Systems and methods for particle separation and concentration

Abstract

A system and method for producing a product enriched in one or more target particles, comprising a cassette to which one or more microfluidic cartridges are releasably coupled and supported, the cassette comprising: (i) a plurality of input receptacles releasably and fluidically coupled thereto, at least one of the plurality of input receptacles containing an incoming sample, (ii) a plurality of outlets having a plurality of output receptacles releasably and fluidically coupled thereto, (iii) one or more microfluidic cartridges for separating one or more target particles from the sample, and (iv) a plurality of fluid channels extending between the plurality of inlets, the plurality of outlets, and the one or more microfluidic cartridges. The plurality of input receptacles, the plurality of output receptacles, and the cassette together provide a closed end-to-end sterile environment that enables in-line continuous processing of the incoming sample without external manual handling or intervention to produce a contaminant-free product enriched in one or more target particles.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the benefits of U.S. Provisional Application No. 63 / 139737 (filed January 20, 2021), U.S. Provisional Application No. 63 / 140196 (filed January 21, 2021), and U.S. Provisional Application No. 63 / 163593 (filed March 19, 2021), each of which is incorporated herein by reference. [Background technology]

[0002] The isolation of target cells from patient samples often requires the use of systems and methods that face several obstacles. These systems and methods may require human intervention during sample processing, impacting the sterility of the environment and the resulting product solution. Current systems and methods can struggle to achieve high throughput of sample solutions. They may also involve moving parts throughout the separation process, introducing further potential for failure and contamination. Therefore, the development of better-performing devices and methods to increase the rate at which biological material can be purified and to enhance the sterility of the environment in which the separation process is carried out is of considerable interest. [Overview of the Initiative] [Means for solving the problem]

[0003] This section describes specific cassettes, microfluidic devices, particle separation units, and components connecting these three elements to provide a system for improved target particle separation time in an end-to-end closed system for improved sterility. A specific cassette may have multiple input and output channels connecting multiple inputs to multiple outputs in a closed end-to-end environment that maintains a sterile operating environment with little to no human intervention to provide continuous in-line processing of incoming samples. The cassette may also have multiple pathways for guiding incoming samples and multiple components for preserving the sterility and viability of the sample solution for gentle mixing and transport of the sample solution along with multiple other input and output solutions.

[0004] The methods and systems described herein can produce therapeutic immune cell populations (including T cell populations) that exhibit beneficial properties compared to other methods for producing therapeutic immune cell populations. These advantages include greater proliferative capacity, increased ability to incorporate and introduce exogenous nucleic acids and express proteins from lentiviral vectors, and increased retention of favorable cell populations, such as naive, quiescent, inactivated, or central memory T cells. The immune cells and T cells produced by the systems and methods described herein also exhibit a lower inflammatory phenotype, as indicated by reduced levels of inflammatory cytokines in culture, suggesting a reduced potential contribution to cytokine release syndrome. The low levels of inflammatory cytokines in the produced T cells are significant. This is because cytokine release syndrome is a major adverse event associated with T cell therapy (e.g., CART cell and recombinant T cell therapy) and is related to the presence of differentiated and activated T cell subsets (e.g., effector memory T cells (Tem) or effector T cells that restore the expression of CD45RA (Temra) that releases IL-IRA, IL-6, and IL-13). As shown here, the cells produced by the systems and methods described also exhibit low release of key inflammatory cytokines that contribute to cytokine release syndrome.

[0005] Herein, in one embodiment, a microfluidic cartridge for purifying target particles or target cells of a predetermined size from contaminants in a sample is described. The cartridge comprises a first planar support and a second planar support, each having a top and bottom surface, and the top surface of the first planar support and / or the second planar support comprises at least one embedding channel extending from one or more inlets to one or more outlets, the at least one embedding channel containing a plurality of obstacles.

[0006] Herein, in one embodiment, a particle separation unit is described that includes a cassette, a microfluidic cartridge, and several components for receiving, supporting, and / or operating input and output solutions.

