Cartridge and method of analysing biological sample

Abstract

To provide a cartridge that can be used to analyze the phenotypic response of cells in a biological sample to various agents, and that is easy to use, can be pre-manufactured with required culture medium, agents and chemicals, thereby only requiring addition of the biological sample and allows a fully automated analysis of the phenotypic response.SOLUTION: The cartridge includes a chip chamber configured to house a microfluidic chip including a plurality of sets of cell channels configured to capture cells from a biological sample. The cartridge also includes a sample chamber configured to receive the biological sample and be in fluid connection with the plurality of sets of cell channels and a plurality of medium reservoirs. Each medium reservoir of the plurality of medium reservoirs is configured to be in fluid connection with a respective set of cell channels of the plurality of sets of cell channels. The cartridge further includes culture medium source in fluid connection with the plurality of medium reservoirs and configured to supply a culture medium to the plurality of medium reservoirs.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to cartridges, and in particular to such cartridges for microfluidic chips.The present invention also relates generally to methods for analyzing biological samples. [Background technology]

[0002] Antibiotic susceptible testing (AST), also known as antibiotic sensitivity testing, is the measurement of bacterial susceptibility to antibiotics. The results of susceptibility testing allow clinicians to select appropriate antibiotics or antibiotic mixtures based on knowledge of the disease caused by the bacteria and their susceptibility and resistance.

[0003] Early AST methods were phenotypic methods involving exposing bacteria to antibiotics on agar plates or in dilutions in agar or broth. Such phenotypic methods include the disk diffusion method, also known as the Kirby-Bauer method, in which bacteria are cultured on an agar plate and their growth observed near an antibiotic-impregnated disk. If the antibiotic inhibits microbial growth, a clear ring, or zone of inhibition, is observed around the disk. Bacteria are then classified as susceptible, intermediate, or resistant to the antibiotic by comparing the diameter of the zone of inhibition to a defined threshold value that correlates with the minimum inhibitory concentration (MIC). Other phenotypic methods include gradient methods, such as Etest, which use plastic strips impregnated with different concentrations of antibiotic placed on agar, and the growth medium is examined after an incubation period. The MIC can be identified based on examination of the teardrop-shaped zone of inhibition using markings on the strip.

[0004] The main drawback of the phenotypic methods exemplified above is that they typically require at least overnight incubation of bacteria to obtain a visual response. Therefore, it takes a relatively long time before AST results are available. Another drawback is that only one or a few antibiotics can generally be tested in a single AST run.

[0005] Another group of AST methods are genetic methods based on polymerase chain reaction (PCR), deoxyribonucleic acid (DNA) microarrays, or DNA chips. These genetic methods do not examine the phenotypic response of bacteria to antibiotics, but rather analyze whether bacteria harbor genes that confer antibiotic resistance. Genetic methods have the advantage over culture-based phenotypic methods in that they are more rapid. Disadvantages include the required knowledge of the resistance genes being tested, which is costly and typically requires specially trained personnel. Furthermore, sometimes the genotypic profile of the detected resistance genes does not always match the resistance profile seen with phenotypic methods.

[0006] Microfluidic devices and chips have been proposed for rapid and parallel monitoring of bacterial phenotypic responses to a set of antibiotics. Microfluidic-based AST benefits from being rapid but does not suffer from the drawbacks associated with many genetic methods.

[0007] A prior art microfluidic device, designated the "Mother Machine," is disclosed in Wang et al., Current Biology 2010, 20:1099-1103. The Mother Machine allows for the monitoring of cells in many different cell channels in parallel.

[0008] Further microfluidic devices useful for the analysis of biological samples are shown in WO 2016 / 007063 and WO 2016 / 007068.

[0009] Baltekin et al., PNAS 2017, 114(34):9170-9175, discloses FASTest, a rapid antibiotic susceptibility testing (AST) test that uses a microfluidic device.

[0010] U.S. Patent Nos. 7,341,841 and 8,071,319 disclose methods for detecting microorganisms in a sample, which include contacting the sample with a biosensor concentration module, allowing the microorganisms to grow for a first period of time, and detecting the growth of distinct microorganisms as an indication of the presence of the microorganisms.

[0011] There remains a need for cartridges for such microfluidic chips that can be used for phenotypic analysis of cells in samples, such as bacterial AST. Summary of the Invention

[0012] It is a general object to provide a cartridge for such a microfluidic chip that can be used for phenotyping of cells in a sample.

[0013] It is also a general object to provide a method for analyzing a biological sample.

[0014] These and other objectives are met by the embodiments disclosed herein.

[0015] The invention is defined in the independent claims. Further embodiments are defined in the dependent claims.

[0016] One aspect of the present invention relates to a cartridge including a chip chamber configured to accommodate a microfluidic chip including a set of multiple cell channels configured to capture cells from a biological sample. The cartridge also includes a sample chamber configured to receive the biological sample and to be in fluid communication with the set of multiple cell channels. The cartridge further includes a plurality of medium reservoirs, each of which is configured to be in fluid communication with a respective one of the sets of cell channels. The cartridge further includes a culture medium source configured to be in fluid communication with the plurality of medium reservoirs and to supply culture medium to the plurality of medium reservoirs.

[0017] Another aspect of the present invention relates to a method for analyzing a biological sample. The method includes transferring a biological sample containing cells to a plurality of sets of cell channels in a microfluidic chip. The method also includes transferring a culture medium to a plurality of medium reservoirs. Each medium reservoir of the plurality of medium reservoirs is configured to be in fluid communication with a respective set of cell channels of the plurality of sets of cell channels. At least one medium reservoir of the plurality of medium reservoirs is pre-loaded with a predetermined amount of a drug configured to be dissolved or dispersed in the medium. The method further includes transferring the culture medium with the dissolved or dispersed drug from each medium reservoir, the drug being pre-loaded into each set of cell channels. The method further includes monitoring a response of the cells to the drug in each set of cell channels.

[0018] The cartridge of the present invention can be used to analyze the phenotypic response of cells in a biological sample to various agents. The cartridge is easy to use and can be pre-manufactured with the required culture media, agents, and chemicals, thereby requiring only the addition of a biological sample and allowing for fully automated analysis of the phenotypic response.

[0019] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken together with the accompanying drawings, in which: [Brief explanation of the drawings]

[0020] [Figure 1] 1A-1B. Figures 1A and 1B are exploded views of a cartridge according to one embodiment from above (1A) and below (1B). [Figure 2] Figures 2A-2H. Figures 2A-2H show schematic diagrams of cartridge assembly according to one embodiment with sample filter attachment (2A), back cover attachment (2B), microfluidic chip assembly (2C) and microfluidic chip attachment (2D), valve and dome pump attachment (2E), media blister attachment (2F), addition of gas permeable membrane (2G), and top cover attachment (2H). [Figure 3] Figures 3A-3B. Figures 3A and 3B show a schematic representation of filling a sample chamber (3A) with a biological sample and closing the sample chamber with a cap (3B) according to one embodiment. [Figure 4] Figures 4A-4B. Figures 4A and 4B illustrate one embodiment of a substrate from above (4A) and below (4B). [Figure 5] FIG. 5 is a close-up view of a portion of a substrate illustrating one embodiment of a blister chamber. [Figure 6] FIG. 6 is a close-up view of a portion of a substrate illustrating one embodiment of a medium valve chamber. [Figure 7] FIG. 7 is a close-up view of a portion of a substrate illustrating one embodiment of a medium reservoir. [Figure 8] FIG. 8 is a close-up view of a portion of a substrate illustrating one embodiment of a sample valve chamber and a surfactant chamber. [Figure 9] FIG. 9 is a close-up view of a portion of a substrate illustrating one embodiment of a back-channel reservoir. [Figure 10] FIG. 10 is a close-up view of a portion of a substrate illustrating one embodiment of a dome pump chamber. [Figure 11]FIG. 11 is a close-up view of a portion of a substrate illustrating one embodiment of a sample reservoir. [Figure 12] FIG. 12 schematically illustrates a microfluidic chip mounted on a chip carrier in a bottom view. [Figure 13] FIG. 13 illustrates schematically a microfluidic chip mounted on a chip carrier in a partial perspective view. [Figure 14] FIG. 14 is a close-up view of a portion of the microfluidic chip. [Figure 15] FIG. 15 is a close-up view of a portion of the blister chamber. [Figure 16] Figure 16 shows a close-up (top) and cross-sectional view (bottom) of the media reservoir. [Figure 17] FIG. 17 illustrates schematically the movement of sample and culture medium within a cartridge according to one embodiment. [Figure 18] 18-27. FIGS. 18-27 schematically illustrate a cartridge during a sample and culture medium filling operation as shown in FIG. [Figure 28] FIG. 28 illustrates a schematic of a microfluidic chip and fluids entering and exiting the inlet holes. [Figure 29] Figures 29-31. Figures 29-31 illustrate schematically the microfluidic chip during sample and culture medium filling operations. [Figure 32] FIG. 32 is a flowchart illustrating a method of analyzing a biological sample according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0021] Throughout the drawings, the same reference numbers are used for similar or corresponding elements.

[0022] The present invention relates to a cartridge for a microfluidic chip that can be used to analyze the phenotypic response of cells in a biological sample to various drugs. The cartridge is easy to use and can be pre-manufactured with the required culture medium, drugs, and chemicals, thereby requiring only the addition of a biological sample and allowing for fully automated analysis of the phenotypic response. The present invention also relates to a method for analyzing a biological sample.

[0023] As used herein, a "biological sample" includes any sample, preferably a fluid sample, more preferably a liquid sample containing cells or microorganisms, such as single-cell microorganisms, captured in the microfluidic chip of the cartridge and analyzed for its phenotypic response to one or more drugs. The biological sample is preferably a bodily fluid sample, such as a bodily fluid sample collected from a patient, including a processed bodily fluid sample collected from the patient. Non-limiting, illustrative examples of such bodily fluid samples include urine, blood, plasma, serum, amniotic fluid, cerebrospinal fluid, lymph, saliva, and spinal fluid. Alternatively, the biological sample may be a biological sample obtained from a solid bodily sample, such as a biopsy, in which the cells of the solid bodily sample are suspended or dispersed in a fluid, preferably a liquid such as a culture medium. As used herein, a processed bodily fluid sample is a bodily fluid sample that has been processed in some way before being loaded into the cartridge of the present invention. For example, the bodily fluid sample may be filtered or separated into different sample portions to obtain a processed bodily fluid sample, such as a serum or plasma sample obtained from blood.

