System and method for bone marrow hematopoesis and leukocyte mobilization

Abstract

Provided herein is a bioreactor system, including a cell growth platform having a hydrogel arranged thereon, at least one first cell received on and / or in the hydrogel, a flow channel configured to receive a flow of a liquid therethrough, a separator arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel, and at least one second cell received on the separator.

Description

SYSTEM AND METHOD FOR BONE MARROW HEMATOPOESIS AND LEUKOCYTE MOBILIZATIONCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 619,015, filed January 9, 2024, the disclosure of which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under EB029085 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTIONField of the Invention

[0003] Provided herein are systems and methods for cellular growth and / or differentiation, screening of potential therapeutic agents, and / or identifying and counting cells.Description of Related Art

[0004] Hematopoiesis is the process through which blood immune (e.g., monocytes, neutrophils, NK cells, T lymphocytes, B lymphocytes, etc.) and non- immune (e.g., platelets, red blood cells [RBCs]) cells, as well as bone marrow (BM) cells, are created. The blood (immune and non-immune) cells upon production need to egress out of BM niche to enter circulation. This allows replenishment of the systemic repertoire and provision of the cells to peripheral organs during infection, inflammation, and fighting against tumors. Dysfunction in hematopoiesis and mobilization of the cells out of the BM can lead to a range of pathologies such as blood cancers and immune disorders. As such, modeling these processes outside of the human body is crucial to understanding the underlying pathophysiology and enabling therapeutic testing, safety evaluation of drugs I drug-like compounds and identification of druggable targets.SUMMARY OF THE INVENTION

[0005] Provided herein is a bioreactor system, including a cell growth platform having a hydrogel arranged thereon; at least one first cell received on and / or in thehydrogel; a flow channel configured to receive a flow of a liquid therethrough; a separator arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel; and at least one second cell received on the separator.

[0006] Also provided herein is a bioreactor system, including a cell growth platform having a fibrin hydrogel arranged thereon; at least one mesenchymal stem cell and at least one CD34+ cell embedded in the fibrin hydrogel; a flow channel configured to receive a flow of a culture medium therethrough; a porous membrane arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel; at least one endothelial cell arranged on the second surface of the porous membrane; and an imaging device arranged about the flow channel downstream of the cell growth platform and having a moveable stage; a flow cell arranged on the moveable stage, the flow cell receiving at least a portion of the flow channel therein; a light source arranged above the flow cell; and an image capture device arranged below the flow cell.

[0007] Also provided herein is a system for imaging and counting cells, including a moveable stage; a flow cell arranged on the moveable stage, the flow cell receiving at least a portion of a flow channel therein; a light source arranged relative to the flow cell; an image capture device arranged relative to the flow cell; and at least one processor in communication with the imaging device, the at least one processor configured to: receive at least one image from the image capture device; perform a cell detection procedure on the at least one image to identify at least one cell object in the at least one image; and perform a cell counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure.

[0008] Also provided herein is a non-transitory, computer-readable medium having stored thereon programming instructions that, when executed by a processor, cause the processor to: receive at least one image from an image capture device; perform a cell detection procedure on the at least one image to identify at least one cell object in the at least one image; and perform a cell counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure.

[0009] The following numbered clauses outline various aspects or embodiments of the present invention:

[0010] 1. A bioreactor system, comprising: a cell growth platform having a hydrogel arranged thereon; at least one first cell received on and / or in the hydrogel; a flow channel configured to receive a flow of a liquid therethrough; a separator arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel; and at least one second cell received on the separator.

[0011] 2. The bioreactor system of clause 1 , wherein the cell growth platform comprises a housing or a well defining an interior, and wherein the hydrogel is received within the interior.

[0012] 3. The bioreactor system of clause 1 or clause 2, wherein the at least one first cell comprises a stem cell, pluripotent cell, and / or multipotent cell.

[0013] 4. The bioreactor system of any of clauses 1 -3, wherein the at least one first cell comprises at least one CD34+ cell.

[0014] 5. The bioreactor system of any of clauses 1 -4, wherein the at least one first cell comprises at least one mesenchymal stem cell (MSC).

[0015] 6. The bioreactor system of any of clauses 1 -5, wherein the at least one first cell comprises at least one CD34+ cell and at least one MSC.

[0016] 7. The bioreactor system of any of clauses 1 -6, wherein the at least one CD34+ cell and the at least one MSC are human cells.

[0017] 8. The bioreactor system of any of clauses 1 -7, wherein the at least one first cell is received within the hydrogel.

[0018] 9. The bioreactor system of any of clauses 1 -8, wherein the at least one second cell comprises an endothelial cell.

[0019] 10. The bioreactor system of any of clauses 1 -9, wherein the endothelial cell is a human umbilical vein endothelial cell.

[0020] 11. The bioreactor system of any of clauses 1 -10, wherein the hydrogel comprises a fibrin hydrogel.

[0021] 12. The bioreactor system of any of clauses 1 -11 , wherein the hydrogel is formed from fibrinogen and thrombin.

[0022] 13. The bioreactor system of any of clauses 1 -12, wherein the flow channel is in fluid communication with a media reservoir and a pump, such that the media flows through the flow channel.

[0023] 14. The bioreactor system of any of clauses 1 -13, wherein the media comprises a cross-linker.

[0024] 15. The bioreactor system of any of clauses 1 -14, wherein the cross-linker is a transglutaminase.

[0025] 16. The bioreactor system of any of clauses 1 -15, wherein the cross-linker is gelatin transglutaminase.

[0026] 17. The bioreactor system of any of clauses 1 -16, wherein the media comprises a proteinase inhibitor.

[0027] 18. The bioreactor system of any of clauses 1 -17, wherein the proteinase inhibitor is a matrix metalloproteinase (MMP) inhibitor.

[0028] 19. The bioreactor system of any of clauses 1 -18, wherein the MMP inhibitor is aprotinin.

[0029] 20. The bioreactor system of any of clauses 1 -19, wherein the MMP inhibitor is a tissue inhibitor of metalloproteinase (TIMP).

[0030] 21. The bioreactor system of any of clauses 1 -20, wherein the media comprises one or more first additives capable of inducing differentiation of the at least one first cell.

[0031] 22. The bioreactor system of any of clauses 1 -21 , wherein the media comprises one or more second additives capable of inducing cellular egress from the cell growth platform.

[0032] 23. The bioreactor system of any of clauses 1 -22, wherein the flow channel is a straight channel.

[0033] 24. The bioreactor system of any of clauses 1 -23, wherein the flow channel is non-linear.

[0034] 25. The bioreactor system of any of clauses 1 -24, wherein the flow channel comprises a plurality of curves.

[0035] 26. The bioreactor system of any of clauses 1 -25, wherein the flow channel comprises a zig zag pattern.

[0036] 27. The bioreactor system of any of clauses 1 -26, wherein the at least one second cell is arranged on the second surface of the separator.

[0037] 28. The bioreactor system of any of clauses 1 -27, wherein the cell growth platform comprises, a thermoplastic polymer or copolymer, a resin, and / or a glass.

[0038] 29. The bioreactor system of any of clauses 1 -28, wherein the cell growth platform comprises polydimethylsiloxane (PDMS).

[0039] 30. The bioreactor system of any of clauses 1 -29, wherein separator comprises a porous membrane.

[0040] 31. The bioreactor system of any of clauses 1 -30, wherein one or more pores of the porous membrane have a diameter of about 3 pm to about 7 pm.

[0041] 32. The bioreactor system of any of clauses 1 -31 , wherein the separator comprises PDMS or polyethylene terephthalate (PET).

[0042] 33. The bioreactor system of any of clauses 1 -32, wherein the cell growth platform is arranged above the flow channel.

[0043] 34. The bioreactor system of any of clauses 1 -33, further comprising: an imaging device comprising: a light source; a moveable stage, wherein the flow channel is received on and / or in the moveable stage; and an image capture device, wherein the imaging device is configured such that the moveable stage is arranged between the light source and the image capture device.

[0044] 35. The bioreactor system of any of clauses 1 -34, wherein the light source is arranged above the moveable stage and the image capture device is arranged below the moveable stage.

[0045] 36. The bioreactor system of any of clauses 1 -35, wherein the imaging device is arranged downstream of the cell growth platform.

[0046] 37. A bioreactor system, comprising: a cell growth platform having a fibrin hydrogel arranged thereon; at least one mesenchymal stem cell and at least one CD34+ cell embedded in the fibrin hydrogel; a flow channel configured to receive a flow of a culture medium therethrough; a porous membrane arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel; at least one endothelial cell arranged on the second surface of the porous membrane; and an imaging device arranged about the flow channel downstream of the cell growth platform and comprising: a moveable stage; a flow cell arranged on the moveable stage, the flow cell receiving at least a portion of the flow channel therein; a light source arranged above the flow cell; and an image capture device arranged below the flow cell.

[0047] 38. A system for imaging and counting cells, comprising: a moveable stage; a flow cell arranged on the moveable stage, the flow cell receiving at least a portion of a flow channel therein; a light source arranged relative to the flow cell; an image capture device arranged relative to the flow cell; and at least one processor incommunication with the imaging device, the at least one processor configured to: receive at least one image from the image capture device; perform a cell detection procedure on the at least one image to identify at least one cell object in the at least one image; and perform a cell counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure.

[0048] 39. The system of clause 38, wherein the image capture device captures a plurality of images and wherein the at least one processor is configured to: receive the plurality of images from the image capture device; perform a concatenating procedure on the plurality of images to provide a sequence of images; and convert the sequence of images into a video file format.

[0049] 40. The system of clause 38 or clause 39, wherein the at least one processor is further configured to: perform another cell detection procedure on the at least one image to identify, in the at least one image, a background, the background data comprising one or more stationary objects in the at least one image; and perform a background subtraction procedure to remove the background from the at least one image to obtain a first processed image, the first processed image comprising only moving objects with the one or more stationary objects removed.

[0050] 41. The system of any of clauses 38-40, wherein the at least one processor is further configured to: perform an additional cell detection procedure on the at least one image to identify, in the first processed image, one or more non-cell objects, the one or more non-cell objects identified based on a predetermined size threshold; and perform an image subtraction procedure to remove the one or more non-cell objects from the first processed image to obtain a second processed image, the second processed image comprising only cell objects with the one or more non- cell objects removed.

[0051] 42. A non-transitory, computer-readable medium having stored thereon programming instructions that, when executed by a processor, cause the processor to: receive at least one image from an image capture device; perform a cell detection procedure on the at least one image to identify at least one cell object in the at least one image; and perform a cell counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure.

[0052] 43. The non-transitory, computer-readable medium of clause 42, further comprising programming instructions that, when executed by a processor, cause the processor to: receive a plurality of images from the image capture device; perform aconcatenating procedure on the plurality of images to provide a sequence of images; and convert the sequence of images into a video file format.

[0053] 44. The non-transitory, computer-readable medium of clause 42 or clause 43, further comprising programming instructions that, when executed by a processor, cause the processor to: perform another cell detection procedure on the at least one image to identify, in the at least one image, a background, the background data comprising one or more stationary objects in the at least one image; and perform a background subtraction procedure to remove the background from the at least one image to obtain a first processed image, the first processed image comprising only moving objects with the one or more stationary objects removed.

[0054] 45. The non-transitory, computer-readable medium of any of clauses 42-44, further comprising programming instructions that, when executed by a processor, cause the processor to: perform an additional cell detection procedure on the at least one image to identify, in the first processed image, one or more non-cell objects, the one or more non-cell objects identified based on a predetermined size threshold; and perform an image subtraction procedure to remove the one or more non-cell objects from the first processed image to obtain a second processed image, the second processed image comprising only cell objects with the one or more non-cell objects removed.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 is a schematic of a cell growth platform and flow channel according to non-limiting embodiments described herein;

[0056] FIGS. 2A-2B show various arrangements of flow channels according to nonlimiting embodiments described herein;

[0057] FIG. 3 is a schematic of a bioreactor system according to non-limiting embodiments described herein;

[0058] FIG. 4 shows a bioreactor system according to non-limiting embodiments described herein;

[0059] FIG. 5 shows an imaging system according to non-limiting embodiments described herein;

[0060] FIGS. 6A-6F show components of an imaging system according to nonlimiting embodiments as described herein;

[0061] FIG. 7 is a schematic diagram of example components of one or more devices useful in non-limiting embodiments of systems and methods according to nonlimiting embodiments described herein;

[0062] FIG. 8 shows a method for growing and / or differentiating cells according to non-limiting embodiments described herein;

[0063] FIGS. 9A-9B show a method for processing images according to non-limiting embodiments described herein;

[0064] FIGS. 10A-10B show a method for processing images according to nonlimiting embodiments described herein;

[0065] FIGS. 11A-11 B show a method for processing images according to nonlimiting embodiments described herein;

[0066] FIG. 12 shows a method for processing images according to non-limiting embodiments described herein;

[0067] FIG. 13 shows (left panel) a plot showing the mobilization rate of egressed cells over time when 500 nM LTB4 was flowed through the vascular channel of hBM- Chips for a period of 8 hrs. The dots are the raw data obtained every 5 seconds, while the red line is the mobilization average over 5 min. Note that the egress of cells (out of the chip BM niche compartment into the vascular channel and exiting the device with flow) is not constant; rather, it is highly time dependent. That is, the mobilization rate increased sharply during the first 1 .5 hrs to a maximum value of ~ 450 cells / min. Fluctuations as high as 700 cells / min were observed during the first 30 min of this period. Subsequently, after reaching the maximum value of ~ 450 cells / min at 1 .5 hrs, the mobilization rate gradually decreased to ~ 200 cells / min at 4 hrs after chemoattractant introduction. From 4 to 8 hrs, the mobilization rate stabilized at ~ 200 cells / min; and (right panel) a plot showing the total number of egressed cells counted by ICD software (green) in pairwise comparisons with those counted manually (red). No significant difference between the ICD and manual counting was observed in any of the tested experiments. The data were analyzed by the nonparametric Mann- Whitney test; n.s.: not significant;

[0068] FIG. 14 shows a plot showing the total number of cells passing through the flow cell at any given time-point versus the chemoattractant treatment time. Four independent experiments were performed; the blue line shows the average, while the shaded area represents the s.e.m. Approximately one-third of the total mobilized cells (~20,000 cells) migrated into the vascular channel during the first 1.5 hrs of theexperiment, with an average of 222 cells / min. For the other two-thirds, i.e., ~40,000 cells, the cells were allowed to migrate for the following 6.5 hrs at an average rate of 102 cells / min;

[0069] FIG. 15 shows leukocyte chemotaxis in response to flowing agents, through which egressed cells enter the vascular channel via a porous membrane and then exit the device according to non-limiting embodiments described herein;

[0070] FIG. 16 shows hydrogel formulations with varying collagen content and protein-crosslinking transglutaminase dose according to non-limiting embodiments described herein;

[0071] FIGS. 17A-17C show flow cytometry analysis of the cellular composition of the BM Niche Compartment at Days 7 and 10. Using sequential gating, the frequencies of live cells, myeloid cells, monocytes, neutrophils, HSCs, and MPPs were determined at each time point. In total, n= 6-8 replicates per condition per time point; each color indicates a different cord blood donor (7 donors were studied each as an independent experiment). The data were analyzed by the nonparametric Mann- Whitney test. *p < 0.05, **p < 0.01 . The data are presented as the mean and s.e.m. BM: bone marrow; HUVECs: human umbilical vein endothelial cells; HSPCs: hematopoietic stem and progenitor cells.

