pump of the oxygen-scavenger. Additionally, the LED470s of the mini-microscopes were connected to the laptop running the MATLAB program. The control system functioned in adjusting the flow rate of the syringe pump, which in return changes the final oxygen level in the culture medium at the output of the oxygen-scavenger. The process of control is shown as follows. First, the MATLAB code was run, which initialized the mini-microscopes and the Arduino that communicated with MATLAB via a serial port. The Arduino triggered the LEDs in the mini-microscopes to turn on, while the MATLAB captured a picture from each camera. Once the pictures were captured, the MATLAB sent a signal to the Arduino to turn the LEDs off. The LEDs were only switched on when capturing a picture to avoid photobleaching of the sensors.

[0067] The image-processing was also done using built-in MATLAB functions. First, each picture was separated into the three RGB channels, which provided the R hue of the sensor. Only the R channel of the picture was used. Each picture was binarized to generate a mask. This mask was then used to isolate the parts of the picture. The program then calculated the mean intensity value of the isolated pixels. For each chip, a base picture was used as a reference. This base picture was obtained by processing a picture of the chip at the oxygen level of 0%. After calculating the ratio between the mean intensity values ( / o / / ) of the study picture and the based picture, the mean oxygen levels were obtained using the Stern-Volmer equation. Given the oxygen value of the microfluidic chip, a classificatory coded in the program decided the signal that was sent to the Arduino to control the pump flow rate. The Arduino was connected to the pumps using the RS485 communication and the MODBUS protocol. The connection between the Arduino and the pump was carried out by a converter (MAX485, Maxim Integrated). The microcontrollerBWH2025-025 controlled the revolutions per minute of the pump, which in turn controlled the speed of the pumped culture medium. The working flow rate for the closed-loop microfluidic system for each organ-on-a-chip was controlled by the peristaltic pump at a constant flow rate of 200 pL h’1, which could also be used to control start / stop, and flowing direction. The minimum overall volume of the three connected multi-organ-on-chips system was 100 mL.

[0068] To facilitate user control, a graphic user-interface (GUI) was also programmed using MATLAB. The interface allows the user to visualize each sample in real-time, along with the mean intensity of each microfluidic chip and the corresponding oxygen levels. Besides, the pump can be directly turned on and off, while the flow can be modified by the user without relying on the programmed classificatory. The program updates each picture every 30 min by default using a timer (also run in MATLAB). The time intervals can be adjusted by the user on the GUI.

[0069] Steps of the algorithm1 ) Initialization of the mini-microscopes and Arduino.2) Activation of the LEDs via Arduino. Capturing and digitalization of the pictures for each mini-microscope via the USB ports. Deactivation of the LED via Arduino.3) Separation of the RGB layers for each picture. Binarization of the images via the R component. Obtaining of the mean value for each binarized R- component.4) Calculation of the oxygen concentration using the Stern-Volmer equation, via comparison to the mean-values of the previously processed base picture of the chip at 0% of oxygen level.5) Application of the classification algorithm to the obtained oxygen concentration, which adjusts the pump flow rate via Arduino.6) Steps 1 -5 are repeated every 30 min until the oxygen level of each sample chip reaches the desired level.

[0070] Classification algorithm (in pseudocode)COUNT=0 DIR=1IF the measured oxygen concentration M is greater than the desired value VIF DIR=0BWH2025-025Increase COUNT by 1Set DIR=1Increase FLOW RATE by 200 / 2ACOUNT pL / hIF the measured oxygen concentration M is greater than the desired value VIF DIR=1Increase COUNT by 1Set DIR=0Decrease FLOW RATE by 200 / 2ACOUNT pL / hEND when M and V coincide or the flow rate increase reaches minimum resolution.The GUI is the interface by which the user can take pictures using the minimicroscopes, process the pictures, visualize the mean oxygen levels, and control the pump flow rates. It uses the following programs to control certain parts of the operation:Initialize Cam 1: The first program, which opens the first webcam, sets its parameters and initializes the serial connection with the Arduino.Initialize Cam 2: Turn on the second webcam and set its parameters.Initialize Cam 3: Turn on the third webcam and set its parameters.Initialize Cam 4; Turn on the fourth webcam and set its parameters.Timer Integration: To control the turning off and on of the LEDs in the minimicroscope, a timer needs to be initialized.Integration: This is the main operating program. It communicates with the Arduino continuously to take pictures from the mini-microscope, process the picture using the Processing Cam function, and control the oxygen level by adjusting the pump flow.Processing Cam: It is the main picture processing program. Using MATLAB image processing functions, it binarizes the picture and isolates the relevant parts, where the beads are located.Finish Integration: This program deletes all variables, clears the Arduino object, and terminates the timer.

[0071] Cell culture

[0072] Human hepatocellular carcinoma cells (HepG2 / C3A), human proximal tubule epithelial cells (HK-2), and human umbilical vein endothelial cells (HUVECs) (ATCC) were maintained in DMEM with 10% FBS and incubated at 37 °C with 5% CO2. For static culture, culture medium was renewed every 2 days. Cell spheroids were formed followed by deposition in the microwells in PDMS molds. Cell spheroidsBWH2025-025 were collected after a 7-day culture in the microwells for the subsequent encapsulation.

[0073] Cell encapsulation

[0074] The collected cell spheroids were blended with a pre-gel solution containing GelMA (7%, w / v) and 0.5 wt% LAP. The microtissues were photocrosslinked on a glass slide by using a photolithography technology exposing to UV light (0.5 W cm-2, 20 s). The uncrosslinked solution was washed, generating the 45-micropillar array on a glass slide. Subsequently, the microtissue was assembled in the PMMA bioreactor with an inlet and an outlet.

[0075] Hypoxia incubator experiments

[0076] For static culture, the 3D microtissue was placed in the wells of 12-well plates and incubated in the hypoxia chamber supplemented with oxygen level at 5%, 13%, or 20%, each at a time. For dynamic culture of the individual microtissue-on-a- chip systems, the 3D microtissues were separately assembled in the bioreactors, and dynamically supplied with fresh culture media using separate syringe pumps. The bioreactors were incubated in the same hypoxia incubator. For dynamic culture of connected multi-organ-on-chips, each microtissue-on-a-chip was connected in parallel and dynamically supplied with a unified fresh culture medium through all modules using a peristatic pump.

