Platforms, cartridges, and methods for isolation and enrichment of target cells

Abstract

Provided are platforms, cartridges, and methods for isolating target cells in a fluid sample. In an aspect, disclosed is a platform for isolating target cells in a fluid sample. That platform can include a cartridge; one or more pumps configured to flow the one or more fluids through the flow chamber of the cartridge; one or more valves configured to stop or start the flow of the one or more fluids through the flow chamber of the cartridge; one or more magnetic arrays configured to apply the magnetic field to the magnetically susceptible nanobeads in the flow chamber; one or more bubble sensors configured to detect bubbles in the one or more fluids of the cartridge; one or more processors configured to control one or more processes for isolating the target cells from the fluid sample in the cartridge; and a power supply configured to supply power to the platform.

Description

PLATFORMS, CARTRIDGES, AND METHODS FOR ISOLATION AND ENRICHMENT OF TARGET CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 63 / 592,285, filed October 23, 2023, and U.S. Provisional Application No. 63 / 564,669, filed March 13, 2024, each of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Physical separation of heterogeneous cells based on biomarker expressions (e.g., proteins, RNA, and the like) can be important for therapeutic applications. There is an unmet need to obtain pure populations of cells that are of interest in diagnostic and therapeutic applications. Microfluidic-based approaches can be used for cell sorting with high specificity and sensitivity. Such approaches have been widely applied to the isolation, recovery, and analysis of various mammalian cells. However, technical problems remain for obtaining large-scale, volumetric, high-recovery, high -purity cell sorting at a reasonable cost (e.g., enrichment of Tumor-Infiltrating Leukocytes). For example, some platforms such as fluorescence-activated cell sorting (FACS) platforms or magnetic-activated cell sorting (MACS) platforms may be unsuitable due to, e.g., poor recovery or purity of the target cells.SUMMARY

[0003] Disclosed herein are platforms, cartridges, and methods for isolation or separation of target cells from fluid samples. The platforms, cartridges, and methods disclosed herein are useful for large-scale, high-throughput processing of cells to recover and enrich a target population of cells from a heterogeneous population of cells. The present disclosure relates to platforms, cartridges, and methods that can use magnetic fields for recovering magnetically-labeled target cells in a flow chamber using magnetically susceptible nanoparticles and principles of microfluidics. For example, complex biological fluid samples containing rare cells of interest can be labeled with antibody-labeled magnetic nanoparticles (MNPs). The fluid samples can be flowed through one or more cartridges at predetermined velocities or flow rates. The cartridges can include low-flow zones generated by flow rate-reducing structures. Different cell populations of the fluid samples with predetermined expression levels (e.g., high, high-medium, medium-low, and low) can be isolated or captured into different subsets of cell populations. These subsets can be further processed by, e.g., sequencing methods, expansion methods, characterization methods, and the like.

[0004] In an aspect, described herein is a cartridge for isolating target cells in a fluid sample, comprising: a flow chamber configured to receive the fluid sample; one or more flow ratereducing structures operatively coupled to the flow chamber configured to reduce a flow rate of the fluid sample proximate to the flow rate-reducing structures; and a degassing region operatively coupled to the flow chamber configured to remove bubbles from the fluid sample, wherein the cartridge is configured to receive one or more fluids comprising the fluid sample and further comprising the target cells labeled with magnetically susceptible nanobeads. In some embodiments, the cartridge further comprises a magnetic field generator configured to generate a magnetic field. In some embodiments, the magnetic field causes a magnetic force to act on the magnetically susceptible nanobeads to isolate the target cells proximate to the flow rate-reducing structures. In some embodiments, the one or more flow-rate reducing structures are configured to reduce the flow rate of the fluid sample sufficiently low for the magnetic force of the magnetic field acting on the nanobeads to overcome a drag force of the reduced flow rate acting on the target cells thereby isolating the target cells proximate to the one or more flow-rate reducing structures. In some embodiments, the magnetic field generator is configured as one or more magnetic arrays. In some embodiments, the one or more magnetic arrays are configured to cause one or more magnetic forces to act on the magnetically susceptible nanobeads. In some embodiments, the flow chamber is configured with one or more isolation zones comprising the one or more flow-rate reducing structures. In some embodiments, the one or more isolation zones are configured as a first zone and a second zone. In some embodiments, the cartridge is configured to flow the one or more fluids through the first zone at a first flow rate and to flow the one or more fluids through the second zone at a second flow rate. In some embodiments, a combination of the first flow rate and the second flow rate is configured based at least on the drag force acting on the target cells and a magnetic force acting on the nanobeads. In some embodiments, a height, a width, or a depth of the one or more flow-rate reducing structures in the first zone is identical as a height, a width, or a depth of the one or more flow-rate reducing structures in the second zone. In some embodiments, the height comprises a range of from about 50 to 150 micrometers (pm). In some embodiments, a height, a width, or a depth of the one or more flow-rate reducing structures in the first zone is different from a height, a width, or a depth of the one or more flow-rate reducing structures in the second zone. In some embodiments, the height comprises a range of from about 50 - 150 micrometers (pm). In some embodiments, the cartridge is further configured to transmit the one or more fluids to one or more flow outlets. In some embodiments, the cartridge further comprises a degasser fluidically coupled to the degassing region, the flow chamber, or the one or more fluids. In some embodiments, the degasser comprises an optical sensor configured to detect the bubbles in the one or more fluids. In some embodiments, the degasser is configured to changea flow of the one or more fluids. In some embodiments, the degasser is configured to change the flow by bypassing the flow chamber and flowing the one or more fluids to one or more outlets. In some embodiments, at least one of the one or more flow-rate reducing structures is configured with one or more trapping surfaces. In some embodiments, at least one of the one or more trapping surfaces is configured as a concave surface defined by two joined arms defining an angle. In some embodiments, the cartridge further comprises a fluid flow generator and one or more valves configured to generate a flow of the one or more fluids through the flow chamber. In some embodiments, a first fluid of the one or more fluids comprises a priming fluid, a second fluid comprises the fluid sample, and a third fluid comprises a washing fluid. In some embodiments, the fluid flow generator comprises a peristaltic pump. In some embodiments, the one or more valves comprises one or more solenoid pinch valves. In some embodiments, the cartridge is configured to perform high-throughput processing of the fluid sample at a rate of at least about 109 cells per hour. In some embodiments, the cartridge further comprises: one or more inlets configured to receive the one or more fluids from one or more input containers; and one or more outlets configured to transmit the one or more fluids to one or more output containers. In some embodiments, the target cells comprise peripheral blood mononuclear cells (PBMCs), tumor- reactive lymphocytes (TRLs), or circulating TRLs. In some embodiments, the magnetically susceptible nanobeads are configured with or attached to one or more antibodies specific to a surface protein of the target cells. In some embodiments, the surface protein comprises CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1 or any combination thereof. In some embodiments, the target cells or the magnetically susceptible nanobeads are configured with or attached to one or more fluorophores.

[0005] In another aspect, described herein is a platform for isolating target cells in a fluid sample, comprising: the cartridge described herein; one or more pumps configured to flow the one or more fluids through the flow chamber of the cartridge; one or more valves configured to stop or start the flow of the one or more fluids through the flow chamber of the cartridge; one or more magnetic arrays configured to apply the magnetic field to the magnetically susceptible nanobeads in the flow chamber; one or more bubble sensors configured to detect bubbles in the one or more fluids of the cartridge; one or more processors configured to control one or more processes for isolating the target cells from the fluid sample in the cartridge; and a power supply configured to supply power to the platform. In some embodiments, the platform further comprises a deck operatively coupled to the one or more pumps, the one or more valves, the one or more magnetic arrays, or the one or more bubble sensors. In some embodiments, at least one pump of the one or more pumps is a peristaltic pump. In some embodiments, at least one pump of the one or more pumps is operatively coupled to the one or more valves, the one or more bubble sensors, and theone or more fluids. In some embodiments, at least one valve of the one or more valves is a solenoid pinch valve. In some embodiments, at least one valve of the one or more valves is operatively coupled to the one or more pumps, the one or more bubble sensors, and the one or more fluids. In some embodiments, at least one magnetic array of the one or more magnetic arrays is a permanent magnet. In some embodiments, at least one magnetic array of the one or more magnetic arrays is magnetically coupled to the one or more fluids, wherein the coupling comprises magnetic field lines of the at least one magnetic array to pass through the one or more fluids. In some embodiments, at least one bubble sensor of the one or more bubble sensors is an optical sensor. In some embodiments, at least one bubble sensor of the one or more bubble sensors is operatively coupled to the one or more pumps, the one or more valves, and the one or more fluids. In some embodiments, the deck further comprises a deck framing and a deck enclosure. In some embodiments, the deck framing is operatively coupled to the deck enclosure. In some embodiments, the deck framing is operatively coupled to the one or more pumps, the one or more valves, the one or more magnetic arrays, or the one or more bubble sensors. In some embodiments, the platform further comprises a magnet actuator assembly operatively coupled to the deck framing. In some embodiments, the magnet actuator assembly is configured to be operatively coupled to the one or more magnetic arrays. In some embodiments, the magnet actuator assembly comprises: a linear actuator configured to generate a movement of the one or more magnetic arrays proximate to the cartridge; a linear motion rail configured to generate a position of the one or more magnetic arrays proximate to the cartridge; and a cartridge interface configured to releasably couple the cartridge to the magnet actuator assembly. In some embodiments, the magnet actuator assembly is configured to move or position the one or more magnetic arrays to generate one or more magnetic capture zones in the flow chamber. In some embodiments, the one or more magnetic capture zones comprises an area circumscribing all or substantially all of the flow-rate reducing structures. In some embodiments, the platform further comprises a motion limit assembly operatively coupled to the deck framing. In some embodiments, the motion limit assembly comprises: one or more optical sensors configured to determine the movement or the position of the magnet actuator assembly; and one or more hard stops configured to stop the movement of the magnet actuator assembly when the movement exceeds a predetermined movement. In some embodiments, the deck enclosure is configured to be compliant with at least contract development and manufacturing organization (CDMO) safety protocols. In some embodiments, the platform further comprises a main body operatively coupled to the deck, a graphical user interface (GUI), or the power supply. In some embodiments, the one or more processors is configured to automatically execute instructions comprising: (a) priming the cartridge with priming fluid; (b) isolating the target cells from the fluid sample; (c) washing the cartridge with washing fluid; (d)recovering the target cells from the cartridge; or (e) any combination of (a) to (d). In some embodiments, the power supply is configured to supply 24 volts (V), 9.2 amps (A), or 221 watts (W) to the platform. In some embodiments, the main body further comprises a body framing and a body enclosure. In some embodiments, the body framing is configured to be operatively coupled to the GUI. In some embodiments, the body framing is configured to be operatively coupled to the power supply. In some embodiments, the body framing is configured to be operatively coupled to one or more poles and wherein the one or more poles are configured to hold one or more containers for the one or more fluids. In some embodiments, the one or more containers are configured as a primer container, a fluid sample container, and a wash container. In some embodiments, the body enclosure further comprises one or more lids operatively coupled to the body enclosure. In some embodiments, the one or more lids are configured to isolate a user from the one or more magnetic arrays. In some embodiments, the body enclosure is configured to be compliant with at least contract development and manufacturing organization (CDMO) safety protocols. In some embodiments, the target cells comprise peripheral blood mononuclear cells (PBMCs). In some embodiments, the target cells comprise tumor-reactive lymphocytes (TRLs). In some embodiments, the platform is configured to process the fluid sample at a rate of at least about 109 cells per hour. In some embodiments, the platform is configured to isolate the target cells from the fluid sample with a purity of at least about 95%. In some embodiments, the platform is configured to recover the target cells from the fluid sample with a recovery rate of at least about 95%. In some embodiments, the platform is configured to flow the one or more fluids at a flow rate of about 1 to 32 milliliters per hour (mL / hr). In some embodiments, the platform further comprises one or more sensors configured to detect or measure one or more fluorescent signals associated with the target cells. In some embodiments, the platform further comprises a graphical user interface (GUI) configured to allow a user to select or interact with the one or more processes.

[0006] In another aspect, described herein is a method for using a platform for isolating target cells in a fluid sample, comprising: (a) providing the cartridge described herein; (b) connecting the cartridge to the platform described herein; (c) priming the cartridge by flowing a priming fluid of the one or more fluids through the cartridge; (d) removing bubbles from the cartridge; (e) flowing the fluid sample of the one or more fluids through the cartridge; (f) applying the magnetic field to isolate the target cells in the fluid sample; (g) washing the cartridge by flowing a washing fluid of the one or more fluids through the cartridge; and (h) recovering the isolated target cells in the fluid sample. In some embodiments, the priming in (c) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. In some embodiments, the removing in (d) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. In some embodiments, the flowing in (e) furthercomprises using the instrument to automatically operate the one or more pumps and the one or more valves. In some embodiments, the applying in (f) further comprises using the instrument to automatically apply the magnetic field by operation of the magnet actuator assembly. In some embodiments, the washing in (g) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. In some embodiments, the recovering in (h) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. In some embodiments, the target cells comprise peripheral blood mononuclear cells (PBMCs).

[0007] In another aspect, described herein is a kit for isolating target cells in a fluid sample, comprising: the cartridge described herein; a priming fluid, wherein the priming fluid comprises one or more priming reagents for priming the cartridge; and a washing fluid, wherein the washing fluid comprises one or more washing reagents for washing the cartridge. In some embodiments, the kit further comprises the fluid sample, wherein the fluid sample comprises the target cells labeled with magnetically susceptible nanobeads. In some embodiments, the one or more priming reagents comprises phosphate-buffered saline with ethylenediaminetetraacetic acid (PBS-EDTA). In some embodiments, the one or more washing reagents comprises phosphate-buffered saline with ethylenediaminetetraacetic acid (PBS-EDTA).

[0008] Additional aspects and advantages of the present disclosure will become readily apparent from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosurewill be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0011] FIG. 1 illustrates an example cell-separation technique using microfluidic approaches.

[0012] FIG. 2 illustrates an example of sorting magnetically-tagged target cells using microfluidic immunomagnetic cell sorting (MICS), in accordance with some embodiments.

[0013] FIGs. 3A-3G illustrate or depict the first and second generations of instruments and cartridges configured to perform MICS, in accordance with some embodiments. FIG. 3A illustrates the first generation instrument and cartridge, which involved manual cell sorting. FIG. 3B illustrates the second generation instrument and cartridge, which has been engineered for high- throughput therapeutics applications. FIG. 3C depicts the second-generation cartridge, which has been engineered for high-throughput therapeutics applications. FIG. 3D depicts a perspective view of different heights of the flow-rate reducing structures of the second-generation cartridge. FIG. 3E depicts a plan view of different heights of the flow-rate reducing structures of the second- generation cartridge. FIG. 3F illustrates a perspective view of the second-generation cartridge, which has been engineered for high-throughput therapeutics applications. FIG. 3G illustrates a plan view of the second-generation cartridge, which has been engineered for high-throughput therapeutics applications.

[0014] FIG. 4 A and FIG. 4B illustrate the high-throughput improvement of MICS compared to fluorescence-activated cell sorting (FACS) techniques, in accordance with some embodiments. FIG. 4A depicts an image of cells captured by FACS and illustrates the throughput of FACS. FIG. 4B depicts an image of cells captured by MICS and illustrates the throughput of MICS, in accordance with some embodiments.

[0015] FIG. 5 lists some advantages of MICS over other cell sorting techniques.

[0016] FIG. 6 illustrates a high-level architecture of MICS including tubing and connector specifications for dry consumables, in accordance with some embodiments.

[0017] FIG. 7 illustrates a high-level architecture of MICS including tubing and connector specifications for wet consumables, in accordance with some embodiments.

[0018] FIG. 8 illustrates an example workflow for MICS including using upstream processing operations for input samples before automated magnetic cell sorting, in accordance with some embodiments.

[0019] FIG. 9 illustrates an example workflow for MICS including automated magnetic cell sorting, in accordance with some embodiments.

[0020] FIGs. 10A-10C depict example equipment used for cartridge activation protocols, in accordance with some embodiments. FIG. 10A depicts a plasma chamber before loadingcartridges. FIG. 10B depicts a plasma chamber while activation is being performed. FIG. IOC depicts a plasma chamber after activation is performed.

[0021] FIG. 11A and FIG. 11B depict cartridge assembly and tube connections therein, in accordance with some embodiments. FIG. 11A depicts the ridge over the connector. FIG. 11B depicts connectors in place.

[0022] FIGs. 12A-12C depict examples of non-degassed and degassed cartridges, in accordance with some embodiments. FIG. 12A depicts an example of a non-degassed cartridge. FIG. 12B depicts a closer view of the non-degassed cartridge. FIG. 12C depicts an example of a degassed cartridge.

[0023] FIG. 13 illustrates an example MICS workflow for cell sorting, in accordance with some embodiments.

[0024] FIGs. 14A-14E depict an example MICS workflow for loading cartridges and consumables on an instrument or platform, in accordance with some embodiments. FIG. 14A depicts starting with consumables laid out left-right in front of the instrument. FIG. 14B depicts hanging input and output containers on poles. FIG. 14C depicts loading a cartridge into the cartridge interface of the instrument. FIG. 14D depicts load tubes into pinch valves and the peristaltic pump. FIG. 14E depicts the instrument with fully loaded consumables.

[0025] FIG. 15 depicts correct and incorrect tubing configurations for connecting the instrument to a cartridge, in accordance with some embodiments.

[0026] FIGs. 16A-16D illustrate example workflows for preparing a cartridge by operation of pinch valves and pumps, in accordance with some embodiments. FIG. 16A illustrates a starting state of the cartridge, e.g., liquids in input containers and air in the cartridge before priming. FIG. 16B illustrates priming of a sample input tube. FIG. 16C illustrates priming of wash or buffer tubing. FIG. 16D illustrates that air slugs, e.g., bubbles, may result from priming.

[0027] FIG. 17A and FIG. 17B illustrate example workflows for priming of Zone 1 and 2 of the cartridge by operation of pinch valves and pumps, in accordance with some embodiments. FIG. 17A illustrates priming of Zone 1 of the cartridge. FIG. 17B illustrates priming of Zone 2 of the cartridge.

[0028] FIG. 18 illustrates example workflows for processing the sample fluid through the cartridge during magnetic capture of immunomagnetic tagged cells by operation of pinch valves and pumps, in accordance with some embodiments.

[0029] FIG. 19 illustrates example workflows for processing the wash fluid through Zone 1 and 2 by operation of pinch valves and pumps, in accordance with some embodiments.

[0030] FIG. 20 illustrates example workflows for processing the wash fluid through Zone 1 for recovery of target cells captured therein into output 1 container by operation of pinch valves and pumps, in accordance with some embodiments.

[0031] FIG. 21 illustrates example workflows for processing the wash fluid flow through Zone 2 for recovery of cells captured therein in output 2 container by operation of pinch valves and pumps, in accordance with some embodiments.

[0032] FIG. 22 illustrates example workflows for downstream applications or processing of the target cells recovered from the cartridge, in accordance with some embodiments.

[0033] FIG. 23 illustrates an example instrument or platform configured to process cells using the cartridge, which can include two major subsystems (e.g., main body and deck), in accordance with some embodiments.

[0034] FIG. 24 depicts a solenoid pinch valve configured to process cells, which can be located in the deck of the instrument, in accordance with some embodiments.