[0072] FIG. 18 shows flow cytometry analysis of the cellular composition of the BM Niche Compartment at Days 7 and 10;

[0073] FIGS. 19A-19C show flow cytometry characterization of cells egressed out of hBM-Chips in response to 500 nM LTB4 chemotactic gradient across the porous membrane of the device. The mobilized cells entered the vascular flow and exited the chip. The LTB4 treatment duration was 8 hrs. Four cord blood donors were studied (each indicated by a different color). In total, n= 9-11 replicates per condition were used. The data were analyzed by the nonparametric Mann-Whitney test. **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . The data represent the mean and s.e.m;

[0074] FIG. 20 shows analysis of mobilized cells by flow cytometry;

[0075] FIG. 21 shows that any difference in cell mobilization between treated groups and control groups was not due to differences in cell density;

[0076] FIGS. 22A-22B show flow cytometry assays performed on cells generated by devices according to non-limiting embodiments described herein and transwell inserts;

[0077] FIGS. 23A-23D show CFU assays performed on cells generated by devices according to non-limiting embodiments described herein and transwell inserts;

[0078] FIG. 24 shows flow cytometry assays performed on cells generated by devices according to non-limiting embodiments described herein; and

[0079] FIG. 25 shows hydrogel thickness within 10 days of culture.DESCRIPTION OF THE INVENTION

[0080] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0081] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.

[0082] Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of "1 to 10" is intended to include all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

[0083] As used herein "a" and "an" refer to one or more.

[0084] As used herein, the term "comprising" is open-ended and may be synonymous with "including", "containing", or "characterized by".

[0085] As used herein, the term "patient" or "subject" refers to members of the animal kingdom including but not limited to human beings, and "mammal" refers to all mammals, including, but not limited to human beings.

[0086] For purposes of the description hereinafter, the terms "end," "upper," "lower," "right," "left," "vertical," "horizontal," "top," "bottom," "lateral," "longitudinal," and derivatives thereof shall relate to the embodiments as they are oriented in the drawing figures. However, it is to be understood that the present disclosure may assume various alternative variations and step sequences, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification, are simply exemplary and non-limiting embodiments or aspects of the disclosed subject matter. Hence, specific dimensions and other physical characteristics related to the embodiments or aspects disclosed herein are not to be considered as limiting.

[0087] Some non-limiting embodiments or aspects are described herein in connection with thresholds. As used herein, satisfying a threshold may refer to a value being greater than the threshold, more than the threshold, higher than the threshold, greater than or equal to the threshold, less than the threshold, fewer than the threshold, lower than the threshold, less than or equal to the threshold, equal to the threshold, etc.

[0088] No aspect, component, element, structure, act, step, function, instruction, and / or the like used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles "a" and "an" are intended to include one or more items and may be used interchangeably with "one or more" and "at least one." Furthermore, as used herein, the term "set" is intended to include one or more items (e.g., related items, unrelated items, a combination of related and unrelated items, and / or the like) and may be used interchangeably with "one or more" or "at least one." Where only one item is intended, the term "one" or similar language is used. Also, as used herein, the terms "has," "have," "having," or the like are intended to be open-ended terms. Further, the phrase "based on" is intended to mean "based at least partially on" unless explicitly stated otherwise. In addition, reference to an action being "based on" a condition may refer to the action being "in response to" the condition. For example, the phrases "based on" and "in response to" may, in some non-limiting embodiments or aspects, refer to a condition for automatically triggeringan action (e.g., a specific operation of an electronic device, such as a computing device, a processor, and / or the like).

[0089] As used herein, the term "communication" may refer to the reception, receipt, transmission, transfer, provision, and / or the like of data (e.g., information, signals, messages, instructions, commands, and / or the like). For one unit (e.g., a device, a system, a component of a device or system, combinations thereof, and / or the like) to be in communication with another unit means that the one unit is able to directly or indirectly receive information from and / or transmit information to the other unit. This may refer to a direct or indirect connection (e.g., a direct communication connection, an indirect communication connection, and / or the like) that is wired and / or wireless in nature. Additionally, two units may be in communication with each other even though the information transmitted may be modified, processed, relayed, and / or routed between the first and second unit. For example, a first unit may be in communication with a second unit even though the first unit passively receives information and does not actively transmit information to the second unit. As another example, a first unit may be in communication with a second unit if at least one intermediary unit processes information received from the first unit and communicates the processed information to the second unit. In some non-limiting embodiments or aspects, a message may refer to a network packet (e.g., a data packet and / or the like) that includes data. It will be appreciated that numerous other arrangements are possible. Communication may include one or more wired and / or wireless networks. For example, communication may include a cellular network (e.g., a long-term evolution (LTE) network, a third-generation (3G) network, a fourth-generation (4G) network, a fifth-generation (5G) network, a code division multiple access (CDMA) network, etc.), a public land mobile network (PLMN), a local area network (LAN), a wide area network (WAN), a metropolitan area network (MAN), a telephone network (e.g., the public switched telephone network (PSTN) and / or the like), a private network, an ad hoc network, an intranet, the Internet, a fiber optic-based network, a cloud computing network, and / or the like, and / or a combination of some or all of these or other types of networks.

[0090] As used herein, the term "computing device" may refer to one or more electronic devices configured to process data. A computing device may, in some examples, include the necessary components to receive, process, and output data, such as a processor, a display, a memory, an input device, a network interface, and / orthe like. A computing device may be a mobile device. As an example, a mobile device may include a cellular phone (e.g., a smartphone or standard cellular phone), a portable computer, a wearable device (e.g., watches, glasses, lenses, clothing, and / or the like), a personal digital assistant (PDA), and / or other like devices. A computing device may also be a desktop computer or other form of non-mobile computer.

[0091] As used herein, the term "server" may refer to or include one or more computing devices that are operated by or facilitate communication and processing for multiple parties in a network environment, such as the Internet, although it will be appreciated that communication may be facilitated over one or more public or private network environments and that various other arrangements are possible. Further, multiple computing devices (e.g., servers, mobile devices, etc.) directly or indirectly communicating in the network environment may constitute a "system."

[0092] As used herein, the term "system" may refer to one or more computing devices or combinations of computing devices (e.g., processors, servers, client devices, software applications, components of such, and / or the like). Reference to "a device," "a server," "a processor," and / or the like, as used herein, may refer to a previously-recited device, server, or processor that is recited as performing a previous step or function, a different device, server, or processor, and / or a combination of devices, servers, and / or processors. For example, as used in the specification and the claims, a first device, a first server, or a first processor that is recited as performing a first step or a first function may refer to the same or different device, server, or processor recited as performing a second step or a second function.

[0093] Provided herein are systems and methods for growing and / or differentiating cells, as well as systems and methods for distinguishing cells from other objects and counting cells in an image. The systems and methods provide the ability to generate various physiological niches for generation of various cell types, for, among other potential uses, testing potential therapeutics. The imaging systems described herein provide savings in terms of computer resources for distinguishing objects and counting objects of interest, by, among other elements, implementing parallel processing of various image frames.

[0094] While this disclosure is focused on human cells, those of skill in the art will appreciate that the same principles can be applied to other species including rodent (e.g., mice and rats), ferret, guinea pig, dog, cat, and non-human primate species. In such cases the source of cells would be from the respective animals. Additionally,while the devices, systems, and methods described herein are exemplified in a bottom-up approach (e.g., where stem cells are guided to differentiate and maintain stem / progenitor function), ex vivo cultures can be performed, where the content of cells (from humans or other animals) may be transferred to cell growth platform as described herein for maintenance (e.g., a top-down approach).

[0095] Turning to FIG. 1 , shown is a non-limiting embodiment of a bioreactor system 1000 for growing and / or differentiating cells. Bioreactor system 1000 may include a cell growth platform 100, which may be a two-dimensional platform and / or a three- dimensional housing, defining an interior. Cell growth platform 100 may have, received thereon (two-dimensional) and / or therein (three-dimensional) a hydrogel 120. One or more first cells 140 may be received on and / or in hydrogel 120. Cell growth platform may be formed of any useful materials. In non-limiting embodiments, the cell growth platform 100 is at least partially formed of a silicon-containing material, for example polydimethylsiloxane (PDMS), for example, in non-limiting embodiments, at least a portion of the cell growth platform 100 adjacent to separator 180 and / or flow channel 160 may be formed at least partially of PDMS. In non-limiting embodiments, cell growth platform 100 may be formed of a thermoplastic polymer or copolymer, a resin, and / or glass.

[0096] Hydrogel 120 may be any useful type of biocompatible hydrogel that can support cellular growth and / or differentiation. Useful hydrogels may be fully natural (or natural molecule(s)-derived), synthetic, or semi-synthetic (a mixture of natural and synthetic (bio)materials). In the “natural” domain, the hydrogels can be, for example, a fibrin network holding natural polymers or derivatives (e.g., collagen, laminin, fibronectin) in place. In other words, fibrin (a natural molecule) may be the scaffold keeping other extracellular matrix proteins of interest (compassion and stiffness / rheology) together. In the “synthetic” domain, the hydrogels can be, for example, biocompatible polymers or their derivatives (e.g., PEGs) to generate a scaffold holding the cells. In the “semi-synthetic” domain, the hydrogels may include a combination of natural and synthetic polymers. This can include, for example, gelatin- methacrylate (GelMA). In non-limiting embodiments, hydrogel 120 is a fibrin hydrogel, for example a hydrogel formed from thrombin and fibrinogen.

[0097] The one or more first cell(s) 140 may be any useful cell type, and may be received on and / or may be embedded within hydrogel 120. In non-limiting embodiments, at least one first cell 140 includes at least one type of multipotent,pluripotent, and / or stem cell. In non-limiting embodiments, the at least one first cell 140 includes a mesenchymal stem cell (MSC), for example a human MSC (hMSC). Such hMSCs can be obtained from umbilical cord tissue, fat tissue, bone marrow itself, or any other organ at any stage of life (e.g., not just fetal tissue). In addition, MSCs can be derived from stem cells such as induced pluripotent stem cells (iPSCs). In non-limiting embodiments, the at least one first cell includes at least one hematopoietic stem and progenitor cells (HSPC), hematopoietic stem cells (HSC) and / or at least one CD34+ cell, for example a human HSPC and / or a human CD34+ cell. Herein, ‘HSPC’, ‘HSC’, and ‘CD34+ cell’ are used interchangeably. In non-limiting embodiments, the HSPCs are obtained from human umbilical cord tissue and / or blood, and / or may be derived from iPSCs. In non-limiting embodiments, at least one first cell 140 includes at least one MSC and at least one HSPC. In non-limiting embodiments, at least one first cell 140 includes at least one MSC and at least one HSPC at a 1 :1 ratio.

[0098] Cells useful in the systems and methods described herein, and / or their culture media, may be genetically modified, or not, as applicable for the purposes for which the systems and methods are employed. For example, and without limitation, to assess efficacy of a potential therapy, one or more of the cells received on and / or within cell growth platform 100 (for example, on and / or within hydrogel 120) may be modified to express one or more genes of interest, and / or modified such that one or more genes of interest are not expressed. Similarly, culture media can by modified to contain certain stimuli or reporter agents to be switched on or off at desired timepoint during culture or with a given challenge.

[0099] Bioreactor system 1000 further may include a flow channel 160, arranged relative to cell growth platform 100, which may be configured to permit flow of a liquid therethrough. Cell growth platform 100 may be separated from an interior of flow channel 160 by a separator 180. At least one second cell 170 may be arranged on and / or in separator 180. In non-limiting embodiments, at least one second cell 170 may be an endothelial cell, for example a human endothelial cell. In non-limiting embodiments, at least one second cell 170 may be a human umbilical vein endothelial cell (HUVEC). Other endothelial cells such as primary endothelial cells obtained from fetal, pediatric or adult tissues (e.g., bone marrow endothelial cells) as well as stem cell (e.g., iPSC)-derived endothelial cells may be used in embodiments. In non-limiting embodiments, separator 180 has at least two surfaces, a first surface facing cell growth platform 100 and a second surface facing an interior of flow channel 160. Innon-limiting embodiments, one or more of second cell(s) 170 may be arrange on a second surface of separator 180. In non-limiting embodiments, separator 180 is formed of a porous material, to allow growth and / or migration of second cell(s) 170 and / or first cell(s) 140 therethrough. In non-limiting embodiments, separator 180 may be formed at least partially of a silicon-containing material, such as PDMS. In nonlimiting embodiments, separator 180 may be formed of one or more biocompatible materials, such as polymers, for example polyethylene terephthalate (PET), non-PET thermoplastic polymers such as polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cyclo-olefin-copolymers (COCs) (e.g., Topas), cyclo-olefin polymers (COPs) (e.g., Zeonor, Zeonex), and / or glass (e.g., etched and / or porous glass). Those of skill in the art will appreciate that any porous material that is biocompatible may be used in separator 180. In non-limiting embodiments, separator 180 may have pores with a diameter of from about 1 pm to about 10 pm, about 2 pm to about 9 pm, about 3 pm to about 7 pm, about 4 pm to about 6 pm, and / or about 5 pm, all values and subranges therebetween inclusive. In non-limiting embodiments, separator 180 has pores with a diameter of from about 3 pm to about 7 pm.

