Abstract
There is provided a microfluidic device comprising a first region configured to hold target cells, e.g., tumor cells, a second region configured to hold effector cells, e.g., immune cells, and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of immune cells, from the second region to an interaction zone that is at least partially disposed within the first region, for interaction with tumor cells in the interaction zone. The array of microstructures can be an array of micropillars. Also provided is a chip comprising a plurality of the device and a method of studying interactions of a first cell type with a second cell type.
Claims
1. A microfluidic device comprising a first region configured to hold tumor cells; a second region configured to hold immune cells; and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of immune cells, from the second region to an interaction zone that is at least partially disposed within the first region, for interaction with tumor cells in the interaction zone.
2. The device as claimed in claim 1, wherein the first and second regions are symmetrical about a same line of symmetry.
3. The device as claimed in claim 1, further comprising one or more third regions, the third region being in fluid communication with the first and second region, wherein the array of microstructures comprises microstructures disposed between the third region and the first region.
4. The device as claimed in claim 3, wherein the array of microstructures comprises microstructures disposed between the third region and the second region.
5. The device as claimed in claim 3, wherein the third region substantially surrounds the first region.
6. The device as claimed in claim 3, wherein the first, second and third regions are symmetrical about a same line of symmetry.
7. The device as claimed in claim 1 wherein the array of microstructures comprises microstructures organised in a radial manner or a grid-like manner.
8. The device as claimed in claim 1 wherein the array of microstructures comprises microstructures organised as substantially concentric rows of microstructures.
9. The device as claimed in claim 8, wherein the distance between the microstructures in the row closest to the first region is smaller than the distance between the microstructures in the row furthest from the first region.
10. The device as claimed in claim 8, wherein the size of the microstructures in the row closest to the first region is smaller than the size of the microstructures in the row furthest from the first region.
11. The device as claimed in claim 1, wherein each of said regions comprises a shape defined by tapering of a bigger area to a smaller area.
12. The device as claimed in claim 1, further comprising ports corresponding to each of said regions for providing access to each of the regions.
13. The device as claimed in claim 12, wherein the device comprises a seeding layer and a support layer, wherein the ports are disposed on the seeding layer and the corresponding regions are disposed on the support layer.
14. The device as claimed in claim 1, wherein the first region has a larger depth than the second region.
15. The device as claimed in claim 1, wherein the second region has substantially the same depth as the third region.
16. A chip comprising a plurality of the device of claim 1.
17. A method of studying interactions of a first cell type with a second cell type, the method comprising: providing a device comprising a first region configured to hold a first cell type; a second region configured to a second cell type; and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of the second cell type from the second region to an interaction zone that is at least partially disposed within the first region, to allow interaction of the first cell type and the second cell type in the interaction zone; seeding the first cell type in the first region of the device; applying a first external force to direct the first cell type to the interaction zone; seeding the second cell type in the second region of the device; allowing the second cell type to migrate from the second region to the interaction zone for interaction with the first cell type in the interaction zone; and monitoring migration of the second cell type and interaction of the second cell type with the first cell type.
18. The method of claim 17, wherein the method further comprises, subsequent to the monitoring step, applying a second external force to direct cells present within the interaction zone away from the interaction zone for retrieval and analysis.
19. The method of claim 17, wherein the monitoring step comprises monitoring the migration and interaction of the cells with an image capturing apparatus.
20. The method of claim 17, wherein the device further comprises one or more third regions, and the method further comprises, prior to the step of applying the first external force, seeding microenvironment materials into the one or more third regions, wherein the third region is in fluid communication with the first and second regions, and wherein the array of microstructures comprises microstructures disposed between the third region and the first region.
Description
BRIEF DESCRIPTION OF FIGURES
[0084] FIG. 1A is a schematic diagram illustrating a three-dimensional exploded view of a single compartmentalised microfluidic screening device, in accordance with various embodiments disclosed herein. FIG. 1B is a schematic drawing illustrating a top view of the compartmentalized microfluidic screening device, in accordance with various embodiments disclosed herein. FIG. 1B shows the designated compartments, device dimensions and depth ranges.