[0077] Microtissue viability, proliferation, and functionality

[0078] The viability of microtissues within the hydrogel constructs was investigated by a Live / Dead assay. Briefly, LIVE / DEAD Viability / Cytotoxicity Kit was diluted with PBS to a final concentration of calcein AM at 0.5 pL mL-1and ethidium homodimer-1 at 2 pL mL-1. The microtissues were dissembled from the bioreactor and incubated with the working probe solution (-100 pL) at 37 °C for 30 min. Fluorescence micrographs were captured by using the inverted fluorescence microscope (Olympus). Live hMSCs were stained in green, while dead cells were stained in red. The viability was quantified by counting live and dead cell numbers using Imaged (National Institutes of Health). The evaluation of proliferative activity was conducted by using a Prestoblue assay. The microtissues were added to the 24- well plate. The PrestoBlue reagent (20 pL) was added to each microwell and mixed with 180 pL of culture medium at 37 °C for 4 h. The supernatants were collected and assessed by using a microplate reader (Bio-Tek Instruments). For the albumin test and the KIM-1 test, the culture media in the liver and kidney microtissuesBWH2025-025(bioreactors) were collected on Days 1 , 3, 5, and 7 at the outlets of the bioreactors respectively. The concentrations of the secreted albumin and KIM-1 in the supernatants of the bulk culture media were assessed by the ELISA assays using the human albumin ELISA kit and the human KIM-1 DuoSet ELISA kit.

[0079] Quantitative PCR

[0080] Total RNA was isolated from cell cultures using the TRIzol reagent. RNA concentration was measured using a NanoDrop spectrophotometer and Purity was determined by A260 / A280. Isolated RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and the purity was determined by A260 to A280. Isolated RNA (1 pg) was reverse transcribed into cDNA using the iScript Reverse Transcription Kit. Quantitative PCR (qPCR) was performed using the iTaq Universal SYBR Green Supermix (BIO-RAD, 1725121 ) on a CFX96 Real-Time System (Bio-Rad). All samples were measured with technical duplicates and normalized against GAPDH. Changes in the mRNA expression were calculated using the AActmethod relative to a control.

[0081] Simulating oxygen-consumption

[0082] Microtissues consume oxygen and are exposed to shear stresses in the microfluidic chips. In general, fluid dynamics in organ-on-a-chip systems can be described by Navier-Strokes equations to simulate oxygen transport through the flowing medium in the bioreactors. Considering lateral flow in a device, the general Navier-Stokes equation is described as: p^ + P& ■ V)u + Vp — Y] V2u — (A + rj)V(V - u) = f, (Eq. 1 ) where p is the dynamic fluid viscosity and -A is a second viscosity coefficient (related to compressibility of the fluid). To model and simulate the fluid flow inside the bioreactor, Navier-Stokes equations are simplified considering the fluid as incompressible, which means that the density p is constant in space as well as in time:where u denotes the velocity vector, p the density of the fluid, p the pressure, p = r] / p is the dynamic viscosity of the fluid and f is a body force term. Equation 4 is the momentum balance, and Equation 5 is the continuity for incompressible fluids. To determine the flow rate and cell density that enable the supplied oxygen level sufficient for the microtissue to maintain metabolic activities in the bioreactor, a mathematicalBWH2025-025 model (COMSOL Multiphysics) describing oxygen-transport in the bioreactor was utilized to estimate the oxygen-consumption rate of the microtissue, assuming that this rate follows the Michaelis-Menten kinetics.where Vmaxrepresents the maximum volumetric oxygen consumption rate, C02is the oxygen concentration in the cell layer, and KMM 02is the Michaelis constant. Therefore:where q is the oxygen-consumption rate per cell (4.8 x10-16mol s-1per cell), Ncellis the cell number, and V is the volume of microtissue. All boundaries were considered to be impermeable to oxygen, since the microtissue was enclosed in a thermoplastic chip. The boundary condition for the solid-fluid interface consisted of no-penetration into system walls and no-slip. The oxygen level at the inset was set to be constant. Ccris the critical limit and considered to be 1 .0x10~4mol nr3, while diffusion coefficient of oxygen in the culture medium at the physiological temperature is 3.8x1 O’9m2s’1.Results

[0083] Chips was separately patterned via direct laser-ablation. To allow the patterns with high precisions and defined geometries, the laser power and speed were optimized for the CO2 laser beam. After surface-smoothing and thermal treatment, the thermoplastic microfluidic chips were assembled and integrated into a closed-loop circuit. To ensure sufficient oxygen and nutrient supplies at the vicinity of microtissues, the on-chip circulation was driven by a peristaltic pump primed at a flow rate of 200 pL h-1powered by programmed MATLAB codes. These codes were also written to drive the optical sensor(s) that monitor oxygen level(s) in the system at predetermined time points and to control the pump(s) integrated with the oxygen- scavenger(s). A flow rate-monitor was also encoded into the program to monitor the flow rate as well as potential channel-blockage or leakage.

[0084] The closed-loop organ-on-a-chip platform was designed to be modular, including a programmable controlling system for microfluidic routing, oxygen levelregulation, and real-time monitoring, bioreactor(s) for housing the microtissue(s), customized mini-microscope(s) integrated with optical oxygen-sensor(s), oxygen- scavenger(s) connected to flow rate-controlling pumps, and an oxygen-generator forBWH2025-025 global oxygen-exchange. To mimic the physiological microenvironments in the human body, the entire closed-loop platform was installed in a customized benchtop incubator maintained at 37 °C, 5% ambient CO2, and 21% ambient oxygen that was connected to an oxygen-controlling and monitoring system. The classificator code allowed the system to automatically alter the flow rate on each chip until the desired oxygen level was reached, based on the optical sensor(s)’ monitoring. Alternatively, a custom-made user-interface enabled direct control over the pumps, and a real-time visualization of the oxygen-sensors. Individual modules were interconnected by using Teflon tubes, which allowed for fluid flow in the circulation. The oxygen level in each microchannel was controlled by “Light Timer Control” (excitation duration of the mini-microscopes), “Pump Control” (syringe pumps connected to the oxygenscavengers), and real-time-monitored in “Microscope Pump”. “Microscope Output” was designed to monitor the oxygen level in the recycled culture medium. To ensure that the platform is compatible with multiple tissues that require different oxygen levels, several branches of the microfluidic channels could be designed to maintain intended oxygen levels (e.g., 5%, 13%, and 20%, respectively) in the desired microtissue modules. As the microtissues normally consume the dissolved oxygen in the culture medium, the oxygen level at the outlet of the microtissues was preset to 3% and monitored in “Microscope Output”.