[0100] In the non-limiting embodiment shown in FIG. 1 , cell growth platform 100 is arranged above flow channel 160, though those of skill in the art will appreciate that other configurations may be possible. In non-limiting embodiments, the cell growth platform 100 is arranged above the flow channel 160. Flow channel 160 may assume any useful configuration. In non-limiting embodiments, for example as shown in FIG. 2A, flow channel 160 is substantially linear. In non-limiting embodiments, for example as shown in FIG. 2B, flow channel 160 includes one or more curves. Without wishing to be bound by the theory, it is believed that the inclusion of one or more curves, for example in a zig-zag pattern, may increase the surface area available for passage of nutrients from a liquid passing through flow channel 160 to cells 140 / 170, hydrogel 120, and / or an interior of cell growth platform 100, and / or allows for greater migration of cells 140 out of cell growth platform 100. In non-limiting embodiments, flow channel 160 includes one or more smooth curves (rather than sharp angles), to reduce turbulence within flow channel 160.

[0101] Turning to FIG. 3, a bioreactor system 2000 may include a cell growth platform 100, a reservoir 400 for liquid that flows through flow channel 260, and an imaging system 300, which will be described in greater detail below. One or more pumps (not shown) may be arranged within reservoir 400 and / or along flow channel260 to cause circulate of liquid. In non-limiting embodiments, reservoir 400 and flow channel 260 form a closed circuit, such that liquid flowing from reservoir 400 through flow channel 260 is recirculated to reservoir 400. In non-limiting embodiments, reservoir 400 and flow channel 260 do not form a closed circuit (e.g., liquid flowing through flow channel is not recirculated to reservoir 400). Liquid flowing through flow channel 260 may include one or more constituents for maintaining the hydrogel on / within cell growth platform 100. For example, the liquid may include one or more cross-linkers and / or proteinase inhibitors, such as aprotinin. Cross-linkers and proteinase inhibitors are known to those of skill in the art; however, in non-limiting embodiments, the liquid may include a transglutaminase (such as a gelatin transglutaminase) and / or a matrix metalloproteinase (MMP) inhibitor. In non-limiting embodiments, the MMP inhibitor is aprotinin. In non-limiting embodiments, the MMP is a tissue inhibitor of metalloproteinase (TIMP).

[0102] In non-limiting embodiments, the liquid stored in reservoir 400 and / or provided in flow channel 260 may include one or more constituents, for example for supporting cellular growth on and / or within cell growth platform 100. For example, and without limitation, the liquid may be a cell medium including one or more of growth and / or differentiation-supporting supplements (e.g., Granulocyte Colony-Stimulating Factor (G-CSF), Macrophage Colony-Stimulating Factor (M-CSF), Granulocyte- Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin 3 (IL-3), IL-6, Stem Cell Factor (SCF), and / or Fms-related Tyrosine Kinase 3 Ligand (Flt3-L) for HSPCs, or Fetal Bovine or Calf Sera (FBS / FCS), Hydrocortisone, Fibroblast Growth Factor- Basic (FGF-B), Vascular Endothelial Growth Factor (VEGF), Recombinant Analog of Insulin-like Growth Factor 1 (R3-IGF-1 ), Ascorbic Acid, Epidermal Growth Factor (EGF), and / or Heparin for endothelial cells and / or MSCs), chemoattractants (including but not limited to protein chemokines and non-protein chemotactic factors), infectious agents (e.g., viruses, bacteria, fungi, or their derivatives), therapeutic agents or candidates (e.g., small molecules, peptides, antibodies, or hybrid compounds at preclinical and / or clinical stage of development), antimicrobial agents, and / or other cells or their derivatives (e.g., circulating tumor cells, cancer neoantigens).

[0103] In non-limiting embodiments, the liquid stored in reservoir 400 and / or provided in flow channel 260 may include one or more constituents, for example for causing differentiation of cells on and / or within cell growth platform 100. For example,and without limitation, the liquid may be a cell medium including one or more of the aforementioned growth and / or differentiation-supporting supplements.

[0104] In non-limiting embodiments, the liquid stored in reservoir 400 and / or provided in flow channel 260 may include one or more constituents, for example for causing migration of one or more cells from cell growth platform 100 to flow channel 260. For example, and without limitation, the liquid may be a cell medium including a chemoattractant, including, but not limited to, a chemokine (e.g., a CXC, a CC, a CX3C, and / or a C chemokine), a lipid mediator, a complement factor, and / or other peptides known to function as chemoattractants. In non-limiting embodiments, the chemoattractant is one or more of the aforementioned growth and / or differentiationsupporting supplements, and / or CCL1 , CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL1 1 , CCL12, CCL13, CCL14, CCL15, CCL16, CCL 17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 , CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL26, CXCL17, XCL1 , XCL2, CX3CL1 , a prostaglandin, a thromboxane, a leukotriene, a lipoxin, a resolvin, an epoxyeicosatrienoic acid, a hydroxyeicosatetraenoic acid, a dihydroxyeicosatetraenoic acid, a dihydroxyeicosatrienoic acid, a hydroxyheptadecatrienoic acid, a fatty acid, a hydroxyeicosapentaenoic acid, a hydroxydocosahexaenoic acid, a specialized pro-resolving mediator (e.g., maresin 1 , maresin 2, MCTR1 , MCTR2, MCTR3, PCTR1 , PCTR2, PCTR3, PD1 , and / or PDX), a dihydroxyeicosapentaenoic acid, an epoxydocosapentaenoic acid, an epoxyeicosatetraenoic acid, and / or a hydroxyoctadienoic acid, a complement component (e.g., complement component 1 and / or complement component 3), C4b- binding protein, a complement factor (e.g., complement factor B and / or complement factor I), a delay-accelerating factor, a ficolin (e.g., ficolin-1 , ficolin-2, and / or ficolin-3), a collectin (e.g., collectin 11 ), and / or a serine protease (e.g., mannose-binding lectin- associated serine protease). In non-limiting embodiments, the chemoattractant is leukotriene B4 (LTB4).

[0105] In non-limiting embodiments, the liquid stored in reservoir 400 and / or provided in flow channel 260 may include one or more constituents, for example one or more potential therapeutic agents, for evaluating efficacy against one or more of the cells on and / or within cell growth platform 100.

[0106] In non-limiting embodiments, the therapeutic agent is one or more of the aforementioned growth and / or differentiation-supporting supplements, therapeutic agents, antimicrobial agents, and / or a hormone and / or pro-hormone, such as estrogen, progestin, progesterone, growth hormone, thyroid-stimulating hormone, oxytocin, follicle-stimulating hormone, luteinizing hormone, testosterone, cortisol, prolactin, corticotropin-releasing hormone, gonadotrophin-releasing hormone, somatostatin, thyrotropin-releasing hormone, antidiuretic hormone, corticotropin, melatonin, thyroxine, triiodothyronine, reverse triiodothyronine, calcitonin, aldosterone, DHEA, epinephrine, norepinephrine, insulin, glucagon, leptin, adiponectin, plasminogen activator inhibitor-1 , angiotensin, angiotensinogen, erythropoietin, renin, vitamin D, insulin-like growth factors (IGFs), such as IGF-1 , ghrelin, somatostatin, glucagon-like peptides (GLPs), such as GLP-1 , and / or the like.

[0107] In non-limiting embodiments, the therapeutic agent is one or more of the aforementioned growth and / or differentiation-supporting supplements, therapeutic agents, antimicrobial agents, and / or a growth-enhancing factor, which as utilized herein means a composition that enhances, accelerates, and / or promotes healing following trauma. In non-limiting embodiments, such growth-enhancing factors include growth factors, such as, without limitation, a neurotrophic or angiogenic factor, which optionally may be prepared using recombinant techniques. Non-limiting examples of growth factors include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), stromal derived factor 1 alpha (SDF-1 alpha), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3, neurotrophin- 4, neurotrophin-5, pleiotrophin protein (neurite growth-promoting factor 1 ), midkine protein (neurite growth-promoting factor 2), brain-derived neurotrophic factor (BDNF), tumor angiogenesis factor (TAF), corticotrophin releasing factor (ORF), transforming growth factors a and [3 (TGF-a and TGF-p), interleukin-8 (IL-8), granulocytemacrophage colony stimulating factor (GM-CSF), interleukins, and interferons. Commercial preparations of various growth factors, including neurotrophic and angiogenic factors, are available from R & D Systems, Minneapolis, Minnesota; Biovision, Inc, Mountain View, California; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel; and Cell Sciences®, Canton, Massachusetts.

[0108] In non-limiting embodiments, the therapeutic agent is one or more of the aforementioned growth and / or differentiation-supporting supplements, therapeutic agents, antimicrobial agents, and / or a cytokine, for example a pro-inflammatory cytokine and / or an anti-inflammatory cytokine. In non-limiting embodiments, the therapeutic agent may be, without limitation, an interleukin, such as IL-1 , IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL- 32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, and / or IL-40, tumor necrosis factors (TNF), such as TNF-a, interferons (IFN), such as IFN-a, IFN-p, and / or IFN-y, colonystimulating factors (CSF), such as G-CSF, chemokines, lymphokines, monokines, and the like known to those of skill in the art.

[0109] In non-limiting embodiments, the therapeutic agent is a drug. As used herein, the terms "drug" and "drugs" refer to any compositions having a preventative or therapeutic effect, including and without limitation, antibiotics, antivirals, antimycotics, peptides, hormones, organic molecules, steroids, NSAIDS, vitamins, supplements, factors (including growth factors), proteins, and chemoattractants.

[0110] In non-limiting embodiments, the drug is an antimicrobial agent, such as, without limitation, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet, penicillin, gentamycin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione, and silver salts such as chloride, bromide, iodide and periodate. Exemplary antimicrobial agents are known to those of skill in the art.

[0111] In non-limiting embodiments, the drug is an antimycotic agent, such as clotrimazole, fluconazole, ketoconazole, posconazole, voriconazole, isavuconazole, nystatin, amphotericin B, flucytosine, echinandins, and / or micafungin. Exemplary antimycotic agents are known to those of skill in the art.

[0112] In non-limiting embodiments, the drug is an antiviral agent (which as used herein includes antiretroviral agents), such as adamantane antivirals, antiviral boosters, antiviral combinations, antiviral interferons, chemokine receptor antagonists, integrase strand transfer inhibitors, miscellaneous antivirals, neuraminidase inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), non-structural protein 5A(NS5A) inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors, and / or purine nucleosides. Exemplary antiviral agents are known to those of skill in the art.

[0113] In non-limiting embodiments, the drug is an anti-inflammatory agent, such as, without limitation, an NSAID, such as salicylic acid, indomethacin, sodium indomethacin trihydrate, salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen, sodium salicylamide; an anti-inflammatory cytokine; an anti-inflammatory protein; a steroidal anti-inflammatory agent; or an anti-clotting agent, such as heparin. Other drugs that may promote wound healing and / or tissue regeneration may also be included.

[0114] In non-limiting embodiments, for example as shown in FIG. 4, cell growth platform 100 is a three-dimensional bioreactor, including a fibrin hydrogel received therein, arranged above a flow channel. One or more CD34+ cells and one or more MSCs are arranged within the fibrin hydrogel. HUVECs are arranged on the separator that separates the interior of the bioreactor from the flow channel, and, depending on the constituents supplied in the liquid flowing through flow channel, the CD34+ cells may be maintained as HSPCs, may be directed to a certain desired lineage (for example, a leukocyte), and may migrate to the flow channel (for example, by providing one or more chemoattractants within the liquid.

[0115] Turning to FIGS. 5-6F, shown are non-limiting embodiments of a bioreactor system 3000, including an imaging system 300 and components thereof, which may be used independently of or in conjunction with a cell growth platform and / or flow channel as described herein. Bioreactor system 3000 may include a computing device 200, for example a computing device as shown in FIG. 7 and described below, a light source 320, and image capture device 340, a flow cell 360, and / or a moveable stage 380. FIG. 5 shows a schematic of components of bioreactor system 3000. For example, in non-limiting embodiments, flow cell 360 may be in fluid communication with a flow channel as described herein and may be arranged downstream of a cell growth platform as described herein, such that cells that migrate out of the cell growth platform pass through flow cell 360. Flow cell 360 may be arranged between light source 320 and image capture device 340. In non-limiting embodiments, for example as shown in FIG. 6A, light source 320 may be arranged above, on a side of, and / or below flow cell 360, and image capture device 340 may be arranged above, on a side of, and / or below flow cell 360. In non-limiting embodiments, light source 320 isarranged above flow cell 360 and image capture device 340 is arranged below flow cell 360. In non-limiting embodiments, for example as shown in FIG. 6A, flow cell 360 may be in fluid communication with flow channel 260, downstream of cell growth platform 100, and may be arranged on and / or may be connected to moveable stage 380. Moveable stage 380 may allow for movement of flow cell 360 along one or more axes, for example along an x-, y-, and / or z-axis, as shown in FIG. 6B. Moveable platform 380 may be in communication with computing device 200 and may be controlled thereby and / or may be manually controlled by a user.

