EP4655586A1

EP4655586A1 - Systems and methods for culturing, immobilizing, and/or imaging of biological samples

Info

Publication number: EP4655586A1
Application number: EP24747939.7A
Authority: EP
Inventors: Adela Ben-Yakar; Sudip Mondal; Evan HEGARTY; Khashayar MOSHKSAYAN; Anirudha HARIHARA
Original assignee: University of Texas System; University of Texas at Austin
Current assignee: University of Texas System; University of Texas at Austin
Priority date: 2023-01-27
Filing date: 2024-01-29
Publication date: 2025-12-03
Also published as: WO2024159214A1

Abstract

A microfluidic device is disclosed including at least one trapping device. A culture chamber adjacent the first end of the trapping device has a first height between the upper surface and the lower surface of the trapping device and a maximum width. First and second perfusion walls extend into the culture chamber from an upper surface towards a lower surface of the trapping device. First and second perfusion channels are defined by the perfusion walls and an adjacent side wall. A staging chamber is defined at a second end of the culture chamber having the same first height and a smaller width. An immobilization chamber is in fluid communication with the staging chamber and has a height that is less than the first height. The immobilization chamber is configured to retain a target biological subject while a fluid flows through the trapping device.

Description

SYSTEMS AND METHODS FOR CULTURING, IMMOBILIZING, AND/OR IMAGING

OF BIOLOGICAL SAMPLES

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Grant No. R43 ES029890 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/441,653, filed January 27, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] In recent years, organoid technology has emerged as an advanced in vitro model that can recapitulate the inherent characteristics of their corresponding organ tissue. Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. Interrogating various cell types and understanding the phenomena occurring within the organoid microenvironment requires high-content imaging (HCI). To achieve this goal, platforms are needed to enable high-resolution and high-throughput imaging of the samples in an automated, efficient, and fast manner.

[0003] Monitoring and end-point high-content imaging (HCI) of organoids are the key to better understanding the impact of drug administration on their development and morphology. Currently, platforms that enable both monitoring and HCI of organoids in a fast manner are missing. Therefore, a need exists for improved systems for imaging organoids.

SUMMARY

[0004] According to one implementation of the present disclosure, a microfluidic device includes at least one trapping device. The at least one trapping device includes a first end, a second end, at least one side wall extending between the first and second ends, a lower surface, and an upper surface opposite and spaced apart from the lower surface. The trapping device further includes a fluid inlet, a culture chamber, first and second perfusion walls, at least one staging chamber, at least one immobilization chamber, and an exit chamber. The fluid inlet is defined at the first end of the trapping device. The culture chamber is defined adjacent the first end of the trapping device and extends from the first end of the trapping device towards the second end of the trapping device. A first end of the culture chamber is in fluid communication with the fluid inlet, and a second end of the culture chamber is opposite of the first end of the culture chamber along a first axis of the trapping device. A first height of the culture chamber is defined between the upper surface and the lower surface of the trapping device. The first height is measured in a direction that is parallel to a second axis that is orthogonal to the first axis. A maximum width of the culture chamber is measured in a direction that is parallel to a third axis and that is orthogonal to each of the first axis and the second axis. The first and second perfusion walls extend into the culture chamber from the upper surface towards the lower surface of the trapping device. A free end of the perfusion wall is spaced apart from the lower surface of the trapping device. The perfusion walls extend from the first end of the culture chamber towards the second end of the culture chamber. First and second perfusion channels are respectively defined between each perfusion wall and an adjacent side wall of the at least one side wall of the trapping device. The at least one staging chamber is defined at the second end of the culture chamber having a height that is equal to the first height and a width that is less than the maximum width. The at least one immobilization chamber is in fluid communication with the at least one staging chamber and has a height that is less than the first height. The at least one immobilization chamber is configured to retain a target biological subject while a fluid flows through the trapping device towards the second end of the trapping device. The exit chamber is in fluid communication with the at least one immobilization chamber and is disposed between the at least one immobilization chamber and the second end of the trapping device, The trapping device is configured such that the fluid may flow from the fluid inlet to the exit chamber through the culture chamber, the at least one staging chamber, and the at least one immobilization chamber. [0005] In some implementations, the trapping device defines an opening associated with the fluid inlet defined in a first plane spaced apart from and above a second plane that extends through the culture chamber and the at least one staging chamber. [0006] In some implementations, the microfluidic device includes two or more trapping devices. In some implementations, the trapping device includes at least one layer of a thermoplastic material.

[0007] In some implementations, the trapping device further includes third and fourth perfusion channels respectively defined between third and fourth perfusion walls extending adjacent to the at least one staging chamber and the adjacent side wall of the at least one side wall of the trapping device. The third perfusion channel is in fluid communication with the first perfusion channel and the fourth perfusion channel is in fluid communication with the second perfusion channel. In some implementations, the first and second perfusion walls extend past the at least one staging chamber.

[0008] In some implementations, the trapping device further includes a serpentine exit pathway on the second end of the trapping device and in fluid communication with the exit chamber, the serpentine exit pathway configured to provide a flow resistance in the culture chamber.

[0009] In some implementations, the at least one immobilization chamber has a width that is greater than the width of the at least one staging chamber. In some implementations, the maximum width of the culture chamber is defined across the second end of the culture chamber. [0010] In some implementations, the at least one staging chamber includes a plurality of staging chambers arranged along the second end of the culture chamber. The at least one immobilization chamber includes a plurality of immobilization chambers corresponding to the plurality of staging chambers.

[0011] In some implementations, the trapping device further includes an immobilization wall extending between the upper and lower surfaces of the trapping device at an end of the at least one immobilization chamber adjacent to the exit chamber. The immobilization wall and the at least one immobilization chamber define a filter channel through which the fluid may flow from the at least one immobilization chamber towards the exit chamber.

[0012] In some implementations, the trapping device further includes at least one baffle extending into the culture chamber from the upper surface of the trapping device at a portion of the culture chamber adjacent the fluid inlet and inward of the perfusion wall.

[0013] In some implementations, the culture chamber includes a pocket defined by the at least one side wall of the trapping device at the first end of the culture chamber adjacent to the fluid inlet, the pocket configured to aggregate biological subjects during formation. In some implementations, the trapping device is disposed on a tilter capable of tilting the trapping device to a desired angle.

[0014] In some implementations, the trapping device further includes a central divider wall extending into the culture chamber between the upper and lower surfaces of the trapping device to define a first portion and a second portion of the culture chamber, the central divider wall extending from the first end of the culture chamber to the second end of the culture chamber. [0015] According to another implementation, a system is disclosed including the microfluidic device of this disclosure. The system includes a first trapping device, a second trapping device, and a connection conduit coupled to and extending between (i) the exit chamber and an adjacent fluid outlet of the first trapping device and (ii) a fluid inlet of the second trapping device.

[0016] In some implementations, the system further includes a camera arranged to view the trapping device such that the at least one immobilization chamber is within a field of view of the camera. The system further includes a controller in communication with the camera, the controller configured to capture images by the camera and store a plurality of images from the camera.

[0017] In some implementations, the system further includes a first fluid reservoir, a second fluid reservoir, a first fluid conduit coupled between the first fluid reservoir and the fluid inlet of the trapping device, a second fluid conduit coupled between the second fluid reservoir and an outlet of the trapping device, and a pump in fluid communication with the first fluid conduit. The pump configured to direct a fluid from the first fluid reservoir along the first fluid conduit to the fluid inlet of the trapping device.

[0018] In some implementations, the system further includes a controller coupled to the pump and configured to control the flow of the fluid into the trapping device.

[0019] According to another implementation, a method is disclosed, the method including first providing a microfluidic device of this disclosure. The method further includes depositing a biological subject-laden gel into the culture chamber via the fluid inlet of the trapping device. The biological subject-laden gel includes a gel and a plurality of cells suspended therein. The biological subject-laden gel coalesces or solidifies in the culture chamber between the first and second perfusion walls. The method further includes providing a flow of a culture medium through the culture chamber via the fluid inlet. The culture medium flows (i) directly to a first portion of the biological subject-laden gel adjacent the fluid inlet (ii) through the first perfusion channel to a second portion of the biological subject-laden gel adjacent the first perfusion channel, (iii) through the second perfusion channel to a third portion of the biological subjectladen gel adjacent the second perfusion channel, and (iv) through a temporary perfusion channel to a fourth portion of the biological subject-laden gel,. The temporary perfusion channel is defined between the fourth portion of the biological subject-laden gel on a second end of the culture chamber and the at least one staging chamber. The culture medium is configured to facilitate growth of the plurality of cells. The method further includes dissolving the gel of the biological subject-laden gel to release the plurality of cells. The method further includes moving at least one of the plurality of cells into the at least one immobilization chamber of the trapping device.

[0020] In some implementations, the method further includes imaging the plurality of cells via a camera adjacent to the trapping device. In some implementations, the method further includes injecting a staining fluid into the trapping device at the fluid inlet such that the plurality of cells are stained with the staining fluid.

[0021] According to another implementation, a microfluidic device is disclosed. The microfluidic device includes at least one trapping device including a first end, a second end, at least one side wall extending between the first and second ends, a lower surface, and an upper surface opposite and spaced apart from the lower surface. The trapping device further includes a fluid inlet defined at the first end of the trapping device. The trapping device further includes an inlet chamber defined adjacent the first end of the trapping device and extending towards the second end of the trapping device. The inlet chamber includes: (i) a first end of the inlet chamber in fluid communication with the fluid inlet and a second end of the inlet chamber opposite of the first end of the inlet chamber along a first axis of the trapping device, (ii) a first height defined between the upper surface and the lower surface of the trapping device, the first height being measured in a direction that is parallel to a second axis that is orthogonal to the first axis, and (iii) a maximum width as measured in a direction that is parallel to a third axis that is orthogonal to each of the first axis and the second axis. The trapping device further includes at least one staging chamber defined at the second end of the inlet chamber having a height that is equal to the first height and a first width that is less than the maximum width. The trapping device further includes at least one immobilization chamber in fluid communication with the at least one staging chamber and having a height that is less than the first height and a second width that is greater than the first width of the at least one staging chamber. The at least one immobilization chamber is configured to retain a target biological subject while a fluid flows through the trapping device toward the second end of the trapping device. The trapping device further includes an exit chamber in fluid communication with the at least one immobilization chamber and disposed between the at least one immobilization chamber and the second end of the trapping device. The trapping device is configured such that the fluid may flow from the fluid inlet to the exit chamber through the inlet chamber, the at least one staging chamber, and the at least one immobilization chamber.

[0022] According to another implementation, a microfluidic device is disclosed. The microfluidic device includes at least one trapping device including a first end, a second end, at least one side wall extending between the first and second ends, a lower surface, and an upper surface opposite and spaced apart from the lower surface. The trapping device further includes a fluid inlet defined at the first end of the trapping device. The trapping device further includes a culture chamber defined adjacent the first end of the trapping device and extending from the first end of the trapping device towards the second end of the trapping device. The culture chamber includes: (i) a first end of the culture chamber in fluid communication with the fluid inlet and a second end of the culture chamber opposite of the first end of the culture chamber along a first axis of the trapping device, (ii) a first height defined between the upper surface and the lower surface of the trapping device, the first height being measured in a direction that is parallel to a second axis that is orthogonal to the first axis, and (iii) a maximum width as measured in a direction that is parallel to a third axis that is orthogonal to each of the first axis and the second axis. The trapping device further includes first and second perfusion walls that extend into the culture chamber from the upper surface towards the lower surface of the trapping device, a free end of the perfusion wall being spaced apart from the lower surface of the trapping device, the perfusion walls extending from the first end of the culture chamber towards the second end of the culture chamber. First and second perfusion channels are respectively defined between each perfusion wall and an adjacent side wall of the at least one side wall of the trapping device. The trapping device further includes a fluid exit defined at the second end of the trapping device in fluid communication with the second end of the culture chamber. The trapping device is configured such that the fluid may flow from the fluid inlet to the fluid exit through the culture chamber.

[0023] According to another implementation, a method is disclosed the method including first providing a microfluidic device of this disclosure. The method further includes depositing a biological subject-laden gel into the culture chamber via the fluid inlet of the trapping device. The biological subject-laden gel includes a gel and a plurality of cells suspended therein. The biological subject- laden gel coalesces or solidifies in the culture chamber between the first and second perfusion walls. The method further includes providing a flow of a culture medium through the culture chamber via the fluid inlet. The culture medium flows (i) directly to a first portion of the biological subject-laden gel adjacent the fluid inlet (ii) through the first perfusion channel to a second portion of the biological subject-laden gel adjacent the first perfusion channel, (iii) through the second perfusion channel to a third portion of the biological subjectladen gel adjacent the second perfusion channel, and (iv) through a third perfusion channel to a fourth portion of the biological subject-laden gel, The third perfusion channel is defined between the fourth portion of the biological subject-laden gel on a second end of the culture chamber and the at least one side wall of the trapping device. The culture medium is configured to facilitate growth of the plurality of cells.