[0085] Real-time oxygen-monitoring and controlling systems

[0086] To achieve real-time monitoring of oxygen levels within the system, a customized microfluidic oxygen-sensor was integrated, where the fluorescence emission intensity of the sensing microbeads was observed and converted to the dissolved oxygen level in the programmed software. The fluorescent oxygen-sensing beads were produced by using a flow-focusing microfluidic droplet-generator, which could encapsulate oxygen-sensitive fluorescent dye (tris(4,7-diphenyl-1 ,10- phenanthroline)ruthenium(ll) dichloride) in a PDMS matrix. These as-synthesized oxygen-sensing microbeads were well-dispersed, where the average diameter was approximately 20 pm. To ensure the real-time monitoring of the oxygen level of the flowing culture medium in the microchannel, the fluorescent microbeads were fixed on the surface of the microchannel. The microfluidic oxygen-sensing chip was installed in a customized mini-microscope, where the emission of the sensing microbeads could be observed under the excitation of a high-intensity 470-nm lightemitting diode (LED) (FIG. 6A and B) and directly monitored by a colorBWH2025-025 complementary metal-oxide-semiconductor (CMOS) chip. The CMOS chip integrated with the custom-coded program could then split a captured image into the red (R), green (G), and blue (B) channels and analyze the emission intensities. As expected, a high emission intensity signal was observed in the R channel of the captured image, while the signals of G and B were negligible (FIG. 6C).

[0087] Stability is an important parameter for long-term oxygen-monitoring by using the fluorescent oxygen-sensor. One approach is to decrease the decay time of the fluorophores. The intrinsic fluorescence-decay as the exposure time was prolonged, was assessed. The A / of the oxygen-sensor decay decreased as the exposure time was prolonged to 180 min. The fluorescence decay was stable after 150 min. The fluorescence-decay is described by the Stern-Volmer equation:where T0represents sensor lifetime at 0% of the oxygen level, znis the sensor lifetime at the measured oxygen level [O2]„, and Kqis the Stern-Volmer quenching constant (2.7x103pmol L1). Following the 180-min light exposure, the response time and reversibility of the oxygen-sensor were evaluated. The result in FIG. 6D shows that as the oxygen level was decreased from 20% to 5%, the measured response time was 140 s and the emission intensity increased 1.2-fold. When the oxygen level was increased from 5% to 20%, the response time was 60 s and the emission intensity decreased by 1 .2-fold . After six cycles, the emission intensity could be maintained at the original level, demonstrating high stability and reversibility of the microfluidic oxygen-sensor. The standard curve of the oxygen-sensor is shown in FIG. 6E. The effective detection range of the oxygen-sensor was between 20% to 0%, where the emission intensity increased linearly. The sensitivity of the sensor was calculated to be 0.02 %’1. Therefore, the developed microfluidic oxygen-sensor could be used for real-time monitoring of oxygen levels in the microfluidic chip within the physiological range.

[0088] Dynamic oxygen-scavenging and mixing systems

[0089] To rapidly deplete excessive oxygen from the culture medium, an oxygen-scavenger was designed to integrate with the organ-on-a-chip platform. Sodium sulfite (Na2SO3) is a promising reagent that can irreversibly consume dissolved oxygen in the solution, producing harmless sodium sulfate (NasSC ). ToBWH2025-025 accelerate the rate of reaction, cobalt sulfate (CoSO4) was added as a catalyst following the reactions specified below:

[0090] However, the direct addition of Na2SOs and CoSO4 to the circulating medium is unrealistic to microtissue culture due to the toxicity of the scavenging solution and the unnecessary need for alteration of the shear force acting on the microtissues when changing the oxygen levels based on scavenging solution flow rate. Thus, a thermoplastic microfluidic chip was fabricated with three layers, where a PDMS membrane with a thickness of 20 pm was sandwiched between the engraved PMMA layers to seal the open microchannels and separate the two flow layers (FIG. 7A). The culture medium flows within the microchannel of the top layer in the scavenger (FIG. 7B-i and ii), while the mixture of Na2SO3 and C0SO4 flows within the bottom microchannel. Since PDMS has high selectivity to oxygen and poor permeability to the aqueous solution and the chemical species, dissolved oxygen molecules in the culture medium could diffuse into the scavenging solution through the PDMS membrane and rapidly diminished there. The optimization of the PDMS membrane in terms of the ratio of polymer base and curing agent was investigated to achieve the highest oxygen permeability. PDMS membranes with different ratios at 8:1 , 10:1 , and 12:1 were utilized to fabricate oxygen-scavengers. The oxygendepletion effect of the scavengers was assessed by real-time measurements of the oxygen levels at the outlet of the main channel in each oxygen-scavenger while the inlet of the scavenger was supplied with fresh culture medium (20-21 % of oxygen). It was observed that the oxygen-depletion effect increased 51 .2% as the ratio was elevated from 8:1 to 12:1 . As the flow rate was ramped up from 1 ,000 to 2,000 pL IT1, the oxygen-depletion effect increased from 26.7% to 38.7% at different ratios. The result demonstrated that the oxygen-permeability of PDMS membrane was improved as the ratio was increased since the higher ratio resulting in a less concentrated polymer network would allow for oxygen-diffusion more easily. However, when the ratio was higher than 13:1 , the PDMS membrane was too soft to use. Therefore, the ratio of polymer base and curing agent at 12:1 was applied for the following experiments.