[0024] As used herein, "drug" refers to any molecule, compound, composition, or other agent capable of affecting cells captured in a microfluidic chip. Typical, but non-limiting, examples of such agents include drugs or pharmaceuticals, including drug candidates. Agents do not necessarily have to be drugs or pharmaceuticals, but can be, for example, other chemicals, and it may be of interest to determine whether the agent has any effect on cells. Illustrative, but non-limiting, examples of such agents include antimicrobial agents, such as antibacterial, antifungal, antiviral, or antiparasitic agents. Specific examples include antibacterial agents, i.e., antibiotics, including, but not limited to, beta-lactams, cephalosporins, sulfonamides, aminoglycosides, chloramphenicol, tetracyclines, macrolides, glycoproteins, ansamycins, quinolones, streptogramins, oxazolidinones, and lipopeptides.

[0025] As used herein, a "cell" includes any cell type or population or mixture of cells or cell population that is present in a biological sample and can be captured in a microfluidic chip. The cell can be, for example, a pathogen that can cause a disease or disorder in a subject. Non-limiting, illustrative examples of such pathogens include bacteria, algae, fungi, protozoa, and other parasites. Alternatively, the cell can be a pathogenic host for a pathogen such as a viroid, virus, or prion. The cell does not necessarily have to be a pathogen, but can alternatively be any cell type or population to be captured and monitored, including microorganisms, particularly single-celled microorganisms.

[0026] In an illustrative example, the cartridge can be used for AST of bacteria in a biological sample, particularly a urine sample collected from a subject suffering from a urinary tract infection (UTI), using an antibiotic as an example of a drug. However, the cartridge of the present invention is not limited to the use of a urine sample as the biological sample. In fact, the cartridge can be used to load bacteria, or indeed any type of cell or microorganism, into a microfluidic chip for analysis, thereby allowing it to be used with other biological samples.

[0027] One aspect of the present invention relates to a cartridge 1 including a chip chamber 105 configured to house a microfluidic chip 500 including a plurality of sets 531 of cell channels 530 configured to capture cells from a biological sample. The cartridge 1 also includes a sample chamber 140 configured to receive the biological sample and to be in fluid communication with the plurality of sets 531 of cell channels 530. The cartridge 1 includes a plurality of medium reservoirs 170. Each medium reservoir 170 of the plurality of medium reservoirs 170 is configured to be in fluid communication with a respective set 531 of cell channels 530 of the plurality of sets 531 of cell channels 530. The cartridge 1 further includes a culture medium source 110, 400 in fluid communication with the plurality of medium reservoirs 170 and configured to supply culture medium to the plurality of medium reservoirs 170.

[0028] The cartridge 1 of the present invention thereby enables the capture of cells from a biological sample in multiple sets 531 of cell channels 530 in a microfluidic chip 500 housed within the chip chamber 105. The cells can then be manipulated, such as by exposing them to drugs at various concentrations and / or multiple different drugs, while monitoring the cells' response to the manipulation, such as the response of the cells to drugs at different concentrations and / or to different drugs, in parallel. Thus, a single source of cells, i.e., a biological sample, can be applied to the cartridge 1 to distribute the cells provided therein to the different sets 531 of cell channels 530.

[0029] The cartridge 1 also includes a plurality of medium reservoirs 170, such that each set 531 of cell channels 530 is fluidly connected to a respective medium reservoir 170. Thus, if the microfluidic chip 500 includes N sets of cell channels 530, the cartridge 1 includes N medium fluids 170. This means that each set 531 of cell channels 530 is fluidly connected to its own medium reservoir 170 from which it receives culture medium. In other words, each medium reservoir 170 of the plurality of medium reservoirs 170 only supplies culture medium to a single set 531 of cell channels 530 in the microfluidic chip 500, i.e., is only fluidly connected to a single set 531 of cell channels 530. The placement of the medium reservoirs 170 relative to the set 531 of cell channels 530 within the microfluidic chip 500 prevents mixing of culture media from different medium reservoirs 170 within the microfluidic chip 500, thereby preventing cells captured within the set 531 of cell channels 530 from being exposed to culture media from different medium reservoirs 170.

[0030] The cartridge 1 further includes a culture medium source 110, 400 in fluid communication with the plurality of medium reservoirs 170. The culture medium source 110, 400 supplies culture medium to the plurality of medium reservoirs 170. Thus, in a preferred embodiment, culture medium is supplied from the culture medium source 110, 400 and distributed to the plurality of culture reservoirs 170.

[0031] In one embodiment, multiple medium reservoirs 170 are arranged symmetrically with respect to the chip chamber 105 .

[0032] 4A, the chip chamber 105 is preferably provided between a plurality of medium reservoirs 170. Preferably, half of the medium reservoirs 170 are located on one side of the chip chamber 105, with the other half of the medium reservoirs 170 located on the other side of the chip chamber 105. Thus, the chip chamber 105 is preferably located between two sets or rows of medium reservoirs 170. This distribution of the medium reservoirs 170 relative to the central chip chamber 105 allows for the placement of a plurality of medium reservoirs 170 in a relatively small space within the cartridge 1.

[0033] In one embodiment, the multiple medium reservoirs 170 have substantially the same shape and / or internal volume.

[0034] In a currently preferred embodiment, the multiple medium reservoirs 170 have substantially the same internal volume and preferably substantially the same shape. In such a case, each medium reservoir 170 of the multiple medium reservoirs 170 is preferably filled with substantially the same volume of culture medium from the culture medium source 110, 400. Such a design of the medium reservoir 170 makes it possible to achieve a precise and controlled concentration of any agent dissolved or dispersed in the culture medium within the medium reservoir 170. For example, the medium reservoir 170 can be preloaded with a defined amount of the agent. Filling the medium reservoir 170 with a defined volume of culture medium provided from the culture medium source 110, 400 thereby enables the formation of a well-defined concentration of the agent dissolved or dispersed in the culture medium within the medium reservoir 170. Thus, precise and well-defined concentrations of one or more agents can be achieved in different medium reservoirs 170 when the different medium reservoirs 170 are preloaded with different amounts of an agent or agents and when the medium reservoirs 170 have substantially the same internal volume.

[0035] In one embodiment, at least some of the multiple media reservoirs 170 are pre-loaded with different amounts of drugs or different drugs configured to be dissolved or dispersed in the culture medium.

[0036] As described above, one or more drugs pre-loaded in the medium reservoir 170 are dissolved or dispersed in culture medium supplied from the culture medium source 110, 400 to the medium reservoir 170. The culture medium with the dissolved or dispersed drugs is then supplied from the medium reservoir 170 to its connected set 531 of cell channels 530 in the microfluidic chip 500, thereby exposing cells captured in the cell channels 530 of the set 531 of cell channels 530 to the drug dissolved or dispersed in the culture medium. The response of the cells in the cell channels 530 to the drug can then be monitored and determined.

[0037] Multiple media reservoirs 170 can be pre-loaded with different amounts of the same drug, thereby providing different concentrations of the drug in the culture medium contained within media reservoirs 170. Cells trapped in different sets 531 of cell channels 530 are thereby exposed to different concentrations of the drug, and the cellular responses to these different concentrations can be monitored.

[0038] Alternatively or additionally, different agents can be pre-loaded into different media reservoirs 170, thereby allowing the response of the captured cells to these different agents to be monitored.

[0039] In certain embodiments, a first set of the plurality of media reservoirs 170 are pre-loaded with different amounts of drugs or different agents, and a second set of the plurality of media reservoirs 170 are not pre-loaded with any drugs. In preferred embodiments, a first set of the plurality of media reservoirs 170 are pre-loaded with different amounts of drugs or different agents, and a remaining set of the plurality of media reservoirs 170 are not pre-loaded with any drugs.

[0040] For example, in a cartridge 1 having 32 different media reservoirs 170, five different drugs at five different concentrations can be pre-loaded into 25 of the 32 media reservoirs 170, with the remaining seven media reservoirs 170 being used as various controls, such as not pre-loaded with any drugs or pre-loaded with one or more control chemicals or drugs.

[0041] Thereby, the media reservoir 170 without any drug contains only culture medium. Such media reservoir 170 can be used as a control to enable monitoring of the response of cells captured within a connected set of cell channels 530 to the culture medium. In such a case, the response of the cells to one or more drugs can be determined relative to the response of the cells to the culture medium.

[0042] In one embodiment, the agent is an antibacterial agent. Currently preferred examples of antibacterial agents are antibiotics.

[0043] In one embodiment, each medium reservoir 170 of the plurality of medium reservoirs 170 includes two separate interconnected chambers 170A, 170B.

[0044] In one such embodiment, culture medium supplied from the culture medium source 110, 400 is preferably delivered to the first or rear chamber 170A of each of the medium reservoirs 170. The culture medium is then pushed from the first or rear chamber 170A to the second or front chamber 170B of each of the medium reservoirs 170. Such an approach allows for the metering of a clearly defined volume of culture medium into the first or rear chamber 170A and the subsequent delivery of this volume of culture medium to the second or front chamber 170B. In one such embodiment, any drug preloaded into the medium reservoir 170 is preferably preloaded into the second or front chamber 170B. If a relatively large amount of drug is required to preload the medium reservoir 170 and / or the drug requires a relatively long time to become dissolved or dispersed in the culture medium, the major portion of the drug is preferably preloaded into the second or front chamber 170B with an amount complementary to the drug preloaded into the first or rear chamber 170A.

[0045] In one embodiment, the cartridge 1 further includes a medium valve chamber 120. The medium valve chamber 120 includes an inlet hole 121 that is in fluid communication with a culture medium source 110, 400. The medium valve chamber 120 also includes a plurality of medium reservoir holes 123. Each of the plurality of medium reservoir holes 123 is in fluid communication with a respective medium reservoir 170 of the plurality of medium reservoirs 170. The cartridge 1 also includes a culture medium valve 420 configured to direct culture medium supplied from the culture medium source 110, 400 through the inlet hole 121 to the plurality of medium reservoir holes 123.

[0046] In certain embodiments, the culture medium valve 420 is configured to direct culture medium supplied from the culture medium source 110, 400 through the inlet hole 121 and through the plurality of medium reservoir holes 123 to the plurality of medium reservoirs 170.

[0047] The medium chamber 120 and culture medium valve 420 are configured to redirect the inflow of culture medium from the culture medium source 110, 400 through the inlet hole 121 to the outflow of culture medium through the plurality of medium reservoir holes 123 to the plurality of medium reservoirs 170.

[0048] In one embodiment, the culture medium valve 420 is configured to block the plurality of medium reservoir holes 123 when culture medium is supplied to the plurality of medium reservoirs 170. The culture medium valve 420 thereby prevents or limits any backflow of culture medium from one medium reservoir 170 into the medium chamber 120 and into another medium reservoir 170 of the plurality of medium reservoirs 170.