[0024] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive, as claimed.

[0025]

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows a top view of a microfluidic device with a trapping device, according to one implementation.

[0027] FIG. 2A shows a top view of the trapping device of FIG. 1. FIG. 2B shows a corresponding cross-sectional view along line 2B-2B shown in FIG. 2A. FIG. 2C shows corresponding cross-sectional view along line 2C-2C shown in FIG. 2A. FIG. 2D shows a corresponding cross-sectional view along line 2D-2D shown in FIG. 2A. FIG. 2E shows a corresponding cross-sectional view along line 2E-2E shown in FIG. 2A. [0028] FIGS. 3A-3C show top views of trapping devices according to alternative implementations.

[0029] FIG. 4A shows a top view of the trapping device of FIG. 1 having a biological subjectladen gel deposited therein. FIG. 4B is a cross-sectional view along line 4B-4B in FIG. 4A through the culture chamber.

[0030] FIG. 5 shows a cross-sectional side view of the trapping device of FIG. 1 showing biological subjects within various chambers of the trapping device.

[0031] FIGS. 6A-6C show top views of the trapping device shown in FIG. 3B and the progression of flow of biological subjects through the trapping device.

[0032] FIGS. 7A and 7B show representative images that depict a device with trapped biological subjects, according to one implementation.

[0033] FIG. 8 shows a top view of a trapping device, according to one implementation.

[0034] FIG. 9 shows a top view of a trapping device, according to one implementation.

[0035] FIG. 10A shows a top view of a trapping device, according to one implementation. FIG. 10B is a cross-sectional view along line 10B-10B in FIG. 10A through the culture chamber. [0036] FIGS. 11 A and 1 IB show trapping devices according to other implementations.

[0037] FIG. 12A shows the trapping device of FIG. 3C with a suspension of biological subjects therein, according to one implementation. FIG. 12B shows a trapping device having a pocket for the aggregation of cells, according to one implementation.

[0038] FIG. 12C shows the trapping device of FIG. 12A from a side view on a tilter.

[0039] FIG. 13 shows a diagram of a system that includes a microfluidic device, pressure system, and imaging system, according to one implementation.

[0040] FIGS. 14A-14C show diagrams depicting the difference in the number of z-images to image the entire organoid, according to various implementations.

[0041] FIGS. 15A and 15B shows systems having microfluidic devices each having multiple trapping devices, according to one implementation.

[0042] FIG. 16 presents a comparative flowchart showing the DOX treatment and calcium imaging of cardiac organoids on chamber slides and in a microfluidic device, according to one implementation.

[0043] Various objects, aspects, features, and advantages of the disclosure will become more apparent and better understood by referring to the detailed description taken in conjunction with the accompanying drawings, in which like reference characters identify corresponding elements throughout. In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements.

DETAILED DESCRIPTION

[0044] To address the challenges faced by existing systems, methods, and devices, various implementations described herein include a microfluidic device and imaging system for high- resolution, and/or high-content, imaging (HCI). High-resolution imaging includes a high magnification and/or a high numerical aperture objective. High-resolution imaging may enable high-content imaging. High-content imaging further includes multiple color options and multiple objective options. High-content imaging (HCI) is crucial for studying various cell types and phenomena within the organoid microenvironment. To facilitate HCI, various implementations include flat-bottom platforms that enable full immobilization and restrict the distribution of the organoids to predetermined areas and distances from the microscope objective. Along with automation, such devices facilitate fast and blur-free high-resolution imaging of organoid samples, according to some implementations. Furthermore, the systems, methods, and devices of this disclosure provide for various imaging modalities. Various implementations of imaging modalities can include, but are not limited to, bright field microscopy, phase microscopy (DIC or Phase-contrast), fluorescence microscopy, confocal microscopy, structured illumination microscopy, computational microscopy, and/or microscopy with point-spread function engineering.

[0045] Fluorescence live and dead staining and immunostaining of organoids is challenging when organoids are embedded in hydrogels because large staining molecules have reduced diffusion rates limited by the hydrogel porosity. This problem is more significant for immunostaining due to the multiplicity of the needed reagents and secondary issues such as nonspecific binding. Various implementations of the microfluidic devices and systems described herein have the ability to facilitate staining and imaging of immobilized organoids upon hydrogel digestion while enabling organoid culture and monitoring.

[0046] Various implementations of the microfluidic device have a culture chamber for organoid culturing and growth monitoring. The microfluidic device also has staging and immobilization chambers for organoid immobilization and fluorescence live and dead imaging upon digestion of the hydrogel (or other extracellular matrix scaffold), such as MATRIGEL, which is a trademark owned by DISCOVERY LABWARE, INC.

[0047] In various implementations, the microfluidic device includes at least one trapping device that receives organoids and holds them steadily within its immobilization chambers to facilitate end-point calcium transient and live/dead imaging. Each trapping device has one or more trapping channels (e.g., between one and fifty trapping channels, such as six trapping channels as shown in FIG. 1) that are arranged parallel to each other. Each trapping channel includes two consecutive chambers relative to the flow direction: the staging chamber (SC) and the immobilization chamber (IC). The chambers slightly compress the organoids laterally and/or vertically to provide sufficient friction to fully immobilize organoids during imaging and to avoid blurring due to any organoid movements on the device. The predetermined locations of the immobilization chambers accelerate the imaging process by eliminating the need for scanning across a large field of view to locate the organoids and changing between low and high magnification objectives during high-resolution imaging.

[0048] In some implementations, each trapping channel receives organoids transferred from their native culture plates to anchor them within the immobilization chamber of that trapping channel. In other implementations, each trapping channel receives organoids grown within a culture chamber of the trapping device. The trapping channel can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. In addition, the trapping channels allow for assessing dose-dependent responses of organoids’ viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-device experiments, according to some implementations. Importantly, the various implementations of the trapping channels enable organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevent the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the trapping device according to various implementations is a microfluidic platform that can serve as a building block for a multi-well plate, or microfluidic device, that can provide high-throughput and high-resolution imaging of organoids.

Trapping Device Structure

[0049] Referring generally to the figures, a microfluidic device comprising at least one trapping device is shown, according to various implementations. Various implementations of the trapping device address challenges associated with organoids movements through a unique geometry to facilitate immobilization of organoids in pre-determined locations. While immobilization of organoids prevents any movements, the known locations of immobilized organoids eliminate the laborious search for organoids during imaging.

[0050] Various implementations of the trapping device allow for immobilization of organoids to prevent undesired movement during fast motorized imaging, thus eliminating the risk of an organoid drifting out of focus and field of view. Pre- determined locations of the immobilization chambers eliminate the cumbersome and time-consuming task of searching across an entire well or hydrogel pad to locate organoids. The trapping device according to various implementations is also advantageous for volumetric imaging of organoids. For larger organoids, the limited height of the trapping channels allows for imaging using a fewer number of z-slices. Specifically, the crypts and villi structures inherent to intestinal organoids require many optical sections to completely image the organoid morphology (e.g., FIG. 14). Taking fewer images reduces the required time and minimizes data storage space.

[0051] FIG. 1 shows a microfluidic device 100, according to one implementation. The microfluidic device 100 includes one or more thermoplastic materials (e.g., polymers, COC (cyclic olefin copolymer), ABS (Acrylonitrile butadiene styrene), Acrylic, or Polystyrene). The microfluidic device 100 is manufactured using a layered molding process.

[0052] FIG. 1 shows a top view of the microfluidic device 100. The microfluidic device 100 includes a trapping device 102. FIG. 2A also shows a top view of the trapping device 102. FIG. 2B shows a corresponding cross-sectional side view along line 2B-2B shown in FIG. 2A. FIG. 2C similarly shows a corresponding cross-sectional view along line 2C-2C shown in FIG. 2A. FIG. 2D shows corresponding cross-sectional view along line 2D-2D shown in FIG. 2A. FIG. 2E shows a corresponding cross-sectional view along line 2E-2E shown in FIG. 2A. The dimensions described in relation to FIGS. 2A-2E (and elsewhere, unless noted otherwise) are in microns, or micrometers, denoted generally as “pm”.

[0053] The trapping device 102 includes a first end 104 and a second end 106 opposite and spaced apart from the first end 104. The trapping device 102 further includes at least one side wall 108 extending between the first end 104 and the second end 106 of the trapping device 102. For example, the trapping device 102 shown in FIG. 1 includes at least a first side wall 108a and a second side wall 108b. The trapping device 102 also includes an end side wall 108c that extends between the first side wall 108a and the second side wall 108b and defines an opening on one end of the serpentine exit pathway 170. However, in other implementations, the trapping device may include only a single side wall (e.g., an arcuate shaped wall extending from the fluid inlet 114) or a plurality of side walls defining various chambers and sections of the trapping device.

[0054] The trapping device 102 further includes a lower surface 110 and an upper surface 112 opposite and spaced apart from the lower surface 110. While the lower surface 110 and the upper surface 112 are not clearly visible in the top view of FIG. 1, the cross-sectional side views of FIGS. 2B and 2D-2F show the structure of the lower surface 110 and the upper surface 112.

[0055] The trapping device 102 further includes a fluid inlet 114, a culture chamber 120, a first perfusion wall 130, a second perfusion wall 132, staging chambers 140, immobilization chambers 150, and an exit chamber 160. The trapping device 102 is configured such that a fluid may flow substantially in a first direction and through the various sections of the trapping device 102, as further described herein. The first direction is flow in the direction from the culture chamber 120 toward the exit chamber 160 along a first axis 101, which extends between the first end 104 and the second end 106 of the trapping device 102.

[0056] The fluid inlet 114 is defined at the first end 104 of the trapping device 102. A fluid may flow into the trapping device 102 through the fluid inlet 114 (e.g., via a pipette, tubing, or other conduit in fluid communication with the fluid inlet 114). As shown in FIG. 2A and 2B, an opening 116 to the fluid inlet 114 is defined in a first plane 117 that is spaced apart from and above a second plane 118. The first plane 117 and the second plane 118 extend parallel to each other. The first plane 117 extends along a top surface of the microfluidic device 100, and the second plane 118 extends through the culture chamber 120 and the staging chambers 140.

[0057] The culture chamber 120 is defined adjacent the first end 104 and extends from the first end 104 of the trapping device 102 towards the second end 106 of the trapping device 102 in a direction parallel to the first axis 101. The culture chamber 120 includes a first end 122 and a second end 124 opposite and spaced apart from the first end 122 along the first axis 101. The first end 122 of the culture chamber 120 is adjacent to and in fluid communication with the fluid inlet 114. The culture chamber 120 is generally bound and defined by the fluid inlet 114, the first side wall 108a, second side wall 108b, and the staging chambers 140 of the trapping channels. [0058] The culture chamber 120 has a first height (Hcc) defined between the lower surface 110 and the upper surface 112 of the trapping device 102 as measured in a direction parallel to a second axis, which is designated by axis 103. The second axis is orthogonal to the first axis 101 and can be seen in the cross-sectional side view of FIG. 2B. The height of the culture chamber 120 in FIG. 2B is 550pm. However, in other implementations, the culture chamber has a height in the range of 50pm to 2cm.

[0059] The culture chamber 120 also has a width (Wcc) defined in a third axis, which is designated by axis 105, that is orthogonal to each of the first axis 101 and the second axis 103. A maximum width of the culture chamber 120 (Wccmax) is measured in a direction that is parallel to the third axis 105, and the maximum width of the culture chamber 120 of FIG. 1 is at the second end 124 of the culture chamber 120 such that that the width of the culture chamber 120 generally increases from the first end 122 to the second end 124 of the culture chamber 120 (e.g., gradually, as shown in FIGS. 1, and 2A, or stepped (not shown). In FIG. 2 A, the maximum width (Wccmax) of the culture chamber 120 is 5223 pm (0.5223 cm). However, the maximum width of the culture chamber may vary in a range of 50 to 10,000 microns. In other implementations, the width of the culture chamber may be substantially the same from the first end to the second end. In other implementations, the maximum width of the culture chamber may be in between the first and second ends, or adjacent to the first end of the culture chamber.

[0060] The culture chamber 120 acts as a diffuser by expanding the flow from the first end 122 to the second end 124 of the culture chamber 120, facilitating the distribution of organoids amongst the trapping channels (i.e., the staging chamber 140 and the corresponding immobilization chamber 150). This design ensures that each trapping channel will trap only a single organoid. When one trapping channel receives an organoid, its hydraulic resistance increases. Therefore the flow directs the other organoids toward the unoccupied trapping channels.