[0035] FIG. 25 illustrates a high-level architecture of an instrument or platform configured to perform workflows associated with MICS, in accordance with some embodiments.

[0036] FIG. 26 illustrates a high-level architecture of an instrument or platform configured to enable independent flow of liquid through the cartridge from input containers by operation of pinch valves and pumps, in accordance with some embodiments.

[0037] FIG. 27 illustrates a high-level architecture of an instrument or platform configured to enable flow of liquids across Zone 1 and 2 of the cartridge in series by operation of pinch valves and pumps, in accordance with some embodiments.

[0038] FIG. 28 illustrates a high-level architecture of an instrument or platform configured to direct fluid across Zones 1 and 2 of the cartridge independently by operation of pinch valves and pumps, in accordance with some embodiments.

[0039] FIG. 29 illustrates a high-level architecture of an instrument or platform configured to direct fluids into any of the output containers independently by operation of pinch valves and pumps, in accordance with some embodiments.

[0040] FIG. 30 illustrates a high-level architecture of an instrument or platform configured to direct fluids from one side of the cartridge to the other side without passing (e.g., bypass) through the cartridge by operation of pinch valves and pumps, in accordance with some embodiments.

[0041] FIG. 31 illustrates a high-level architecture of an instrument or platform configured to perform cartridge priming before a fluid sample is introduced into the cartridge by operation of pinch valves and pumps, in accordance with some embodiments.

[0042] FIG. 32A and FIG. 32B depict or illustrate examples of fluid sample containers used in the instrument or platform, in accordance with some embodiments. FIG. 32A depicts anexample of a fluid sample container. FIG. 32B illustrates an example of tubing connections associated with a fluid sample container.

[0043] FIG. 33 illustrates a perspective view of an instrument or platform configured to perform cell processing with a cartridge, which can include a deck framing panel that is mounted on the front of the deck framing of the instrument, in accordance with some embodiments.

[0044] FIG. 34 illustrates a front view of FIG. 33, in accordance with some embodiments.

[0045] FIG. 35 illustrates an instrument or platform configured to perform cell processing with a cartridge, which can include a deck enclosure with threaded inserts that align and mount to the deck framing, in accordance with some embodiments.

[0046] FIG. 36 illustrates the back of the deck enclosure with threaded inserts that align and mount to the deck framing, in accordance with some embodiments.

[0047] FIG. 37 illustrates the back of the deck framing panel configured with a pinch valve and flange that are mounted to the back of the deck framing and the deck enclosure, in accordance with some embodiments.

[0048] FIGs. 38A-38C illustrate an instrument or platform configured to perform cell processing with a cartridge, which can include peristaltic pumps, in accordance with some embodiments. FIG. 38A illustrates a peristaltic pump configured to be installed from the front of the deck enclosure. FIG. 38B depicts a peristaltic pump (e.g., a 6-roller pump and head structure) tested and utilized in the instrument. FIG. 38C illustrates performance of the peristaltic pump.

[0049] FIGs. 39A-39C illustrate an instrument or platform configured to perform cell processing with a cartridge, which can include bubble sensors, in accordance with some embodiments. FIG. 39A illustrates the location of the bubble sensor mounted to threaded inserts in the underside of the deck enclosure. FIG. 39B depicts the bubble sensor, e.g., optical bubble sensor. FIG. 39C depicts the bubble sensor operatively coupled to tubing.

[0050] FIG. 40 illustrates an instrument or platform configured to perform cell processing with a cartridge, which can include a magnet actuator subassembly (MSA) mounted to the underside of the deck framing or deck enclosure, in accordance with some embodiments.

[0051] FIG. 41 illustrates the MSA, which can include a baseplate, in accordance with some embodiments.

[0052] FIGs. 42A-42C illustrate the MSA, which can include features for actuating magnet arrays, in accordance with some embodiments. FIG. 42A illustrates features of the MSA, which can include a linear actuator, linear motion rail, and a cartridge interface. FIG. 42B illustrates features of the MSA, which can include one or two magnet arrays. FIG. 42C illustrates features of the MSA, which can include a cartridge interface.

[0053] FIG. 43A and FIG. 43B illustrate the MSA, which can include safety features while actuating magnet arrays, in accordance with some embodiments. FIG. 43A illustrates safety features of the MSA, which can include the deck enclosure configured around the MSA. FIG. 43B illustrates motion stop features, which can include optical switches mounted to the baseplate of the MSA or hard stops mounted to the linear motion rail.

[0054] FIG. 44A and FIG. 44B illustrate the MSA, which can include features for actuating magnet arrays, in accordance with some embodiments. FIG. 44A illustrates the positioning of the magnet array in the ON position, where it is engaged with a face of the cartridge, and can include optical switches for determining the position of the magnetic array. FIG. 44B illustrates the positioning of the magnet array in the OFF position, where it is disengaged with a face of the cartridge, and can include optical switches for determining the position of the magnetic array.

[0055] FIGs. 45A-45D depict the MSA, which can include features for actuating magnet arrays, in accordance with some embodiments. FIG. 45A depicts the MSA configured to disengage (e.g., “Off’) the magnet array from a face of the cartridge. FIG. 45B depicts the MSA configured to engage (e.g., “On”) the magnet array in contact with or proximate to a face of the cartridge. FIG. 45C illustrates an alignment of the magnet array when engaging with the cartridge capture area. FIG. 45D depicts the magnet array configured with a gap in alignment (e.g., about 0.0015”) between the magnet array and a face of the cartridge for successful isolation or processing of cells.

[0056] FIG. 46A and FIG. 46B illustrate an instrument or platform configured to perform cell processing with a cartridge, which can include a main body of the instrument, in accordance with some embodiments. FIG. 46A illustrates the framing of the main body. FIG. 46B illustrates the enclosure of the main body.

[0057] FIG. 47 illustrates an instrument or platform configured to perform cell processing with a cartridge, which can include the framing of the main body and an enclosure configured to provide mounting interfaces for cartridges associated with instrument electronics, in accordance with some embodiments.

[0058] FIG. 48 illustrates an instrument or platform configured to perform cell processing with a cartridge, which can include mounting interfaces for input and output container poles on the framing of the main body, in accordance with some embodiments.

[0059] FIG. 49A and FIG. 49B illustrate a thermal management module of the instrument configured to meet predetermined performance characteristics, in accordance with some embodiments. FIG. 49A depicts the thermal management module of the instrument. FIG. 49B illustrates performance characteristics of the thermal management module, e.g., temperatures of different instrument components plotted against duration of operation of the instrument.

[0060] FIGs. 50A-50C illustrate a thermal management module of the instrument configured to meet predetermined performance characteristics, which can include features located in the main body of the instrument, in accordance with some embodiments. FIG. 50A illustrates the enclosure back part mounted to the framing of the main body. FIG. 50B illustrates a location of the thermal management intake or exhaust vents in the instrument. FIG. 50C illustrates a location of the thermal management circulation or cooling fans in the instrument.

[0061] FIG. 51A and FIG. 51B illustrate an instrument or platform configured to perform cell processing with a cartridge, which can include a lid attached to the main body of the instrument, in accordance with some embodiments. FIG. 51A illustrates the main body configured with a lid in the closed position. FIG. 51B illustrates the main body configured with a lid in the open position.

[0062] FIG. 52A and FIG. 52B illustrate an instrument or platform configured to perform cell processing with a cartridge, which can include a graphical user interface (GUI), in accordance with some embodiments. FIG. 52A illustrates mounting of the GUI (e.g., configured as a tablet) to the framing or enclosure of the main body. FIG. 52B illustrates a bracket configured to allow mounting of the GUI, e.g., configured as a tablet.

[0063] FIG. 53A and FIG. 53B illustrate an instrument or platform configured to perform cell processing with a cartridge, which can include a power supply and communications features, in accordance with some embodiments. FIG. 53A illustrates a power switch configured to turn the power supply on or off. FIG. 53B illustrates a USB connector configured to provide communications associated with operation of the instrument.

[0064] FIG. 54 illustrates a high-level architecture of an electrical system configured to perform operations associated with the instrument, in accordance with some embodiments.

[0065] FIG. 55 illustrates a GUI configured to perform operations associated with a development module of the instrument, in accordance with some embodiments.

[0066] FIG. 56 illustrates example workflows when using preprogrammed modules for cell processing or sorting with the instrument, in accordance with some embodiments.

[0067] FIGs. 57A-57G illustrate example GUI instructions or interactions with a user during consumable loading of the instrument before cell processing or sorting, in accordance with some embodiments. FIG. 57A illustrates loading consumables, e.g., positioning input and output containers. FIG. 57B illustrates loading consumables, e.g., inserting the cartridge into the instrument. FIG. 57C illustrates routing tubing, e.g., loading tubing into pinch valves. FIG. 57D illustrates routing tubing, e.g., loading tubing into peristaltic pumps. FIG. 57E illustrates routing tubing, e.g., loading tubing into bubble sensors. FIG. 57F illustrates routing tubing, e.g., releasingpinch clamps on input fluid containers. FIG. 57G illustrates unloading operations, e.g., closing pinch clamps on output fluid containers.

[0068] FIG. 58 illustrates a non-limiting example of a computing device, in accordance with some embodiments.

[0069] FIG. 59 illustrates a non-limiting example of a web / mobile application provision system, in accordance with some embodiments.

[0070] FIG. 60 illustrates a non-limiting example of a cloud-based web / mobile application provision system, in accordance with some embodiments.

[0071] FIG. 61A and FIG. 61B illustrate example enrichment of CD8+and CD103+cells using the instrument, cartridge, and methods herein. FIG. 61 A illustrates the frequency of CD103+from live cells. FIG. 61B illustrates frequency of CD8+and CD103+cells and the enrichmentfold achieved after isolation.DETAILED DESCRIPTION

[0072] While various embodiments of the disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, or substitutions may occur without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0073] The platforms, cartridges, and methods disclosed herein can provide at least a technical solution for large-scale, diagnostic and therapeutic applications of cell processing utilizing microfluidic immunomagnetic cell sorting (MICS). The platforms, cartridges, and methods described herein can offer technical improvements over existing techniques of processing cells, such as fluorescence-activated cell sorting (FACS), including faster and more consistent cell processing at a much lower cost, throughput of at least about 109cells per hour, and higher purity and recovery rates than existing techniques. For example, FIGs. 3A-3G, 4A, 4B, and 5 illustrate that the platforms, cartridges, and methods described herein can significantly outperform other methods of cell processing such as FACS or MACS by generating higher throughput, better recovery and purity of target cells, and improved resolution into multiple subpopulations of cells.

[0074] FIG. 3A and FIG. 3B compare improved design and performance characteristics of second-generation cartridges (e.g., cartridge 600) versus first generation cartridges (e.g., cartridge 500). For example, as illustrated in FIG. 3B, cartridge 600, described herein elsewhere, can process cells at a rate of at least about 109cells per hour; can be injection molded for high consistency and low cost; can be fabricated from USP VI grade medical plastics; can be configured as a closed loop system; and can be plasma treated for consistent fluid flow. Compared to cartridge 600 and as illustrated in FIG. 3A, first generation cartridges may only process cells at a rate of atmost about 107cells per hour; may be fabricated with a high level of variation; may be fabricated from silicone elastomers; and may be configured as a manual, open loop system. FIG. 4A and FIG. 4B compare improved design and performance characteristics of cartridge 600 versus FACS. For example, as depicted in FIG. 4B, cartridge 600, described herein elsewhere, can include flowrate reducing structures with trapping surfaces to process cells at a rate of at least about 109cells per hour. Compared to cartridge 600 and as illustrated in FIG. 4A, FACS may process cells one at a time through a narrow flow channel at a rate of at most about 106cells per hour. FIG. 5 compares improved design and performance characteristics of cartridge 600 versus FACS and MACS. For example, cartridge 600, described herein elsewhere, can process cells at a rate of at least about 109cells per hour; can achieve cell recovery rates of at least about 95%; can achieve purity rates of at least about 95%; and can achieve better resolution with at least 4 subpopulations of cells.

[0075] The present disclosure also provides use cases of platforms, cartridges, and methods that can automate cell processing for diagnostic applications (e.g., diagnosing patients with chronic lymphocytic leukemia (CLL)) or therapeutic applications (e.g., treating patients CLL using leukapheresis or treating patients with an immunotherapy). In some cases, platforms, cartridges, and methods herein can be used to automate cell processing for treating patients with an immunotherapy. For example, the immunotherapy can include autologous T-cell based immunotherapy. In some cases, T-cell based immunotherapy can include CD8+or CD103+T-cells for the treatment of recurrent or refractory solid tumors. In some cases, said T-cells can be prepared from the patient’s peripheral blood mononuclear cells (PBMCs). In some cases, said T- cells or PBMCs can be obtained via a leukapheresis procedure using platforms, cartridges, and methods herein. In some cases, said T-cells can be expanded in cell culture, washed, and formulated as a cell suspension and cryopreserved. In some cases, said cell suspension can be used as a drug in immunotherapy to treat certain cancers, e.g., bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancers, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, or stomach cancer. In some cases, said cell suspensions can be used as a drug in immunotherapy to treat certain autoimmune diseases, e.g., rheumatoid arthritis (RA) or multiple sclerosis (MS).

[0076] FIG. 1 illustrates the principle of physical separation of heterogeneous cells based on biomarker expressions, e.g., proteins, RNA, and the like. Such separation can be important for therapeutic applications. There is an unmet need to obtain pure populations of cells (e.g., target cells) that are of interest in diagnostic and therapeutic engineering. FIG. 2 illustrates that, in microfluidic immunomagnetic cell sorting methods (MICS) described herein, complex fluidsamples containing rare cells of interest can be labeled with antibody-labeled magnetic nanoparticles (MNPs). The fluid samples can be flowed through one or more modular cartridges at predetermined flow velocities. The modular cartridges can include low-flow zones generated by flow rate-reducing structures (e.g., X-shaped structures). Different cell populations of the fluid samples with predetermined expression levels (e.g., high, high-medium, medium-low, and low) can be isolated or captured into different subsets of cell populations. These subsets can be further processed by, e.g., sequencing methods, expansion methods, characterization methods, and the like. The target cells can be captured on the cartridge according to their surface marker expression as described in U.S. Patents 10,073,079, 10,809,180, and 11,807,898 and U.S. Patent Application 18 / 547,832, each of which is incorporated by reference herein in its entirety.Cartridges for processing cellsOverview

[0077] The present disclosure provides cartridges for isolating or separating magnetically labeled cells (e.g., target cells) into one or more zones of a cartridge based on surface marker expression of the target cells. The target cells may be suspended in fluid samples collected from or prepared by a user, e.g., a subject, patient, doctor, healthcare technician, or healthcare provider.

[0078] Disclosed herein, in some embodiments, are cartridges for isolating target cells in a fluid sample. In some embodiments, the cartridge comprises a flow chamber configured to receive one or more fluids from one or more flow inlets. In some embodiments, the cartridge comprises one or more flow rate-reducing structures operatively coupled to the flow chamber configured to reduce a flow rate of the fluid sample proximate to the one or more flow rate-reducing structures. In some embodiments, the cartridge comprises a magnetic field magnetically coupled to the flow chamber configured to isolate the target cells proximate to the one or more flow rate-reducing structures. In some embodiments, the cartridge comprises a degassing region operatively coupled to the one or more flow inlets configured to remove bubbles from the one or more fluids.

[0079] In some embodiments, the reduced flow rate of the fluid sample is configured sufficiently low for a magnetic force of the magnetic field acting on the nanobeads to overcome a drag force of the reduce flow rate acting on the target cells thereby isolating the target cells proximate to the one or more flow-rate reducing structures. In some embodiments, a first fluid of the one or more fluids comprises a priming fluid, a second fluid comprises the fluid sample, and a third fluid comprises a washing fluid. In some cases, the terms cartridge and modular cartridge can be used interchangeably. In some cases, the cartridge can include cartridge 600 depicted in FIGs. 3B-3G

[0080] In some embodiments, the cartridge further comprises a magnetic field generator configured to generate the magnetic field. In some embodiments, the magnetic field causes amagnetic force to act on the magnetically susceptible nanobeads to isolate the target cells proximate to the flow rate-reducing structures. For example, the one or more magnetic arrays, e.g., comprising one or more permanent magnets, can create magnetic field lines that extend through (e.g., magnetically coupled) the cartridge and the one or more fluids of the cartridge. The magnetic field lines can couple with the one or more fluids comprising magnetically susceptible nanoparticles. The magnetic field lines can cause a magnetic force to act on the magnetically susceptible nanoparticles. In some cases, the magnetic field generator may be external to the cartridge, e.g., operatively coupled to the instrument, and configured to generate the magnetic field. For example, the magnetic field generator can be configured using the magnet actuator assembly described herein elsewhere. In some embodiments, the magnetic field generator is configured as one or more magnetic arrays. In some embodiments, at least one of the one or more magnetic arrays is a permanent magnet. In some cases, a magnetic array can be configured with a plurality of small magnets (e.g., micro-magnets) instead of a large magnet. The plurality of micromagnets can include at least 1 to 180 micro-magnetics. In some cases, the plurality of micromagnets can include more than 180 micro-magnetics. In some cases, each one of the plurality of micro-magnets can be sized to align with each one of the plurality of flow-rate reducing structures. In some cases, a magnetic array can be configured with magnets fabricated from alnico (AlNiCo), samarium cobalt (SmCo), neodymium iron boron (NdFeB), ferrite, or any combination thereof.

[0081] In some embodiments, the one or more magnetic arrays are configured to cause one or more magnetic forces to act on the magnetically susceptible nanobeads. In some cases, the magnetically coupling generates a magnetic force on the nanobeads to attract or aggregate the nanobeads towards the flow-rate reducing structures. In some cases, the magnetic field generator may generate a magnetic field of about 0.1 Tesla (T) to 10 T. In some cases, the magnetic field generator may generate a magnetic field of about 0.5 T to 1 T. In some cases, the magnetic field generator may generate a magnetic field of less than about 0.5 T. In some cases, the magnetic field generator may generate a magnetic field of greater than about 1 T. In some cases, the magnetic field may be substantially uniform across a thickness the cartridge or along a height of the plurality of flow-rate reducing structures. In some cases, the magnetic field may be substantially nonuniform (e.g., field gradients) across a thickness of the cartridge or along a height of the plurality of flow-rate reducing structures. In some cases, the magnetic field may be substantially uniform across one or more regions of the cartridge and substantially nonuniform across one or more other regions of the cartridge.

[0082] In some cases, the magnetic field generator may be integrated into the cartridge and configured to generate the magnetic field. For example, the magnetic field generator can be configured with planar microelectrodes arranged as one or more coils to generate the magneticfield. The planar microelectrodes can be configured to receive an electrical alternating current (AC) at a predetermined frequency and strength to generate the magnetic field. The magnetic field may be an electromagnetic field (e.g., alternating magnetic field) generated by the AC current. The magnetic field may generate a magnetic force on the magnetically susceptible nanobeads, which can be coupled to the target cells. The planar microelectrodes can be positioned proximate to the flow-rate reducing structures. The magnetic force may trap the target cells via the nanobeads proximate to the flow-rate reducing structures.