[0091] To generate homogenous oxygen levels in the flowing culture medium, a chaotic mixer was designed and connected with the outlet of the scavenger. TheBWH2025-025 mixer with patterns of grooves fabricated by direct laser-writing could generate transverse flow in the microchannel, making it possible to apply efficient microscale flow-mixing (FIG. 7C). The manipulation of oxygen levels in the culture medium was investigated by adjusting the flow rate and scavenging reagent concentration in the bottom microchannel of the scavenger. The oxygen level of the culture medium downstream was monitored by the abovementioned oxygen-sensor. As the flow rate was increased from 1 ,000 pL h-1to 2,000 pL IT1, the oxygen level decreased linearly. The highest value of oxygen-depletion was 63.0% when the NaaSOs concentration was 20 wt%. As the NasSOs concentration was decreased to 2 wt%, the oxygendepletion dropped to 24.3%. To ensure that the oxygen-scavenger could supply the culture medium with a wide range of oxygen levels, NaaSOs at the concentration of 20 wt% was chosen for the subsequent studies. C0SO4 at a concentration of 0.02 wt% was used as the catalyst to accelerate oxygen-depletion by the scavenging agent. Thus, the oxygen level in the outlet was designed to be controlled by adjusting the flow rate of the scavenger. The result showed that the oxygen level was approximately 13% when the flow rate was set to 1 ,000-1 ,250 pL IT1. TO obtain the oxygen level at 5%, the flow rate was set to 1 ,750-2,000 pL h-1. The stability of the system was estimated by monitoring the oxygen level every 30 min. After a 3-h observation, the oxygen level was maintained compared to the initial measurement (FIG. 7D). Cropped micrographs from the user-interface further evidenced that as the flow rate was increased from 1 ,000 to 2,000 pL h-1, the emission intensity increased (FIG. 7E).

[0092] Dynamic oxygen-generating system

[0093] A closed-loop modular organ-on-a-chip platform requires the culture medium to be ideally circulated in the entire system. Since oxygen is constantly consumed by microtissues in the platform, the increase in oxygen level from a low level to 20-21 % should be considered, and in fact, is essential. Accordingly, an oxygen-generator was designed that was composed of a microfluidic chip sealed with a PDMS membrane with a thickness of 50 pm (FIG. 8A). Again, since the PDMS membrane has a high permeability to oxygen molecules at 35-37 °C (800x10-10cm3(STP) cm2s-1cmHg-1), the dissolved oxygen level could be effectively increased by exchanging with oxygen in the ambient air, functioning as an artificial breathing ‘lung’ that oxygenates the ‘blood’ (medium). For well-mixing the flowing culture medium in the oxygen-generator, staggered herringbone patterns were fabricated atBWH2025-025 the bottom of the microchannel, where the culture medium could perfuse without any noticeable leakage (FIG. 8B). The oxygen levels at the inlet and the outlet of the oxygen-generator were measured to be <4% and 20%, respectively (FIG. 80). Longer-term monitoring of oxygen levels at the outlet shows that as the time was prolonged to 3 h, the measured oxygen level was still maintained at approximately 20% (FIG. 80), which demonstrated the high stability of the oxygen-generator.

[0094] The geometry of the bioreactor was adopted from our previous reports to ensure a uniform fluid flow distribution (FIG. 9A). A configuration featuring an array of 45 cell-encapsulating 3D microstructured gelatin methacryloyl (GelMA) dots was designed to emulate the microtissue as a proof-of-concept (FIG. 9B). The cell- encapsulated GelMA hydrogel was micropatterned to allow for culture medium flow and oxygen-exchange. The oxygen level at the inlet was kept constant at 13%. To ensure the oxygen level at the outlet to remain at 3%, which is comparable to the oxygen level in the venous blood in general scenarios, cell number and flow rate were optimized in the simulation. As the cell number was increased from 0.4x106to 1 xl o6cell mL-1(the flow rate was set to 200 pL IT1) and the flow rate was decreased from 800 pL h-1to 200 pL h-1(the cell density was set to 1 x106cell mL-1), the numerically simulated oxygen level decreased from 8% to 3% (FIG. 9C).

[0095] The fabricated thermoplastic bioreactor shown in FIG. 9D contained a microtissue, which was fabricated by photopatterning. To guarantee sufficient oxygen-diffusion and culture medium flowing within the microtissue, 3D GelMA- encapsulated cells were patterned into a dot array as the microtissue at the bottom of the bioreactor. The bioreactor was assembled with resealability that allowed for convenient post-analyses of the microtissue (FIG. 9D). Higher flow rates of the culture medium would increase laminar shear force exerted across the microtissue, while lower flow rates could not provide sufficient oxygen-diffusion within the microtissue. Therefore, 200 pL h-1and 1 x106cell mL-1were applied throughout the subsequent experiments.

[0096] Effects of oxygen levels on biological behaviors and drug responses

[0097] To investigate the oxygen effects on physiological and metabolic activities of our multi-organ-on-chips platform, a static 3D microtissue culture as a conventional method was first developed. The liver is the main organ for drug metabolism, and the hepatic artery is the major source of oxygen for the hepaticBWH2025-025 cells. The oxygen availability would be expected to differ at different parts of the liver, according to the supply of blood, where the most common range is 10-13% and can be lowered in certain other areas. The 3D liver microtissues were fabricated by applying the photolithography technique to GelMA hydrogel-encapsulated human hepatic spheroids. A static culture placed the 3D liver microtissues in the hypoxia incubator setting the oxygen levels to 5%, 13%, or 20%, separately, and supplemented with fresh standard culture medium every 2 days. The immortal human hepatocellular carcinoma (HepG2 / C3A) cells have been shown to exhibit the main functions of liver tissues. Accordingly, the 3D GelMA-encapsulated HepG2 / C3A cells were used to create liver microtissues. The viability of the liver microtissues was evaluated by a Live / Dead assay, in which the live cells were stained in green, while dead cells in red. It was shown that the viabilities of the liver microtissues at the oxygen level of 13% were 31% and 23% higher than the counterparts at the oxygen levels at 5% and 20% on Day 7, respectively. Proliferation evaluations using the Prestoblue assay further validated that the liver microtissues were more amenable to survival at the oxygen level of 13%. Albumin is synthesized in the liver, where its functions include maintaining osmotic pressure and transporting a variety of circulating molecules. Serum albumin levels are manipulated via hepatic tissue secretion, mass-exchange among the intra- and extravascular compartments, protein levels, lymphatic uptake, and body changes. The functions of the liver microtissues were investigated by the assessments of albumin-production. After a 7- day incubation, albumin-production of the liver microtissue at the oxygen level of 13% was 2.1 -fold and 0.8-fold higher than the counterparts at the oxygen levels of 5% and 20%, respectively. These results demonstrated that the liver microtissues exhibited favorable activities at the oxygen level of 13%, studies while 5% was hypoxic and 20% was hyperoxic to the liver microtissues.