[0061] The first perfusion wall 130 and the second perfusion wall 132 of the trapping device 102 each extend from the first end 122 of the culture chamber 120 towards the second end 124 of the culture chamber 120. The perfusions walls 130, 132 also extend in a direction parallel to the second axis 103 into the culture chamber 120 from the upper surface 112 towards the lower surface 110 of the trapping device 102. The first perfusion wall 130 has a free end 131, and the second perfusion wall 132 has a free end 133. Each of the free ends 131, 133 are spaced apart from the lower surface 110 of the culture chamber 120 to define perfusion gaps 134, 135. The perfusion gap 134 is defined between the free end 131 of the first perfusion wall 130 and the lower surface 110. The perfusion gap 135 is defined between the free end 133 of the second perfusion wall 132 and the lower surface 110.

[0062] A first perfusion channel 136 is defined between the first perfusion wall 130 and the adjacent first side wall 108a of the trapping device 102. A second perfusion channel 138 is defined between the second perfusion wall 132 and the adjacent second side wall 108b of the trapping device 102. The first perfusion channel 136 is in fluid communication with the culture chamber 120 at (i) the first end 122 of the culture chamber 120, (ii) the second end 124 of the culture chamber 120, and (iii) via the perfusion gap 134. Similarly, the perfusion channel 138 is in fluid communication with the culture chamber 120 at (i) the first end 122 of the culture chamber 120, (ii) the second end 124 of the culture chamber 120, and (iii) via the perfusion gap 135.

[0063] The perfusion gaps 134, 135 have dimensions of 100 microns in height (HPG), as shown in FIG. 2C. However, in other implementations, the height of the perfusion gaps (HPG) is in a range of 1 -400 pm. In other implementations, the height of the perfusion gaps depends on the height of the culture chamber. In some implementations, the height of the perfusion gaps is 5% - 50% of the height of the culture chamber.

[0064] Each trapping channel includes a staging chamber 140, followed by an immobilization chamber 150. The staging chambers 140 have the same height (Hsc) as the culture chamber 120 to inflict minimal resistance during the initial organoid loading. Subsequently, the organoids move into immobilization chambers 150 having reduced heights (Hie) that allow a slight compression and flattening of the organoids. The immobilization chamber width (Wic) (e.g., 650 pm) is larger than that of the staging chamber (Wsc) (e.g., 430 pm) to account for any expansion that may arise from this vertical compression. The resulting static friction secures the organoids in place and renders them stationary during fast motorized imaging. The dimensions of the immobilization chamber 150 accommodate organoids across various sizes, provided they can enter the immobilization chamber 150. Organoids with diameters larger than the width of staging chambers 140 commonly do not enter the immobilization chambers 150 and are held in place by slight lateral compression of the side walls of the staging chamber 140, still enabling immobilization for imaging. [0065] The staging chambers 140 of the trapping device 102 are defined at the second end 124 of the culture chamber 120. The trapping device 102 of FIG. 1 includes six staging chambers 140 arranged substantially parallel to each other and the first axis 101. However, in other implementations, the trapping device may include anywhere from one to fifty staging chambers. In other implementations, the staging chambers may not be parallel to each other (e.g., arranged circumferentially around a culture chamber or radially extending relative to the culture chamber). [0066] The staging chambers 140 have a height (Hsc) that is equal to the first height of the culture chamber 120. For example, FIGS. 2C and 2D show the cross-sectional side view wherein the heights of the culture chamber 120 (Hcc) and the staging chambers 140 (Hsc) are shown, both equal to 550 pm. The staging chambers 140 have a width (Wsc) that is less than the maximum width of the culture chamber 120 (Wccmax). In FIG. 2D, each staging chamber 140 has a width (Wsc) of 430 pm, compared to the maximum width of the culture chamber 120 (Wccmax) of 5223 pm. However, in other implementations, the staging chambers may have a width in the range of 10 to 1500 microns.

[0067] The immobilization chambers 150 of the trapping device 102 are defined between the staging chambers 140 and the exit chamber 160, in a direction parallel to the first axis 101. The trapping device 102 of FIG. 1 includes six immobilization chambers 150 arranged substantially parallel to each other along the first axis 101. Each immobilization chamber 150 is downstream in the first axis 101 of a corresponding staging chamber 140. However, in other implementations, the trapping device may include anywhere from one to fifty immobilization chambers. In other implementations, the immobilization chambers may not be strictly parallel to each other (e.g., arranged circumferentially around a culture chamber or radially extending relative to the culture chamber).

[0068] The immobilization chambers 150 are in fluid communication with each of the corresponding staging chambers 140. The immobilization chambers 150 have a height (Hie) less than the first height of the culture chamber 120 (Hcc) and the staging chambers 140 (Hsc). For example, as shown in FIG. 2E, each immobilization chamber 150 has a height of 290 pm. However, in other implementations, the immobilization chamber may have a height in the range of 5 to 1200 microns. The lower height of the immobilization chambers 150 corresponds to a smaller distance between the lower surface 110 and the upper surface 112 of the trapping device 102. The upper surface 112 of the trapping device 102 is stepped down. However, in other implementations, the upper surface can taper downwards.

[0069] Each immobilization chamber 150 has a width (Wic) that is larger than the width of the corresponding staging chamber 140 (Wsc). For example, as shown in FIGS. 2D and 2Eand 2F, each immobilization chamber 150 has a width (Wic) of 650 pm. However, in other implementations, the immobilization chambers have a width in the range of 15 to 2500 microns. In other implementations, the width of the immobilization chambers is the same or less than that of the staging chamber.

[0070] The immobilization chambers 150 are configured to retain a biological subject (e.g., an organoid, a three-dimensional cell, or a target group of spheroid cells) while a fluid flows through the trapping device 102 from the first end 104 to the second end 106 of the trapping device 102. An immobilization wall 156 is disposed at a second end 154 of each immobilization chamber 150 and is adjacent to the exit chamber 160. The immobilization wall 156 extends between the upper surface 112 and the lower surface 110 of the trapping device 102. Each immobilization chamber 150 defines, along with at least a portion of the immobilization wall 156, at least one filter channel 152 at the second end 154 of the immobilization chamber 150. The immobilization wall 156 of the immobilization chamber 150 retains the biological subject, while the at least one filter channel 152 allows fluid to flow through the immobilization chamber 150. The exit chamber 160 is adjacent to and in fluid communication with the immobilization chambers 150. Specifically, the exit chamber 160 receives fluid from each of the filter channels 152 of the immobilization chambers 150. The exit chamber 160 is disposed between the immobilization chambers 150 and the second end 106 of the trapping device 102.

[0071] A serpentine exit pathway 170 is in fluid communication with the exit chamber 160 on one end. The serpentine exit pathway 170 is in fluid communication with a fluid outlet 180 on an opposite end. A width of the exit chamber 160 narrows towards the serpentine exit pathway 170 (e.g., gradually or stepped). The serpentine exit pathway 170 has an overall length much larger than the width across the serpentine exit pathway 170. For example, the length, width, and height of the serpentine exit pathway 170 are related to each other by the following equation: Where L is the length, w is the width, h is the height, p is the dynamic viscosity of the fluid, and R is the hydraulic resistance.

[0072] The serpentine exit pathway 170 may be sized such that hydraulic resistance of the serpentine exit pathway 170 limits the flow rate of fluid through the trapping device 102. For example, viscous forces of the fluid therein produce a hydraulic resistance in the serpentine exit pathway 170, the exit chamber 160, and other portions of the trapping device 102.

[0073] The exit chamber 160 is connected to the fluid outlet 180 through the 71 -mm long serpentine exit pathway 170 with reduced height (100 pm). This design provides a hydraulic resistance to control and maintain the low flow rates. A height reduction within the wide portion of the exit chamber 160 is implemented to prevent small aggregates of cellular debris, which might escape the immobilization chambers 150, from clogging the exit chamber 160.

[0074] The fluid outlet 180 is defined on the second end 106 of the trapping device 102. The outlet 180 is similar in shape and structure as the fluid inlet 114. For example, the trapping device 102 defines an opening 182 associated with the fluid outlet 180 such that a fluid may flow out of the trapping device 102 (e.g., via a pipette, tubing, or other conduit coupled to the fluid outlet 180). The opening 182 is defined in the first plane 117 spaced apart from and above the second plane 118, as shown in FIG. 2A.

[0075] The trapping device 102 is configured such that the fluid from the first end 104 to the second end 106 of the trapping device 102. Specifically, the fluid flows from the fluid inlet 114, into the culture chamber 120, through the staging chambers 140, immobilization chambers 150, through the exit chamber 160, through the serpentine exit pathway 170, and out through the fluid outlet 180. However, fluid can be caused to flow in the opposite direction.

[0076] In other implementations, the microfluidic device may include two or more trapping devices (e.g., two trapping devices coupled to the same inlet, two trapping devices coupled in series, two trapping devices coupled in parallel, or an array of a plurality of trapping devices). In addition, in other implementations, other manufacturing methods may be used (e.g., additive manufacturing such as 3D printing). In other implementations, other materials may be used to manufacture the microfluidic device and/or trapping devices such as PDMS and/or glass, as described herein in the section “Examples, Experimental Testing, and Results.”

[0077] In other implementations, the microfluidic device may contain a culture chamber with the perfusion channels but without the staging and immobilization chambers. Such a device may be used to efficiently grow organoids such that culture medium is efficiently distributed throughout the culture chamber. In other implementations, the microfluidic device may contain the staging and the trapping chambers but without the culture chamber. Such a device may be used to trap grown organoids inserted into an inlet chamber of the device.

[0078] FIGS. 3A-3C show example images of alternative implementations of trapping devices. FIGS. 3A-3C show images of trapping devices with various alternative structures and sizes compared to that of the microfluidic device 100 having the trapping device 102 of FIGS. 1-2E. [0079] For example, FIG. 3 A shows an image of a trapping device 300a that does not include perfusion walls or perfusion channels in the culture chamber 301. FIG. 3B shows an image of a trapping device 300b having a structure substantially similar to that of trapping device 300a. However, the exit chamber 309 of trapping device 300b is coupled directly to the fluid outlet 310, rather than through a serpentine exit pathway.

[0080] FIG. 3C shows a trapping device 300c having a single inlet chamber 302. The inlet chamber 302 is in fluid communication with a single staging chamber 303 having a width smaller than that of the inlet chamber 302. The staging chamber 303 is in fluid communication with a single immobilization chamber 304 having a width larger than that of the single staging chamber 303 and a height smaller than that of the single staging chamber 303. The single immobilization chamber 304 is in fluid communication with a single exit chamber 305 leading to a fluid outlet 306.

[0081] Other implementations of trapping devices are shown in FIGS. 4A-6C. For example, FIG. 4A shows a top view of the trapping device 102 shown in FIGS. 1-2E along with a biological subject-laden gel 402. FIG. 4B includes a cross section along the line 4B in FIG. 4A. FIG. 5 shows a cross-sectional view of the trapping device 102 of FIG. 1. FIGS. 6A-6C show the trapping device 300b of FIG. 3B and the progression of organoid growth and trapping within the trapping device 300b.

[0082] FIG. 8 shows a top view of a microfluidic device 800 having a trapping device 802. The microfluidic device 800 is substantially similar to the microfluidic device 100 of FIGS. 1- 2E. The trapping device 802 includes immobilization chambers 850 similar in structure and function to the immobilization chambers 150 of the trapping device 102. However, while the immobilization chambers 150 of the trapping device 102 of FIGS. 1-2E each includes two filter channels 152 along either side of an immobilization wall 156, the immobilization chambers 850 include a single, centralized filter channel 852. The immobilization chambers 850 are thus defined by adjacent immobilization walls 856 that extend along a side and a second end 854 of the immobilization chambers 850. Adjacent immobilization walls 856 each have a shoulder 858, and each pair of shoulders 858 define a respective filter channel 852 corresponding to each immobilization chamber 850.

[0083] FIG. 9 shows a top view of a microfluidic device 900 having a trapping device 902. The microfluidic device 900 is substantially similar to the microfluidic device 100 of FIGS. 1- 2E. However, the trapping device 902 includes a first perfusion channel 911, a second perfusion channel 912, a third perfusion channel 913, and a fourth perfusion channel 914. The first perfusion channel 911 is defined by the first perfusion wall 921 and the adjacent first side wall 903a of the trapping device 902. The second perfusion channel 912 is defined by the second perfusion wall 922 and the adjacent second side wall 903b of the trapping device 902.

[0084] The third perfusion channel 913 is defined by a third perfusion wall 923 and the adjacent first side wall 903a of the trapping device 902. The fourth perfusion channel 914 is defined by a fourth perfusion wall 924 and the adjacent second side wall 903b of the trapping device 902.

[0085] The third and fourth perfusion walls 923, 924 each extend adjacent to the staging chambers 940. Thus, the third perfusion channel 913 is in fluid communication with the first perfusion channel 911, and the fourth perfusion channel 914 is in fluid communication with the second perfusion channel 912.

[0086] The trapping device 902 allows culture medium to flow around a biological subjectladen gel to contact at least three portions of the periphery of the biological subject-laden gel. In the trapping device 902, the culture medium can flow through first and second perfusion gaps associated with the first and second perfusion walls 921, 922 to side portions of the biological subject- laden gel. This structure provides the above-described advantages of providing several sides of the perimeter of the biological subject-laden gel with growth or culture medium.