[0083] In some embodiments, the flow chamber is configured with one or more isolation zones comprising the one or more flow-rate reducing structures. In some embodiments, the one or more isolation zones are configured as a first zone and a second zone. For example, as illustrated in FIG. 3B, the flow chamber of the cartridge, e.g., cartridge 600, can include two isolation zones, e.g., Zone 1 and Zone 2. For example, as illustrated in FIG. 4B, each zone can include flow-rate reducing structures. The target cells may be trapped proximate to the flow-rate reducing structures by a combination of the magnetic field, trapping surfaces of the flow-rate reducing structures, or flow rate of the fluid sample.

[0084] In some embodiments, the cartridge is configured to flow the one or more fluids through the first zone at a first flow rate and to flow the one or more fluids through the second zone at a second flow rate. For example, as described herein elsewhere, the one or more fluids can include prime or priming fluids, fluid samples, and wash or washing fluids. The prime fluid may be used to prime the cartridge before cell processing. The fluid sample may contain the target cells to be isolated from the fluid sample. The wash fluid may be used to remove contaminants (e.g., non-target cells) from the cartridge and to recover target cells from the cartridge.

[0085] In some embodiments, a combination of the first flow rate and the second flow rate is configured based at least on the drag force acting on the target cells and the magnetic force acting on the nanobeads. For example, each of the flow-rate reducing structures may be configured to determine the first flow rate through the first zone, e.g., Zone 1, and the second flow rate through the second zone, e.g., Zone 2. Each flow rate may generate a drag force on the target cells. The drag force acting on the target cells can be overcome by the magnetic force acting on the target cells, via the nanobeads, to trap or isolate the target cells near the flow-rate reducing structures.

[0086] In some embodiments, a height or a width of the one or more flow-rate reducing structures in the first zone is identical as a height or a width of the one or more flow-rate reducing structures in the second zone. For example, flow-rate reducing structures in Zone 1 may be sized the same as flow-rate reducing structures in Zone 2. The drag force on the target cells in each zone may be similar or the same. In some cases, the height of each flow-rate reducing structure may be the principal determinant of the drag force on the target cells. In some embodiments, the height isabout 50 to about 150 micrometers (pm). In some embodiments, the height is about 55 gm to about 140 gm, 60 gm to 130 gm, about 65 gm to about 120 gm, about 70 gm to about 110 gm, about 75 gm to about 100 gm, or about 80 gm to about 90 gm. In some cases, the height of the flow rate-reducing structures is about 100 gm. In some cases, the height of the flow rate-reducing structures is about 100 gm. In some cases, the width is about 2 mm. A detailed description of cartridges (e.g., structures, materials, and sizes) is described herein elsewhere.

[0087] In some embodiments, a height or a width of the one or more flow-rate reducing structures in the first zone is different from a height or a width of the one or more flow-rate reducing structures in the second zone. For example, the flow-rate reducing structures in Zone 1 may be sized differently from the flow-rate reducing structures in Zone 2. The drag force on the target cells in each zone may be different. In some cases, the height of each flow-rate reducing structure may be a principal determinant of the drag force on the target cells. In some embodiments, the height comprises a range of from about 100 to 800 micrometers (gm). In some embodiments, the height is about 50 to about 150 micrometers (gm). In some embodiments, the height is about 55 gm to about 140 gm, 60 gm to 130 gm, about 65 gm to about 120 gm, about 70 gm to about 110 gm, about 75 gm to about 100 gm, or about 80 gm to about 90 gm. In some cases, the height of the flow rate-reducing structures in Zone 1 is about 100 gm. In some cases, the height of the flow rate-reducing structures in Zone 2 is about 400 gm. In some cases, the width is about 2 mm. A detailed description of cartridges (e.g., structures, materials, and sizes) is described herein elsewhere.

[0088] In some embodiments, the cartridge is further configured to transmit the one or more fluids to one or more flow outlets. For example, as described herein elsewhere, the cartridge may have one or more flow outlets. The one or more fluids can include prime fluids, fluid samples, and wash fluids. The prime fluid may be used to prime the cartridge before cell processing and then flowed out of a flow outlet by operation of pumps and valves described herein elsewhere. The fluid sample may contain the target cells during cell processing. The wash fluid may be used to remove contaminants (e.g., non-target cells) from the cartridge by flowing the contaminants out of a flow outlet by operation of pumps and valves described herein elsewhere. The wash fluid may be used to recover or remove target cells from the cartridge by flowing the target cells out of a flow outlet by operation of pumps and valves described herein elsewhere.

[0089] In some embodiments, the cartridge further comprises a degasser fluidically coupled to the degassing region, the flow chamber, or the one or more fluids. For example, as described herein elsewhere, bubbles may form in the tubesets of the cartridge when preparing the cartridge for cell processing. The degasser may be configured with a degassing region, which can include a bypass line. In some embodiments, the degasser is configured to change a flow of the one ormore fluids. In some embodiments, the degasser is configured to change the flow by bypassing the flow chamber and flowing the one or more fluids to one or more outlets. For example, the degasser may configure the bypass line to direct or change the flow of the one or more fluids around Zone 1 and Zone 2 thereby bypassing Zone 1 and Zone 2. By operation of the degasser in combination with valves and pumps, as described herein elsewhere, the bubbles may be removed from the tubesets or the cartridge before cell processing. In some cases, the bypass line can be configured as an external fluid line coupled to the tubesets on the input side of the cartridge and the output side of the cartridge. In some cases, the bypass line can be fabricated internal to the cartridge as a fluid channel (e.g., microfluidic channel) which couples the input side of the cartridge and the output side of the cartridge.

[0090] In some embodiments, the degasser comprises an optical sensor configured to detect the bubbles in the one or more fluids. For example, as described herein elsewhere, the optical sensor may be operatively coupled to the deck of the instrument and configured to detect bubbles in the tubesets or the cartridge. In some cases, the optical sensor may be integrated into the cartridge and configured to detect bubbles. For example, the optical sensor may be configured using a microelectronic optical sensor that can be sized to integrate into the cartridge.

[0091] Optical sensors can generally include through-beam sensors, retro-reflective sensors, or diffuse reflection sensors. In some cases, the optical sensor may be configured as a through- beam sensor. The sensor may use an infrared (IR) transmitter and receiver to detect optical differences (e.g., refractive index differences) between air bubbles and fluid. Through-beam sensors can be configured with two separate components. The components can include a transmitter and a receiver placed opposite to each other. The transmitter can project a light beam onto the receiver. An interruption of the light beam caused by a bubble, e.g., an air bubble, may be interpreted as a switch signal by the receiver. Retro-reflective sensors can be configured with a transmitter and a receiver in the same structure and with a reflector. Through the reflector, the transmitted light beam can be reflected back to the receiver. An interruption of the light beam caused by a bubble, e.g., an air bubble, may be interpreted as a switch signal by the receiver. Diffuse reflection sensors can be configured with transmitter and receiver in the same housing. The transmitted light is reflected by the object to be detected, e.g., air bubbles or fluid. Sensors for detecting bubbles can include non-optical sensors. For example, non-optical sensors can include ultrasonic sensors and capacitive sensors. Ultrasonic sensors can be configured to use an ultrasonic transmitter and receiver to detect changes or differences in strains in the walls of tubing caused by the passage of air bubbles and fluids. Capacitive sensors can be configured to detect changes or differences between the dielectric mediums of air bubbles and fluid.

[0092] In some embodiments, at least one of the one or more flow-rate reducing structures is configured with one or more trapping surfaces. In some cases, a shape of each flow-rate reducing structure may be a principal determinant to trap or retain the target cells when the magnetic force exceeds the drag force thereby pulling the target cells towards each flow-rate reducing structure. As illustrated in FIG. 4B, the shape of each flow-rate reducing structure may include one or more trapping surfaces. In some embodiments, at least one of the one or more trapping surfaces is configured as a concave surface defined by two joined arms defining an angle. For example, a concave surface of each flow-rate reducing structure can be concave towards the direction of flow of the one or more fluids. This can result in an arc-shaped flow rate-reducing structure. In some cases, each trapping surface can be shaped to reduce a flow rate of the fluid sample in a vicinity of the trapping surface.

[0093] In some embodiments, the cartridge further comprises a fluid flow generator and one or more valves configured to generate a flow of the one or more fluids through the flow chamber. For example, the fluid flow generator can be configured as a fluidic pump external to the cartridge. In some embodiments, the fluid flow generator comprises a peristaltic pump. For example, as described herein elsewhere, the fluid flow generator can be configured as a peristaltic pump. By operation of valves and the peristaltic pump, the one or more fluids can flow through the cartridge. In some cases, the fluid flow generator may be configured as a micromachining-based micropump, which can be sized and integrated into the cartridge. In some embodiments, the one or more valves comprises one or more solenoid pinch valves. For example, as described herein elsewhere, valves can be configured as solenoid pinch valves external to the cartridge. In some cases, valves may be configured as microfluidic valves sized and integrated into the cartridge.

[0094] In some cases, the cartridge can be configured to perform high-throughput processing of cells. In some cases, the cartridge can be configured to flow the one or more fluids at a flow rate of about 0.1 to 30 milliliters per hour (mL / hr). In some cases, the cartridge can be configured to flow the one or more fluids at a flow rate of about 0.5 to 3 mL / hr. In some cases, the cartridge can be configured to flow the one or more fluids at a flow rate determined by the magnetic force acting on the target cells via the magnetic nanobeads. For example, isolation of target cells based on messenger ribonucleic acid (mRNA) expression can include flow rates of about 0.5 to 1 mL / hr. For example, isolation of target cells based on protein or antibody expression can include flow rates of about 1 to 3 mL / hr.

[0095] In some embodiments, the cartridge is configured to perform high-throughput processing of the fluid sample at a rate of greater than or equal to about 109cells per hour. In some cases, the cartridge can be configured to perform high-throughput processing of the fluid sample at a rate of about 106to about 109or about 107to about 108cells per hour. In some cases, thecartridge can be configured to perform high-throughput processing of the fluid sample at a rate of less than or equal to about 106cells per hour. In some cases, the cartridge can be configured to isolate the target cells from the fluid sample with a purity of about 65% to about 95%, about 70% to about 90%, or about 75% to about 85%. In some embodiments, the cartridge can be configured to recover the target cells from the fluid sample with a purity of greater than or equal to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the cartridge can be configured to recover the target cells from the fluid sample with a purity of less than or equal to about 99%, 95%, 90%, 85%, 80%, 75%, or 70%. In some cases, the cartridge can be configured to isolate the target cells from the fluid sample with a purity of greater than 95%. In some embodiments, the cartridge can be configured to recover the target cells from the fluid sample with a recovery rate of about 65% to about 95%, about 70% to about 90%, or about 75% to about 85%. In some embodiments, the cartridge can be configured to recover the target cells from the fluid sample with a recovery rate of greater than or equal to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the cartridge can be configured to recover the target cells from the fluid sample with a recovery rate of less than or equal to about 99%, 95%, 90%, 85%, 80%, 75%, or 70%. In some embodiments, the recovery rate is the percentage of target cells input into the cartridge that are recovered by the methods and systems of the present disclosure. In some cases, the cartridge can be configured to recover the target cells from the fluid sample with a recovery rate of greater than 95%.Cartridge structures

[0096] FIGs. 3C, 3F and 3G depict or illustrate a cartridge, which can include a base portion, flow chamber walls, and a top portion. As depicted in FIG. 3C, the flow chamber walls, base portion, and top portion can collectively form one or more flow chambers of the cartridge. FIG. 3F illustrates a perspective view of a cartridge, e.g., cartridge 600. FIG. 3G illustrates a plan view of a cartridge, e.g., cartridge 600. In some cases, the cartridge can be configured with two flow chambers. The cartridge can further include one or more flow inlets. In some cases, the cartridge can be configured with 2 flow inlets. The cartridge can further include one or more flow outlets. In some cases, the cartridge can be configured with 2 flow outlets. The cartridge can further include one or more zones of the one or more flow chambers. In some cases, the cartridge can be configured with two zones, e.g., Zone 1 and Zone 2. The one or more zones can include a plurality of flow-rate reducing structures. The plurality of flow-rate reducing structures can be configured with different sizes or shapes to generate different flow rates of the one or more fluids. In some cases, the plurality of flow-rate reducing structures can include 1 to 200 structures. In some cases, the plurality of flow-rate reducing structures can include more than 200 structures.

[0097] In some cases, the base portion can be fabricated from silicon, quartz, fused silica, glass, a ceramic, a polymer, a metal, or any combination thereof. In some cases, the base portion can be surface activated. In some cases, the top portion can be fabricated from silicon, quartz, fused silica, glass, a ceramic, a polymer, a metal, or any combination thereof. In some cases, the top portion can be surface activated. In some cases, the flow chamber walls can be fabricated from silicon, quartz, fused silica, glass, a ceramic, a polymer, a metal, or any combination thereof. In some cases, the flow chamber walls can be surface activated. In some cases, the flow rate-reducing structures of the flow chamber can be surface activated. In some cases, the flow-rate reducing structures can be fabricated from silicon, quartz, fused silica, glass, a ceramic, a polymer, a metal, or any combination thereof. In some cases, surface activation can include activation by oxygen plasma treatment.

[0098] In some cases, the base portion may have a thickness of about 0.05 mm to 0.5 mm. In some cases, the base portion may have a thickness of about 0.1 mm. In some cases, the base portion may have a length of about 30 mm to 300 mm. In some cases, the base portion may have a length of about 75 mm. In some cases, the base portion may have a width of about 12.5 mm to 125 mm. In some cases, the base portion may have a width of about 50 mm.

[0099] In some cases, the top portion may have a thickness of about 0.05 mm to 0.5 mm. In some cases, the top portion may have a thickness of about 0.1 mm. In some cases, the top portion may have a length of about 30 mm to 300 mm. In some cases, the top portion may have a length of about 75 mm. In some cases, the top portion may have a width of about 12.5 mm to 125 mm. In some cases, the top portion may have a width of about 50 mm.

[0100] In some cases, a flow chamber may have a thickness (or height) of about 10 pm to 1000 pm. In some cases, the first flow chamber (e.g., Zone 1) may have a thickness of about 100 pm. In some cases, the second flow chamber (e.g., Zone 2) may have a thickness of about 400 pm. In some cases, the thickness may be determined by a height of the plurality of flow-rate reducing structures. In some cases, a flow chamber may have a length of about 30 mm to 300 mm. In some cases, the flow chamber may have a length of about 75 mm. In some cases, a flow chamber may have a length that extends substantially from an inlet of the cartridge to an outlet of the cartridge. In some cases, a flow chamber may have a width of about 12.5 mm to 125 mm. In some cases, the flow chamber may have a width of about 25 mm. In some cases, a flow chamber may have a width that extends substantially from an edge of the cartridge to an opposite edge of the cartridge.

[0101] FIG. 3D and FIG. 3E depict different structures of the plurality of flow-rate reducing structures. FIG. 3C depicts the second-generation cartridge, which has been engineered for high- throughput therapeutics applications. FIG. 3D depicts a perspective view of different heights ofthe flow-rate reducing structures of the second-generation cartridge. In some cases, a flow-rate reducing structure can be fabricated as an X-shaped structure. In some cases, described herein elsewhere, the flow-rate reducing structure can include one or more concave trapping surfaces to isolate or trap the target cells. In some cases, a flow-rate reducing structure can be fabricated as a V-shaped structure or a C-shaped structure. As depicted in FIG. 3D, a flow-rate reducing structure may have a height. In some cases, a flow-rate reducing structure may have a height of about 10 pm to 1000 pm. In some cases, a flow-rate reducing structure may have a height of less than about 10 pm. In some cases, a flow-rate reducing structure may have a height of greater than about 1000 pm. In some cases, a flow-rate reducing structure may have a height of about 100 pm, 200 pm, 400 pm, or 800 pm. In some cases, the flow-rate reducing structures of Zone 1 may have a height of about 100 pm. In some cases, the flow-rate reducing structures of Zone 2 may have a height of about 400 pm. In some cases, the plurality of flow-rate reducing structures may have a same or uniform height. In some cases, the plurality of flow-rate reducing structures may have a plurality of different or nonuniform heights.

[0102] Further depicted in FIG. 3D, a flow-rate reducing structure may have a length. The length may be determined from an end of the flow-rate reducing structure to an opposite end of the flow-rate reducing structure. In some cases, a flow-rate reducing structure may have a length of about 0.5 mm to 5 mm. In some cases, a flow-rate reducing structure may have a length of less than about 0.5 mm. In some cases, a flow-rate reducing structure may have a length of greater than about 5 mm. In some cases, a flow rate-reducing structure may have a length of about 1 mm. In some cases, a flow-rate reducing structure may have two or more lengths. For example, a first arm of the X-shaped structure may be a different length than a second arm of the X-shaped structure. In some cases, a first arm of a flow-rate reducing structure may have a length of about 0.25 mm to 2.5 mm. In some cases, a first arm of a flow-rate reducing structure may have a length of less than about 0.25 mm. In some cases, a first arm of a flow-rate reducing structure may have a length of greater than about 2.5 mm. In some cases, a first arm of a flow rate-reducing structure may have a length of about 0.25 mm. In some cases, a second arm of a flow-rate reducing structure may have a length of about 0.25 mm to 2.5 mm. In some cases, a second arm of a flow-rate reducing structure may have a length of less than about 0.25 mm. In some cases, a second arm of a flow-rate reducing structure may have a length of greater than about 2.5 mm. In some cases, a second arm of a flow rate-reducing structure may have a length of about 0.25 mm. In some cases, the plurality of flow-rate reducing structures may have a same or uniform length. In some cases, the plurality of flow-rate reducing structures may have a plurality of different or nonuniform lengths.

[0103] Further depicted in FIG. 3D, a flow-rate reducing structure may be positioned with a distance or pitch from another flow-rate reducing structure. The distance may be determined from a center of a flow-rate reducing structure to a center of another flow-rate reducing structure. In some cases, a distance from one flow-rate reducing structure to another flow-rate reducing structure may have a distance of about 0.5 mm to 5 mm. In some cases, a distance from one flowrate reducing structure to another flow-rate reducing structure may have a distance of less than about 0.5 mm. In some cases, a distance from one flow-rate reducing structure to another flowrate reducing structure may have a distance of greater than about 5 mm. In some cases, a distance from one flow-rate reducing structure to another flow-rate reducing structure may have a distance of about 1 mm. In some cases, the plurality of flow-rate reducing structures may be spaced at a same or uniform distance. In some cases, the plurality of flow-rate reducing structures may be spaced at a plurality of different or nonuniform distances.

[0104] In some cases, the one or more flow chambers of the cartridge can be configured as one or more microfluidic channels. In some cases, the one or more microfluidic channels can include the degasser and the degassing zone disposed therein. In some cases, the cartridge can be configured as two or more cartridges, e.g., a modular cartridge. In some cases, the two or more cartridges can be fluidically connected to each other in parallel. In some cases, the two or more cartridges can be fluidically connected to each other in series. In some cases, the two or more cartridges can be configured as a first modular cartridge and a second modular cartridge. In some cases, the flow chamber of the first cartridge comprises an outlet and the flow chamber of the second cartridge comprises an inlet. In some cases, the outlet of the flow chamber of the first cartridge is in direct fluid communication with the inlet of the flow chamber of the second modular cartridge.Methods for processing cells

[0105] The present disclosure provides methods for isolating or separating magnetically labeled cells (e.g., target cells) into one or more zones of the cartridge based on surface marker expression of the target cells. The target cells may be suspended in fluid samples collected from or prepared by a user, e.g., a subject, patient, doctor, healthcare technician, or healthcare provider.