[0007] In one embodiment, this describes a system for generating a product enriched in one or more target particles, comprising: a cassette having (i) one or more cartridge ports, (ii) at least one inlet for receiving a sample, (iii) at least one outlet for outputting the product, and (iv) at least one recirculation path; and one or more microfluidic cartridges operably coupled to one or more cartridge ports and establishing fluid communication with the cassette, wherein one or more microfluidic cartridges are used to separate one or more target particles from a sample, and at least one recirculation path is used to recirculate one or more target particles through the cassette to concentrate one or more target particles to a predetermined volume of medium or a predetermined concentration, and the one or more microfluidic cartridges and at least one recirculation path operate in parallel to optimize the run time for generating the product. In certain embodiments, the one or more microfluidic cartridges and at least one recirculation path operate independently of each other. In certain embodiments, one or more microfluidic cartridges are configured to separate one or more target particles from a sample without affecting the recirculation process by at least one recirculation path. In certain embodiments, at least one recirculation path is configured to recirculate one or more target particles through a cassette without affecting the separation process by one or more microfluidic cartridges. In certain embodiments, one or more microfluidic cartridges and at least one recirculation path are individually controllable in real time to achieve a desired concentration of one or more target particles in the product. In certain embodiments, at least one recirculation path extends between at least one inlet and at least one outlet. In certain embodiments, at least one recirculation path is configured to recirculate one or more target particles clockwise on the cassette. In certain embodiments, at least one recirculation path is configured to recirculate one or more target particles counterclockwise on the cassette. In certain embodiments, the cassette further includes multiple paths for loading and unloading samples into and from one or more microfluidic cartridges.In certain embodiments, at least one recirculation path is provided separately from multiple paths. In certain embodiments, at least one recirculation path is adjacent to or connected to one or more of the multiple paths. In certain embodiments, the system further comprises one or more mass sensors, and the recirculation of one or more target particles through the cassette is controlled based on one or more readings obtained from one or more mass sensors.

[0008] In other embodiments, this describes a system for generating a product enriched with one or more target particles, comprising: a cassette on which one or more microfluidic cartridges are releasably coupled and supported, the cassette including a plurality of fluid channels extending longitudinally and spaced apart on the cassette; and a plurality of pumps peristaltically coupled to the plurality of fluid channels and controlling the flow of a fluid-containing material through the plurality of fluid channels downstream of the one or more microfluidic cartridges for the separation of one or more target particles from a sample, wherein none of the moving parts from the pumps are in direct contact with the fluid-containing material during its flow. In certain embodiments, the plurality of fluid channels include flexible tubes. In certain embodiments, each of the plurality of pumps includes a set of pump heads peristaltically coupled to each subset of the plurality of fluid channels. In certain embodiments, the set of pump heads in each pump includes two or more pump heads. In certain embodiments, the two or more pump heads include two or more roller sets. In certain embodiments, each subset of the plurality of fluid channels includes two or more fluid channels. In certain embodiments, the fluid-containing material includes a sample, a medium solution, a diluent, and waste generated by one or more microfluidic cartridges after one or more target particles have been separated from the sample. In certain embodiments, the fluid-containing material further includes a priming solution. In certain embodiments, the fluid-containing material further includes a recirculating solution containing a concentrated amount of one or more target particles. In certain embodiments, the pumps include a first pump for controlling the flow of the sample, a second pump for controlling the flow of the medium solution, a third pump for controlling the flow of the diluent, and a fourth pump for controlling the flow of the waste. In certain embodiments, the pumps are individually controllable to adjust the relative flow rates between the sample, medium solution, diluent, and waste. In certain embodiments, the set of pump heads in each pump are configured to operate in opposite phases to each other. In certain embodiments, the set of pump heads in each pump operate in opposite phases by only about 180 degrees or less. In certain embodiments, the set of pump heads in each pump have fixed movements relative to each other.In certain embodiments, fixed motion includes the movement of a set of pump heads in each pump in the same direction at the same velocity relative to each other. In certain embodiments, for each pump and each subset of fluid channels, the fluid components transported by the set of pump heads merge together into a single fluid path at the outlet of each subset of fluid channels. In certain embodiments, the pulsation of each pump is reduced by moving the set of pump heads in opposite phases. In certain embodiments, multiple pumps can be individually controlled to (a) control the ratio of waste volume to sample volume, (b) control the ratio of sample volume to diluent volume, or (c) adjust the dilution ratio. In certain embodiments, multiple pumps can be individually controlled to be in phase or out of phase relative to each other. In certain embodiments, multiple pumps can be individually controlled to achieve the same flow rate, different flow rates, the same flow direction, or different flow directions. In certain embodiments, multiple pumps can be individually controlled to adjust the flow of fluid components in real time when one or more microfluidic cartridges separate one or more target particles from a sample. In certain embodiments, multiple pumps can be individually controlled to allow a desired concentration of one or more target particles in the product. In certain embodiments, the multiple pumps include peristaltic pumps. In certain embodiments, the multiple pumps include at least two, three, four, five, six, seven, or eight pumps.