[0087] However, in the trapping device 902, the fourth portion of the biological subject-laden gel that is adjacent to the staging chambers 940 does not receive as much culture medium as compared to the trapping device 102 of FIG. 1. The third perfusion wall 923 and the fourth perfusion wall 924 essentially bypass the staging chambers 940. This structure avoids excess flow of culture medium to the staging chambers 940 that are closest to the sides of the trapping device 902 near the perfusion walls 921, 922. Because those “outside” staging chambers 940 may receive excess flow, a non-uniform flow of culture medium to the staging chambers 940 may be produced. The trapping device 902 addresses this problem by providing perfusion walls that extend past the staging chambers 940.

[0088] FIG. 10A shows a top view of a trapping device 1002. The trapping device 1002 includes a central divider wall 1004 extending into the culture chamber between the lower and upper surfaces of the trapping device 1002. The central divider wall 1004 extends from the first end of the culture chamber to the second end of the culture chamber. The central divider wall 1004 splits the culture chamber into a first portion 1006 and a second portion 1008 opposite the first portion 1006.

[0089] As shown in FIG. 10A and the corresponding cross-sectional view in FIG. 10B, the first portion 1006 of the trapping device 1002 includes the first perfusion wall 1030 as well as a third perfusion wall 1010 adjacent to the central divider wall 1004. The second portion 1008 of the trapping device 1002 includes the second perfusion wall 1032 as well as a fourth perfusion wall 1012 adjacent to the central divider wall 1004.

[0090] Essentially, each of the first and second portions 1006, 1008 formed by the central divider wall 1004 are each smaller versions of the culture chamber having perfusion walls 1030, 1032 on opposing sides. The trapping device 1002 thus provides a uniform nutrient distribution (e.g., culture medium) into the culture chamber. For example, two smaller portions of biological subject- laden gel may be deposited into the trapping device 1002 - one in each of the first portion 1006 and the second portion 1008. Then, culture medium flowing into the fluid inlet may split into each portion 1006, 1008 to feed the corresponding biological subject-laden gel. The culture medium may flow to all sides of the biological subject- laden gel within each portion 1006, 1008 due to the perfusion walls (and the associated perfusion channels and perfusion gaps) on each side. The result may be that more nutrients reach more cells of the biological subjectladen gel in a more efficient manner.

[0091] FIGS. 11A and 1 IB show trapping devices 1102a and 1102b that are substantially similar to one another. Each trapping devices 1102a and 1102b is substantially similar to the trapping device 300b of FIG. 3B. The trapping devices 1102a 1102b includes at least one baffle extending into the culture chamber 1120 from the upper surface of the trapping device 1102 at a portion of the culture chamber 1120 adjacent to the fluid inlet 1114 and inward of the perfusion walls (not shown in FIGS. 11 A and 1 IB). For example, trapping device 1102a includes a plurality of walls 1104 extending into the culture chamber 1120 from the upper surface of the trapping device 1102a. Similarly, trapping device 1102b include a plurality of pillars 1106 extending into the culture chamber 1120 from the upper surface of the trapping device 1102b. Each of the walls 1104 and the pillars 1106 are considered to be the “at least one baffle” of FIGS. 11 A and 1 IB. However, in other implementations, different shapes and structures of the bafflers are contemplated by this disclosure (e.g., curved walls, rectangular pillars, multiple rows of pillars, or pillars and walls in combination).

[0092] The baffles of FIGS. 11 A and 1 IB provide structure to distribute the cells throughout the culture chamber 1120 more evenly as they flow towards the staging chambers. In other implementations, the culture chamber includes a chemical treatment (e.g., a surface-based chemical deposited onto one or more of the upper and lower surface of the trapping device), the chemical treatment performing a similar distribution function as the at least one baffle.

[0093] FIG. 12A shows the trapping device 300c of FIG. 3C. The trapping device 300c of FIG. 12A includes a suspension of cells therein. FIG. 12B shows a trapping device 1202 having a pocket for the aggregation of cells. FIG. 12C shows the trapping device 330c of FIG. 12A from a side view on a tilter 1201.

[0094] The trapping device 1202 includes a culture chamber 1204 adjacent to the inlet. However, the culture chamber 1204 includes a pocket 1206 defined by the sidewall 1208 of the trapping device 1202 (which may be substantially coincident with the inlet wall). The pocket 1206 is configured to aggregate biological subjects (e.g., a plurality of cells) during formation and/or growth. The pocket 1206 includes a semi-spherical or semi-ovoid shape, but in other implementations the pocket may have a different shape (e.g., rectangular prism or cube).

[0095] However, the pocket 1206 is not the only aggregation method. FIG. 12C shows the trapping device 300c disposed on a tilter 1201. The tilter 1201 is a device capable of tilting trapping device 300c in a variety of angles with respect to an initial, neutral plane. As shown, the tilter 1201 is tilting trapping device 300c at an angle such that the first end of the trapping device 300c is lower than the second end of the trapping device 300c.

[0096] In use, the titled trapping device 300c allows for a plurality of biological subjects that have been deposited into the culture chamber to aggregate on a first side of the trapping device 300c, driven by gravity. A culture medium may flow through the trapping device 1202 as elsewhere described, feeding the plurality of biological subjects as they grow into an organoid/spheroid. In other implementations, the cells may be similarly forced into a corner or a pocket via centrifugal forces.

[0097] The trapping device 300c with the tilter 1201 may be used when forming cardiac organoids, which may have difficulty forming and/or growing within a gel. By injecting individual cells or groups of cells into the culture chamber and tilting the trapping device 300c, the individual cells or groups of cells congregate at the lowest point in the trapping device (e.g., a pocket such as the pocket 1206). As the cells grow to form an organoid, their close proximity allows a singular organoid to form. After such organoid is formed, the tilter 1201 can return the trapping device to the initial, neutral plane and proceed with trapping the organoid in the immobilization chamber.

[0098] In use, the trapping device 102 of FIGS. 1-2E is configured for growing three- dimensional biological subjects (e.g., target organoids or a plurality of cells). For example, the trapping device 102 is configured to grow organoids in place in the culture chamber 120 for imaging, staining, and/or analyte testing. The trapping channels of the microfluidic device 100 of FIGS. 1-2E trap a target biological subject (e.g., an organoid, spheroid, a plurality of cells, a three-dimensional cell group, or any group of cells of interest). Trapping the target biological subject (e.g., in the immobilization chambers 150) provides a consistent location for analyzing the biological subject and performing tasks concerning the biological subject (e.g., imaging, staining, and analyte testing).

[0099] Various implementations include a method of growing and trapping biological subjects. Various steps of this method are described below in relation to the structure shown in FIGS. 1- 2E and 4A-4B, but it should be understood that this method can be used with any of the trapping device implementations described herein. The method of growing and trapping biological subjects begins with providing a microfluidic device 100 having a trapping device 102. Next, the method includes depositing a biological subject-laden gel 402 into the culture chamber 120 of the trapping device 102 via the fluid inlet 114 of the trapping device 102. The biological subjectladen gel 402 includes a gel 403 (e.g., hydrogel) and a plurality of biological subjects 404 suspended therein. The biological subject-laden gel 402 coalesces or solidifies in the culture chamber 120 between the first and second perfusion walls 130, 132. For example, the biological subject- laden gel 402 solidifies generally in the middle of the culture chamber 120. 1 [0100] The gel 403 is a viscous substance that, even when partially un-solidified, cannot flow through relatively small gaps. For example, the gel 403 is viscous enough that the gel 403 cannot flow through the perfusion gaps 134, 135. Thus, the biological subject-laden gel 402 cannot flow through the perfusion gaps 134, 135 into the first perfusion channel 136 nor the second perfusion channel 138. Therefore, once the biological subject-laden gel 402 is deposited into the culture chamber 120 of the trapping device 102, it remains in-place within the culture chamber 120.

[0101] A first portion 411 of the biological subject-laden gel 402 is defined adjacent to the fluid inlet 114, as shown in FIG. 4A. A second portion 412 of the biological subject- laden gel402 is defined adjacent to the first perfusion channel 136, and a third portion 413 of the biological subject- laden gel 402 is defined adjacent to the second perfusion channel 138. A fourth portion 414 is defined adjacent to a temporary perfusion channel 406 that is defined between the second end 124 of the culture chamber 120 and the fourth portion 414 of the biological subject-laden gel 402. In other words, the first portion 411 of the biological subject-laden gel 402 is adjacent to the fluid inlet 114, the second and third portions 412, 413 of the biological subject-laden gel 402 are adjacent to the first and second perfusion channels 136, 138, respectively, and the fourth portion 414 of the biological subject-laden gel 402 is adjacent to the staging chambers 140.

[0102] After the biological subject- laden gel 402 is placed in the culture chamber 120, the method further includes providing a flow of a culture medium 420 into and through the culture chamber 120 via the fluid inlet 114. The culture medium 420 (shown as arrows extending from the fluid inlet 114 and spreading throughout the culture chamber 120) is a liquid or semi-solid, flowable substance designed to support growth of a population of the biological subjects, such as microorganisms or cells (e.g., a growth medium for the plurality of biological subjects 404). [0103] The flow of the culture medium 420 includes flowing the culture medium 420 to each of the first, second, third, and fourth portions 411, 412, 413, and 414 of the biological subjectladen gel 402. The culture medium 420 flows directly from the fluid inlet 114 into the culture chamber 120 to contact the first portion 411 of the biological subject- laden gel 402. The culture medium 420 flows along the first perfusion channel 136 to contact the second portion 412 of the biological subject-laden gel 402 via the first perfusion gap 134. The culture medium 420 flows along the second perfusion channel 138 to contact the third portion 413 of the biological subjectladen gel 402 via the second perfusion gap 135. The culture medium 420 flows along either one of the first and second perfusion channels 138 to the temporary perfusion channel 406, contacting the fourth portion 414 of the biological subject-laden gel 402.

[0104] Therefore, the systems, methods, and devices described herein provide a flow of culture medium to more than one portion of a biological subject-laden gel configured for growing cells (e.g., organoids). Rather than flowing only to a portion adjacent to the fluid inlet, as in previous systems, the systems, methods, and devices disclosed herein provide a flow of culture medium to all portions of the biological subject- laden gel at once (e.g., around a periphery of the biological subject- laden gel to surround a circumference of the biological subject- laden gel). In this way, the biological subjects in the first portion of the biological subject-laden gel are not growing at a substantially faster rate than those in other portions. Because the culture medium is more evenly distributed about the biological subject-laden gel, the biological subjects therein will grow at a rate more in sync with each other.

[0105] The method further includes observing, monitoring, imaging, and measuring the plurality of biological subjects 404 while they grow in the biological subject-laden gel 402 in the culture chamber 120. For example, the method may include capturing images of the trapping device 102 at various points in time to track the growth of the plurality of biological subjects 404 therein. In some implementations, the culture medium 420 may include a target analyte or drug used for drug screening, and the capturing of images may investigate the effect that the drug has on cell growth rate.

[0106] Once the plurality of biological subjects 404 have reached a target growth size or time period, the gel 403 is dissolved with a biological subject recovery solution. The biological subject- laden gel 402 is broken down by the biological subject recovery solution into a dissolved fluid gel 403 and the plurality of biological subjects 404 now grown. Thus, the plurality of biological subjects 404 are released to flow into other areas of the trapping device 102.

[0107] The method further includes urging the plurality of biological subjects 404 into the immobilization chambers 150 of the trapping device 102. As shown in FIGS. 6B and 6C, urging the released plurality of biological subjects (shown as biological subjects 600 in FIGS. 6A-6C) includes causing the flow of a fluid (e.g., the fluid culture medium) through the trapping device, similar to the growth process. However, now released, the plurality of biological subjects flow with the fluid culture medium towards the second end of the culture chamber. [0108] The plurality of biological subjects distribute throughout the culture chamber 120 as they approach the staging chambers 140. Each staging chambers 140 then accepts a single biological subject 405 of the plurality of biological subjects 404 (e.g., as shown in FIG. 6C as biological subject 600). Other biological subjects flowing into an occupied staging chamber 140 will be unable to enter due to the presence of an existing biological subject 405. In other implementations, the staging chambers may be sized and configured to accept more than one biological subject. Essentially, the staging chambers 140 provide a method of indexing individual biological subject 405.

[0109] Once held within the staging chambers 140, the individual biological subjects 405 are urged by the fluid flow into the immobilization chambers. As discussed above, the immobilization chambers 150 have a smaller height compared to that of the staging chambers 140. The biological subject 405 entering the immobilization chamber 150 is thus “squished” to have an overall smaller height (e.g., the biological subject 405 becomes shorter and wider than it was prior to entering the immobilization chamber). However, the immobilization chambers 150 have a larger width, enabling the volume of the biological subject 405 to spread out to a greater width corresponding to the height reduction. This shape difference may be seen in the cross- sectional diagram of FIG. 5.

