Systems and methods for manipulation or evaluation of a biological sample

Abstract

Disclosed herein are fluidic devices configured for bioparticle processing, systems comprising said fluidic devices, and methods of bioparticle processing using said systems.

Description

WSGR Docket No. 65809-702.601SYSTEMS AND METHODS FOR MANIPULATION OR EVALUATION OF A BIOLOGICAL SAMPLECROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 684,200, filed August 16, 2024, and U.S. Provisional Application 63 / 723,956, filed November 22, 2024, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Cell therapies have the potential to revolutionize treatments across multiple medical domains such as oncology, autoimmune diseases, regenerative medicine, and neurodegenerative diseases. However, this potential has been constrained by a host of flaws within the pharmaceutical industry’s cunent paradigm for therapeutic production that impede accessibility. Shortfalls in manufacturing scalability, the trained workforce, global pharmaceutical models, and technological deficiencies have resulted in a lack of sufficient access to curative medicines, particularly autologous cell therapies. Current approaches to cell therapy manufacturing entail expensive brick- and-mortar clean-room facilities, highly trained technical staff, and lengthy manufacturing times, which is further exacerbated by complex logistics for the collection and transportation of biological materials derived from the patient. Risk of failure can be high and is often due to complications with the manufacturing process. Accordingly, improved biomanufacturing processes and systems are needed. The present disclosure provides technological solutions that offer enhanced scalability', reduced workforce requirements, more affordable pricing, improved safety’, and greater therapeutic success.SUMMARY

[0003] In some aspects, described herein are fluidic systems and devices. In some embodiments, the fluidic systems and devices comprise fluidic devices for cell processing. In some embodiments, the fluidic systems and devices herein provide label-free cell processing and / or ey'aluation including, for example, one or more of label-free cell identification, characterization, and / or measurement. In some embodiments, the label-free cell processing and / or evaluation applies variable amounts of force to modulate bioparticle interactions with a functionalized surface and captures the resulting interactions or interaction changes between the bioparticle and the functionalized surface for evaluating the bioparticle. In some embodiments, the fluidic devices for cell processing comprise one or more bioprocessing flow paths, each comprising one or more functionalized surfaces configured to bind a target bioparticle. In some embodiments, the fluidicWSGR Docket No. 65809-702.601 devices for cell processing comprise an actuation module configured to apply variable amounts of force to one or more bioparticles passing through one or more bioparticle processing flow paths. In some embodiments, the actuation module comprises a fluid flow distribution module (e.g., a dynamically configurable manifold) that is configured to apply variable force through adjusting a fraction of a fluid flow within the device passing through one or more cell processing flow path(s) fluidically coupled to the flow distribution module. Accordingly, the variable force can be applied through the force of the liquid medium flow that passes through the flow paths whereby a stronger rate of fluidic flow applies a stronger motive force upon the bioparticles in the flow paths. A slow or paused flow can allow bioparticles to settle (e.g., via gravity) and bind or interact with the functionalized surface. An increasing flow rate can gradually apply increasing amounts of motive force upon a bioparticle until the force overcomes the binding force and causes the bioparticle to detach from or move across the functionalized surface. Alternatively or in combination, in some embodiments, the actuation module comprises one or more actuators (e.g., ultrasonic transducers) positioned along the one or more bioprocessing flow paths that are configured to apply force upon one or more bioparticles passing through the flow paths. In some embodiments, an actuator is positioned to direct force (e.g., a pressure wave or standing wave) perpendicular to the flow path to push or pull a bioparticle towards a functionalized surface of a bioprocessing flow path, thereby forcing the bioparticle into contact with the functionalized surface. In some embodiments, another actuator is positioned to direct force perpendicular to the flow path to push or pull a bioparticle away from a functionalized surface of a bioprocessing flow path.

[0004] In some embodiments, the fluidic devices for cell processing comprise a dynamically configurable manifold, configured to adjust a fraction of a fluid flow within the device passing through one or more cell processing flow path(s) fluidically coupled to the configurable manifold. In some embodiments, the one or more cell processing flow path(s) each comprise an optical channel having a functionalized surface configured to interact with one or more of the individual cells passing through the optical channel. In some embodiments, the functionalized surface interacts with an individual cell to form transient or temporary interactions that results in a modification of the cell's movement through the optical channel. In some embodiments, the functionalized surface interacts with an individual cell to form stable interactions that results in immobilization or stopping of the cell’s movement through the optical channel. In some embodiments, the fluidic devices for cell processing comprise an input manifold, fluidically coupled to, or integrated with, at least one inlet of the dynamically configurable fluidic manifold. In some embodiments, the fluidic devices for cell processing comprise an outlet manifold, fluidically coupled to one or more outlets of the cell processing flow path(s).WSGR Docket No. 65809-702.601

[0005] In some embodiments, fluidic coupling of the input manifold, the dynamically configurable manifold, and the outlet manifold forms a recurrent loop. In some embodiments, the plurality of parallel cell processing flow paths comprise two or more flow paths selected from the group of: an identification flow path, a characterization flow path, a cell sorting flow path, a cell selection flow path, a cell deselection flow path, a cell engineering flow path, a cell culturing flow path, a cell activating flow path, a cell washing flow path, and a cell expanding flow path.

[0006] In some embodiments, the recurrent loop is configured to allow at least a portion of cells flowing through the plurality of cell processing flow paths to recirculate from the outlet manifold to the at least one inlet of the dynamically configurable manifold. In some embodiments, the recirculation follows a continuous, monodirectional flow path. In some embodiments, recirculation comprises flowing of the same fluid or the same bioparticles along one or more reversals in flow direction within the same channel. In some embodiments, recirculation comprises alternating flow direction along 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fluidic channels to direct bioparticles.

[0007] In some embodiments, the plurality of parallel cell processing flow paths comprise a cell culturing flow path, a cell activation flow path, and: (i) a cell measurement flow path; or (ii) a cell characterization flow path. In some embodiments, the cell measurement flow path comprises a cell counting module, and / or the cell characterization flow path comprises a functional screening module.

[0008] In some embodiments, the plurality of parallel cell processing flow paths comprise (i) a cell measurement module or a cell characterization module; and (ii) a cell sorting module in the same flow path. In some embodiments, the cell sorting flow path comprises a label-free cell sorting module. In some embodiments, the cell sorting flow path comprises a cell concentrating module. In some embodiments, the cell sorting flow path comprises a cell dilution module.

[0009] In some embodiments, the cell sorting flow path comprises an acoustic sorting module comprising one or more acoustic transducers and a plurality of acoustic sorting channels. In some embodiments, the acoustic sorting module comprises at least 2 parallel acoustic sorting channels. In some embodiments, the acoustic sorting module comprises a plurality7of capacitive micromachined ultrasonic transducers (CMUT) and / or an array of piezoelectric transducers (PMUT).

[0010] In some embodiments, cells within the at least 2 parallel acoustic sorting channels are diverted in sorting channels that are positioned in a different axis to the upstream flow path, such as vertically above or below the upstream cell sorting flow path.

[0011] In some embodiments, the input manifold comprises a plurality7of inlets and / or the outlet manifold comprises a plurality of outlets.WSGR Docket No. 65809-702.601

[0012] In some embodiments, the input manifold comprises a plurality of inlets and a single outlet fluidically coupled to the at least one inlet of the dynamically configurable manifold, and / or the outlet manifold comprises: a plurality of inlets, each fluidically coupled to a corresponding outlet of the plurality of cell processing flow paths; and a single outlet. In some embodiments, the fluidic device comprises one or more inline pH sensors, one or more inline pyruvate concentration sensors, one or more lactate concentration sensors, one or more pCO2 sensors, and / or one or more inline dissolved oxygen sensors.

[0013] In some embodiments, at least one of the one or more inline pH sensors, at least one of the one or more inline dissolved oxygen sensors, at least one of the one or more inline pyruvate concentration sensors, at least one of the one or more lactate concentration sensors, or at least one of the one or more pCO2 sensors, is comprised in a cell characterization module. In some embodiments, the input manifold comprises an inline mixer. In some embodiments, the inline mixer is and / or comprises a zero dead-volume mixer.

[0014] In some embodiments, the input manifold and / or the outlet manifold comprise one or more zero dead-volume switching valves. In some embodiments, the fluidic device comprises one or more positive displacement pumps, such as peristaltic, syringe, or other continuous flow pumps.

[0015] In some embodiments, the input manifold comprises at least three input manifold inlets. In some embodiments, the outlet manifold comprises at least three input manifold inlets. In some embodiments, the plurality of parallel flow paths comprises at least three parallel flow paths each corresponding to at least one cell processing application. In some embodiments, the cell characterization flow path, and / or the cell measurement flow path comprises an optical channel comprising an optical sensor.

[0016] In some embodiments, the optical sensor is a complementary metal oxide semiconductor sensor, a photomultiplier or photodiode, and / or a camera. In some embodiments, the optical channel comprises a functionalized surface configured to interact with one or more cells passing through the optical channel. In some embodiments, the optical channel further comprises an acoustic transducer. In some embodiments, the acoustic transducer is configured to promote and / or increase interaction of the one or more cells passing through the optical channel with the functionalized surface.

[0017] In some embodiments, the functionalized surface is functionalized using an antibody, an aptamer, a cell adhesion molecule, and / or an adhesion protein. In some embodiments, the fluidic device is: a microfluidic device, and / or a device configured for manufacture of engineered cells for use in a cell therapeutic. In some embodiments, the engineered cells are leukocytes. In some embodiments, the engineered cells are CAR-T cells, CAR-NK cells, modified B-cells, tumorWSGR Docket No. 65809-702.601 infiltrating lymphocyte cells, induced pluripotent stem cells (iPSC) and / or hematopoietic stem cells. In some embodiments, the cells processed are allogenic to a patient.

[0018] In some embodiments, one or more components of the fluidic device are comprised in a cartridge. In some embodiments, the cartridge is a disposable cartridge.

[0019] In some embodiments, the cartridge comprises the input manifold, the output manifold, the fluidic couplings, and / or the outlet manifold. In some embodiments, a majority of the fluidic volume of the fluidic system is comprised within the cartridge, and / or is comprised within a plurality of cartridges of the system.

[0020] In some embodiments, systems and devices described herein further comprise a controller configured to dynamically adjust an operation of the dynamically controllable manifold. In some embodiments, the controller further comprises a user interface which allows for user selection of two or more parallel cell processing operations. In some embodiments, the controller is configured to perform at least a subset of a plurality of pre-programmed cell processing operations on cells present in the device in parallel. In some embodiments, a plurality of cell processing operations are automatically performed by the controller using the fluidic device. In some embodiments, the controller is operated remotely and / or is remotely programmable or reprogrammable.

[0021] In some embodiments, the controller is configured to dynamically adjust the operation of a plurality of dynamically adjustable manifolds of a plurality of microfluidic devices. In some embodiments, the plurality of parallel cell processing flow paths comprise a cell delivery and / or a cell acquisition flow path. In some embodiments, the cell delivery and / or cell acquisition flow path is configured to be fluidically couplable to a blood vessel of a subject.

[0022] In some embodiments, the device is a point-of-care cell therapeutic device, and the subject is a patient suffering from a disease or condition in need of treatment by a cell therapy .

[0023] In some aspects, disclosed herein is a system comprising: a fluidic device for bioparticle processing, comprising: a chamber defining one or more bioparticle processing flow paths, each bioparticle processing flow path comprising a functionalized surface configured to specifically interact with a target bioparticle; an actuation module configured to apply variable amounts of force to one or more bioparticles within the one or more bioparticle processing flow paths; and a sensor module configured to detect one or more changes in interaction between the one or more bioparticles and the functionalized surface associated with the variable amounts of force. In some embodiments, the system further comprises a controller configured to dynamically adjust an operation of the actuation module, optionally wherein the actuation module comprises a dynamically configurable fluidic manifold. In some embodiments, the controller is configured toWSGR Docket No. 65809-702.601 dynamically adjust the operation of the actuation module to apply the variable amounts of force upon one or more bioparticles located within the one or more bioparticle processing flow paths.

[0024] In some embodiments, the controller further comprises a user interface which allows for user selection of two or more parallel bioparticle processing operations. In some embodiments, the controller is configured to perform at least a subset of a plurality of pre-programmed bioparticle characterization operations on the one or more bioparticles located within the one or more bioparticle processing flow paths. In some embodiments, the controller is configured to perform at least a subset of a plurality of pre-programmed bioparticle characterization operations on a plurality of the one or more bioparticles located within the one or more bioparticle processing flow paths in parallel.

[0025] In some embodiments, the plurality of pre-programmed bioparticle characterization operations is automatically performed by the controller using the fluidic device. In some embodiments, the controller is operated remotely and / or is remotely programmable or reprogrammable. In some embodiments, the controller is configured to dynamically adjust the operation of a plurality of dynamically configurable fluidic manifolds of a plurality of microfluidic devices. In some embodiments, the one or more pre-programmed bioparticle characterization operations comprise instructions for the actuation module to generate force acting upon one or more bioparticles located within the one or more bioparticle processing flow paths. In some embodiments, the one or more pre-programmed bioparticle characterization operations comprise instructions for the actuation module to carry out one or more of: increase the force, optionally until a threshold amount is reached; decrease force, optionally until a threshold amount is reached; maintain the force at a current amount; reduce the force to zero; adjust the force according to a linear model; adjust the force according to a non-linear model; and / or adjust the force to a preset amount. In some embodiments, the actuation module comprises a fluid flow distribution module, an actuator, or a combination thereof. In some embodiments, the actuator utilizes an actuation mechanism selected from a pneumatic, thermal-pneumatic, piezoelectric, thermal-electric, and / or shape memory' alloy.

[0026] In some embodiments, the actuator is an acoustic actuator configured to increase and / or decrease interaction between the one or more bioparticles and the functionalized surface within the one or more bioparticle processing flow paths. In some embodiments, the acoustic actuator is configured to generate a force that pushes or pulls the one or more bioparticles toward or against the functionalized surface, thereby enabling binders on the functionalized surface to interact with any of the one or more bioparticles expressing a target molecule. In some embodiments, the flow distribution module generates fluidic flow through the one or more bioparticle processing flowWSGR Docket No. 65809-702.601 paths. In some embodiments, the flow distribution module comprises one or more of a flow diverter, one or more valves, and / or a manifold fluidically coupled to the one or more bioparticle processing flow paths.

[0027] In some embodiments, the flow distribution module comprises a dynamically configurable fluidic manifold fluidically coupled to the one or more bioparticle processing flow paths. In some embodiments, the dynamically configurable fluidic manifold is configured to cany’ out instructions from a controller to adjust a rate of fluidic flow through the one or more bioparticle processing flow paths, optionally according to one or more pre-programmed bioparticle characterization operations. In some embodiments, the one or more pre-programmed bioparticle characterization operations comprise instructions to cany' out one or more of: increase the rate of fluidic flow, optionally until a threshold flow rate is reached; decrease the rate of fluidic flow, optionally until a threshold flow rate is reached; maintain the rate of fluidic flow at a cunent flow rate; reduce the rate of fluidic flow to zero; adjust the rate of fluidic flow according to a linear model; adjust the rate of fluidic flow according to a non-linear model; and / or adjust the rate of fluidic flow to a preset flow rate. In some embodiments, the rate of fluidic flow is adjusted according to a sinusoidal curve, a continuous and gradual rate change, step-wise rate change, or any combination thereof.

[0028] In some embodiments, the fluidic device comprises: an input manifold, fluidically coupled to, or integrated with, at least one inlet of the flow distribution module, optionally wherein the flow distribution module is a dynamically configurable fluidic manifold; and an outlet manifold, fluidically coupled to one or more outlets of the one or more bioparticle processing flow paths. In some embodiments, fluidic coupling of the input manifold, the flow distribution module, and the outlet manifold forms a recurrent loop. In some embodiments, the input manifold comprises a plurality of inlets and / or the outlet manifold comprises a plurality’ of outlets. In some embodiments, the input manifold comprises a plurality of inlets and a single outlet fluidically coupled to the at least one inlet of the dynamically configurable manifold, and / or the outlet manifold comprises: a plurality of inlets, each fluidically coupled to a corresponding outlet of the plurality of cell processing flow paths; and a single outlet. In some embodiments recurrent loops are configured to allow reversal of flow direction over time along the same fluidic channel.

[0029] In some embodiments, the fluidic device comprises one or more inline pH sensors, one or more inline glucose concentration sensors, one or more inline pyruvate concentration sensors, one or more lactate concentration sensors, one or more pCO2 sensors, and / or one or more inline dissolved oxygen sensors. In some embodiments, at least one of the one or more inline pH sensors, at least one of the inline dissolved oxygen sensors, at least one or more inline glucoseWSGR Docket No. 65809-702.601 concentration sensors, at least one of the one or more inline pyruvate concentration sensors, at least one of the one or more lactate concentration sensors, or at least one of the one or more pCO2 sensors, is comprised in a cell characterization module. In some embodiments, the input manifold comprises an inline mixer. In some embodiments, the inline mixer comprises a zero dead-volume mixer. In some embodiments, the input manifold and / or the outlet manifold comprise one or more zero dead-volume switching valves.

[0030] In some embodiments, the input manifold comprises at least three input manifold inlets. In some embodiments, the outlet manifold comprises at least three input manifold inlets. In some embodiments, the fluidic device comprises one or more of peristaltic pumps and / or continuous flow pumps. In some embodiments, the sensor module is positioned to detect and / or track one or more interactions between the functionalized surface and the one or more bioparticles within the one or more bioprocessing flow paths. In some embodiments, each bioparticle processing flow path comprises one or more of a bioparticle identification flow path, a bioparticle characterization flow path, and / or a bioparticle measurement flow path.

[0031] In some embodiments, one or more of the bioparticle identification flow path, the bioparticle characterization flow path, and / or the bioparticle measurement flow path comprises an optical channel configured to allow the sensor module to capture sensor readings or measurements within the optical channel. In some embodiments, the optical channel comprises the functionalized surface configured to specifically interact with the target bioparticle. In some embodiments, the sensor module comprises an optical sensor positioned to capture sensor readings or measurements of the one or more bioparticles within the optical channel. In some embodiments, the optical channel comprises a region that is transparent to the optical sensor to enable capture of the sensor readings or measurements of the one or more bioparticles within the optical channel.

[0032] In some embodiments, the optical sensor is a complementary metal oxide semiconductor sensor, a photomultiplier or photodiode, and / or a camera. In some embodiments, the optical sensor is configured to individually track the position and / or movement of at least a subset of the one or more bioparticles. In some embodiments, the optical sensor is configured to obtain one or more image(s) or video(s) capturing at least a subset of the one or more bioparticles. In some embodiments, the optical sensor captures a field of view comprising at least 10%. at least 20%. at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the functionalized surface.

[0033] In some embodiments, the one or more bioparticle processing flow paths comprise a plurality of parallel bioparticle processing flow paths. In some embodiments, the plurality of parallel bioparticle processing flow paths comprise at least two parallel flow' paths eachWSGR Docket No. 65809-702.601 corresponding to at least one cell processing application. In some embodiments, the plurality of parallel bioparticle processing flow paths comprise at least 2, at least 3, at least 4, at least 5. at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 flow paths. In some embodiments, the plurality of parallel bioparticle processing flow paths comprise two or more flow paths selected from the group consisting of: a bioparticle identification flow path, a bioparticle characterization flow path, a bioparticle measurement flow path, a bioparticle sorting flow path, a bioparticle selection flow path, a bioparticle deselection flow path, a bioparticle engineering flow path, a bioparticle culturing flow path, a bioparticle activating flow path, a bioparticle washing flow path, and a bioparticle expanding flow path.

[0034] In some embodiments, each bioparticle processing flow path comprises a bioparticle characterization flow path, wherein the bioparticle characterization flow path comprises the functionalized surface configured to physically interact with the target bioparticle. In some embodiments, the plurality of parallel bioparticle processing flow paths comprise a plurality of parallel bioparticle characterization flow paths. In some embodiments, the target bioparticle is a cell, a cell fragment, a vesicle, an organoid, a liposome, a lipid nanoparticle, a virus, pollen, or spore. In some embodiments, the functionalized surface comprises a binder configured to interact with and / or bind to a surface marker on the target bioparticle. In some embodiments, the functionalized surface comprises two or more binders that specifically bind different surface markers. In some embodiments, the different surface markers are different types of cell surface biomarkers or receptors. In some embodiments, the two or more binders are segregated into discrete regions of the functionalized surface. In some embodiments, the one or more bioparticle processing flow paths are fluidically coupled, directly and / or indirectly, to an upstream inlet and a downstream outlet, and wherein the discrete regions are positioned in sequence along the one or more bioparticle processing flow paths between the upstream inlet and the downstream outlet.

[0035] In some embodiments, the functionalized surface is functionalized using an antibody or fragment thereof, an aptamer, a cell adhesion molecule, and / or an adhesion protein. In some embodiments, the binder comprises one or more of an antibody or fragment thereof, an aptamer, a cell adhesion molecule, and / or an adhesion protein. In some embodiments, the binder specifically binds a native cell surface receptor or a modified cell surface receptor of the target bioparticle, optionally wherein the modified cell surface receptor is a chimeric antigen receptor. In some embodiments, the binder specifically binds one or more stem cells, progenitor cells, erythrocytes, lymphocytes, platelets, epithelial cells, endothelial cells, hepatocytes, neuronal cells, glial cells, interstitial cells, adipocytes, dermal fibroblasts, myocytes, osteoclasts, osteoblasts, and / or cancerWSGR Docket No. 65809-702.601 cells. In some embodiments, the binder specifically binds one or more of neutrophils, eosinophils, basophiles, monocytes, mast cells, dendritic cells, and / or macrophages.

[0036] In some embodiments, the binder specifically binds one or more of T-cells, NK cells, NKT cells, B cells, plasma cells, and / or CAR-T cells. In some embodiments, the system comprises a processor configured to analyze the one or more movements and / or positional changes and the variable amounts of force applied to the one or more bioparticles, thereby generating an evaluation of at least a subset of the one or more bioparticles, optionally wherein the processor is a component of a controller configured to control the fluidic device. In some embodiments, the processor is configured to analyze at least a subset of the one or more movements and / or positional changes and the variable amounts of force applied to the one or more bioparticles using a statistical model or a trained machine learning model. In some embodiments, the trained machine learning model is trained using an algorithm selected from support vector machine, linear regression, artificial neural network, naive Bayes classifier algorithm, decision tree, random forests, or nearest neighbors. In some embodiments, the evaluation comprises one or more of an identification, a characterization, and / or a measurement.