[0009] In other embodiments, this describes a system for producing a product enriched with one or more target particles, comprising: one or more microfluidic cartridges configured to separate one or more target particles from a sample; one or more sensors for detecting the presence of bubbles in the sample or other solution; one or more controllable valves; and a cassette to which one or more microfluidic cartridges are releasably coupled and supported, the cassette including one or more bypass channels downstream of one or more controllable valves for redirecting portions of the sample or other solution containing bubbles away from one or more microfluidic cartridges based on the detection of the presence of air by one or more sensors. In certain embodiments, one or more sensors are used to detect the presence of bubbles before circulating the sample through one or more microfluidic cartridges. In certain embodiments, the system is configured to generate one or more alerts when one or more sensors detect the presence of bubbles in the sample or other solution. In certain embodiments, one or more sensors and one or more bypass channels work together to prevent bubbles from entering and reducing the efficiency of one or more microfluidic cartridges. In certain embodiments, one or more sensors and one or more bypass channels work together to reduce or eliminate contamination in the product.

[0010] This also describes a system for generating a product enriched with one or more target particles, comprising one or more microfluidic cartridges configured to separate one or more target particles from a sample; a cassette on which one or more microfluidic cartridges are releasably coupled and supported; and at least one degassing unit for removing dissolved gases and preventing bubble formation before the sample circulates through one or more microfluidic cartridges. In certain embodiments, at least one degassing unit is integrated onto the cassette. In certain embodiments, at least one degassing unit is fabricated as part of the cassette. In certain embodiments, at least one degassing unit is mounted on the cassette in close proximity to one or more microfluidic cartridges. In certain embodiments, at least one degassing unit is in fluid communication with multiple pathways introducing fluid into one or more microfluidic cartridges. In certain embodiments, the system further comprises a panel operably coupled to the cassette, and at least one degassing unit is integrated onto the panel. In some embodiments, the degassing unit is a vacuum degassing unit. In one embodiment, an integrated vacuum pump is connected to a vacuum degassing unit, which applies a vacuum to the vacuum chamber of the vacuum degassing unit. In another embodiment, the fluid entering the degassing unit passes through a tube made of a porous membrane that separates the fluid path of the degassing unit from the vacuum chamber of the degassing unit. In yet another embodiment, dissolved gases in the fluid contained in the fluid path of the degassing unit pass through the membrane, while the fluid does not, thereby removing the dissolved gases from the fluid into the vacuum of the vacuum chamber.

[0011] In another embodiment, here we describe a system for producing a product enriched with one or more target particles, comprising a cassette on which one or more microfluidic cartridges are releasably coupled and supported, the cassette comprising: (i) a plurality of inlets having a plurality of input vessels thereto releasably and fluidly coupled thereto, at least one of the plurality of input vessels containing an incoming sample; (ii) a plurality of outlets having a plurality of output vessels thereto releasably and fluidly coupled thereto; (iii) one or more microfluidic cartridges for separating one or more target particles from the sample; and (iv) a plurality of fluid channels extending between the plurality of inlets, a plurality of outlets and one or more microfluidic cartridges, wherein the cassette having the plurality of input vessels, a plurality of output vessels and one or more microfluidic cartridges thereto, as a whole, provides a closed end-to-end sterile environment that enables in-line continuous processing of incoming samples without external manual operation or intervention, and produces a product enriched with one or more target particles and free from contamination. In a particular embodiment, the plurality of inlets and a plurality of input vessels are releasably and fluidly coupled using a plurality of sterile coupling mechanisms. In certain embodiments, the multiple sterile coupling mechanisms include at least one sterile spike and at least one spike port. In certain embodiments, the multiple sterile coupling mechanisms include at least one sterile Luer fitting. In certain embodiments, the multiple sterile coupling mechanisms include at least one sterile tube weld. In certain embodiments, the multiple sterile coupling mechanisms include at least one sterile quick connector fitting. In certain embodiments, the product is collected in at least one of a plurality of output containers. In certain embodiments, the product is collected in at least one of a plurality of output containers without exposing the product to an external non-sterile environment. In certain embodiments, the product is collected in at least one of a plurality of output containers without exposing the product to an external non-sterile environment. In certain embodiments, the sample is fed into the cassette from at least one of a plurality of input containers without exposing the sample to an external non-sterile environment.In certain embodiments, the system does not require any intermediate reagents to be added externally from outside the closed end-to-end sterile environment during in-line continuous processing of incoming samples. In certain embodiments, the system does not require any byproducts to be removed outside the closed end-to-end sterile environment during in-line continuous processing of incoming samples. In certain embodiments, the sample has a volume of at least about 200 mL, and the system is configured to process the sample to produce an enriched product with the recovery of at least about 70% of one or more target particles in less than one hour. In certain embodiments, the system is configured to process the sample at a rate of about 300 mL / hour or more.