[0098] The kidney is the main organ to eliminate drugs from the body. To mimic the kidney, the similar fabrication process was also applied to produce nephric microtissues by 3D encapsulation of nephric spheroids. HK-2 cells, immortalized proximal tubule epithelial cells from normal adult human kidney, were used to produce nephric spheroids and encapsulated within the 3D micro-GelMA dots. The viabilities and proliferation activities of the statically cultured kidney microtissues in the hypoxia incubator confirmed that the oxygen level at 5% was the most functional for kidney microtissues as compared with the higher oxygen levels at 13% and 20%.BWH2025-025Kidney injury molecule-1 (KIM-1 ) is a biomarker that can be upregulated in acutely and chronically injured kidney tissues. The functions of the kidney microtissues were investigated by the detection of the secreted KIM-1 levels. The KIM-1 -expression of the kidney microtissues at 5% of the oxygen level was 76% and 93% lower than the counterparts at 13% and 20% of the oxygen levels after a 7-day incubation, respectively. Thus, the oxygen level of 5% was deemed the most suitable for static culture of kidney microtissues in the conventional hypoxia incubator.

[0099] Blood vessel is an organ that transports blood throughout the body by circulatory system. To mimic the blood vessel, the same photolithography technique was utilized to produce GelMA hydrogel-encapsulated human umbilical vein endothelial cells (HUVECs), which is a typical model cell for studying arterial blood vessels. The vessel microtissues exhibited the optimal behaviors at the oxygen level of 13% in the viability and proliferation activity tests as compared to the oxygen levels at 5% and 20% (FIGS. 10B-i-iii). Endothelin 1 (ET-1 ) is a potent endogenous vasoconstrictor that is secreted from dysfunctional endothelial cells. As compared to the secretion of ET-1 in the vessel microtissues at the oxygen levels of 5% and 20%, the ET-1 level in the vessel microtissues at the oxygen level of 13% was the lowest throughout the 7-day incubation. The results demonstrated that the oxygen level at 13% was suitable for the static culture of the 3D arterial vessel microtissue in the hypoxia incubator. Taken all together, the liver, kidney, and arterial vessel microtissues are sensitive to both hypoxia and hyperoxia. Lower oxygen levels are more likely insufficient to provide enough oxygen to mitochondria for aerobic metabolism of microtissues, while higher oxygen levels would accelerate the progression of pathological phenotypes.

[0100] To reconfirm the optimal oxygen level of each microtissue-on-a-chip model, the 3D liver, kidney, and arterial vessel microtissues were separately placed in their bioreactors and integrated to the oxygen-controlling systems to form three individual organ-on-a-chip platforms, where the culture medium was driven by syringe pumps. By adjusting the parameters of the oxygen-scavengers, the oxygen levels in the flowing culture media were controlled at 5%, 13%, and 20%, respectively, optimized levels for the respective organ types. The viability test of the individual liver-on-a-chip showed that the viability at 13% of the oxygen level was 22% and 12% higher than those at 5% and 20% of the oxygen levels, respectively. The proliferation test further demonstrated that the liver microtissue-on-a-chip atBWH2025-02513% of the oxygen level showed 47% and 17% higher proliferation than the counterparts at 5% and 20% of the oxygen levels, respectively, after the 7-day incubation. Moreover, the liver microtissue-on-a-chip produced 40% and 18% higher of albumin at the oxygen level of 13% than the counterparts at 5% and 20%, respectively. Compared to the conventional static culture, the individual liver-on-a- chip integrated with the built-in oxygen-controlling systems showed similar results that the oxygen level at 13% in the culture medium was the most beneficial for the liver microtissue. Similar assembling processes of bioreactors, oxygen-scavengers, and oxygen-sensors were also applied to individual kidney microtissue-on-a-chip and individual arterial vessel microtissue-on-a-chip cultures. The viabilities, proliferation, and metabolic activities of the kidney microtissue-on-a-chip and vessel microtissue- on-a-chip proved that the optimal oxygen levels for the kidney microtissue was 5% and for the vessel microtissue was 13%, which again, were consistent with the static culture studies.

[0101] All the modules were subsequently integrated to form a closed-loop modular multi-organ-on-chips platform, where the culture medium was circulated and driven by a peristaltic pump. Each branch of the microtissue (liver, kidney, and arterial vessel)-on-a-chip connected to an oxygen-scavenger and a real-time- monitoring oxygen-sensor, together with a global oxygen-generator, all embedded with programmed automation controls, was assembled in parallel. The automation system was controlled in the user-interface. Whether the developed closed-loop modular multi-organ-on-chips platform could independently control the dissolved oxygen in the flowing culture medium at the optimal level for each connected microtissue-on-a-chip was investigated (Figure 10). To this end, based on the optimal oxygen level for each microtissue, the oxygen levels were independently controlled and monitored at 13% in both the connected liver microtissue-on-a-chip and the connected arterial vessel microtissue-on-a-chip, and at 5% in the connected kidney microtissue-on-a-chip (FIGS. 10A-i). The viabilities as well as proliferation and metabolic activities of the connected liver, kidney, and arterial vessel microtissue-on-chips at their respective optimal oxygen levels were studied (FIG.10A-ii-iv).

[0102] For the connected liver microtissue-on-a-chip at the optimal oxygen level of 13%, the viability was calculated to be 94% after the 7-day incubation where no significant differences were observed from the hypoxia incubator-based (95%) andBWH2025-025 individual microtissue-on-a-chip-based (92%) cultures (FIGS. 10A-ii, iii). In addition, the viability test of the liver microtissue in FIGS. 10A-ii and iii showed that dead cells were mainly distributed in the center of each dot (0: 1 mm, h: 0.5 mm). As the flow rate of the culture medium was maintained at 200 pL h’1, this observation was possibly due to distribution differences of oxygen and nutrient concentrations within the hydrogel matrix caused by the diffusion effect, which is also a good mimicry of native tissues since the liver tissues show zonation. Similarly, the proliferation activities and albumin-production of the connected liver microtissue-on-a-chip was 0.93 and 911 ng IT1, which was consistent with 0.88 and 890 ng h-1of the conventional static culture after the 7-day incubation (FIGS. 10A-iv and 1 OB-i).