[0049] In one embodiment, the cartridge 1 includes a pump 410 configured to move the biological sample from the sample chamber 140 to the multiple sets 531 of cell channels 530 .

[0050] In one embodiment, the cartridge 1 includes a surfactant chamber 128 configured to be in fluid connection or communication with a plurality of sets 531 of cell channels 530 and a culture medium source 110, 400. In such an embodiment, the surfactant chamber 128 is pre-loaded with a surfactant configured to be dissolved or dispersed in the culture medium provided by the culture medium source 110, 400. The surfactant dissolved or dispersed in the culture medium is configured to be provided to the plurality of sets 531 of cell channels 530.

[0051] In certain embodiments, the surfactant chamber 128 is pre-loaded with a surfactant configured to be dissolved or dispersed in the culture medium provided from the culture medium source 110, 400. The surfactant dissolved or dispersed in the culture medium is configured to be provided to the multiple sets 531 of cell channels 530 prior to the biological sample.

[0052] In one such embodiment, the cell channel 530 and any fluid channels of the microfluidic chip 500 are preferably wetted with a surfactant-supplemented culture medium prior to loading the biological sample into the microfluidic chip 500. The surfactant preferably coats the inner surfaces of the cell channel 530 and any other fluid channels of the microfluidic chip 500, thereby facilitating loading of the biological sample into the microfluidic chip 500. Such wetting thereby reduces any pressure required to push the biological sample into the microfluidic chip 500.

[0053] In one embodiment, the culture medium source 110, 400 includes a culture medium container 400 pre-packaged with culture medium. The culture medium source 100, 400 also includes a chamber 110 configured to accommodate the culture medium container 400. The chamber 110 includes a drain hole 113 in fluid connection or communication with a plurality of medium reservoirs 170. The chamber 110 also includes a cutter 114 configured to cut a surface of the culture medium container 400 to provide culture medium to the drain hole 113.

[0054] In certain embodiments, the culture medium container 400 is pressed by application of fluid pressure, such as gas pressure and preferably air pressure, onto the culture medium container 400, pressing the culture medium container 400 onto the cutter 114, thereby cutting the surface of the culture medium container 400.

[0055] In one embodiment, cartridge 1 includes a chip carrier 550 attached to microfluidic chip 500. Chip carrier 550 includes a first set of holes 554 configured to provide fluid connection between multiple sets 531 of cell channels 530 and sample chamber 140. Chip carrier 550 also includes a second set of holes 558. Each hole 558 in second set of holes 558 is configured to provide fluid connection between a respective set 531 of cell channels 530 in multiple sets 531 of cell channels 530 and a respective medium reservoir 170 in multiple medium reservoirs 170.

[0056] In certain embodiments, each set 531 of cell channels 530 of the multiple sets 531 of cell channels 530 includes a first port 514 in fluid communication with holes 554 of a first set of holes 554 and a second port 512 in fluid communication with holes 558 of a second set of holes 558.

[0057] In one embodiment, cartridge 1 further includes a substrate 100 that includes a chip chamber 105, a sample chamber 140, a plurality of media reservoirs 170, and a culture media source 110, 400. Cartridge 1 also includes a lid 200 attached to substrate 100 and includes a window 210 configured to allow imaging of a plurality of sets 531 of cell channels 530.

[0058] In one embodiment, each set 531 of cell channels 530 of the multiple sets 531 of cell channels 530 is formed as a compartment within the microfluidic chip 500 that is separated from other sets 531 of cell channels 530 of the multiple sets 531 of cell channels 530.

[0059] The cartridge and its contained components will now be described in more detail with reference to the accompanying drawings, followed by a description of a typical use of the cartridge.

[0060] 1A and 1B are exploded views of one embodiment of cartridge 1 from above (FIG. 1A) and below (FIG. 1B). Cartridge 1 typically includes a microfluidic chip 500 attached to or coupled with a chip carrier 550 and a substrate 100, also referred to as a cartridge substrate, designed to accommodate media blisters 400 containing culture media for cells captured within microfluidic chip 500. Cartridge 1 also includes valves 420, 430, which function as check valves for the culture media and biological sample, respectively, and a dome-shaped pump 410 for accurate metering of the biological sample volume. A sample filter 630 is preferably included in cartridge 1 to filter the biological sample before loading it into microfluidic chip 500. Cartridge 1 further includes multiple gas-permeable membranes 600, 610 that are permeable to gases such as air but impermeable to liquids at operating pressures. Cartridge 1 optionally includes a membrane compressor 620 aligned with gas-permeable membrane 600 and maintaining gas-permeable membrane 600 in sealing connection with substrate 100. Top lid 200 is designed to attach to the upper side or top surface 102 of substrate 100, with a corresponding back lid 300 designed to attach to the lower or bottom surface 103 of substrate 100. Cap 235 is designed to fit onto top lid 200 to close the sample chamber within substrate 100 once a biological sample is added to the sample chamber.

[0061] The cartridge 1 can be assembled according to various embodiments. In an exemplary assembly embodiment, as shown in FIG. 2A , the sample filter 630 is inserted, preferably pressed, into a filter chamber or well 146 having a matching opening in the bottom surface 103 of the substrate 100. As shown in FIG. 2B , the back cover 300 is then attached to the bottom surface 103 of the substrate 100, for example, by welding the back cover 300 to the substrate 100, for example, by laser welding. As suggested in FIG. 2B , optional guide pins on the fixture can be used to align the back cover 300 and the substrate 100. The figure also shows that the back cover 300 includes openings or windows 310 configured to align with corresponding openings 104 in the substrate 100. These openings 104, 310 are, in turn, configured to align, during use, with a portion of the microfluidic chip 500 that includes a cell channel 530 in which cells present in a biological sample are captured and cultured. Thus, the openings 104, 310 in the substrate 100 and the back cover 300, respectively, allow visual access to a portion of the microfluidic chip 500 as disposed within the chip chamber 105, in particular to the set 531 of cell channels 530 within the microfluidic chip 500.

[0062] As shown in FIG. 2C , the microfluidic chip 500 is attached to the chip carrier 550, for example, by bonding the microfluidic chip 500 to the chip carrier 550. As suggested in FIG. 2C , optional guide pins on a fixture can be used to align the microfluidic chip 500 and the chip carrier 550. As suggested in FIG. 2D , the chip carrier 550 with the attached microfluidic chip 500 is then inserted into a matching chip chamber or well 105 in the substrate 100. The chip carrier 550 is preferably attached to the substrate 100, for example, by welding the chip carrier 550 to the substrate 100, preferably by laser welding. As shown in FIG. 2D , optional guide pins on a fixture can be used to align the chip carrier 550 and the substrate 100.

[0063] As shown in FIG. 2E, dome-shaped pump 410, culture medium valve 420, and sample valve 430 are then inserted into each of the chambers or wells 120, 150, 180 in substrate 100. Dome-shaped pump 410 and valves 420, 430 are preferably welded, e.g., laser welded, to substrate 100, e.g., by applying welding, preferably laser welding, along the circumference of each of dome-shaped pump 410 and valves 420, 430 and along the bottom surfaces of chambers or wells 120, 150, 180. FIG. 2F schematically illustrates the insertion of medium blister 400 into blister chambers or wells 110 in substrate 100. Medium blister 400 is preferably attached to substrate 100 by adhesive, e.g., adhesive tape, applied along the periphery of the bottom surface of medium blister 400, e.g., to at least a portion of the bottom surface of medium blister 400 and / or at least a portion of the bottom surface 112 of blister chamber or well 110. As shown in FIG. 2G, a gas-permeable membrane 600 is then placed within each membrane chamber or well 106 in the substrate 100. In an optional embodiment, a membrane compressor 620 can be used to hermetically attach the gas-permeable membrane 600 within the membrane chamber 106. Other techniques for securing the gas-permeable membrane 600 within the membrane chamber 106 can be used instead, such as welding or gluing. A gas-permeable membrane 610 is inserted into each pressure port 108A to 108E in the substrate 100. Finally, as shown in FIG. 2H, the top cover 200 is attached to the top surface 102 of the substrate 100, for example, by welding, preferably laser welding, the top cover 200 to the substrate 100. As suggested in FIG. 2H, optional guide pins on a fixture can be used to align the top cover 200 and the substrate 100. The figure also shows schematically that top cover 200 includes multiple openings or windows 210, 220, 240, 250 configured to align with microfluidic chip 500, dome pump 410, culture medium valve 420, and sample valve 430, respectively. Top cover 200 also preferably includes a raised portion that serves as a blister cover 260 for medium blister 400.

[0064] The order in which the different components of cartridge 1 are assembled may differ from the order described above and shown in Figures 2A to 2H.

[0065] The cartridge 1 of the present invention can be easily handled by the user because all components of the cartridge 1 can be assembled during manufacturing and then provided as a cartridge 1 having components surrounded by a base 100 and a top cover 200 and a back cover 300.

[0066] In actual use, a user adds a biological sample to a sample chamber or well 140 in the substrate 100 through a sample entry port 230 in the top cover 200, as shown in FIG. 3A (see also FIGS. 17 and 18). In one embodiment, a predetermined volume of the biological sample is preferably added to the sample chamber 140 through the sample entry port 230. In a preferred embodiment, a cap or lid 235 is attached to the sample entry port 230 in the top cover 200, enclosing the biological sample within the sample chamber or well 140, as shown in FIG. 3B. The cartridge 1 with the biological sample can then be inserted into an instrument (not shown) configured to load the biological sample into a microfluidic chip 500 and capture cells present in the biological sample within the microfluidic chip 500. The instrument is also preferably configured to expose the cells captured in the microfluidic chip 500 to one or more agents, preferably pre-loaded within the substrate 100, as further described herein. The response, preferably the phenotypic response, of the cells in the microfluidic chip 500 to one or more agents can then be monitored and analyzed by an instrument.

[0067] Substrate 100 optionally, but preferably, includes a grip or handle 101 that facilitates manual handling of cartridge 1, such as when inserting and removing cartridge 1 from an instrument.

[0068] The device is preferably configured to operate by applying pressurized liquid, preferably pressurized gas, more preferably pressurized air, to pressure ports 108A through 108H in substrate 100 (see FIG. 4A). Application of pressurized fluid to these pressure ports 108A through 108H is used to open media blisters and transport culture media across substrate 100 and toward microfluidic chip 500, and transport biological samples across substrate 100 and toward microfluidic chip 500, where it captures any cells present in the biological sample.