[0037] In some embodiments, the identification comprises a determination that the bioparticle is positive or negative for a cell type selected from stem cells, progenitor cells, erythrocytes, lymphocytes, platelets, epithelial cells, endothelial cells, hepatocytes, neuronal cells, glial cells, interstitial cells, adipocytes, dermal fibroblasts, myocytes, osteoclasts, osteoblasts, and cancer cells. In some embodiments, the characterization comprises a determination that the bioparticle is positive or negative for a cell subtype selected from T-cells, NK cells, NKT cells, B cells, plasma cells, and CAR-T cells. In some embodiments, the measurement comprises a determination of an expression level of a surface biomarker corresponding to a T-cell. NK cell, NKT cell, B cell, plasma cell, or CAR-T cell, or a subtype thereof. In some embodiments, the one or more bioparticle processing flow paths comprise (i) one or more of a bioparticle identification module, a bioparticle measurement module and / or a bioparticle characterization module; and (ii) a bioparticle sorting module in the same flow path. In some embodiments, the bioparticle sorting flow path comprises a label-free cell sorting module.

[0038] In some embodiments, the bioparticle sorting module is positioned downstream of one or more of the bioparticle identification module, the bioparticle measurement module, and / or the bioparticle characterization module in the same flow path. In some embodiments, the bioparticle sorting module is configured to exert a force upon, displace, or cause at least a subset of the one or more bioparticles to separate the one or more bioparticles into two or more distinct flow paths. In some embodiments, the two or more distinct flow paths comprise at least 2 parallel sortingWSGR Docket No. 65809-702.601 channels that have at least one fluidic connection enabling the subset of the one or more bioparticles to be moved between the at least 2 parallel sorting channels. In some embodiments, the bioparticle sorting module comprises an acoustic sorting module comprising one or more acoustic transducers. In some embodiments, the acoustic sorting module comprises at least 2 parallel acoustic sorting channels. In some embodiments, the acoustic sorting module comprises a plurality of capacitive micromachined ultrasonic transducers (CMUT) and / or an array of piezoelectric micromechanical ultrasonic transducers (PMUT).

[0039] In some embodiments, the fluidic device is: a microfluidic device, and / or a device configured for manufacture of engineered cells for use in a cell therapeutic. In some embodiments, the engineered cells are leukocytes. In some embodiments, the engineered cells are CAR-T cells, CAR-NK cells, modified B-cells. tumor infiltrating lymphocyte cells, and / or induced pluripotent stem cells and hematopoietic stem cells. In some embodiments, the bioparticles processed by the system comprise cells that are allogenic to a patient. In some embodiments, one or more components of the fluidic device are comprised in a cartridge. In some embodiments, the cartridge is a disposable cartridge. In some embodiments, the cartridge comprises the input manifold, the output manifold, the fluidic couplings, and / or the outlet manifold.

[0040] In some embodiments, a majority of the fluidic volume of the system is comprised within the cartridge, and / or is comprised within a plurality7of cartridges of the system. In some embodiments, the one or more bioparticle processing flow paths are a plurality of parallel cell processing flow paths comprising a cell delivery and / or a cell acquisition flow path. In some embodiments, the cell delivery and / or cell acquisition flow7path is configured to be fluidically couplable to a blood vessel of a patient. In some embodiments, the device is a point-of-care cell therapeutic device, and the subject is a patient suffering from a disease or condition in need of treatment by a cell therapy.

[0041] In some aspects, disclosed herein is a method of processing and / or manufacturing bioparticles, the method comprising: preparing a plurality of engineered bioparticles using the device or system of any of the preceding claims. In some embodiments, bioparticles are circulated through the one or more cell processing flow paths until a bioparticle population in the device possess one or more bioparticle processing target metrics. In some embodiments, the bioparticle population is administered to a subject or patient after the one or more cell or bioparticle processing target metrics are met. In some embodiments, the actuation module comprises a dynamically configurable manifold that is adjusted to maintain one or more cell processing target metrics in a cell population flowing through the device or system. In some embodiments, the cell population is directly administered to a subject or patient during the cell processing operations. In someWSGR Docket No. 65809-702.601 embodiments, the one or more cell processing target metrics comprise: a threshold cell concentration of a target cell type or population thereof, a target expression level of one or more target proteins of a target cell type, a threshold level of one or more contaminants (e.g., of nontarget cell types) in fluid flowing through the device or system, a purity of a target cell or population thereof, a viability of a target cell or population thereof, a number of a target cell or population thereof, a phenotype of a target cell or population and / or a presence of a threshold level of one or more detectable biomarkers present in a cell characterization module of the system.

[0042] In some aspects, disclosed herein is a system comprising: a fluidic device for characterizing individual cells, the device comprising: a dynamically configurable manifold configured to control a rate of fluidic flow within the device: a chamber fluidically coupled to the manifold and defining one or more cell characterization flow path(s), each cell characterization flow path comprising an optical channel having a functionalized surface configured to interact with one or more of the individual cells passing through the optical channel; and an optical sensor configured to obtain one or more sensor reading(s) of the one or more of the individual cells passing through the optical channel; and a controller comprising a processor configured to: dynamically adjust the dynamically configurable manifold to control the rate of fluidic flow within the one or more cell characterization flow path(s) in order to facilitate one or more interactions between the one or more of the individual cells and the functionalized surface in the optical channel: cause the optical sensor to obtain the one or more sensor reading(s) of the one or more individual cells passing through the optical channel; and evaluate the one or more of the individual cells based on the one or more sensor reading(s).

[0043] In some aspects, disclosed herein is a system comprising: a fluidic device for characterizing individual cells, the device comprising: a manifold configured to control a rate of fluidic flow within the device; a chamber fluidically coupled to the manifold and defining one or more cell characterization flow path(s), each cell characterization flow path comprising an optical channel having a functionalized surface configured to interact with one or more of the individual cells passing through the optical channel; and an optical sensor configured to obtain one or more sensor reading(s) of the one or more of the individual cells passing through the optical channel; and a controller comprising a processor configured to: dynamically adjust the manifold to control the rate of fluidic flow within the one or more cell characterization flow path(s) to facilitate one or more interactions between the one or more of the individual cells and the functionalized surface in the optical channel.

[0044] In some aspects, disclosed herein is a system comprising: a fluidic device for characterizing individual bioparticles comprising target bioparticles and non-target bioparticles, theWSGR Docket No. 65809-702.601 device comprising: a dynamically configurable manifold configured to control a rate of fluidic flow within the device; a chamber fluidically coupled to the manifold and defining one or more bioparticle characterization flow path(s), each bioparticle characterization flow path comprising an optical channel having a functionalized surface configured to interact with one or more of the individual bioparticles passing through the optical channel; and an optical sensor configured to obtain one or more sensor reading(s) of the one or more of the individual bioparticles passing through the optical channel; and a controller comprising a processor configured to: dynamically control the dynamically configurable manifold to produce a first flow rate within the one or more bioparticle processing flow path(s) that allows one or more of the individual bioparticles to physically interact with the functionalized surface in the optical channel; dynamically control the dynamically configurable manifold to produce a second flow rate within the one or more bioparticle processing flow path(s) that provides sufficient motive force to overcome interactions between one or more of the individual bioparticles and the functionalized surface in the optical channel; cause the optical sensor to obtain the one or more sensor reading(s) of the one or more individual bioparticles passing through the optical channel during at least the first flow rate and the second flow rate; and analyze the one or more sensor reading(s) to identify, characterize, or measure the one or more of the individual bioparticles based on an indication of the sufficient motive force, wherein the target bioparticles and non-target bioparticles are differentiated based on the one or more sensor reading(s). In some embodiments, the processor is configured to increase fluidic flow from the first flow rate to the second flow rate.

[0045] In some embodiments, the increase in fluidic flow has a peristaltic flow profile characterized by at least a partial sinusoidal flow rate over time. In some embodiments, the processor is configured to increase fluidic flow at a steady rate of increase from the first flow rate to the second flow rate, wherein the sufficient motive force is achieved between the first rate of fluidic flow and the second rate of fluidic flow. In some embodiments, the processor is configured to perform at least a subset of a plurality of pre-programmed bioparticle processing operations on bioparticles present in the device in parallel. In some embodiments, a plurality of bioparticle processing operations is automatically performed by the controller using the fluidic device. In some embodiments, the processor is operated remotely and / or is remotely programmable or reprogrammable. In some embodiments, the processor is configured to dynamically adjust the operation of a plurality of dynamically adjustable manifolds of a plurality of microfluidic devices. In some embodiments, the processor is configured to dynamically control the dynamically configurable manifold to modulate flow rate according to a pre-programmed protocol. In some embodiments, the pre-programmed protocol is configured based on one or more of the targetWSGR Docket No. 65809-702.601 bioparticles, the non-target bioparticles, the functionalized surface, or any combination thereof. In some embodiments, the functionalized surface is functionalized using an antibody, an aptamer, a cell adhesion molecule, and / or an adhesion protein. In some embodiments, the optical channel comprises a plurality of functionalized surfaces.

[0046] In some embodiments, the plurality of functionalized surfaces is organized into discrete regions. In some embodiments, the plurality of functionalized surfaces is arranged in sequence along the one or more bioparticle processing flow path(s). In some embodiments, the plurality of functionalized surfaces comprises a first region configured to interact with a first category' of target bioparticles and a second region configured to interact with a second category of target bioparticles. In some embodiments, the first area comprises binders configured to interact with a substrate expressed on a surface of the first category of bioparticles, and the second area comprises binders configured to interact with a substrate expressed on a surface of the second category' of bioparticles. In some embodiments, the first area comprises antibodies configured to bind to a bioparticle surface marker expressed in the first category of bioparticles, and the second area comprises antibodies configured to bind to a bioparticle surface marker expressed in the second category of bioparticles. In some embodiments, the interactions between the one or more individual bioparticles with the functionalized surface comprises attachment or immobilization of the one or more individual bioparticles upon the functionalized surface.

[0047] In some embodiments, the sufficient motive force overcomes the interactions by causing the one or more of the individual bioparticles to detach from and / or move across the functionalized surface. In some embodiments, the processor is configured to determine a fluidic flow rate or indication thereof that corresponds to a change in interaction between an individual bioparticle and the functionalized surface based on the sensor reading(s). In some embodiments, the change in interaction comprises a detachment or indication thereof of the individual bioparticle from the functionalized surface after being immobilized upon the functionalized surface. In some embodiments, the one or more bioparticle processing flow path(s) comprises a plurality of bioparticle processing flow paths. In some embodiments, the plurality of bioparticle processing flow paths forms a plurality of parallel flow paths. In some embodiments, the chamber is configured to facilitate fluidic dispersal of a plurality of bioparticles entering the chamber into the plurality of parallel flow paths. In some embodiments, the optical sensor comprises an imaging sensor. In some embodiments, the imaging sensor is configured to track each of the individual bioparticles.

[0048] In some embodiments, the imaging sensor is a camera. In some embodiments, the imaging sensor captures a field of view comprising at least 10%, at least 20%, at least 30%, at leastWSGR Docket No. 65809-702.60140%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the optical channel. In some embodiments, the one or more sensor reading(s) comprises one or more image(s) or a video for one or more of the individual bioparticles. In some embodiments, the sensor reading(s) are used to generate at least one bioparticle measurement corresponding to a level of expression of a surface marker on the individual bioparticle. In some embodiments, differentiation of target bioparticles from non-target bioparticles is label-free. In some embodiments, the fluidic device further comprises a sorting mechanism for separating individual bioparticles. In some embodiments, the sorting mechanism comprises a sorting module comprising an acoustic actuator. In some embodiments, the controller is configured to dynamically control the sorting mechanism to separate target and non-target bioparticles. In some embodiments, the sorting mechanism is positioned downstream of the optical channel.

[0049] In some aspects, disclosed herein is a method for characterizing individual bioparticles, comprising: disposing a fluid comprising a plurality of bioparticles comprising target bioparticles and non-target bioparticles on a functionalized surface comprising binders configured to interact with target bioparticles, wherein the target bioparticles and non-target bioparticles have differential interactions with the functionalized surface; providing a variable fluid flow across the functionalized surface that produces a corresponding variable motive force acting upon the plurality of bioparticles on the functionalized surface, wherein the variable motive force overcomes interactions between the functionalized surface and one or more of the plurality of bioparticles; using one or more sensor(s) to obtain one or more sensor reading(s) of the at least a portion of the plurality of bioparticles during the variable fluid flow across the functionalized surface; and generating a bioparticle evaluation for the one or more of the plurality of bioparticles based on the one or more sensor reading(s) obtained by the one or more sensor(s) and a one or more fluid flow rate(s) corresponding to the variable motive force overcoming one or more interaction(s) of the one or more of the plurality of biopartici e(s) with the functionalized surface.

[0050] In some aspects, disclosed herein is a method for characterizing individual bioparticles, comprising: disposing a fluid comprising a plurality of bioparticles comprising a target bioparticle and a non-target bioparticle on a functionalized surface comprising binders configured to interact with the target bioparticle, wherein the target bioparticle has a stronger affinity for the functionalized surface than the non-target bioparticle; providing an increasing fluid flow rate across the functionalized surface that produces an increasing motive force acting upon the plurality of bioparticles on the functionalized surface; using an optical sensor to obtain sensor readings of the target bioparticle and the non-target bioparticle during the variable fluid flow across the functionalized surface; and identifying the target bioparticle and the non-target bioparticle based onWSGR Docket No. 65809-702.601 the variable fluid flow rate and the corresponding sensor readings obtained by the optical sensor, wherein the sensor readings are indicative of (i) a higher motive force required to overcome interactions between the target bioparticle and the functionalized surface and (ii) a lower motive force required to overcome interactions between the non-target bioparticle and the functionalized surface.

[0051] In some aspects, disclosed herein is a method for characterizing individual bioparticles, comprising: providing a functionalized surface configured to interact with individual bioparticles; disposing a fluid comprising one or more bioparticle(s) on the functionalized surface, wherein the one or more bioparticle(s) become at least partially attached to the functionalized surface; providing a variable fluid flow across the functionalized surface that produces a corresponding variable motive force acting upon the one or more bioparticle(s) that are at least partially attached to the functionalized surface; using one or more sensor(s) to obtain one or more sensor reading(s) corresponding to a release or movement of the one or more bioparticle(s) from the functionalized surface; determining one or more fluid flow rate(s) corresponding to the release or movement of the one or more bioparticle(s) based on an association of the one or more sensor reading(s) and the variable fluid flow; and generating one or more bioparticle measurement(s) for the one or more bioparticles based on the one or more fluid flow rate(s) corresponding to the release or movement of the one or more bioparticle(s).

[0052] In some aspects, described herein are methods of processing and / or manufacturing cells. In some embodiments, the methods comprise preparing a plurality of engineered cells using any device or system described herein.

[0053] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0054] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art 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 disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0055] 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 patentWSGR Docket No. 65809-702.601 application was 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 contradictor}' material.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also ‘‘Figure'’ and “FIG.” herein), of which:

[0057] FIG. 1 illustrates a diagram of a microfluidic device configured as a cartridge and the usage of this cartridge within a system for cell processing according to embodiments of the systems, devices, and methods described herein.

[0058] FIGs. 2-3 illustrate schematic diagrams of alternate embodiments of a microfluidic device which can be implemented in a single module or a plurality of modules according to embodiments described herein.

[0059] FIG. 4 illustrates an overview of a bioparticle bioprocessing flow path for bioparticle phenotyping (e.g.. identification, characterization, and measurement for various biomarkers) and sorting and an illustrative design for components of the fluidic device according to embodiments described herein.

[0060] FIG. 5 illustrates an example flow cell module for two of bioparti cl e / cell identification, characterization and / or measurement according to embodiments described herein.

[0061] FIG. 6 illustrates an example workflow for cell evaluation by the flow cell module of FIG. 5, according to some embodiments of the device described herein.

[0062] FIG. 7 illustrates an example workflow for cell characterization by the flow cell module of FIG. 5, according to embodiments described herein.

[0063] FIG. 8 illustrates an example cell selection and / or cell sorting module having parallel flow paths, channels, or lanes according to embodiments described herein.

[0064] FIG. 9 illustrates a top panel showing an image of a flow path containing a plurality of bioparticles / cells, a center panel showing algorithmic detection of bioparticles, and a bottom panel showing flow paths determined for the detected bioparticles.

[0065] FIG. 10 illustrates a side view of the example cell selection and / or cell sorting module illustrated in FIG. 9.WSGR Docket No. 65809-702.601

[0066] FIG. 11 illustrates an example of a customized flow profile which can be used to enhance cell sorting, selection, identification, characterization, and / or measurement according to embodiments described herein.

[0067] FIG. 12 illustrates an example cell identification and / or characterization and / or cell measurement module utilizing acoustic force pull down according to embodiments described herein.

[0068] FIG. 13 illustrates a further example of a microfluidic cell processing device and / or system according to embodiments described herein.

[0069] FIG. 14 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0070] FIG. 15 illustrate a schematic diagram of an embodiment of a microfluidic device having a dynamically configurable manifold designed to allow one or more reversals of flow direction along one or more recurrent loops comprised therein, which can be implemented in a single module or a plurality of modules according to embodiments described herein.

[0071] FIG. 16 illustrates example flow profiles that can be produced using dynamically configurable manifolds described herein, for example, for label free characterization followed by label free sorting of positive and negatively selected bioparticles to different destinations.

[0072] FIGs. 17A-17B illustrate a prototype bioparticle processing and characterization system according to embodiments described herein.

[0073] FIGs. 18A-18B illustrate modules of the prototype system of FIGs. 17A-17B in operation.

[0074] FIGs. 19A-19D illustrate results of cell growth using Jurkat cells in a cell processing device according to embodiments described herein.

[0075] FIGs. 20A-20D illustrate results of cell culture and expansion using Jurkat cells in a cell processing device according to embodiments described herein.

[0076] FIGs. 21A-21D illustrate results of a 4+ day cell expansion using Jurkat cells in a cell processing device according to embodiments described herein.DETAILED DESCRIPTION

[0077] While various embodiments of the invention have been show n and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.WSGR Docket No. 65809-702.601Definitions

[0078] Whenever the term "‘at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values, respectively. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0079] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values, respectively. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0080] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g.. the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed. If the acceptable error range is unclear or subject to debate, then the default value of the error range may be presumed to mean a range from 10% below to 10% above the particular value.

[0081] Point-of-care: The term point-of-care (POC) in reference to cell processing by a system, device, and / or method according to the disclosure, generally refers to cell processing which occurs in close proximity to: a patient undergoing a cell therapy treatment, a subject from whom donor cells are obtained, or both. For example, a cell processing system for processing cells from a donor and or patient may be located within the same room as the donor or patient, within the same building as a donor or patient, or within the same treatment facility (such as being located in a laboratory building of a hospital campus having plurality of buildings). In contrast, methods which require cryogenic preservation of cells for long distance transport would not be point-of-care within the meaning used herein.

[0082] Bioparticle: the term bioparticle refers to a microscopic particle composed of one or more biological material(s) and ranges from about 20 nanometers to about 5 millimeters in diameter as measured along the longest axis of the bioparticle. The bioparticle is typically a substance found inWSGR Docket No. 65809-702.601 living organisms, although it can be an artificial composition of biomolecules, for example lipid nanoparticles. Non-limiting examples of bioparticles include a cell, a cell fragment, a vesicle, an organoid, a liposome, a lipid nanoparticle, a virus, pollen, or spore. A population of bioparticles may be evaluated and sorted by the systems, microfluidic devices, and methods disclosed herein according to their surface expression of specific biomarkers or receptors.

[0083] Cell or Bioparticle identification, characterization, and measurement: as used herein, bioparticle identification generally refers to a determination of a broad class parameter about a bioparticle such as a cell (for example, a determination that a cell is a leukocyte v. an erythrocyte), whereas characterization generally involves a more specific subclass determination (for example, a determination that a cell or a cell population expresses a particular surface marker). Bioparticle measurement generally involves a quantitative determination concerning a bioparticle such as a cell or cell population (e.g., measurement of a level of expression of a surface marker, measurement of a concentration of cells in a fluid, measurement of a ratio of a target cell to a contaminant cell, etc.). Cell identification may be performed based on label-free gross morphological analysis, for example, the size and shape of cells such as convexity and / or elongation, number of nuclei, nuclei shape, and other features. As used herein, each of these functions may be performed together in a single module or method step or may be divided into a plurality of separate modules and / or method steps.

[0084] Cell evaluation: as used herein, cell evaluation generally refers to a determination of some parameter, characteristic, or phenotypic feature of a cell, which can comprise cell identification, cell characterization, or cell measurement, or any combination thereof. As an illustrative non-limiting example, a cell evaluation can include characterization of a cell as being CD4‘ CD8+cytotoxic T-cells based on sensor reading(s) indicative of the individual cell’s physical interactions with a surface functionalized with anti-CD8 antibodies and lack of interaction with a surface functionalized with anti-CD4 antibodies, respectively, combined with a measurement of the expression level of a specific chimeric antigen receptor based on the cell’s strength of interactions with a surface functionalized with anti-CAR antibodies.

[0085] Cell sorting, cell selection, and cell deselection: as used herein, cell sorting generally refers to a broad process of resolving one or more cells from other cells in time and / or space (for example through cell selection or deselection). Cell selection generally refers to a process of sorting a mixed cell population to isolate or enrich a target cell to the exclusion of all other cells, whereas cell deselection generally refers to sorting a mixed cell population to discard a particular contaminant cell while leaving other cells (which may include one or more target cells) in the mixed cell population. As used herein each of these functions may be performed together in aWSGR Docket No. 65809-702.601 single module or method step or may be divided into a plurality' of separate modules and / or method steps.

[0086] Binder: as used herein, a binder refers to a molecule that has an affinity' and specificity for a target molecule located on a target bioparticle. Functionalized surfaces described herein can be functionalized using one or more binders to create a surface coating of binders suitable for engaging in interactions with target molecules of target bioparticles. Illustrative examples of binders include antibodies or fragments thereof, aptamers, cell adhesion molecules, and adhesion proteins.

[0087] Antibody: as used herein, an antibody is a type of binder that refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive toward, a specific antigen. Antibodies can be polyclonal, monoclonal, genetically engineered, and can include antigen binding fragments thereof. An antibody can be, for example, murine, chimeric, humanized, conjugate, bispecific, diabody, triabody, or tetrabody. The antigen binding fragment may include a Fab1, F(ab')2, Fab, Fv, reduced IgG, or scFv.