[0085] FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H are schematic drawings of the array of barriers (or microstructures) which creates a tumor trap inside the tumor-immune cell interaction chamber of the compartmentalized microfluidic device in accordance with various embodiments disclosed herein. Different microstructure geometries and organizations as shown in FIGS. 2A to 2H can be employed depending on need. FIG. 2I is an enlarged schematic drawing of the dotted square portion shown in FIG. 2A. In FIG. 2I, the narrowest gap between structures (on the innermost row of any barrier) is 5 μm while the largest gap is 25 μm (on the outermost row) to accommodate human T-Cell size and migration patterns.
[0086] FIG. 3A is a schematic diagram illustrating a three-dimensional exploded view of another single compartmentalised microfluidic screening device, in accordance with various embodiments disclosed herein. FIG. 3B is a schematic drawing illustrating a top view of the another compartmentalized microfluidic screening device, in accordance with various embodiments disclosed herein. FIG. 3B shows the designated compartments, device dimensions and depth ranges.
[0087] FIGS. 4A, 4B and 4C are schematic drawings of the tumor immune interaction chamber in the another compartmentalized microfluidic screening device of FIGS. 3A and 3B, in accordance with various embodiments disclosed herein. FIG. 4B is an enlarged schematic drawing of the dashed and rounded square portion shown in FIG. 4A. FIG. 4C is a schematic drawing of micropillars shown in FIGS. 4A and 4B. As shown in FIGS. 4A and 4B for example, the area containing the 3D tumor spheroid aggregates is surrounded by two or more concentric rows of rectangular pillars creating a tumor trap which holds the spheroid aggregate in place. Different materials (i.e., hydrogels or cell laden matrices) are centrifuged into position and held in place by the organized rows of pillars. The pillar sizes vary in each concentric row (see FIG. 4C) and the gaps between pillars (see FIG. 4B) can also vary while still allowing effector (immune) cells to be recruited (via chemotaxis) from the outermost layer—the effector cell region—to the tumor spheroid region.
[0088] FIG. 5 is a schematic drawing illustrating an experimental workflow using a compartmentalised microfluidic screening device, in accordance with various embodiments disclosed herein.
[0089] FIG. 6 is a schematic drawing illustrating an experimental workflow using another compartmentalised microfluidic screening device, in accordance with various embodiments disclosed herein. In FIG. 6, in the first step, tumor spheroids and tumor microenvironment (TME) materials are seeded into the device via an automated pipette. The device is then centrifuged to position the tumor cells and TME materials in the tumor-immune interaction zone (Step 2). Effector (immune) cells are then added (Step 3) and the migration and interaction of the effector cells with the tumor aggregate is continuously monitored and analyzed using microscopy and computer vision algorithms which track cell events of interest (Step 4). Post-experiment, the device is centrifuged again (reversing the direction of apparent centripetal force) to pellet the tumor cells with tumor infiltrating lymphocytes (TILs) as well as the TME materials with any immune cells that were located in those regions (Step 5). Finally, the cells and TME materials are retrieved via (automated) pipette and are analyzed further downstream (e.g., sequencing).
[0090] FIG. 7 is a schematic drawing illustrating a three-dimensional exploded view of a multiplexed (6×6) chip device conforming to the standard dimensions of a microscope slide, in accordance with various embodiments disclosed herein.
[0091] FIG. 8 is a schematic drawing illustrating a three-dimensional exploded view of a multiplexed (21×10) plate device conforming to the standard dimensions of a well plate, in accordance with various embodiments disclosed herein.
[0092] FIG. 9 is a schematic drawing illustrating a three-dimensional exploded view of a multiplexed (5×5) chip device conforming to the standard dimensions of a microscope slide, in accordance with various embodiments disclosed herein.