[0110] Once the individual biological subjects 405 are immobilized within the immobilization chambers 150, a variety of tests, experiments, and observations may be performed. Because the biological subjects 405 are isolated and fixed in location, such tests, experiments, and observations are more efficient. In some implementations, the method further includes capturing images of the biological subjects 405 of the plurality of biological subjects 404 that are within the immobilization chambers 150. For example, a camera adjacent to the trapping device 102 may capture images over time (see also FIGS. 13 and 14).

[0111] In other implementations, a staining fluid may be injected into the trapping device at the fluid inlet such that the individual biological subjects 405 of the plurality of biological subjects 404 are stained with the staining fluid. Such a process may target a specific portion of the biological subjects 405, making it easier to observe changes in those structures and/or visualize the biological subjects 405.

[0112] An example of individual biological subjects (e.g., organoids) trapped in an immobilization chamber is shown in FIGS. 7A and 7B, each showing a representative image of trapped organoids. FIG. 7A shows cardiac organoids, and FIG. 7B shows intestinal organoids that are uniformly distributed between the trapping channels (i.e., staging chambers and immobilization chambers) of the device.

Imaging Systems

[0113] FIG. 13 shows a system 1300 for using the microfluidic device 100 having the trapping device 102. It should be understood that system 1300 is described as using the microfluidic device 100 shown in FIGS. 1-2E and 4A-4B, but other microfluidic devices and trapping channels described herein may be used with system 1300. The system includes a camera 1302 positioned such that the bottom side of the microfluidic device 100 is in the field of view of the camera 1302 (e.g., via a mirror and lens system). The camera 1302 is arranged to view the trapping device 102 such that one or more of the immobilization chambers 150 is within the field of view of the camera 1302. The camera 1302 is in electrical communication with a controller 1304 and a data acquisition device (DAQ) 1306. The controller 1304 is configured to capture images, via the camera 1302, a store a plurality of images (e.g., on the DAQ 1306 or a memory/storage unit associated with the controller 1304).

[0114] The microfluidic device 100 is disposed on a motorized stage 1308 used for positioning the trapping device 102 and individual immobilization chambers 150 thereof in one or more desired positions within the field of view of the camera 1302. The immobilization chambers 150 offer predetermined locations recognizable by the camera 1302 and the controller 1304 that facilitate efficient and fast paced imaging of the organoids therein (e.g., including z-stack or depth imaging).

[0115] The camera 1302 is configured to capture images at several layers in the height of the immobilization chambers 150, made easier by the fact that the organoid’s height is “squished” in the immobilization chambers 150. For example, FIGS. 14A-14C show the difference in the number of z-images required to image the entire organoid, depending on its height. The schematic of FIGS. 14A-14C illustrates the difference in the shape of an intestinal organoid when it is within the Matrigel dome and as it moves through the trapping channel of the device. The z-images are shown with horizontal dashed lines. The overall number of images is reduced in the disclosed device, as shown in the schematic in FIG. 14B, as compared to a free-floating gel (e.g., hydrogel dome), shown in FIG. 14A. A comparison diagram showing the number of images for the hydrogel dome, the staging chamber, and the immobilization chamber is shown in FIG. 14C. While the distance between the z-images is the same, the number of z-images required for the hydrogel dome is significantly higher than when the organoid resides in the immobilization chamber. Thus, the device will reduce the number of images, the storage space for them, and the total imaging time.

[0116] Multiple organoids housed within two adjacent trapping channels can fit within a single field of view of a camera, which facilitates the imaging of at least two organoids per field of view (with at least one organoid per trapping channel). Because the trapping channels hold the organoids in place at known locations, the trapping device eliminates the cumbersome task of repeated scanning across an entire well to locate the samples, proving beneficial for larger screens.

[0117] The system 1300 further includes a first solenoid valve 1310 in fluid communication with the fluid inlet 114 and a second solenoid valve 1312 in fluid communication with the fluid outlet 180. The first solenoid valve 1310 is also in fluid communication with a first fluid reservoir 1314, and the second solenoid valve 1312 is in fluid communication with a second fluid reservoir 1316. Each of the first and second solenoid valves 1310, 1312 are also in fluid communication with a waste reservoir 1318.

[0118] A pressure device 1320 (e.g., a pump or air pressure controller) is in fluid communication with each of the first fluid reservoir 1314 and the second fluid reservoir 1316. The controller 1304 and the DAQ 1306 are in electrical communication with each of the pressure device 1320, the first solenoid valve 1310, and the second solenoid valve 1312. Generally, the controller 1304 can control fluid flow through the system 1300 as desired for a specific growing, imaging, washing, staining, clearing, moving, trapping, or other process. For example, the controller 1304 can control the flow and pressure on the fluid flowing through the system 1300. The controller 1304 can also control the direction of the flow of fluid through the system 1300. [0119] The first and second solenoid valves 1310, 1312 are coupled to create a fluidic Id- bridge, which enables the reversing of the flow direction within the trapping device 102. For example, in one configuration, a fluid can flow from the first fluid reservoir 1314, through the first solenoid valve 1310, through the trapping device 102 of the microfluidic device 100 (e.g., via a glass barrel at the fluid inlet), through the second solenoid valve 1312, and into the waste reservoir 1318. [0120] However, in a second configuration, a fluid can flow from the second fluid reservoir 1316, through the second solenoid valve 1312, through the trapping device 102 of the microfluidic device 100, through the first solenoid valve 1310, and into the waste reservoir 1318. The reversing of the flow may be used for redistributing the biological subjects within the culture chamber to ensure that only a single biological subject enters each of the staging chambers and immobilization chambers.

[0121] In some implementations, the trapping device operates under pressure-driven flow, as opposed to using constant flow rates (e.g., a syringe pump), to ensure that the flow rate decreases when the hydraulic resistance in the trapping device rises. A pressure build-up can arise when all trapping channels are occupied by organoids, potentially exposing them to undesirably high shear stress in a flow rate-driven system. Therefore, the device and associated system 1300 use pressure-driven flow that can adjust the flow rate within the trapping device as the trapping channels are loaded with organoids. Once all trapping channels are loaded with organoids, there will still be a small flow rate around the organoids to provide sufficient nutrient exchange.

[0122] FIGS. 15A and 15B show systems 1500a and 1500b, respectively, using a microfluidic device 1500 having a trapping device 1502, according to another implementation. Each of the systems of FIGS. 15A and 15B include a first trapping device 1502a and a second trapping device 1502b. The systems 1500a, 1500b further include a connection conduit 1510 coupled to and extending between the fluid outlet 1580a of the first trapping device 1502a and the fluid inlet 1514b of the second trapping device 1502b. Thus, a fluid flowing into the first trapping device 1502a via the fluid inlet 1514a of the first trapping device 1502a may then flow through the first trapping device 1502a to the fluid outlet 1580a of the first trapping device 1502a, through the connection conduit 1510, and into the fluid inlet 1514b of the second trapping device 1502b.

[0123] The system 1500a shows an implementation wherein, once the fluid leaves the fluid outlet 1580b of the second trapping device 1502b, the fluid enters a sample collection reservoir 1504. The sample collection reservoir 1504 is lower than the microfluidic device 1500a such that a hydrostatic flow is induced. However, in other implementations, a pump may be used.

[0124] The system 1500b shows an implementation wherein, once the fluid leaves the fluid outlet 1580b of the second trapping device 1502b, the fluid enters either a sample collection reservoir 1504 or a recirculation conduit 1506. The recirculation conduit 1506 is coupled between the fluid outlet 1580b of the second trapping device 1502b and the fluid inlet 1514a of the first trapping device 1502a. A pump is disposed along the recirculation conduit 1506 to induce the recirculation flow.

[0125] By connecting several trapping devices 1502 together in systems 1500a, 1500b, multiorgan study may be facilitated. For example, the first trapping device 1502a may contain an intestinal organoid, while the second trapping device 1502b may contain bladder tumor organoids. Such a system allows for the interaction between target analytes or drug interactions to be studied for a certain organoid and for inter-organ interactions. For example, a certain drug may behave a certain way when introduced to a first organoid, but that same drug may treat the first organoid differently when introduced to a second organoid before the first organoid. In other implementations, more than two trapping channels and/or microfluidic devices may be used in the associated system.

Examples, Experimental Testing, and Results

Device fabrication

[0126] An exemplary microfluidic device was fabricated by making a three-layered mold produced by photolithography of negative photoresists SU8-2050 and SU8-2100, followed by soft lithography of poly dimethylsiloxane (PDMS). Three photoresist layers (e.g., having heights of 100, 190, and 260 pm) were stacked during fabrication to define channels (e.g., channels having heights of 100, 290, and 550 pm, respectively) in the device. A 4-inch silicon wafer (STK9671-1, Nova Electronic Materials) was dehydrated on a hot plate at 120 °C for 30 minutes and installed on a spin coater. Depending on the desired layer thickness, 4 ml of either SU8-2050 or SU8-2100 was poured at the center and spun. The wafer was carefully removed from the spincoater and soft-baked. After cooling down to room temperature, the wafer was exposed to UV through the printed high-resolution mylar mask of the specific layer. The wafer photoresist was developed only after the first and third UV exposures. The mold was salinized using (TRIDECAFLUORO- 1,1, 2, 2-TETRAHYDROOCTYL) TRICHLOROSIL- ANE (Gelest, Inc.) for 72 hours to facilitate proper separation of the mold from the PDMS.

[0127] For the soft lithography, the base polymer and the curing agent were mixed at a 10: 1 (w/w) ratio. The mixture was degassed in a vacuum chamber and was then slowly poured onto the mold, followed by oven-baking at 75 °C for 6 hours. The PDMS was peeled off, punched for inlet and outlet tubing, and bonded to a #1.5 cover glass using oxygen plasma treatment to form the chip. Fluidic setup and its operation

[0128] FIG. 13 presents the microfluidic setup, according to one implementation. The flow in the device was regulated by an air pressure controller (ITV0010-3UBL, SMC) capable of delivering pressures between 0.01 - 1 bar. The regulator output was connected to two reservoirs filled with culture medium to establish a pressure-driven flow. The reservoirs and the device were connected to two solenoid valves to create a fluidic H-bridge. With this design, the flow direction within the device could be reversed at any time by controlling the solenoid valves using a DAQ card (NI USB-6009) and a Lab VIEW program (version 13.0f2).

Prior to loading the organoids, the device underwent several perfusion steps. The device was first perfused with deionized water to eliminate air bubbles, followed by sterilization with ethylalcohol, and lastly with culture medium. A glass barrel (Precigenome LLC) was connected via Luer lock to the tubing that supplies culture medium, and was mounted on the device inlet, serving as a nutrient reservoir. The glass barrel also served as an access point for loading the organoids and the fluorescent dyes onto the device. To establish a hydrostatically driven flow within the device, which was needed when using an open-top glass barrel, the waste reservoir was placed 10 cm below the device level. This height difference provided a flow rate of 16 pl/min in the device. The hydrostatically driven flow was initiated by opening the solenoid valve that connected the device outlet to the waste reservoir. Cardiac organoid culture and maintenance

[0129] CMV-GCaMP2 transfected human induced pluripotent stem cells (hiPSCs) were used to generate cardiac organoids and visualize spontaneous intracellular calcium transients. hiPSCs were maintained in Complete Essential 8 Media (E8, StemCell Technologies) on vitronectin- coated culture dishes. Prior to cardiac differentiation, hiPSCs were seeded onto Matrigel-coated multiwell plates at a seeding density of 3.16xl0⁵ cells/cm². Upon reaching an 80% confluence, cardiac differentiation was initiated by WNT/ P-catenin pathway modulation.

[0130] Spontaneously beating cardiomyocytes were harvested using Accutase (StemCell Technologies) 21 days after differentiation initiation. Cells were seeded at varying cell densities into ultra-low attachment round bottom 96-well plates (Nexcelom Biosciences). Next, seeded plates were centrifuged at 300g for 5 minutes to facilitate cell aggregation. The organoids were cultured in suspension for 48 hours in RPMI-1640 (Hyclone™) media supplemented with B-27 with insulin (Gibco™) and 10 pM ROCK inhibitor, to promote cell viability and organoid formation. Thereafter, media was replaced with media devoid of ROCK inhibitor and organoids were maintained in individual wells until they were transferred to chamber slides for DOX treatment followed by device loading.

Ethical Animal Use

[0131] The collection and analysis of intestinal biopsy samples from dogs were previously approved by the Iowa State University (ISU) Institutional Animal Care and Use Committee (IACUC-19-102; PI: Albert E. Jergens). All methods were performed in accordance with the relevant guidelines and regulations of IACUC as required by U.S. federal regulations (Fish, 2004). The study is reported in accordance with ARRIVE guidelines (https : // arriveguidelines . org) .