[0103] For the connected kidney microtissue-on-a-chip at the optimal oxygen level of 5%, the viabilities were calculated to be 97% and 96% on Days 3 and 7, which were similar to 96% and 89% of the conventional static culture in the hypoxia incubator and 97% and 96% of the individual kidney microtissue-on-a-chip culture (FIGS. 10A-ii, iii). The proliferation activities in the connected kidney microtissue-on- a-chip were 11 % higher than the conventional static and the individual microtissue- on-a-chip cultures, respectively (FIG. 10A-iv). In addition, KIM-1 levels of the connected kidney microtissue-on-a-chip culture were 15% and 12% lower than the corresponding levels of the static culture in the hypoxia incubator and the individual kidney microtissue-on-a-chip culture (FIG. 10B-ii), which demonstrated that the connected kidney microtissue-on-a-chip had lower injury.

[0104] For the connected vessel microtissue-on-a-chip at the optimal oxygen level of 13%, the viability was 92%, which was similar to the viabilities of 98% in the hypoxia incubator and 95% in the individual vessel microtissue-on-a-chip (FIG. 10A- ii, iii). The proliferation activity and the ET-1 level of the connected vessel microtissue-on-a-chip were also comparable to those of the static cultured vessel microtissue and the individual vessel microtissue-on-a-chip cultures (FIG. 10B-ii). Other oxygen levels were further applied to each connected microtissue-on-a-chip in the closed-loop multi-organ-on-chips platform, such as 5%, 13%, and 5% for liver, kidney, and arterial vessel microtissues, respectively. Conventional multi-organ-on- chips cultures were also used as a comparison by adopting the standard culture medium at the oxygen level of 20%.

[0105] The viabilities as well as proliferation and metabolic activities of these two scenarios were both lower than the multi-organ-on-chips platform supplementedBWH2025-025 with oxygen at the optimal levels to the intended organ types. Altogether, the develop modular closed-loop multi-organ-on-chips platform could independently and tightly control the oxygen level in each connected microtissue-on-a-chip to continuously supply culture medium for each microtissue at its own optimal concentration.

[0106] Since the conventional approach to control oxygen levels of multi-organ- on-chips is to incubate them collectively in a hypoxia incubator that could only control the oxygen in the culture medium at a single level, the oxygen effects on all microtissue-on-chips in this configuration were further observed. The connected liver, kidney, and arterial vessel microtissue-on-chips system without oxygencontrolling units were placed altogether in the hypoxia incubator providing the oxygen level at 5%, 13%, or 20%, each at a time (FIG. 1 1 ). It was shown that after the 7-day incubation, the viabilities of liver, kidney, and vessel microtissues at the unified oxygen level of 5% were 70%, 94%, and 80%, respectively, while those at 13% were 96%, 80%, and 98%, and those at 20% were 74%, 64%, and 85% (FIG.11 A-ii). The liver and arterial vessel microtissues showed highest viabilities at the oxygen level of 13%, while the kidney microtissues showed a low viability at this oxygen level. Similarly, the proliferation activities of all the connected microtissue-on- chips in the hypoxia incubator at 13% of the oxygen level showed the highest levels in the liver microtissue and vessel microtissue (FIG. 1 1 B-iii) as compared with the counterparts at the oxygen level of 5% (FIG. 1 1 A-iii) and 20%, whereas the proliferation activity was higher in kidney microtissue-on-a-chip at 5% (FIG. 11 A-iii) than those at the oxygen levels of 13% and 20% (FIG. 11 B-iii). Albumin-productions were 91 % and 31 % higher at the oxygen level of 13% (FIG. 11 B-iv) than those at the oxygen levels of 5% (FIG. 11 A-iv) and 20%, respectively. The metabolic activities of the kidney microtissue confirmed that it favored the oxygen level of 5%, while this level was not suitable for the liver and vessel microtissues (FIG. 1 1 A-v). Similarly, 13% of the oxygen level was favorable for the vessel microtissue (FIG. 1 1 B-v), and the oxygen level at 20% was not amenable to any of the three microtissues based on the metabolic activity assays. The results clearly illustrated that the conventional hypoxia incubator-based approach could provide only one oxygen level during each experiment, where not all tissue types would be exposed to their optimal oxygen levels but only one or two at the maximum.BWH2025-025

[0107] To investigate whether there was a difference in the mechanism of drugpoisoning when the microtissues were cultured under different oxygen levels at 5%, 13%, and 20%, acetaminophen (ARAP) (5 mmol L-1) was separately applied to the conventional hypoxia incubator-, individual organ-on-a-chip-, and multi-organ-on- chips-based systems as described above. APAP is the most commonly used antipyretic and analgesic. When taken at therapeutic doses, it is considered a safe drug. Overdose can result in injuries of multiple organs, especially the liver and the kidney. The toxicity of APAP on the microtissues was first evaluated by the Live / Dead assay, the proliferation assay, and metabolic activity test in static culture and individual microtissue-on-a-chip systems separately. With APAP applied, substantial losses of viability in all the liver, kidney, and arterial vessel microtissues from Day 1 to 7 at all oxygen levels were observed. It is known that APAP is metabolically activated by the CYP enzymes to A / -acetyl-p-benzoquinone imine, which is normally detoxified by glutathione. With APAP-poisoning, the highest decreases in viability as well as proliferative and metabolic activities after the 7-day incubation were found in liver microtissues as compared with kidney and arterial vessel microtissues. For example, after APAP-poisoning at the overdose concentration of 5 mmol L-1for 7 days, the viability decreases of the liver microtissues in the static and individual microtissue-on-a-chip cultures were 71% and 78%, respectively, at the optimal oxygen level of 13%, while the viability decreases of the kidney microtissues in the static and individual microtissue-on-a-chip cultures were 50% and 35%, respectively, at the optimal oxygen level of 5%, and those in the individual arterial vessel microtissue-on-a-chip culture were 15% and 21%, respectively, at the optimal oxygen level of 13%. Similar results were also found in proliferation and metabolic activity evaluations. Moreover, the toxicities of APAP- poisoning on the vessel microtissue were lower than that on the liver and kidney microtissues, indicating that APAP was less toxic to vessel than liver at the dose assessed. These findings indicate that APAP-poisoning on liver, kidney, and arterial vessel microtissues could be affected by oxygen levels.