[0106] Disclosed herein, in some embodiments, are methods for using a platform for isolating target cells in a fluid sample. In some embodiments, the method comprises (a) providing the cartridge described herein elsewhere. In some embodiments, the method comprises (b) connecting the cartridge to the platform described herein elsewhere. In some embodiments, the method comprises (c) priming the cartridge by flowing a priming fluid of the one or more fluids through the cartridge. In some embodiments, the method comprises (d) removing bubbles from the cartridge. In some embodiments, the method comprises (e) flowing the fluid sample of the one ormore fluids through the cartridge. In some embodiments, the method comprises (f) applying the magnetic field to isolate the target cells in the fluid sample. In some embodiments, the method comprises (g) washing the cartridge by flowing a washing fluid of the one or more fluids through the cartridge. In some embodiments, the method comprises (h) recovering the isolated target cells in the fluid sample. In some embodiments, the target cells comprise peripheral blood mononuclear cells (PBMCs), tumor-reactive lymphocytes (TRLs), circulating TRLs, or tumor-infiltrating leukocytes (TILs). In some cases, the cartridge can include cartridge 600 depicted in FIGs. 3B- 3G and described herein elsewhere.

[0107] FIG. 8 illustrates that the fluid samples may be initially processed upstream of workflows associated with the platform described herein. For example, a sample may be obtained from a patient, the sample may be transported to a contract development and manufacturing organization- (CDMO) facility, the CDMO-facility may process the sample to obtain a fluid sample with magnetically-labelled target cells, and the fluid sample may be provided to the platform described herein.

[0108] FIG. 9 illustrates a high-level overview of workflows for processing cells using the platform described herein. Workflows can include preparation of the cartridge before target cell isolation protocols. Preparation can include, e.g., cartridge fabrication, cartridge activation using plasma treatment, cartridge priming, or cartridge degassing. Workflows can include magnetically labeling the target cells, isolating the target cells, removing (e.g., washing) non-target cells, and recovering (e.g., washing) the target cells for downstream applications.

[0109] FIGs. 10A-10C depict example equipment, e.g., plasma chambers, configured to perform cartridge activation workflows. For example, a cartridge can be placed in the plasma chamber without blocking access to the cartridge, e.g., inlets and outlets of the cartridge. FIG. 10A depicts that, after closing the door to the chamber, the plasma chamber swaglok can be turned until the arrow on the swaglok is facing up to close air circuit to the chamber for vacuum buildup. The power switch can be turned on followed by the vacuum pump switch. The power and pump can be left on for about 30 minutes to build sufficient vacuum in the chamber. FIG. 10B depicts that, after about 30 minutes, the radio frequency (RF) level knob can be turned to high, and the plasma can be left on for about 15 minutes. FIG. 10C depict that, after about 30 minutes, the radio frequency (RF) level knob can be turned to high, and the plasma can be left on for about 15 minutes. The chamber may have a glow, e.g., a white or blue glow. After about 15 minutes, the RF level can be turned down. The swaglok can be turned so that the arrow is facing left towards the short end to let air enter the chamber for pressurization. The chamber can be opened to retrieve the cartridge for further processing workflows, e.g., cartridge assembly. The equipment can beshut down by turning the RF level to OFF, the pump off, and the power off. Cartridges can be plasma treated in bulk and used for at least up to a week or more.

[0110] FIG. 11A and FIG. 11B depict cartridge assembly workflows that may be performed before degassing protocols. For example, FIG. 11B depicts that male luer lock-to-barb connectors can be coupled to inlets and outlets of the cartridge. FIG. 11A depicts that placing the tubing over the ridge on the connector and extending by about 0.5 to 5 millimeters (mm) over the ridge can avoid the tubing popping off during degassing protocols and other workflows. In some cases, placing the tubing over the ridge on the connector and extending by about 1 mm over the ridge can avoid the tubing popping off during degassing protocols and other workflows.

[0111] FIGs. 12A-12C depict degassing workflows that can include connecting the inlet of the cartridge (e.g., inlet next to the notch, shorter tubing length) to the peristaltic pump. The outlet of the cartridge (e.g., longer tubing length) can be connected to the valve. Protocols can include ensuring that no extra tubing, or substantially no extra tubing, is present. Additional tubing can increase volume of the degassing, which can lead to an increase in pressure causing the tubing to ‘pop off or become detached from the connector. Control software or routines of the instrument can be run on the cartridge while the inlets and outlets of the cartridge are kept facing up. Degassing protocols can be run for about 6 minutes per cartridge. The cartridge can be inspected for bubbles, e.g., air bubbles. FIG. 12A depicts examples of non-degassed cartridges. FIG. 12B depicts examples of non-degassed cartridges. FIG. 12C depicts an example of degassed cartridge. If bubbles are observed, the degassing protocol can be run again. Pinch clamps can be put on the inlet and outlet tubing of the cartridge before the tubing is disconnected. The de-gassed cartridge can be used for automated cell processing of the fluid samples.

[0112] The cartridge can include inlets (e.g., 3 inlets) for flowing different fluids during cell processing, e.g., prime fluids, sample fluids, and wash fluids. The different fluids can be prepared and filled into containers upstream of the cell processing workflow. The cartridge can use workflows that may include priming of the tubing and the cartridge, which can be carried out by flowing priming (or prime) fluids through the flow chamber. Sample fluid containers can be filled in a sterile environment by the operator at the time of use. Wash and prime fluid containers may be filled in a sterile environment by the operator at the time of use or by a qualified sterile filling vendor prior to use. The input containers (e.g., prime, sample, and wash fluid containers) may be separate from the rest of the consumable tubesets until time of use. Open ends of the input tubes can be sealed with a plug to maintain sterility.

[0113] In some cases, the platform can be configured with tubing connecting the input fluid containers to the inlet of the flow chamber of the cartridge. In some cases, the tubing can be operatively coupled to the pinch valves for the controlled release of wash buffer from the washcontainer, the fluid sample from the fluid sample container, and the priming solution from the prime container. In some cases, the one or more output containers can be configured to be in fluidic communication with an outlet of the flow chamber of the cartridge. In some cases, the platform can be configured with tubing connecting the one or more output containers to the outlet of the flow chamber of the cartridge. In some cases, the tubing can be operatively coupled to the pump and pinch valves for controlled release of wash buffer, fluid sample, or priming solution from the outlet of the flow chamber of the cartridge. In some cases, the tubing can be operatively coupled to the pump and pinch valves for the controlled release of wash buffer, fluid sample, or priming solution from the outlet of the flow chamber of the cartridge to the pump, e.g., peristaltic pump. In some cases, the tubing can be operatively coupled to the pump and pinch valves configured for the controlled release of wash buffer, fluid sample, or priming solution from the pump to the one or more output containers. In some cases, the tubing can be operatively coupled to the pump and pinch valves for the controlled release of wash buffer, fluid sample, or priming solution from the pump to the one or more waste containers.

[0114] FIG. 13 illustrates a non-limiting example of a workflow for using the instrument 2300 described herein. In some embodiments, the workflow comprises (1) connecting one or more input containers to the dry consumable by sterile tube welding; (2) hanging the input and output containers on the poles of the instrument 2300, (3) inserting the cartridge into the cartridge interface of the instrument 2300, (4) loading the tubing into the pinch valves and peristaltic pump, (5) releasing the manual pinch clamps on the input fluids, (6) running the cell processing procedure, (7) closing the manual pinch clamps on the one or more output containers, (8) unloading the consumable from the pinch valves and peristaltic pump, (9) disconnecting the one or more output containers by sterile tube sealing, and (10) disposing of the consumable as a biohazard waste.

[0115] For example, FIGs. 14A-14E depict workflows that may require loading consumables for cell processing. FIG. 14A depicts that a user can start with consumables laid out from left to right in front of the instrument. FIG. 14B depicts that the output and input containers can be placed on poles. FIG. 14C depicts that the cartridge can then be loaded into the cartridge interface of the platform. FIG. 14D depicts that tubes can be connected to valves 2340 and a peristaltic pump 2360 to complete the loading of consumables. FIG. 14E depicts the platform configured to isolate target cells from fluid samples. A graphical user interface (GUI) application on the platform can also provide step-by-step instructions for loading consumables. FIG. 15 depicts an example of correct tube loading and incorrect tube loading. The GUI application can also enable the user to perform pre-flight checks before starting a run or to do them automatically, e.g., a connection testto a controller (e.g., AllMotion® Controller), whether a valid script is loaded, or whether the tube is properly loaded in bubble sensor.

[0116] FIGs. 16A-16D illustrate example platform workflows associated with preparing the cartridge to perform other workflows, e.g., protocols for isolating or sorting target cells based on surface marker expression. In some cases, as illustrated in FIGs. 16A-16D, the terms cartridge and chip can be used interchangeably. Workflows can be performed by operation of valves (e.g., valves 1 through 11) and the pump. In some embodiments, the priming in (c) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. For example, FIG. 16A illustrates workflows for preparing the cartridge, which can include a starting state of the cartridge. For example, input containers and associated tubing can be filled with fluid, e.g., prime, sample, and wash fluids. The remainder of the tubeset, including the cartridge, can be filled with air. FIG. 16B illustrates workflows for priming the tubeset. For example, workflows can include priming the entire tubeset, or substantially all of the tubeset, upstream of the pump with fluid. FIG. 16C illustrates workflows for priming the tubeset. For example, workflows can include priming the entire tubeset, or substantially all of the tubeset, upstream of the pump with fluid. By operation of valves (e.g., valves 2-5 and 8-9) and the pump, fluid can be primed from the input containers (e.g., sample and wash containers) through the bypass line to prevent air or fluid slug trains (e.g., bubbles) from passing through the cartridge in downstream operations. FIG. 16D illustrates that bubbles upstream of Zone 1 may still exist and the bypass tube between Zones 1 and 2 may be filled with fluid. Workflows can include priming the cartridge when bubbles are present, which may occur if Zones 1 and 2 in series are primed using the bypass line. In some cases, fluid-air-fluid will pass through Zone 2, which can disrupt priming and introduce air bubbles into the tubesets or the cartridge.

[0117] FIGs. 17A-17D illustrate example platform workflows associated with preparing the cartridge to perform other workflows, e.g., protocols for isolating or sorting target cells based on surface marker expression. In some cases, as illustrated in FIG. 17A and FIG. 17B, the terms cartridge and chip can be used interchangeably. In some embodiments, the removing in (c) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. FIG. 17A illustrates workflows for priming Zone 1, which can include preventing or removing bubbles, e.g., degassing by the degasser. By operation of valves (e.g., valves 1, 6, 8, and 9) and the pump, Zone 1 can be primed independently of Zone 2 to move the bubbles downstream of the pump without passing through Zone 2. FIG. 17B illustrates workflows for priming Zone 2, which can include preventing or removing bubbles, e.g., degassing by the degasser. By operation of valves (e.g., valves 1, 4, 6-7, and 9) and the pump, Zone 1 and all tubingup and downstream may already be primed with fluid. Zone 2 can be primed in series using the bypass line without passing bubbles after fluid is introduced.

[0118] FIG. 18 illustrates example platform workflows associated with operating the cartridge to perform isolating or sorting of target cells based on surface marker expression. In some cases, as illustrated in FIG. 18, the terms cartridge and chip can be used interchangeably. In some embodiments, the flowing in (e) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. By operation of valves (e.g., valves 2, 4, 6-7, and 9) and the pump, sample fluid flows through Zones 1 and 2 in series at a specified flow rate, which can be based on user input or a preset program via the GUI describe herein elsewhere. In some embodiments, the applying in (f) further comprises using the instrument to automatically apply the magnetic field by operation of the magnet actuator assembly. The sample fluid can be flowed through Zones 1 and 2 in series to capture magnetically tagged cells, e.g., the target cells. The target cells can be captured on the cartridge according to their surface marker expression as described in U.S. Patents 10,073,079, 10,809,180, and 11,807,898, and U.S. Patent Application 18 / 547,832, each of which is incorporated by reference herein in its entirety.

[0119] FIGs. 19-21 illustrate example platform workflows associated with operating the cartridge, e.g., protocols for recovering or collecting target cells from the cartridge. In some cases, as illustrated in FIGs. 19-21, the terms cartridge and chip can be used interchangeably. In some embodiments, the washing in (g) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. FIG. 19 illustrates workflows for washing fluid flows through Zones 1 and 2 in series to remove any contaminating cells (e.g., non-target cells) while the magnet array is engaged. By operation of valves (e.g., valves 3-4, 6-7, and 9) and the pump, wash flow solution can be flowed through Zones 1 and 2 in series to remove contaminating cells. In some embodiments, the recovering in (h) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves. FIG. 20 illustrates workflows to recover the target cells from Zone 1 while the magnet array is disengaged. By operation of valves (e.g., valves 3, 6, 8, and 10) and the pump, wash fluid can be flowed through the zones individually, e.g., Zone 1. In some cases, the magnet array can be engaged or disengaged. Wash fluid can flow through Zone 1 of the cartridge to resuspend the captured target cells and flow them into a dedicated output container (e.g., output 1). FIG. 21 illustrates workflows to recover the target cells from Zone 2 while the magnet array is disengaged. By operation of valves (e.g., valves 3, 5, 7, and 11) and the pump, wash fluid can be flowed through the zones individually, e.g., Zone 2. In some cases, the magnet array can be engaged or disengaged. Wash fluid can flow through Zone 2 of the cartridge to resuspend the captured target cells and flow them into a dedicated output container (e.g., output 2).

[0120] The GUI can allow the user to cancel the run at any point in the workflows. If the run is canceled and a script is currently running, the software can automatically terminate all commands on the controller, e.g., AllMotion® controller. In some cases, the user may be instructed to manually close the input and output container pinch clamps, in which case, the software can send a command to open all the valves and instruct the user to remove consumables. Upon completion, the software can return to the Start or Home Screen. FIG. 22 depicts that, after cell processing in the cartridge, the target cells may be further processed for characterization or therapeutic purposes.Fluid sample preparation

[0121] In some cases, the one or more fluids comprises a fluid sample. In some cases, the fluid sample is or comprises a bodily fluid. Non-limiting examples of bodily fluids include whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph, fecal extract, or cheek swab. In some cases, the one or more fluids comprises a cell or tissue suspension comprising tissue obtained through surgical biopsy, primary patient derived cell lines, or archived patient samples in the form of preserved samples, or fresh frozen samples. In some cases, the tissue is tumor tissue. Suitable methods for preparing a cell suspension from tissue are provided in Reichard A, Asosingh K. Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry. Cytometry A. 2019 Feb;95(2):219-226, which is hereby incorporated by reference in its entirety.

[0122] In some cases, the fluid is a priming fluid. In some cases, the priming fluid comprises phosphate-buffered saline (PBS). In some cases, the priming fluid comprises PBS with ethylenediaminetetraacetic acid (PBS-EDTA). In some cases, the priming fluid comprises Pluronic® F-68 1% solution. In some cases, the priming fluid comprises EDTA (50%) plus ImmunoCult™ media (50%). In some cases, the priming fluid comprises 100% EDTA. In some embodiments, the priming fluid comprises Dulbecco’s PBS formula, which can include a buffer solution for maintaining cell media in the physiological pH range of 7.0 to 7.6. In some embodiments, the priming fluid comprises Hank’s buffer, which can include a buffer solution for maintaining cell media in the physiological pH range of 7.0 to 7.6.

[0123] In some cases, the fluid is a wash fluid. In some cases, the wash fluid comprises phosphate-buffered saline (PBS). In some cases, the wash fluid comprises PBS with ethylenediaminetetraacetic acid (PBS-EDTA). In some cases, the wash fluid comprises EDTA (100%). In some cases, the wash fluid comprises EDTA (50%) and ImmunoCult™ media (50%). In some embodiments, the wash fluid comprises Dulbecco’s PBS formula, which can include a buffer solution for maintaining cell media in the physiological pH range of 7.0 to 7.6. In some embodiments, the wash fluid comprises Hank’s buffer, which can include a buffer solution for maintaining cell media in the physiological pH range of 7.0 to 7.6.

[0124] In some cases, the one or more fluids is the fluid sample comprising the target cells labeled with magnetically susceptible nanoparticles. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nanometers (nm) to 700 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 30 nm to about 600 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 40 nm to about 500 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 50 nm to about 400 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 60 nm to about 300 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 70 nm to about 200 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 80 nm to about 100 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 90 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 80 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 70 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 60 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 50 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 40 nm. In cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm to about 30 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 20 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 30 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 400 nm. In some cases, the magnetically susceptible nanoparticles have a diameter of about 700 nm.

[0125] In some cases, the magnetically susceptible nanoparticles comprise magnetic beads. In some cases, the magnetic beads are superparamagnetic beads. In some cases, the magnetic beads are modified. In some cases, the magnetic beads are carboxylate-modified magnetic beads, amine- blocked magnetic beads, oligo(dT)-coated magnetic beads, streptavidin-coated magnetic beads, streptavidin-blocked magnetic beads, NeutrAvidin™ coated magnetic beads, IgA and IgG protein magnetic beads, silica-coated magnetic beads, Mag-sepharose® magnetic beads, or any combination thereof. Non-limiting examples of superparamagnetic beads include Dynabeads® (Thermo Fisher Scientific).

[0126] In some cases, the modified magnetic beads (e.g., superparamagnetic beads) are coupled to an antibody or antigen-binding fragment specific to a surface protein of a target cell. In some cases, the antibody or antigen-binding fragment comprises a Fab, F(ab)2, a single-domain antibody, a single chain variable fragment (scFv), or a nanobody. In some cases, the antibody or antigen-binding fragment are coupled to the magnetic bead through conjugation. In some cases, the conjugation is covalent. In some cases, the magnetic bead is hydrophobic. In some cases, thehydrophobic magnetic bead comprises a carboxylic acid that binds amine groups at pH 5-6 at room temperature. In some cases, the hydrophobic magnetic bead comprises toluenesulfonyl groups that bind amine and sulfhydryl groups at neutral pH and 37 degrees Celsius. In some cases, the magnetic bead is hydrophilic. In some cases, the hydrophilic magnetic bead comprises amine groups that bind aldehydes at neutral to high pH at room temperature. In some cases, the hydrophilic magnetic bead comprises epoxy that binds amine and sulfhydryl groups at neutral pH at a range of temperatures. In some cases, the conjugation is noncovalent.