Intestinal organoid culture and maintenance

[0132] The study used canine intestinal crypts containing LGR5⁺ stem cells to facilitate the fast creation of mature intestinal organoids. Canine intestinal crypts were obtained from endoscopic biopsies of healthy adult canines at Iowa State University (IACUC-22-050). Mature organoids containing villi, crypts, and lumen could be produced typically within 3 to 6 days after a passage. At the time of seeding, canine intestinal crypts were suspended in Matrigel (Corning® Matrigel® GFR) and dispensed in 20 pl droplets onto a 24-well plate. Typically, every Matrigel pad contained 20 - 25 organoids. When organoids became larger than 400 pm, the study employed a splitting ratio of 1 :3. After a 10-minute incubation at 37 °C and 5% CO2, 0.5 ml of the Complete Medium with Growth Factors (CMGF⁺) containing 10 pM ROCK inhibitor and 2.5 pM GSK30 inhibitor was added to each well and maintained for 48 hours. Next, the medium was replaced with fresh CMGF⁺ without ROCK and GSK30 inhibitors and continuously refreshed every 48 hours for 7 days. The organoids in the Matrigel pads were passaged and expanded on day 7.

[0133] To passage the organoids, they were first recovered from the Matrigel pads. Briefly, the Matrigel pads were dissociated by first removing the spent media and adding 0.5 ml of Complete Medium without Growth Factors (CMGF ) at 4 °C to each well to pipette the entire Matrigel pad until it was broken and separated from the well plate. Then, the suspension was centrifuged at 100g for 5 minutes at 4 °C, and the supernatant was removed.

[0134] For passaging, 1 ml of TrypLE Express (Gibco, ThermoFisher Scientific) was added to the pellet and incubated at 37 °C and 5% CO2 for 10 minutes. Following centrifugation at 100g for 5 minutes at 4 °C, the supernatant was removed and replaced with 5 ml of CMGF- to stop the dissociation. An additional centrifugation step replaced the CMGF- with the necessary amount of 4 °C Matrigel (depending on the number of cells). The suspension was dispensed onto a 24-well plate as 15 to 20 pl droplets. The plate was incubated at 37 °C and 5% CO2 for 10 minutes to allow Matrigel solidification; then, 500 pl CMGF⁺ was added to each well for further culture. The Matrigel pads were cleaned up 3 or 4 days after each passage to remove the dead and degenerative cellular debris, as well as very small organoids. The clean-up process included all the steps described above for organoid recovery from Matrigel pads except for the TrypLE Express treatment.

[0135] Prior to DOX treatment, intestinal organoids were passaged 3 times and subsequently cultured for 7 days, with a clean-up performed on Day 5 to obtain the suitable organoid size and morphology. For device loading, intestinal organoids were first recovered from the hydrogel pads, as described above. An additional step was required to fully dissociate any remaining hydrogel on the organoids. This step was essential to prevent any organoid agglomeration. Specifically, we added 1 ml of the Cell Recovery Solution (Corning Inc.) to the centrifuged pellet, pipetted a few times, and then incubated the recovered organoids at 4 °C for 30 minutes to completely dissociate any Matrigel that was still attached to the organoids. Following an additional centrifugation at 100g for 5 minutes at 4 °C, the pellet with clean organoids was recovered in CMGF’ and transferred to a petri dish for loading into the chip.

Doxorubicin treatment of organoids

[0136] The study used 300 to 500 pm diameter organoids for treatment with DOX (0.1% (v/v) in DMSO for 48 hours. Cardiac organoids were first transferred from round bottom 96-well plates to chamber slides (Ibidi Inc.) with a 170 pm thick glass coverslip to enable aberration- free imaging. Before DOX treatment, cardiac organoids were incubated overnight in 8-well chamber slides and imaged for brightfield and calcium transients. These measurements served as baseline for analysing the cardiotoxicity of DOX. Cardiac organoids were treated with 0, 0.1, 0.5, 1, 2, 5, and 10 pM DOX. Intestinal organoids were subjected to 0, 0.1, 0.3, 1, and 2 pM DOX within Matrigel pads on their native 24-well plate. The maximum concentration that the intestinal organoids were subjected to was slightly lower due to their higher sensitivity to DOX.

Cardiac organoids beating kinetics analysis [0137] The study performed 10-second-long, calcium time- lapse imaging of cardiac organoids pre- and 48 hours post-DOX treatment to monitor the change in the beating kinetics of organoids in chamber slides (off-chip beating kinetics data). Then, the same organoids were transferred to the device and allowed to habituate in the TAs for 100 minutes before on-chip imaging. During imaging sessions, cardiac organoids in the chamber slides and the chips were maintained at 37 °C in a humidified mini-incubator (TA-MI-20x46, Bioscience tools). FIG. 16 presents a detailed flowchart describing the entire DOX treatment and calcium imaging of the cardiac organoids on chamber slides and the device.

[0138] To analyse changes in cardiac organoids’ beating kinetics, regions within the organoid that demonstrated calcium transients were identified, and their average signals were monitored across time-lapse images. The beating waveforms, extracted from these regions, were used to estimate various beating kinetics parameters using an in-house MATLAB code. Specifically, the study measured the beating rate (BR) and the ratio of the change in calcium fluorescence intensity to the baseline intensity (AF/Fo). The study also estimated the beating time percentage BT75) and beating power (FBT75) defined by: where T_BR represents the average beating period of an organoid and T₇₅ represents the average duration of beating until the GCaMP fluorescence amplitude drops by 75%. The study then calculated the fractional changes (4(X)) of various parameters for each individual organoid according to: where X_{pre tx} represents the pre-treatment and X_{post tx} represents the post-treatment values of the specific parameter. Measuring pre- and post-treatment values required us to track individual organoids as they were transferred from the chamber slide to the device by looking at their shape, area, and brightness.

Live/dead staining, imaging, and analysis [0139] The study stained the trapped organoids on-chip using Calcein AM (Invitrogen™), Ethidium Homodimer-1 (EthD-1) (Invitrogen™, Waltham, MA), and Hoechst 33342 (ThermoFisher Scientific Inc.) to label live cells, dead cells, and the cell nuclei, respectively. All dyes were administered at 2 pM, according to the manufacturers’ protocol, in culture media. The dyes were introduced into the device through the glass barrel by replacing culture media with dye solutions and applying hydrostatically driven flow in an on-off cycle (1 minute on and 10 minutes off) for a total of 50 minutes. The study used a flow rate of 16 pl/min, which was sufficient to replace the entire device volume of ~7.7 pl in less than 1 minute. Prior to imaging, we perfused the device with fresh culture medium for 2 minutes to remove any unbound dye and to minimize imaging background signal. The study imaged the organoids using widefield and confocal fluorescence microscopies.

[0140] To control for the impact of the device, a subset of cardiac and intestinal organoids was stained and imaged off the device (termed “off-chip” - in contrast to activities on the device, which are termed “on-chip”). Off-chip organoids were treated similarly with 2 pM dyes, followed by a 50-minute incubation at 37 °C. The off-chip cardiac and intestinal organoids were imaged on 8-well flat bottom chamber slides and within the Matrigel pads in 24-well plates, respectively.

[0141] For viability analysis, the study first created a mask, using the brightfield image, to define the boundaries of the organoids. The study used the mask on the maximum intensity projection images to calculate the mean intensity of the live (Sgreen) and the dead Sred) signals of the pixels within the mask and defined the viability ratio (VR) of each organoid according to:

[0142] For the DOX treatment experiments, the study normalized the VR of each organoid by the average VR of the vehicle control organoids to obtain the viability percentage with respect to control.

Image acquisition and processing

[0143] An ORCA-Flash4.0 V2 Digital CMOS camera Cl 1440-22CU (Hamamatsu Photonics K.K.) was used along with an 1X73 Inverted Microscope (Olympus) for widefield and fluorescence imaging. A PZU-2000 SERIES XYZ automated stage with an ASI MS-2000-WK multi-axis stage controller was used for automated imaging and positioning of the well plates and the device during imaging on the inverted microscope. Time-lapse images were obtained at 100 fps to capture the calcium transients (10 , 0.3NA objective, Olympus). The whole imaging setup was controlled using a homebuilt Lab VIEW (NI, USA) program.

[0144] Confocal imaging of trapped organoids on-chip was performed (Leica TAS SP8) immediately after the live/dead imaging on the widefield microscope. The z-images of Calcein AM (excitation: 488 nm, bandpass filter: 493-547 nm), EthD-1 (excitation: 552 nm, bandpass filter: 557-784 nm), and Hoechst (excitation: 405 nm, bandpass filter: 410-483 nm) were captured with step sizes of 5 and 3 pm using the 10x and 40/ objectives, respectively. All the image processing was performed with ImageJ (NIH).

Statistical analysis

[0145] The study used OriginPro (OriginLab Corporation, version 9.9.0.225) to perform statistical analyses, curve fitting for the dose-response assessments, and ICso calculations. The study calculated the /?- values for group comparisons using t-tests or F-tests where p < 0.05 (*) and p < 0.005 (**) were considered significant, after verifying assumptions of normality and checking for the equality of variances.

Undesired organoid motion and image blurring in conventional culture formats

[0146] By contrast, the study observed, in brightfield and fluorescence videos, that a strongly contracting organoid that is remained immobilized in a trapping area (TA), demonstrating the device’s advantage over conventional imaging platforms.

Organoid loading and device trapping efficiency

[0147] Various implementations include a loading method that accommodates the specific properties of each organoid type. The loading process begins by removing a tube connected to the top of a glass barrel and pipetting organoids into the glass barrel. The number of available organoids depends on the size of the organoids. For example, for cardiac organoids, eight organoids may be loaded on average. The cardiac organoids are then transferred from the chamber slides to the trapping device by allowing them to settle to the base of the glass barrel without the presence of flow. Then, organoids are gently directed into the trapping channels using pressures up to 0.15 bar, corresponding to a maximum flow rate of 13 pl/min per trapping channel. Under these flow rates, negligible or no cell shedding is observed from the organoids, indicating that the imposed shear stress is not physically detrimental to the organoids. FIGS. 7A and 7B show representative images that depict a trapping device with trapped organoids. FIG. 7A shows cardiac organoids, and FIG. 7B shows intestinal organoids that are uniformly distributed between the trapping channels of the device.

[0148] Intestinal organoid loading may be more prone to adhering to one another during device loading. Therefore, after Matrigel digestion in their native 24-well plate, intestinal organoids are transferred into a petri-dish and then pipetted one by one into the glass barrel while applying a hydrostatically driven flow. When an organoid gravitates to the bottom of the glass barrel and enters the device, the flow directs the organoid to an empty trapping channel. The organoids are constantly loaded one after another until all the trapping channels are filled with at least one organoid.

[0149] The trapping efficiency of organoids to be 68% on average during the initial loading in the device. The study tested another device with a larger number of trapping channels and found that the organoids did not distribute uniformly among the trapping channels due to the device’s large width, resulting in low trapping efficiency. Therefore, the study decreased the overall width and the tapering angle of the main area by reducing the number of trapping channels to six, that increased the overall trapping efficiency. Occasionally, two or more organoids entered a single trapping channel, leaving some other trapping channels empty. The study implemented an assisted loading strategy to improve the trapping efficiency and reduce the risk of multiple organoids being trapped in a single chamber. After the initial loading, the study reversed the flow to redirect the organoids into the main area, laterally tilted the device to uniformly redistribute them amongst the trapping channels, and resumed the forward flow to guide organoids into the empty trapping channels. Using this assistive loading procedure, the average trapping efficiency for both intestinal and cardiac organoids significantly increased to 85%.

Device does not affect the beating rate of cardiac organoids

[0150] To study the effect of the device flow dynamics and the loading process on the functionality of cardiac organoids, the study measured the changes in organoid beating rates using time-lapse imaging. Their average native off-chip beating rates were 55 beats per minute, as measured in 96- well plates using the BioTek Cytation 3. When the organoids were subsequently transferred into the device and distributed amongst the staging chambers, their beating rates initially decreased because of pipetting, temporary changes in temperature, and exposure to flow during loading. Yet, within 70 minutes of habituation inside the mini-incubator, with no flow in the chip, the organoids’ beating rates showed no statistically significant differences from their native values. During the following 80 minutes in the SCs, their beating rates remained unchanged.

[0151] The study then moved the organoids into the immobilization chambers. Even after 30 minutes of compression in the immobilization chambers, the organoids’ beating rates were similar to those measured off the chip. To further investigate the isolated effect of compression on cardiac organoids, the study performed a separate experiment on a petri-dish. Specifically, the study compressed the organoids to a height of 230 pm using a cover glass and monitored their beating rates over time. The results showed that the beating rates of compressed organoids approached pre-compression levels in less than 20 minutes, consistent with our on-chip observations.

Device does not affect the viability of organoids

[0152] Next, the study tested if loading and flowing organoids into the device affected their viability. The study stained and imaged organoids both off and on the chip. The organoids imaged off-chip served as the controls.