EXAMPLES
[0093] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural and biological changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.
[0094] The examples describe a platform suitable for use for in-vitro chemotherapeutic and immunotherapeutic combinatorial drug testing using at least two different types of cells: tumor cells and immune cells (e.g., CD8+ T cells, Natural Killer (NK) cells, etc.) with the option of including other cell types to create a complex three-dimensional microenvironment surrounding a tumor aggregate and more accurately physiologically mimic the tumor microenvironment in vivo. The platform is comprised of a physical device, an assay protocol, and an analysis procedure designed to be amenable to process automation and scale-up.
The Physical Device
[0095] In the following examples, a microfluidic device (i.e., a compartmentalized tumor trapping microfluidic device) for observation of immune-tumor interactions is provided. In some of the following examples, additional compartmentalized layers are provided for placement of cell-laden hydrogels or artificial extra-cellular matrix material surrounding the trapped tumor to create complex three-dimensional microenvironments.
[0096] A three-dimensional illustration of an exemplary physical device is shown in FIG. 1A, and a view of the device from the top is shown in FIG. 1B to highlight the different compartments.
[0097] In FIG. 1A, the microfluidic device 100 described is a microfluidic device designed specifically with four different zones or compartments, each with a specific purpose: tumor cell seeding zone/compartment, immune cell seeding zone/compartment, controlled tumor-immune cell interaction and observation zone/compartment, and cell recovery zone/compartment.
[0098] In FIG. 1A, the single physical sealed device 100 is comprised of: 1) a support layer in the form of a fluidic layer 102 (or a cell-interaction layer) that contains an array of microstructures 102E (i.e., micro-structured architecture) necessary for partitioning the different zones/compartments as well as controlling the positioning of the different cell populations throughout the entire experimental assay, and 2) a seeding layer 104 (in the form of a cap layer or a cell seeding layer) approximately 1 mm thick that seals the fluidic layer except for the cell seeding ports (i.e., there are openings for the tumor spheroid seeding port 102G, immune cell seeding port 102H, and cell extraction port/bubble trap 102I), and encloses the tumor-immune cell interaction zone/compartment (i.e., in the fluidic layer 102) completely. The device 100 is therefore an open-well format type of device with the ports remaining open to the outside environment.
[0099] The fluidic layer 102 ranges between 25 and 200 μm in depth depending on the type of device and the region/zone/compartment area (i.e., depending on the application and type of cells being used, and the region/zone/compartment area) (see FIG. 1B). In the fluidic layer 102, a first region 102A configured to hold tumor cells (at least partially within the tumor cell seeding port zone/compartment 106) is used for the initial placement of a first cell type (i.e., tumor cells) into the device 100, in either singularized or spheroid form. The tumor cell seeding port zone/compartment 106 is large enough to accommodate the initial seeding of cells in liquid or gel media, and acts as a holding area until the cells can be directed later into a different zone/compartment. The first region 102A has a depth of from 50-200 μm. A second region 102B configured to hold immune cells (at least partially within the immune cell seeding port zone/compartment 108) serves a similar function to the first region 102A. A second cell type (i.e., immune cells) are seeded into this area in a liquid medium and allowed to move freely in the 25-50 μm in depth area shown in FIG. 1B. An interaction zone 102C (at least partially within the tumor—immune cell interaction zone/chamber 110) is a compound zone/compartment in that it is dual-tiered (each tier with different depths) and contains an array of microstructures 102E (i.e., specific microstructure architectures) for controlling the interaction between the different cell populations. The center region of the tumor—immune cell interaction zone/chamber 110 is comprised of a tumor trap, which holds the tumor cells/spheroids in place at the center of a grid or radially arranged matrix of microstructures acting as a barrier between the tumor cells and the immune cells. The fluidic layer 102 also comprises an additional obstacle 102F that reduces the likelihood of non-interacting immune cells from entering the first region 102A during centrifugation to move the interacting tumor/immune cells away from the interacting zone 102C for retrieval and analysis at the tumor cell seeding port 102G.