[0108] APAP (5 mmol L-1) was also applied to the closed-loop multi-organ-on- chips platform (FIG. 10). Fluorescence micrographs evidenced the effect of oxygen levels on APAP-poisoning (FIG. 10A-ii). Comparing the connected liver, kidney, and arterial vessel microtissue-on-chips, the APAP toxicity to the liver microtissue was 60%, which was 2.1 - and 3.5-fold higher in viabilities than the APAP toxicity to theBWH2025-025 kidney and vessel microtissues on Day 7 (FIGS. 10A-ii and iii). The proliferation test demonstrated that APAP affected 1 .1 -fold and 1 .5-fold higher in proliferation activities of the liver microtissue as compared to the activities of the kidney and arterial vessel microtissues (FIG. 10B-ii). The oxygen effect on APAP toxicity was also clearly shown in the metabolic activity test. For example, albumin-production decreased by 93% in the connected liver microtissue chip after the 7-day APAP- poisoning at the oxygen level of 13%, while they were decreased by 92% and 93% at the oxygen level of 5% and 20%, respectively (FIG. 10B-i). In contrast, the APAP toxicity to the liver microtissue was much lower at 5% and 20% in the hypoxia incubator as compared to that at the oxygen level of 13%. Therefore, the conventional hypoxia incubator-based multi-organ-on-chips system failed to clearly reflect drug metabolism within the connected culture.

[0109] The influence of oxygen level on the expressions of hypoxia and hyperoxia-induced genes was finally investigated. The mRNA levels of hypoxiainducible factor-1 a (HIF-1 a), lactate dehydrogenase A (LDHA), and glucose transporter 1 (GLUT1 ) were measured in liver, kidney, and arterial vessel microtissues cultured at oxygen levels of 5%, 13%, and 20% in all the configurations (FIG. 10C). HIF is a widely used marker for hypoxia. HIF-1 a is ubiquitously expressed and can be rapidly degraded in the presence of oxygen by the von Hippel-Lindau ubiquitin pathway. When the oxygen level decreases below physiologic levels, HIF-1 a does not undergo the initial hydroxylation required for the degradation, and protein levels increase. In the studies of the static culture, the HIF- 1 cr-expression of the liver microtissue was at a higher level in the oxygen level at 5%, which was 15% and 5% higher as compared to 13% and 20% of oxygen, respectively, on Day 3. Compared to the oxygen level at 13% on Day 7, the expression of the HIF-1 a was 21 % higher than at the oxygen level at 20%. It is assumed that microtissues exposed to hyperoxic conditions would generate more reactive oxygen species by mitochondria. The oxidative stress may promote HIF-1 a- induction. Together with the viability, proliferation, and metabolic activity assays, the results proved that the oxygen level at 13% was the most favorable for the liver microtissue among the three different oxygen levels. Similar results could also be observed in the liver microtissue-on-a-chip culture where the HIF-1 a expressions were 26% and 21% higher at 5% and 20% than those at 13% of the oxygen levels on Day 7. In addition, it was observed that the oxygen level affects the expressionsBWH2025-025 of HIF-1 a upon APAP-poisoning in the developed liver microtissue-on-a-chip culture. For example, HIF-1 a-expression increased by 11%, 12%, and 8% on Day 1 , while it increased to 13%, 18%, and 10% on Day 7 at the oxygen levels of 5%, 13%, and 20% after APAP-poisoning, respectively. The oxygen effects on HIF-1 cr-expressions in the multi-organ-on-chips platform were also evaluated (FIG. 10C-i). The expressions of HIF-1 a showed the lowest level in the connected liver microtissue-on- a-chip and the connected kidney microtissue-on-a-chip when exposed to 13% of the oxygen level as compared to the counterparts at oxygen levels of 5% and 20%, while the lowest level in the kidney microtissue-on-a-chip was at 5%.

[0110] The LDHA level is closely related to glucose metabolism, which can reduce pyruvate to lactate. This process allows glycolysis to proceed in an anoxic condition. Among the lactate dehydrogenase protein subunits, LDHA has been associated with oxygen levels. As the oxygen levels varied at 5%, 13%, and 20%, the LDHA expressions after APAP-poisoning were different. As shown in FIG. 10G-ii, the lowest expression of LDHA was found at the oxygen level of 5% as compared to 13% and 20% in the connected kidney microtissue-on-a-chip. This observation indicated that lower oxygen contents would be the more relevant oxygen levels for the cultured kidney microtissues, while the 13% and 20% oxygen levels are generally high for the kidney microtissue. The applied oxygen levels could affect the kidney microtissue responses to APAP-poisoning, with oxygen level at 5% delaying the microtissue death. In addition, the expression of LDHA was lower when the liver microtissues were exposed under 13% of the oxygen condition than hypoxia and hyperoxia. APAP-poisoning enhanced the expressions of HIF-1 a and LDHA at all oxygen levels. GLUT1 is the most studied subtype for basal glucose-uptake.Furthermore, GLUT1 is directly responsible for glucose-metabolism and is commonly upregulated by both HIF-1 a and HIF-2cr. GLLJT1 was selected as an indirect biomarker of HIF-1 function. It was shown that the lowest expressions of GLUT1 occurred at 5% in the connected kidney microtissue-on-a-chip and at 13% in the connected liver microtissue-on-a-chip and arterial vessel microtissue-on-a-chip cultures (FIG. 10C-iii). The expressions of HIF-1 a, LDHA, and GLUT1 in the connected kidney microtissue-on-a-chip revealed similar trends as those in the connected liver and arterial vessel microtissue-on-chips. Therefore, it can be concluded that the oxygen level affects the expressions of HIF-1 a, LDHA, and GLUT1 in the liver, kidney, and arterial vessel microtissues. Together with cellBWH2025-025 viability as well as proliferation and metabolic activity tests, the results again proved that the oxygen level at 13% would be the most favorable for liver and vessel microtissues and the kidney microtissues would be more amenable to survive at 5% among the three different oxygen levels. To conclude, the developed closed-loop modular multi-organ-on-chips platform could not only enable individual culture of multiple microtissues at their respective physiological oxygen microenvironments but also be used for studies on oxygen-induced drug metabolism.

[0111] From the above description, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes and modifications are within the skill of one in the art and are intended to be covered by the appended claims.

Claims

BWH2025-025The following is claimed:1 . A system comprising: at least one organ-on-a-chip module, each of the at least one organ-on-a-chip module hosting at least one type of biological material within a culture medium; each of the at least one organ-on-a-chip module comprises: an oxygen sensor; and at least one oxygen scavenger configured to decrease an oxygen level within at least a portion of the culture medium surrounding the at least one type of biological material; and each of the at least one organ-on-a-chip module connected to a controller, the controller comprising: a memory storing instructions; and a processor configured to access the memory to execute the instructions to at least: for each of the at least one organ-on-a-chip module, ensure that the oxygen level of at least the portion of the culture medium surrounding the at least one type of biological material is at least within a predefined range from greater than 0% to less than 21% based on recordings from the oxygen sensor and control of the at least one oxygen scavenger.