[0153] The viability ratios, calculated by applying Eq. 4 on live/dead images, show no statistically significant differences between the off- and on-device organoids. These results indicate that the device loading and immobilization processes have no notable adverse effects on organoid viability. Additionally, these results highlight that our device enables staining in addition to imaging of organoids. Despite the additional processes that the intestinal organoids were subjected to, such as the pipetting and resuspension processes required for dissolving the Matrigel for on-device imaging, their viability ratios are not significantly different than those imaged off-chip. These results further confirm that the additional steps required for deviceloading are also not detrimental to the cells.

Dose-response to Doxorubicin treatment using live/dead assay

[0154] The study evaluated the performance of the device in assessing the dose-dependent toxicity response of organoids using live and dead imaging. Most organoids were successfully immobilized in the immobilization chambers. Since intestinal organoids were more prone to agglomeration, regardless of DOX concentration, occasionally two or more of them adhered to each other and remained as large clumps in the staging chambers (0.1 and 1.0 pM treatment cases). At high DOX concentrations, cardiac organoids were significantly smaller in size (< 350 pm) and appeared to adhere to each other, as was seen in the 10 pM case. The study speculates that dead cells shedding off the organoids surface (observed during off-chip DOX exposure) contributed to size reduction while changes in the surface properties and presence of free DNA from the dissociating cells caused organoid agglomeration. Having multiple organoids in the trapping channels (in immobilization chambers or staging chambers) did not adversely affect the device functionality. Organoids were successfully imaged while immobilized in either chamber of the trapping channels.

[0155] The live/dead dose- response curves were obtained by fitting a sigmoidal Imax model (S-curve) to viability data points. Overall, the shape of the fitted S-curve was steeper for cardiac organoids vs. intestinal organoids. The viability starts dropping in the 10s of nM range for the intestinal organoids while the drop begins in the 100s of nM for cardiac organoids, implying that intestinal organoids are more sensitive to DOX treatment. For example, cardiac organoids treated with 0.1 pM DOX experience marginal effects on cell viability and these organoids exhibiting nearly the same viability as the DMSO-treated condition. On the other hand, treating intestinal organoids with the same DOX concentration reduces their viability to -65%. At higher DOX concentration treatments, such as 2 pM, the viability of both types of organoids significantly drops to below 10%.

[0156] Based on these curves, the study determined that the off- and on-chip DOX ICso values are 0.22 and 0.18 pM for the intestinal organoids, and 0.73 and 0.69 pM for cardiac organoids, respectively. These results further indicate that intestinal organoids are more sensitive to DOX treatment than cardiac organoids, showing toxicity at lower concentrations. The fitted doseresponse curves for the off- and on-chip tests are not statistically different, indicating that the loading, immobilization, and staining processes on the device do not alter the viability results for the DOX-treated organoids.

[0157] In the literature, there is a very limited number of studies presenting viability-based ICso values for DOX-treated intestinal organoids using human-based cells and none for caninebased organoids. One of the human-based studies analyzed images (4x objective) of cells stained with Phalloidin-FITC and Hoechst to find cellular viability. The study estimated their IC50 values to be lower than 1 pM, comparable to this study’s results. Viability analysis of DOX- treated cardiac organoids has been reported using different methods such as the absorbancebased assays, live/dead fluorescence staining, flow cytometry of cells dissociated from organoids, and beating kinetics parameters (i.e. beating rate, area change, etc.). However, many of these studies did not test over enough DOX concentrations to generate exposure-response curves and, ultimately, could not estimate any IC50 values. Among those studies that reported IC50 values, they relied on beating parameters. For example, Richards el al. reported an IC50 value for cardiac organoids exposed to DOX for 48 hours as measured from the fractional area change of the organoids during beating. They found an IC50 of 0.41 pM, comparable to our findings.

[0158] To show the capability of the device in enabling higher-resolution imaging, the study used confocal microscopy for several organoids immobilized on the chip. Representative images show improved optical segmentation and the ability to resolve single cells within an organoid. The co-localized EthD-1- and Hoechst-stained nuclei are resolvable and can potentially be used to increase the accuracy of viability measurements. Future implementation of 3D-segmentation using Al-assisted algorithms in the analysis pipeline can provide more accurate estimations of cellular viability in larger screens.

Calcium transience imaging revealed dose-dependent changes in beating kinetics parameters of cardiac organoids

[0159] Next, the study measured the effect of DOX treatment on the beating kinetics of cardiac organoids. To do this, the study relied on calcium fluorescence imaging, as it has been shown to be a good approximation of the cardiomyocytes’ action potentials. Calcium imaging proved beneficial for beating and contraction parameters since smaller beating portions cannot necessarily be detected from brightfield images, particularly when organoids have been compromised as a result of drug treatment.

[0160] When assessing drug effects, the study observed some degree of variability in the spontaneous contractile behavior and beating kinetics between cardiac organoids. Such variability often skews any averaged parameter value across organoids and does not reflect the effect of the treatment conditions on organoid health. To address this challenge, the study tracked each individual organoid’s beating off- and on-device. The drug-induced functionality results are therefore reported as averages of fractional changes of each individual organoid’s beating kinetics parameters, measured at 48 hours post-treatment, on both the chamber slide and on the chip, relative to its pre-treatment value (Eq. 3).

[0161] In assessing cardiotoxicity, the study studied the well-reported beating kinetics parameters, BR and AF/Fo. Additionally, the study proposes two new parameters: the beating time percentage (BT75) and the beating power (FBT75) to evaluate less-understood aspects of organoid beating kinetics (Eqs. 1 and 2). By fitting S-curves to the dose-dependent responses of organoids, the study estimated IC50 levels of off- and on-chip conditions for each of these parameters.

[0162] Interestingly, all IC50 levels estimated from beating kinetics parameters measured on- device are nearly identical to their off-chip counterparts and to those reported in the literature. Additionally, the off- and on-device S-curve fits are not statistically different. Taken together, these results indicate that toxicity levels obtained from on-chip beating analyses are similar to off-chip measurements, further demonstrating that the device does not introduce bias when assessing organoid viability or functionality. Furthermore, the estimated dose-response curves across all the beating kinetics parameters are similar to each other. Moreover, their resulting IC50 values are within the bounds of the standard error of the IC50 values obtained from the viability analysis. These results indicate that calcium imaging can be used interchangeably with fluorescence live/dead imaging to obtain IC50 values. In some implementations, the device successfully immobilized cardiac and intestinal organoids that were treated with a chemotherapeutic drug, Doxorubicin (DOX), at increasing concentrations. This platform enables image-based analysis of DOX treatment, demonstrating 50% inhibitory concentrations (IC50) values that are comparable to off-device results.

Device facilitates fast imaging

[0163] The study developed a Lab VIEW program to control the camera and our widefield microscope stage for fast and automated imaging and stage movements. The program moves the stage across three fields of view on the device, each containing two trapping channels (corresponding to two organoids), to image the six trapping channels with a high-speed camera. The study achieved imaging times of 10.4 and 70.0 seconds for on- vs. off-device calcium transients imaging and 5.5 and 89.5 seconds for on- vs. off-device live and dead imaging of two cardiac organoids. Eliminating the required times for finding an organoid in a well, changing to high-NA objective, and locating the correct ROIs and focal planes for the on-device imaging, significantly reduced the imaging time. The measurements show that the device is a promising tool for fast imaging to accelerate drug toxicity assessments if designed in a higher throughput manner. [0164] Finally, the study emphasizes that imaging calcium transients of spontaneously contracting cardiac organoids proves challenging as the fluorescing region of interest is subject to significant motion when imaging off-chip. Lots of post-processing are necessary for monitoring the same region of interest using cross-correlation metrics of the organoid’s position if the organoid is still within the field of view. If the organoid propels itself outside of the field of view, the calcium transient data is commonly lost in conventional imaging methods. Minimizing post-processing steps to account for organoid movements during timelapse imaging will prove to save time and processing power.

[0165] The study successfully loaded, immobilized, stained, and imaged organoids with the device and showed that these processes do not affect the viability or functionality of the organoids. Specifically, the study studied cardiac and intestinal organoids that were exposed to DOX, a standard chemotherapeutic drug, for 48 hours in a dose-dependent manner. The effects of DOX toxicity on the viability of both organoid types and functionality of cardiac organoids were found to be nearly identical between the off- and on-device tests. The estimated ICso values were similar to published data from the literature, especially those performed using image-based analysis. These studies have used low-resolution microscopy (i.e., 4 objective) to reduce image blurring and eliminate time-consuming scans for locating individual organoids within a well for viability assessment. Higher resolution imaging (i.e., > 20 x confocal imaging) might potentially provide more accurate and sensitive detection of drug-induced cytotoxic effects which can be implemented using the device.

[0166] Due to the organoids’ tendency to agglomerate and the stochasticity of organoid distribution among trapping channels, the loading process needs manual tilting and flow reversal to achieve high trapping efficiency (for this example testing device of the study only). Although such manual interventions are feasible for small-scale studies, they are not practical for large- scale studies using a multiwell chip. This challenge can be addressed by tilting the device steadily on an automated rocker during the loading process to promote a better organoid distribution among the trapping channels. Otherwise, we can also design the device with a single trapping channel per well, in a 384- well plate format, to eliminate organoid agglomeration since there will be only one organoid per device and, thus, the trapping process will be deterministic. [0167] The dimensions of our device are designed to be compatible with cost-effective fabrication methods, such as hot embossing and injection molding. These features can be replicated into a hot embossing mold by means of elastomeric transfer from the SU-8/Si wafer. The minimum feature size of the microchannels is on the order of ~ 0.1 mm to ensure that the chip’s features are compatible with CNC micro-milling, making it an ideal candidate for scaling up production via injection molding, in the future.

[0168] Other implementations of the device incorporate wells for culturing organoids, specifically cardiac organoids, within the imaging device. The automatic introduction of organoids into the trapping areas significantly reduces the challenges associated with pipetting during the organoid transfer process. Cultured organoids can also be optically cleared, fixed, immune-stained, imaged on device, and retrieved from the device for further analysis.

[0169] By and large, the data indicate that the device eliminates many of the current technologies’ shortcomings for imaging organoids. Furthermore, the recent FDA modernization Act 2.0, approving the use of organoid data for novel drug IND submission instead of in vivo animal testing, highlights the potential of the device in the preclinical evaluation of therapeutic drug candidates.

Culture Chamber Study

[0170] A Matrigel-embedded intestinal organoids sourced directly from canine intestinal biopsy samples were cultured in a culture chamber of the device. Organoids grew in the culture chamber with a limited height up to 11 days. Following this step, a transfer-less end-point fluorescence live and dead viability assay was performed by digesting the Matrigel and immobilizing the organoids within the trapping chambers of the device. Similar growth rates and cell viability were observed between the organoids grown on the device and outside of the device. Furthermore, the device saves an enormous amount of time that is otherwise required in the conventional platforms for locating the organoids within the multiwell plate and various steps of fluorescence staining. Thus, the device is a state-of-the-art device that enables both organoid culturing and high-resolution imaging for imaging-based drug testing assays.

Methods and Materials

[0171] The device was fabricated by soft lithography of PDMS using an SU-8 mold made via photolithography on a silicon wafer. Enteroids were thawed from liquid Nitrogen and grown on a conventional 24-well plate for 3 days. After that, the enteroids were trypsinized into single cells, embedded in Matrigel and the suspension was dispensed within the culture chamber of the device using a bent 22 gage Luer stub. Alternatively, a pipette containing the cell-laden Matrigel may be used. The tip of the pipette can be placed near the inlet of the device (e.g., the trapping device) so that capillary action automatically directs the cell-laden Matrigel into the culture chamber. The device was transferred to a 37 °C incubator for 15 minutes for the Matrigel to solidify. A glass barrel was mounted at the inlet to serve as a media reservoir whose content was refreshed every 48 hours. Automated imaging of the device and the plates was performed using a house-built Lab VIEW program to control the camera and the microscope stage. Brightfield images were taken every day for 7 days to monitor enteroid growth. The device was maintained in a humidified mini-incubator (TA-MI-20x46, Bioscience tools) during imaging sessions. On day 7, the Matrigel was digested with Cell Recovery Solution (Corning Inc.) while kept at 4 °C. Enteroids were then flown into the trapping channels for immobilization followed by live, dead and nuclei staining with Calcein AM, EthD-1 and Hoechst, respectively. The organoid viability was obtained as described previously.