[0069] As shown schematically in Figures 4A, 4B, 5, and 19, a medium blister 400 is provided within a blister chamber or well 110, also referred to herein as a blister pocket. In a preferred embodiment, the blister chamber 110 includes a central depression 111 and a surrounding, substantially flat chamber bottom 112. In such a case, the medium blister 400 is preferably attached to the surrounding, flat chamber bottom 112 by welding, e.g., laser welding, or by an adhesive, such as adhesive tape, applied along a peripheral portion of the bottom surface of the medium blister 400. The central depression 111 includes a drainage hole 113 and one or more cutters 114. The central depression 111 is sufficiently deep so that the cutters 114 are positioned away from the bottom surface of the medium blister 400 when the medium blister 400 is placed within the blister chamber 110.

[0070] Media blister 400 contains the culture medium that is transported to microfluidic chip 500 and used therein for culturing cells captured within microfluidic chip 500 from a biological sample. Thus, the type of culture medium contained within media blister 400 is preferably selected based on the type of cells being captured.

[0071] Media blister 400 can be manufactured from a variety of materials, including metal and / or plastic. A typical example of media blister 400 is a plastic-coated aluminum blister. The bottom of media blister 400 is preferably manufactured from a thin foil configured to be pierced by cutter 114. An illustrative, but non-limiting, example of such a foil is aluminum foil.

[0072] The medium blister 400 is opened by applying fluid pressure, preferably gas pressure, at pressure port 108E, which is in fluid communication with blister chamber 110. More specifically, pressurized fluid, preferably pressurized gas, more preferably pressurized air, is introduced by an instrument into pressure port 108E and flows into blister chamber 110 via pressure channel 116, which interconnects pressure port 108E and blister chamber 110 (see FIG. 5 ). The pressurized fluid is introduced into blister chamber 110 between top cover 200 and the top surface of medium blister 400. The pressurized fluid thereby pressurizes medium blister 400 downward toward central recess 111, causing cutter 114 to engage and pierce the bottom of medium blister 400. The culture medium contained within the open medium blister 400 is forced through the drain hole 113 by the applied pressure and transported into the channel 115 in the bottom surface 103 of the substrate 100 toward the inlet hole 121 in the medium valve chamber or well 120, also referred to herein as the medium valve pocket (see Figures 4B, 6, 17 and 21).

[0073] 15 is a close-up view of a portion of blister chamber 110 showing one embodiment of drain hole 113 and cutter 114. In this embodiment, cutter 114 preferably includes a recess or channel 118 in a side 117 of cutter 114 facing drain hole 113. This recess or channel 118 guides culture medium from the opened medium blister 400 to drain hole 113. Thus, more efficient emptying of medium blister 400 and pushing of culture medium to drain hole 113 is achieved when cutter 114 includes such a guide or drain recess or channel 118.

[0074] Another feature of cutter 114 that facilitates the evacuation of culture medium into drain hole 113 is that cutter 114 has a curved side 117 that faces drain hole 113. As shown schematically in FIG. 15 , side 117 that faces drain hole 113 is slightly curved or arced around a portion of drain hole 113. This shape of side 117 promotes the flow of culture medium from open medium blisters 118 along side 117 of cutter 114 down into drain hole 113.

[0075] 4A, 4B, 6, and 21, medium valve chamber 120 is designed to distribute culture medium entering inlet hole 121 to multiple medium reservoirs 170 and rear channel reservoirs 130, and to prevent backflow of culture medium through substrate 100. The bottom of medium valve chamber 120 includes central inlet hole 121 and rear channel hole 122, and multiple medium reservoir holes 123 arranged circumferentially around inlet hole 121. Medium valve chamber 120 preferably includes one medium reservoir hole 123 for each medium reservoir 170 in substrate 100.

[0076] A peripheral or circumferential portion 124 of the bottom surface of the medium valve chamber 120 is preferably flat, allowing the culture medium valve 420 to be attached to this peripheral portion 124 of the bottom surface, for example, by welding, preferably by laser welding. The culture medium valve 420 is preferably in the shape of a disk made of a substantially flexible material, such as a thermoplastic elastomer (TPE). When culture medium enters the inlet hole 121, the culture medium is forced between the bottom surface of the medium valve chamber 120 and the culture medium valve 420, which is bent upward by pressure applied to the pressure port 108E. The culture medium is further forced into the medium reservoir hole 123 and the rear channel hole 122.

[0077] In one embodiment, redirection of culture medium from inlet hole 121 to medium reservoir hole 123 and rear channel hole 122 is facilitated by applying low pressure (approximately 2 bar) to the top side of culture medium valve 420, i.e., between culture medium valve 420 and top cover 200. This low pressure is applied by an instrument through pressure port 108G and pressure channel 129A to bottom surface 103 of substrate 100 and pressure inlet 129B of medium valve chamber 120 (see FIGS. 4A and 4B).

[0078] In one embodiment, the bottom surface of medium valve chamber 120 includes at least one recessed circular channel 129 connected to rear channel hole 122. This at least one recessed circular channel 129 guides the culture medium into rear channel hole 122 and facilitates the culture medium exiting medium valve chamber 120 (see FIG. 6).

[0079] In one embodiment, culture medium is first transported from medium valve chamber 120 to medium reservoirs 170, filling each medium reservoir 170 with an equal, defined volume of culture medium before transporting the culture medium to rear channel reservoirs 130 (see FIGS. 7, 17, and 21). This sequential filling of rear channel reservoirs 130 after filling medium reservoirs 170 can be controlled by applying fluid pressure, preferably gas pressure, and more preferably air pressure, to pressure port 108A, which is in fluid communication with rear channel reservoirs 130. Initially, the pressure applied to pressure port 108A means that the culture medium is exposed to a higher flow resistance in the channel interconnecting rear channel holes 122 with rear channel reservoirs 130 compared to any flow resistance in the channel interconnecting each medium reservoir hole 123 with medium reservoir 170.

[0080] The medium valve chamber 120 with the culture medium valve 420 provides several functions during the filling process of the substrate 100. Initially, the medium valve chamber 120 and the culture medium valve 420 operate as a 1-to-(N+1) valve, where N represents the number of medium reservoir holes 123 and the number of medium reservoirs 170 in the substrate 100. In other words, the medium valve chamber 120 redirects the inflow of culture medium through the inlet hole 121 to the N medium reservoir holes 123 and rear channel hole 122.

[0081] Culture medium valve 420 further operates as a check valve, preventing the flow of culture medium from medium reservoir 170 and / or rear channel reservoir 130 back into medium valve chamber 120 upon application of fluid pressure, preferably gas pressure, more preferably air pressure, above culture medium valve 420. This pressure forces culture medium valve 420 toward the bottom of medium valve chamber 120, thereby closing holes 121, 122, 123 in the bottom of medium valve chamber 120. As a result, holes 121, 122, 123 become sealed, thereby preventing any undesired crosstalk between medium reservoir 170 and rear channel reservoir 130. This is important to prevent culture medium from entering one medium reservoir 170, which may contain one agent from mixing with another agent or culture medium entering another medium reservoir, which may contain the same agent but in a different amount.

[0082] Closure of the medium valve chamber 120 using the culture medium valve 420 is achieved by applying pressure at pressure port 108G, causing pressurized fluid, preferably pressurized gas, more preferably pressurized air, to be forced through pressure channel 129A and into the medium valve chamber 120 through pressure inlet 129B (see Figures 4A, 4B and 6).

[0083] 4A, 4B and 7, each medium reservoir hole 123 is connected to a respective inlet hole 171A, 171B of medium reservoir 170 through a respective medium channel 126 disposed in bottom surface 103 of substrate 100. Culture medium is pushed through medium channels 126 and enters medium reservoir 170 through these inlet holes 171A (see also FIG. 21).

[0084] The medium reservoirs 170 are designed to accommodate a predetermined volume of culture medium, and in particular so that each medium reservoir 170 of the plurality of medium reservoirs 170 contains the same, or at least substantially the same, predetermined volume of culture medium. The medium reservoirs 170 are therefore designed to facilitate the removal of air inside the medium reservoirs 170 during filling, thereby preventing or at least greatly reducing the risk of trapping any air bubbles within the medium reservoirs 170 that, if present, would prevent the medium reservoirs 170 from being filled with the predetermined volume of culture medium.

[0085] The media reservoirs 170 are further designed to be pre-loaded with drugs to be mixed with the culture medium, thereby dissolving or dispersing the drugs in the culture medium. By having multiple different media reservoirs 170, it is possible to include different drugs in different media reservoirs 170 and / or include the same drug in different amounts in different media reservoirs 170, thereby achieving different concentrations of the drug when dissolved or dispersed in a given volume of culture medium. For example, as shown schematically in FIG. 4A, a substrate 100 having 32 different media reservoirs 170 can have five different drugs pre-loaded in 25 of the 32 media reservoirs 170 at five different concentrations, with the remaining seven media reservoirs 170 used as various controls, such as not pre-loaded with any drugs or pre-loaded with one or more control chemicals or drugs.

[0086] In one embodiment, each medium reservoir 170 includes two separate but interconnected chambers or wells, herein designated rear chamber or well 170A and front chamber or well 170B. As can be seen more clearly in FIG. 4B , each medium channel 126 from the medium valve chamber 120 is preferably connected to two inlet holes 171A, 171B, one to the rear chamber 170A and one to the front chamber 170B. The front chamber 170B is fluidly connected to the microfluidic chip 500, thereby exerting a higher flow resistance on the culture medium in the medium channel 126 than the flow resistance exerted by the rear chamber 170A due to the minute dimensions of the microfluidic channels in the microfluidic chip 500. This difference in flow resistance causes culture medium flowing in the medium channel 126 to be forced into the inlet hole 171A of the rear chamber 170A but not into the inlet hole 171B of the front chamber 170B.

[0087] Rear chamber 170A of medium reservoir 170 preferably includes a pillar chamber or well 172A with an inlet hole 171A. Rear chamber 170A also includes a rear chamber or well portion 174A with a waist 173A or narrow passage between pillar chamber 172A and rear chamber portion 174A. Waist 173A restricts the flow of culture medium within rear chamber 170A, so that when culture medium enters through inlet hole 171A, it first fills pillar chamber 172A before flowing through waist 173A into rear chamber portion 174A. This initially fills the rear chamber 170A, creating a pillar of culture medium within pillar chamber 172A before continuing to fill rear chamber portion 174A. This sequential filling of rear chamber 170A in two stages facilitates the evacuation of any air present in rear chamber 170A from pillar chamber 172A to rear chamber portion 174A and, together with channels 176 disposed at bottom surface 103 of substrate 100, to passages 175 interconnecting rear chamber 170A with membrane chamber 106. Thus, the risk of unintentional trapping of small air bubbles in rear chamber 170A during the filling process is minimized, thereby enabling accurate filling of rear chamber 170A with a predetermined volume of culture medium.