[0088] Interaction: as used herein, an interaction refers to physical contact between a bioparticle and a binder of a functionalized surface that affects the movement and / or position of the bioparticle. An interaction can include specific binding between one or more surface molecules of the bioparticle and one or more binders of the functionalized surface such as, for example, antibody-antigen binding. Non-limiting examples of such interactions include bioparticle immobilization upon a functionalized surface, partial attachment to the functionalized surface (e.g.. movement is slowed when moving across the surface relative to fluidic flow through the flow path), detachment from the functionalized surface (e.g., when a motive force applied upon an immobilized bioparticle causes it to move across the functionalized surface at a speed or rate commensurate with fluidic flow rate), and increased movement (e.g., a partially attached bioparticle is moving across or rolling along the functionalized surface slower than the fluidic flow rate in the flow path, but an increasing motive force causes the bioparticle to roll or move faster).

[0089] CAR T cells: The term "CAR" is an acronym for "chimeric antigen receptor." A "CAR T cell" is therefore a T cell that has been genetically engineered to express a chimeric antigen receptor.

[0090] CAR T cell therapy: This term refers to any procedure in which a disease or condition is treated with CAR T cells. Diseases that may be treated include hematological and solid tumor cancers, autoimmune diseases and infectious diseases.

[0091] Target bioparticles: As used herein "target bioparticles" are the bioparticles that various procedures described herein require or are designed to purify, collect, engineer etc. WhatWSGR Docket No. 65809-702.601 the specific bioparticles are will depend on the context in which the term is used. For example, if the objective of a procedure is to isolate a particular kind of stem cell, that cell would be the target bioparticle or target cell of the procedure.

[0092] Non-target bioparticle: as used herein, “non-target bioparticles” are the bioparticles that the various procedures described herein require or are designed to remove, discard, or separate from the target bioparticles. As an illustrative example, when processing a patient’s blood sample to isolate leukocytes from other blood cells, erythrocytes may be identified and sorted for removal from the bioprocessing flow paths by a bioparticle sorting module as an initial first step before subsequent step(s) to characterize the remaining leukocytes according to lymphocyte subtype to isolate / enrich for T-cells.Cell Processing Devices and Systems for Bioparticle Evaluation

[0093] In general, the microfluidic devices will be used to evaluate and optionally enrich target bioparticles or target cells relative to contaminants such as other types of cells (e.g., non- target cells) or debris contained within a sample. When a sample containing the target cells or bioparticles is introduced into a device or a module of a device through a sample inlet and fluidically passed through a channel of the device, the target cells or target bioparticles can flow through one or more processing flow paths for bioparticle processing and / or evaluation to one or more product outlets where a product enriched in target cells or target bioparticles is obtained. The term "enriched" as used in this context means that the ratio of target cells or bioparticles to contaminants is higher in the product than in the sample. Contaminants may flow predominantly to one more waste outlets where they may be either collected or discarded or may be recirculated through one or more manifolds of the device. Combinations of two or more separation or cell sorting modules may be employed to accomplish positive and / or negative selection of target cells or contaminants. For example, a first module may separate T cells from granulocytes and monocytes and a second module may separate T cells from platelets and red blood cells.

[0094] In some embodiments, disclosed herein is a system comprising: a fluidic device for bioparticle processing, comprising: (a) a chamber defining one or more bioparticle processing flow paths, each bioparticle processing flow path comprising a functionalized surface configured to specifically interact with a target bioparticle; (b) an actuation module configured to apply variable amounts of force to one or more bioparticles within the one or more bioparticle processing flowpaths; and (c) a sensor module configured to detect one or more changes in interaction between the one or more bioparticles and the functionalized surface associated with the variable amounts of force.WSGR Docket No. 65809-702.601

[0095] In some embodiments, the fluidic device comprises one or more bioprocessing modules configured for performing one or more bioparticle processing tasks. A bioprocessing module can include one or more bioprocessing flow paths. A bioprocessing module can include modules for performing subtasks or operations. For example, one or more bioprocessing modules can include one or more of a bioparticle identification module, a bioparticle characterization module, and / or a bioparticle measurement module. A bioparticle identification, characterization, or measurement module can include one or more flow paths (optionally arranged parallel) comprising one or more channels (e.g., optical channels) configured for detection or measurement by a sensor module. The channels may comprise one or more functionalized surfaces comprising one or more binders that specifically bind one or more bioparticle surface molecules or biomarkers (e.g.. CD4 and CD8 biomarkers for T cell subtypes). In some embodiments, the system or fluidic device comprises a bioparticle sorting module configured to sort, separate, and / or enrich target bioparticles from non-target bioparticles. In some embodiments, the system or fluidic device comprises a bioparticle culturing module configured for culturing bioparticles (e.g., cell culturing module for growing and / or maintaining cells in a controlled environment). In some embodiments, the system or fluidic device comprises a bioparticle engineering module configured for engineering or modifying bioparticles (e.g., genetic engineering or modification of T cells to express a CAR). In some embodiments, the system or fluidic device comprises a bioparticle activation module configured for activating bioparticles (e.g., T cell activation). In some embodiments, the system or fluidic device comprises a bioparticle washing module configured to remove unwanted components or contaminants from a bioparticle liquid composition (e.g., bioparticle in an aqueous suspension). In some embodiments, the system or fluidic device comprises a bioparticle expanding module configured to expand a population or plurality of bioparticles (e.g., CAR-T cell expansion of target cells expressing the appropriate markers as one of a series of steps for preparing a medical composition to be administered to a patient).

[0096] In some embodiments, the fluidic device comprises an actuation module configured to apply variable amounts of force upon a portion of one or more bioprocessing flow paths. In some cases, the actuation module is positioned in proximity to an optical channel portion of a bioprocessing flow path, wherein the actuation module is configured to apply force upon the optical channel to push or pull one or more bioparticles into contact with a functionalized surface within the optical channel. This application of external force can be used to encourage binding between target bioparticles and the functionalized surface. Alternatively or in combination, gravitational force may be utilized whereby bioparticles are allowed to settle onto a bottom surface of the optical channel that has been functionalized with one or more binders (e.g., fluidic flow rate is paused toWSGR Docket No. 65809-702.601 allow bioparticles to settle). In some embodiments, the actuation module comprises one or more actuators configured to apply an external force to force the interaction between bioparticles and the functionalized surface. In some embodiments, an actuator utilizes an actuation mechanism selected from a pneumatic, thermal-pneumatic, piezoelectric, thermal-electric, electrophoretic or dielectrophoretic, and / or shape memory' alloy. In some embodiments, the actuation module comprises a flow distribution module configured to direct fluidic flow through one or more bioparticle processing flow paths. In some embodiments, the flow distribution module comprises a dynamically configurable fluidic manifold configured to adjust fluidic flow through the one or more bioparticle processing flow paths.

[0097] Different mechanisms and combinations of mechanisms may be used to exert variable force upon bioparticles within a bioparticle processing flow path to generate evaluations based on interactions or changes in interactions with a functionalized surface. In some embodiments, variable fluidic flow is used to increase or force bioparticle-functionalized surface interactions (e.g., decreasing or pausing fluidic flow to allow bioparticles to settle onto a functionalized surface). In some embodiments, an additional force generator such as an actuator is used, for example, an acoustic transducer generating one or more pressure waves that push or pull bioparticles onto the functionalized surface. In some embodiments, after one or more bioparticles are bound or immobilized to the functionalized surface, fluidic flow is increased according to various functions or models until the bioparticles are detached or pushed / pulled away from the surface (or any other interaction change).

[0098] In some aspects, the systems and devices are configured to evaluate bioparticles using utilize fluidic actuator(s) to exert variable force to drive changes in interactions between bioparticles and functionalized surface(s). In some embodiments, the systems and devices comprise an actuation module comprising one or more actuators and a flow distribution module. The one or more actuators and the flow distribution module can work in coordination apply a variable amount of force upon one or more bioparticles in order to provoke different interactions with the functionalized surface. In some embodiments, the actuation module comprises a flow distribution module (e g., dynamically configurable fluidic manifold) configured or otherwise programmatically instructed to provide variable fluidic flow rates through the one or more bioprocessing flow paths within the chamber of the fluidic device. Bioparticles can be manipulated within the flow path(s) by stopping, pausing, or slowing fluidic flow to enable interactions with the functionalized surface (e.g., allowing bioparticles to settle onto a functionalized surface positioned on the bottom of the flow path(s)). The manipulation of fluidic flow can be optionally combined with one or more actuators (e.g., acoustic transducers) positioned in proximity to the flow path(s) that apply force toWSGR Docket No. 65809-702.601 push or pull the one or more bioparticles into contact with the functionalized surface (e.g., using a preset / standard amount of force in order to provide a consistent bioparticle-binder interaction).

[0099] Following bioparticle-functionalized surface interactions (e.g., binding and / or immobilization), the flow distribution module can increase fluidic flow rate through the flow path(s) to increase the motive force exerted upon bioparticle(s) located therein. As the fluidic flow rate increases, those bioparticles attached to or immobilized upon the functionalized surface will undergo changes in interactions, for example, detaching and moving with the liquid medium flowing through the flow path(s). A sensor module can capture sensor readings or measurements of the change in interactions. The sensor readings or measurements can be used to determine the corresponding fluidic flow rate that presumably caused the change in interactions (e.g., using a camera image timestamp at the measured point of bioparticle detachment). The measured fluidic flow rate corresponding to the time or moment of detachment can be used as an indicator of the bioparticle-surface binding strength that the force of the fluidic flow rate overcame. Similarly, the measured fluidic flow rate can be used as an indicator of surface molecule or biomarker levels of a bioparticle (e.g., surface biomarker density) since higher levels should correspond to more surface molecules available to interact with binders on the functionalized surface. The measured fluidic flow rate or the corresponding indication of binding strength or surface molecule / biomarker level / expression / density can make up at least a part of an evaluation of an individual bioparticle. For example, a bioparticle may be evaluated to be a positive CAR-T cell for enrichment based on an indirect measurement of the CAR expression to be sufficient based on the fluidic flow rate being higher than a predetermined threshold demarcating positive and negative categories (optionally including additional categories such as a category for bioparticle re-analysis based on ambiguous or intermediate / low CAR expression measurements).

[0100] Alternatively, in some embodiments, a different mechanism from variable fluidic flow is used to detach or push or pull bioparticles away from the functionalized surface. This different mechanism may utilize one or more actuators that apply varying amounts of force (e.g., via pressure waves) directed away from the functionalized while the bioparticles are under a constant and continuous fluidic flow (or alternatively, applying a constant amount of force from the actuators for an increasing amount of time continuously until interactions are disrupted or modified). For example, immobilized bioparticles may be under a constant fluidic flow that is insufficient to detach them from the surface such that the force generated by the one or more actuators is what produces the detachment or change in interaction. The amount of force that caused the change in interaction can be used to generate an evaluation of the bioparticle (e.g.. as an indication of target molecule expression).WSGR Docket No. 65809-702.601

[0101] In some aspects, the systems and devices are configured to evaluate bioparticles using utilize non-fluidic actuator(s) to exert variable force to drive changes in interactions between bioparticles and functionalized surface(s). In some embodiments, the system or device comprises an actuation module comprising a flow distribution module and one or more actuators configured to apply variable force for promoting detachment or modification of binding interactions betw een bioparticles and the functionalized surface. In some embodiments, the flow distribution module is configured to or otherwise programmatically instructed to apply a constant continuous fluidic flow while the one or more actuators exert variable forces to detach or modify binding interactions between bioparticles and the functionalized surface. In some embodiments, the force used to detach bioparticles from the functionalized surface is not supplied by a flow distribution module, but instead is provided by one or more actuators such as an acoustic transducer positioned to direct force perpendicular to the fluid flow along the bioprocessing flow paths in a direction that pushes or pulls bioparticles away from the functionalized surface.

[0102] In some embodiments, the flow distribution module is configured to pause or slow fluidic flow for bioparticles positioned within one or more bioprocessing flow paths (e.g.. within the optical channel(s) comprising the functionalized surface(s)). One or more actuators (e.g., ultrasonic transducers) can be activated to push or pull the one or more bioparticles into contact with the functionalized surface to promote target bioparticle binding. In some embodiments, the flow distribution module then commences or increases fluidic flow rate to a constant and continuous flow through the one or more bioprocessing flow paths comprising the one or more optical channels. Those non-target bioparticles that failed to bind or establish sufficient strength of binding are carried away in the liquid medium flowing through the one or more bioprocessing flow paths, while target bioparticles remain attached or immobilized on the functionalized surface.

[0103] Accordingly, in some embodiments, instead of increasing the fluidic flow rate until it overcomes bioparticle-surface binding interactions, one or more actuators of the actuation module apply or exert a force directed to weaken interactions between the one or more bioparticles and the functionalized surface. For example, an actuator may be positioned perpendicular to the flow paths in order to direct force that pushes or pulls one or more bioparticles away from the functionalized surface. In some embodiments, the actuator configured to weaken or reduce interactions is positioned opposite from the actuator configured to drive interactions between one or more bioparticles and the functionalized surface. In some embodiments, the actuator configured to weaken or reduce interactions adjusts the amount of force directed towards the one or more bioparticles for purposes of determining an amount that is sufficient to overcome the binding interaction, for example, gradually increasing the amount of force. Alternatively, the actuator mayWSGR Docket No. 65809-702.601 apply a continuous and constant amount of force until bioparticle-functionalized surface interactions change. This approach allows an evaluation of the one or more bioparticles (e.g., indicator of binding strength and / or surface biomarker expression or density) based on an approximation or calculation of the amount of force necessary and / or sufficient to detach or modify surface binding of the bioparticles.

[0104] Combinations of two or more bioparticle processing modules may be employed to accomplish one or more of identification, characterization, and / or measurement of bioparticles or cells and / or contaminants in order to identify, characterize, and / or measure them for enrichment for specific target bioparticles or target cells. As an illustrative and non-limiting example, a bioparticle processing module configured to process a patient blood sample comprises a bioparticle identification module configured to identify T cells from erythrocytes and platelets, a bioparticle characterization module configured to determine a T cell subtype (e.g., helper T cell or cytotoxic T cell), and a bioparticle measurement module configured to measure an expression level of a chimeric antigen receptor (e.g., an indicator of expression such as fluidic flow rate or acoustic transducer force measured as sufficiently powerful to detach a bioparticle immobilized to a functionalized surface comprising a CAR binder). Processing of a blood sample on such a device allows for the collection of a product in which target cells (e.g., T cells) have been separated from other cell ty pes present in the sample (e.g., granulocytes, monocytes, platelets and red blood cells).

[0105] One or more bioparticle identification, characterization, and / or measurement modules can be used in combination with one or more separation or bioparticle sorting modules. For example, after a bioparticle has undergone one or more of identification, characterization, and / or measurement within those modules, which are optionally positioned in sequence along one or more bioparticle processing flow paths, the bioparticle may be then sorted and / or enriched using one or more bioparticle sorting modules positioned downstream of the preceding modules along the flow paths in accordance with the evaluations results for the bioparticle. As an illustrative example, a CD3+CD4+CARlowbioparticle may be marked for recirculation, while CD3+CD4+CARhlghis marked for enrichment according to evaluations using 3 bioprocessing modules arranged in sequence, and these bioparticles are then sorted into distinct parallel sorting channels by one or more downstream sorting modules. Alternatively, in some embodiments, a bioparticle sorting module is positioned in sequence after each preceding bioprocessing module (e.g., identification, characterization, and / or measurement module). As an illustrative example, the CD3+CD4+CARlowbioparticle may be positively sorted into parallel sorting channels on a flow path towards enrichment after being evaluated for each of CD3+and CD4+by different bioprocessing modules, and then negatively sorted into a recirculation channel or flow path based the CARlowevaluationWSGR Docket No. 65809-702.601(e.g., for re-analysis via recirculation through the fluidic device). It is understood by a person of ordinary skill in the art that various alternative combinations, permutations, and sequences of these modules are fully supported by the present disclosure.

[0106] Combinations of two or more separation or cell sorting modules may be employed to accomplish positive and / or negative selection of target bioparticles or cells and / or contaminants in order to enrich for specific target bioparticles or target cells. For example, a first module may separate T cells from granulocytes and monocytes and a second module may separate T cells from platelets and red blood cells. Processing of a blood sample on such a device allows for the collection of a product in which target cells (e.g., T cells) have been separated from other cell types present in the sample (e g., granulocytes, monocytes, platelets and red blood cells).

[0107] The sorting and / or selection of individual bioparticles or cells can be performed based on the sensor readings or measurements of these bioparticles or cells obtained by a sensor module comprising one or more sensors or detectors positioned in proximity’ to one or more bioparticle processing flow path(s) such as, for example, within one or more of bioparticle identification, bioparticle characterization, and / or bioparticle measurement flow path(s). The sensor or detector can be an imaging sensor such as a camera configured to measure or detect one or more bioparticles within the flow path(s), for example, with one or more optical channels comprising one or more functionalized surfaces or regions of functionalized surfaces positioned within the flow path(s).

[0108] The fluidic device can include an actuation module configured to exert a force upon or within one or more optical channels, for example, which forms a portion of one or more bioparticle characterization flow paths and comprises one or more functionalized surfaces or regions of functionalized surfaces which are configured to bind a target bioparticle. The actuation module can include a flow distribution mechanism, which can provide a constant fluidic flow (or is configured or programmatically instructed to provide constant and continuous fluidic flow), or alternatively, is dynamically configurable to adjust the rate of fluidic flow through the bioparticle characterization flow path to exert variable force upon one or more bioparticles in the flow path.

[0109] As an illustrative example, in some embodiments, as bioparticles are carried in a liquid medium through the processing flow paths of within a chamber of the fluidic device, the actuation module comprises an ultrasonic transducer that exerts a force upon the bioparticles to push or pull or otherwise force them into physical contact with one or more functionalized surfaces or discrete regions thereof (which may be functionalized with one or more different binders). In certain cases, the target bioparticle expressing sufficient density surface molecules or biomarkers that interact with the binder become immobilized upon the functionalized surface, whereas a non-WSGR Docket No. 65809-702.601 target bioparticle that does not express sufficient density of surface molecules or biomarkers will not engage in significant or detectable interactions (i.e., detectable using the sensor module) with the functionalized surface.

[0110] In some embodiments, a sufficiently powerful or high rate of fluidic flow can provide a motive force that overcomes the binding interactions between a bioparticle and the functionalized surface, resulting in detachment and the free movement of the bioparticle with the liquid medium flowing through the flow path. In some cases, the bioparticle may only partially detach and move along the functionalized surface in a rolling adhesion manner.

[0111] The sample may be obtained from an individual or a patient, especially a patient with cancer, an autoimmune disease or an infectious disease. In some embodiments, the sample comprises bioparticles such as eukaryotic cells, bacteria, fungi, spores, and / or parasites. In some embodiments, the bioparticles are not cells. In some embodiments, the bioparticles are drug delivery vehicles such as liposomes, microspheres, nanoparticles (e.g., lipid nanoparticles), exosomes, polymeric micelles, or other suitable bioparticles. In a certain embodiment, the sample is blood or is derived from blood (e.g., an apheresis or leukapheresis sample), and the target cells are dendritic cells, leukocytes (especially T cells), stem cells, B-cells, NK-cells. monocytes or progenitor cells. The contaminants in these instances may include red blood cells, white blood cells, and / or platelets, depending on the target cell ty pe selected and / or the condition of the patient or individual. The purification can result in a product enriched in target cells and in which at least 80% (e.g.. at least 90%. or at least 95%) of the contaminant cells from the sample have been removed or in which the target cell type is enriched at least 80% (e.g., at least 90%, or at least 95%) more than the contaminant cells.

[0112] In another embodiment, the sample is a liquid tumor biopsy or a solid tumor biopsy (which has been disassociated into individual cells before being processed by a system and / or device disclosed herein), and the target cells are cancer cells or a subset of cancer cells present within the tumor biopsy. The target cancer cells can undergo multiple rounds of characterization and sorting as they flow through one or more bioprocessing fluidic flow paths comprising characterization and sorting modules in a recurrent flow. With each round of recurrent flow, one or more target cells may be positively selected and / or non-target cells or contaminants are negatively selected, wherein the repeated cycles of characterization and sorting produce a product enriched in target cancer cells. In some cases, each round of recurrent flow comprises transfer of cells or fluid along a same fluidic channel using one or more reversals in flow direction. These cells may be enriched for purposes for further downstream analysis, for example, next generation sequencing (NGS), which may be used to identify cancer mutations for purposes of classifying cancer subtypeWSGR Docket No. 65809-702.601 and / or predicting suitable treatments. Non-limiting examples of such downstream analysis include evaluating the tumor microenvironment by using RNA-seq to characterize the transcriptome, whole genome sequencing to characterize the genome, epigenomics sequencing to characterize the epigenome, and mass spectrometric analysis to characterize the proteome and / or the metabolome.

[0113] The systems, devices, and methods disclosed herein allow for biological samples to be processed, characterized, and enriched for downstream uses relating to cell therapy and disease evaluation including, for example, blood cancers, such as leukemia (e.g., Acute Lymphoblastic Leukemia, Chronic Lymphocytic Leukemia), Lymphomas (e.g., Diffuse Large B-Cell Lymphoma, Follicular Lymphoma), Multiple Myeloma; Skin and Soft Tissue Damage; Autoimmune Disorders such as Rheumatoid Arthritis and Systemic Lupus Erythematosus; Solid Tumors in lung, pancreatic, breast, and ovarian cancers; neurodegenerative diseases such as Parkinson's Disease and Amyotrophic Lateral Sclerosis (ALS); Cardiovascular Diseases, such as Heart Failure and Myocardial Infarction; Diabetes (Type 1 Diabetes); Liver Diseases; Genetic Disorders, such as but not limited to Sickle Cell Disease and Beta-Thalassemia and Muscular Dystrophies like Duchenne Muscular Dystrophy. Samples pertaining to these diseases, disorders, and conditions may be processed and enriched for downstream characterization (e.g., molecular profiling, genomic sequencing, etc.).

[0114] The present disclosure provides microfluidic cartridges (i.e. devices, chips, plates, microfluidic devices, cartridges, etc.) and methods for purifying particles, bioparticles, or cells, which may comprise chimeric antigen receptor (CAR) T and NK cells. The microfluidic cartridges (i.e. devices, chips, plates, microfluidic devices, cartridges, etc.) may be any of those described herein. The use of the described cartridges may allow for production of more highly effective CAR T or NK cells by providing an integrated system for acquisition and downstream genetic engineering of therapeutic cells such as CAR T or CAR NK cells.