Results

[0172] The organoids grew on the device for up to 11 days. However, they can grow until they reach the ideal size for study. They have been grown to 800 pm projected diameter, while limited by the height of the chip (550 pm). The study preoduced high-resolution images that were taken daily using a 20 x, 0.75 NA Olympus objective to track the organoids’ growth from day 7 to 11 after culture initiation. The study tested two initial cell densities off- and on-device to understand the effect of cell density on the growth rates of the organoids. Because of the limited space inside the chip or the Matrigel dome in the multiwell plate and proximity of the organoids, they face nutrient deficiency at higher cell densities which leads to decreased growth rates, typically from day 4 onwards. The study observed that the growth rate of organoids in terms of their projected area that is normalized by their initial size at day 0. It is evident that the organoid can reach larger sizes when starting from low initial cell densities (- 5x104 cells/ml). At high cell densities (- 5x105 cells/ml), the organoid size might start to decrease due to cell shedding and necrosis. However, the study observed similar growth rates for both the off- and on-chip conditions up to day 4, regardless of the cell density. This observation can be justified based on the fact that at the beginning of the organoid culture, the nutrient consumption of the organoids are low which allows them to grow at normal rates for a few days. The growth rate and viability of organoids cultured (or grown) in this device are comparable to those grown in a multiwell plate, which indicates that the device can serve as a useful tool for organoid studies. To understand the effects of on-chip organoid growth on the cellular viability, the study performed a fluorescence live and dead imaging. The Matrigel was first digested and then the organoids were flown to the trapping channels for imaging. The results indicate no statistically significant difference between the off- and on-device conditions.

Configuration of Certain Implementations

[0173] The construction and arrangement of the systems and methods as shown in the various implementations are illustrative only. Although only a few implementations have been described in detail in this disclosure, many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes, and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.). For example, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. Accordingly, all such modifications are intended to be included within the scope of the present disclosure. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative implementations. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of the implementations without departing from the scope of the present disclosure. [0174] Although the figures show a specific order of method steps, the order of the steps may differ from what is depicted. Also, two or more steps may be performed concurrently or with partial concurrence. Such variation will depend on the software and hardware systems chosen and on designer choice. All such variations are within the scope of the disclosure. Likewise, software implementations could be accomplished with standard programming techniques with rule-based logic and other logic to accomplish the various connection steps, processing steps, comparison steps and decision steps.

[0175] It is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting.

[0176] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another implementation includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another implementation. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0177] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal implementation. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0178] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific implementation or combination of implementations of the disclosed methods.

Claims

WHAT IS CLAIMED IS:

1. A microfluidic device comprising: at least one trapping device comprising a first end, a second end, at least one side wall extending between the first and second ends, a lower surface, and an upper surface opposite and spaced apart from the lower surface, the trapping device further comprising: a fluid inlet defined at the first end of the trapping device; a culture chamber defined adjacent the first end of the trapping device and extending from the first end of the trapping device towards the second end of the trapping device, the culture chamber comprising: (i) a first end of the culture chamber in fluid communication with the fluid inlet and a second end of the culture chamber opposite of the first end of the culture chamber along a first axis of the trapping device, (ii) a first height defined between the upper surface and the lower surface of the trapping device, the first height being measured in a direction that is parallel to a second axis that is orthogonal to the first axis, and (iii) a maximum width as measured in a direction that is parallel to a third axis that is orthogonal to each of the first axis and the second axis; first and second perfusion walls that extend into the culture chamber from the upper surface towards the lower surface of the trapping device, a free end of the perfusion wall being spaced apart from the lower surface of the trapping device, the perfusion walls extending from the first end of the culture chamber towards the second end of the culture chamber, wherein first and second perfusion channels are respectively defined between each perfusion wall and an adjacent side wall of the at least one side wall of the trapping device; at least one staging chamber defined at the second end of the culture chamber having a height that is equal to the first height and a width that is less than the maximum width; at least one immobilization chamber in fluid communication with the at least one staging chamber and having a height that is less than the first height, the at least one immobilization chamber configured to retain a target biological subject while a fluid flows through the trapping device towards the second end of the trapping device; and an exit chamber in fluid communication with the at least one immobilization chamber and disposed between the at least one immobilization chamber and the second end of the trapping device, wherein the trapping device is configured such that the fluid may flow from the fluid inlet to the exit chamber through the culture chamber, the at least one staging chamber, and the at least one immobilization chamber.

2. The microfluidic device of claim 1 , wherein the trapping device defines an opening associated with the fluid inlet defined in a first plane spaced apart from and above a second plane that extends through the culture chamber and the at least one staging chamber.

3. The microfluidic device of claim 1, wherein the microfluidic device comprises two or more trapping devices.

4. The microfluidic device of claim 1, wherein the trapping device further comprises third and fourth perfusion channels respectively defined between third and fourth perfusion walls extending adjacent to the at least one staging chamber and the adjacent side wall of the at least one side wall of the trapping device such that the third perfusion channel is in fluid communication with the first perfusion channel and the fourth perfusion channel is in fluid communication with the second perfusion channel.

5. The microfluidic device of claim 1, wherein the first and second perfusion walls extend past the at least one staging chamber.

6. The microfluidic device of claim 1, the trapping device further comprising a serpentine exit pathway on the second end of the trapping device and in fluid communication with the exit chamber, the serpentine exit pathway configured to provide a flow resistance in the culture chamber.

7. The microfluidic device of claim 1 , wherein the at least one immobilization chamber has a width that is greater than the width of the at least one staging chamber.

8. The microfluidic device of claim 1, wherein the maximum width of the culture chamber is defined across the second end of the culture chamber.

9. The microfluidic device of claim 8, wherein the at least one staging chamber comprises a plurality of staging chambers arranged along the second end of the culture chamber, wherein the at least one immobilization chamber comprises a plurality of immobilization chambers corresponding to the plurality of staging chambers.

10. The microfluidic device of claim 1, the trapping device further comprising an immobilization wall extending between the upper and lower surfaces of the trapping device at an end of the at least one immobilization chamber adjacent to the exit chamber, the immobilization wall and the at least one immobilization chamber defining a filter channel through which the fluid may flow from the at least one immobilization chamber towards the exit chamber.

11. The microfluidic device of claim 1 , the trapping device further comprising at least one baffle extending into the culture chamber from the upper surface of the trapping device at a portion of the culture chamber adjacent the fluid inlet and inward of the perfusion wall.

12. The microfluidic device of claim 1, wherein the trapping device comprises at least one layer of a thermoplastic material.

13. The microfluidic device of claim 1, wherein the culture chamber comprises a pocket defined by the at least one side wall of the trapping device at the first end of the culture chamber adjacent to the fluid inlet, the pocket configured to aggregate biological subjects during formation.

14. The microfluidic device of claim 1, wherein the trapping device is disposed on a tilter capable of tilting the trapping device to a desired angle.

15. The microfluidic device of claim 1, the trapping device further comprising a central divider wall extending into the culture chamber between the upper and lower surfaces of the trapping device to define a first portion and a second portion of the culture chamber, the central divider wall extending from the first end of the culture chamber to the second end of the culture chamber.

16. A system comprising: the microfluidic device of claim 1 , wherein the trapping device is a first trapping device; a second trapping device; and a connection conduit coupled to and extending between (i) the exit chamber and an adjacent fluid outlet of the first trapping device and (ii) a fluid inlet of the second trapping device.

17. A system comprising at least one of the microfluidic device of claim 1, the system further comprising: a camera arranged to view the trapping device such that the at least one immobilization chamber is within a field of view of the camera; and a controller in communication with the camera, the controller configured to capture images by the camera and store a plurality of images from the camera.

18. A system comprising at least one of the microfluidic device of claim 1, the system further comprising: a first fluid reservoir; a second fluid reservoir; a first fluid conduit coupled between the first fluid reservoir and the fluid inlet of the trapping device; a second fluid conduit coupled between the second fluid reservoir and an outlet of the trapping device; and a pump in fluid communication with the first fluid conduit, the pump configured to direct a fluid from the first fluid reservoir along the first fluid conduit to the fluid inlet of the trapping device.

19. The system of claim 18, further comprising: a controller coupled to the pump and configured to control the flow of the fluid into the trapping device.

20. A method of growing three-dimensional cells comprising: providing the microfluidic device of claim 1 ; depositing a biological subject-laden gel into the culture chamber via the fluid inlet of the trapping device, the biological subject-laden gel comprising a gel and a plurality of cells suspended therein, the biological subject-laden gel coalescing or solidifying in the culture chamber between the first and second perfusion walls; providing a flow of a culture medium through the culture chamber via the fluid inlet, wherein the culture medium flows (i) directly to a first portion of the biological subject-laden gel adjacent the fluid inlet (ii) through the first perfusion channel to a second portion of the biological subject-laden gel adjacent the first perfusion channel, (iii) through the second perfusion channel to a third portion of the biological subject-laden gel adjacent the second perfusion channel, and (iv) through a temporary perfusion channel to a fourth portion of the biological subject- laden gel, the temporary perfusion channel defined between the fourth portion of the biological subject-laden gel on a second end of the culture chamber and the at least one staging chamber, wherein the culture medium configured to facilitate growth of the plurality of cells; dissolving the gel of the biological subject- laden gel to release the plurality of cells; and moving at least one of the plurality of cells into the at least one immobilization chamber of the trapping device.

21. The method of claim 20, further comprising: imaging the plurality of cells via a camera adjacent to the trapping device.

22. The method of claim 20, further comprising: injecting a staining fluid into the trapping device at the fluid inlet such that the plurality of cells are stained with the staining fluid.

23. A microfluidic device comprising: at least one trapping device comprising a first end, a second end, at least one side wall extending between the first and second ends, a lower surface, and an upper surface opposite and spaced apart from the lower surface, the trapping device further comprising: a fluid inlet defined at the first end of the trapping device; an inlet chamber defined adjacent the first end of the trapping device and extending towards the second end of the trapping device, the inlet chamber comprising: (i) a first end of the inlet chamber in fluid communication with the fluid inlet and a second end of the inlet chamber opposite of the first end of the inlet chamber along a first axis of the trapping device, (ii) a first height defined between the upper surface and the lower surface of the trapping device, the first height being measured in a direction that is parallel to a second axis that is orthogonal to the first axis, and (iii) a maximum width as measured in a direction that is parallel to a third axis that is orthogonal to each of the first axis and the second axis; at least one staging chamber defined at the second end of the inlet chamber having a height that is equal to the first height and a first width that is less than the maximum width; at least one immobilization chamber in fluid communication with the at least one staging chamber and having a height that is less than the first height and a second width that is greater than the first width of the at least one staging chamber, the at least one immobilization chamber configured to retain a target biological subject while a fluid flows through the trapping device toward the second end of the trapping device; and an exit chamber in fluid communication with the at least one immobilization chamber and disposed between the at least one immobilization chamber and the second end of the trapping device, wherein the trapping device is configured such that the fluid may flow from the fluid inlet to the exit chamber through the inlet chamber, the at least one staging chamber, and the at least one immobilization chamber.

24. A microfluidic device comprising: at least one trapping device comprising a first end, a second end, at least one side wall extending between the first and second ends, a lower surface, and an upper surface opposite and spaced apart from the lower surface, the trapping device further comprising: a fluid inlet defined at the first end of the trapping device; a culture chamber defined adjacent the first end of the trapping device and extending from the first end of the trapping device towards the second end of the trapping device, the culture chamber comprising: (i) a first end of the culture chamber in fluid communication with the fluid inlet and a second end of the culture chamber opposite of the first end of the culture chamber along a first axis of the trapping device, (ii) a first height defined between the upper surface and the lower surface of the trapping device, the first height being measured in a direction that is parallel to a second axis that is orthogonal to the first axis, and (iii) a maximum width as measured in a direction that is parallel to a third axis that is orthogonal to each of the first axis and the second axis; first and second perfusion walls that extend into the culture chamber from the upper surface towards the lower surface of the trapping device, a free end of the perfusion wall being spaced apart from the lower surface of the trapping device, the perfusion walls extending from the first end of the culture chamber towards the second end of the culture chamber, wherein first and second perfusion channels are respectively defined between each perfusion wall and an adjacent side wall of the at least one side wall of the trapping device; and a fluid exit defined at the second end of the trapping device in fluid communication with the second end of the culture chamber, wherein the trapping device is configured such that the fluid may flow from the fluid inlet to the fluid exit through the culture chamber.

25. A method of growing three-dimensional cells comprising: providing the microfluidic device of claim 24; depositing a biological subject-laden gel into the culture chamber via the fluid inlet of the trapping device, the biological subject-laden gel comprising a gel and a plurality of cells suspended therein, the biological subject-laden gel coalescing or solidifying in the culture chamber between the first and second perfusion walls; and providing a flow of a culture medium through the culture chamber via the fluid inlet, wherein the culture medium flows (i) directly to a first portion of the biological subject-laden gel adjacent the fluid inlet (ii) through the first perfusion channel to a second portion of the biological subject-laden gel adjacent the first perfusion channel, (iii) through the second perfusion channel to a third portion of the biological subject-laden gel adjacent the second perfusion channel, and (iv) through a third perfusion channel to a fourth portion of the biological subject- laden gel, the third perfusion channel defined between the fourth portion of the biological subject- laden gel on a second end of the culture chamber and the at least one side wall of the trapping device, wherein the culture medium configured to facilitate growth of the plurality of cells.