[0088] When pillar chamber 172A is filled with culture medium, the culture medium is pushed through waist 173A into rear chamber portion 174A. When pillar chamber 172A and rear chamber portion 174A are both filled with culture medium, any excess culture medium is pushed over partition or wall 270 between rear chamber portion 174A and passage 175 and into channel 271 interconnecting rear chamber portion 174A and passage 175 (see FIG. 16 ). The excess culture medium is then pushed further through passage 175 and channel 176 toward membrane chamber 106.

[0089] Each membrane chamber 106 includes a gas-permeable membrane 600 configured to allow gases, such as air, to pass through the gas-permeable membrane 600 but restrict the passage of culture medium therethrough. Thus, during filling with culture medium, air present in rear chamber 170A is forced through gas-permeable membrane 600 and through pressure ports 108B, 108D, which are fluidly connected to membrane chamber 106. This means that any air present in rear chamber 170A is expelled from it during the filling process. Once rear chamber 170A is filled with culture medium, any excess culture medium is forced through passage 175 and channel 176 into membrane chamber 106. However, when the culture medium contacts gas-permeable membrane 600 present in membrane chamber 106, further flow is prevented, and the filling process of rear chamber 170A is completed. Thus, gas-permeable membrane 106 exerts a counterforce on the culture medium that is greater than the force caused by applying pressure to culture medium valve 420 in medium valve chamber 120. Gas permeable membrane 600 also prevents culture medium in one rear chamber 170A from passing through membrane chamber 106 and passages 175 and channels 176 into another rear chamber 170A.

[0090] When rear chamber 170A within medium reservoir 170 is filled with a predetermined volume of culture medium, the counterpressure applied to rear channel reservoir 130 via pressure port 108A is released (see FIG. 17). This causes the remaining culture medium to be pushed from medium valve chamber 120 through rear channel hole 122 and medium channel 125 in bottom surface 103 of substrate 100 (see FIG. 4B). Additionally, once medium reservoir 170 is filled, a certain volume of culture medium may still be present in medium blister 400. This volume of culture medium is then expelled through medium valve chamber 120 into rear channel reservoir 130.

[0091] 4A, 4B, 8, 17, and 22, the culture medium passes through a surfactant chamber or well 128 before reaching the rear channel reservoir 130. This surfactant chamber 128 is preferably pre-loaded with a surfactant that is mixed and dissolved with the culture medium within the surfactant chamber 128. The surfactant is preferably dissolved or at least dispersed in the culture medium and is used to wet the microfluidic channels within the microfluidic chip 500 prior to transporting the biological sample to the microfluidic chip 500.

[0092] Any surfactant that can be dissolved or at least dispersed in culture medium and that can wet the microfluidic channels in the microfluidic chip 500 can be used in accordance with the present invention and can be pre-loaded into the surfactant well 128. An illustrative, but non-limiting, example of a surfactant is a non-ionic surfactant such as poloxamer. Poloxamers are triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Examples of poloxamers that can be used as surfactants include those sold under the trademark PLURONIC®, e.g., PLURONIC® F108.

[0093] The surfactant chamber 128 is preferably designed with an inlet hole 127A and an outlet hole 127B positioned at the top of the surfactant chamber 128, such that the culture medium entering the surfactant chamber 128 is forced to enter from the medium channel 125 through the inlet hole 127A and contact the pre-loaded surfactant at the bottom of the surfactant chamber 128 before exiting the surfactant chamber 128 through the outlet hole 127B. This allows the surfactant to be dissolved or dispersed in the culture medium before exiting the surfactant chamber 128. The culture medium with surfactant is then transported through the channel system 127C, 127D to the bottom surface 103 of the cartridge 103 and into the first channel 551 in the carrier chip 550 before reaching the inlet hole 131 to the rear channel reservoir 130.

[0094] 4A, 4B, and 9, rear channel reservoir 130 is designed in a similar manner to rear chamber 170A of medium reservoir 170, with inlet hole 131 leading to pillar chamber or well 132. Pillar chamber 132 is connected to rear chamber or well 134 through narrow waist 133. Rear chamber 134 is also connected to rear channel waste 136 through passage 135.

[0095] The culture medium with surfactant enters rear channel reservoir 130 through inlet hole 131 and initially fills pillar chamber 132 while expelling air through waist 133 into rear chamber 134 and passage 135 and into rear channel waste 136. Rear channel waist 136 is fluidly connected to pressure port 108A, allowing air to enter rear channel waste 136 and exit through pressure port 108A. Once the culture medium with surfactant forms a pillar and completely fills pillar chamber 132, the culture medium with surfactant is pushed through waist 133 into rear chamber 134. Any excess culture medium with surfactant also enters rear channel waste 136 through passage 135 when rear chamber 134 is filled. Thus, rear channel waste 136 is designed to have a volume capable of accommodating all excess culture medium with surfactant that is pushed into rear channel reservoir 130.

[0096] Substrate 100 preferably includes a dome pump chamber or well 180 configured to contain a dome pump 410 (see FIGS. 4A, 4B, and 10). Dome pump 410 is attached to the bottom surface of dome pump chamber 180, for example, by welding, preferably laser welding, a circumferential portion of dome pump 410 to a peripheral portion 181 of the bottom surface of dome pump chamber 180. When attached to the bottom surface of dome pump chamber 180, dome pump 410 encloses a defined volume of air.

[0097] When pressure is applied to the medium blister 400 in the medium chamber 110 through pressure port 108E, pressure is preferably simultaneously applied to the top of the dome-shaped pump 410 through pressure port 108H via pressure channel 187 and pressure inlet 183 in the dome pump chamber 180 (see FIG. 17 ). The applied pressure pushes the dome-shaped pump 410 downward toward the bottom surface of the dome pump chamber 180. The volume of air contained by the dome-shaped pump 410 in the dome pump chamber 180 is then forced through a drain hole 182, which is preferably located in the center of the dome pump chamber 180. The bottom surface optionally includes a coiled recess 188 for guiding the compressed air to the drain hole 182.

[0098] Drain hole 182 is fluidly connected to inlet hole 185 in sample chamber or well 140 via air channel 184 in the bottom surface 103 of substrate 100. A predetermined volume of air entering sample chamber 140 through inlet hole 185 and air passage 186 in the top surface 102 of substrate 100 pushes a controlled volume of biological sample from sample reservoir 140 through drain hole 141 and a sample transport system toward filter chamber or well 146, also referred to herein as a filter pocket. In one embodiment, the transport system includes channel 142 in bottom surface 103 of substrate 100, channel 143 from bottom surface 103 to top surface 102 of substrate 100, and channel 144 in top surface 102 of substrate 100. The biological sample is thereby pushed into inlet hole 145, into filter chamber 146, and through a sample filter 630 disposed within filter chamber 146 (see also FIGS. 17 and 20). A sample filter 630 is disposed within the filter chamber 146 and filters out any large debris, dust, or dirt present in the biological sample, while allowing any cells present in the biological sample to pass through the sample filter 630. The filtered biological sample is further transported through a channel 147 in the bottom surface 103 of the substrate 100 to an inlet hole 148 of a sample valve chamber or well 150, also referred to herein as a sample valve pocket (see also FIG. 20).

[0099] 4A, 4B, and 8, sample valve chamber 150 includes a sample valve 430 that is attached to sample valve chamber 150, preferably by welding a circumferential portion of sample valve 430 to a peripheral portion of the bottom surface of sample valve chamber 150, e.g., by laser welding. Sample valve 430 is fabricated from a flexible material, such as in the form of a TPE disk, and functions as a check valve. In addition to inlet hole 148, sample valve chamber 150 includes drain hole 151 in the bottom surface of sample valve chamber 150. Filtered biological sample entering the valve chamber through inlet hole 148 is further pushed through drain hole 151 and transported by a transport system to sample reservoir 160. In one embodiment, the transport system includes a first T-shaped channel 152 disposed on the bottom surface 103 of the substrate 100, a second channel 553 in the chip carrier 550, and a second T-shaped channel 153 disposed on the bottom surface 103 of the substrate 100 and entering the sample reservoir 160 at an inlet hole 154 (see Figures 17 and 20).

[0100] 4A, 4B, and 11, sample reservoir 160 is designed similarly to rear channel reservoir 130, with inlet hole 154 in pillar chamber or well 161 initially filled with filtered biological sample, while venting air through waist 162 to rear chamber 163 and passageway 164 and into sample waste 165. Sample waste 165 is fluidly connected to pressure port 108F, allowing air to enter sample waste 165 and exit through pressure port 108F. Once the filtered biological sample forms a pillar and completely fills pillar chamber 161, the filtered biological sample is pushed through waist 162 into rear chamber 163. When rear chamber 163 is also filled, any excess filtered biological sample enters sample waste 165 through passageway 164. Thus, sample waste 165 is designed to have a volume capable of accommodating all excess filtered biological sample pushed into sample reservoir 160.

[0101] As previously discussed herein, sample valve chamber 150 is fluidly connected to medium valve chamber 120 via passage 155 between chambers 120, 150. Thus, when pressure is applied to medium valve chamber 120, urging culture medium valve 420 therein toward the bottom of medium valve chamber 120 and preventing backflow of culture medium from back channel reservoir 130 and medium reservoir 170 toward medium valve chamber 120, pressure is also applied to sample valve chamber 150, urging sample valve 430 therein toward the bottom of sample valve chamber 150. Sample valve 430 thereby prevents backflow of filtered biological sample from sample reservoir 160 toward sample valve chamber 150.

[0102] Referring to Figures 4A, 4B and 7, a predetermined volume of culture medium contained in rear chamber 170A and passage 175 and channel 176 is then forced through inlet hole 171A in rear chamber 170A and into inlet hole 171B in front chamber 170B by application of excess pressure to membrane chamber 106 through pressure ports 108B, 108D (see Figures 17 and 23).

[0103] In one embodiment, anterior chamber 170B is designed in a similar manner to posterior chamber 170A, having pillar chambers or wells 172B, waist 173B, and anterior chamber portion 174B. Culture medium enters anterior chamber 170B through inlet hole 171B and begins to fill pillar chamber 172B while evacuating air through waist 173B, anterior chamber portion 174B, and channels 178 on the bottom surface 103 of substrate 100 and draining through drain hole 177 connected to microfluidic chip 500. Culture medium first fills pillar chamber 172B, which forms a pillar, and then flows into anterior chamber portion 174B through waist 173B.