[0100] Some examples of the different types of microstructures arrays and resulting barriers are shown in FIGS. 2A to 2H. The microstructure arrays have the same height as the fluidic layer 102 and can thus range from 25 μm to 50 μm in height. The distance between each type of microstructure (i.e., barrier porosity) depends on the organization of the array. Radially-organized microstructure arrays (see FIGS. 2A, 2B, 2E and 2F) exhibit a gradient of distances between microstructures. Here, the distance between microstructures in the outermost row (farthest from the center) is equal to 25 μm whereas the distances between microstructures decrease incrementally as they come closer to the center until reaching the innermost row (closest to the center) which has a distance of 5 μm between structures. As an example, see FIG. 2I for an expanded view of the dotted section in FIG. 2A. This gradient was designed with the size distribution of a population of immune cells in mind (ranging from 5 to 20 μm). The gradient also reflects an increasing challenge posed to the immune cells in terms of the narrowness of the channel pathway that they must navigate through to move from the outside of the barrier to the inside. Grid-organized microstructure arrays (see FIGS. 2C, 2D, 2G and 2H) have a uniform distance between microstructures throughout the entire barrier, independent of their distance from the radial center. Although the type of structure may vary, the distance between structures can range from 5 to 25 μm. The challenge posed to the immune cell population in this case is consistent throughout the entire barrier.
[0101] Returning to FIG. 1B, the device 100 further comprises a third region 102D (at least partially within the cell recovery port and air bubble trap zone/compartment 112). The third region 102D and the cell recovery port and air bubble trap zone/compartment 112 of the device 100 serve two purposes: as an open exit port in contact with the surrounding atmosphere for equilibration and also so that any incident air bubbles can be pushed through the device 100. The third region 102D and the cell recovery port and air bubble trap zone/compartment 112 also serve as a cell recovery port to recover immune cell populations that did not migrate through the barrier in the tumor.
[0102] A three-dimensional illustration of another exemplary physical device 300 is shown in FIG. 3A, while a view of the device from the top is shown in FIG. 3B to highlight the different compartments.
[0103] The microfluidic device 300 is a physical device designed specifically with four or more different zones or compartments, each with a specific purpose: a tumor cell seeding port zone which can also function as a cell recovery port zone, an immune cell seeding port zone, a controlled tumor-immune cell interaction and observation zone, and a variable number of tumor microenvironment (TME) material (such as hydrogels, cellular material, or both) seeding port zones which can also function as cell recovery port zones.
[0104] Similar to the device 100 described with reference to FIG. 1A, the device 300 of FIG. 3A is comprised of: 1) a support layer in the form of a fluidic layer 302 (or cell-interaction layer) that contains an array of microstructures 302F (i.e., micro-structured architecture) necessary for partitioning the different compartments as well as controlling the positioning of the different cell populations throughout the entire experimental assay, and 2) a seeding layer 304 (or a cap layer) approximately 1 mm thick that seals the fluidic layer 302 with openings for the cell seeding ports (e.g., tumor spheroid seeding port 302G and immune cell seeding port 302H), and encloses the tumor-immune cell interaction zone/compartment completely. The device 300 is therefore an open-well format type of device with the ports remaining open to the outside environment.
[0105] Also similar to the device 100 described with reference to FIG. 1A, the fluidic layer 302 as shown in FIG. 3B comprises a first region 302A for holding tumor cells/spheroids with a depth of between 50-250 μm (that is at least partially within a tumor cell seeding port zone/compartment 306), a second region 302B for holding immune cells with a depth of between 25-50 μm (that is at least partially within an immune cell seeding port zone/compartment 308) and an interaction zone 302C (that is at least partially within a tumor-immune cell interaction zone/chamber 310). In FIG. 3B, the fluidic layer 302 further comprises at least two third regions 302D (that are at least partially within the Matrigel/fibroblast seeding port zones/compartments 312). The third regions 302D and the Matrigel/fibroblast seeding port zones/compartments 312 allow tumor microenvironment (TME) materials to be provided and to be removed. TME materials include cancer associated fibroblasts or artificial extra cellular matrices such as Matrigel.