[0116] Turning to FIG. 6C, shown is an image capture device 340 useful in bioreactor system 3000 and / or imaging system 300 as described herein. Those of skill in the art will appreciate that image capture devices suitable for capturing images of cells, are known and are commercially available from, for example and without limitation, Zeiss, Leica Microsystems, Teledyne Vision Solutions, Olympus Life Sciences, Thorlabs, Inc., Keyence Corp., Excelitas Technology Corp, and Nikon Instruments. Suitable image capture devices 340 as described herein may be provided in conjunction with suitable magnification devices, for example with microscopy devices and systems as are known in the art. Image capture device 340 may be in communication with computing device 200 and may be controlled thereby and / or may be manually controlled by a user. Image capture device 340 may be configured to capture one or more discrete images or may be configured for continuous image capture (e.g., video capture).

[0117] Turning to FIG. 6D, shown is a light source 320 useful in bioreactor system 3000 and / or imaging system 300 as described herein. Light source 320 may be configured to emit any type of light useful for capturing one or more images with image capture device 340, including, without limitation, visible light, infrared light, and / or ultraviolet light. Light source 320 may be in communication with computing device 200, and may be controlled thereby and / or may be manually controlled by a user.

[0118] Turning to FIGS. 6E-6F, shown is a flow cell 360 useful in bioreactor system 3000 and / or imaging system 300 as described herein. As shown in FIG. 6E, flow cell 360 may be a two-piece arrangement, where a disposable flow cell may be inserted into and / or on and secured to a platform and may be removed and discarded after used. As may be appreciated from the non-limiting embodiment shown in FIG. 6F, flow cell 360 may include an inlet and an outlet, in fluid communication with flow channel 260 and a reservoir (e.g., reservoir 400), respectively. Flow cell may beformed of any suitable material to allow image capture device 340 to capture one or more images of one or more objects flowing through flow cell 360.

[0119] With continuing reference to FIGS. 5-6F, in non-limiting embodiments, a bioreactor system 3000 may include a cell growth platform 100 having a fibrin hydrogel arranged thereon and / or therein. The cell growth platform 100 may include at least one mesenchymal stem cell and at least one CD34+ cell embedded in the fibrin hydrogel. Bioreactor system 3000 may further include a flow channel 260 configured to receive a flow of a culture medium therethrough and a porous membrane (not shown) arranged between the cell growth platform 100 and the flow channel 260 and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel. In non-limiting embodiments, at least one endothelial cell may be arranged on the second surface of the porous membrane. With continuing reference to the drawings, a bioreactor system 3000 may include an imaging device arranged about the flow channel downstream of the cell growth platform, wherein the imaging device may include a moveable stage 380, a flow cell 360 arranged on the moveable stage 380, where the flow cell may receive at least a portion of the flow channel 260 thereon and / or therein. Imaging device may further include a light source 320 arranged above, on a side of, and / or below the flow cell 360 and an image capture device 340 arranged above, on a side of, and / or below the flow cell.

[0120] Referring now to FIG. 7, FIG. 7 is a diagram of example components of a device 200. Device 200 may correspond to imaging system 300 (e.g., one or more devices of imaging system 300, such as computing device 500). In some non-limiting embodiments or aspects, imaging system 300 may include at least one device 200 and / or at least one component of device 200.

[0121] Device 200 may correspond to any element of a system. In some nonlimiting embodiments, such systems or devices may include at least one device 200 and / or at least one component of device 200. The number and arrangement of components shown are provided as an example. In some non-limiting embodiments, device 200 may include additional components, fewer components, different components, or differently arranged components than those shown. Additionally, or alternatively, a set of components (e.g., one or more components) of device 200 may perform one or more functions described as being performed by another set of components of device 200.

[0122] As shown in FIG. 7, device 200 may include a bus 202, a processor 204, memory 206, a storage component 208, an input component 210, an output component 212, and a communication interface 214. Bus 202 may include a component that permits communication among the components of device 200. In some non-limiting embodiments, processor 204 may be implemented in hardware, firmware, or a combination of hardware and software. For example, processor 204 may include a processor (e.g., a central processing unit (CPU), a graphics processing unit (GPU), an accelerated processing unit (APU), etc.), a microprocessor, a digital signal processor (DSP), and / or any processing component (e.g., a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), etc.) that can be programmed to perform a function. Memory 206 may include random access memory (RAM), read only memory (ROM), and / or another type of dynamic or static storage device (e.g., flash memory, magnetic memory, optical memory, etc.) that stores information and / or instructions for use by processor 204.

[0123] With continued reference to FIG. 7, storage component 208 may store information and / or software related to the operation and use of device 200. For example, storage component 208 may include a hard disk (e.g., a magnetic disk, an optical disk, a magneto-optic disk, a solid-state disk, etc.) and / or another type of computer-readable medium. Input component 210 may include a component that permits device 200 to receive information, such as via user input (e.g., a touch screen display, a keyboard, a keypad, a mouse, a button, a switch, a microphone, etc.). Additionally, or alternatively, input component 210 may include a sensor for sensing information (e.g., a global positioning system (GPS) component, an accelerometer, a gyroscope, an actuator, etc.). Sensors useful here may include biochemical sensors, electrochemical sensors, sensors for detecting autonomic tone, sensors for detecting sympathetic tone, and / or the like. Output component 212 may include a component that provides output information from device 200 (e.g., a display, a speaker, one or more light-emitting diodes (LEDs), etc.). Communication interface 214 may include a transceiver-like component (e.g., a transceiver, a separate receiver and transmitter, etc.) that enables device 200 to communicate with other devices, such as via a wired connection, a wireless connection, or a combination of wired and wireless connections. Communication interface 214 may permit device 200 to receive information from another device and / or provide information to another device. For example, communication interface 214 may include an Ethernet interface, an opticalinterface, a coaxial interface, an infrared interface, a radio frequency (RF) interface, a universal serial bus (USB) interface, a Wi-Fi® interface, a cellular network interface, and / or the like.

[0124] Device 200 may perform one or more processes described herein. Device 200 may perform these processes based on processor 204 executing software instructions stored by a computer-readable medium, such as memory 206 and / or storage component 208. A computer-readable medium may include any non-transitory memory device. A memory device includes memory space located inside of a single physical storage device or memory space spread across multiple physical storage devices. Software instructions may be read into memory 206 and / or storage component 208 from another computer-readable medium or from another device via communication interface 214. When executed, software instructions stored in memory 206 and / or storage component 208 may cause processor 204 to perform one or more processes described herein. Additionally, or alternatively, hardwired circuitry may be used in place of or in combination with software instructions to perform one or more processes described herein. Thus, embodiments described herein are not limited to any specific combination of hardware circuitry and software. The term "configured to," as used herein, may refer to an arrangement of software, device(s), and / or hardware for performing and / or enabling one or more functions (e.g., actions, processes, steps of a process, and / or the like). For example, "a processor configured to" may refer to a processor that executes software instructions (e.g., program code) that cause the processor to perform one or more functions.

[0125] In non-limiting embodiments, provided herein is an imaging system 300. Imaging system 300 may include a moveable stage 380 and a flow cell 360 arranged in and / or on the moveable stage 380 and receiving at least a portion of flow 260 channel therein. Imaging system 300 may further include a light source 320 arranged relative (e.g., above, on a side of, and / or below) to the flow cell 360 and an image capture device 340 arranged relative (e.g., above, on a side of, and / or below) to the flow cell 360 and at least one processor in communication with the moveable stage 380, the light source 320, and / or the image capture device 340. The at least one processor 200 may be configured to receive at least one image from the image capture device 340, perform a cell detection procedure (e.g., a feature detection procedure performed on one or more images to determine a number of cells in the one or more images) on the at least one image to identify at least one cell object in the at least oneimage, and perform a cell counting procedure (e.g., a feature counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure).

[0126] Turning to FIG. 9A, shown is a method for processing images. In nonliming embodiments, the method may be performed by imaging system 300 (e.g., computing device 500 of imaging system 300) (e.g., a component of imaging system 300 and / or may be one or more systems or devices outside of imaging system 300) . In a first step, imaging system 300 may receive data associated with one or more images. For example, imaging system 300 may receive data associated with one or more images captured by image capture device 340. In a second step, imaging system 300 may process the data associated with the one or more images. In a third step, imaging system 300 may output data associated with the one or more processed images. In non-limiting embodiments, the output data may include one or more graphs and / or one or more files (e.g., CSV files) containing cell count data. In non-limiting embodiments, cell count data many including a number of cells that pass through a flow cell 360, for example cells that have migrated out of cell growth platform 100, in a given time period.

[0127] In non-limiting embodiments, for example as shown in FIG. 10A, the step of processing the images may include imaging system 300 performing a cell detection process to isolate cells in an image from background aspects of an image. In nonlimiting embodiments, in a first step of a cell detection process, imaging system 300 may identify and one or more stationary objects in one or more images.

[0128] In non-limiting embodiments, imaging system 300 may generate stationary object data associated with the one or more stationary objects in the one or more images. In non-limiting embodiments, imaging system 300 may generate the stationary object data by applying a Gaussian process and / or an advanced motion detection process (e.g., a process by which imaging system 300 compares objects in a first image to objects in a second image and determines which objects are in the same position in both images), to all moving objects, including cells, bubbles, and hydrogel debris, in an image. In non-limiting embodiments, the advanced motion detection process is one or more of a contrast enhancement-based process e.g., Otsu’s method or a Layered Difference Representation (LDR), a region selectionbased process such as a Region-Based Convolutional Neural Network (R-CNN), FastR-CNN, and Faster R-CNN. In non-limiting embodiments, the stationary object data may include a background with pixels representing stationary objects.

[0129] In non-limiting embodiments, imaging system 300 may subtract the stationary object data (e.g., the background with pixels representing stationary objects) from the one or more images (e.g., data associated with one or more images) to generate a semi-processed image (e.g., data associated with one or more semiprocessed images). In non-limiting embodiments, a semi-processed image (e.g., an image that is not fully processed, such as an image that has not undergone size filtration) may include an image with stationary objects removed. In non-limiting embodiments, imaging system 300 may subtract the stationary object data (e.g., the background with pixels representing stationary objects) from the one or more images to provide one or more images that include moving objects (e.g., non-stationary objects). In non-limiting embodiments, the semi-processed image (e.g., data associated with one or more semi-processed images) may include data relating only to moving objects. In non-limiting embodiments, imaging system 300 may convert the semi-processed image data associated with one or more processed images into binary image data. In non-limiting embodiments, imaging system 300 may convert the data associated with one or more processed images into binary image data using any suitable process known to those of skill in the art, for example using Otsu’s thresholding method.

[0130] With continuing reference to FIG. 10A, in a second step of a cell detection process, imaging system 300 may filter moving objects based on size (e.g., based on the number of pixels that the object occupies), thereby generating a fully processed image. In non-limiting embodiments, imaging system 300 may distinguish cells from non-cellular artifacts, for example bubbles and / or hydrogel debris, in a semi-processed image. In non-limiting embodiments, imaging system 300 may distinguish cells from non-cellular artifacts using binary image data. In non-limiting embodiments, imaging system 300 may apply a filter to the semi-processed image (e.g., data associated with the one or more semi-processed images and / or binary data). In non-limiting embodiments, the filter may be a size exclusion filter. For example, and without limitation, a first exclusion size filter may be used to identify bubbles and / or other objects of a first size, and a second size exclusion filter may be used to identify hydrogel debris and / or other objects of a second size. In such non-limiting embodiments, objects having a size between the first and second size exclusion filtersmay be identified as cells. In non-limiting embodiments, the first size exclusion filter may be set at about 5 gm, for example objects less 5 pm maybe classified by device 200 as bubbles. In non-limiting embodiments, the second size exclusion filter may be set at about 20 pm, for example objects greater than 20 pm maybe classified by device 200 as hydrogel debris. As a result of such filtering, in non-limiting embodiments, imaging system 300 may classify objects between the first and second size exclusion filters (e.g., between about 5 pm and about 20 pm) as cells. In non-limiting embodiments, an output of filtering may include objects classified as cells. For example, an output may include an image with only objects that are classified as cells (e.g., data associated only with objects that are classified as cells).

[0131] In a third step of a cell detection process, imaging system 300 may output a processed image (e.g., an image that has objects below a first specified size, e.g., equal to and / or below a first size exclusion filter, and / or an image that has objects above a second specified size, e.g., equal to and / or greater than a second size exclusion filter) that is based on filtering the moving objects. For example, imaging system 300 may output a processed image (e.g., data associated with a processed image) that includes only objects (e.g., objects of a specified size, such as cells) that remain after the moving objects have been filtered.

[0132] In non-limiting embodiments, for example as shown in FIG. 11A, the step of processing the images may include imaging system 300 counting objects classified as cells through a cell detection process (e.g., the cell detection process shown in FIG. 10A). In non-limiting embodiments, N processed images may be further processed by imaging system 300. In non-limiting embodiments, imaging system 300 may extract a line from each processed image, to generate a gate (e.g., a line removed from a processed image). The width of the generated gate may be a set parameter determined based on displacement of the objects classified as cells in the processed image, thereby ensuring that every cell is captured, and no cell is captured (and thus counted) more than once. In non-limiting embodiments, imaging system 300 may concatenate N gates from / V processed images into one single concatenated image. Each object in the concatenated image may represent a cell passing through the field of view of the image capture device 340. The number of objects may then be processed by imaging system 300 to obtain the number of cells passing through the flow cell 360 (e.g., data associated with a cell count). In non-limiting embodiments, imaging system 300 may concatenate / V gates from A / processed images into a video(e.g, a plurality of images, a plurality of N gates). The number of objects may then be counted by imaging system 300 to obtain the number of cells passing through the flow cell 360 (e.g., data associated with a cell count).