[0115] A method for producing chimeric antigen receptor (CAR) T or NK cells may comprise obtaining a sample comprising T or NK cells and enriching the T or NK cells relative to contaminants. Separating contaminants may comprise applying the sample to the one or more sample inlets of any of the cartridges or devices described herein, flowing the sample to the outlets of the cartridge, obtaining a product enriched in T or NK cells from the product outlet, and genetically engineering the T cells in the enriched product to product chimeric antigen receptors on the surface of the T or NK cells. The genetically engineering of the method may comprise genetic engineering methods as described herein and / or are known to a person of ordinary skill in cell therapeutic manufacturing. The method may further comprise expanding the CAR T or NK cells by growing the cells in vitro.WSGR Docket No. 65809-702.601

[0116] In some cases, a lentiviral vector is used for CAR-T manufacturing, and CAR expression on the cell is measured during operation of devices of the disclosure. In some embodiments, devices of the disclosure are used to manufacture a therapeutic dose containing from 1 million to 50 million therapeutic cells. Some commercial examples of CAR T cell therapeutics that can be engineered according to the devices, systems, and methods herein include axicabtagene ciloleucel. tisagenlecleucel, and brexucabtagene autoleucel.

[0117] The target particles, target cells, or target bioparticles of the systems, devices, and methods thereof described herein may comprise target cells such as stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells. The method may comprise enriching target particles or target cells to produce enriched target cells comprising stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells. DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells. The contaminants of the method may comprise stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells. DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells. For example, the target cells may be peripheral blood mononuclear cells and the contaminants may be platelets. For example, the target cells may be CD3+ cells and the contaminants may be platelets.

[0118] The bioparticles disclosed herein can have a variety of sizes and dimensions. As described herein, the size of a bioparticle may range from about 20 nanometers to about 5 millimeters in diameter as measured along the longest axis of the bioparticle. In some embodiments, a bioparticle has a diameter of at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1 micron, at least 2 microns, at least 3 microns, at least 4 microns, at least 5 microns, at least 10 microns, at least 15 microns, at least 20 microns, at least 25 microns, at least 30 microns, at least 35 microns, at least 40 microns, at least 45 microns, at least 50 microns, at least 60 microns, at least 70 microns, at least 80WSGR Docket No. 65809-702.601 microns, at least 90 microns, at least 100 microns, at least 200 microns, at least 300 microns, at least 400 microns, at least 500 microns, at least 600 microns, at least 700 microns, at least 800 microns, at least 900 microns, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, or at least 5 mm, and / or no more than 10 nm, no more than 20 nm, no more than 30 nm, no more than 40 nm, no more than 50 nm, no more than 100 nm, no more than 200 nm, no more than 300 nm, no more than 400 nm, no more than 500 nm. no more than 600 nm, no more than 700 nm, no more than 800 nm, no more than 900 nm, no more than 1 micron, no more than 2 microns, no more than 3 microns, no more than 4 microns, no more than 5 microns, no more than 10 microns, no more than 15 microns, no more than 20 microns, no more than 25 microns, no more than 30 microns, no more than 35 microns, no more than 40 microns, no more than 45 microns, no more than 50 microns, no more than 60 microns, no more than 70 microns, no more than 80 microns, no more than 90 microns, no more than 100 microns, no more than 200 microns, no more than 300 microns, no more than 400 microns, no more than 500 microns, no more than 600 microns, no more than 700 microns, no more than 800 microns, no more than 900 microns, no more than 1 mm, no more than 2 mm. no more than 3 mm, no more than 4 mm, or no more than 5 mm.

[0119] The methods described herein may comprise modifying the enriched target cells. The method may comprise genetically engineering the enriched target cells to obtain genetically engineered target cells. Genetically engineering includes transfecting or transducing the target cells with a recombinant nucleic acid. Methods of genetic engineering may include the use of TALENs, Zinc Finger Nucleases, CRISPR-Cas associated proteins, homologous recombination, viral vectors, or heterologous plasmids. The method may also include expanding the enriched target cells or genetically engineered cells by culturing them in vitro.

[0120] Samples used to manufacture therapeutic cells by methods, systems, and / or devices of this disclosure may be autologous (e.g., where therapeutic cells are manufactured from a sample obtained from the patient to be treated) or allogenic (e.g., where a donor sample is obtained from a separate subject prior to cell engineering).

[0121] Devices and systems of this disclosure comprise a fluidic manipulation instrument that can perform all of the key elements of a cell therapy manufacturing process in a single, integrated, highly automated, closed system. By consolidating complex workflows into an integrated architecture, the instrument can be configured in a physical form suitable for distributed use, meaning, it is suitable for installations in research, clinical, and point-of-care or near-point-of- care facilities without a need for building out specialized processing facilities. The systems and device can be automated with preloaded operating procedures and / or disposable single-use manufacturing cartridges, allowing operators with a limited skill level to use and / or maintain theWSGR Docket No. 65809-702.601 systems. Lowering the skill level necessary to create a cell therapeutic can result in a commensurate reduction in staffing costs (or, in the alternative allows more patients to be treated at the same cost.)

[0122] Methods, systems, and devices of this disclosure permit researchers to undertake drug product research on a market-enabling platform. By working with such methods, devices, and systems, the burdens associated with scaling and regulatory' compliance, from pre-clinical through phased trials, are reduced and streamlined.

[0123] Methods, systems, and devices described herein provide substantial improvements in cell manufacturing by providing a paradigm shift away from the standard workflow which often includes several complex and time-consuming manual processes such as cell characterization, cell sorting, cell genome engineering, cell expansion, and related analytics. Each of the typical cell manufacturing steps is labor intensive, time consuming, and highly regulated due to risk of human error and variability in precursor products, however, as described herein, it is possible to take advantage of constant flow processes that permit deep, multi-pass refinement of the cell engineering work product through a recurrent flow architecture, which maximizes efficiency, and / or a microfluidic process that utilizes an actuator module that is configured to apply varying amounts of force in order to modulate bioparticle movement and / or positional changes and then generates an evaluation of bioparticles based on analysis of the relationship between the amount of force and interactions or changes in interactions (e.g., bioparticle movement and / or positional changes relative to a functionalized surface), which enables improved reproducibility and efficiency.

[0124] The present disclosure changes the cell processing paradigm by enabling an end-to- end cell manufacturing process in a closed, benchtop instrument. Methods, systems, and device of the disclosure are also more broadly suitable for applications where recurrent flows are advantageous to allow for the sequential build of molecular complexes or structures utilizing stepwise chemical and biochemical synthesis.

[0125] As illustrated in FIG. 1 and FIG. 4, some embodiments of the devices and systems disclosed herein are capable of carrying out a plurality of cell identification, characterization, and / or measurement steps that enable the efficient sorting or selection of target cells based on multiple parameters. FIG. 1 shows an illustrative and non-hmiting cell therapy production method suitable for point-of-care use that shows (1) loading of a fluidic cartridge with a patient’s biological sample, (2) loading the cartridge into a system for processing, (3) addition of a therapeutic agent (e.g., LLV or LNP added to create a modified cell product), and (4) removal of a product module from the system for downstream use such as. for example, administration or infusion into the patient. FIG. 4 shows an illustrative and non-limiting example of bioparticle bioprocessing processWSGR Docket No. 65809-702.601(and corresponding illustrative fluidic device chip design) in which cells are subjected to multiple rounds of evaluation for various parameters such as viability, CD4+, CD8+, CAR+, or other suitable marker (marked as CDX in the drawing). However, whereas some approaches to bioparticle characterization may entail circulating bioparticles across antibody-coated surfaces at a constant flow rate from the source or inlet and detecting changes in bioparticle movement upon contact with these surfaces, embodiments of the devices and systems disclosed herein are configured to vary fluidic flow and then detect the corresponding bioparticle-surface interactions or changes thereof. Although there may be a concern that modulating flow rate to allow bioparticles to attach or immobilize upon the functionalized substrates creates a risk of blockage or clogging in microfluidic devices that could impair the rate of bioparticle evaluation, careful control of the fluidic flow rate or other mechanisms for exerting force combined with individual cell tracking can mitigate such challenges. Moreover, the use of parallel flow channels can further help prevent the buildup of bioparticle “traffic” along the bioprocessing flow paths.

[0126] Example embodiments of devices and system capable of use in methods of the disclosure are illustrated in FIGs. 2-3, 6, 13, and 15 which each describe a closed recirculating fluid flow path with such flow driven by a motive source such as a pump (e.g., a peristaltic pump or a syringe pump) or compressed gas, such fluid flow first encountering an upstream multi-position valve (upstream manifold) that permits reagents or other ingredients to flow from individual reservoirs into the process stream. Such process stream then encounters a second flow-directing array of individually controllable valves (on-chip, the Dynamic Bus) which directs the output from the upstream manifold into any one or more channel of a multi-channel microfluidic flow network (each channel of the flow network a Functional Channel and, collectively, the Flow Module). The separate Functional Channels may be dedicated to enabling the performance of specific biochemical reactions or biophysical processes. Such Functional Channels may have sub-channels directing flow between the separate Functional Channels for mixing or other convenience as specified by a unique process recipe or method for using the Device. In some cases, the recirculation involves flow in a first direction down a channel at a first time followed by flow in a second direction down the same channel at a second time. Downstream from the Function Module is a second manifold in which the output(s) from the Functional Channels join a common flow path leading to ports for sample collection, waste, or the recurrent loop. In addition to programmatic commands to the active flow channels, the operation is concurrently or optionally influenced by closed-loop dynamic flow control.

[0127] FIG. 3 shows an expanded representation of a system implementing a fluidic device of the disclosure (also shown in FIG. 2), wherein the fluidic device is divided into a plurality ofWSGR Docket No. 65809-702.601 modules. Each numbered module in the example is as follows: (1) The recurrent loop, which includes the motive force (alternative types of pumps suitable for the specific application) driving the matrix fluid (typically a buffer) through the device. In certain instances, a direction of the motive force may be reversed in an alternating fashion to transfer bioparticles from one functional channel to another. Also in the Recurrent stream are sensors for both monitoring of general conditions and dynamic control of the recurrent environment. The Input Manifold (2) is a valve array that permits fluid from one or more reagent reservoirs to join the recurrent flow stream. Each valve in the Input Manifold is individually controllable for metering of the specific reagent. Collective output from the Input Manifold flows to the Dynamic Bus (3) which, as a one-into-many flow diversion valve, delivers the recurrent flow stream to one or more of the Functional Channels within the Functional Module (4). The Functional Module is a sophisticated portion of the integrated microfluidic chip where the various biochemical and biophysical actions occur. In a stepwise sequence, the recurrent flow stream is directed around the recurrent loop to, in turn, deliver the cells of interest to the specific Functional Channel responsible for the corresponding reaction or process in the protocol. When the cells leave the Functional Channel, sensors will assess the output and determine if the cells will recirculate through the recurrent loop for additional pass(es) through the same Functional Channel (deeper exposure to that specific step) or, if the threshold parameters are met, the stream and cells are directed around the recurrent loop to the next Functional Channel step in the protocol. The post-Functional Module stream flows to a collection manifold (5) which receives the flow streams from all Functional Channels and directs them as a single stream in a Zero Dead Volume (ZDV) switching valve (6). The ZDV valve responds to the sensors that instruct whether the stream shall recirculate, provide aliquots for at-line or off-line testing, provide process retains for future testing, or deliver some fraction to waste. A person of ordinary skill in the art will understand that the modules illustrated in the drawings and described herein are illustrative and non-limiting embodiments of the systems and devices, and that other variations of these modules for performing one or more tasks or operations are fully supported by the present disclosure.

[0128] FIG. 5 is an illustration of a top-down view and a side view of a flow cell design for a fluidic device or chamber thereof. As shown, there are two areas of functionalized surfaces positioned in sequence along a flow path from a fluidic inlet and a fluidic outlet. Area 1 has a surface functionalized with a first binder, while area 2 has a surface functionalized with a second binder. As bioparticles flow as a suspension within a liquid medium across the functionalized surfaces, various mechanisms described herein may be employed to promote or force interactions between the bioparticles with the functionalized surfaces. These interactions or indications thereofWSGR Docket No. 65809-702.601 can be captured using a sensor module such as a CMOS sensor positioned to measure an area encompassing both area 1 and area 2 as well as a downstream sorting region. For example, individual bioparticles’ interactions with each of the area 1 and 2 binders may be captured and individually tracked as they pass through those areas into the sorting region where actuation mechanisms are employed to sort or push / pull individual bioparticles into the appropriate sorting channels.

[0129] FIG. 6 is an expanded representation of an alternative configuration of the Device, demonstrating the flexible utility of the modular design. In this instantiation of the Device, the Recurrent Loop (1) consists of a continuous fluidic path driven by the motive force of an inline pump (e.g., syringe, diaphragm, or peristaltic) to carry cells or other particles suspended in a carrier fluid (e.g.. buffer or cell-culture medium) from the Culture Chamber (7) through a series of sensors within the Environment Module (8), routing to one or more of the Functional Channels (9) within the Process Module (10), and then routing back to the Culture Chamber via polishing actions (removal of impurities, evaluating consistency) within the Output Module (11). In this instantiation of the Device, loading of the Recurrent Loop with metered volumes of alternative input reagents, media, cells, buffer, etc. is achieved by a syringe pump (12) coupled to a distribution valve, the Dynamic Bus (3) within the SPDModule (13). During recurrent flow through the Device, the Concentration Manifold (14) within the Output Module is used to maintain a consistent volume within the Recurrent Loop. The Concentration Manifold may consist of a semi-permeable membrane filter or another separation modality (e.g. acoustic forces) to separate large particles (e.g., biological cells) from smaller particles (debris) and to remove excess fluid volume. An optional inline mixer (15) ensures input fluid from the SPDModule combines fully with the fluid in the Recurrent Loop exiting the Concentration Manifold. Sensors in the Environment Module monitor cellular parameters (e.g., count, viability, morphology) and physical and biochemical characteristics of the carrier fluid (pH, dissolved oxygen, glucose, lactate, etc.) in order to enable dynamic control of the Recurrent Loop and its directed interaction with one or more of the Functional Channels within the LFCManifold (16) of the Process Module, allowing for label free characterization of particles or bioparticles passing through the LFCManifold. The Functional Channels can comprise of a simple pass-through flow path, as well as sophisticated microfluidics to carry out complex biochemical / biophysical interactions acting upon the cells (e.g., bioparticle characterization). Additional bespoke sensors can be integrated to assess these interactions to determine if the bioparticles should recirculate through the Recurrent Loop for additional pass(es) through the same Functional Channel (deeper exposure to that specific step) or, if the threshold parameters are met, the stream and cells can be directed around the Recunent Loop whereby theWSGR Docket No. 65809-702.601 cells will undergo the action of next Functional Channel in the protocol. The Output Manifold receives the flow streams from all Functional Channels in the LFCManifold and directs the fluid to a ZDV Selector Manifold (17). The ZDV Selector Manifold responds to the sensors that instruct whether the stream shall, in the alternative, recirculate around d the Recurrent Loop for further processing in the Process Module, exit the system for final processing as a therapeutic dose, provide sampling aliquots for at-line or off-line analytical testing, or deliver some fraction of the fluid and its contents to waste. Inline Bubble Traps (18) are placed at critical locations within the Recurrent Loop to ensure bubble-free operation of downstream microfluidic features and Check Valves (19) are placed in specific locations in the flow circuit to prevent backflow. Pressure sensors located at selector manifolds as well as inline flow sensors at critical junctions enable further monitoring to ensure fine-tuning of flow control throughout the system.

[0130] The valves in the manifolds, dynamic bus, and recurrent control loop may be of one or more types (pinch, rotary, solenoid membrane) and may be separate modules or incorporated directly into the microfluidic chips. Each valve is individually controllable and may alternatively operate as binary (on / off) or gradient (variable orifice) to precisely regulate flow.

[0131] The motive force in the recurrent flow segment that drives the flow of the matrix fluid through (and around) the device may be any one or more of syringe / piston pump, gravity pump, peristaltic or other cam pump, compressed gas, or other means providing controllable flow parameters.

[0132] Devices, methods, and systems of this disclosure generally provide a multifunctional recurrent flow architecture. Where existing devices and processes are often strictly linear in workflow, with attempted improvements in output through more intensified single steps (which can often be detrimental to the cells), devices and processes of the disclosure move cells around the reaction cycle to iteratively enhance the performance (e.g.. identification, selection, expansion, yield, etc. may all occur at least partially in parallel according to the disclosure). The disclosure provides a multi-element device that may incorporate several external components such as pumps, optical systems and multimodality' sensors, however, in many cases the core microfluidic chip will be made from a biologically compatible material such as glass, silicon or one or more non-leaching polymer(s).

[0133] FIG. 7 shows an illustrative example of a process for evaluating a bioparticle using variable fluidic flow. In this example, flow rate is varied over time to enable bioparticles to settle onto the functionalized surface and become attached or immobilized when fluidic flow is low or zero, weakly binding or non-binding bioparticles to be pushed away from the surface when the fluidic flow is low or intermediate in strength, and more strongly binding bioparticles to requireWSGR Docket No. 65809-702.601 strong or high fluidic flow to be detached. Ambiguously binding bioparticles may be individually marked for recirculation and the process repeated multiple times to process the sample until sufficient bioparticles have been enriched to generate a product (e.g., further engineering, activation, expansion, or other operations for cell therapy production).

[0134] FIG. 8 shows an illustrative diagram of areas 1 and 2 and the sorting region of FIG. 5 in which the bioprocessing flow path is divided into a plurality of parallel bioprocessing lanes. This design is for illustrative purposes only and not intended to limit the design of parallel lanes. In this example, individual cells may be more easily tracked by use of the parallel lanes, which can also assist in preventing cells from clumping together and / or clogging the flow path and / or outlet manifold.

[0135] FIG. 9 shows a top panel of an image captured of bioparticles moving across a surface such as in an area of FIG. 8, a middle panel showing an algorithmically constructed image representation of the bioparticles with background noise removed, and a bottom panel showing algorithmically detected flow paths for the individually tracked bioparticles. Individual bioparticle tracking may be performed using various software tools and / or algorithms including machine learning models for object detection and tracking.

[0136] FIG. 10 shows an illustrative diagram of parallel lanes in a sorting module and an acoustic transducer positioned to push individual bioparticles from one lane into a parallel lane for purposes of sorting and / or enrichment. The sorting may be carried out based on results of bioparticle-functionalized surface interactions detected in response to variable force applied to said bioparticle while moving across the functionalized surface upstream of the sorting area where the parallel channels are fluidically connected.

[0137] FIG. 11 shows an illustrative diagram of one model or function for the modulation or adjustment of flow rate (or any other amount of force to be produced by an actuation module). As an example, a sinusoidal model may be used or alternatively another linear model or a nonlinear model may be suitable.

[0138] FIG. 12 shows an illustrative diagram for the use of an actuation mechanism such as an acoustic transducer for promoting the settling of bioparticles onto the functionalized surface. For example, it may be determined that gravity -induced settling is insufficient or not fast enough, so an acoustic transducer may be positioned in proximity to the bioprocessing flow path to push or pull bioparticles onto the functionalized surface. In some embodiments, this approach may be preferred if it provides greater uniformity and consistency of cell-surface interactions.

[0139] Devices, systems and methods described herein can provide a laboratory instrument for automated cell identification, cell characterization, cell selection or deselection, intracellularWSGR Docket No. 65809-702.601 molecular engineering, cell population expansion, and process analytics, including output quality control. For example, FIG. 13 provides a schematic of general flow paths of a basic microfluidic recurrent or variable flow architecture for biological characterizations.

[0140] FIG. 15 provides a schematic of general flow paths of an alternate embodiment to that shown in FIG. 13, which is configured for recirculation comprising a plurality of alternating direction changes, whereby fluid flowing between one functional flow path and an alternate function flow path (e.g.. between any of the flow paths described herein) are recirculated along the same channel in alternating directions.

[0141] In some embodiments, membrane-based separation modules and / or label-free cell sorting are employed. For example, label free multi marker single cell phenotyping and selection and observation of interaction of surface phenotyping with a functionalized surface may each be performed in one or more modules of systems described herein.

[0142] In some embodiments, deflection within a channel from a current stream into a parallel stream is observed for characterization and / or is used for sorting of cells. In some cases, optical tracking of cells is used to modulate actuation of a dynamic flow manifold to shunt contaminants off to the waste or to cause recirculation of target cells through the manifold.

[0143] In some embodiments, target cells comprise leukocytes such as B-cells, T-cells, progenitor stem cells, NK-cells, and / or combinations thereof.

[0144] In some embodiments, described herein is an autologous manufacturing and characterization system. Such systems may be tied to one or more recirculating loop operably coupled to a dynamically configurable manifold. In some embodiments, systems include a flow cytometer configured as a process analytical tool for real time monitoring of a cell manufacturing process.

[0145] In some embodiments surfaces of an analytical and / or sorting channel of the disclosure may be functionalized with a binder such as an antibody. In some embodiments, the antibody is selected based on the type of target cell selected. In some embodiments, the antibody is an anti-CD4, anit-CD19, or anti-CD8 antibody. Categories of binders that can be used to functionalize the surfaces of the devices disclosed herein include antibodies and modifications and fragments thereof, cell adhesion molecules, and adhesion proteins. Non-hmiting embodiments of antibodies and modifications and fragments thereof can include antigen-binding fragments such as Fab, F(ab')2, Fab', Fv, and single-chain variable fragments (scFv), and bispecific and trispecific antibodies, bifunctional antibodies, minibodies, diabodies, triabodies, and single-domain antibodies. The substrates that the binders disclosed herein can specifically bind include various antigens and biomarkers such as immune cell markers, cancer cell markers, neurodegenerativeWSGR Docket No. 65809-702.601 disease cell markers, autoimmune disease cell markers, cardiovascular disease cell markers, liver disease cell markers, and other suitable biomarkers. Illustrative and non-limiting examples of cell markers include CD45 for leukocytes, CD3 for T-cells, CD4 for T-helper cells, CD8 for cytotoxic T-cells, CD56 for natural killer (NK) cells, CD19 and CD20 for B-cells, CD11c for dendritic cells, CD14 and CD33 for monocytes and macrophages, CD66b for granulocytes, CD34 for hematopoietic stem cells. Those skilled in the art will recognize that a variety of molecular targets and their antibodies and other suitable binders may be obtained from the scientific literature and commercial vendors, and the biomarkers described above are intended only to be illustrative.

[0146] In some embodiments, cells flow down a channel having more than one functionalized surface (e.g., a surface functionalized with two or more different antibodies). Depending on the proteins expressed on a cell’s surface and the same cell’s resulting interaction with the functionalized channel surface, the cell’s transit time in the channel can be measured to provide cell characterization. Acoustic force can then be used to direct target cells to a selected channel (e.g., back through the recurrent loop or out of the system to be administered to a subject) or to remove contaminants (e.g., by directing them to waste).