[0104] The internal volume of the anterior chamber 170B is preferably approximately equal to, and more preferably smaller than, the internal volume of the posterior chamber 170A, which means that the anterior chamber 170B can be completely filled with culture medium before the posterior chamber 170A is filled.

[0105] As previously disclosed herein, at least some of the multiple media reservoirs 170 contain one or more drugs. In one embodiment, the drug is preloaded into the front chamber 170B of the media reservoir 170, preferably into the front chamber portion 174B of the front chamber 170B. In another embodiment, the drug is preloaded into the rear chamber 170A of the media reservoir 170, preferably into the rear chamber portion 174A of the rear chamber 170A. It is also possible to preload a portion of the drug into the front chamber 170B, e.g., into the front chamber portion 174B, and the remaining portion of the drug into the rear chamber 170A, e.g., into the rear chamber portion 174A. Preloading a drug into the front chamber 170B of the media reservoir 170 is generally preferred. However, some drugs require a relatively long period of time to dissolve or at least disperse in the culture medium. In such cases, it may be advantageous to preload all or at least a portion of the drug into the rear chamber 170A, thereby extending the time the drug is in contact with the culture medium. Thus, in one embodiment, a drug is pre-loaded in the front chamber 170B, eg, in the front chamber portion 174B, and optionally a supplemental or additional drug is pre-loaded in the rear chamber 170A, eg, in the rear chamber portion 174A.

[0106] In one embodiment, the antechamber 170B includes a mixing device 273 configured to facilitate mixing of any pre-loaded drugs and culture medium within the antechamber 170B to obtain culture medium with a substantially homogenous drug concentration (see FIG. 16 ). In one embodiment, this mixing device 273 or function is achieved by having a step 273 disposed between the drain hole 177 and the antechamber portion 174A. This means that the bottom 272 of the antechamber portion 174A is lowered relative to this step 273 and relative to the pillar chamber 172B. Thus, as shown in FIG. 16 , there is a depression within the antechamber portion 174A into which drugs can be loaded. When culture medium is pushed into the antechamber 170B, the step 273 creates turbulence inside the antechamber portion 174B, facilitating efficient mixing of the drugs and culture medium pre-loaded at the bottom 272.

[0107] At this point, the filling operation of cartridge 1 is completed with the filtered biological sample present in sample reservoir 160, the culture medium with surfactant in media reservoir 170 including the rear channel reservoir 130 and the culture medium in anterior chamber 170B, the culture medium in at least a portion of these anterior chambers 170B including a dissolved or dispersed drug.

[0108] 12-14 and 28 illustrate the microfluidic chip 500 and chip carrier 550 in detail. The chip carrier 550 includes a matrix or array of inlet holes 552, 554, 556, 558 that provide access to the microfluidic chip 500. The matrix preferably includes four columns of holes and N rows of holes. The holes 552 in the first column 550A, closest to a central window 559 in the chip carrier 550, are preferably interconnected via a first channel 551 that runs along the bottom surface of the chip carrier 550. This first channel 551, in turn, is fluidly connected to the rear channel reservoir 130 via T-shaped channel 127D and to the media valve chamber 120 via the transport system 127. The holes 554 in the second column 550B are also preferably interconnected via a second channel 553 in the bottom surface of the chip carrier 550. This second channel 553 is fluidly connected to the sample reservoir 160 via T-channel 153 and to the sample valve chamber 150 via T-channel 152. The holes 556 in the third row 550C are preferably interconnected via channels 555 in the bottom surface of the chip carrier 550. This channel is in turn fluidly connected to the waste chamber 190 via channels 191 in the bottom surface 103 of the substrate 100. The holes 558 in the outermost fourth row 550D are not interconnected to each other. In sharp contrast, each hole 558 in this fourth row is connected to a respective medium reservoir 170 via a channel 178.

[0109] Each of the N rows of four holes 552, 554, 556, 558 is fluidly connected to ports 512, 514, 522 of a set 531 of cell channels 530 (see FIGS. 14, 29-31). Thus, the microfluidic chip 500 includes a plurality of such sets 531 of cell channels 530 configured to capture cells present in a biological sample. Each set 531 of cell channels 530 includes an input channel 510 having first and second input ports 512, 514, respectively, and an output channel 520 having an output port 522 at either end of the input channel 510. The cell channels 530 are then preferably arranged in parallel between the input channel 510 and the output channel 520. Thus, each cell channel 530 in a set has a first end 532 fluidly connected to the input channel 510 and a second end 534 fluidly connected to the output channel 520. At least a portion of the cell channel 530 further includes a cell block 535 configured to block cells entering the cell channel 530 from the input channel 510 from exiting the cell channel 530 and entering the output channel 520. The cell block 535 thus traps and captures any cells that enter the cell channel 530. Detailed information on the design of the cell channel 530 and the set 531 of input and output channels 510, 520 can be found in U.S. Patent No. 10,041,104, which is incorporated herein by reference.

[0110] The holes 552 in the first row 550A are fluidly connected to the output port 522 of the output channel 520 (see Figure 29), the holes 554 in the second row 550B are fluidly connected to the second input port 514 at one end of the input channel 510 (see Figure 30), while the holes 556, 558 in the third and fourth rows 550C, 550D are fluidly connected to the first input port 512 at the other end of the input channel 510 (see Figure 31).

[0111] Before loading the filtered biological sample into the microfluidic chip 500, the microfluidic channels of the microfluidic chip 500 are preferably wetted to remove any air trapped in these microfluidic channels (see FIGS. 17 and 24). Wetting of the microfluidic chip 500 is performed with a surfactant-containing culture medium contained in the rear channel reservoir 130. Thus, pressure is applied to the pressure port 108A, which is fluidly connected to the rear channel reservoir 130 through the rear channel waste 136 and the narrow channel 135. This forces the surfactant-containing culture medium from the rear channel reservoir 130 into the holes 552 and the first channel 551 in the first row 550A in the chip carrier 550 (see FIG. 29). This causes the surfactant-containing culture medium to enter the set 531 of cell channels 520 in the microfluidic chip 500 through the output port 522 and the output channel 520, flow through the cell channel 530 to the input channel 510, and exit through the second input port 512. The excess culture medium with surfactant then flows into holes 556 of the third row 550C and further into the waste chamber 190 via channel 191. This initial wetting of the microfluidic chip 500 removes any air trapped within the microfluidic chip 500, i.e., within the set 531 of cell channels 530 and the input and output channels 510, 520. Furthermore, the surfactant dissolved or dispersed in the culture medium coats the surfaces of the cell channels 530 and the input and output channels 510, 520, thereby reducing the flow resistance of the culture medium with the biological sample and dissolved or dispersed drugs.

[0112] Once wetting of the microfluidic chip 500 is complete, the filtered biological sample is pushed into the microfluidic chip 500, trapping therein any cells present in the filtered biological sample in the cell channel 530 (see FIGS. 17, 26, and 30). The instrument then applies pressure to pressure port 108F, which is fluidly connected to the sample reservoir 160, via the sample waste chamber 165 and passageway 164. The filtered biological sample in the sample reservoir 160 is then pushed through the inlet hole 154 into the T-shaped channel 153, and further into the holes 554 and second channel 553 in the second row 550B in the chip carrier 550. The filtered biological sample is further pushed into the input channel 510 via the second input port 514 and then into the cell channel 530. The filtered biological sample further flows out through the second end 534 of the cell channel 530, enters the output channel 520, and exits through the output port 522 before leaving the microfluidic chip 500. Cells present in the filtered biological sample are trapped within the cell channel 530 by the cell block 535 provided therein. Excess filtered biological sample is allowed to flow into the holes 552 in the first row 550A and the first channel 511, and further into the rear channel reservoir 130 and the rear channel waste chamber 136. During the filling process, excess filtered biological sample may also leave the input channel 510 through the first input port 512 and then into the holes 556 in the third row 550C and the third channel 555, thereby entering the waste chamber 190.

[0113] In this regard, cells present in a biological sample are trapped within cell channel 530 in microfluidic chip 500, where they may be exposed to one or more drugs dissolved or dispersed in culture medium in loaded medium reservoir 170 (see FIGS. 17 and 27). Accordingly, the device applies pressure at pressure ports 108B, 108D, which are fluidly connected to medium reservoir 170 via channel 176 and membrane chamber 106. The applied pressure forces the culture medium with the dissolved or dispersed drug, dissolved control chemical, or undissolved chemical or drug to exit through drain hole 177 and channel 178 at bottom surface 103 of substrate 100 and into each hole 558 in fourth row 550D of chip carrier 550 (see FIG. 31). The culture medium with the dissolved or dispersed drug or chemical is further forced into first input port 512 of input channel 520, flows into cell channel 530, and exits through output channel 520 and output port 522. This flow of culture medium means that cells trapped in one set 531 of cell channels 530 are exposed to a drug at a predetermined concentration, while cells trapped in another set 531 of cell channels 530 can be exposed to a different drug at a predetermined concentration, a different drug at a predetermined concentration, a control chemical, or simply culture medium. By having multiple sets 531 of cell channels 530, such as 2×N or 32 as shown, multiple different drugs and various concentrations of these different drugs can be tested for a particular biological sample while still allowing an internal control to be present in one or more sets 531 of cell channels 530. Excess culture medium with any dissolved or dispersed drug or control chemical flows from output port 522 into holes 552 in first row 550A and first channel 551, then out through rear channel reservoir 130 and rear channel waste chamber 136. Excess culture medium may also leave input channel 510 through second input port 514 and exit into holes 554 in third row 550C and third channel 555, and then into waste chamber 190.

[0114] The response of cells trapped in cell channel 530 to various agents can then be monitored and analyzed by the instrument. In certain embodiments, the phenotypic response of cells to various agents is monitored and analyzed by the instrument. Various types of phenotypic responses and phenotypic characteristics can be monitored and analyzed, including, but not limited to, growth rate, shape, size, the shape of a growth rate curve defining growth rate over time, the shape of a length curve defining cell length over time, the shape of an area curve defining cell area over time, color, optical density, electrical conductivity, heat production, surface antigen composition as observed by affinity reagents, absorbance spectrum, and a mixture of at least two such phenotypic characteristics.

[0115] Proliferation rate is a phenotypic characteristic or trait that can be conveniently used to determine the response of captured cells to various agents. Proliferation rate can be determined, for example, by monitoring the number of cells in each cell channel 530 as the number increases over time for proliferating cells. Alternatively, or in addition, proliferation rate can be determined by monitoring the length of the portion of cell channel 530 occupied by cells. This length increases over time for proliferating cells but remains the same for nonviable and non-proliferating cells. Alternatively, or in addition, proliferation rate can be determined by monitoring the area divided or the length of the cells within an image of cell channel 530.