[0133] In non-limiting embodiments, imaging system 300 may convert the data associated with a cell count to data associated with a mobilization rate (e.g., mobilization rate data) of cells (e.g., a number of mobilized cells per unit time, such as second, minute, hour, and / or day) using the frames per second (FPS) of the video image. In non-limiting embodiments, imaging system 300 may process the mobilization rate data to obtain a total number of mobilized cells and / or an accumulated cell count at specified time (e.g., data associated with total cell count). In some non-limiting embodiments, imaging system 300 may display the total number of mobilized cells and / or the accumulated cell count on a user interface.

[0134] In non-limiting embodiments, image capture device 340 may capture a plurality of images. In non-limiting embodiments, the at least one processor 200 may be configured to receive the plurality of images from the image capture device 340, perform a frame sequencing procedure on the plurality of images to provide a sequence of images, and convert the sequence of images into a video file format.Example 1Materials and MethodsBiochip Fabrication

[0135] The design for hBM-Chips were created using the 3D CAD software SolidWorks. The biochip molds were 3D-printed in VeroClear™ using a PolyJet printer. The chips were fabricated as previously described using PDMS (Sylgard 184 Silicone Elastomer Kit). Briefly, PDMS was mixed at a 10:1 ratio of base:curing agent and degassed before being poured into the prefabricated molds. These were cured for 1 hr at 60 ' before chip assembly. PET membranes with a 9 pm thickness, 3 pm pore size, and 8 x 105cm-2 pore density (IT4IP, Cat. # 2000M12 / 580M303) were treated with 2% bis[3-(trimethoxysilyl)propyl]amine (Santa Cruz, Cat. # 82985-35-1 ), 1% H20, and 97% isopropanol solution at 80 °C for 20 min. The membranes were rinsed with 100% isopropanol and then allowed to cure at 70 °C for 20 min before being transferred to a 70% ethanol solution for an additional 20 min. The top and bottom halves of the PDMS chips were plasma treated with 100% O2 at 80% power for 3 min, after which the treated PET membranes were bonded first to the bottom half of thechip and then to the top half of the chip. The assembled chips were left at 70 °C overnight to ensure strong chip bonding.Isolation of CD34+ Cells

[0136] De-identified human cord blood samples were collected from Clinlmmune and Vitalant Biobanks. Mononuclear cells were first isolated from the blood by density gradient centrifugation. For this purpose, the blood was mixed at a ratio of 1 :1 with room temperature RPMI-1640 (Gibco, Cat. # 61870-036), 30 ml_ of the diluted blood was layered over 15 mL of Lymphoprep (StemCell Technologies, Cat. # 07801 ) in a 50 mL conical tube, and centrifugation at 800xg for 30 min with no acceleration or brakes was performed. Mononuclear cells were collected from the top band of cells and transferred to a fresh 50 mL conical tube, after which the cells were washed with RPMI-1640. CD34+ HSPCs were extracted from the mononuclear fraction using positive (magnetic) microbead selection according to the manufacturer’s protocol (Miltenyi Biotec, Cat. # 130-046-703).Hydrogel Preparation and Biochip and Static Transwell Insert Seeding

[0137] To prepare for cell attachment, biochips were sterilized, and their surface was functionalized by plasma treatment with 100% O2 at 80% power for 3 min. Within 5 min of plasma treatment, the apical and basal channels were filled with 300 pg / ml rat tail collagen type I (Corning, Cat. # CB-40236). Collagen-coated chips were incubated overnight at 37^ in a humidified incubator. The following day, the excess collagen solution was removed, and the device channels and chambers were flushed with 37°C endothelial growth media (EGM-2, PromoCell, Cat. # C-22111 ). HUVECs (PromoCell; Cat. # C-12200, Lot # 434Z003 and 445Z004) were resuspended at a density of 2.5 x 106 cells / mL in EGM-2 media, and 100 pl of the cell suspension was pipetted into the vascular channel of each hBM-Chip. The chips were turned upsidedown so that the HUVECs would adhere to the porous PET membrane, after which the cells were incubated for 2 hrs at 37 °C. Next, the chips were turned upright, the vascular channel was connected to a reservoir of EGM-2, and the culture medium was perfused using a peristaltic pump set at 2.5-10 pL / min. While a monolayer of endothelial cells was achieved at this point, the chips were left overnight at 37 °C. The static culture counterparts were prepared in a similar fashion, where transwell inserts (Thincerts, Grenier, Cat. # 657630) were coated with the same collagen coating solution and HUVECs were seeded at the same density on the undersurface of the insert porous membrane and incubated for 2 hrs. Following cell attachment, the insertswere placed in a 6-well plate, and endothelial cells were cultured with 2 ml_ of EGM-2 on the basal side of the transwell insert and 1 mL of EGM-2 on the apical side.

[0138] The next day, single-cell suspensions of CD34+ cord blood cells and mesenchymal stem cells (PromoCell, Cat. # C-12971 , Lot # 453Z018.1 ) were prepared first. Then, the hydrogels were mixed with fibrinogen (Sigma, Cat. # F3879- 1 G), aprotinin (Sigma, Cat. #A6106), collagen (Sigma, Cat. #C2249-20ML), transglutaminase (Modernist Pantry, Cat. #1203-50), CaCL, and thrombin (Sigma, Cat. #T6884) to reach final concentrations of 30 mg / mL, 0.04 mg / mL, 0.5 mg / mL, 10 mg / mL, 2.5 mM, and 1 .5 U / mL, respectively. Next, the hMSCs and HSPCs were mixed at a 1 :1 ratio, where 105cells of each cell type were added to the hydrogel. To prevent premature gelation, the cells were mixed first with fibrinogen, aprotinin, collagen, and transglutaminase. CaCb and thrombin were added last, and the hydrogel solution was gently mixed using a pipet before being transferred into the BM niche chamber of the biochips or the transwell inserts. The apical media was removed from the biochips and transwell inserts immediately prior to the addition of CaCl2 and thrombin, so that the hydrogel could be cast directly on top of the porous membranes. The chips and the inserts were then incubated for 1 hr at room temperature to allow gelation. Once the gels had polymerized, HSC culture media was added to the top of the hydrogels to feed the HSPCs. The HSC culture media used consisted of StemSpan SFEM II (StemCell, Cat. # 09655) supplemented with 10 ng / mL FI13-L (PeproTech, Cat. # 300- 19), 50 ng / mL GCSF (PeproTech, Cat. # 300-23), 50 ng / mL GMCSF (PeproTech, Cat.# 300-03), 10 ng / mL IL3 (PeproTech, Cat. # 200-03), 50 ng / mL IL6 (PeproTech, Cat.# 200-06), 25 ng / mL MCSF (PeproTech, Cat. # 300-25), 10 ng / mL SCF (PeproTech, Cat. # 300-07), and 40 pg / mL aprotinin (Sigma, Cat. #A6106). The culture media used for the HSCs in the BM niche compartment was changed every 48-72 hrs, while the media used for the biochips were changed to EGM-2 media in the basal channel every 48 hrs for static culture.Flow Cytometry Analysis

[0139] The hydrogels were degraded after 7 or 10 days of culture to collect cells for flow cytometric analysis. To degrade the gels, the apical media were removed and replaced with 5 mg / mL collagen / dispase blend II (Millipore, Cat. # SCR140), which was dissolved in DPBS and supplemented with 150 mM CaCh. The biochips were then incubated for 1 hr at 37 °C with gel-dissociation solution with gentle agitation every 15 min. Next, the cell suspension was recovered from each chip and transferred to a15 mL conical tube, after which the cells were pelleted for counting and staining. Viability was determined using live / dead near-IR staining (Thermo Fisher Scientific, Cat. # L10119) according to the manufacturer’s protocol. The following cell surface stains were used: Brilliant Violet 421 anti-human CD34 (BioLegend, Cat. # 343609), Brilliant Violet 510 anti-human CD45 (BioLegend, Cat. # 368526), ARC anti-human CD38 (eBioscience, Cat. # 17038941 ), Brilliant Violet 605 anti-human CD45RA (BioLegend, Cat. # 304133), Brilliant Violet 711 anti-human CD90 (BioLegend, Cat. # 328140), PE anti-human CD123 (eBioscience, Cat. # 12123941 ), Brilliant Ultra Violet 396 anti-human CD16 (BD Horizon, Cat. # 563784), Alexa Fluor 488 anti-human CD15 (Invitrogen, Cat. # 53881341 ), PE / ef610 anti-human CD14 (eBioscience, Cat. # 61014941 ), PE / Cy5.5 anti-human CD33 (eBioscience, Cat. # 15033942), PE / Cy7 antihuman CD11 b (eBioscience, Cat. # 2501 1841 ), and Alexa Fluor 700 anti-human CD19 (BioLegend, Cat. #302225). Flow cytometry data were acquired using a Beckman CytoFLEX LX cytometer.Immunofluorescence Microscopy

[0140] To capture images of the HUVEC monolayer in the hBM-Chip, immunofluorescence staining was performed after the cells were fixed in situ on the PET membrane. Once the monolayer was established, as described above, the cells were fixed by flushing the vascular channel with 4% PFA (Microscopy Sciences, Cat. # 157-4) and incubating for 15 min at 37'0. The cells were then washed with DPBS and blocked for 1 hr at room temperature with 5% FBS / 1 % BSA solution in DPBS. The cells were subsequently stained overnight with Alexa Fluor 488-conjugated antihuman CD31 (BioLegend, Cat. # 3031 10). Afterwards, the membrane was cut from the chips and mounted on a glass slide in VectaShield mounting medium with DAPI (Fisher Scientific, Cat. # NC1848444). Images were acquired on a Leica SP8 confocal microscope.

[0141] To capture images of the hydrogel-embedded cells in the BM niche, a protocol reported previously was used. Briefly, after 7 days of differentiation, the BM niche and the vascular channel were washed with DPBS after removing the media. Then, 10% buffered formalin (Sigma Aldrich, Cat. # HT501128) was added to the BM niche, which was incubated for 2 hrs at room temperature, followed by washing with DPBS overnight at 4^ . Flash phalloidin (BioLegend, Cat. # 424203) and DAPI (Invitrogen, Cat. # D1306) were used for staining F-actin and nuclei, respectively, within 2 hrs of incubation. The hydrogel was then washed for 24 hrs with DPBS. Themembrane containing the hydrogel was removed from the chip and mounted on glass slides using silicone separators (Electron Microscopy Sciences, Cat. # 70346-74) and VectaShield mounting medium (Fisher Scientific, Cat. # NC9265087). Images were acquired on a Leica SP8 confocal microscope.Chemoattractant Treatment and Terminal Leukocyte Egress Evaluation

[0142] For chemotaxis studies, the design of the hBM-Chip vascular channel was modified to increase the surface area contact between the BM niche compartment and the vascular channel (from 30 mm2to 82 mm2) (FIG. 2B) and excluded the endothelial cell seeding step. At five days post-cell seeding into the hBM-Chips, the HSPC medium in the BM niche compartment was replaced with supplement-free StemSpan SFEM II base medium for 48 hrs to prime the cells. Next, we initiated mobilization studies. For this purpose, we removed the media in the vascular channel and rinsed it gently twice with DPBS. Next, we flowed 500 nM LTB4 (Sigma, Cat. # 434625)- supplemented StemSpan SFEM II medium through the vascular channel for 8 hrs at a rate of 2.5 pL / min. During this period, the effluent of the channel was collected. At the end of the experiment, we stopped the flow, spun down cells collected in the effluent tube at 300xg for 10 min, resuspended the cells in DPBS, and manually counted the egressed cells. We then immunostained the egressed cells for flow cytometry analysis. Additionally, in parallel, we digested the BM niche hydrogel and counted the cells.ICD Hardware

[0143] The ICD, which includes five major components - i.e., a motorized 3D-stage module (FIG. 6B), a camera (FIG. 6C), a light source (FIG. 6D), a flow cell (FIG. 6E), and a control computer - was designed and developed as follows. The 3D-stages module comprised three stages to adjust the flow cell in the X, Y, and Z axes (FIG. 6B). The X and Y manual stages (Thorlabs, Inc., DT12XY) were used to adjust the imaging position of the flow cell. These stages were directly mounted onto the Z motorized linear stage (Thorlabs, Inc., KMTS25E / M) via a 90-degree adaptor (Thorlabs, Inc., AB90A / M). The motorized stage was controlled by a controller (Thorlabs, Inc., KDC101 ), which can receive user commands from motion control software provided by the manufacturer (Thorlabs, Inc., APT Ver 3.21 .6). The resolution of the motorized stage was 100 nm, and it had a travel range of 25 mm and was capable of accurately adjusting the focus of the camera remotely.

[0144] A CMOS high-speed camera (Thorlabs, Inc., CS165CU) running at a preset speed (typically 200 FPS) was used for acquiring images (FIG. 6C). The camera was connected to an infinity-corrected lens tube (Thorlabs, Inc., MVR10A) for image correction. The lens tube was then connected to a brightfield 4X objective (Olympus, UPLanFLN 4X) with a depth of field of 16.27 pm, sufficient to visualize cells flowing inside the flow cell channel. The total magnification of the camera module gave a 1 pm / pixel resolution of the images, which was sufficient to resolve cells as small as a few microns in diameter. The camera was connected to the control computer to stream high-quality real-time videos during the experiments. A white-light LED (Thorlabs, Inc., WFA1 010) was used to perform brightfield illumination for imaging (FIG. 6D). The light source was coupled with a condenser to focus the light to the imaging region.

[0145] To mount the flow cells onto the 3D-stages module for imaging, a flow cell holder was used, which was fabricated by 3D-printing (FIG. 6E). The holder had a slim track to house the flow cell. After the flow cell was inserted into the holder, the flow cell was secured using two screws at the two ends to minimize drifting and vibrating during the experiments. The holder was mounted directly on top of the XY part of the 3D-stages module and placed between the objective and the light condenser (see FIG. 6A).