[0127] In some cases, the antibody or antigen binding fragment is specific to a marker on a mononuclear cell. In some cases, the mononuclear cell is a tumor-infiltrating lymphocyte (TIL). In some cases, the TIL is an intratumoral TIL. In some cases, the TIL is a stromal TIL. In some cases, the TIL is tumor-reactive. In some cases, the marker comprises T-Cell Surface Glycoprotein CD3 (“CD3” including CD3-gamma, CD3-epsilon, CD3-delta and / or or CD3-zeta subunits), T- Cell Surface Glycoprotein CD4 (“CD4”- NCBI Gene: 920; UniProtKB / Swiss-Prot: P01730), cluster of differentiation 8 (“CD8” including CD8 Subunit Alpha - NCBI Gene: 925, UniProtKB / Swiss-Prot: P01732 and / or CD8 Subunit Beta - NCBI Gene: 926, UniProtKB / Swiss- Prot: P10966), Ectonucleoside Triphosphate Diphosphohydrolase 1 (“CD39” - NCBI Gene: 953, UniProtKB / Swiss-Prot: P49961), Integrin Subunit Alpha E (“CD103” - NCBI Gene: 3682, UniProtKB / Swiss-Prot: P38570), Solute Carrier Family 6 Member 19 (“SLC6A19” - NCBI Gene: 340024, UniProtKB / Swiss-Prot: Q695T7), or SID1 Transmembrane Family Member 1 (“SIDT1” - NCBI Gene: 54847, UniProtKB / Swiss-Prot: Q9NXL6), Programmed cell death protein 1 (“PDF’ - NCBI Gene: 5133, UniProtKB / Swiss-Prot: Q15116), Hepatitis A Virus Cellular Receptor 2 (“TIM-3” - NCBI Gene: 84868, UniProtKB / Swiss-Prot: Q8TDQ0), Lymphocyte Activating 3 (“LAG-3” - NCBI Gene: 3902, UniProtKB / Swiss-Prot: P18627), T Cell Immunoreceptor With Ig And ITIMDomains (“TIGIT”-NCBI Gene: 201633, UniProtKB / Swiss-Prot: Q495A1), CD244 Molecule (“CD244”- NCBI Gene: 51744, UniProtKB / Swiss-Prot: Q9BZW8), Cytotoxic T- Lymphocyte Associated Protein 4 (“CTLA-4” - NCBI Gene: 1493, UniProtKB / Swiss-Prot: P16410), B And T Lymphocyte Associated (“BTLA” - NCBI Gene: 151888, UniProtKB / Swiss- Prot: Q7Z6A9), or any combination thereof. In some cases, the antibody or antigen-binding fragment is specific to components of the sample other than the target marker and depletes or substantially depletes the sample of the other components so that the target cell expressing the marker of interest is isolated by negative selection. In some cases, other components of the sample include, but are not limited to CD8+ T cells, B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes and erythrocytes.

[0128] The fluid sample can include the target cells conjugated to magnetically-labeled antibodies and magnetic nanoparticles (MNPs), which is further described herein elsewhere. Insome cases, the magnetically-labeled antibody can be conjugated to the magnetic nanoparticle. In some cases, the conjugation can include contacting the fluid sample with a second magnetically- labeled antibody specific to a second target cell. In some cases, the second magnetically-labeled antibody can be conjugated to the magnetic nanoparticle. In some cases, the fluid sample can be contacted with a plurality of major histocompatibility complex (MHC) multimers mimicking a tumor epitope. In some cases, at least one of the plurality of MHC multimers is conjugated to a linker molecule. In some cases, the linker molecule comprises a fluorophore coupled thereto. In some cases, the linker molecule comprises a magnetic nanoparticle coupled thereto. In some cases, the linker molecule is conjugated to the magnetic nanoparticle, the fluorophore, or both. In some cases, the fluid sample can be obtained by dissolving a patient sample (e.g., a tumor sample) into a solution. In some cases, the fluid sample can include peripheral blood. In some cases, the fluid sample can include vascularized tumors, malignant pleural effusion, lymphatic fluid, a fluid portion of bone marrow, or another cell-carrying fluid.Instruments for processing cells

[0129] The present disclosure provides instruments or platforms for isolating or separating magnetically labeled cells (e.g., target cells) into one or more zones of the cartridge based on surface marker expression of the target cells or messenger RNA (mRNA) of the target cells. The target cells may be suspended in fluid samples collected from or prepared by a user, e.g., a subject, patient, doctor, healthcare technician, or healthcare provider.

[0130] Disclosed herein, in some embodiments, are platforms for isolating target cells in a fluid sample. In some embodiments, the platform comprises the cartridge described herein elsewhere. In some embodiments, the platform comprises one or more pumps configured to flow the one or more fluids through the flow chamber of the cartridge. In some embodiments, the platform comprises one or more valves configured to stop or start the flow of the one or more fluids through the flow chamber of the cartridge. In some embodiments, the platform comprises one or more magnetic arrays configured to apply the magnetic field to the magnetically susceptible nanobeads in the flow chamber. In some embodiments, the platform comprises one or more bubble sensors configured to detect bubbles in the one or more fluids of the cartridge. In some embodiments, the platform comprises one or more processors configured to control one or more processes for isolating the target cells from the fluid sample in the cartridge. In some embodiments, the platform comprises a power supply configured to supply power to the platform. In some cases, the terms platform and instrument can be used interchangeably. In some cases, the cartridge can include cartridge 600 depicted in FIGs. 3B-3G and described herein elsewhere.Deck of the platform

[0131] In some embodiments, the platform further comprises a deck operatively coupled to the one or more pumps, the one or more valves, the one or more magnetic arrays, or the one or more bubble sensors. In some embodiments, the deck further comprises a deck framing and a deck enclosure. In some embodiments, the deck framing is operatively coupled to the deck enclosure.

[0132] FIG. 23 illustrates an instrument 2300 (or alternatively platform) that can be configured to perform operations or workflows associated with cell processing. In some cases, instrument 2300 can perform operations with no user intervention outside of a sterile environment. In some embodiments, the deck framing is operatively coupled to the one or more pumps, the one or more valves, the one or more magnetic arrays, or the one or more bubble sensors. Instrument 2300 configured for automated cell processing can include one or more valves 2340 (e.g., solenoid pinch valves), one or more bubble sensors 2350 (e.g., optical sensors), one or more pumps 2360 (e.g., peristaltic pumps), a magnet actuator subassembly (MSA) 4000, one or more displays 2370 (e.g., a GUI or touch-screen displays), framing and enclosures, one or more container poles 2330, one or more power supplies, and one or more cooling fans. Instrument 2300 can be configured as two major subsystems, e.g., main body 2310 and deck 2320. Main body 2310 can include at least include framing, enclosures, lids, displays 2370, control software, and control electronics. Deck 2320 can include at least framing, enclosures, MSA 4000, one or more pumps 2360 (e.g., peristaltic pumps), one or more valves 2340 (e.g., solenoid pinch valves), and one or more bubble sensors 2350 (e.g., optical sensors). Instrument 2300, including main body 2310, deck 2320, and the cartridge, can be configured as a fully closed platform so that no in-process materials are accessible from the outside surfaces of the consumable.

[0133] FIG. 23 illustrates instrument 2300, which can include control electronics and software for operating valves 2340 (e.g., 11 pinch valves), bubble sensors 2350 (e.g., 1 bubble sensor), and pumps 2360 (e.g., 1 peristaltic pump) configured to perform operations associated with cell processing using the cartridge. Consumables associated with instrument 2300 can comprise input and output containers, manual pinch clamps, tubing with connectors, or the cartridge. Consumables associated with instrument 2300 can include removable containers for downstream workflows, e.g., output containers. Dry consumables can comprise tubing, connectors, the cartridge, or the empty input or output containers. Dry consumables may be sterilized by gamma irradiation after assembly. Wet consumables can include the dry consumables filled with prime fluids, sample fluids, and wash fluids, which can be attached (e.g., sterile- welded) to predetermined locations on instrument 2300 using container poles 2330 (e.g., 6 posts). Individual input containers can include containers provided in a sterile state. The tubing and connector specifications for the dry consumables are depicted in FIG. 6. The tubing and connectorspecifications for the wet consumables are depicted in FIG. 7. Instrument 2300 can be configured to include mechanical, electrical, and software hooks to populate spare valves.

[0134] FIGs. 24-31 illustrate instrument 2300, which can include controls for operating valves 2340 configured to perform operations associated with cell processing. In some embodiments, at least one of the one or more valves is operatively coupled to the one or more pumps, the one or more bubble sensors, and the one or more fluids. In some embodiments, at least one of the one or more valves is a solenoid pinch valve. For example, FIG. 24 depicts a valve 2340 (e.g., a solenoid pinch valve) that can be configured to stop or stop flow of fluids by control of instrument 2300. Instrument 2300 can direct different fluids (e.g., wash, sample, and prime fluids) from any of the input fluid containers independently. For example, FIG. 25 illustrates instrument 2300 in a starting state with cartridge 600, a pump 2360, valves 2340, a bubble sensor 2350, input containers (e.g., wash, sample, and fluid), output containers (waste), and tubesets. By operation of valves 2340 and pump 2360, pump 2360 can cause one or more of the fluids to flow out of the input containers, through cartridge 600 (e.g., the flow channel), and out to the output containers. For example, FIG. 26 illustrates valves 2340 that can be configured to interface with each of the input lines (e.g., three input lines or tubesets) to allow each line to be opened and closed independently by control of instrument 2300. Valves 2340 can be positioned to interface with tubing connecting to each cartridge port (e.g., inputs and outputs of the cartridge) as well as the bypass line. For example, FIG. 27 illustrates that Zone 1 outlet and Zone 2 inlet valves 2340 can be configured (e.g., closed) to direct fluid through Zones 1 and 2 in series using the bypass line. For example, FIG. 28 illustrates that valves 2340 can be configured to interface with tubing connecting to each cartridge port as well as the bypass line causing Zone 2 inlet and outlet valves 2340 to be closed thereby directing fluid through Zone 1 independently and vice versa. For example, FIG. 29 illustrates that valves 2340 can be configured to interface with each of the output lines (e.g., three output lines) to allow each line to be opened and closed independently. For example, FIG. 30 illustrates that valves 2340 can be configured to direct fluid from one side of the cartridge to the other side without passing through Zones 1 and 2 of the cartridge. Valves 2340 can be configured to interface with tubing connecting to each cartridge port as well as the bypass line causing Zone 1 inlet and Zone 2 outlet valves 2340 to be closed thereby directing fluid through the cartridge bypass line without passing through either zone. For example, FIG. 31 illustrates that instrument 2300 can be configured to perform cartridge priming before fluid samples are introduced or flowed into the cartridge. The prime container, prime input line, and valves 2340 can be configured to move priming fluid into the cartridge independent of other fluids. Ingress protection for the valves 2340 can include sealing grommets mounted to enclosures.

[0135] FIG. 32A and FIG. 32B depict or illustrate input containers, e.g., bags, dispensers, and the like, for the one or more fluids, e.g., the fluid sample, which can be configured to be gas permeable for cell viability and certified for US Plastic® (USP) Class VI biocompatibility (e.g., CellBios® EVA Transfer Bags). In some cases, containers can include bags such as cleanroom bags, amber ultraviolet (UV) resistant protective bags, biohazard specimen poly bags, tamper evident plastic bags, healthcare bag liners, pliable bags, soft bags, hard bags, and the like. In some cases, the output containers can be the same or different than the input containers. In some cases, as illustrated in FIG. 32A, the containers may hold a fluid volume of about 50 to 200 mL. In some cases, the containers may hold a fluid volume of less than 50 mL. In some cases, the containers may hold a fluid volume of more than 200 mL. The containers may be gamma stable and may include polyvinyl chloride (PVC) tubing or thermoplastic elastomers (TPE) tubing with a predetermined size, e.g., about 3 mm inner diameter (mmID). The tubing may be compatible with sterile welding and sealing and available in a di(2-ethylhexyl) phthalate- (DEHP) free formulation. FIG. 32B illustrates that the containers and tubing can include a choice of large spike port, small spike port, or twist-off port, as well as manual pinch clamps. Wash and prime containers (e.g., about 50 mL volume) can be non-gas permeable for storing degassed fluids and USP® Class VI certified. Containers can include C-Flex 374 tubing (e.g., about 1 / 8” ID) that is compatible with sterile welding, sealing, and gamma irradiation. Containers can include luer-lock ports (e.g., female luers, male luers, spikes, and the like) and manual pinch clamps.

[0136] FIGs. 39A-39C illustrate instrument 2300, which can include controls for operating bubble sensors 2350 configured to detect bubbles (e.g., air or fluid bubbles) associated with cell processing. In some embodiments, at least one of the one or more bubble sensors is operatively coupled to the one or more pumps, the one or more valves, and the one or more fluids. In some embodiments, at least the one or more bubble sensors is an optical sensor. FIG. 39A illustrates that bubble sensors 2350 can be mounted onto threaded inserts in the deck 2320 enclosure (e.g., the underside) due to their compact size. In some cases, one bubble sensor 2350 can be included to detect bubbles on the fluid sample line. In some cases, bubble sensors 2350 can be included on any of the fluid lines, e.g., wash fluid line, sample fluid line, or prime fluid line. Bubble sensors 2350 can be ingress protected by gasket tape applied around the outside faces of the protruding part before installation. Deck 2320 can be configured with an access hole in the framing panel to allow for installation or maintenance of bubbles sensors 2350 from the back of instrument 2300. Bubble sensor 2350 can be configured to accept 1 / 16” ID x 1 / 8” OD clear soft-wall tubing. FIG. 39B illustrates that bubble sensor 2350 can be configured to use a signal (e.g., infrared (IR) signal) emitter and sensor to detect differences between air and fluid in the tubing. Integrated circuitry of the bubble sensor 2350 can be configured to generate both analog and digital signal outputs forintegration flexibility. The digital signal output can be determined by pre-set or predetermined thresholds. FIG. 39C illustrates that bubble sensor 2350 can be configured to operatively couple to tubing for detecting bubbles in the tubing.

[0137] FIG. 38A and FIG. 38B illustrate instrument 2300, which can include controls for operating pumps 2360 (e.g., peristaltic pumps) associated with cell processing. In some embodiments, at least one pump of the one or more pumps is a peristaltic pump. In some embodiments, at least one pump of the one or more pumps is operatively coupled to the one or more valves, the one or more bubble sensors, and the one or more fluids. FIG. 38A illustrates that deck 2320 can include a mounting interface for pump 2360 with a stepper motor. Pump 2360 can be installed from the front of the enclosure by fasteners (e.g., mounting screws) that pass through the enclosure and thread into PEM nuts in the framing for rigidity. In some cases, fasteners described here and throughout the present disclosure can include screws, bolts, nuts, washers, rivets, snap fits, slip fits, pressure fits, adhesives, welding joints, and the like. Pump 2360 can include a face seal gasket that can provide ingress protection around pump 2360 mount interface in the enclosure. Pump 2360 can be configured to be driven by a stepper motor for accurate flow rate and volume control. Pump 2360 can be configured to run open-loop for the majority of the processing workflow. Bubble sensors 2350 can be configured to close the loop in the sample capture operation to maximize sample throughput. FIG. 38B illustrates that pump 2360 can be configured with a 6-roller head to minimize flow pulsatility and for compatibility with 1 mm ID x 3 mm OD line tubing. Pump 2360 can be configured to produce flow rates from about 0.1 to 30 milliliters per minute (mL / minute) and include a flip-top design for easy tube loading. Pump 2360 segments can generate flow rates within about + / -10% accuracy range across a 90 mL volume dispense. A control board of instrument 2300 can control the stepper motor to spin in either direction causing pump 2360 to move fluid in both directions through the cartridge. FIG. 38C illustrates performance of the peristaltic pump.

[0138] FIG. 40 and FIG. 41 illustrate instrument 2300, which can include a magnet actuator subassembly (MSA) 4000 configured to perform operations associated with cell processing. In some embodiments, the deck framing further comprises a magnet actuator assembly operatively coupled to the deck framing. In some embodiments, the magnet actuator assembly is configured to be operatively coupled to the one or more magnetic arrays. In some embodiments, the magnet actuator assembly comprises: a linear actuator configured to generate a movement of the one or more magnetic arrays proximate to the cartridge; a linear motion rail configured to generate a position of the one or more magnetic arrays proximate to the cartridge; and a cartridge interface configured to releasably couple the cartridge to the magnet actuator assembly.

[0139] FIG. 40 depicts that MSA 4000 can be mounted to the underside of deck 2320 and main body 2310 framing panels of instrument 2300. FIG. 41 illustrates a magnet actuator baseplate 4010 configured to mount or attach MSA 4000 to instrument 2300 and can be a formed and welded sheet metal component with threaded inserts. MSA 4000 can include mounting locations for cartridge interface 4050, linear motion rail 4020, optical switches 4060, hard stops 4070, and linear actuator 4030. MSA 4000 can be configured with a drip tray 4040 in baseplate 4010 to contain any fluids that enter the enclosure at cartridge interface 4050.

[0140] FIGs. 42A-42C illustrate that MSA 4000 can be configured to constrain the cartridge in one or more directions and to engage or disengage magnet array 4080 during cell processing. In some embodiments, at least one of the one or more magnetic arrays is magnetically coupled to the one or more fluids. In some embodiments, at least the one or more magnetic arrays is a permanent magnet. For example, the one or more magnetic arrays, e.g., comprising one or more permanent magnets, can create magnetic field lines that extend through (e.g., magnetically coupled) the cartridge and the one or more fluids of the cartridge. The magnetic field lines can couple with the one or more fluids comprising magnetically susceptible nanoparticles. The magnetic field lines can cause a magnetic force to act on the magnetically susceptible nanoparticles.

[0141] FIG. 42A illustrates that the combination of linear motion rail 4020 and linear actuator 4030 can be configured to position magnet array 4080 relative to the cartridge. In some embodiments, the magnet actuator assembly is configured to move or position the one or more magnetic arrays to generate one or more magnetic capture zones in the flow chamber. 3D printed and sheet metal components can be configured to create a slot to constrain the edges of the cartridge while leaving the top and bottom faces uncovered during operation. Magnet array 4080 can be secured using a bracket and operatively coupled to linear motion rail 4020 and linear actuator 4030. Linear motion rail 4020 can be configured to constrain the movement of magnet array 4080 in one direction and can be driven by linear actuator 4030 to engage and disengage magnet array 4080. FIG. 42B illustrates that MSA 4000 can be configured with one or more magnet arrays 4080 (e.g., one above the cartridge and one below the cartridge). Magnet array 4080 can be fastened (e.g., permanently glued) into place in 3D-printed magnet holders. The holders can be mounted to a bracket and spring-loaded to ensure magnet array 4080 contacts, or is substantially near, the top and bottom surfaces of the cartridge when engaged. In some cases, one magnet array 4080 may be positioned below the cartridge without a magnet array 4080 above the cartridge; the top (above) position may be populated with a ‘dummy’ plastic array to reduce cartridge flexing when the bottom (below) magnet array 4080 is engaged, e.g., positioned in the “on” position for cell processing. FIG. 42C depicts that cartridge interface 4050 may include oneor more assemblies or brackets configured to secure the cartridge during cell processing. The brackets can include a slot for inserting the cartridge and a tensioning means (e.g., tensioning brackets) to further secure the cartridge. Cartridge interface 4050 can be configured to align cartridge 600 to instrument 2300. Alignment can include fluidic alignment, magnetic alignment, or optical alignment.

[0142] FIGs. 43A-43B illustrate that MSA 4000 can be configured to limit the motion of magnet array 4080 during positioning (e.g., engaging or disengaging) relative to the cartridge during cell processing. In some embodiments, the magnet actuator assembly further comprises a motion limit assembly operatively coupled to the deck framing. In some embodiment, in the motion limit assembly comprises: one or more optical sensors configured to determine the movement or the position of the magnet actuator assembly; and one or more hard stops configured to stop the movement of the magnet actuator assembly when the movement exceeds a predetermined movement.