[0147] Such devices and systems allow use of individual operations that are not 100% efficient in every instance (e g., an activation process that is 30% efficient but is less detrimental to the target cells may be used to produce larger populations of activated cells by sending the cells which aren’t yet activated back through until they become activated). Culturing can also be configured to happen in parallel by adjusting the dynamic bus so a fraction (or in some cases, the majority) of the fluid (and therefore the cells) are circulated through the cell culturing module.

[0148] In some cases, delivery of manufactured cells to a patient can comprise a mixture of cells in different states. In some embodiments, devices and systems of the disclosure measure and select the actual cells which comprise a delivered dose itself. In some embodiments, devices, systems, and methods described herein can dial in an exact ratio of cells to tightly control the contents of a dose which is administered to a patient (e.g., by selectively removing anything that may have a negative effect). In some embodiments, software controlling a system described herein can provide an ability for a user to specifically select what cells will (or will not) be present in the manufactured therapeutic.

[0149] In some embodiments, a cell therapeutic can be manufactured from start to finish using methods, systems, and / or devices described herein over a period of less than about 14 days. In some embodiments, the cell therapeutic is manufactured in less than about 3 days. In some embodiments, the cell therapeutic is manufactured in less than about 1 day.WSGR Docket No. 65809-702.601

[0150] In some aspects, described herein are fluidic systems and devices. In some embodiments, the fluidic systems and devices comprise fluidic devices for cell processing. In some embodiments, the fluidic devices for cell processing comprise a dynamically configurable manifold, configured to adjust a fraction of a fluid flow within the device passing through one or more cell processing flow path(s) fluidically coupled to the configurable manifold. In some embodiments, the fluidic devices for cell processing comprise an input manifold, fluidically coupled to, or integrated with, at least one inlet of the dynamically configurable fluidic manifold. In some embodiments, the fluidic devices for cell processing comprise an outlet manifold, fluidically coupled to one or more outlets of the cell processing flow path(s).

[0151] In some embodiments, fluidic coupling of the input manifold, the dynamically configurable manifold, and the outlet manifold forms a recurrent loop. In some embodiments, the plurality of parallel cell processing flow paths comprise two or more flow paths selected from the group of: an identification flow path, a characterization flow path, a cell sorting flow path, a cell selection flow path, a cell deselection flow path, a cell wash flow path, a cell engineering flow path, a cell culturing flow path, a cell activating flow path, and a cell expanding flow path. In some cases, one or more flow paths may have a corresponding module (e.g.. bioparticle characterization module for a bioparticle characterization flow path).

[0152] In some embodiments, the systems and devices described herein comprise a recurrent loop configured to allow at least a portion of cells flowing through the plurality of cell processing flow paths to recirculate. For example, the liquid medium may flow from the outlet manifold to the at least one inlet of the dynamically configurable manifold. In some embodiments, the plurality of parallel cell processing flow paths comprise a cell culturing flow path, a cell activation flow path, and: (i) a cell measurement flow path; or (ii) a cell characterization flow path.

[0153] In some embodiments, the cell measurement flow path comprises a cell counting module, and / or the cell characterization flow path comprises a functional screening module.

[0154] In some embodiments, the plurality of parallel cell processing flow paths comprise (i) a cell measurement module or a cell characterization module; and (ii) a cell sorting module in the same flow path. In some embodiments, the cell sorting flow path comprises a label-free cell sorting module. In some embodiments, the cell sorting flow path comprises a cell concentrating module. In some embodiments, the cell sorting flow path comprises a cell dilution module.

[0155] In some embodiments, the bioparticle sorting flow path comprises a sorting module such as an acoustic sorting module positioned along the bioparticle sorting flow path. In some embodiments, the acoustic sorting module comprises one or more acoustic transducers and a plurality of acoustic sorting channels. In some embodiments, the acoustic sorting module comprisesWSGR Docket No. 65809-702.601 at least 2 parallel acoustic sorting channels. In some embodiments, bioparticles within the at least 2 parallel acoustic sorting channels are diverted into parallel sorting channels that are positioned vertically above the bioparticle sorting flow path. In some embodiments, the acoustic sorting module comprises a plurality of capacitive micromachined ultrasonic transducers (CMUT) and / or an array of piezoelectric transducers. In some embodiments, the acoustic sorting module comprises one or more piezoelectric micromachined ultrasonic transducers (PMUT) and / or capacitive micromachined ultrasonic transducers (CMUT).

[0156] In some embodiments, the bioparticle sorting flow path comprises a sorting module such as an electrophoretic module positioned along the bioparticle sorting flow path. In some embodiments, the sorting module comprises one or more electrophoretic transducers and a plurality of electrophoretic sorting channels. In some embodiments, the electrophoretic sorting module comprises at least 2 parallel electrophoretic sorting channels. In some embodiments, bioparticles within the at least 2 parallel electrophoretic sorting channels are diverted into parallel sorting channels that are positioned vertically above the bioparticle sorting flow path. In some embodiments, the acoustic sorting module comprises one or more piezoelectric micromachined ultrasonic transducers (PMUT) and / or capacitive micromachined ultrasonic transducers (CMUT).

[0157] In some embodiments, the input manifold comprises a plurality of inlets and / or the outlet manifold comprises a plurality of outlets.

[0158] In some embodiments, the input manifold comprises a plurality of inlets and a single outlet fluidically coupled to the at least one inlet of the dynamically configurable manifold, and / or the outlet manifold comprises: a plurality of inlets, each fluidically coupled to a corresponding outlet of the plurality of cell processing flow paths; and a single outlet. In some embodiments, the fluidic device comprises one or more inline pH sensors, one or more glucose concentration sensors, one or more inline pyruvate concentration sensors, one or more lactate concentration sensors, one or more pCO2 sensors, one or more glucose sensors, and / or one or more inline dissolved oxygen sensors.

[0159] In some embodiments, at least one of the one or more inline pH sensors, at least one of the inline dissolved oxygen sensors, at least one of the one or more inline pyruvate concentration sensors, at least one of the one or more lactate concentration sensors, or at least one of the one or more pCO2 sensors, is comprised in a cell characterization module. In some embodiments, the input manifold comprises an inline mixer. In some embodiments, the inline mixer is and / or comprises a zero dead-volume mixer.WSGR Docket No. 65809-702.601

[0160] In some embodiments, the input manifold and / or the outlet manifold comprise one or more zero dead-volume switching valves. In some embodiments, the fluidic device comprises one or more of peristaltic pumps and / or continuous flow pumps.

[0161] In some embodiments, the input manifold comprises at least three input manifold inlets. In some embodiments, the outlet manifold comprises at least three input manifold inlets. In some embodiments, the plurality of parallel flow paths comprises at least three parallel flow paths each corresponding to at least one cell processing application. In some embodiments, the cell characterization flow path, and / or the cell measurement flow path comprises an optical channel comprising an optical sensor.

[0162] In some embodiments, the optical sensor is a complementary metal oxide semiconductor sensor (CMOS), a photomultiplier or photodiode, and / or a camera. In some embodiments, the optical channel comprises a functionalized surface configured to interact with one or more cells passing through the optical channel. For example, FIG. 3 illustrates an example analytical channel comprising a CMOS sensor together with a functionalized surface. A process for characterization of cells using such a channel in combination with a positive displacement pump (peristaltic, diaphragm, or other pump providing a similar flow profile with respect to time) is illustrated in FIG. 4.

[0163] In some embodiments, the optical channel further comprises an acoustic transducer. In some embodiments, the acoustic transducer can be used to facilitate sorting or enrichment of cells, particularly when used in combination with a plurality of parallel channels (e.g., as illustrated in FIGs. 5-6). In some embodiments, the acoustic transducer is configured to promote and / or increase interaction of the one or more cells passing through the optical channel with the functionalized surface. In some embodiments, a flow profile through the device over time is configured specifically to facilitate interaction of cells with the functionalized surface, (for example as shown in FIGs. 7-8).

[0164] In some embodiments, the functionalized surface is functionalized using an antibody, an aptamer, a cell adhesion molecule, and / or an adhesion protein. In some embodiments, the fluidic device is: a microfluidic device, and / or a device configured for manufacture of engineered cells for use in a cell therapeutic. In some embodiments, the engineered cells are leukocytes. In some embodiments, the engineered cells are CAR-T cells, CAR-NK cells, modified B-cells, tumor infiltrating lymphocyte cells, and / or hematopoietic stem cells. In some embodiments, the cells processed are allogenic to a patient.

[0165] In some embodiments, one or more components of the fluidic device are comprised in a cartridge. In some embodiments, the cartridge is a disposable cartridge.WSGR Docket No. 65809-702.601

[0166] In some embodiments, the cartridge comprises the input manifold, the output manifold, the fluidic couplings, and / or the outlet manifold. In some embodiments, a majority of the fluidic volume of the fluidic system is comprised within the cartridge, and / or is comprised within a plurality of cartridges of the system.

[0167] In some embodiments, systems and devices described herein further comprise a controller configured to control or modify operations of one or more modules including, for example, the actuation module and / or the sensor module. In some embodiments, the controller comprises one or more processors for carrying out one or more instructions or operations. In some embodiments, the one or more instructions are executable to perform one or more operations such as a bioparticle processing operation, a bioparticle identification operation, a bioparticle characterization operation, a bioparticle measurement operation, a bioparticle culturing operation, a bioparticle sorting operation, a bioparticle selection operation, a bioparticle deselection flow path, a bioparticle engineering operation, a bioparticle culturing operation, a bioparticle activating operation, a bioparticle washing operation, and a bioparticle expanding operation. In some embodiments, the controller is configured to dynamically adjust an operation of the actuation module, for example, a dynamically controllable manifold. In some embodiments, the controller further comprises a user interface which allows for user selection of two or more parallel cell processing operations. In some embodiments, the controller is configured to perform at least a subset of a plurality of pre-programmed cell processing operations on cells present in the device in parallel. In some embodiments, a plurality of cell processing operations are automatically performed by the controller using the fluidic device. In some embodiments, the controller is operated remotely and / or is remotely programmable or reprogrammable.

[0168] In some embodiments, the controller is configured to dynamically adjust the operation of a plurality of dynamically adjustable manifolds of a plurality of microfluidic devices. In some embodiments, the plurality of parallel cell processing flow paths comprise a cell delivery and / or a cell acquisition flow path. In some embodiments, the cell delivery and / or cell acquisition flow path is configured to be fluidically couplable to a blood vessel of a subject.

[0169] In some embodiments, the device is a point-of-care cell therapeutic device, and the subject is a patient suffering from a disease or condition in need of treatment by a cell therapy.

[0170] In some embodiments, cells are circulated through the one or more cell processing flow paths until a cell population in the device possess one or more cell processing target metrics. In some embodiments, the cell population is administered to a subject after the one or more cell processing target metrics. In some embodiments, the dynamically configurable manifold is adjusted to maintain one or more cell processing target metrics in a cell population flowing through theWSGR Docket No. 65809-702.601 device or system. In some embodiments, the cell population is directly administered to a subject during the cell processing operations.

[0171] In some embodiments, the one or more cell processing target metrics comprise: a threshold cell concentration of a target cell type, a target expression level of one or more target proteins of a target cell type, a threshold level of one or more contaminants (e.g., of non-target cell types) in fluid flowing through the device or system, and / or a presence of a threshold level of one or more detectable biomarkers present in a cell characterization module of the system.

[0172] In some aspects, described herein are methods of processing and / or manufacturing cells. In some embodiments, the methods comprise preparing a plurality7of engineered cells using any device or system described herein.

[0173] In some embodiments, manufacturing of a cell therapeutic by a system or device described herein involves circulation of at least a portion of the cells of a sample through a recurrent loop of the system or device. In some embodiments, the recurrent loop has at least 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 15 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles. 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles. 700 cycles. 800 cycles. 900 cycles, or 1000 cycles, and / or no more than 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 15 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles, or no more than 1000 cycles.

[0174] In some embodiments, manufacturing of a cell therapeutic by a system or device described herein involves circulation of at least a portion of the cells of a sample through a recurrent loop of the system or device over about 2 cycles to about 1,000 cycles. In some embodiments, manufacturing of a cell therapeutic by a system or device described herein involves circulation of at least a portion of the cells of a sample through a recurrent loop of the system or device over about 2 cycles to about 3 cycles, about 2 cycles to about 5 cycles, about 2 cycles to about 10 cycles, about 2 cycles to about 20 cycles, about 2 cycles to about 50 cycles, about 2 cycles to about 100 cycles, about 2 cycles to about 300 cycles, about 2 cycles to about 500 cycles, about 2 cycles to about 1,000 cycles, about 3 cycles to about 5 cycles, about 3 cycles to about 10 cycles, about 3 cycles to about 20 cycles, about 3 cycles to about 50 cycles, about 3 cycles to about 100 cycles, about 3 cycles to about 300 cycles, about 3 cycles to about 500 cycles, about 3 cycles to about 1,000 cycles, about 5 cycles to about 10 cycles, about 5 cycles to about 20 cycles, about 5 cycles to about 50 cycles, about 5 cycles to about 100 cycles, about 5 cycles to about 300 cycles, about 5 cycles to about 500 cycles, about 5 cycles to about 1,000 cycles, about 10 cycles to aboutWSGR Docket No. 65809-702.60120 cycles, about 10 cycles to about 50 cycles, about 10 cycles to about 100 cycles, about 10 cycles to about 300 cycles, about 10 cycles to about 500 cycles, about 10 cycles to about 1,000 cycles, about 20 cycles to about 50 cycles, about 20 cycles to about 100 cycles, about 20 cycles to about 300 cycles, about 20 cycles to about 500 cycles, about 20 cycles to about 1,000 cycles, about 50 cycles to about 100 cycles, about 50 cycles to about 300 cycles, about 50 cycles to about 500 cycles, about 50 cycles to about 1,000 cycles, about 100 cycles to about 300 cycles, about 100 cycles to about 500 cycles, about 100 cycles to about 1,000 cycles, about 300 cycles to about 500 cycles, about 300 cycles to about 1,000 cycles, or about 500 cycles to about 1,000 cycles. In some embodiments, manufacturing of a cell therapeutic by a system or device described herein involves circulation of at least a portion of the cells of a sample through a recurrent loop of the system or device over about 2 cycles, about 3 cycles, about 5 cycles, about 10 cycles, about 20 cycles, about 50 cycles, about 100 cycles, about 300 cycles, about 500 cycles, or about 1,000 cycles. In some embodiments, manufacturing of a cell therapeutic by a system or device described herein involves circulation of at least a portion of the cells of a sample through a recurrent loop of the system or device over at least about 2 cycles, about 3 cycles, about 5 cycles, about 10 cycles, about 20 cycles, about 50 cycles, about 100 cycles, about 300 cycles, or about 500 cycles. In some embodiments, manufacturing of a cell therapeutic by a system or device described herein involves circulation of at least a portion of the cells of a sample through a recurrent loop of the system or device over at most about 3 cycles, about 5 cycles, about 10 cycles, about 20 cycles, about 50 cycles, about 100 cycles, about 300 cycles, about 500 cycles, or about 1.000.Computer systems

[0175] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. In some embodiments, systems and devices comprise a controller that comprises a computer system and / or processor(s) configured for carrying out various bioprocessing operations described herein. Alternatively, in some embodiments, a controller is a computer system comprising one or more processors configured for carrying out various bioprocessing operations described herein. FIG. 14 shows a computer system 1401 that is programmed or otherwise configured to control any or each of the modules of a fluidic system according to the disclosure. The computer system 1401 can regulate various aspects of fluid flow within fluidic devices of the present disclosure, such as, for example, by dynamically controlling a fraction of fluid flowing through each of a plurality of flow paths using one of more dynamically configurable manifolds of the disclosure. The computer system 1401 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.WSGR Docket No. 65809-702.601

[0176] The computer system 1401 includes a central processing unit (CPU, also "processor" and “computer processor’ herein) 1405, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1401 also includes memory or memory location 1414 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1415 (e.g., hard disk), communication interface 1420 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1425, such as cache, other memory, data storage and / or electronic display adapters. The memory 1410, storage unit 1415, interface 1420 and peripheral devices 1425 are in communication with the CPU 1405 through a communication bus (solid lines), such as a motherboard. The storage unit 1415 can be a data storage unit (or data repository ) for storing data. The computer system 1401 can be operatively coupled to a computer network (“network”) 1430 with the aid of the communication interface 1420. The network 1430 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 1430 in some cases is a telecommunication and / or data network. The network 1430 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1430, in some cases with the aid of the computer system 1401, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1401 to behave as a client or a server.

[0177] The CPU 1405 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory’ 1410. The instructions can be directed to the CPU 1405, which can subsequently’ program or otherwise configure the CPU 1405 to implement methods of the present disclosure. Examples of operations performed by the CPU 1405 can include fetch, decode, execute, and writeback.

[0178] The CPU 1405 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1401 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0179] The storage unit 1415 can store files, such as drivers, libraries and saved programs. The storage unit 1415 can store user data, e.g., user preferences and user programs. The computer system 1401 in some cases can include one or more additional data storage units that are external to the computer system 1401, such as located on a remote server that is in communication with the computer system 1401 through an intranet or the Internet.

[0180] The computer system 1401 can communicate with one or more remote computer systems through the network 1430. For instance, the computer system 1401 can communicate with a remote computer system of a user (e.g., a computer system of a physician monitoring a point-of-WSGR Docket No. 65809-702.601 care or near point- of- care real-time cell manufacturing process). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1401 via the network 1430.

[0181] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1401. such as, for example, on the memory 1410 or electronic storage unit 1415. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 1405. In some cases, the code can be retrieved from the storage unit 1415 and stored on the memory 1410 for ready access by the processor 1405. In some situations, the electronic storage unit 1415 can be precluded, and machine-executable instructions are stored on memory 1410.

[0182] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0183] Aspects of the systems and methods provided herein, such as the computer system 1401, can be embodied in programming. Various aspects of the technology may be thought of as "products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory' (e.g., readonly memory, random-access memory', flash memory ) or a hard disk. “Storage” ty pe media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein,WSGR Docket No. 65809-702.601 unless restricted to non-transitory, tangible “storage’' media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0184] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory' of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM. a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0185] The computer system 1401 can include or be in communication with an electronic display 1435 that comprises a user interface (UI) 1440 for providing, for example, controls for configuration of fluidic flow, cell processing thresholds, and / or cell process function. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0186] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1405. The algorithm can, for example, provide dynamic adjustment of cell processing operations to optimize, reach or maintain one or more system parameters for a cell population produced or maintained by systems and methods described herein.Algorithms and Machine Learning Methods

[0187] Various algorithms can be used to carry out the processes disclosed herein such as, for example, bioparticle or cell detection within sensor data (e.g., computer vision as applied to video feed, images, etc. captured by a sensor module’s camera), bioparticle or cell movementWSGR Docket No. 65809-702.601 detection, bioparticle or cell classification (e.g., cell identification, cell characterization, etc.), and regression analysis for biomarkers (e.g., cell measurement for CAR expression level). In some instances, machine learning methods are applied to the generation of models for evaluating information in carrying out these processes. Such models can be generated by providing a machine learning algorithm with training data in which the expected output is known in advance, e.g., the correct labeling are known for the image in the case of computer vision detection of a bioparticle or cell.

[0188] The model, classifier, or trained algorithm of the present disclosure can comprise one feature space. In some cases, the classifier comprises two or more feature spaces. The two or more feature spaces may be distinct from one another. Each feature space can comprise types of information about a case, such as biomarker expression or genetic mutations. The training data is fed into the machine learning algorithm, which processes the input features and associated outcomes to generate a model or trained algorithm. In some cases, the machine learning algorithm is provided with training data that includes the classification, thus enabling the algorithm to “learn’' by comparing its output with the actual output to modify and improve the model. This is often referred to as supervised learning. Alternatively, in some instances, the machine learning algorithm is provided with unlabeled or unclassified data, which leaves the algorithm to identify hidden structure amongst the cases (e.g., clustering). This is referred to as unsupervised learning. Sometimes, unsupervised learning is useful for identifying the representations that are most useful for classifying raw data (e.g., clustering populations of bioparticles into distinct clusters).

[0189] An algorithm may utilize a predictive model such as a neural network, a decision tree, a support vector machine, or other applicable model. Using the training data, an algorithm can form a classifier for classifying the case according to relevant features. The features selected for classification can be classified using a variety of viable methods. In some instances, the trained algorithm comprises a machine learning algorithm. The machine learning algorithm may be selected from the group consisting of a supervised, semi-supervised and unsupervised learning, such as, for example, a support vector machine (SVM), a Naive Bayes classification, a random forest, an artificial neural network, a decision tree, a K-means, learning vector quantization (LVQ), self-organizing map (SOM), graphical model, regression algorithm (e.g., linear, logistic, multivariate, association rule learning, deep learning, dimensionality reduction and ensemble selection algorithms. In some embodiments, the machine learning algorithm is selected from the group consisting of: a support vector machine (SVM), a Naive Bayes classification, a random forest, and an artificial neural network. Machine learning techniques include bagging procedures, boosting procedures, random forest algorithms, and combinations thereof. Illustrative algorithmsWSGR Docket No. 65809-702.601 for analyzing the data include but are not limited to methods that handle large numbers of variables directly such as statistical methods and methods based on machine learning techniques. Statistical methods include penalized logistic regression, prediction analysis of microarrays (PAM), methods based on shrunken centroids, support vector machine analysis, and regularized linear discriminant analysis.

[0190] Machine learning algorithms that are useful for image analysis may include artificial neural networks, specifically convolutional neural networks (CNN). Artificial neural networks mimic networks of neurons based on the neural structure of the brain. They process records one at a time, or in a batch mode, and “learn” by comparing their classification of the case (which can be at the case level or at a pixel level) (which, at the outset, may be largely arbitrary) with the known actual classification of the case. Artificial neural networks are typically organized in layers which comprise an input layer, an output layer, and at least one hidden layer, wherein each layer comprises one or more neurons. Deep learning neural networks tend to include many layers. Each node in a given layer is usually connected to the nodes in the preceding layer and the nodes in the subsequent layer. Typically, a node receives input from the neurons in the preceding layer, changes its internal state (activation) based on the value of the received input, and generates an output based on the input and activation that is then sent towards the node in the subsequent layer. The connections between neurons or nodes are represented by a number (weight) which can be positive (indicative of activating or exciting the subsequent node) or negative (indicative of suppression or inhibition of the subsequent node). A larger weight value indicates a stronger influence the node in a preceding layer has on the node in the subsequent layer. Accordingly, the input propagates through the layers of the neural network to generate a final output.ExamplesExample 1: Production of a cell therapeutic using a cell manufacturing system described herein using variable fluidic flow rate.