[0146] A microfluidic channel with a rectangular cross-section of 1 .2 mm x 60 pm and a length of 20 mm was used for the flow cell (FIG. 6F). The flow cell was fabricated following the standard procedure of the soft-lithography technique. Briefly, first, a channel mold with the same dimensions as the flow cell channel was fabricated by photopolymerizing an SU-8 3050 photoresist (KAYAKU Advanced Materials, Inc., Y311075)-coated silicon wafer in a maskless aligner (MLA100, Heidelberg Instruments, Inc., Germany). After the standard baking steps, the wafer was developed using an SU-8 developer (KAYAKU Advanced Materials, Inc., Y020100) to remove non-polymerized regions and reveal the channel structure. Finally, the mold was ramp / step hard baked at 150 °C on a hot plate to further cross-link the material. Then, the wafer was put into a petri dish filled with a layer of 1 cm-thick PDMS at a 10:1 base:curing agent. After degassing, the peri-dish containing PDMS and the wafer were placed in an oven at 70 °C for curing overnight. The next day, the PDMS was removed from the wafer, revealing the channel. Inlets and outlets of the flow cell were created using a 1 mm biopsy punch. The PDMS was then cut into appropriately smallpieces and bonded onto a standard 25 x 75 mm glass slide using the plasma- enhanced bonding technique.

[0147] All the components of the ICD were securely mounted on a breadboard (Thorlabs, Inc., MB3045 / M), which was then screwed to one side of a cell culture incubator wall (see FIG. 6A). This position of the system minimized the gravitational effect on the cell trajectory when flowing through the flow cell, providing more accurate cell quantification. During the experiments, cells that migrated from the hBM-Chip were pushed to the flow cell using a syringe pump (Chemyx, Inc., Fusion 200) at a preset flow rate. The syringe pump also ensured a fixed flow rate, an important requirement for accurate cell quantification analysis.ICD Analytical Software

[0148] To determine the number of cells that were mobilized, software that can process the images captured by the ICD in real-time (FIG. 9B) was developed. The software was written in Python and can be executed on any system that allows the use of a Python interpreter. The software analyzed the input videos and outputs a set of graphs and CSV files containing the number of cells passing through the flow cell in each video. Using the FPS of the videos, these data were converted to the number of mobilized cells per minute. The mobilization rate data could be further processed to obtain total mobilized cells or the accumulated cell count at any moment in time. A user-interface was developed to display the results and receive input from the user.

[0149] The core of the software relied on two custom-developed algorithms: a cell detection algorithm (FIG. 10B) and a cell quantification algorithm (FIG. 11 B). The purpose of the cell detection algorithm was to isolate cells from the image background and other non-cell moving objects, such as bubbles and hydrogel debris (FIG. 10B). For this purpose, the software first calculated the background of each image by applying the mixture of Gaussian algorithm 2, an advanced motion detection algorithm, to all moving objects in the image, creating a background with all the stationary pixels. The algorithm then subtracted the original image from the background to obtain an image containing only moving objects. The image was then converted into a binary image using Otsu’s thresholding method. Next, a morphological bandpass filter was applied to remove all the objects that were smaller than 5 pm or larger than 20 pm. The outcome was an image that contains only moving objects 5-20 pm in length, which were predominantly cells.

[0150] Using the cell quantification algorithm, an analytical method called the gating algorithm was applied (FIG. 11 B). By this approach, a line image was first extracted with a pre-determined height from the filtered image after the cell detection step; this image was called the gate. Then, N gates extracted from the N consecutive images were concatenated into one. Next, the number of spots in the concatenated image was counted to obtain the number of cells passing through the flow cells in N frames (FIG. 11 B). Cell accumulation over time was calculated by summing the number of cells that passed through each analyzed N image; typically, N = 1200 in these experiments. The cell count data were saved in a CSV file in real time. The software also calculated the total number of cells accumulated at every timepoint in any given experiment and created a series of plots showing the mobilization rate as well as the cumulative cell mobilization over time (FIGS. 13-14).Chemoattractant Treatment and Real-time Leukocyte Egress Evaluation by ICD

[0151] Real-time leukocyte egress evaluation was performed using the ICD system as detailed below. First, the hBM-Chip was prepared and primed following the protocols described above. On the day of the experiment, the vascular channel was first flushed with the appropriate media several times to wash away any cells that might have resided inside the channel during culture. The hBM-Chip outlet was then connected to the flow cell inlet, and the flow cell outlet was connected to a collecting tube using polytetrafluoroethylene (PTFE) tubing.

[0152] Cell culture medium (StemSpan SFEM II) was added to the cells in a humidified CO2 incubator for at least 30 min prior to each experiment to reach 37 °C and stabilize the pH. Ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Cat. # 15575-020) was added to the medium at a final concentration of 2 mM to reduce the adhesion of cells to the chip and tubing inner surface surfaces during the mobilization study. The chemoattractant LTB4 was also added to the medium at a concentration of 500 nM. The EDTA- and LTB4-supplemented media were then degassed using a sterile vacuum desiccator and loaded into a 5 mL syringe, which was subsequently connected to the inlet of the hBM-Chip via PTFE tubing. Next, the flow cell was inserted into the flow cell holder and secured it by tightening the screws at the two ends of the holder (FIG. 6E). The field of view of the camera was then adjusted using the XY 3D-stages module. To focus the camera, the motorized stage Z was used to adjust the focal point of the objective to the glass-PDMS interface in the flow cell. The focal point was then moved up to ~20 pm using the motorized 3D-stagesmodule via motion control software. Note that as the channel height is 60 gm, this position of the focal point is optimal for acquiring high-contrast images of flowing cells inside the channel. After finishing the flow cell setup, the syringe pump was started at a fixed flow rate of 50 pL / min, forcing the medium to slowly fill the tubing and vascular channel of the hBM-Chip. This method helped minimize the entrapment of bubbles inside the channel, which is crucial for obtaining accurate cell quantification downstream. Once the medium filled the entire vascular channel, the flow rate was set to the desired mobilization flow rate of 2.5 pL / min, and the recording was started for 8 hrs to generate the data presented in FIGS. 13-14.

[0153] The mobilized cells were also manually counted for comparison against the digital quantification data, as shown in FIG. 13, right panel. To do this, at the end of each mobilization experiment, the media that passed through the flow cells was collected, including the media inside the flow cell, into a centrifuge tube. The cells were then centrifuged at 300xg for 5 min and manually counted the cells.

[0154] In the mobilization validation studies, 293T cells were suspended in culture media at a density of 105cells / mL and were allowed to flow through the flow cell at a rate of 2.5 pL / min.ResultsEmulating Hematopoietically Active Human BM Niche On-Chip

[0155] The human BM comprises a variety of cellular niches that support the function of HSPCs; these niches include early lineage-committed immune cell populations, endothelial cells (EnCs), and mesenchymal stromal cells (MSCs), among other cell types. In addition, a key feature of hematopoiesis is the maintenance of HSPCs such that they retain the capacity for self-renewal and the ability to give rise to immune cell populations on demand. Thus, in recreating the BM niche using Organ- on-a-Chip technology, a goal was to design a model that would allow for the co-culture of these unique cell types while simultaneously supporting the maintenance of HSPC multipotency and the production of mature myeloid cell populations (FIG. 4).

[0156] The hBM-Chip was fabricated using biocompatible, transparent, gas- permeable polymer polydimethylsiloxane (PDMS) as previously described. The device was designed such that it has a chamber on top that houses the BM niche components juxtaposed to a vascular channel lined with EnCs at the bottom (FIG. 2A). The vascular channel and the BM niche compartment were separated by a thin, semi-porous (polyethylene terephthalate [PET]) membrane with pores 3 pm in diameter. Followingchip assembly, sterilization, and coating with a thin layer of collagen, primary human umbilical vascular endothelial cells (HUVECs) were seeded on the undersurface of the membrane in the vascular channel and allowed to form a monolayer (FIG. 2A, FIG. 15). HUVECs were used for two reasons. First, in early studies, it was found that the quantity of human bone marrow endothelial cells (hBMEnCs) obtained from (deidentified) clinical bone aspirates was very low, and these cells were difficult to culture in vitro (data not shown). In addition, these cells were not readily available from commercial sources. Second, HUVEC donors were identified for the expression of several markers, such as CD29, CD31 , CD44, CD49b, CD49c, CD61 and CD144, which have been previously reported to be present on hBMEnCs (data not shown). Following HUVEC monolayer formation (~ 24 hrs after seeding), a fibrin gel was cast containing cord blood-derived human CD34+ HSPCs and umbilical cord-derived human mesenchymal stem cells (hMSCs) mixed 1 :1 at a density of 105cells / chip in the BM niche compartment (FIGS. 2A and 4). For this purpose, a hydrogel formulation was used (by adjusting the collagen content and protein-crosslinking transglutaminase dose) that maintained structural integrity throughout the culture (FIG. 16). Once the cell-laden fibrin gel was polymerized on-chip, HSPC growth and differentiation cell culture media were added to the BM niche compartment (on top of the gel) and replaced every 48-72 hrs. Meanwhile, the HUVECs in the vascular channel were fed through constant perfusion of endothelial cell culture media using a peristaltic pump.

[0157] Next, the cells in the BM niche compartment were collected and analyzed by flow cytometry (FIGS. 17A-17C and 18). It was observed that as early as 7 days (D7) after fibrin gel casting, the cells considerably proliferated and formed myeloid lineage-committed cells (live CD45+ CD34- CD11 b+) and differentiated monocytes (live CD45+ CD34- CD11 b+ CD14++ CD16-, live CD45+ CD34- CD1 1 b+ CD14++ CD16++, live CD45+ CD34- CD11 b+ CD14+ CD16++) and neutrophils (live CD45+ CD34- CD11 b+ CD14-, CD15+), while retaining stem (live CD45+ CD34+ CD38- CD90+) and multi-potent progenitor (MPP) (live CD45+ CD34+ CD38- CD90-) cell populations (FIGS. 17A-17C and 18). Note that different gating strategies using other markers (additional or instead of ones used here) and / or sequence of gating and subgating can be applied. A similar trend was also observed on day 10 (D10) after HSPC / hMSC seeding (FIGS. 17A-C and 18). Interestingly, compared with their static culture counterparts, in which fibrin gel-embedded HSPCs / hMSCs were placed over the porous membrane of transwell inserts and HUVECs were coated on theundersurface of the membrane, it was found that hBM-Chips produced significantly greater numbers of total live cells, myeloid cells, neutrophils, stem cells and MPPs at D7 (FIGS. 17A-17C and 18).Recreating Device-Existing Cell Egress Out of BM Niche On-Chip

[0158] Having established the haematopoietically active hBM-Chip system, the platform was then utilized to study leukocyte chemotaxis in response to flowing agents, through which egressed cells enter the vascular channel via a porous membrane and then exit the device (FIGS. 19A-20). Leukotriene B4(LTB4) is pro-inflammatory lipid mediator that has been shown to be a potent chemoattractant for neutrophils and T cells. Therefore, in these studies, LTB4 was chosen as a representative chemotactic agent. Two other modifications to the mobilization experiments were also made. First, the contact surface area between the BM niche compartment and the vascular channel was increased (from 30 mm2to 82 mm2) to maximize the opportunity for cells embedded in the fibrin gel to respond to the test chemoattractant (FIG. 2B). Second, early platform validation studies revealed that the permeability of the endothelial monolayer needs to be increased by treatment with a pro-inflammatory molecule, such as TNFa, to allow cells to enter the vascular channel (data not shown). In addition, it has been reported that BM sinusoidal endothelial cells constitutively express celltethering CD62E in vivo, whereas in other types of EnCs, the expression of this protein is induced upon inflammation. Thus, for immune cell egress studies to minimize the potential cofounding impact of treatment with EnC-permeabilizing agents, the chips were cultures endothelial cell-free. After 8 hrs of treatment with 500 nM LTB4, a significant increase in the number of egressed cells was observed when LTB4 flowed through the vascular channel (FIGS. 19A-20). Notably, when the mobilized cells were analyzed by flow cytometry, it was found that in addition to neutrophils (live CD45+ CD34- CD11 b+ CD14-, CD15+), monocytes (live CD45+ CD34- CD11 b+ CD14+) and HSPCs (live CD45+ CD34+) also responded to the LTB4-induced chemotactic gradient (FIGS. 19A-20). The number of cells in the BM niche compartment was quantified to ensure that the difference in cell mobilization between the LTB4-treated group and the control group was not due to differences in cell density. As shown in FIG. 21 , there was no significant difference between these two groups across all the donors tested.Design and Development of Inline Cyto-Descriptor (ICD)

[0159] To monitor cell mobilization out of the BM niche into the vascular channel in real-time, the ICD (FIGS. 5-6F, 9B, 10B, 11 B, and 12) was designed, developed, and validated. The ICD is a microf luidically adapted precise microscope that identifies and quantifies flowing cells in microfluidic channels with high specificity and sensitivity. It is an electro-optical-mechanical engineered system with custom-developed software for operation, user-selected hardware-related adjustments, and cell mobilization analysis. The ICD was designed using the optical principle of brightfield inverted microscopy, where the samples (moving cells that egress out of the BM niche in the hBM-Chips and enter the ICD flow cell) are illuminated from the top, and images are recorded from the bottom. The ICD consists of five major parts: (1 ) a translational 3D- stages module, (2) a camera, (3) a light source, (4) a flow cell, and (5) a computer equipped with a multi-component (operation and analysis) software. All these components are securely mounted on a breadboard, which itself is mounted to one of the sidewalls within a cell culture incubator.

[0160] The translational 3D-stages module (FIG. 6B) controls the position of the flow cell in the X, Y, and Z axes. The X- and Y-stages are adjusted manually at the beginning of each experiment, and the Z-stage, which serves to bring cellular movements into focus, is motorized and adjusted digitally via operation software installed on a linked computer. This setup allows remote control of the focal plane from outside the cell culture incubator, at the beginning or if needed during the experiment, minimizing unwanted physical system movements that may negatively impact the experiment.