[0116] The growth rate over time can typically vary depending on the presence and / or concentration of any drug in culture medium.In some cases, cells grow exponentially, while in other cases, cells grow in a more cyclical manner.Therefore, the shape or form of the growth rate curve can be used to determine the phenotypic response of cells to various drugs.

[0117] Other phenotypic characteristics that can be altered by cells in the absence or presence of a drug include shape, size, color, and optical density. Optical density, color, or other spectral properties can vary depending on the cell's contents, cell shape, etc. Optical properties can therefore be used to determine the cell's response to various drugs. Electrical conductivity and heat production depend on the cell's chemical composition and metabolic state and can therefore form the basis for determining the cell's response to drugs.

[0118] The device is preferably configured to monitor cells trapped within cell channels 530 either more or less continuously, or at multiple time points as culture medium with dissolved or dispersed agents flows through cell channels 530. For example, the device can include one or more video cameras to monitor the response of cells to various agents, or one or more cameras that take pictures of the set of cell channels 531 at selected time points.

[0119] In certain embodiments, the instrument uses a microscope, such as a phase-contrast microscope connected to a camera such as a charge-coupled device (CCD) and complementary metal-oxide semiconductor (CMOS) camera, or a confocal scanning system for fluorescence, Raman imaging, coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS), and similar chemically sensitive techniques that give spectral changes for dead and live cells, to take pictures of the cells in the cell channel 530. This includes measurements at one or several wavelengths with and without contrast-enhancing additives to the growth medium, such as chemically specific probes and dyes.

[0120] The electrical conductivity and / or heat production can be measured by electrodes or sensors in the device placed within or connected to the cell channel.

[0121] In certain embodiments, the cartridge 1 and device are used for AST of bacteria present in a biological sample, such as a urine sample. The substrate 100, designed as shown, can then be preloaded with, for example, five different antibiotics at five different concentrations in 25 of the 32 media reservoirs 170. The remaining seven media reservoirs 170 can be empty, i.e., not preloaded with any chemicals, or at least some of them can be preloaded with one or more control chemicals. Phenotypic responses, such as growth rates of bacteria in a biological sample, such as a urine sample, can then be determined using the cartridge 1 and device in a very short timeframe, typically within one or a few hours, or even within one hour. This is in comparison to traditional culture-based AST methods, which require at least overnight bacterial culture.

[0122] Another aspect of the present invention relates to a method for analyzing a biological sample (see FIG. 32). The method includes, in step S1, transferring a biological sample containing cells to a plurality of sets 531 of cell channels 530 in a microfluidic chip 500. The method also includes, in step S2, transferring a culture medium to a plurality of medium reservoirs 170. Each medium reservoir 170 of the plurality of medium reservoirs 170 is configured to be fluidly connected to a respective set 531 of cell channels 530 of the plurality of sets 531 of cell channels 530. At least one of the medium reservoirs 170 of the plurality of medium reservoirs 170 is pre-loaded with a predetermined amount of a drug configured to be dissolved or dispersed in the culture medium. The method further includes, in step S3, transferring the culture medium with the dissolved or dispersed drug from each pre-loaded medium reservoir 170 to each set 531 of cell channels 530. The method further includes, in step S4, monitoring the response of cells to the drug in each set 531 of cell channels 530.

[0123] In one embodiment, step S3 includes moving culture medium with dissolved or dispersed drugs or culture medium without any drugs from each culture medium reservoir 170 of the plurality of culture reservoirs 170 to each set 531 of cell channels 530.

[0124] In one embodiment, at least some of the plurality of media reservoirs 170 are pre-loaded with different amounts of or different drugs configured to be dissolved or dispersed in the culture medium.

[0125] In one embodiment, step S1 includes transferring a biological sample containing cells to a plurality of sets 531 of cell channels 530 in a microfluidic chip 500 disposed in a chip chamber 105 of the cartridge 1, according to any embodiment as disclosed herein. In this embodiment, step S2 includes transferring culture medium to a plurality of medium reservoirs 170 of the cartridge 1, according to any embodiment as disclosed herein.

[0126] Various additional aspects of the invention will be described in more detail herein, any of which may be combined with each other and / or with previously described aspects of the invention.

[0127] A first additional embodiment relates to a cartridge 1 including a substrate 100. The substrate 100 includes a blister chamber 110 configured to accommodate a medium blister 400 containing a culture medium. The substrate 100 also includes a pressure port 108E configured to fluidly connect with a pressure source configured to apply a pressurized fluid, preferably pressurized gas, more preferably pressurized air, at the pressure port 108E. The substrate 100 further includes a pressure channel 116 interconnecting the pressure port 108E and the blister chamber 110. The cartridge 1 also includes a top cover 200 attached to the top surface 102 of the substrate 100 and configured to enclose the medium blister 400 within the blister chamber 110. According to this first additional embodiment, the blister chamber 110 includes a central recess 111 and a surrounding chamber bottom 112. The medium blister 400 is attached to the surrounding chamber bottom 112. The central recess 111 includes a drain hole 113 and at least one cutter 114, and has a depth deep enough to space the at least one cutter 114 a distance from the bottom surface of the medium blister 400. The at least one cutter 114 is configured to pierce the bottom surface of the medium blister 400 upon application of pressurized fluid, preferably pressurized gas, more preferably pressurized air, at pressure port 108E and flow through pressure channel 116 into blister chamber 110 between top cover 200 and the top surface of the medium blister 400, thereby pushing the medium blister 400 downward toward the central recess 111, opening the bottom surface of the medium blister 400, and pushing the culture medium within the medium blister 400 through the drain hole 113.

[0128] In one embodiment, cutter 114 includes a drainage channel 118 on a side 117 of cutter 114 facing drainage hole 113. In this embodiment, drainage channel 118 is configured to direct culture medium from medium blister 400 toward the drainage hole.

[0129] In one embodiment, the side 117 of the cutter facing the drain hole 113 is arced around a portion of the drain hole 113 .

[0130] A second additional embodiment relates to a cartridge 1 including a substrate 100. The substrate 100 includes a medium valve chamber 120 including, at a bottom surface of the medium valve chamber 120, an inlet hole 121 in fluid communication with a culture medium source 110, 400, a rear channel hole 122 in fluid communication with a rear channel reservoir 130, and a plurality of medium reservoir holes 123, each in fluid communication with a respective medium reservoir 170. The substrate 100 also includes a culture medium valve 420 attached to a peripheral portion 124 of the bottom surface of the medium valve chamber 120. The substrate 100 further includes a pressure port 108G configured to fluidly connect with a pressure source configured to apply pressurized fluid, preferably pressurized gas, more preferably pressurized air, at the pressure port 108G, and a pressure channel 129A interconnecting the pressure port 108G and the medium valve chamber 120. The culture medium valve 420 is configured to redirect the inflow of culture medium from the inlet hole 121 to the rear channel hole 122 and the plurality of medium reservoir holes 123 and toward the rear channel reservoir 130 and the medium reservoir 170 upon application of a first fluid pressure, preferably a gas pressure, more preferably an air pressure, at the pressure port 108G. The culture medium valve 420 is also configured to press near the inlet hole 121, the rear channel hole 122, and the plurality of medium reservoir holes 123 against a bottom surface of the medium valve chamber 120 to prevent any flow of culture medium between the medium reservoir 170 and the rear channel reservoir 130 upon application of a second fluid pressure, preferably a gas pressure, more preferably an air pressure, at the pressure port 108G, that is higher than the first fluid pressure.

[0131] In one embodiment, the medium valve chamber 120 includes a central inlet hole 121, a rear channel hole, and a plurality of medium reservoir holes 123 arranged circumferentially around the central inlet hole 121 in the bottom surface of the medium valve chamber 120.

[0132] In one embodiment, the culture medium valve 420 is a disk made of a flexible material, preferably a thermoplastic elastomer.

[0133] In one embodiment, pressure port 108E is the first pressure port 108. In this embodiment, substrate 100 further includes a second pressure port 108A in fluid communication with rear channel reservoir 130. Culture medium valve 420 is configured, upon application of a first fluid pressure, preferably gas pressure, more preferably air pressure, at first pressure port 108G, and upon application of a fluid pressure, preferably gas pressure, more preferably air pressure, at second pressure port 108A, to first redirect the inflow of culture medium from inlet hole 121 to the plurality of medium reservoir holes 123 toward medium reservoir 170, and, when medium reservoir 170 is filled with culture medium, to sequentially redirect the inflow of culture medium from inlet hole 121 to rear channel holes 122 toward rear channel reservoir 130.

[0134] In one embodiment, the culture medium valve 420 is configured to first redirect the inflow of culture medium from the inlet hole 121 to the plurality of medium reservoir holes 123 toward the medium reservoir 170 upon application of a first fluid pressure, preferably gas pressure, more preferably air pressure, at the first pressure port 108G, and upon application of a fluid pressure, preferably gas pressure, more preferably air pressure, at the second pressure port 108A, and then sequentially redirect the inflow of culture medium from the inlet hole 121 to the rear channel hole 122 toward the rear channel reservoir 130 upon release of the fluid pressure, preferably gas pressure, more preferably air pressure, at the second pressure port 108A.

[0135] A third additional aspect relates to a cartridge 1 including a substrate 100. The substrate includes a plurality of medium reservoirs 170. At least some of the plurality of medium reservoirs 170 contain different amounts of a drug and / or different drugs. The cartridge 1 also includes a microfluidic chip 500 including a plurality of sets 531 of cell channels 530 configured to capture cells from a biological sample. Each set 531 of cell channels 530 is fluidly connected to a respective one of the plurality of medium reservoirs 170. The cartridge 1 further includes a culture medium source 110, 400 in fluid communication with the plurality of medium reservoirs 170. Each medium reservoir 170 includes an inlet hole 171A, 171B in fluid communication with the culture medium source 110, 400, as well as pillar chambers 172A, 172B including the inlet holes 171A, 171B. Each medium reservoir 170 also includes chamber portions 174A, 174B interconnected with inlet pillar chambers 172A, 172B through waists 173A, 174B configured to exert a flow resistance on the culture medium, and drain holes 177 fluidly connected to each set 531 of cell channels 530 of the multiple sets 531 of cell channels 530. In this embodiment, the culture medium entering the culture medium reservoir 170 through the inlet holes 171A, 171B is configured to first fill the pillar chambers 172A, 172B due to the flow resistance exerted by the waists 173A, 173B, expelling any air present in the pillar chambers 172A, 172B through the waists 173A, 173B, and when the pillar chambers 172A, 172B are filled with the culture medium, sequentially filling the chamber portions 174A, 174B, thereby filling the culture medium reservoir 170 with each predetermined volume of culture medium and obtaining each predetermined concentration of the drug or different drugs.