[0191] A sample derived from blood of a donor containing a starting population of bioparticles (in this case, cells) is obtained and introduced to a system such as the device described in any of FIGs. 1-3, 6, 13, 15, or 17A-17B. The cells are subject to a genetic engineering process within a cell engineering module to generate CAR-T cells. Cells are circulated through a plurality of parallel bioprocessing flow paths within the device automatically by a controller which adjusts an actuation module comprising a dynamically configurable fluidic manifold to apply varying amounts of motive force upon a liquid medium carrying a plurality of cells through the flow paths according to a pre-programmed bioparticle characterization protocol.WSGR Docket No. 65809-702.601

[0192] (a)(1) The controller first causes the manifold to apply motive force to propel the liquid medium carrying the cells into the chamber through the bioprocessing flow paths until at least a subset of the cells are positioned within the optical channels of the parallel bioprocessing flow paths at a first bioparticle identification module for general identification of bioparticle or cell ty pe. A sensor module contains a camera that captures video of the optical channels, which is analyzed by the controller to detect the presence of cells within the optical channels. After detection of cells in the optical channels, the controller instructs the manifold to stop applying motive force, thereby stopping the fluidic flow through the parallel bioprocessing flow paths and leaving the cells suspended in the liquid medium within the optical channel. This operation is dependent upon the pre-programmed protocol which sets a threshold amount or number of cells in the optical channels to trigger the operation to stop applying motive force.

[0193] (a)(2) The controller then briefly activates the actuation module’s acoustic transducer positioned in proximity to the optical channels to apply a pre-defined amount of force perpendicular to the fluidic flow path in the direction of the functionalized surface (e.g., via generation of an acoustic standing wave), thereby pushing or pulling the subset of cells suspended in the liquid medium into contact with the functionalized surface of the optical channels. The subset of cells contains target cells that express a surface lymphocyte biomarker, and the functionalized surface is functionalized with antibodies that specifically bind the surface lymphocyte biomarker. The contact between the target cells and the functionalized surface allows the antibodies to bind the surface lymphocyte biomarkers on the surface of the target cells while this binding does not occur with non-target cells (at least not to a relatively significant degree).

[0194] (a)(3) The controller then causes the dynamically configurable fluidic manifold to gradually and continuously increase the rate of fluidic flow- through the parallel bioprocessing flow paths. The increasing fluidic flow provides an increasing motive force against the cells. Those nontarget cells that do not express surface lymphocyte biomarker or express relatively low amounts of the biomarker are pushed across the functionalized surface at relatively low fluidic flow rates. By comparison, the target cells that express higher levels of the surface lymphocyte biomarker require higher fluidic flow rates to sufficiently overcome the immobilization caused by the binding of its lymphocyte biomarker with the functionalized surface.

[0195] The sensor module’s camera captures video of the entire process, and this data is provided to the controller for real-time analysis. The controller is configured with a machine learning algorithm that uses a convolutional neural network trained for object detection and tracking to analyze the video in order to automatically detect the initial immobilization and subsequent detachment from the functionalized surface and movement of target cells across theWSGR Docket No. 65809-702.601 optical channels. Individual cells are tracked as they move across the optical channels of the first bioparticle identification module. These cells are individually labeled by the controller’s software algorithm as positive or negative for identification as a lymphocyte based on the amount of motive force (as indicated by the fluidic flow rate) necessary' to detach and propel each cell across the functionalized surface. In this example, the timestamp of the video frame that marks the beginning of a given cell’s detachment from the functionalized surface is used to determine the corresponding fluidic flow rate that produced the detachment.

[0196] (b)(1) The motive force generated by the manifold pushes the bioparticles from the first bioparticle identification module downstream through the flow paths into a second bioparticle characterization module comprising parallel bioparticle characterization flow paths that have parallel optical channels comprising a functionalized surface that is functionalized with antibodies specific for the T-cell subtype.

[0197] (b)(2) The controller causes the actuation module to activate an acoustic transducer that pushes or pulls the cells against the functionalized surface within the parallel optical channels of this bioparticle characterization module. Similar to step (a)(2), target cells expressing the appropriate T-cell surface biomarkers will bind to the functionalized surface, while non-target cells do not bind.

[0198] (b)(3) The controller then causes the dynamically configurable fluidic manifold to gradually and continuously increase the rate of fluidic flow through the parallel bioprocessing flow paths according to the pre-programmed protocol. The camera of the sensor module captures video of this process as cells detach from the functionalized surface and move across the optical channel in response to the increasing motive force created by the fluidic flow' through the parallel bioprocessing flow paths. The controller analyzes the video in order to characterize individual cells as positive or negative for the T-cell subtype based on the relative amounts of force required to force the cells off the functionalized surface and move with the liquid medium.

[0199] (c)(1) The motive force generated by the manifold pushes the cells from the second bioparticle characterization module downstream through the flow' paths into a third cells measurement module comprising parallel cells measurement flow paths that have parallel optical channels comprising a functionalized surface that is functionalized with antibodies specific for a chimeric antigen receptor (CAR).

[0200] (c)(2) The controller instructs the actuation module to activate an acoustic transducer that exerts force (e.g., via generation of an acoustic standing wave) upon and pushes or pulls the bioparticles against the functionalized surface within the parallel optical channels of this bioparticleWSGR Docket No. 65809-702.601 characterization module. Similar to step (b)(2), target cells expressing the appropriate chimeric antigen receptor bind to the functionalized surface, while non-target cells do not bind.

[0201] (c)(3) The controller then causes the dynamically configurable fluidic manifold to gradually and continuously increase the rate of fluidic flow through the parallel bioprocessing flow paths. The camera of the sensor module captures video of this process as cells detach from the functionalized surface and move across the optical channel in response to the increasing motive force created by the accelerating fluidic flow through the parallel bioprocessing flow paths. The controller analyzes the video in order to measure the expression level (or an indicator thereof) of the chimeric antigen receptor for individual cells based on the amount of force / fluidic flow rate required to force the cells off the functionalized surface and move with the liquid medium. In this case, the pre-programmed protocol establishes threshold expression levels and ranges for cells to be labeled as a high, medium, and low expression CAR-T cells.

[0202] (d)(1) The motive force generated by the manifold pushes the cells from the third bioparticle measurement module downstream through the flow paths into a bioparticle sorting module comprising parallel bioparticle sorting flow paths that are each organized as parallel sorting channels. In this example, the parallel bioparticle sorting flow paths are each organized as a sorting channel positioned along the bottom surface of the chamber of the fluidic device and a corresponding parallel sorting channel positioned above the bottom surface sorting channel. Each bottom sorting channel is positioned in fluidic connection with one of the parallel cell measurement flow paths such that bioparticles passing through the flow paths move from the cell measurement flow path into the bottom sorting channel as they are propelled by the motive force of the manifold. Based on the video data analysis, the controller determines if a specific individual cell is to be enriched, discarded, or recirculated for additional evaluation in accordance with the preprogrammed protocol. In this example, individual cells that are negative for the lymphocyte biomarker or the T-cell subtype biomarker are marked for discard, individual cells that have low and medium levels of expression of CAR are recirculated for further analysis, and individual cells that have high levels of CAR expression are marked for enrichment.

[0203] (d)(2) As individual bioparticles move across the sorting module, the controller activates an acoustic transducer positioned in proximity to the bottom sorting channel to exert a force upon or otherwise propel the individual cells marked for discard from the initial bottom sorting channel through a shared fluidic connection into the parallel sorting channel that has a flow path towards a discard outlet. This is achieved by using the camera to continuously track individual cells as they move through the sorting channels. It is noted that the particular arrangement of parallel sorting channels can be reconfigured. For example, an alternative arrangement is thatWSGR Docket No. 65809-702.601 individual cells marked for discard remain in the initial bottom sorting channel, while cells marked for enrichment are sorted into the parallel sorting channel that have a flow path towards a product collection outlet.

[0204] (d)(3) The individual bioparticles remaining in the initial main sorting channel are those that have been marked for recirculation or enrichment. As the cells are further propelled through the sorting channels, the controller activates an acoustic transducer to propel or push or pull individual cells marked for enrichment into a second parallel sorting channel that has a flow path towards a product collection outlet. The remaining cells marked for recirculation continue through the initial main sorting channels into the outlet manifold where they are recirculated for re- evaluation.

[0205] The controller controls fluidic device and various modules of the system to repeat this process in a recurrent loop, including other necessary processes such as, for example, cell culturing, until sufficient target cells have been evaluated to satisfy all the parameters and pass qualify control targets to be administered to a patient for treatment. Upon detection that a target cell has reached all required target parameters (e.g., the specific cell to be administered passes all qualify control targets), the cell is sorted into a delivery module by the device, which can be utilized by a healthcare professional for administration to the patient or cryogenically preserved for future administration to the patient.Example 2: Production of a cell therapeutic using a cell manufacturing system described herein using constant fluidic flow rate and variable acoustic force.

[0206] A sample derived from blood of a donor containing a starting population of bioparticles (in this case, cells) is obtained and introduced to a system such as the device described in any of FIGs. 1-3, 6, 13, 15, or 17A-17B. The cells are subject to a genetic engineering process within a cell engineering module to generate CAR-T cells. Cells are circulated through a plurality of parallel bioprocessing flow paths within the device automatically by a controller which uses a fluidic manifold to provide a constant fluidic flow rate and adjusts an actuation module comprising a pair of acoustic transducers positioned in opposition around the flow paths and configured to apply varying amounts of motive force upon a plurality of bioparticles according to a preprogrammed bioparticle characterization protocol.

[0207] (a)(1) The controller first causes the manifold to apply a constant motive force to propel the liquid medium carrying the cells into the chamber through the bioprocessing flow paths until at least a subset of the cells are positioned within the optical channels of the parallel bioprocessing flow paths at a bioparticle measurement module for measuring expression levels ofWSGR Docket No. 65809-702.601 chimeric antigen receptor. A sensor module contains a camera that captures video of the optical channels, which is analyzed by the controller to detect the presence of cells within the optical channels.

[0208] (a)(2) After detection of cells in the optical channels, the controller briefly activates one of the two acoustic transducers, which are positioned in proximity to the optical channels, to apply a pre-defined amount of force perpendicular to the fluidic flow path in the direction of the functionalized surface, thereby pushing or pulling the subset of cells suspended in the liquid medium into contact with the functionalized surface of the optical channels. The subset of cells contains target cells that express a varying levels of a T-cell subtype surface biomarker, and the functionalized surface is functionalized with antibodies that specifically bind the surface biomarker. The contact between the target cells and the functionalized surface allows the antibodies to bind the surface biomarkers on the surface of the target cells, and the extent of the binding will vary depending on the level of expression of the surface biomarker. The fluidic manifold continues to provide a constant rate of fluidic flow throughout the process, thereby providing a consistent motive force acting against the cells in the optical channels. Those cells that do not express the CAR will not bind to the functionalized surface and continue to move through the optical channels. Those cells that express CAR at sufficient levels to establish antibody binding that is stronger than the motive force provided by the fluidic manifold will become immobilized or at least partially attached to the functionalized surface.

[0209] (a)(3) While some cells that do not express CAR are carried away through the optical channels by the fluidic flow of the liquid medium, those target cells that do express sufficient levels of CAR will remain immobilized or at least partially attached to the functionalized surface. The controller then activates a second acoustic transducer positioned opposite to the first acoustic transducer. The second acoustic transducer is positioned to exert a motive force against the cells in the optical channel that pushes or pulls or otherwise propels them away from the functionalized surface. The controller instructs the second acoustic transducer to gradually increase the motive force according to a pre-programmed protocol.

[0210] The camera of the sensor module captures video of this process as cells detach from the functionalized surface and move across the optical channel in response to the increasing motive force created by the second acoustic transducer. The controller analyzes the video in order to measure the expression level of the chimeric antigen receptor for individual cells based on the relative amounts of force required to force the cells off the functionalized surface and move with the liquid medium. In this case, the pre-programmed protocol establishes a threshold expression levels and ranges for cells to be labeled as a high, medium, and low expression CAR-T cells.WSGR Docket No. 65809-702.601

[0211] (d)(1) As all of the cells eventually detach from the functionalized surface, the constant motive force generated by the fluidic manifold pushes the cells from the bioparticle measurement module downstream through the flow paths into a bioparticle sorting module comprising parallel bioparticle sorting flow paths that each have a parallel sorting channels. Based on the video data analysis, the controller determines if a specific individual cell is to be enriched, discarded, or recirculated for additional evaluation in accordance with the pre-programmed protocol. In this example, individual cells that are low or medium levels of expression of CAR are recirculated for further analysis, and individual cells that have high levels of CAR expression are marked for enrichment.

[0212] (d)(2) As individual bioparticles move across the sorting module, the controller activates an acoustic transducer positioned in proximity to the parallel sorting channels to exert a force upon or otherwise propel the individual cells marked for discard from the initial main sorting channel through a shared fluidic connection into a parallel sorting channel that has a flow path towards a discard outlet. This is achieved by using the camera to continuously track individual cells as they move through the sorting channels.

[0213] (d)(3) The individual bioparticles remaining in the initial main sorting channel are those that have been marked for recirculation or enrichment. As the cells are further propelled through the sorting channels, the controller activates an acoustic transducer to propel or push or pull individual cells marked for enrichment into a second parallel sorting channel that has a flow path towards a product collection outlet. The remaining cells marked for recirculation continue through the initial main sorting channels into the outlet manifold where they are recirculated for re- evaluation.

[0214] The controller controls fluidic device and various modules of the system to repeat this process in a recurrent loop, including other necessary processes such as. for example, cell culturing, until sufficient target cells have been evaluated to satisfy all the parameters and pass qualify control targets to be administered to a patient for treatment. Upon detection that a target cell has reached all required target parameters (e.g., the specific cell to be administered passes all qualify control targets), the cell is sorted into a delivery’ module by the device, which can be utilized by a healthcare professional for administration to the patient or cryogenically preserved for future administration to the patient.Example 3: Cancer stem cell characterization and enrichment for RNA-seq analysis using a cell manufacturing system described herein using constant fluidic flow rate and variable acoustic force.WSGR Docket No. 65809-702.601

[0215] Doctors seek to isolate cancer stem cells from a solid tumor biopsy obtained from a subject for genetic characterization to help guide treatment target these cells, which are suspected of conferring resistance to chemotherapy and radiation therapeutic modalities and increased tumorgenicity. The solid tumor biopsy is subjected to mechanical and chemical dissociation according to standard protocols to produce a single-cell suspension. The single-cell suspension is introduced to the system such as the device described in any of FIGs. 1-3, 6, 13, 15, or 17A-17B and Example 1 for processing - with the main distinction being the binders used on the functionalized surfaces. Cells are circulated through a plurality of parallel bioprocessing flow paths within the device automatically by a controller which adjusts an actuation module comprising a dynamically configurable fluidic manifold to apply varying amounts of motive force upon a liquid medium carrying a plurality of cells through the flow paths according to a pre-programmed bioparticle characterization protocol. The cells are first processed through a series of cell characterization modules that assess surface expression of known cancer stem cell markers (e.g., CD44, CD 134) using the same mechanisms as described in Example 1. The cells are identified and characterized according to marker expression based on the strength of interaction / binding to the functionalized surfaces that have been functionalized with anti-CSC markers. The cells are then sorted into distinct fluidic outlets based on their estimated / predicted biomarker expression level using strength of the fluidic flow rate required to detach cells from the functionalized surface as an indicator or proxy for biomarker expression level). The target cells are marked for enrichment (CD441CD 134 ) and the remaining cells are marked for discard. The cells marked for enrichment are sorted and collected as described in Example 1 .

[0216] The controller controls fluidic device and various modules of the system to repeat this process in a recurrent loop, including other necessary' processes such as, for example, cell culturing, until sufficient target cells have been evaluated to satisfy all the parameters and pass qualify control targets to be administered to a patient for treatment. Upon detection that a target cell has reached all required target parameters (e.g., the specific cell to be genetically analyzed passes all quality control targets), the cell is sorted into a delivery module by the device, which is detachable and portable for a technician to transport for genetic testing.Example 4: Production of a cell therapeutic using a cell manufacturing system described herein comprising one or more dynamically configurable manifolds for recurrent cell processing.

[0217] A sample derived from blood of a donor containing a starting population of cells is obtained and introduced to a system such as the device described in any of FIGs. 1-3, 6, 13, 15, or 17A-17B. Cells are recirculated through the device automatically by a controller which adjusts aWSGR Docket No. 65809-702.601 dynamically configurable manifold to subject each cell from the sample at least one time to each of a cell activation module, a cell engineering module, a cell measurement module, a cell culture module, and / or sorting module prior to delivery to a patient. Many cells from the sample are circulated a plurality of times (e.g., up to hundreds of times) through each of the said modules via a recurrent loop until they reach a target parameter for administration to the patient. Upon detection that a target cell has reached all required target parameters (e.g., the specific cell to be administered passes all quality control targets), the cell is sorted into a delivery module by the device and optionally administered to the patient or cryogenically preserved for future administration to the patient.Example 5: Batch-based cell processing using 2-plex recurrent flow architecture comprising recirculation by flow direction reversal

[0218] An example device was constructed according to the schematic shown in FIG. 15, designed to shift from single cell-based to population-based methods according to methods described herein. Such embodiments may removes the need for a) acoustic array-based sorting, b) acoustic pull-down, c) multi-marker chip patterning, d) real-time inference at scale, and / or e) complex multi-systems fabrication.

[0219] The example device used a label free characterization (LFC)-based assay & sort method according to this disclosure to perform inline phenotyping and selection. The LFC assay was run using a ramped flow profile to characterize the cell population. This created a flow release profile (detachments vs. flow rate). The protocol (or user) of such a device can set the selection flow' rate(s). The system then generates stepped flow profiles and switching instructions, directing positive and negative cells to different destinations, as illustrated by the flow' profiles shown in FIG. 16. The example recurrent bioreactor constructed allow s for software-defined process flows. Illustrations of the protype device constructed are shown in FIGs. 17A-17B. The example system is comprised of a module-based architecture (1), biocompatible valve manifolds and switching hardware, inline pumps, and physiological sensors with online telemetry. A breadboard prototype (2) that incorporates a plurality of unit operations and sensing operations required to maintain and expand a suspension cell population was constructed. A standalone incubator was used for temperature and humidity control, though alternate embodiments can incorporate full environment management into the system. Cells w'ere cultured using the prototy pe system over a three day span w'hile maintaining cell viability. FIGs. 18A-18B illustrate the example device in operation.

[0220] The example device provide a recurrent bioreactor comprising a continuous closed- loop flow of cells in culture environment enables exposing every cell to LFC interrogation and sorting, which is controlled by a single control module that manages capture, fluidic control, andWSGR Docket No. 65809-702.601 fast sensor readouts. The example recurrent bioreactor supports in-line cell washing / concentration, programmatic and reagent introduction. The prototype breadboard design enables de-risking of bioreactor unit operations for alternate embodiments utilizing single-use cartridges. The shared control scheme with label-free characterization allows for in-line phenotyping capability.

[0221] The prototype system comprised: 24v and 12v power supply w / power distribution blocks; an output manifold to pull samples during culture duration; a 16 channel relay to control valves, shakers, and fans; webcam-based cell tracking and counting; integrated fluidics w / inline fluidic and syringe-based pumps; a PDMS culture environment; inline flow, pH, dissolve oxygen, and optional metabolic sensors; and a plurality of valves and check valves to route fluid flows. Example 6: Culture and expansion of Jurkat cells using a system implemented according to embodiments described herein.