2. The system of claim 1 , wherein for each of the at least one organ-on-a-chip module, the processor ensures that the oxygen level of at least the culture medium surrounding the at least one type of biological material is within the predefined range from greater than 0% to less than 21 % by: receiving a recording of the oxygen level of at least the the culture medium surrounding the at least one type of biological material at a time; determining the oxygen level of the culture medium surrounding the at least one type of biological material at the time based on the recording; and determining whether the oxygen level of the culture medium surrounding the at least one type of biological material at the time is within a target oxygen level range for the at least one type of biological material.BWH2025-0253. The system of claim 2, wherein: when the oxygen level of the culture medium surrounding the at least one type of biological material at the time matches the target oxygen level for the at least one type of biological material, receive another recording at another time; and when the oxygen level of the culture medium surrounding the at least one type of biological material at the time does not match the target oxygen levels for the at least one type of biological material: take a corrective action by regulating the at least one oxygen scavenger to ensure that the oxygen level of the culture medium surrounding the at least one type of biological material matches the target oxygen level for the at least one type of biological material; and receive another recording at the other time.

4. The system of claim 3, wherein the at least one type of biological material comprises alveoli microtissue, arterial microtissue, bone microtissue, liver microtissue, kidney microtissue, gut microtissue, and / or vein microtissue.

5. The system of claim 3, wherein a target oxygen level range related to different types of biological materials comprises alveoli microtissue froml 2.5-13.5%, arterial microtissue from 12.5-13.5%, bone microtissue from 6.5-7.5%, liver microtissue from 12.5-13.5%, kidney microtissue from 5.5-6.5%, gut microtissue from 7.5-8.5%, and / or vein microtissue from 2.5-3.5%.

6. The system of claim 1 , wherein the at least one organ-on-a-chip module comprises at least two organ-on-a-chip modules connected in parallel.

7. The system of claim 6, wherein the at least two organ-on-a-chip modules are used for drug screening and the oxygen level affects the drug screening in each of the at least two organ-on-a-chip modules.

8. The system of claim 1 , wherein the at least one type of biological material comprises a cell and / or a tissue and the at least one organ-on-a-chip simulates a physiological microenvironment for the biological material.BWH2025-0259. The system of claim 1 , wherein the oxygen sensor detects an amount of dissolved oxygen in the culture medium within the at least one organ-on-a-chip module.

10. The system of claim 9, wherein the at least one oxygen scavenger is configured to down-regulate the amount of the dissolved oxygen in the culture medium, wherein at least one oxygen generator is configured to up-regulate the amount of the dissolved oxygen in the culture medium.

11. A method for controlling an oxygen level of a culture medium surrounding at least one type of biological material housed by at least one organ-on-a-chip module, the method comprising: receiving, by a system comprising a processor, a recording from an oxygen sensor within the culture medium indicative of the oxygen level of the culture medium; determining, by the system, whether the oxygen level of the culture medium needs to be adjusted to a value from greater than 0% to less than 21 %; and when the oxygen level of the culture medium needs to be adjusted to a value from greater than 0% to less than 21%, controlling, by the system, at least one oxygen scavenger within the culture medium to ensure that the oxygen level of the culture medium is the value.

12. The method of claim 11 , wherein another at least one organ-on-a-chip module comprising another part of the other medium surrounding another biological material is connected in parallel to the at least one organ-on-a-chip module, the method further comprises: receiving, by the system, another recording from another oxygen sensor within another part of the part of the medium surrounding the other biological material indicative of another oxygen level of the other part of the culture medium; determining, by the system, whether the other oxygen level of the other part of the culture medium needs to be adjusted to another value is from greater than 0% to less than 21 %; and when the other oxygen level of the other part of the culture medium needs to be adjusted to the other value from greater than 0% to less than 21%, controlling, byBWH2025-025 the system, at least another oxygen scavenger within the other part of the culture medium to ensure that the other oxygen level of the other part of the culture medium is the other value.

13. The method of claim 1 1 , wherein the at least one organ-on-a-chip module comprises the oxygen sensor; the at least one oxygen scavenger configured to decrease an oxygen level within the culture medium surrounding the at least one type of biological material; and at least one oxygen generator configured to increase the oxygen level within the culture medium surrounding the at least one type of biological material.

14. The method of claim 1 1 , wherein the controlling the at least one oxygen scavenger within the culture medium to ensure that the oxygen level of the culture medium is the value comprises: determining whether the oxygen level of the culture medium is within a target oxygen level range for the at least one type of biological material at a time.

15. The method of claim 14, wherein: when the oxygen level of the culture medium at the time matches the target oxygen level for the at least one type of biological material, receiving another recording at another time; and when the oxygen level of the culture medium surrounding the at least one type of biological material at the time does not match the target oxygen levels for the at least one type of biological material: take a corrective action by regulating the at least one oxygen scavenger to ensure that the oxygen level of the culture medium matches the target oxygen level for the at least one type of biological material; and receive another recording at the other time.

16. The method of claim 15, wherein the at least one type of biological material comprises alveoli microtissue, arterial microtissue, bone microtissue, liver microtissue, kidney microtissue, gut microtissue, and / or vein microtissue.BWH2025-02517. The method of claim 15, wherein a target oxygen level range related to different types of biological materials comprises alveoli microtissue -12.5-13.5%, arterial microtissue - 12.5-13.5%, bone microtissue - 6.5-7.5%, liver microtissue - 12.5-13.5%, kidney microtissue - 5.5-6.5%, gut microtissue - 7.5-8.5%, and / or vein microtissue - 2.5-3.5%.

18. The method of claim 11 , wherein the at least one type of biological material comprises a cell and / or a tissue and the at least one organ-on-a-chip simulates a physiological microenvironment for the biological material.

19. The method of claim 11 , wherein the oxygen sensor detects an amount of dissolved oxygen in the culture medium within the at least one organ-on-a-chip module.

20. The method of claim 19, wherein the at least one oxygen scavenger is configured to down-regulate the amount of the dissolved oxygen in the culture medium and the at least one oxygen generator configured to up-regulate the amount of the dissolved oxygen in the culture medium.

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