[0136] In one embodiment, each medium reservoir 170 of the plurality of medium reservoirs 170 includes a rear chamber 170A and a front chamber 170B. The rear chamber 170A includes an inlet hole 171A that is fluidly connected to a culture medium source 110, 400. The rear chamber 170A also includes a pillar chamber 172A that includes the inlet hole 171A and a chamber portion 174A that is interconnected with the inlet pillar chamber 172A through a waist 173A that is configured to exert a flow resistance on the culture medium and that is fluidly connected to the membrane chamber 106. The front chamber 170B includes an inlet hole 171B that is fluidly connected to the culture medium source 110, 400 and a pillar chamber 172B that includes the inlet hole 171B. The anterior chamber 170B also includes a chamber portion 174B interconnected with the inlet pillar chamber 172B through a waist 173B configured to exert a flow resistance on the culture medium and a drain hole 177 fluidly connected to each set 531 of the cell channels 530 of the multiple sets 531 of cell channels 530.

[0137] In one embodiment, the front chamber 170B exerts a higher flow resistance on the culture medium than the rear chamber 170A, directing the culture medium towards the inlet holes 171A of the rear chamber 170A.

[0138] In one embodiment, the membrane chamber 106 is in fluid communication with pressure ports 108B, 108D configured to be in fluid communication with a pressure source configured to apply pressurized fluid, preferably pressurized gas, more preferably pressurized air, at the pressure ports 108B, 108D to force the culture medium in the rear chamber 170A into the respective front chamber 170B.

[0139] In one embodiment, antechamber 170B includes steps 273 disposed between drain holes 177 and chamber portion 174B, the steps 273 configured to induce turbulence in the culture medium during filling of chamber portion 174B to promote mixing of the drug or different drugs and media.

[0140] A fourth additional aspect relates to a cartridge 1 including a substrate 100. The substrate 100 is configured to contain a biological sample including cells and includes a sample chamber 140 including a drain hole 141. The substrate 100 also includes a dome pump chamber 180 having a bottom surface including a drain hole 182 in fluid communication with the sample chamber 140. The cartridge 1 also includes a dome pump 410 attached to a peripheral portion 181 of the bottom surface of the dome pump chamber 180 and configured to enclose a defined volume of air. The substrate 100 further includes a pressure port 108E configured to be fluidly connected to a pressure source configured to apply pressurized fluid, preferably pressurized gas, more preferably pressurized air at the pressure port 108E to force the dome-shaped pump 410 downward toward the bottom surface of the dome pump chamber 180, forcing a predetermined volume of air contained by the dome-shaped pump 410 in the dome pump chamber 180 through the drain hole 182 of the dome pump chamber 180 into the sample chamber 140, and forcing a predetermined volume of biological sample into the drain hole 141 of the sample chamber 140.

[0141] In one embodiment, the dome pump 410 is a flexible dome, preferably a thermoplastic elastomer dome.

[0142] In one embodiment, the substrate 100 further includes a filter chamber 146 that includes a sample filter 630 that is in fluid communication with the drain hole 141 of the sample chamber 140 .

[0143] In one embodiment, substrate 100 also includes a sample valve chamber 150 having a bottom surface including an inlet hole 148 and a drain hole 151 in fluid communication with filter chamber 146. Substrate 100 further includes a pressure port 108G configured to fluidly connect with a pressure source configured to apply pressurized fluid, preferably pressurized gas, and more preferably pressurized air, at pressure port 108G, and pressure channels 129A, 155 interconnecting pressure port 108G and sample valve chamber 150. Cartridge 1 further includes a sample valve 430 attached to a peripheral portion of the bottom surface of sample valve chamber 150. In this embodiment, sample valve 430 is configured to press against the bottom surface of sample valve chamber 150 near inlet hole 148 and drain hole 151 upon application of fluid pressure, preferably gas pressure, and more preferably air pressure, at pressure port 108G to prevent any backflow of the biological sample into sample valve chamber 150.

[0144] The above-described embodiments should be understood as some illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined into other configurations where technically possible. However, the scope of the present invention is defined by the appended claims.

Claims

1. A cartridge (1), comprising: a chip chamber (105) configured to accommodate a microfluidic chip (500) including a plurality of sets (531) of cell channels (530) configured to capture cells from a biological sample; a sample chamber (140) configured to receive the biological sample and in fluid communication with the plurality of sets (531) of cell channels (530); a plurality of medium reservoirs (170), each of which is configured to be in fluid communication with a respective set (531) of cell channels (530) of said plurality of sets (531) of cell channels (530); and a culture medium source (110, 400) in fluid communication with said plurality of medium reservoirs (170) and configured to supply culture medium to said plurality of medium reservoirs (170); Including, cartridge.

2. The cartridge of claim 1 , wherein the plurality of medium reservoirs (170) have substantially the same shape and / or internal volume.

3. 3. The cartridge of claim 1 or 2, wherein at least some of the plurality of medium reservoirs (170) are pre-loaded with different amounts or different drugs configured to be dissolved or dispersed in the culture medium.

4. 4. The cartridge of claim 3, wherein a first set of the plurality of medium reservoirs (170) are pre-loaded with the different amounts of the drug or the different drugs, and a second set of the plurality of medium reservoirs (170) are not pre-loaded with any drug.

5. The cartridge of claim 3 , wherein the agent is an antimicrobial agent or the different agents are different antimicrobial agents.

6. moreover: an inlet hole (121) in fluid communication with said culture medium source (110, 400); and a plurality of medium reservoir holes (123), each of which is in fluid communication with a respective medium reservoir (170) of said plurality of medium reservoir holes (123); a medium valve chamber (120); and a culture medium valve (420) configured to direct culture medium supplied from said culture medium source (110, 400) through said inlet hole (121) to said plurality of medium reservoir holes (123); 3. The cartridge of claim 1 or 2, comprising:

7. The culture medium source (110, 400) comprises: a culture medium container (400) prepackaged with the culture medium; and configured to receive said culture medium container (400): a drain hole (113) in fluid communication with said plurality of medium reservoirs (170); and a cutter (114) configured to cut the surface of the culture medium container (400) to supply the culture medium to the drain hole (113); a chamber (110) comprising The cartridge of claim 6 , comprising:

8. The cartridge of claim 1 or 2, further comprising a pump (410) configured to move a biological sample from the sample chamber (140) to the plurality of sets (531) of cell channels (530).

9. further comprising a surfactant chamber (128) configured to be in fluid communication with said plurality of sets (531) of cell channels (530) and said culture medium source (110, 400), wherein: the surfactant chamber (128) is pre-loaded with a surfactant configured to dissolve or disperse in the culture medium provided by the culture medium source (110, 400); and The surfactant dissolved or dispersed in the culture medium is configured to be supplied to the plurality of sets (531) of cell channels (530).

3. The cartridge according to claim 1 or 2.

10. Attached to the microfluidic chip (500): a first set of holes (554) configured to provide fluid communication between the plurality of sets (531) of cell channels (530) and the sample chamber (140); and a second set of holes (558), each hole (558) of the second set of holes (558) configured to provide a fluid connection between a respective set (531) of cell channels (530) of said plurality of sets (531) of cell channels (530) and a respective medium reservoir (170) of said plurality of medium reservoirs (170); A chip carrier (550) comprising 3. The cartridge of claim 1 or 2, further comprising:

11. Each set (531) of cell channels (530) of said plurality of sets (531) of cell channels (530) comprises: a first port (514) in fluid communication with the holes (554) of said first set of holes (554); and a second port (512) in fluid communication with the holes (558) of said second set of holes (558); The cartridge of claim 10, comprising:

12. a substrate (100) comprising the chip chamber (105), the sample chamber (140), the plurality of medium reservoirs (170) and the culture medium source (110, 400); and a lid (200) attached to the substrate (100) and including a window (210) configured to allow imaging of the plurality of sets (531) of cell channels (530); 3. The cartridge of claim 1 or 2, further comprising:

13. 3. The cartridge of claim 1 or 2, wherein each set (531) of cell channels (530) of the plurality of sets (531) of cell channels (530) is formed as a compartment in the microfluidic chip (500) that is separated from other sets (531) of cell channels (530) of the plurality of sets (531) of cell channels (530).

14. 3. The cartridge of claim 1 or 2, wherein the chip chamber (105) comprises the microfluidic chip (500) including the plurality of sets (531) of cell channels (530) configured to capture cells from the biological sample.

15. 1. A method of analyzing a biological sample, comprising: A step (S1) of transferring a biological sample containing cells to a plurality of sets (531) of cell channels (530) in a microfluidic chip (500); a step (S2) of transferring culture medium to a plurality of medium reservoirs (170), each medium reservoir (170) of the plurality of medium reservoirs (170) configured to be in fluid communication with a respective set (531) of cell channels (530) of the plurality of sets (531) of cell channels (530), and at least one of the medium reservoirs (170) of the plurality of medium reservoirs (170) being pre-loaded with a predetermined amount of a drug configured to dissolve or disperse the culture medium; transferring (S3) the culture medium with the dissolved or dispersed drug from each medium reservoir (170) pre-loaded with the drug to each set (531) of cell channels (530); and monitoring (S4) the response of the cells to the agent within each set (531) of cell channels (530); A method comprising:

16. the step (S3) of transferring the culture medium having the dissolved or dispersed drug comprises the step (S3) of transferring the culture medium having the dissolved or dispersed drug or the culture medium not having any drug from each medium reservoir (170) of the plurality of medium reservoirs (170) to each set (531) of cell channels (530); 16. The method of claim 15.

17. at least some of the plurality of medium reservoirs (170) are pre-loaded with different amounts of the drug or different drugs configured to be dissolved or dispersed in the culture medium; 17. The method of claim 15 or 16.

18. The step (S1) of transferring the biological sample includes a step (S1) of transferring the biological sample containing cells to a plurality of sets (531) of cell channels (530) in a microfluidic chip (500) disposed in a chip chamber (105) of the cartridge (1) according to any one of claims 1 to 14; and The step (S2) of transferring the culture medium comprises a step (S2) of transferring the culture medium to a plurality of medium reservoirs (170) of a cartridge (1) according to any one of claims 1 to 14.

17. The method of claim 15 or 16.

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