[0106] In FIG. 3B, the center region of the tumor/immune interaction zone/chamber 310 is comprised of a tumor trap, which holds the tumor cells/spheroids in place at the center of one or more radially arranged rows of micropillars 302F.
[0107] These concentric rows of micropillars keep the tumor spheroid in place, and allow for the organized placement (via centrifugation) of layers of tumor-microenvironment materials. There are spaces between each of the micropillar structures, allowing for effector (immune) cells migrating from the outer regions of the tumor-immune interaction zone to the innermost region (where the tumor spheroid is located) to pass through.
[0108] An example of the arrangement of the micropillar barriers in the tumor-immune interactions zone creating two distinct layers surrounding the tumor spheroid regions is shown in FIGS. 4A to 4C. The micropillar structures form the physical barrier around the tumor spheroid and have the same height as the fluidic layer and can thus range from 25 μm to 50 μm in height. The distance between each type of microstructure (i.e., barrier porosity) can vary. In the example shown in FIG. 4B, the distance between the micropillars in the outermost row (farthest from the center) of radially arranged micropillars is equal to 15 μm whereas the distances between micropillar structures in the rows closer to the tumor-spheroid region decrease incrementally as they come closer to the center until reaching the innermost row (closest to the center) which has a distance of approximately 6 μm between structures. This gradient was designed with the size distribution of a population of immune cells in mind (ranging from 5 to 20 μm). The gradient also reflects an increasing challenge posed to the immune cells in terms of the narrowness of the channel pathway that they must navigate through to move from the outside of the barrier to the inside. As indicated in FIGS. 4A and 4B, in between the radially organized rows of micropillars different materials can be strategically placed to form a complex three-dimensional biological barrier between the effector (immune) cells and their tumor spheroid target. The materials forming these layers can be positioned in an organized manner via centrifugation (see FIG. 4A). Together with the physical barriers, these biological barriers form the complex three-dimensional tumor microenvironment surrounding the tumor through which the effector cells must migrate in order to reach the tumor.
The Experimental Assay Protocol (Workflow)
[0109] In the following examples, processes (involving the devices e.g., device 100 of FIG. 1A and device 300 of FIG. 3A) resulting in directed, controlled interactions between cell populations is described.
[0110] In the following first example, the protocol and workflow described was developed specifically for the device 100 described with reference to FIG. 1A (which conversely was designed for streamlined processing and scale-up) and entails a multi-step process that aims to achieve three general goals: cell seeding and localization, observation of cellular interactions, and cell recovery.