[0161] To continuously record images for generation of videos, a high-speed camera coupled with an infinity-corrected lens tube and a 4X objective (FIG. 6C) was used. The recording speed of the camera is as high as 250 frames per second (FPS), which is sufficient to capture images of cells flowing through the flow cell channel at flow rates of up to 20 pl / min. The camera is controlled directly by the analysis software that was custom-designed and developed to stream the generated videos for real-time processing. Using the 4X objective in the ICD, it was possible to achieve a magnification of 1 pm / pixel, which is sufficient for resolving cells with sizes as small as a few microns. Moreover, the use of an infinity-corrected lens tube allows flexibility of the camera position without affecting its performance or the system’s desired magnification.

[0162] To obtain high-contrast brightfield images, the flow cell was illuminated using a white-light LED coupled with a condenser (FIG. 6D). The flow cell, fabricated new for each experiment, is mounted horizontally on the 3D-stages module using a purpose-built flow cell holder between the light source and the camera (FIG. 6E). Following insertion into its holder, the flow cell is secured using two screws at opposite ends of the holder to minimize drifting and vibration while the experiment is running. The flow cell itself is a narrow microfluidic channel with a rectangular cross-section of 1 .2 mm x 60 pm (FIG. 6F). The width and height of the flow cell channel were designed so that 1 .4 mm2of the field of view (FOV) of the camera as well as the depth of field 16.27 pm of the 4X objective could be used. In other words, the visualized FOV covers the entire width of the channel and all the flowing cells. In addition, the flow cell channel has a length of 20 mm, which is sufficiently long to stabilize the flow before it reaches the imaging region.

[0163] Having developed the ICD, it was validated by flowing live 293T cells at a cell density and flow rate of 105cells / mL and 2.5 pL / min, respectively, and recording a 10-minute video at a speed of 35 FPS. It was observed that the cells were clearly distinguishable from each other and from the background, confirming the ability of ICD to visualize cells flowing through the flow cell channel.

[0164] To quantify the cells in real-time and identify the dynamics of cell mobilization, analysis software was applied (FIGS. 9B, 10B, 11 B, and 12), which allows for analysis of up to 40 videos at the same time. The software directly acquires images from the camera, packages them into a video(s), and transfers them to the computer for processing. Up to 300 images / second can be processed, which is fast enough to comply with the recording speed of the camera in these studies — i.e. , up to 240 FPS. The final output was a series of plots showing the mobilization rate and cumulative mobilization over time.

[0165] The core component of the analysis software is an image processing algorithm that itself comprises two sub-algorithms, one for cell detection (FIG. 10) and the other for cell quantification (FIG. 11 B). The purpose of the cell detection algorithm is to isolate cells from the image background and other non-cell moving objects, such as bubbles and hydrogel debris (FIG. 10B). Given an image ith(i > 400) counted from the beginning of the experiment, the software will initially use the 400 historical images acquired prior to the image ithto calculate the background ithusing the mixture of Gaussian algorithm 2 (MoG2). The advanced motion detection algorithm MoG2identifies all moving objects, including cells, bubbles, and hydrogel debris, in an image; all the stationary pixels are considered the background of the image. The software subsequently subtracts the original image from the background to obtain an image containing only moving objects. The image is subsequently converted to a binary image using Otsu’s thresholding method.

[0166] During an actual experiment, not only did cells pass through the imaging region, but there were also bubbles and hydrogel debris (FIG. 10B, original image). This noise causes challenges when trying to accurately determine and quantify cells. It was found that the key distinguishing factor between cells and non-cell objects is size. Specifically, in these studies, it was observed that while cells typically have sizes in the range of 5 pm to 20 pm, bubbles are usually smaller than 5 pm, and hydrogel debris is typically much larger than 20 pm. This approach allowed for application of a morphological bandpass filter to remove objects smaller than 5 pm or larger than 20 pm. Thus, the analysis software processed the images so that they would only contain moving objects with sizes between 5 pm and 20 pm, which are mostly moving cells (FIG. 10B, filtered image). This approach allowed high accuracy of cell identification so that the cells could be counted in the next step.

[0167] To quantify the cells in real-time, a method referred to as the gating algorithm (FIG. 11 B) was developed. Given an image package of N frames resulting from the cell detection step in FIG. 10B, a line was extracted from each frame, called the gate (FIG. 11 B, Gating). The width of the extracted gate is a set parameter determined based on the displacement of the cells in the images by each frame, ensuring that every cell is captured one and only one time when flowing through the gate. Then, N gates extracted from the N frames were concatenated into one single image (FIG. 11 B, Stacking). Each white spot in this concatenated image represents a cell passing through the field of view in the N-frame video. The number of spots were then counted to obtain the number of cells passing through the flow cells in N frames.

[0168] The algorithm was validated by analyzing the images obtained from the trial runs using live 293T cells as described above. A series of snapshots capturing the main steps of the analysis were obtained from the original image (FIG. 12, panel (i)) to the cell detection (FIG. 12, panels (ii)-(iii)) and cell quantification (FIG. 12, panels (iv)-(vi)). The total cell counts obtained by the software for 10 independent tests were then compared with those counted manually. The good match between the number ofcells counted by the software and by the experimenter validated the developed algorithm.Deciphering Real-time Immune Cell Mobilization Out of the hBM-Chip Under a Chemoattractant Gradient

[0169] The newly developed and validated ICD system and software was applied to quantify cell mobilization in hBM-Chips under LTB4 treatment; this time, the realtime dynamics of immune cell mobilization out of the BM-Chips was characterized rather than terminally counting the cells in the vascular effluent of the chips. It was observed that the cell egress rate, defined as the number of cells passing through the flow cell per minute, did not appear constant; rather, the rate was highly timedependent during the experimental window (FIG. 13, left panel). In other words, cells did not linearly respond to the chemoattractant gradient to leave the BM niche. This is the first time such a discovery has been made on the kinetics of leukocyte egress from a hematopoietically active BM. As illustrated in FIG. 13, left panel, the mobilization rate increased sharply during the first 1.5 hrs following the introduction of the chemoattractant via the vascular channel. High fluctuations in the mobilization rate, up to 700 cells / min, were observed during the first 30 min of this period. These fluctuations were attributed to the cells remaining inside the vascular channel prior to the experiment. Although the vascular channel of the BM-Chip was flushed before the experiment to remove as many cells as possible, some cells were still inside the vascular channel, and the media were allowed to flow out when the experiment was started. Nevertheless, the increasing trend of the mobilization rate is clear during this period. Later, after reaching the maximum value of ~ 450 cells / min at 1.5 hrs, the mobilization rate gradually decreased to ~ 200 cells / min at 4 hrs following chemoattractant treatment. From 4 to 8 hrs, the mobilization rate stabilized and plateaued at ~ 200 cells / min.

[0170] Next, the total number of cells that passed through the flow cell at any given moment was calculated by plotting the cumulative sum of the prior mobilization rate data. As illustrated in FIG. 14, the data clearly reveal time-dependent cell mobilization. Notably, approximately one-third of the total mobilized cells - i.e., ~20,000 cells - migrated to the vascular channel during the first 1 .5 hrs of the experiment, with an average rate of 222 cells / min, indicating a surprisingly fast acute response of cells to chemoattractant treatment. During the following 6.5 hrs of LTB4 treatment - that is,from 1.5 hrs to 8 hrs - two-thirds of the egressed cells - i.e., ~40,000 cells - entered the vascular channel at an average rate of 102 cells / min.

[0171] Finally, the real-time mobilization results were validated by performing a matched comparative analysis of the total number of cells quantified by the ICD against the number of cells manually counted for each of the four independent experiments that were performed. No statistically significant differences between the two quantification methods (FIG. 13, right panel) were observed, confirming the accuracy of the ICD platform.Discussion

[0172] The research presented here signifies the integration of an Organ-on-a- Chip system with advanced cell imaging technology. This approach enabled the concurrent investigation of myelopoiesis and real-time immune cell mobilization in response to an inflammatory chemotactic factor. Utilization of the hBM-Chip allowed faithful replication of several crucial aspects of living myeloid tissue in the BM in vitro, including the intricate 3D organization of immune and stromal cells, generation of lineage-committed cells while maintaining the HSPC population, vascularization, and circulation-like flow for introduction of a test chemoattractant and analysis of leukocyte mobilization leaving the BM niche. Collectively, these distinctive features endow the hBM-Chip with key advantages over traditional 2D models when modeling myelopoiesis. Moreover, the strategic separation of the vascular channel and BM niche compartment by a porous membrane allowed for study of cell trafficking. The modular hBM-Chip-ICD-linked system is a key novel distinguisher of the platform compared with other human BM-on-a-Chip systems (see below).

[0173] The emergence of Organ-on-a-Chip technologies represents a major advance in the ability to preclinically model tissue- and organ-level biology, intricate 3D multicellular architecture, mechanical forces, and vascular perfusion for diverse translational applications. In the context of BM biology and functioning, few microfluidic MPS prototypes of human bone marrow have been developed.

[0174] Chou et al. used a two-parallel-channel BM-on-a-Chip system in which one channel was filled with fibrin gel containing CD34+ and stromal cells and the other was lined with endothelial cells. The model was shown to be effective at recreating myeloerythroid proliferation and differentiation, chemotherapeutic-induced toxicity, and hematopoietic abnormalities in patients with inherited bone marrow failure. However, this platform exhibited major differences compared with the hBM-Chip. (1 )The entire chip was fed only by perfusion medium - a stem cell-based medium supplemented with growth factors and supplements needed for endothelial cell proliferation and culture - through the endothelium-coated channel, whereas the BM niche compartment could be fed separately using its own specific medium directly through the space over the hydrogel (without needing to mix with endothelial cell growth factors and supplements) while feeding the HUVECs cells with endotheliumspecific culture medium. (2) The on-chip cell proliferation plateaued at ~ D21-D28 of culture, whereas this was achieved at a faster pace at D7-D10. (3) Only neutrophils from the myeloid lineage were studied in the hematopoietic niche; no characterization of monocytes was presented. (4) The authors primarily used CD45+ CD34+ population to define HSPCs, with use of CD38 marker in some instances to identify immature progenitor cells. In contrast, by including CD90 in addition to CD45, CD34 and CD38, a clear definition of HSCs and MPPs in these chips was achieved. (5) It was unclear if the 3D gel integrity was fully maintained throughout the culture. In these studies, the gel formulation was designed to establish robustness and continued integrity throughout the culture. (6) The authors reported spontaneous migration of mostly myeloid lineage cells into the vascular channel of the device after two weeks of culture. However, chemoattractant-driven leukocyte mobilization, real-time analysis of such egress, and more detailed profiling of intravasated cells were not demonstrated.

[0175] In an independent study, Nelson and colleagues developed a microfluidic human BM-on-a-Chip system in which a five-channel PDMS device was bonded to a bottomless 96-well plate. The microfluidic PDMS component consisted of a central hydrogel channel (where the fibrin-collagen matrix was filled with hMSCs, HUVECs, and CD34+ cells were layered over a sheet of hMSC-derived osteoblasts), two adjacent channels (for supply of vasculogenic culture media), and two outer (hMSC- laden) hydrogel channels. The wells of the 96-well plate served as culture medium reservoirs and were used for imaging. This platform was principally used to characterize vasculogenesis and cytokine production, evaluate CD34+ cell proliferation, and study the impact of ionizing radiation-related damage on HSPCs. However, this device needed 21-26 days in culture to be ready for experimentation, did not demonstrate myeloid differentiation or lineage commitment, and lacked characterization of HSPC populations. Importantly, no data on the ability of the platform to recreate leukocyte mobilization (at baseline or in response to a chemokine) were presented. In addition, the control and analysis of vascular flow in this modelwere partly limited due to the reliance on vasculogenesis for blood channel formation and the use of tissue culture plate wells as medium reservoirs.

[0176] Most recently, Glaser et al. reported a vascularized model of human BM- on-a-Chip. The device design consisted of two inter-linked hexagonal chambers adjacent to a third bottom chamber. The hexagonal chambers were populated with fibrin hydrogels containing cord blood-derived endothelial colony-forming and CD34+ cells, a fetal osteoblast cell line, and BM stromal cells to model the endosteal and perivascular niches of the BM. The authors reported that their model recapitulates several markers of native BM, supports HSPC maintenance and differentiation, allows cell egress, and can be utilized for cancer cell migration into the BM niche. Nevertheless, this MPS needed 14 days of culture to reconstitute the hematopoietically active BM niche, after which the total number of lineage-committed neutrophils generated in each device was still relatively low. Additionally, immunophenotypic characterization of HSPC populations and the presence of nonneutrophil myeloid cells was not performed. Moreover, the study did not present realtime analysis of immune cell mobilization dynamics or clear profiling of all egressed cells.

[0177] In this study, the egress of HSPCs, in addition to neutrophils and monocytes, to LTB4 in vascular channel was partly unexpected, as the majority of the literature has focused on the chemotaxis of neutrophils in response to LTB4. However, this can be possibility explained given the reported expression of high-affinity LTB4 receptor BLT1 on the surface of monocytes and CD34+ progenitor cells. Therefore, an advantage of the hBM-Chip model in this context would be that it enables the profiling of chemoattractant-responsive cells using a multicellular preclinical tool rather than focusing on a single immune cell type. Notably, LTB4-induced mobilization of cells out of the BM niche serves as a pivotal proof-of-concept for the utility of the system in disease modeling, particularly in the investigation of specific immune cell subsets. For instance, prior research has indicated that neutrophil mobilization contributes to influenza clearance, while the recruitment of inflammatory monocytes is associated with more adverse outcomes. The present system can be employed as a valuable tool for dissecting the factors influencing the preferential mobilization of distinct cellular subsets in scenarios such as these. Furthermore, the adaptability of the hBM-Chip makes it suitable for integration into linked multi-Organ-on-Chips (e.g., to recreate the lung-BM axis) so that the selective egress and mobilization dynamicsof immune cells out of the BM niche following injury / insult in peripheral organs can be studied.