[0222] A culture overgrowth and crash process was performed where Jurkats were washed into fresh medium using an 87 hour culture period. 8 million cells were seeded at 95% viability and 20 million cells were harvested, which had 53% viability after 87 hours, as illustrated in FIGs.19A-19D. An overnight culture test was also performed to evaluate healthy expansion. The results are illustrated in FIGs. 20A-20D. Jurkats were passed into fresh medium and cultured for 24 hours. 5.5 million cells were seeded at 93% viability, while 8 million cells were harvested at 93% viability. The harvested cells exhibited healthy morphology7with no signs of contamination. A further cell processing procedure was carried out using the prototype system at lower seeding density, which showed a healthy 4+ day expansion, as illustrated in FIGs. 21A-21B. Jurkats were washed into fresh medium and cultured for 1 12 hours. 1 million, 96% viable cells were seeded while 19 million, 95% viable cells w ere harvested, resulting in an approximate 26 hour doubling time.Example Embodiments1. A system comprising: a fluidic device for bioparticle processing, comprising: a chamber defining one or more bioparticle processing flow' paths, each bioparticle processing flow path comprising a functionalized surface configured to specifically interact with a target bioparticle; an actuation module configured to apply variable amounts of force to one or more bioparticles within the one or more bioparticle processing flow' paths; and a sensor module configured to detect one or more changes in interaction between the one or more bioparticles and the functionalized surface associated with the variable amounts of force.WSGR Docket No. 65809-702.6012. The system of embodiment 1, further comprising a controller configured to dynamically adjust an operation of the actuation module, optionally wherein the actuation module comprises a dynamically configurable fluidic manifold.3. The system of any one of the preceding embodiments, wherein the controller is configured to dynamically adjust the operation of the actuation module to apply the variable amounts of force upon one or more bioparticles located within the one or more bioparticle processing flow paths.4. The system of any one of the preceding embodiments, wherein the controller further comprises a user interface which allows for user selection of two or more parallel bioparticle processing operations.5. The system of any one of the preceding embodiments, wherein the controller is configured to perform at least a subset of a plurality of pre-programmed bioparticle characterization operations on the one or more bioparticles located within the one or more bioparticle processing flow paths.6. The system of any one of embodiments 2-5, wherein the controller is configured to perform at least a subset of a plurality of pre-programmed bioparticle characterization operations on a plurality of the one or more bioparticles located within the one or more bioparticle processing flow paths in parallel.7. The system of embodiment 6, wherein the plurality of pre-programmed bioparticle characterization operations is automatically performed by the controller using the fluidic device.8. The system of any one of embodiments 2-7, wherein the controller is operated remotely and / or is remotely programmable or reprogrammable.9. The system of any one of embodiments 2-8, wherein the controller is configured to dynamically adjust the operation of a plurality of dynamically configurable fluidic manifolds of a plurality of microfluidic devices.10. The system of embodiments 5-9, wherein the one or more pre-programmed bioparticle characterization operations comprise instructions for the actuation module to generate force acting upon one or more bioparticles located within the one or more bioparticle processing flow paths.11. The system of any one of embodiments 5-10, wherein the one or more pre-programmed bioparticle characterization operations comprise instructions for the actuation module to carry out one or more of increase the force, optionally until a threshold amount is reached;WSGR Docket No. 65809-702.601 decrease force, optionally until a threshold amount is reached; maintain the force at a current amount; reduce the force to zero; adjust the force according to a linear model; adjust the force according to a non-linear model; and / or adjust the force to a preset amount.12. The system of any one of the preceding embodiments, wherein the actuation module comprises a fluid flow distribution module, an actuator, or a combination thereof.13. The system of embodiment 12, wherein the actuator utilizes an actuation mechanism selected from a pneumatic, thermal-pneumatic, piezoelectric, thermal-electric, and / or shape memory alloy.14. The system of any one of embodiments 12-13, wherein the actuator is an acoustic actuator configured to increase and / or decrease interaction between the one or more bioparticles and the functionalized surface within the one or more bioparticle processing flow paths.15. The system of embodiment 14, wherein the acoustic actuator is configured to generate a force that pushes or pulls the one or more bioparticles toward or against the functionalized surface, thereby enabling binders on the functionalized surface to interact with any of the one or more bioparticles expressing a target molecule.16. The system of any one of embodiments 12-15, wherein the flow distribution module generates fluidic flow through the one or more bioparticle processing flow paths.17. The system of any one of embodiments 12-16, wherein the flow distribution module comprises one or more of a flow diverter, one or more valves, and / or a manifold fluidically coupled to the one or more bioparticle processing flow paths.18. The system of any one of embodiments 12-17, wherein the flow distribution module comprises a dynamically configurable fluidic manifold fluidically coupled to the one or more bioparticle processing flow paths.19. The system of embodiment 18, wherein the dynamically configurable fluidic manifold is configured to carry out instructions from a controller to adjust a rate of fluidic flow through the one or more bioparticle processing flow paths, optionally according to one or more preprogrammed bioparticle characterization operations.20. The system of embodiment 19, wherein the one or more pre-programmed bioparticle characterization operations comprise instructions to carry out one or more of: increase the rate of fluidic flow, optionally until a threshold flow rate is reached; decrease the rate of fluidic flow, optionally until a threshold flow rate is reached;WSGR Docket No. 65809-702.601 maintain the rate of fluidic flow at a current flow rate; reduce the rate of fluidic flow to zero; adjust the rate of fluidic flow according to a linear model; adjust the rate of fluidic flow according to a non-linear model; and / or adjust the rate of fluidic flow to a preset flow rate.21. The system of any one of embodiments 19-20, wherein the rate of fluidic flow is adjusted according to a sinusoidal curve, a continuous and gradual rate change, step-wise rate change, or any combination thereof.22. The system of any one of embodiments 12-21, wherein the fluidic device comprises: an input manifold, fluidically coupled to, or integrated with, at least one inlet of the flow distribution module, optionally wherein the flow distribution module is a dynamically configurable fluidic manifold; and an outlet manifold, fluidically coupled to one or more outlets of the one or more bioparticle processing flow paths.23. The system of embodiment 22, wherein fluidic coupling of the input manifold, the flow distribution module, and the outlet manifold forms a recurrent loop.24. The system of any one of embodiments 22-23, wherein the input manifold comprises a plurality7of inlets and / or the outlet manifold comprises a plurality' of outlets.25. The system of any one of embodiments 22-23, wherein the input manifold comprises a plurality of inlets and a single outlet fluidically coupled to the at least one inlet of the dynamically configurable manifold, and / or the outlet manifold comprises: a plurality of inlets, each fluidically coupled to a corresponding outlet of the plurality7of cell processing flow paths; and a single outlet.26. The system of any one of the preceding embodiments, wherein the fluidic device comprises one or more inline pH sensors, one or more inline glucose concentration sensors, one or more inline pyruvate concentration sensors, one or more lactate concentration sensors, one or more pCCh sensors, and / or one or more inline dissolved oxygen sensors.27. The system of embodiment 26, wherein at least one of the one or more inline pH sensors, at least one of the inline dissolved oxygen sensors, at least one or more inline glucose concentration sensors, at least one of the one or more inline pyruvate concentration sensors, at least one of the one or more lactate concentration sensors, or at least one of the one or more pCCh sensors, is comprised in a cell characterization module.28. The system of any one of embodiments 22-27, wherein the input manifold comprises an inline mixer.WSGR Docket No. 65809-702.60129. The system of embodiment 28, wherein the inline mixer comprises a zero dead-volume mixer.30. The system of any one of the embodiments 22-29, wherein the input manifold and / or the outlet manifold comprise one or more zero dead-volume switching valves.31. The system of any one of embodiments 22-30, wherein the input manifold comprises at least three input manifold inlets.32. The system of any one of embodiments 22-31, wherein the outlet manifold comprises at least three input manifold inlets.33. The system of any one of the preceding embodiments, wherein the fluidic device comprises one or more of peristaltic pumps and / or continuous flow pumps.34. The system of any one of the preceding embodiments, wherein the sensor module is positioned to detect and / or track one or more interactions between the functionalized surface and the one or more bioparticles within the one or more bioprocessing flow paths.35. The system of any one of the preceding embodiments, wherein each bioparticle processing flow path comprises one or more of a bioparticle identification flow path, a bioparticle characterization flow path, and / or a bioparticle measurement flow path.36. The system of embodiment 35, wherein one or more of the bioparticle identification flow path, the bioparticle characterization flow path, and / or the bioparticle measurement flow path comprises an optical channel configured to allow the sensor module to capture sensor readings or measurements within the optical channel.37. The system of embodiment 36, wherein the optical channel comprises the functionalized surface configured to specifically interact with the target bioparticle.38. The system of any one of embodiments 36-37, wherein the sensor module comprises an optical sensor positioned to capture sensor readings or measurements of the one or more bioparticles within the optical channel.39. The system of any one of embodiments 36-38, wherein the optical channel comprises a region that is transparent to the optical sensor to enable capture of the sensor readings or measurements of the one or more bioparticles within the optical channel.40. The system of any one of embodiments 38-39, wherein the optical sensor is a complementary metal oxide semiconductor sensor, a photomultiplier or photodiode, and / or a camera.41. The system of any one of embodiments 38-40, wherein the optical sensor is configured to individually track the position and / or movement of at least a subset of the one or more bioparticles.WSGR Docket No. 65809-702.60142. The system of any one of embodiments 38-41, wherein the optical sensor is configured to obtain one or more image(s) or video(s) capturing at least a subset of the one or more bioparticles.43. The system of any one of embodiments 38-42, wherein the optical sensor captures a field of view comprising at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. or 100% of the functionalized surface.44. The system of any one of the preceding embodiments, wherein the one or more bioparticle processing flow paths comprise a plurality of parallel bioparticle processing flow paths.45. The system of embodiment 44, wherein the plurality of parallel bioparticle processing flow paths comprises at least two parallel flow paths each corresponding to at least one cell processing application.46. The system of any one of embodiments 44-45, wherein the plurality of parallel bioparticle processing flow paths comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 flow paths.47. The system of any one of embodiments 44-46, wherein the plurality of parallel bioparticle processing flow paths comprise two or more flow paths selected from the group consisting of: a bioparticle identification flow path, a bioparticle characterization flow path, a bioparticle measurement flow path, a bioparticle sorting flow path, a bioparticle selection flow path, a bioparticle deselection flow path, a bioparticle engineering flow path, a bioparticle culturing flow path, a bioparticle activating flow path, a bioparticle washing flow path, and a bioparticle expanding flow- path.48. The system of any one of the preceding embodiments, wherein each bioparticle processing flow path comprises a bioparticle characterization flow path, wherein the bioparticle characterization flow path comprises the functionalized surface configured to physically interact with the target bioparticle.49. The system of any one of embodiments 44-48, w erein the plurality of parallel bioparticle processing flow paths comprises a plurality of parallel bioparticle characterization flow7paths.50. The system of any one of the preceding embodiments, wherein the target bioparticle is a cell, a cell fragment, a vesicle, an organoid, a liposome, a lipid nanoparticle, a virus, pollen, or spore.51. The system of any one of the preceding embodiments, wherein the functionalized surface comprises a binder configured to interact with and / or bind to a surface marker on the target bioparticle.WSGR Docket No. 65809-702.60152. The system of any one of the preceding embodiments, wherein the functionalized surface comprises two or more binders that specifically bind different surface markers.53. The system of embodiment 52, wherein the different surface markers are different types of cell surface biomarkers or receptors.54. The system of any one of embodiments 52-53, wherein the two or more binders are segregated into discrete regions of the functionalized surface.55. The system of embodiment 54, wherein the one or more bioparticle processing flow paths are fluidically coupled, directly and / or indirectly, to an upstream inlet and a downstream outlet, and wherein the discrete regions are positioned in sequence along the one or more bioparticle processing flow paths between the upstream inlet and the downstream outlet.56. The system of any one of the preceding embodiments, wherein the functionalized surface is functionalized using an antibody or fragment thereof, an aptamer, a cell adhesion molecule, and / or an adhesion protein.57. The system of any one of embodiments 51-56, wherein the binder comprises one or more of an antibody or fragment thereof, an aptamer, a cell adhesion molecule, and / or an adhesion protein.58. The system of any one of embodiments 51-57, wherein the binder specifically binds a native cell surface receptor or a modified cell surface receptor of the target bioparticle, optionally wherein the modified cell surface receptor is a chimeric antigen receptor.59. The system of any one of embodiments 51-58, wherein the binder specifically binds one or more stem cells, progenitor cells, erythrocytes, lymphocytes, platelets, epithelial cells, endothelial cells, hepatocytes, neuronal cells, glial cells, interstitial cells, adipocytes, dermal fibroblasts, myocytes, osteoclasts, osteoblasts, and / or cancer cells.60. The system of any one of embodiments 51-59, wherein the binder specifically binds one or more of neutrophils, eosinophils, basophiles, monocytes, mast cells, dendritic cells, and / or macrophages.61. The system of any one of embodiments 51-60, wherein the binder specifically binds one or more of T-cells, NK cells. NKT cells. B cells, plasma cells, and / or CAR-T cells.62. The system of any one of the preceding embodiments, wherein the system comprises a processor configured to analyze the one or more movements and / or positional changes and the variable amounts of force applied to the one or more bioparticles, thereby generating an evaluation of at least a subset of the one or more bioparticles, optionally wherein the processor is a component of a controller configured to control the fluidic device.WSGR Docket No. 65809-702.60163. The system of embodiment 62, wherein the processor is configured to analyze at least a subset of the one or more movements and / or positional changes and the variable amounts of force applied to the one or more bioparticles using a statistical model or a trained machine learning model.64. The system of embodiment 63, wherein the trained machine learning model is trained using an algorithm selected from support vector machine, linear regression, artificial neural network, naive Bayes classifier algorithm, decision tree, random forests, or nearest neighbors.65. The system of any one of embodiments 62-64, wherein the evaluation comprises one or more of an identification, a characterization, and / or a measurement.66. The system of embodiment 65, wherein the identification comprises a determination that the bioparticle is positive or negative for a cell type selected from stem cells, progenitor cells, erythrocytes, lymphocytes, platelets, epithelial cells, endothelial cells, hepatocytes, neuronal cells, glial cells, interstitial cells, adipocytes, dermal fibroblasts, myocytes, osteoclasts, osteoblasts, and cancer cells.67. The system of any one of embodiments 65-66, wherein the characterization comprises a determination that the bioparticle is positive or negative for a cell subtype selected from T- cells, NK cells, NKT cells, B cells, plasma cells, and CAR-T cells.68. The system of any one of embodiments 65-67, wherein the measurement comprises a determination of an expression level of a surface biomarker corresponding to a T-cell. NK cell, NKT cell, B cell, plasma cell, or CAR-T cell, or a subtype thereof.69. The system of any one of the preceding embodiments, wherein the one or more bioparticle processing flow paths comprise (i) one or more of a bioparticle identification module, a bioparticle measurement module and / or a bioparticle characterization module; and (ii) a bioparticle sorting module in the same flow path.70. The system of embodiment 69, wherein the bioparticle sorting flow path comprises a label- free cell sorting module.71. The system of any one of embodiments 69-70, wherein the bioparticle sorting module is positioned downstream of one or more of the bioparticle identification module, the bioparticle measurement module, and / or the bioparticle characterization module in the same flow path.72. The system of any one of embodiments 69-71, wherein the bioparticle sorting module is configured to exert a force upon, displace, or cause at least a subset of the one or more bioparticles to separate the one or more bioparticles into two or more distinct flow paths.WSGR Docket No. 65809-702.60173. The system of embodiment 72, wherein the two or more distinct flow paths comprise at least 2 parallel sorting channels that have at least one fluidic connection enabling the subset of the one or more bioparticles to be moved between the at least 2 parallel sorting channels.74. The system of any one of embodiments 69-73, wherein the bioparticle sorting module comprises an acoustic sorting module comprising one or more acoustic transducers.75. The system of embodiment 74, wherein the acoustic sorting module comprises at least 2 parallel acoustic sorting channels.76. The system of any one of embodiments 74-75, wherein the acoustic sorting module comprises a plurality' of capacitive micromachined ultrasonic transducers (CMUT) and / or an array of piezoelectric micromechanical ultrasonic transducers (PMUT).77. The system of any one of the preceding embodiments, wherein the fluidic device is: a microfluidic device, and / or a device configured for manufacture of engineered cells for use in a cell therapeutic.78. The system of embodiment 77, wherein the engineered cells are leukocytes.79. The system of embodiment 78, wherein the engineered cells are CAR-T cells, CAR-NK cells, modified B-cells, tumor infiltrating lymphocyte cells, and / or induced pluripotent stem cells and hematopoietic stem cells.80. The system of any one of the preceding embodiments, wherein the bioparticles processed by the system comprise cells that are allogenic to a patient.81. The system of any one of the preceding embodiments, wherein one or more components of the fluidic device are comprised in a cartridge.82. The system of embodiment 81, wherein the cartridge is a disposable cartridge.83. The system of any one of embodiments 81-82, wherein the cartridge comprises the input manifold, the output manifold, the fluidic couplings, and / or the outlet manifold.84. The system of any one of embodiments 81-83, wherein a majority of the fluidic volume of the system is comprised within the cartridge, and / or is comprised within a plurality of cartridges of the system.85. The system of any one of the preceding embodiments, wherein the one or more bioparticle processing flow paths are a plurality of parallel cell processing flow paths comprising a cell delivery and / or a cell acquisition flow path.86. The system of embodiment 85, wherein the cell deliver}' and / or cell acquisition flow path is configured to be fluidically couplable to a blood vessel of a patient.WSGR Docket No. 65809-702.60187. The system of any one of the preceding embodiments, wherein the device is a point-of-care cell therapeutic device, and the subject is a patient suffering from a disease or condition in need of treatment by a cell therapy.88. A method of processing and / or manufacturing bioparticles, the method comprising: preparing a plurality of engineered bioparticles using the device or system of any of the preceding embodiments.89. The method of embodiment 88. wherein bioparticles are circulated through the one or more cell processing flow paths until a bioparticle population in the device possess one or more bioparticle processing target metrics.90. The method of embodiment 89, wherein the bioparticle population is administered to a patient after the one or more bioparticle processing target metrics.91. The method embodiment any one of embodiments 88-90, wherein the actuation module comprises a dynamically configurable manifold that is adjusted to maintain one or more cell processing target metrics in a cell population flowing through the device or system.92. The method of any one of embodiments 88-91, wherein the cell population is directly administered to a patient during the cell processing operations.93. The method of any one of embodiments 91-92, wherein the one or more cell processing target metrics comprise: a threshold cell concentration of a target cell type or population thereof, a target expression level of one or more target proteins of a target cell type, a threshold level of one or more contaminants in fluid flowing through the device or system, a purity of a target cell or population thereof, a viability of a target cell or population thereof, a number of a target cell or population thereof, a phenoty pe of a target cell or population and / or a presence of a threshold level of one or more detectable biomarkers present in a cell characterization module of the system.94. A system comprising: a fluidic device for characterizing individual cells, the device comprising: a dynamically configurable manifold configured to control a rate of fluidic flow within the device; a chamber fluidically coupled to the manifold and defining one or more cell characterization flow path(s), each cell characterization flow path comprising an optical channel having a functionalized surface configured to interact with one or more of the individual cells passing through the optical channel; and an optical sensor configured to obtain one or more sensor reading(s) of the one or more of the individual cells passing through the optical channel; andWSGR Docket No. 65809-702.601 a controller comprising a processor configured to: dynamically adjust the dynamically configurable manifold to control the rate of fluidic flow within the one or more cell characterization flow path(s) in order to facilitate one or more interactions between the one or more of the individual cells and the functionalized surface in the optical channel; cause the optical sensor to obtain the one or more sensor reading(s) of the one or more individual cells passing through the optical channel; and evaluate the one or more of the individual cells based on the one or more sensor reading(s).95. A system comprising: a fluidic device for characterizing individual cells, the device comprising: a manifold configured to control a rate of fluidic flow within the device; a chamber fluidically coupled to the manifold and defining one or more cell characterization flow path(s), each cell characterization flow path comprising an optical channel having a functionalized surface configured to interact with one or more of the individual cells passing through the optical channel; and an optical sensor configured to obtain one or more sensor reading(s) of the one or more of the individual cells passing through the optical channel; and a controller comprising a processor configured to: dynamically adjust the manifold to control the rate of fluidic flow within the one or more cell characterization flow path(s) to facilitate one or more interactions between the one or more of the individual cells and the functionalized surface in the optical channel.96. A system comprising: a fluidic device for characterizing individual bioparticles comprising target bioparticles and non-target bioparticles, the device comprising: a dynamically configurable manifold configured to control a rate of fluidic flow within the device; a chamber fluidically coupled to the manifold and defining one or more bioparticle characterization flow path(s), each bioparticle characterization flow path comprising an optical channel having a functionalized surface configured to interact with one or more of the individual bioparticles passing through the optical channel; and an optical sensor configured to obtain one or more sensor reading(s) of the one or more of the individual bioparticles passing through the optical channel; and a controller comprising a processor configured to:WSGR Docket No. 65809-702.601 dynamically control the dynamically configurable manifold to produce a first flow rate within the one or more bioparticle processing flow path(s) that allows one or more of the individual bioparticles to physically interact with the functionalized surface in the optical channel; dynamically control the dynamically configurable manifold to produce a second flow rate within the one or more bioparticle processing flow path(s) that provides sufficient motive force to overcome interactions between one or more of the individual bioparticles and the functionalized surface in the optical channel; cause the optical sensor to obtain the one or more sensor reading(s) of the one or more individual bioparticles passing through the optical channel during at least the first flow rate and the second flow rate; and analyze the one or more sensor reading(s) to identify, characterize, or measure the one or more of the individual bioparticles based on an indication of the sufficient motive force, wherein the target bioparticles and non-target bioparticles are differentiated based on the one or more sensor reading(s).97. The system of embodiment 96, wherein the processor is configured to increase fluidic flow from the first flow rate to the second flow rate.98. The system of embodiment 97, wherein the increase in fluidic flow has a peristaltic flow profile characterized by at least a partial sinusoidal flow rate over time.99. The system of any one of embodiments 97-98, wherein the processor is configured to increase fluidic flow at a steady rate of increase from the first flow rate to the second flow rate, wherein the sufficient motive force is achieved between the first rate of fluidic flow and the second rate of fluidic flow.100. The system of any one of embodiments 96-99, wherein the processor is configured to perform at least a subset of a plurality of pre-programmed bioparticle processing operations on bioparticles present in the device in parallel.101. The system of any one of embodiments 96-100, wherein a plurality of bioparticle processing operations is automatically performed by the controller using the fluidic device.102. The system of any one of embodiments 96-101. wherein the processor is operated remotely and / or is remotely programmable or reprogrammable.103. The system of any one of embodiments 96-102, wherein the processor is configured to dynamically adjust the operation of a plurality of dynamically adjustable manifolds of a plurality of microfluidic devices.WSGR Docket No. 65809-702.601104. The system of any one of embodiments 96-103, wherein the processor is configured to dynamically control the dynamically configurable manifold to modulate flow rate according to a pre-programmed protocol.105. The system of embodiment 104, wherein the pre-programmed protocol is configured based on one or more of the target bioparticles, the non-target bioparticles, the functionalized surface, or any combination thereof.106. The system of any one of embodiments 96-105. wherein the functionalized surface is functionalized using an antibody, an aptamer, a cell adhesion molecule, and / or an adhesion protein.107. The system of any one of embodiments 96-106. wherein the optical channel comprises a plurality of functionalized surfaces.108. The system of embodiment 107, wherein the plurality of functionalized surfaces is organized into discrete regions.109. The system of any one of embodiments 107-108, wherein the plurality of functionalized surfaces is arranged in sequence along the one or more bioparticle processing flow path(s).110. The system of any one of embodiments 107-109, wherein the plurality of functionalized surfaces comprises a first region configured to interact with a first category' of target bioparticles and a second region configured to interact with a second category of target bioparticles.1 1 1. The system of embodiment 1 10, wherein the first area comprises binders configured to interact with a substrate expressed on a surface of the first category of bioparticles, and the second area comprises binders configured to interact with a substrate expressed on a surface of the second category’ of bioparticles.112. The system of any one of embodiments 110-1 11, wherein the first area comprises antibodies configured to bind to a bioparticle surface marker expressed in the first category of bioparticles, and the second area comprises antibodies configured to bind to a bioparticle surface marker expressed in the second category of bioparticles.113. The system of any one of embodiments 96-112. wherein the interactions between the one or more individual bioparticles with the functionalized surface comprises attachment or immobilization of the one or more individual bioparticles upon the functionalized surface.114. The system of any one of embodiments 96-113. wherein the sufficient motive force overcomes the interactions by causing the one or more of the individual bioparticles to detach from and / or move across the functionalized surface.WSGR Docket No. 65809-702.601115. The system of any one of embodiments 96-114, wherein the processor is configured to determine a fluidic flow rate or indication thereof that corresponds to a change in interaction between an individual bioparticle and the functionalized surface based on the sensor reading(s).116. The system of embodiment 115, wherein the change in interaction comprises a detachment or indication thereof of the individual bioparticle from the functionalized surface after being immobilized upon the functionalized surface.117. The system of any one of embodiments 96- 116, wherein the one or more bioparticle processing flow path(s) comprises a plurality of bioparticle processing flow paths.118. The system of embodiment 117. wherein the plurality of bioparticle processing flow paths forms a plurality of parallel flow paths.119. The system of any one of embodiments 96- 118, wherein the chamber is configured to facilitate fluidic dispersal of a plurality of bioparticles entering the chamber into the plurality of parallel flow paths.120. The system of any one of embodiments 96-119. wherein the optical sensor comprises an imaging sensor.121. The system of embodiment 120, wherein the imaging sensor is configured to track each of the individual bioparticles.122. The system of embodiment 121. wherein the imaging sensor is a camera.123. The system of any one of embodiments 120-122, wherein the imaging sensor captures a field of view comprising at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the optical channel.124. The system of any one of embodiments 96-123. wherein the one or more sensor reading(s) comprises one or more image(s) or a video for one or more of the individual bioparticles.125. The system of any one of embodiments 92-124, wherein the sensor reading(s) are used to generate at least one bioparticle measurement corresponding to a level of expression of a surface marker on the individual bioparticle.126. The system of any one of embodiments 96-125, wherein differentiation of target bioparticles from non-target bioparticles is label-free.127. The system of any one of embodiments 96-126. wherein the fluidic device further comprises a sorting mechanism for separating individual bioparticles.WSGR Docket No. 65809-702.601128. The system of embodiment 127. wherein the sorting mechanism comprises a sorting module comprising an acoustic actuator.129. The system of any one of embodiments 127-128, wherein the controller is configured to dynamically control the sorting mechanism to separate target and non-target bioparticles.130. The system of any one of embodiments 127-129, wherein the sorting mechanism is positioned downstream of the optical channel.131. A method for characterizing individual bioparticles, comprising: disposing a fluid comprising a plurality of bioparticles comprising target bioparticles and non-target bioparticles on a functionalized surface comprising binders configured to interact with target bioparticles, wherein the target bioparticles and non-target bioparticles have differential interactions with the functionalized surface; providing a variable fluid flow across the functionalized surface that produces a corresponding variable motive force acting upon the plurality of bioparticles on the functionalized surface, wherein the variable motive force overcomes interactions between the functionalized surface and one or more of the plurality of bioparticles; using one or more sensor(s) to obtain one or more sensor reading(s) of the at least a portion of the plurality' of bioparticles during the variable fluid flow across the functionalized surface; and generating a bioparticle evaluation for the one or more of the plurality of bioparticles based on the one or more sensor reading(s) obtained by the one or more sensor(s) and a one or more fluid flow rate(s) corresponding to the variable motive force overcoming one or more interact! on(s) of the one or more of the plurality of bioparticle(s) with the functionalized surface.132. A method for characterizing individual bioparticles, comprising: disposing a fluid comprising a plurality of bioparticles comprising a target bioparticle and a non-target bioparticle on a functionalized surface comprising binders configured to interact with the target bioparticle, wherein the target bioparticle has a stronger affinity for the functionalized surface than the non-target bioparticle; providing an increasing fluid flow rate across the functionalized surface that produces an increasing motive force acting upon the plurality' of bioparticles on the functionalized surface; using an optical sensor to obtain sensor readings of the target bioparticle and the non-target bioparticle during the variable fluid flow across the functionalized surface; andWSGR Docket No. 65809-702.601 identifying the target bioparticle and the non-target bioparticle based on the variable fluid flow rate and the corresponding sensor readings obtained by the optical sensor, wherein the sensor readings are indicative of (i) a higher motive force required to overcome interactions between the target bioparticle and the functionalized surface and (ii) a lower motive force required to overcome interactions between the non-target bioparticle and the functionalized surface.133. A method for characterizing individual bioparticles, comprising: providing a functionalized surface configured to interact with individual bioparticles; disposing a fluid comprising one or more bioparticle(s) on the functionalized surface, wherein the one or more bioparticle(s) become at least partially attached to the functionalized surface; providing a variable fluid flow across the functionalized surface that produces a corresponding variable motive force acting upon the one or more bioparticle(s) that are at least partially attached to the functionalized surface; using one or more sensor(s) to obtain one or more sensor reading(s) corresponding to a release or movement of the one or more bioparticle(s) from the functionalized surface; determining one or more fluid flow rate(s) corresponding to the release or movement of the one or more bioparticle(s) based on an association of the one or more sensor reading(s) and the variable fluid flow; and generating one or more bioparticle measurement(s) for the one or more bioparticles based on the one or more fluid flow rate(s) corresponding to the release or movement of the one or more bioparticle(s).134. A system comprising: a fluidic device for cell processing, the device comprising: a dynamically configurable manifold, configured to adjust a fraction of a fluid flow within the device passing through one or more cell processing flow path(s) fluidically coupled to the dynamically configurable manifold; an input manifold, fluidically coupled to. or integrated with, at least one inlet of the dynamically configurable fluidic manifold; an outlet manifold, fluidically coupled to one or more outlets of the cell processing flow path(s); wherein fluidic coupling of the input manifold, the dynamically configurable manifold, and the outlet manifold forms a recunent loop.WSGR Docket No. 65809-702.601135. The system of embodiment 134, wherein the plurality of parallel cell processing flow paths comprise two or more flow paths selected from the group of an identification flow path, a characterization flow path, a cell sorting flow path, a cell selection flow path, a cell deselection flow path, a cell engineering flow path, a cell culturing flow path, a cell activating flow path, a cell washing flow path, and a cell expanding flow path.136. The system of embodiment 135, wherein the recurrent loop is configured to allow at least a portion of cells flowing through the plurality of cell processing flow paths to recirculate from the outlet manifold to the at least one inlet of the dynamically configurable manifold.137. The system of embodiment 135 or 136, wherein the recurrent loop is configured to recirculate, isolate, or transfer fluid and / or cells between one or more of the two or more flow paths in a cyclic flow path which is monodirectional through each respective flow path.138. The system of embodiment 135 or 136, wherein the recurrent loop is configured to recirculate, isolate, or transfer fluid and / or cells between one or more of the two or more flow paths by reversing a direction of flow within a same fluidic channel.139. The system of embodiment 137, wherein the system comprises at least 3 fluidic channels configured for reversal of fluid flow direction.140. The system of embodiment 138, wherein the system comprises at least 6 fluidic channels configured for reversal of fluid flow direction.141. The system of any of the preceding embodiments, wherein the plurality of parallel cell processing flow paths comprise a cell culturing flow path, a cell activation flow path, and: (i) a cell measurement flow path; or (ii) a cell characterization flow path.142. The system of any of the preceding embodiments, wherein the cell measurement flow path comprises a cell counting module, and / or the cell characterization flow path comprises a functional screening module.143. The system of any of the preceding embodiments, wherein the plurality of parallel cell processing flow paths comprise (i) a cell measurement module or a cell characterization module; and (ii) a cell sorting module in the same flow path.144. The system of any one of embodiments 134-143, wherein the cell sorting flow path comprises a label-free cell sorting module.WSGR Docket No. 65809-702.601145. The system of any one of embodiments 134-144, wherein the cell sorting flow path comprises a cell concentrating module wherein such concentration may be affected by one or more of filtration, inertial, acoustic or similar modalities.146. The system of any one of embodiments 134-145, wherein the cell sorting flow path comprises a cell dilution module.147. The system of any one of embodiments 134-146, wherein the cell sorting flow path comprises an acoustic sorting module comprising one or more acoustic transducers and a plurality of acoustic sorting channels.148. The system of embodiment 147, wherein the acoustic sorting module comprises at least 2 parallel acoustic sorting channels.149. The system of any one of embodiments 146-148, wherein the acoustic sorting module comprises a plurality of capacitive micromachined ultrasonic transducers (CMUT) and / or an array of piezoelectric micromechanical ultrasonic transducers (PMUT).150. The system of any of the preceding embodiments, wherein the input manifold comprises a plurality of inlets and / or the outlet manifold comprises a plurality’ of outlets.151. The system of embodiment 150, wherein the input manifold comprises a plurality of inlets and a single outlet fluidically coupled to the at least one inlet of the dynamically configurable manifold, and / or the outlet manifold comprises: a plurality of inlets, each fluidically coupled to a corresponding outlet of the plurality of cell processing flow paths; and a single outlet.152. The system of any of the preceding embodiments, wherein the fluidic device comprises one or more inline pH sensors, one or more inline glucose concentration sensors, one or more inline pyruvate concentration sensors, one or more lactate concentration sensors, one or more pCO2 sensors, and / or one or more inline dissolved oxygen sensors.153. The system of embodiment 152, wherein at least one of the one or more inline pH sensors, at least one of the inline dissolved oxygen sensors, at least one or more inline glucose concentration sensors, at least one of the one or more inline pyruvate concentration sensors, at least one of the one or more lactate concentration sensors, or at least one of the one or more pCO2 sensors, is comprised in a cell characterization module.154. The system of any of the preceding embodiments, wherein the input manifold comprises an inline mixer.155. The system of embodiment 154, wherein the inline mixer is and / or comprises a zero dead-volume mixer.WSGR Docket No. 65809-702.601156. The system of any of the preceding embodiments, wherein the input manifold and / or the outlet manifold comprise one or more zero dead-volume switching valves.157. The system of any of the preceding embodiments, wherein the fluidic device comprises one or more of peristaltic pumps and / or continuous flow pumps.158. The system of any of the preceding embodiments, wherein the input manifold comprises at least three input manifold inlets.159. The system of any of the preceding embodiments, wherein the outlet manifold comprises at least three input manifold inlets.160. The system of any of the preceding embodiments, wherein the plurality' of parallel flow paths comprises at least three parallel flow paths each corresponding to at least one cell processing application.161. The system of any of the preceding embodiments, wherein the cell characterization flow path, and / or the cell measurement flow path comprises an optical channel comprising an optical sensor.162. The system of embodiment 161, wherein the optical sensor is a complementary metal oxide semiconductor sensor, a photomultiplier or photodiode, and / or a camera.163. The system of any one of embodiments 160-162, wherein the optical channel comprises a functionalized surface configured to interact with one or more cells passing through the optical channel.164. The system of any one of embodiments 160-163, wherein the optical channel further comprises an acoustic transducer.165. The system of embodiment 164, wherein the acoustic transducer is configured to promote and / or increase interaction of the one or more cells passing through the optical channel with the functionalized surface.166. The system of any one of embodiments 160-165, wherein the functionalized surface is functionalized using an antibody, an aptamer, a cell adhesion molecule, and / or an adhesion protein.167. The system of any of the preceding embodiments, wherein the fluidic device is: a microfluidic device, and / or a device configured for manufacture of engineered cells for use in a cell therapeutic.168. The system of embodiment 167, wherein the engineered cells are leukocytes.169. The system of embodiment 168, wherein the engineered cells are CAR-T cells, CAR- NK cells, modified B-cells, tumor infiltrating lymphocyte cells, and / or induced pluripotent stem cells and hematopoietic stem cells.WSGR Docket No. 65809-702.601170. The system of any of the preceding embodiments, wherein the cells processed are allogenic to a patient.171. The system of any of the preceding embodiments, wherein one or more components of the fluidic device are comprised in a cartridge.172. The system of embodiment 171, wherein the cartridge is a disposable cartridge.173. The system of any one of embodiments 170-172, wherein the cartridge comprises the input manifold, the output manifold, the fluidic couplings, and / or the outlet manifold.174. The system of any one of embodiments 170-173, wherein a majority of the fluidic volume of the fluidic system is comprised within the cartridge, and / or is comprised within a plurality of cartridges of the system.175. The system of any of the preceding embodiments, further comprising a controller configured to dynamically adjust an operation of the dynamically controllable manifold.176. The system of embodiment 175, wherein the controller further comprises a user interface which allows for user selection of two or more parallel cell processing operations.177. The system of embodiment 176, wherein the controller is configured to perform at least a subset of a plurality of pre-programmed cell processing operations on cells present in the device in parallel.178. The system of any one of embodiments 175-177, wherein a plurality7of cell processing operations are automatically performed by the controller using the fluidic device.179. The system of any one of embodiments 175-178, wherein the controller is operated remotely and / or is remotely programmable or reprogrammable.180. The system of any one of embodiments 175-179, wherein the controller is configured to dynamically adjust the operation of a plurality7of dynamically adjustable manifolds of a plurality of microfluidic devices.181. The system of any of the preceding embodiments, wherein the plurality of parallel cell processing flow paths comprise a cell delivery and / or a cell acquisition flow path.182. The system of embodiment 181, wherein the cell delivery7and / or cell acquisition flow path is configured to be fluidically couplable to a blood vessel of a patient.183. The system of embodiment 182, wherein the device is a point-of-care cell therapeutic device, and the subject is a patient suffering from a disease or condition in need of treatment by a cell therapy.184. A method of processing and / or manufacturing cells, the method comprising: preparing a plurality of engineered cells using the device or system of any of the preceding embodiments.WSGR Docket No. 65809-702.601185. The method of embodiment 184, wherein cells are circulated through the one or more cell processing flow paths until a cell population in the device possess one or more cell processing target metrics.186. The method of embodiment 185, wherein the cell population is administered to a patient after the one or more cell processing target metrics.187. The method any one of embodiments 179-186, wherein the dynamically configurable manifold is adjusted to maintain one or more cell processing target metrics in a cell population flowing through the device or system.188. The method of embodiment 187, wherein the cell population is directly administered to a patient during the cell processing operations.