[0111] FIG. 5 provides an overview of the protocol and workflow 500 of the assay directly involving the device 100 of FIG. 1A. The entire experimental process, including obtaining patient samples and/or patient-derived cell lines, the culturing and maintenance of these samples prior to and after the experiment and the identification of biomarkers is implicit and thus not included in the figure. In the first step (FIG. 5, step 502), a population of tumor cells/spheroids 502B are seeded into the sealed device via the tumor seeding zone/compartment using an automated handler 502A (such as a bioprinter). The device 100 is then quickly placed into a special adapter 504A (FIG. 5, Step 504) for a multiplexed chip 504B (e.g., 36 plex chip) or plate 504C (e.g., 210-plex plate) (i.e., comprising a plurality of the device 100 described with reference to FIG. 1A), and then centrifuged (FIG. 5, Step 506). As the device 100 is symmetric along a central axis, it can be arranged with this axis pointing radially towards or away from the center of the centrifuge (central axis and directionality of central axis shown in FIG. 1B). In FIG. 5, Step 506, the device 100 is oriented with its central axis pointing towards the center of the centrifuge and the resulting apparent centrifugal force thus acts in the opposite direction. This apparent centrifugal force (shown by the downward arrow) directs the recently seeded tumor cells/spheroids 506A towards the tumor-immune cell interaction zone/chamber and into the trap area where they are trapped by the array of microstructures, which act as a barrier in this compartment, hold the tumor cells in a centralized position. After centrifugation, the device 100 is removed and the immune cell population 508A is added via a port 102B in the immune cell seeding port zone/compartment (FIG. 5, Step 508) of the sealed device, again using an automated handler. The device, with both cell populations seeded and in position, is now primed for observation via microscopy (FIG. 5, Step 510). Immune-tumor interactions are observed throughout the duration of the experiment and immune cells that are successfully recruited to the tumor mass by migrating through the artificial barrier separating the two cell populations are separated from the cells that are not recruited. Immune cells located inside the tumor trap region can infiltrate the tumor mass, potentially resulting in tumor cell death. In FIG. 5, Step 512, the cells located inside the tumor mass (successful infiltration) and in the tumor trap region as well as other immune cells located at various positions inside and along the artificial barrier are tracked, monitored and recorded using an automated computer vision analysis procedure which includes using a computer vision algorithm to detect and identify targeted interactions. In FIG. 5, Step 514, the device 100 is centrifuged once more but with its central axis pointing away from the center of the centrifuge (i.e., in the opposite direction as Step 506) to recover cell populations in devices which exhibited interesting cellular interactions. The apparent centrifugal force (shown by the upwards arrow) now directs the cells which are located in the tumor trapping region (both tumor and recruited immune cells 514A) back into the tumor cell seeding port zone/compartment. Any non-recruited (non-migratory) immune cells 514B will be directed to the opposite end of the device 100 towards the cell recovery port zone/compartment. In the last step (FIG. 5, Step 516), the separated, phenotypically different cell populations are recovered with an automated handler and analyzed to identify genetic biomarkers that can be used to determine responsiveness to treatment.
[0112] In the following second example, the protocol and workflow described was developed specifically for the device 300 described with reference to FIG. 3A (which conversely was designed for streamlined processing and scale-up) and entails a multi-step process that aims to achieve three general goals: 1) the creation of a complex three dimensional tumor microenvironment (involving cell seeding and organization of TME materials in stratified layers, 2) observation of cellular interactions, and 3) cell recovery.
[0113] FIG. 6 provides an overview of the protocol and workflow 600 of the assay directly involving the device 300 of FIG. 3A. The entire experimental process, including obtaining patient samples and/or patient-derived cell lines, the culturing and maintenance of these samples prior to and after the experiment and the identification of biomarkers is implicit and thus not included in the figure. In the first step (FIG. 6A, Step 602), a population of tumor cells/spheroids 602A as well as tumor microenvironment materials 602B such as cancer associated fibroblasts or artificial extra cellular matrices such as Matrigel, are seeded into the device via different seeding ports (ports 302G and 302I) using an automated handler (such as a bioprinter or a pipette). A multiplexed chip or plate (i.e., comprising a plurality of the device 300 described with reference to FIG. 3A) is prepared, and the device 300 is then centrifuged (FIG. 6A, Step 604). At Step 604, if a multiplexed chip is used, to fit the multiplexed chip into centrifuges, the chip is placed inside an adaptor, for example, a plastic falcon tube. If a multiplexed plate is used at step 604, the multiplexed plate with its