[0143] FIG. 43B illustrates that MSA 400 can be configured with optical switches or sensors 4060 to determine the engaged and disengaged positions of magnet array 4080. In some cases, optical switches 4060 can be fastened (e.g., mounting screws) to baseplate 4040. MSA 400 can also be configured with hard stops 4070 to limit the motion of magnet array 4080. Hard stops 4070 can be positioned about 0.5 mm to about 20 mm beyond each optical switch 4060 trigger point to ensure magnet array 4080 will not damage instrument 2300 if the optical switches 4060 fail. In some cases, hard stops 4070 can be positioned about 1 mm beyond each optical switch 4060.

[0144] FIG. 43A illustrates instrument 2300, which can be configured with safety features near the MSA 4000. In some embodiments, the deck enclosure is configured to be compliant with at least contract development and manufacturing organization (CDMO) safety protocols. Instrument 2300 can include a deck 2320 enclosure configured to wrap around the moving components of MSA 4000 to prevent, e.g., a pinch hazard, an electrical hazard, an optical hazard, or a fluid hazard, to users. In some cases, moving parts can include magnet arrays 4080, cartridge interface 4050, magnet actuator baseplate 4010, or drip tray 4010. Deck 2320 enclosure can be shaped such that the cartridge ports are exposed on the outside of instrument 2300 when the cartridge is installed to allow for consumable tube routing. Baseplate 4040 can be configured with mounting slots to allow it to be precisely aligned to deck 2320 enclosure at the time of assembly to prevent interference.

[0145] FIGs. 44A, 44B, 45A, and 45B illustrate or depict that MSA 4000 can be configured to position magnet array 4080 relative to the cartridge during cell processing. FIG. 44A illustrates or depicts that MSA 4000 can be configured to actuate the magnet array 4080 into contact (e.g.,engage or turn on) with the cartridge. FIG. 45B illustrates or depicts that MSA 4000 can be configured to actuate the magnet array 4080 into contact (e.g., engage or turn on) with the cartridge. In some cases, MSA 4000 can be configured to actuate the magnet array 4080 into proximity or near contact with the cartridge. MSA 4000 can include magnet array 4080 and an actuator mechanism (e.g., linear actuator 4030) configured to engage and disengage magnet array 4080 with the bottom side or the top side of the cartridge. MSA 4000 can be configured with at least one magnet array 4080, which can be actuated to engage and disengage with the bottom side of the cartridge. In some cases, MSA 4000 can be configured with a second magnet array 4080 positioned in contact with or proximate (e.g., near contact) to the top side of the cartridge. In some cases, MSA 4000 can include more than two magnet arrays. In some cases, as described herein elsewhere, the two or more magnet arrays can be configured with a plurality of small magnets, e.g., micro-magnets, to form the two or magnet arrays. MSA 4000 can be configured to actuate magnet array 4080 using a combination of linear actuator 4030, linear motion rail 4020, optical switches 4060, and hard stops 4070 to control the position of the magnet array 4080 with respect to the cartridge. FIG. 44B illustrates or depicts that MSA 4000 can be configured to actuate the magnet array 4080 out of contact (e.g., disengage or turn off) with the cartridge in a similar manner as described in FIGs. 44A and 45B. FIG. 45A illustrates or depicts that MSA 4000 can be configured to actuate the magnet array 4080 out of contact (e.g., disengage or turn off) with the cartridge in a similar manner as described in FIGs. 44A and 45B.

[0146] FIG. 45C and FIG. 45D illustrate MSA 4000, which can be configured to position magnet array 4080 so that magnet array 4080 contacts with or is in near proximity to the bottom side of the cartridge. In some cases, magnet array 4080 may be positioned proximate (e.g., a gap) to the bottom side of the cartridge, e.g., a gap of about 0.01 mm to about 5 mm from the bottom side of the cartridge. In some cases, MSA 400 can be configured to position magnet array 4080 so that magnet array 4080 contacts with or is in near proximity to the top side of the cartridge. In some cases, magnet array 4080 may be positioned proximate (e.g., a gap) to the top side of the cartridge, e.g., a gap of about 0.01 mm to about 5 mm from the top side of the cartridge. In some embodiments, the magnet actuator assembly is configured to move or position the one or more magnetic arrays to generate one or more magnetic capture zones in the flow chamber. In some embodiments, the one or more magnetic capture zones comprises an area circumscribing all or substantially all of the flow-rate reducing structures. FIG. 45C depicts that when magnet array 4080 is in contact or near proximity (e.g., engaged or turn on) with the cartridge, the bottom magnet array 4080 may cover the entire capture area of the cartridge between the outermost edges of the cylindrical post features in the channels. The bottom magnet array 4080 may reproducibly cover a sufficient area of Zone 1 or Zone 2 during cell processing. Sufficient area coverage canhelp to ensure that the construction of instrument 2300 allows clear evaluation of cell processing performance. MSA 4000 can be aligned to instrument 2300 such that the edge of magnet array 4080 will reach the outermost edge of the cylindrical posts, even at extremes of part tolerance ranges. Magnet array 4080 can be configured to overlap the outermost edges of the post features by about 1 mm on each side. FIG. 45D further depicts that when magnet array 4080 is engaged, the MSA 4000 can be configured to allow a maximum gap of about 0.0015” or less in the Z- direction between the cartridge and the bottom magnet array 4080. The flatness of the cartridge against magnet array 4080 can sample capture performance over the entire capture areas of Zone 1 or Zone 2.

[0147] FIG. 33 and FIG. 34 illustrate that deck 2320 framing panel can be configured to mount to threaded inserts in the front of main body 2310 framing. FIG. 33 illustrates a perspective view of deck 2320 framing panel that can be configured separate from main body 2310 framing to increase modularity in deck 2320 area to accommodate routine design changes. Deck 2320 framing panel can include a flat sheet metal panel with threaded inserts. Deck 2320 faming panel can be configured to provide mounting interfaces for valves 2340 (e.g., solenoid pinch valves), pumps 2360 (e.g., peristaltic pumps), bubble sensors 2350 (e.g., optical sensors), MSA 400, and deck 2320 enclosures. Mounting locations can be predetermined by consumable designs to ensure the components interface with the appropriate tube segments on the consumables. FIG. 34 illustrates a plan view of FIG. 33.

[0148] FIG. 35 illustrates that deck 2320 enclosure can be fabricated from a single 3D-printed plastic part with threaded inserts that align and mount to deck 2320 framing. Deck 2320 enclosure can be configured to provide user access and ingress protection for the consumable loading touchpoints including pinch valve 2340 heads, pump 2360 heads, bubble sensors 2350, and MSA 4000. Deck 2320 enclosure can be separate from instrument 2300 enclosure to increase modularity in deck 2320 area to accommodate routine design changes.

[0149] FIG. 36 illustrates that the back of the deck 2320 enclosure can be configured with fasteners (e.g., threaded inserts) for valves 2340 gasket mounting, bubble sensors 2350 mounting, and enclosure to framing mounting. Pinch valve 2340 gaskets can be assembled to the enclosure prior to enclosure mounting to instrument 2300. Bubble sensors 2350 may be mounted after the enclosure is mounted to instrument 2300.

[0150] FIG. 37 illustrates that deck 2320 framing panel can be configured to use custom mount flanges that secure each pinch valve 2340 with plastic spring tabs and set screws. The custom flanges and valve mounts can secure each pinch valve 2340 to the back of deck 2320 framing panel. Each pinch valve 2340 head can be configured to pass through a sealing grommet mounted to the underside of deck 2320 enclosure for ingress protection. Each pinch valve 2340power and communications cable can pass through a cutout in deck 2320 framing to route to the control board.Main body of the platform

[0151] In some embodiments, the instrument further comprises a main body operatively coupled to the deck, a graphical user interface (GUI), or the power supply. In some embodiments, the main body further comprises a body framing and a body enclosure. In some embodiments, the body framing is configured to be operatively coupled to the body enclosure. In some cases, the main body can be operatively coupled to the deck, the GUI, and the power supply.

[0152] FIG. 46A and FIG. 46B illustrate the framing and enclosure of main body 2310 of instrument 2300. FIG. 46A illustrates main body 2310 framing, which can be a formed and welded sheet metal part with threaded inserts. Main body 2310 framing can be configured to provide mounting interfaces for the control electronics, deck 2320, and main body 2310 enclosure. Main body 2310 framing can be mounted to a baseplate configured to enclose the bottom face of instrument 2300 and to provide mounting locations for rubber feet. FIG. 46B illustrates main body 2310 enclosure, which can be fabricated from one or more 3D-printed plastic parts, e.g., a front enclosure and a back enclosure. The front enclosure can be configured to mount to main body 2310 framing at the beginning of the assembly process before deck 2320 is mounted. The front enclosure mounting locations can be accessible from inside instrument 2300 through a back access panel. The back enclosure (not shown) can be mounted at the end of the assembly process.

[0153] FIG. 47 illustrates main body 2310 framing and enclosure, which can be configured with a back panel to provide mounting interfaces and access for instrument 2300 software and control electronics, power, communications, and cooling. For example, instrument 2300 can be configured with a terminal block (1), a stepper motor controller and drives (2), a USB communications hub (3), a cooling fan (4), a power connector (5), and a power switch (6). A tablet and USB-C panel mount connector (7) can be mounted on the front of main body 2310 enclosure.

[0154] FIG. 48 illustrates main body 2310, which can include bag poles 2330 configured to hold fluid bags. In some embodiments, the body framing is configured to be operatively coupled to one or more poles and wherein the one or more poles are configured to hold one or more containers for the one or more fluids. In some embodiments, the one or more containers are configured as a primer container, a fluid sample container, and a wash container. Fluid containers (e.g., bags) can include wash, sample, and prime fluid bags on the input side and waste and output fluid bags on the output side. Main body 2310 framing can be configured to provide mounting interfaces for the input and output bag poles 2330. Each bag pole 2330 can be mounted via shaft collars internal to main body 2310 framing. Each bag pole 2330 can be configured with adjustable thumb screws to adjust the vertical pole height and bag hanging position.

[0155] FIG. 49 A and FIG. 49B illustrate cooling performance of instrument 2300. Instrument 2300 can be configured with a cooling module to maintain predetermined temperatures during cell processing. FIG. 49A illustrates the cooling module, which can include a combination of fans (e.g., cooling fans or circulation fans) and vents (e.g., intake vents or exhaust vents) to increase the rate of heat transfer to ambient air and to exchange warm internal air for cooler ambient air. The cooling module can decrease power consumption and heat generation during operation of instrument 2300. FIG. 49B illustrates cooling performance of the cooling module at different locations or components of instrument 2300. The cooling module can control or maintain each temperature to remain below about 40 degrees Celsius for at least 8 hours of continuous operation of instrument 2300. In some cases, the cooling module can control or maintain each temperature within a range of about 15 to 40 degrees Celsius during the entire period of continuous operation of instrument 2300. In some cases, the period of continuous operation can include at least about 1 minute, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, or more. In some cases, the period of continuous operation can include at most about 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 1 minute, or less. Instrument 2300 can be configured with a C-slot loading design for easy tube loading.

[0156] FIGs. 50A-50C illustrate instrument 2300, which can be configured with the cooling module to maintain predetermined temperatures during cell processing. The cooling module can include a combination of fans (e.g., cooling or circulation fans) and vents (e.g., intake or exhaust vents). FIG. 50A illustrates how main body 2310 enclosure can be mounted to main body 2310 framing and to close the back access panel once all deck 2320 components and electronics have been mounted. FIG. 50B illustrates locations of cooling or circulation fans and intake or exhaust vents on the back of instrument 2300 to maintain predetermined temperatures during cell processing. FIG. 50C illustrates locations of cooling or circulation fans and intake or exhaust vents on the back of instrument 2300 to maintain predetermined temperatures during cell processing.

[0157] FIG. 51A and FIG. 51B illustrate instrument 2300, which can be configured or sized to fit on a lab bench. In some embodiments, the body enclosure is configured to be compliant with at least contract development and manufacturing organization (CDMO) safety protocols. Instrument 2300 can be configured to use contract development and manufacturing organization- (CDMO) approved sterile docking or sealing procedures to prevent in-process materials from contacting instrument 2300. In some embodiments, the body enclosure further comprises one or more lids operatively coupled to the body enclosure. In some embodiments, the one or more lids are configured to isolate a user from the one or more magnetic arrays. FIG. 51A illustrates instrument 2300, which can be configured to allow a user to load a wet consumable when mainbody 2310 enclosure lid is open and cell processing is not in progress. Main body 2310 enclosure lid can include hinges and be 3D-printed to cover deck 2320 when closed. Main body 2310 enclosure lid can be configured to mount to main body 2310 enclosure with torque hinges and concealed when closed. FIG. 51B illustrates that valves 2340 and other touchpoints can all be accessible when main body 2310 enclosure lid is open. In some cases, instrument 2300 can have a width ranging from about 1 foot to 5 feet. In some cases, instrument 2300 can have depth ranging from about 1 foot to 5 feet. In some cases, instrument 2300 can have a height ranging from about 1 foot to 5 feet. In some cases, instrument 2300 can be configured or sized to fit on a lab bench. In some cases, instrument 2300 can be configured or sized (e.g., lid open) to have a width about 3 feet, a depth of about 1.5 feet, and a height of about 2 feet. In some cases, instrument 300 can be configured or sized (e.g., lid closed) to have a width about 3 feet, a depth of about 2 feet, and a height of about 2 feet.

[0158] FIG. 52A and FIG. 52B illustrate instrument 2300, which can be configured with display 2370. In some embodiments, the platform further comprises a graphical user interface (GUI) configured to allow a user to select or interact with the one or more processes. In some cases, a combination of the one or more processors and the GUI, described herein elsewhere, is configured to allow a user to select or interact with the one or more processes. Display 2370 can be configured as a GUI to allow users to operate or control instrument 2300. Display 2370 can be operable when a user wears gloves to interact with display 2370. In some embodiments, the body framing is configured to be operatively coupled to the GUI. FIG. 52A illustrates that display 2370 can be configured as a tablet and mounted with a bracket. FIG. 52B illustrates that display 2370 can be configured to attach to instrument 2300 via the bracket and fasteners (e.g., threaded inserts) in the front of main body 2310 enclosure.

[0159] FIG. 53A and FIG. 53B illustrate instrument 2300, which can be configured to include mounting locations for one or more power switches to control power to all components within instrument 2300. In some cases, instrument 2300 may be configured to use a power supply. In some cases, the power supply can be configured with a voltage and electrical current to supply at least or equal to about 10, 20, 30, 40, 50, 100, 200, 300 or more watts of power to instrument 2300. In some cases, the power supply can be configured with a voltage or electrical current to supply at most or equal to about 300, 200, 100, 50, 40, 30, 20, 10, or less watts of power to instrument 2300. In some cases, the power supply can be configured with a voltage or electrical current to supply at about 10-300, about 20-200, about 30-100, or about 40-50 watts of power to instrument 2300. In some embodiments, the power supply is configured to supply 24 volts (V), 9.2 amps (A), or 221 watts (W) to the platform. In some embodiments, the body framing is configured to be operatively coupled to the power supply. FIG. 53A illustrates instrument 2300can include a cutout in the back left corner to provide access to the power switch and power connector. FIG. 53B illustrates instrument 2300 can include a mounting location for a USB-C connector. Display 2370 power cord can plug into the panel-mount USB-C connector in main body 2310 enclosure.

[0160] FIG. 54 illustrates a block diagram of the electrical system architecture that may be used to operate all aspects of instrument 2300 during cell processing. For example, the architecture can be configured for power flow, communications flow, motor control for pumps 2360, and motor control for MSA 400. Power flow can include the power supply configured to convert power from an AC power outlet to different DC voltages and currents. The power supply may supply 24 V for operation of components such as a communications hub (e.g., 24 V), the cooling module (e.g., 24 V), the cooling or recirculation fans (e.g., 24 V), motor control and power for pumps 2360 (e.g., 24 V), and motor control and power for MSA 4000 (e.g., 24 V). A separate power converter may receive and convert the 24 volts of the power supply to 5 V to operate optical sensors 2350 (e.g., 5 V). Motor control and power for pumps 2360 and MSA 400 may be via a stepper controller (e.g., AllMotion® EZQuad Stepper). The stepper controller may have at least 1, 2, 3, 4, or more channels for controlling pumps 2360, MSA 4000, and other peripherals. Communications flow can include the communications hub, USB-C communications protocols, and a display, e.g., a GUI.

[0161] FIG. 55 and FIG. 56 illustrate that instrument 2300 can include software applications configured to allow a user to build and run scripts for cell processing. In some embodiments, the one or more processors is configured to automatically execute instructions comprising: (a) priming the cartridge with priming fluid; (b) isolating the target cells from the fluid sample; (c) washing the cartridge with washing fluid; (d) recovering the target cells from the cartridge; or (e) any combination of (a) to (d). Software application can execute entire scripts, execute functions containing a subset of commands, or execute individual commands. Communication status of instrument 2300 can be monitored using display 2370, including real-time status of certain components, such as valves 2340, magnet array 4080 position, and flow direction. Software applications can also include a development mode for users to test low-level commands and to develop scripts, which may provide flexible control and debug information in display 2370. Command sets can be configured to allow a user, e.g., to configure the flow rate, move volume, pump 2360 direction, pump 2360 acceleration factor, user action or pause, or valve states. In the development mode, the run status can be indicated by the system block diagram and console bar. Also, a Cell Processing Protocol can provide a locked-down interface that guides a user through a workflow. Software applications can guide a user through workflows. Software applications can collect user input such as date and time, operator name, cartridge ID, reagent ID, consumable ID,or sample ID. An output summary or log file can include run data, timing analysis, and log files. The log file can record all communication traffic associated with instrument 2300 including commands, parameter values, and errors. A timestamp can be associated with each log file entry. A script can be locked to prevent editing in the development mode and in the Cell Processing Protocol mode.

[0162] FIG. 55 and FIG. 56 illustrate a GUI or workflow that may be executed on display 2370 to provide instructions for a user during cell processing. For example, FIG. 56 illustrates instructions for consumable loading by a user in accordance with workflows described herein elsewhere. The GUI software can enable the user to perform pre-flight checks before starting a run or do them automatically, e.g., a connection test to a controller (e.g., AllMotion® Controller), determine whether a valid script is loaded, or determine whether a tube is properly loaded in bubble sensor 2350. FIGs. 57A-57G illustrate example GUI instructions or interactions with a user during consumable loading of the instrument before cell processing or sorting, in accordance with some embodiments. FIG. 57A illustrates loading consumables, e.g., positioning input and output containers. FIG. 57B illustrates loading consumables, e.g., inserting the cartridge into the instrument. FIG. 57C illustrates routing tubing, e.g., loading tubing into pinch valves. FIG. 57D illustrates routing tubing, e.g., loading tubing into peristaltic pumps. FIG. 57E illustrates routing tubing, e.g., loading tubing into bubble sensors. FIG. 57F illustrates routing tubing, e.g., releasing pinch clamps on input fluid containers. FIG. 57G illustrates unloading operations, e.g., closing pinch clamps on output fluid containers.

[0163] The GUI software can allow the user to cancel the run at any point in the workflow. If the run is canceled and the script is currently running, the GUI software can automatically terminate all commands on a controller, e.g., AllMotion® Controller. After the user is instructed to manually close the input and output bag pinch clamps, the GUI software can send a command to open all valves 2340 and to instruct the user to remove the consumables. Upon completion, the GUI software may return to the start or home screen and allow the user to perform other operations associated with instrument 2300.