[0223] While preferred embodiments of the present invention have been show n and described herein, it will be obvious to those skilled in the art that such embodiments are provided by w ay of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 65809-702.601CLAIMSWHAT IS CLAIMED IS:

1. A system comprising: a fluidic device for cell processing, the device comprising: a dynamically configurable manifold, configured to adjust a fraction of a fluid flow within the device passing through one or more cell processing flow path(s) fluidically coupled to the dynamically configurable manifold; an input manifold, fluidically coupled to, or integrated with, at least one inlet of the dynamically configurable fluidic manifold; an outlet manifold, fluidically coupled to one or more outlets of the cell processing flow path(s); wherein fluidic coupling of the input manifold, the dynamically configurable manifold, and the outlet manifold forms a recurrent loop.

2. The system of claim 1, wherein the plurality' of parallel cell processing flow paths comprise two or more flow paths selected from the group of: an identification flow path, a characterization flow path, a cell sorting flow path, a cell selection flow path, a cell deselection flow path, a cell engineering flow path, a cell culturing flow path, a cell activating flow path, a cell washing flow path, and a cell expanding flow path.

3. The system of claim 2, wherein the recurrent loop is configured to allow at least a portion of cells flowing through the plurality of cell processing flow paths to recirculate from the outlet manifold to the at least one inlet of the dynamically configurable manifold.

4. The system of claim 2 or 3, wherein the recurrent loop is configured to recirculate, isolate, or transfer fluid and / or cells between one or more of the two or more flow paths in a cyclic flow path which is monodirectional through each respective flow path.

5. The system of claim 2 or 3, wherein the recurrent loop is configured to recirculate, isolate, or transfer fluid and / or cells between one or more of the two or more flow paths by reversing a direction of flow within a same fluidic channel.

6. The system of claim 5, yvherein the system comprises at least 3 fluidic channels configured for reversal of fluid flow direction.

7. The system of claim 6, wherein the system comprises at least 6 fluidic channels configured for reversal of fluid flow direction.

8. The system of any of the preceding claims, wherein the plurality of parallel cell processing flow paths comprise a cell culturing flow path, a cell activation flow path, and: (i) a cell measurement flow path; or (ii) a cell characterization flow path.WSGR Docket No. 65809-702.6019. The system of any of the preceding claims, wherein the cell measurement flow path comprises a cell counting module, and / or the cell characterization flow path comprises a functional screening module.

10. The system of any of the preceding claims, wherein the plurality of parallel cell processing flow paths comprise (i) a cell measurement module or a cell characterization module; and (ii) a cell sorting module in the same flow path.

11. The system of any one of claims 2-10, wherein the cell sorting flow path comprises a label- free cell sorting module.

12. The system of any one of claims 2-11, wherein the cell sorting flow path comprises a cell concentrating module wherein such concentration may be affected by one or more of filtration, inertial, acoustic or similar modalities.

13. The system of any one of claims 2-12, wherein the cell sorting flow path comprises a cell dilution module.

14. The system of any one of claims 2-13, wherein the cell sorting flow path comprises an acoustic sorting module comprising one or more acoustic transducers and a plurality of acoustic sorting channels.

15. The system of claim 14, wherein the acoustic sorting module comprises at least 2 parallel acoustic sorting channels.

16. The system of any one of claims 14-15, wherein the acoustic sorting module comprises a plurality of capacitive micromachined ultrasonic transducers (CMUT) and / or an array of piezoelectric micromechanical ultrasonic transducers (PMUT).

17. The system of any of the preceding claims, wherein the input manifold comprises a plurality' of inlets and / or the outlet manifold comprises a plurality of outlets.

18. The system of claim 17. wherein the input manifold comprises a plurality of inlets and a single outlet fluidically coupled to the at least one inlet of the dynamically configurable manifold, and / or the outlet manifold comprises: a plurality of inlets, each fluidically coupled to a corresponding outlet of the plurality of cell processing flow paths; and a single outlet.

19. The system of any of the preceding claims, wherein the fluidic device comprises one or more inline pH sensors, one or more inline glucose concentration sensors, one or more inline pyruvate concentration sensors, one or more lactate concentration sensors, one or more pCCh sensors, and / or one or more inline dissolved oxygen sensors.

20. The system of claim 19. wherein at least one of the one or more inline pH sensors, at least one of the inline dissolved oxygen sensors, at least one or more inline glucose concentrationWSGR Docket No. 65809-702.601 sensors, at least one of the one or more inline pyruvate concentration sensors, at least one of the one or more lactate concentration sensors, or at least one of the one or more pCCh sensors, is comprised in a cell characterization module.

21. The system of any of the preceding claims, wherein the input manifold comprises an inline mixer.

22. The system of claim 21. wherein the inline mixer is and / or comprises a zero dead-volume mixer.

23. The system of any of the preceding claims, wherein the input manifold and / or the outlet manifold comprise one or more zero dead-volume switching valves.

24. The system of any of the preceding claims, wherein the fluidic device comprises one or more of peristaltic pumps and / or continuous flow pumps.

25. The system of any of the preceding claims, wherein the input manifold comprises at least three input manifold inlets.

26. The system of any of the preceding claims, wherein the outlet manifold comprises at least three input manifold inlets.

27. The system of any of the preceding claims, wherein the plurality of parallel flow paths comprises at least three parallel flow paths each corresponding to at least one cell processing application.

28. The system of any of the preceding claims, wherein the cell characterization flow path, and / or the cell measurement flow path comprises an optical channel comprising an optical sensor.

29. The system of claim 28, wherein the optical sensor is a complementary metal oxide semiconductor sensor, a photomultiplier or photodiode, and / or a camera.

30. The system of any one of claims 28-29. wherein the optical channel comprises a functionalized surface configured to interact with one or more cells passing through the optical channel.

31. The system of any one of claims 28-30, wherein the optical channel further comprises an acoustic transducer.

32. The system of claim 31. w herein the acoustic transducer is configured to promote and / or increase interaction of the one or more cells passing through the optical channel w ith the functionalized surface.

33. The system of any one of claims 30-32, w herein the functionalized surface is functionalized using an antibody, an aptamer, a cell adhesion molecule, and / or an adhesion protein.WSGR Docket No. 65809-702.60134. The system of any of the preceding claims, wherein the fluidic device is: a microfluidic device, and / or a device configured for manufacture of engineered cells for use in a cell therapeutic.

35. The system of claim 34, wherein the engineered cells are leukocytes.

36. The system of claim 35, wherein the engineered cells are CAR-T cells, CAR-NK cells, modified B-cells, tumor infiltrating lymphocyte cells, and / or induced pluripotent stem cells and hematopoietic stem cells.

37. The system of any of the preceding claims, wherein the cells processed are allogenic to a patient.

38. The system of any of the preceding claims, wherein one or more components of the fluidic device are comprised in a cartridge.

39. The system of claim 38, wherein the cartridge is a disposable cartridge.

40. The system of any one of claims 38-39, wherein the cartridge comprises the input manifold, the output manifold, the fluidic couplings, and / or the outlet manifold.

41. The system of any one of claims 38-40, wherein a majority of the fluidic volume of the fluidic system is comprised within the cartridge, and / or is comprised within a plurality of cartridges of the system.

42. The system of any of the preceding claims, further comprising a controller configured to dynamically adjust an operation of the dynamically controllable manifold.

43. The system of claim 42. wherein the controller further comprises a user interface which allows for user selection of two or more parallel cell processing operations.

44. The system of any one of claims 42-43, wherein the controller is configured to perform at least a subset of a plurality of pre-programmed cell processing operations on cells present in the device in parallel.

45. The system of any one of claims 43-44, wherein a plurality of cell processing operations are automatically performed by the controller using the fluidic device.

46. The system of any one of claims 42-45, wherein the controller is operated remotely and / or is remotely programmable or reprogrammable.

47. The system of any one of claims 44-46. wherein the controller is configured to dynamically adjust the operation of a plurality of dynamically adjustable manifolds of a plurality of microfluidic devices.

48. The system of any of the preceding claims, wherein the plurality of parallel cell processing flow paths comprise a cell delivery and / or a cell acquisition flow path.WSGR Docket No. 65809-702.60149. The system of claim 48, wherein the cell delivery and / or cell acquisition flow path is configured to be fluidically couplable to a blood vessel of a patient50. The system of claim 49, wherein the device is a point-of-care cell therapeutic device, and the subject is a patient suffering from a disease or condition in need of treatment by a cell therapy.

51. A method of processing and / or manufacturing cells, the method comprising: preparing a plurality of engineered cells using the device or system of any of the preceding claims.

52. The method of claim 51, wherein cells are circulated through the one or more cell processing flow paths until a cell population in the device possess one or more cell processing target metrics.

53. The method of claim 52, wherein the cell population is administered to a patient after the one or more cell processing target metrics.

54. The method any one of claims 51-53, wherein the dynamically configurable manifold is adjusted to maintain one or more cell processing target metrics in a cell population flowing through the device or system.

55. The method of claim 54, wherein the cell population is directly administered to a patient during the cell processing operations.

56. The method of any one of claims 44-55, wherein the one or more cell processing target metrics comprise: a threshold cell concentration of a target cell type or population thereof, a target expression level of one or more target proteins of a target cell type, a threshold level of one or more contaminants (e.g., of non-target cell types) in fluid flowing through the device or system, a purity of a target cell or population thereof, a viability of a target cell or population thereof, a number of a target cell or population thereof, a phenotype of a target cell or population and / or a presence of a threshold level of one or more detectable biomarkers present in a cell characterization module of the system.

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