[0178] Also of note, as shown here, the hBM-Chip and ICD allow for: 1 ) endothelial cell verification and validation by flow cytometry to demonstrate expression of bone marrow sinusoidal endothelial cells, including but not limited to CD29, CD31 , CD44, CD49b, CD49c, CD61 , and CD144 (in the case of human cells); and 2) application of flow cytometry (as a representative analytical tool) to confirm identify of cells (e.g., including, but not limited to, via immunostaining for CD45, CD34, CD38, CD90, CD11 b, CD14, CD15, CD16, and CD33).

[0179] From a technological development perspective, the ICD system has important advantages not only in the context of BM-Chip cell mobilization experiments but also for various applications requiring real-time quantification of flowing cells. The imaging principle employed in the system, namely, brightfield inverted microscopy, not only ensures high image quality for visualizing cells in microfluidic channels but also offers simplicity and compactness — two crucial characteristics facilitating real-time cell tracking. The compact dimensions of the ICD (25 x 15 x 10 cm, H x W x D) make it small enough to fit seamlessly inside a standard cell culture incubator. Moreover, the system exhibited robust performance for extended periods under cell culture and environmental conditions, ensuring its functionality for weeks without any failures. This capability is particularly valuable for conducting long-term observations, a critical aspect of numerous in-culture cell-quantification experiments. Additionally, with the utilization of a high-speed camera featuring an adjustable frame rate, the system can visualize cells flowing through the flow cell across a wide range of user-defined flow rates. The trackable cell velocity spans from near-zero to as high as 12.5 mm / s. The ICD system is flexible and easy to upgrade. Specifically, the use of an infinity-corrected lens tube enables flexible positioning of the camera without compromising the system's specifications. This flexibility permits the integration of additional optical equipment to enhance the system's functionality. For instance, the immediate implementation of a fluorescent function can be achieved by coupling the current optics with a laser source and appropriate filters placed between the camera and the infinity-corrected lens tube, all without impacting the camera's field of view or magnification.

[0180] The real-time analysis software that was developed empowers users to scrutinize ongoing experimental results, facilitating on-the-fly adjustments ifnecessary. This capability is achieved through the integration of two crucial algorithms into the software. First, the MoG2 algorithm allows for the reliable differentiation of cells from image backgrounds across a diverse range of image contrasts and qualities, significantly enhancing analysis accuracy. Second, the cell quantification algorithm, utilizing the gating method coupled with parallel processing, enables high-speed processing, reaching up to 300 images / second. This high-speed processing is pivotal for real-time analysis, supporting a recording rate of up to 250 FPS, as demonstrated in these studies. The gating algorithm exhibits remarkable versatility in handling substantial data volumes, as evidenced by the 8-hour experiment spanning 7.2 million images and generating 6,000 data points (utilizing 1 ,200 images for each data point). Despite the use of this large dataset, the analysis was conducted in real-time, underscoring the rapidity and robustness of the implemented algorithms.

[0181] In summary, the above experiments present a proof-of-concept set of technologies designed for investigating myeloid hematopoiesis and mobilization of immune cells in response to a pro-inflammatory chemoattractant. The integration of the ICD system with the hBM-Chip introduces a real-time dimension to data acquisition, crucial for a precise comprehension of the kinetics governing the egress of leukocytes from the BM niche. Overall, the integrated system can pave the way for diverse future applications in disease modeling, pre-clinical drug testing, and personalized medicine.Example 2

[0182] As shown in FIGS. 22A-22B, at day 10 of culture extensive analysis (by flow cytometry) of cells (other than total myeloid cells, monocytes and neutrophils) and in the BM niche compartment of chip were compared with static culture controls (Transwell Insert (TWI)).

[0183] The results show that devices as described herein can generate mast cells and eosinophils (two important myeloid immune cell types), T and B cells and NK cells (of lymphoid lineage differentiation), and platelets and erythroid cells. That is, devices as described herein can generate all expected immune and non-immune cells and the production of these cells is significantly higher in almost all cases in the chip compared with TWI.

[0184] As shown in FIGS. 23A-23D, at day 10 of culture colony forming unit (CFU) assays were performed on cells from a device a described herein and TWIs. Different colony types and their numbers are shown.

[0185] A key point is that different kinds of colonies are produced and maintained in devices as described herein at higher quantities compared with TWIs. This shows high stem / progenitor potency of the devices described herein to give rise to various immune and non-immune cells.

[0186] As shown in FIG. 24, when CD34+ HSCs from cord blood were cultured along with MSCs in a hydrogel in a device as described herein, and then analyzed by flow cytometry on Days 7, 10 and 28 post-culture, full maturity (in terms of cell expansion, generation of hematopoietic cells including myeloid cells, and maintenance of HSPCs) is seen as early as Day 7 and Day 10. Of interest, this functionality and phenotype can be maintained through at least Day 28 of culture.

[0187] To assess effects of various treatments on membranes, hBM-Chips (e.g., devices as described herein) made with PET membranes were treated with oxygen plasma (100% O2, 80% power, 3 min treatment time), followed by collagen coating (300 ug / mL, overnight incubation) and dried at room temperature. After rinsing the membrane, TG solution (120 mg / mL) was added onto the membrane and incubated for 1 -2 hrs until it is mostly dried. The excess was removed, and the fibrinogen-based hydrogel was casted on top of the treated membrane.

[0188] As can be seen in FIG. 25, double coating of the thermoplastic (TP) membranes, including but not limited to polyethylene terephthalate (PET; polyester) membranes, with collagen and transglutaminase (TG) helps with both attachment of the hydrogel to the membrane as well as maintenance of the hydrogel thickness during the culture time (up to 28 days in culture). It is expected that non-TP membranes (such as PDMS, glass, etc.) will respond similarly. Moreover, if collagen and / or TG were to be replaced with alternative agents (such as another natural or derivative extracellular matrix (ECM) protein such as fibronectin or laminin in place of collagen). It is expected that synthetic materials (e.g., PEG) or semi-synthetic materials would be useful as well as replacements for collagen.

[0189] It is believed that the cross-linking function of TG between the proteins likely helps with binding the fibrinogen in the hydrogel and the collagen coating on top of the membrane. In other words, this process likely helps maintaining the hydrogel attached to the membrane (as opposed to being gradually dislodged with culture time).

[0190] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions,modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.

Claims

THE INVENTION CLAIMED IS1 . A bioreactor system, comprising: a cell growth platform having a hydrogel arranged thereon; at least one first cell received on and / or in the hydrogel; a flow channel configured to receive a flow of a liquid therethrough; a separator arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel; and at least one second cell received on the separator.

2. The bioreactor system of claim 1 , wherein the cell growth platform comprises a housing or a well defining an interior, and wherein the hydrogel is received within the interior.

3. The bioreactor system of claim 1 or claim 2, wherein the at least one first cell comprises a stem cell, pluripotent cell, and / or multipotent cell.

4. The bioreactor system of claim 1 , wherein the at least one first cell comprises at least one CD34+ cell.

5. The bioreactor system of claim 1 , wherein the at least one first cell comprises at least one mesenchymal stem cell (MSC).

6. The bioreactor system of claim 1 , wherein the at least one first cell comprises at least one CD34+ cell and at least one MSC.

7. The bioreactor system of claim 6, wherein the at least one CD34+ cell and the at least one MSC are human cells.

8. The bioreactor system of claim 1 , wherein the at least one first cell is received within the hydrogel.

9. The bioreactor system of claim 1 , wherein the at least one second cell comprises an endothelial cell.

10. The bioreactor system of claim 9, wherein the endothelial cell is a human umbilical vein endothelial cell.11 . The bioreactor system of claim 1 , wherein the hydrogel comprises a fibrin hydrogel.

12. The bioreactor system of claim 1 , wherein the hydrogel is formed from fibrinogen and thrombin.

13. The bioreactor system of claim 1 , wherein the flow channel is in fluid communication with a media reservoir and a pump, such that the media flows through the flow channel.

14. The bioreactor system of claim 13, wherein the media comprises a cross-linker.

15. The bioreactor system of claim 14, wherein the cross-linker is a transglutaminase.

16. The bioreactor system of claim 14, wherein the cross-linker is gelatin transglutaminase.

17. The bioreactor system of claim 13, wherein the media comprises a proteinase inhibitor.

18. The bioreactor system of claim 17, wherein the proteinase inhibitor is a matrix metalloproteinase (MMP) inhibitor.

19. The bioreactor system of claim 18, wherein the MMP inhibitor is aprotinin.

20. The bioreactor system of claim 18, wherein the MMP inhibitor is a tissue inhibitor of metalloproteinase (TIMP).21 . The bioreactor system of claim 13, wherein the media comprises one or more first additives capable of inducing differentiation of the at least one first cell.

22. The bioreactor system of claim 13, wherein the media comprises one or more second additives capable of inducing cellular egress from the cell growth platform.

23. The bioreactor system of claim 1 , wherein the flow channel is a straight channel.

24. The bioreactor system of claim 1 , wherein the flow channel is nonlinear.

25. The bioreactor system of claim 24, wherein the flow channel comprises a plurality of curves.

26. The bioreactor system of claim 24, wherein the flow channel comprises a zig zag pattern.

27. The bioreactor system of claim 1 , wherein the at least one second cell is arranged on the second surface of the separator.

28. The bioreactor system of claim 1 , wherein the cell growth platform comprises, a thermoplastic polymer or copolymer, a resin, and / or a glass.

29. The bioreactor system of claim 1 , wherein the cell growth platform comprises polydimethylsiloxane (PDMS).

30. The bioreactor system of claim 1 , wherein separator comprises a porous membrane.31 . The bioreactor system of claim 30, wherein one or more pores of the porous membrane have a diameter of about 3 pm to about 7 pm.

32. The bioreactor system of claim 30, wherein the separator comprises PDMS or polyethylene terephthalate (PET).

33. The bioreactor system of claim 1 , wherein the cell growth platform is arranged above the flow channel.

34. The bioreactor system of claim 1 , further comprising: an imaging device comprising: a light source; a moveable stage, wherein the flow channel is received on and / or in the moveable stage; and an image capture device, wherein the imaging device is configured such that the moveable stage is arranged between the light source and the image capture device.

35. The bioreactor system of claim 34, wherein the light source is arranged above the moveable stage and the image capture device is arranged below the moveable stage.

36. The bioreactor system of claim 34, wherein the imaging device is arranged downstream of the cell growth platform.

37. A bioreactor system, comprising: a cell growth platform having a fibrin hydrogel arranged thereon; at least one mesenchymal stem cell and at least one CD34+ cell embedded in the fibrin hydrogel; a flow channel configured to receive a flow of a culture medium therethrough;a porous membrane arranged between the cell growth platform and the flow channel and having a first surface that faces the cell growth platform and a second surface in fluid communication with the flow channel; at least one endothelial cell arranged on the second surface of the porous membrane; and an imaging device arranged about the flow channel downstream of the cell growth platform and comprising: a moveable stage; a flow cell arranged on the moveable stage, the flow cell receiving at least a portion of the flow channel therein; a light source arranged above the flow cell; and an image capture device arranged below the flow cell.

38. A system for imaging and counting cells, comprising: a moveable stage; a flow cell arranged on the moveable stage, the flow cell receiving at least a portion of a flow channel therein; a light source arranged relative to the flow cell; an image capture device arranged relative to the flow cell; and at least one processor in communication with the imaging device, the at least one processor configured to: receive at least one image from the image capture device; perform a cell detection procedure on the at least one image to identify at least one cell object in the at least one image; and perform a cell counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure.

39. The system of claim 38, wherein the image capture device captures a plurality of images and wherein the at least one processor is configured to: receive the plurality of images from the image capture device; perform a concatenating procedure on the plurality of images to provide a sequence of images; and convert the sequence of images into a video file format.

40. The system of claim 38, wherein the at least one processor is further configured to: perform another cell detection procedure on the at least one image to identify, in the at least one image, a background, the background data comprising one or more stationary objects in the at least one image; and perform a background subtraction procedure to remove the background from the at least one image to obtain a first processed image, the first processed image comprising only moving objects with the one or more stationary objects removed.41 . The system of claim 40, wherein the at least one processor is further configured to: perform an additional cell detection procedure on the at least one image to identify, in the first processed image, one or more non-cell objects, the one or more non-cell objects identified based on a predetermined size threshold; and perform an image subtraction procedure to remove the one or more non- cell objects from the first processed image to obtain a second processed image, the second processed image comprising only cell objects with the one or more non-cell objects removed.

42. A non-transitory, computer-readable medium having stored thereon programming instructions that, when executed by a processor, cause the processor to: receive at least one image from an image capture device; perform a cell detection procedure on the at least one image to identify at least one cell object in the at least one image; and perform a cell counting procedure to determine a number of cell objects in the at least one image based on a result of the cell detection procedure.

43. The non-transitory, computer-readable medium of claim 42, further comprising programming instructions that, when executed by a processor, cause the processor to: receive a plurality of images from the image capture device;perform a concatenating procedure on the plurality of images to provide a sequence of images; and convert the sequence of images into a video file format.

44. The non-transitory, computer-readable medium of claim 42, further comprising programming instructions that, when executed by a processor, cause the processor to: perform another cell detection procedure on the at least one image to identify, in the at least one image, a background, the background data comprising one or more stationary objects in the at least one image; and perform a background subtraction procedure to remove the background from the at least one image to obtain a first processed image, the first processed image comprising only moving objects with the one or more stationary objects removed.

45. The non-transitory, computer-readable medium of claim 43, further comprising programming instructions that, when executed by a processor, cause the processor to: perform an additional cell detection procedure on the at least one image to identify, in the first processed image, one or more non-cell objects, the one or more non-cell objects identified based on a predetermined size threshold; and perform an image subtraction procedure to remove the one or more non- cell objects from the first processed image to obtain a second processed image, the second processed image comprising only cell objects with the one or more non-cell objects removed.

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