larger dimensions would require a different type of specialized adaptor (e.g., an adaptor that can accommodate the larger dimensions of the multiplexed plate). As the device 300 is symmetric along a central axis, it can be arranged with this axis pointing radially towards or away from the center of the centrifuge (central axis and directionality of central axis shown in FIG. 3B). The device 300 is oriented with its central axis pointing towards the center of the centrifuge and the resulting apparent centrifugal force (shown by the downward pointing arrow) thus acts in the opposite direction. The apparent centrifugal force directs the recently seeded tumor cells and TME materials towards the tumor-immune cell interaction zone/chamber where they are trapped by the array of microstructures. These micropillar microstructures serve as a barrier in this compartment (i.e., the tumor-immune cell interaction zone/chamber) and hold the tumor cells in a centralized position while also creating stratified layers of materials to which create the complex three-dimensional TME (i.e., a biological barrier). After centrifugation, the device 300 is removed and the immune cell (effector cell) population 606A is added via the port 302H in the immune cell seeding port zone/compartment (FIG. 6A, Step 606), again using an automated handler. The device 300, with both the tumor and immune cell populations seeded and in position, is now primed for observation via microscopy (FIG. 6A, Step 608). Immune-tumor interactions are observed throughout the duration of the experiment and immune cells are allowed to migrate through the different barriers (both physical and biological). The immune cells are thus separated into different populations based on the extent to which they can migrate through barriers. Immune cells located inside the tumor trap region can infiltrate the tumor mass, potentially resulting in tumor cell death. The cells located inside (successful infiltration) and around the tumor mass as well as other immune cells located at various positions within the tumor-immune interaction zone are tracked, monitored and recorded using an automated computer vision analysis procedure, which includes an automated machine learning computer vision algorithm that identifies devices of interest with tumor infiltrating lymphocytes (TILs). In FIG. 6A, Step 610, the device 300 is centrifuged once more but with its central axis pointing away from the center of the centrifuge. The apparent centrifugal force (shown by downward pointing arrow) now directs the cells which are located in the tumor trapping region (both tumor and recruited immune cells 610A, such as infiltrating lymphocytes (TIL) that penetrated into the tumor area) back into the tumor cell seeding zone/compartment. The apparent centrifugal force also directs the TME materials 610B (e.g., cancer associated fibroblasts, Matrigel, etc.) that were surrounding the tumor spheroid back into their seeding port regions along with any immune cells that might have been positioned within those barriers at the end of the experiment. In the last step (FIG. 6A, Step 612), the separated, phenotypically different cell populations are recovered with an automated handler and can be analyzed further to identify genetic biomarkers that can be used to determine responsiveness to treatment.
The Automated Analysis Procedure
[0114] In the examples described above e.g., with reference to FIGS. 5 and 6, a computer vision algorithm to identify, track and monitor cellular interactions is described.
[0115] In the examples described above, microscopic images captured at a frequency of 2 to 4 images per hour are recorded throughout the duration of the experiment and analyzed using a computer vision algorithm written specifically to identify, track and flag cellular interactions of interest, namely: tumor cell viability, immune cell viability and immune cell count (within the barrier and inside the tumor trap area).
[0116] In the examples described above, the algorithm is designed to simplify and streamline the analysis process (e.g., see FIG. 5, Step 512, and FIG. 6, Step 608) thereby expediting the rate at which devices of interest in an array of devices can be identified (e.g., see FIG. 5, Step 514, and FIG. 6, Step 608) and methodically recovered (e.g., see FIG. 5, Step 516, and FIG. 6, Step 612).
Platform Flexibility
[0117] In the examples described e.g., with reference to FIGS. 5 and 6, a multiplexed chip or plate device has been described.
[0118] Two different exemplary multiplexed devices (based on the device 100 described with reference to FIG. 1A) that conform to industry standard dimensions (the size of a standard microscope slide or a well plate) are presented in FIGS. 7 and 8, respectively. In FIG. 7, a multiplexed (6×6) chip device 700 conforming to the standard dimensions of a microscope slide is shown. The chip device 700 comprises a fluidic layer 702, which contains numerous micro-structured architecture, and a cap layer 704, which seals the fluidic layer 702 except for the numerous cell seeding ports. In FIG. 8, a multiplexed (21×10) plate device 800 conforming to the standard dimensions of a well plate is shown. The plate device 800 similarly comprises a fluidic layer 802, which contains numerous micro-structured architecture, and a cap layer 804, which seals the fluidic layer 802 except for the numerous cell seeding ports.