[0164] Instrument 2300 can be configured to detect loading errors. Loading errors can include input bags welded to wrong tubes, input bags input bags welded outside of specified weld location range, failure to open manual pinch clamps before run, tubes loaded in the wrong valves 2340, tubes not fully seated in valves 2340, kinked or twisted tubing, tubing not loaded properly in pump 2360, the cartridge not loaded properly into cartridge interface 4050, failure to close manual pinch clamps after run. Sample throughput based on dead volume estimates can be greater than about 90% of initial sample volume. Pump 2360 parameters and valve 2340 states can be configurable to tune capture and recovery performance, e.g., flow rate, acceleration, direction, and flow path.Instrument 2300 can be configured so that cell viability is not affected by mechanical stress introduced by pump 2360 rollers.

[0165] In some cases, instrument 2300 can be configured to perform high-throughput processing of cells with the cartridge. In some cases, instrument 2300 can be configured to flow the one or more fluids through the cartridge at a flow rate of about 0.1 to 30 milliliters per hour (mL / hr). In some cases, instrument 2300 can be configured to flow the one or more fluids through the cartridge at a flow rate of about 0.5 to 3 mL / hr. In some cases, instrument 2300 can be configured to flow the one or more fluids through the cartridge at a flow rate determined by the magnetic force acting on the target cells via the magnetic nanobeads. For example, isolation of target cells based on messenger ribonucleic acid (mRNA) expression can include flow rates of about 0.50 to about 1 mL / hr, about 0.60 to about 0.90 mL / hr, or about 0.70 to about 0.80 mL / hr. For example, isolation of target cells based on protein or antibody expression can include flow rates of about 1 to about 3 mL / hr or about 1.50 to about 2.5 mL / hr.

[0166] In some cases, instrument 2300 can be configured to perform high-throughput processing of the fluid sample through the cartridge at a rate of at least about 109cells per hour. In some cases, instrument 2300 can be configured to perform high-throughput processing of the fluid sample through the cartridge at a rate of about 106to 109cells per hour. In some cases, instrument 2300 can be configured to perform high-throughput processing of the fluid sample through the cartridge at a rate of less than about 106cells per hour. In some cases, instrument 2300 can be configured to isolate the target cells from the fluid sample of the cartridge with a purity of at least about 65% to 95%. In some cases, instrument 2300 can be configured to isolate the target cells from the fluid sample of the cartridge with a purity of greater than about 95%. In some cases, instrument 2300 can be configured to recover the target cells from the fluid sample of the cartridge with a recovery rate of at least about 65% to 95%. In some cases, instrument 2300 can be configured to recover the target cells from the fluid sample of the cartridge with a recovery rate of greater than about 95%.Computing systems

[0167] In some cases, platforms, cartridges, and methods disclosed herein may utilize one or more computing systems to perform operations associated with isolating target cells in fluid samples.

[0168] Referring to FIG. 58, a block diagram is shown depicting an exemplary machine that includes a computer system 5800 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and / or methodologies for static code scheduling of the present disclosure. The components in FIG. 58 are examples only and do not limit the scope of use or functionality of any hardware,software, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0169] Computer system 5800 may include one or more processors 5801, a memory 5803, and a storage 5808 that communicate with each other, and with other components, via a bus 5840. The bus 5840 may also link a display 5832, one or more input devices 5833 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 5834, one or more storage devices 5835, and various tangible storage media 5836. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 5840. For instance, the various tangible storage media 5836 can interface with the bus 5840 via storage medium interface 5826. Computer system 5800 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.

[0170] Computer system 5800 includes one or more processor(s) 5801 (e.g., central processing units (CPUs) or general-purpose graphics processing units (GPGPUs)) that carry out functions. Processor(s) 5801 optionally contains a cache memory unit 5802 for temporary local storage of instructions, data, or computer addresses. Processor(s) 5801 are configured to assist in execution of computer readable instructions. Computer system 5800 may provide functionality for the components depicted in FIG. 58 as a result of the processor(s) 5801 executing non- transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 5803, storage 5808, storage devices 5835, and / or storage medium 5836. The computer-readable media may store software that implements particular embodiments, and processor(s) 5801 may execute the software. Memory 5803 may read the software from one or more other computer-readable media (such as mass storage device(s) 5835, 5836) or from one or more other sources through a suitable interface, such as network interface 5820. The software may cause processor(s) 5801 to carry out one or more processes or one or more steps of one or more processes described or illustrated herein. Carrying out such processes or steps may include defining data structures stored in memory 5803 and modifying the data structures as directed by the software.

[0171] The memory 5803 may include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 5804) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phasechange random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 5805), and any combinations thereof. ROM 5805 may act to communicate data and instructions unidirectionally to processor(s) 5801, and RAM 5804 may act to communicate data andinstructions bidirectionally with processor(s) 5801. ROM 5805 and RAM 5804 may include any suitable tangible computer-readable media described below. In one example, a basic input / output system 5806 (BIOS), including basic routines that help to transfer information between elements within computer system 5800, such as during start-up, may be stored in the memory 5803.

[0172] Fixed storage 5808 is connected bidirectionally to processor(s) 5801, optionally through storage control unit 5807. Fixed storage 5808 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 5808 may be used to store operating system 5809, executable(s) 5810, data 5811, applications 5812 (application programs), and the like. Storage 5808 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above. Information in storage 5808 may, in appropriate cases, be incorporated as virtual memory in memory 5803.

[0173] In one example, storage device(s) 5835 may be removably interfaced with computer system 5800 (e.g., via an external port connector (not shown)) via a storage device interface 5825. Particularly, storage device(s) 5835 and an associated machine-readable medium may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 5800. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 5835. In another example, software may reside, completely or partially, within processor(s) 5801.

[0174] Bus 5840 connects a wide variety of subsystems. Herein, reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate. Bus 5840 may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example and not by way of limitation, such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.

[0175] Computer system 5800 may also include an input device 5833. In one example, a user of computer system 5800 may enter commands and / or other information into computer system 5800 via input device(s) 5833. Examples of an input device(s) 5833 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still imagecapture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device is a Kinect®, Leap Motion®, or the like. Input device(s) 5833 may be interfaced to bus 5840 via any of a variety of input interfaces 5823 (e.g., input interface 5823) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.

[0176] In particular embodiments, when computer system 5800 is connected to network 5830, computer system 5800 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 5830. Communications to and from computer system 5800 may be sent through network interface 5820. For example, network interface 5820 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 5830, and computer system 5800 may store the incoming communications in memory 5803 for processing. Computer system 5800 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 5803 and communicated to network 5830 from network interface 5820. Processor(s) 5801 may access these communication packets stored in memory 5803 for processing.

[0177] Examples of the network interface 5820 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 5830 or network segment 5830 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 5830, may employ a wired and / or a wireless mode of communication. In general, any network topology may be used.

[0178] Information and data can be displayed through a display 5832. Examples of a display 5832 include, but are not limited to, a graphical user interface (GUI), a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 5832 can interface to the processor(s) 5801, memory 5803, and fixed storage 5808, as well as other devices, such as input device(s) 5833, via the bus 5840. The display 5832 is linked to the bus 5840 via a video interface 5822, and transport of data between the display 5832 and the bus 5840 can be controlled via the graphics control 5821. In some embodiments, the display is a video projector.In some embodiments, the display is a head-mounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive®, Oculus Rift®, Samsung Gear VR®, Microsoft HoloLens®, Razer OSVR®, FOVE VR®, Zeiss VR One®, Avegant Glyph®, Freefly VR® headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.

[0179] In addition to a display 5832, computer system 5800 may include one or more other peripheral output devices 5834 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus 5840 via an output interface 5824. Examples of an output interface 5824 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

[0180] In addition or as an alternative, computer system 5800 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein. Reference to software in this present disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to a computer-readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both.

[0181] Various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality.

[0182] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general-purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0183] The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium. An exemplary storage medium is coupled to the processor such that the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0184] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers, in various embodiments, include those with booklet, slate, and convertible configurations.

[0185] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Suitable server operating systems include, by way of non-limiting examples, FreeBSD®, OpenBSD®, NetBSD®, Linux®, Apple® Mac OS X Server®, Oracle Solaris®, Windows Server®, and Novell NetWare®. Suitable personal computer operating systems include, by way of non-limiting examples, Microsoft Windows®, Apple Mac® OS X, UNIX®, and UNIX- like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia Symbian® OS, Apple® iOS, Research In Motion BlackBerry® OS, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile OS, Linux®, and Palm® WebOS. Suitable media streaming device operating systems include, by way of nonlimiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Suitable video game console operating systems include, by way of non-limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One®, Nintendo Wii®, Nintendo Wii U®, and Ouya®. Suitable virtual reality headset systems include, by way of non-limiting example, Meta Oculus®.Non-transitory computer readable storage mediums

[0186] In some cases, platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device. In further embodiments, a computer readable storage medium is a tangible component of a computing device. In still further embodiments, a computer readable storage medium is optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non- transitorily encoded on the media.Computer programs

[0187] In some cases, computer programs can be configured to implement any one or more methods disclosed herein. In another aspect, disclosed herein is a computer program product for isolating target cells in a fluid sample, the computer program product comprising at least one non- transitory computer-readable medium having computer-readable program code portions embodied therein, the computer-readable program code portions comprising: (a) an executable portion configured to prime the device by flowing a priming fluid of the one or more fluids through the device; (b) an executable portion configured to remove bubbles from the device; (c) an executable portion configured to flow the fluid sample of the one or more fluids through the device; (d) an executable portion configured to apply the magnetic field to isolate the target cells in the fluid sample; and (e) an executable portion configured to wash the device by flowing a washing fluid of the one or more fluids through the device; and (f) an executable portion configured to recover the isolated target cells in the fluid sample.

[0188] In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types. In light of the present disclosure provided herein, a computer program may be written in various versions of various languages.

[0189] The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer programcomprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.Web applications

[0190] In some cases, a computer program includes a web application. In light of the disclosure provided herein, a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft® .NET or Ruby on Rails® (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of nonlimiting examples, relational, non-relational, object oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, by way of nonlimiting examples, Microsoft® structured query language (SQL) Server, mySQL™, and Oracle®. A web application, in various embodiments, is written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML® (AJAX), Flash Actionscript, Javascript®, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages® (ASP), ColdFusion®, Perl®, Java®, JavaServer Pages® (JSP), Hypertext Preprocessor® (PHP), Python®, Ruby®, Tel®, Smalltalk®, WebDNA®, or Groovy®. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM Lotus Domino®. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft Silverlight®, Java®, and Unity®.

[0191] Referring to FIG. 59, in a particular embodiment, an application provision system comprises one or more databases 5900 accessed by a relational database management system (RDBMS) 5910. Suitable RDBMSs include Firebird®, MySQL®, PostgreSQL®, SQLite®, Oracle Database®, Microsoft SQL Server®, IBM DB2®, IBM Informix®, SAP Sybase®, SAP Sybase®, Teradata®, PostGIS®, time-series databases, graph databases, and the like. In this embodiment, the application provision system further comprises one or more application servers 5920 (such as Java® servers, .NET® servers, PHP® servers, and the like) and one or more web servers 5930 (such as Apache®, IIS®, GWS® and the like). The web server(s) optionally expose one or more web services via app application programming interfaces (APIs) 5940. Via a network, such as the Internet, the system provides browser-based and / or mobile native user interfaces.

[0192] Referring to FIG. 60, in a particular embodiment, an application provision system alternatively has a distributed, cloud-based architecture 6000 and comprises elastically load balanced, auto-scaling web server resources 6010 and application server resources 6020 as well synchronously replicated databases 6030.Mobile applications

[0193] In some cases, a computer program includes a mobile application provided to a mobile computing device. In some embodiments, the mobile application is provided to a mobile computing device at the time it is manufactured. In other embodiments, the mobile application is provided to a mobile computing device via the computer network described herein.

[0194] In view of the disclosure provided herein, a mobile application is created by techniques using hardware, languages, and development environments. Mobile applications are written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, Java®, Javascript®, Pascal®, Object Pascal®, Python™, Ruby®, VB.NET®, WML®, and XHTML / HTML with or without CSS, or combinations thereof.

[0195] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK®, alcheMo®, Appcelerator®, Celsius®, Bedrock®, Flash Lite®, .NET Compact Framework®, Rhomobile®, and WorkLight Mobile Platform®. Other development environments are available without cost including, by way of non-limiting examples, Lazarus®, MobiFlex®, MoSync®, and Phonegap®. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone® and iPad® (iOS) SDK, Android® SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian® SDK, webOS® SDK, and Windows® Mobile SDK.

[0196] Several commercial forums are available for distribution of mobile applications including, by way of non-limiting examples, Apple® App Store, Google® Play, Chrome®WebStore, BlackBerry® App World, App Store® for Palm devices, App Catalog® for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, and Nintendo® DSi Shop.Standalone applications

[0197] In some cases, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Standalone applications are often compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C®, COBOL®, Delphi®, Eiffel®, Java®, Lisp®, Python®, Visual Basic®, and VB .NET®, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable compiled applications. Additionally, microservices related to Python® and JavaScript® may be used.Web browser plug-ins

[0198] In some cases, the computer program includes a web browser plug-in (e.g., web extension, etc.). In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Makers of software applications support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. Several web browser plug-ins may include Adobe Flash Player®, Microsoft Silverlight®, and Apple QuickTime®. In some embodiments, the toolbar comprises one or more web browser extensions, add-ins, or add-ons. In some embodiments, the toolbar comprises one or more explorer bars, tool bands, or desk bands.

[0199] In view of the disclosure provided herein, several plug-in frameworks are available that enable development of plug-ins in various programming languages, including, by way of nonlimiting examples, C++, Delphi®, Java®, PHP®, Python®, and VB .NET®, or combinations thereof.

[0200] Web browsers (also called Internet browsers) are software applications, designed for use with network-connected computing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of nonlimiting examples, Microsoft Internet Explorer®, Mozilla Firefox®, Google Chrome®, Apple Safari®, Opera Software Opera®, and KDE Konqueror®. In some embodiments, the web browser is a mobile web browser. Mobile web browsers (also called microbrowsers, mini -browsers, andwireless browsers) are designed for use on mobile computing devices including, by way of nonlimiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting examples, Google Android® browser, RIM BlackBerry® Browser, Apple Safari®, Palm Blazer®, Palm WebOS® Browser, Mozilla Firefox® for mobile, Microsoft Internet Explorer Mobile®, Amazon Kindle Basic Web®, Nokia Browser®, Opera Software Opera Mobile®, and Sony PSP® browser.Software modules

[0201] In some cases, platforms, devices, and methods disclosed herein include software, server, and / or database modules, or use of the same. In view of the disclosure provided herein, software modules are created by techniques using machines, software, and languages. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.Databases

[0202] In some cases, platforms, devices, and methods disclosed herein include one or more databases (DB), or use of the same. In view of the disclosure provided herein, many databases are suitable for storage and retrieval data. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity -relationship model databases, associative databases, XML databases, time-series databases, graph databases, and the like. Further non-limiting examples include SQL, PostgreSQL®, MySQL®, Oracle®, DB2®, and Sybase. In some embodiments, a database is internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing-based. In a particular embodiment, a database is adistributed database. In other embodiments, a database is based on one or more local computer storage devices.Certain definitions

[0203] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present subject matter belongs.

[0204] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0205] Reference throughout this specification to “some embodiments,” “further embodiments,” or “a particular embodiment,” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “in some embodiments,” or “in further embodiments,” or “in a particular embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0206] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example. Numerous variations, changes, and substitutions will now occur without departing from the present disclosure. It can be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLES

[0207] The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concepts.Example 1 : Fabrication of cartridges and dry consumables

[0208] A cartridge disclosed herein can be fabricated using a plurality of methods. An example fabrication method of the disclosed cartridge is now described. The separability of the cartridge from the instrument can allow for at least the cartridge to be fabricated more quickly and cheaply (e.g., using 3D printing or injection molding techniques) than using lithography techniques. Further, by enabling a low cost, high volume fabrication process for the cartridge (e.g., using 3D printing), the cartridge can be configured as a disposable component of the instrument. Since the cartridge may be the component that is most in direct contact with the fluid sample, thedisposability of the cartridge may be beneficial for hygienic reasons or for use in healthcare settings. In some cases, the cartridge can be fabricated using injection molding techniques with U.S. Plastics® (USP) class VI polycarbonates. In some cases, the cartridge can be fabricated using injection molding techniques with plastics comprising suitability for medical devices.

[0209] In this example, the mold for fabricating the cartridge was 3D printed by a stereolithographic 3D printer (e.g., Microfluidics® Edition 3D Printer and Creative® CADworks) using the “CCW master mold for PDMS” resin (Resinworks® 3D). Other 3D printing resins may be used in this process such as resins designed for ultraviolet (UV) treatment or thermal treatment. The cartridge was made by casting PDMS (Dow Chemical Sylgard® 184, 182 or 186) on printed molds followed by 30 minutes to 4 hours of incubation time at about 50 to 100 degrees Celsius. Cured PDMS replicas of the cartridge were peeled off, punched, and plasma bonded to glass (e.g., thickness no. 1 glass coverslips) to complete assembly of the cartridge. Before using the cartridge, the cartridge was treated by about 0.01% to 1% Pluronic® F68 in phosphate-buffered saline (PBS) for about 30 minutes to 24 hours to reduce non-specific binding between cells (e.g., target cells and nontarget cells) and cartridges. During operation of the instrument, each cartridge can be inserted into the instrument.

[0210] Fabricated cartridges can be sputter-coated with 15 nm gold (Au) for characterization (e.g., imaging) under a field-emission scanning electron microscope using 5 kV accelerating voltage and high-vacuum mode. In other applications, the fabricated cartridges may not be sputter- coated when used in the instrument as described elsewhere herein.

[0211] With regards to 3D printing of the flow-rate reducing structures, the ability of a 3D printer to print positive and negative structures was first characterized. Such characterization can be useful for accurately and precisely 3D printing the flow-rate reducing structures. It was found that the 3D printer in this example (e.g., Microfluidics® Edition 3D) has better resolution in printing positive structures compared to negative structures. The lower resolution of negative structures may come from the residue of resins due to surface tension or the high viscosity of resins. In that regard, the 3D printer may print high-resolution patterns such as the trapping surfaces of the flow-rate reducing structures. The 3D printer was found to support a fine structure with a height of at least up to 1 mm, which lithography techniques may not be able to achieve. In addition, the 3D printer can generate multi-depth structures in one operation or pass while lithography techniques may require multiple operations or passes since lithography techniques can only produce one thickness per operation or pass.

[0212] A challenge of limited resolution in 3D printing of negative structures may be addressed through the use of a specially-designed, double replica procedure of casting PDMS. A double replicate procedure can generate a PDMS mold comprising negative structures. Thenegative mold can then be treated with saturated detergent in 70% ethanol solution for about 20 minutes to 72 hours. This detergent treatment allows newly cured PDMS to be separated from the treated one. This protocol can enable the use of negative PDMS structures as a mold to generate positive structures during cartridge fabrication, e.g., the flow-rate reducing structures.