[0119] Another exemplary multiplexed device (based on the device 300 described with reference to FIG. 3A) that conform to industry standard dimensions (the size of a standard microscope slide) is presented in FIG. 9. In FIG. 9, a multiplexed (5×5) chip device 900 conforming to the standard dimensions of a microscope slide is shown. Similar to the chip device 700 described with reference to FIG. 9, the chip device 900 comprises a fluidic layer 902, which contains numerous micro-structured architecture, and a cap layer 904, which seals the fluidic layer 902 except for the numerous cell seeding ports.
[0120] In FIGS. 7, 8 and 9, the devices shown have been designed such that the orientation of the individual microfluidic compartments maintain their alignment and can still be centrifuged with nearly identical centrifugal force. The devices can be used in existing imaging solutions present in the laboratory ecosystem (e.g., confocal microscopes, plate readers, etc.) and exhibit sufficient spacing between individual cell seeding/recovery ports such that automated handlers (e.g., bioprinters) can still be used.
APPLICATIONS
[0121] Advantageously, various embodiments of the method/process (including the use of the device) disclosed herein provide an in vitro assay that can be scaled-up easily and used for high-throughput screening that would allow pharmaceutical companies and clinical researchers for example to study patient responses to specific therapies without posing danger to the patient, as well as provide an early-stage screening for healthcare providers for example to determine which patients may or may not respond well to a particular combination therapy.
[0122] Various embodiments of the present disclosure provide an OncoMiMIC (Onco-Multi-Metric Immuno-Combinatorial) testing platform. In various embodiments, the OncoMiMIC testing platform is advantageously adapted for forming a complex 3D Tumor Microenvironments (TME). In various embodiments, such platforms may be referred to as an OncoMiMIC-CTM (Onco-Multi-Metric Immuno-Combinatorial Complex Microenvironment) testing platform.
[0123] Various embodiments of the present disclosure provide a testing platform that comprises a class of microfluidic chips that are inexpensive. In various embodiments of the present disclosure, the comprehensive in-vitro testing platforms can screen individual tumor sample responses to new and existing combinatorial cancer therapies, thereby potentially reducing the cost of identifying and testing target drugs in the development and discovery stage.
[0124] In various embodiments of the present disclosure, the OncoMiMIC-CTM testing platform advantageously allows for an end user to place, in a precise and well-organized manner, one or several surrounding layers of tissues or artificial extra-cellular matrix (ECM) material around a tumor sample, thereby creating a more physiologically relevant, complex, three-dimensional microenvironment for the tumor. Then, in various embodiments, similar to the way in which the OncoMiMIC platform functions, the OncoMiMIC-CTM platform allows for the interaction of effector cells (immune cells) to interact with this complex tumor structure.
[0125] In various embodiments of the present disclosure, the testing platforms provide platform flexibility in that the platforms are amenable to automation and scale-up for an industry requiring high-throughput and big data.
[0126] In various embodiments of the testing platform disclosed herein, the platform provides an easily scalable design, which allows the platform to be highly amenable to automation. It has been recognized that a single screening test can take place in an individual device (e.g., see FIGS. 1A and 3A), but the screening of tumor-immune interactions requires a very high number of repeatable experiments to generate statistically relevant data. Furthermore, there are often many combination therapies and/or drug candidates which require testing. Therefore, various embodiments of the testing platform disclosed herein advantageously provide an ideal platform (e.g., the multiplexed devices) that would be able to conduct screening experiments quickly with the same experimental conditions and with minimal human intervention.
[0127] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.