[0213] The use of this protocol has at least three major advantages. First, the protocol can allow any structure (e.g., flow-rate reducing structures) to be fabricated with a sufficiently high resolution. Since the 3D printer was found to have a higher resolution when printing positive structures, it may be useful to use this protocol to have the ability to make both positive and negative structures for cartridge fabrication. Second, this protocol can generate multiple negative molds from one 3D-printed piece, which can allow for scaling the fabrication of cartridges. Because the molds can be formed from the exact same piece, it can also reduce or minimize the batch-to-batch variation of cartridges compared to other 3D printing techniques or lithography techniques. Third, this protocol can be cost-effective and straightforward as it does not involve the use of other types of resins.

[0214] In some cases or additionally, the cartridge can be fabricated using a 2-part manufacturing process. For example, with reference to FIG. 3C, the top portion can include the flow-rate reducing structures designed for cell capture. The base portion can include a thin plastic film configured to seal the flow-rate reducing structures thereby forming one or more microfluidic channels, e.g., Zone 1 and Zone 2. In some cases, the film’s thickness can be less than about 250 pm to ensure close or proximate contact with the magnetic array underneath used to generate a strong magnetic field and capture force. In some cases, the top portion can be manufactured using standard injection molding with Lexan™ HPS1 resin, medical -grade polycarbonate (PC) that is biocompatible (ISO 10993 or United States Pharmacopeia [USP] VI) grade for medical and pharmaceutical applications. In some cases, the bottom portion thin film can be manufactured using Lexan 8040, a PC film that complies with USP Class VI criteria. In some cases, the top portion and the bottom portion can be bonded together by laser welding to complete the fabrication or sealing of the cartridge. In some cases, the materials used to manufacture the cartridge can include gamma stable materials. Gamma irradiation can then be used to sterilize the materials of the cartridge. Laser welding can be an advantageous process in manufacturing the cartridge because said welding is a physical process. For example, laser welding may not use adhesives or chemicals that can contaminate samples due to leaching of the adhesives or chemicals into the sample during operation of the instrument.

[0215] In some cases, materials for manufacturing the cartridge can be as shown in Table 1.Table 1

[0216] In some cases, materials for manufacturing dry consumables of the instrument can be as shown in Table 2.Table 2Abbreviations: EVOH = ethylene-vinyl alcohol; FEP = fluorinated ethylene propylene; LLDPE = linear low density; polyethylene; TPE = thermoplastic elastomers.Example 2: Use of the instrument and cartridge for therapeutic applications in leukapheresis

[0217] A fluid sample obtained from a subject is analyzed with the instrument of the present disclosure for a therapeutic application. In some cases, the therapeutic application is leukapheresis. Leukapheresis can include procedures for treating subjects with chronic lymphocytic leukemia (CLL). Subjects with CLL can have very high white blood cell counts. A goal of leukapheresis can include quickly reducing the number of white blood cells, e.g., leukemiacells. During the procedure, white blood cells can be removed from a subject’s blood. Other blood cells and plasma can then be returned to the subject’s bloodstream.

[0218] The instrument of the present disclosure can be used to treat a subject with leukapheresis. For example, the instrument herein can utilize a sample comprising peripheral blood mononuclear cells (PBMCs) obtained from the subject. By methods herein, the instrument may isolate or separate target cells (e.g., white blood cells such CD8+ cells or cTRLs) from the sample. For example, the sample comprising PBMCs can be labeled with antibodies, e.g., antibodies herein. The sample comprising PBMCs can be labeled with magnetic nanoparticles. The sample comprising PBMCs, the antibodies, and the magnetic nanoparticles can be processed by the instrument herein to isolate or separate target cells (e.g., white blood cells) from the sample by methods herein. The remaining sample can be returned to the bloodstream of the subject. The instrument, cartridges, and methods herein can preserve the inherent, highly desired features of cTRLs and may improve healthcare outcomes for subjects undergoing leukapheresis treatment.

[0219] For example, as illustrated in Table 3, PBMCs can be obtained from a subject and provided in a leukopak. In some cases, the leukopak can include at least 6 billion PBMCs. In some cases, a full leukopak, a half leukopak, or a quarter leukopak can be used by the instrument herein. In some cases, the full leukopak can have at least 900 million cells, e.g., CD8+ cells, the half leukopak can have at least 450 million cells, e.g., CD8+ cells, or the quarter leukopak can have at least 220 million cells, e.g., CD8+ cells. In some cases, the instrument herein can isolate or separate circulating tumor-reactive lymphocytes (cTRLs) from the population of the PBMCs or the CD8+ cells. For example, the instrument herein can isolate at least 7 million cTRLs (about 0.8%) from a population of about 900 million CD8+ cells, at least 3.5 million cTRLs (about 0.8%) from a population of about 450 million CD8+ cells, or at least 1.5 million cTRLs (about 0.7%) from a population of about 220 million CD8+ cells. In some cases, the cTRLs may be returned to the bloodstream of the subject by the instrument herein for performing leukapheresis. In some cases, the CD8+ cells may be returned to the bloodstream of the subject by the instrument herein for performing leukapheresis.Table 3Example 3: Use of the instrument and cartridge for therapeutic applications in immunotherapy

[0220] A fluid sample obtained from a subject is analyzed with the instrument of the present disclosure for a therapeutic application. In some cases, the therapeutic application is immunotherapy. Immunotherapy can include a treatment or therapy that uses a patient’s own immune system to fight a disease, e.g., cancer or autoimmune diseases. For example, immunotherapy can boost or change how the immune system works so it can find and attack cancer cells. ). In some cases, platforms, cartridges, and methods herein can be used to automate cell processing for treating patients with an immunotherapy. For example, the immunotherapy can include autologous T-cell based immunotherapy. In some cases, T-cell based immunotherapy can include CD8+or CD103+T-cells for the treatment of recurrent or refractory solid tumors. In some cases, said T-cells can be prepared from the patient’s peripheral blood mononuclear cells (PBMCs). In some cases, said T-cells or PBMCs can be obtained via a leukapheresis procedure using platforms, cartridges, and methods herein. In some cases, said T-cells can be expanded in cell culture, washed, and formulated as a cell suspension and cryopreserved. In some cases, said cell suspension can be used as a drug in immunotherapy to treat certain cancers or autoimmune diseases. For example, as illustrated in FIG. 61A, the instrument and cartridge herein were used to isolate CD8+and CD103+from patient samples. Live cells from patient samples included melanoma, head and neck cancer, cervical cancer, and healthy PBMCs. The starting frequency was 0.1% to 5.0% for CD8+and CD103+cells in the samples tested. As illustrated in FIG. 61B, the increase in the frequency of the CD8+and CD103+cells ranged between 5% to 90% thereby achieving an enrichment-fold of about 10-fold to 150-fold.

Claims

CLAIMSWhat is claimed is:

1. A cartridge for isolating target cells in a fluid sample, comprising: a flow chamber configured to receive the fluid sample; one or more flow rate-reducing structures operatively coupled to the flow chamber configured to reduce a flow rate of the fluid sample proximate to the flow rate-reducing structures; and a degassing region operatively coupled to the flow chamber, wherein the degassing region is configured to remove bubbles from the fluid sample, wherein the cartridge is configured to receive one or more fluids comprising the fluid sample and further comprising the target cells, wherein the target cells are labeled with magnetically susceptible nanobeads.

2. The cartridge of claim 1, further comprising a magnetic field generator configured to generate a magnetic field.

3. The cartridge of claim 2, wherein the magnetic field causes a magnetic force to act on the magnetically susceptible nanobeads to isolate the target cells proximate to the one or more flow rate-reducing structures.

4. The cartridge of claim 3, wherein the one or more flow-rate reducing structures are configured to reduce the flow rate of the fluid sample sufficiently low for the magnetic force of the magnetic field acting on the nanobeads to overcome a drag force of the reduced flow rate acting on the target cells thereby isolating the target cells proximate to the one or more flow-rate reducing structures.

5. The cartridge of claim 2, wherein the magnetic field generator is configured as one or more magnetic arrays.

6. The cartridge of claim 5, wherein the one or more magnetic arrays are configured to cause one or more magnetic forces to act on the magnetically susceptible nanobeads.

7. The cartridge of claim 1, wherein the flow chamber is configured with one or more isolation zones comprising the one or more flow-rate reducing structures.

8. The cartridge of claim 7, wherein the one or more isolation zones are configured as a first zone and a second zone.

9. The cartridge of claim 8, wherein the cartridge is configured to flow the one or more fluids through the first zone at a first flow rate and to flow the one or more fluids through the second zone at a second flow rate.

10. The cartridge of claim 9, wherein a combination of the first flow rate and the second flow rate is configured based at least on the drag force acting on the target cells and a magnetic force acting on the nanobeads.

11. The cartridge of claim 8, wherein a height, a width, or a depth of the one or more flow-rate reducing structures in the first zone is identical as a height, a width, or a depth of the one or more flow-rate reducing structures in the second zone.

12. The cartridge of claim 11, wherein the height comprises a range of from about 50 to 150 micrometers (pm).

13. The cartridge of claim 8, wherein a height, a width, or a depth of the one or more flow-rate reducing structures in the first zone is different from a height, a width, or a depth of the one or more flow-rate reducing structures in the second zone.

14. The cartridge of claim 13, wherein the height comprises a range of from about 50 - 150 micrometers (pm).

15. The cartridge of claim 1, wherein the cartridge is further configured to transmit the one or more fluids to one or more flow outlets.

16. The cartridge of claim 1, further comprising a degasser fluidically coupled to the degassing region, the flow chamber, or the one or more fluids.

17. The cartridge of claim 16, wherein the degasser comprises an optical sensor configured to detect the bubbles in the one or more fluids.

18. The cartridge of claim 16, wherein the degasser is configured to change a flow of the one or more fluids.

19. The cartridge of claim 18, wherein the degasser is configured to change the flow by bypassing the flow chamber and flowing the one or more fluids to one or more outlets.

20. The cartridge of claim 1, wherein at least one of the one or more flow-rate reducing structures is configured with one or more trapping surfaces.

21. The cartridge of claim 20, wherein at least one of the one or more trapping surfaces is configured as a concave surface defined by two joined arms defining an angle.

22. The cartridge of claim 1, further comprising a fluid flow generator and one or more valves configured to generate a flow of the one or more fluids through the flow chamber.

23. The cartridge of claim 22, wherein a first fluid of the one or more fluids comprises a priming fluid, a second fluid comprises the fluid sample, and a third fluid comprises a washing fluid.

24. The cartridge of claim 22, wherein the fluid flow generator comprises a peristaltic pump.

25. The cartridge of claim 22, wherein the one or more valves comprises one or more solenoid pinch valves.

26. The cartridge of claim 1, wherein the cartridge is configured to perform high-throughput processing of the fluid sample at a rate of at least about 109cells per hour.

27. The cartridge of claim 1, further comprising: one or more inlets configured to receive the one or more fluids from one or more input containers; and one or more outlets configured transmit the one or more fluids to one or more output containers.

28. The cartridge of claim 1, wherein the target cells comprise peripheral blood mononuclear cells (PBMCs), tumor-reactive lymphocytes (TRLs), or circulating TRLs.

29. The cartridge of claim 1, wherein the magnetically susceptible nanobeads are configured with or attached to one or more antibodies specific to a surface protein of the target cells.

30. The cartridge of claim 29, wherein the surface protein comprises CD3, CD4, CD8, CD39, CD 103, SLC6A19, SIDT1 or any combination thereof.

31. The cartridge of claim 1, wherein the target cells or the magnetically susceptible nanobeads are configured with or attached to one or more fluorophores.

32. A platform for isolating target cells in a fluid sample, comprising: the cartridge of any one of claims 1-31; one or more pumps configured to flow the one or more fluids through the flow chamber of the cartridge; one or more valves configured to stop or start the flow of the one or more fluids through the flow chamber of the cartridge; one or more magnetic arrays configured to apply the magnetic field to the magnetically susceptible nanobeads in the flow chamber; one or more bubble sensors configured to detect bubbles in the one or more fluids of the cartridge; one or more processors configured to control one or more processes for isolating the target cells from the fluid sample in the cartridge; and a power supply configured to supply power to the platform.

33. The platform of claim 32, further comprising a deck operatively coupled to the one or more pumps, the one or more valves, the one or more magnetic arrays, or the one or more bubble sensors.

34. The platform of claim 32, wherein at least one pump of the one or more pumps is a peristaltic pump.

35. The platform of claim 32, wherein at least one pump of the one or more pumps is operatively coupled to the one or more valves, the one or more bubble sensors, and the one or more fluids.

36. The platform of claim 32, wherein at least one valve of the one or more valves is a solenoid pinch valve.

37. The platform of claim 32, wherein at least one valve of the one or more valves is operatively coupled to the one or more pumps, the one or more bubble sensors, and the one or more fluids.

38. The platform of claim 32, wherein at least one magnetic array of the one or more magnetic arrays is a permanent magnet.

39. The platform of claim 32, wherein at least one magnetic array of the one or more magnetic arrays is magnetically coupled to the one or more fluids, wherein the coupling comprises magnetic field lines of the at least one magnetic array to pass through the one or more fluids.

40. The platform of claim 32, wherein at least one bubble sensor of the one or more bubble sensors is an optical sensor.

41. The platform of claim 32, wherein at least one bubble sensor of the one or more bubble sensors is operatively coupled the one or more pumps, the one or more valves, and the one or more fluids.

42. The platform of claim 33, wherein the deck further comprises a deck framing and a deck enclosure.

43. The platform of claim 42, wherein the deck framing is operatively coupled to the deck enclosure.

44. The platform of claim 42, wherein the deck framing is operatively coupled to the one or more pumps, the one or more valves, the one or more magnetic arrays, or the one or more bubble sensors.

45. The platform of claim 42, further comprising a magnet actuator assembly operatively coupled to the deck framing.

46. The platform of claim 45, where the magnet actuator assembly is configured to be operatively coupled to the one or more magnetic arrays.

47. The platform of claim 45, wherein the magnet actuator assembly comprises: a linear actuator configured to generate a movement of the one or more magnetic arrays proximate to the cartridge; a linear motion rail configured to generate a position of the one or more magnetic arrays proximate to the cartridge; and a cartridge interface configured to releasably couple the cartridge to the magnet actuator assembly.

48. The platform of claim 47, wherein the magnet actuator assembly is configured to move or position the one or more magnetic arrays to generate one or more magnetic capture zones in the flow chamber.

49. The platform of claim 48, wherein the one or more magnetic capture zones comprises an area circumscribing all or substantially all of the flow-rate reducing structures.

50. The platform of claim 47, further comprising a motion limit assembly operatively coupled to the deck framing.

51. The platform of claim 50, where in the motion limit assembly comprises: one or more optical sensors configured to determine the movement or the position of the magnet actuator assembly; and one or more hard stops configured to stop the movement of the magnet actuator assembly when the movement exceeds a predetermined movement.

52. The platform of claim 42, wherein the deck enclosure is configured to be compliant with at least contract development and manufacturing organization (CDMO) safety protocols.

53. The platform of claim 33, further comprising a main body operatively coupled to the deck, a graphical user interface (GUI), or the power supply.

54. The platform of claim 32, wherein the one or more processors is configured to automatically execute instructions comprising:(a) priming the cartridge with priming fluid;(b) isolating the target cells from the fluid sample;(c) washing the cartridge with washing fluid;(d) recovering the target cells from the cartridge; or(e) any combination of (a) to (d).

55. The platform of claim 32, wherein the power supply is configured to supply 24 volts (V), 9.2 amps (A), or 221 watts (W) to the platform.

56. The platform of claim 53, wherein the main body further comprises a body framing and a body enclosure.

57. The platform of claim 56, wherein the body framing is configured to be operatively coupled to the GUI.

58. The platform of claim 56, wherein the body framing is configured to be operatively coupled to the power supply.

59. The platform of claim 56, wherein the body framing is configured to be operatively coupled to one or more poles and wherein the one or more poles are configured to hold one or more containers for the one or more fluids.

60. The platform of claim 59, wherein the one or more containers are configured as a primer container, a fluid sample container, and a wash container.

61. The platform of claim 56, wherein the body enclosure further comprises one or more lids operatively coupled to the body enclosure.

62. The platform of claim 61, wherein the one or more lids are configured to isolate a user from the one or more magnetic arrays.

63. The platform of claim 56, wherein the body enclosure is configured to be compliant with at least contract development and manufacturing organization (CDMO) safety protocols.

64. The platform of claim 32, wherein the target cells comprise peripheral blood mononuclear cells (PBMCs).

65. The platform of claim 32, wherein the target cells comprise tumor-reactive lymphocytes (TRLs).

66. The platform of claim 32, wherein the platform is configured to process the fluid sample at a rate of at least about 109cells per hour.

67. The platform of claim 32, wherein the platform is configured to isolate the target cells from the fluid sample with a purity of at least about 95%.

68. The platform of claim 32, wherein the platform is configured to recover the target cells from the fluid sample with a recovery rate of at least about 95%.

69. The platform of claim 32, wherein the platform is configured to flow the one or more fluids at a flow rate of about 1 to 32 milliliters per hour (mL / hr).

70. The platform of claim 32, further comprising one or more sensors configured to detect or measure one or more fluorescent signals associated with the target cells.

71. The platform of claim 32, further comprising a graphical user interface (GUI) configured to allow a user to select or interact with the one or more processes.

72. A method for using a platform for isolating target cells in a fluid sample, comprising:(a) providing the cartridge of any one of claims 1-31;(b) connecting the cartridge to the platform of any one of claims 32-71;(c) priming the cartridge by flowing a priming fluid of the one or more fluids through the cartridge;(d) removing bubbles from the cartridge;(e) flowing the fluid sample of the one or more fluids through the cartridge;(f) applying the magnetic field to isolate the target cells in the fluid sample;(g) washing the cartridge by flowing a washing fluid of the one or more fluids through the cartridge; and(h) recovering the isolated target cells in the fluid sample.

73. The method of claim 72, wherein the priming in (c) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves.

74. The method of claim 72, wherein the removing in (d) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves.

75. The method of claim 72, wherein the flowing in (e) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves.

76. The method of claim 72, wherein the applying in (f) further comprises using the instrument to automatically apply the magnetic field by operation of the magnet actuator assembly.

77. The method of claim 72, wherein the washing in (g) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves.

78. The method of claim 72, wherein the recovering in (h) further comprises using the instrument to automatically operate the one or more pumps and the one or more valves.

79. The method of claim 72, wherein the target cells comprise peripheral blood mononuclear cells (PBMCs).

80. A kit for isolating target cells in a fluid sample, comprising: the cartridge of any one of claims 1-31; a priming fluid, wherein the priming fluid comprises one or more priming reagents for priming the cartridge; and a washing fluid, wherein the washing fluid comprises one or more washing reagents for washing the cartridge.

81. The kit of claim 80, further comprising the fluid sample, wherein the fluid sample comprises the target cells labeled with magnetically susceptible nanobeads.

82. The kit of claim 80, wherein the one or more priming reagents comprises phosphate-buffered saline with ethylenediaminetetraacetic acid (PBS-EDTA), Pluronic F-68® 1% solution, EDTA (50%) plus ImmunoCult™ media (50%), 100% EDTA, Dulbecco’s PBS buffer, or Hank’s buffer.

83. The kit of claim 80, wherein the one or more washing reagents comprises phosphate- buffered saline with ethylenediaminetetraacetic acid (PBS-EDTA), Pluronic F-68® 1% solution, EDTA (50%) plus ImmunoCult™ media (50%), 100% EDTA, Dulbecco’s PBS buffer, or Hank’ s buffer.

Images