[0012] Also described here is a system for generating a product enriched with one or more target particles, comprising one or more microfluidic cartridges configured to separate one or more target particles from a sample, and a cassette comprising a mixer configured to releasely connect and support one or more microfluidic cartridges, and which includes a mixer configured to mix the sample inline on the cassette with a diluent without using any moving parts before the sample circulates through one or more microfluidic cartridges. In certain embodiments, the mixer includes a first fluid channel for the sample and a second fluid channel for the diluent. In certain embodiments, the first and second fluid channels merge to enable mixing of the sample and the diluent. In certain embodiments, the first and second fluid channels include a plurality of structural elements to facilitate inline mixing of the sample and the diluent.

[0013] Here, we also describe a method comprising the steps of (a) preparing a system including a cassette on which one or more microfluidic cartridges are releasably coupled and supported; (b) priming the cassette by flowing a priming solution through the system; (c) processing the sample by flowing the sample through the system and separating one or more target particles from the system using one or more microfluidic cartridges; and (d) recovering a product containing one or more target particles separated from the sample, wherein the sample has a volume of at least about 40 mL, and the product is enriched with the recovery of at least about 70% of one or more target particles, and steps (b) to (d) are completed continuously inline in a closed sterile environment for about 1 hour or less. In certain embodiments, step (b) is completed in about 20 minutes or less. In certain embodiments, steps (c) and (d) are completed in about 40 minutes or less. In certain embodiments, the cassette and one or more microfluidic cartridges are configured for single use. In certain embodiments, step (b) allows the cassette to be reusable for multiple uses. In certain embodiments, step (b) allows one or more microfluidic cartridges to be reusable for multiple uses. In certain embodiments, the sample is a human sample. In certain embodiments, the sample contains blood-related products. In certain embodiments, the blood-related products contain apheresis products. In certain embodiments, the apheresis products are leukocyte apheresis products. In certain embodiments, one or more target particles isolated from the sample contain cells. In certain embodiments, the cells are human cells. In certain embodiments, the cells have a viability greater than about 90% at recovery. In certain embodiments, the cells contain peripheral blood mononuclear cells. In certain embodiments, the cells contain CD3+ T cells. In certain embodiments, the T cells exhibit a naive or central memory phenotype. In certain embodiments, the method further includes the step of culturing or growing the cells in vitro. In certain embodiments, the method further includes the step of transgenicizing the cells using exogenous nucleic acids.In certain embodiments, the exogenous nucleic acid encodes a chimeric antigen receptor or a recombinant T cell receptor. In certain embodiments, the sample has a volume of at least about 300 mL. In certain embodiments, the sample has a volume of at least about 100 mL. In certain embodiments, the product is enriched with a recovery of at least about 80% of one or more target particles. In certain embodiments, the product is enriched with a recovery of at least about 90% of one or more target particles. In certain embodiments, the product is enriched with a recovery of at least about 95% of one or more target particles. In certain embodiments, the sample has a volume of at least about 100 mL. In certain embodiments, the sample has a volume of at least about 150 mL. In certain embodiments, the sample has a volume of at least about 200 mL.

[0014] This also describes a method comprising the steps of: displaying a graphical user interface (GUI) on a computer, the GUI including (i) a control panel and (ii) a visual representation of the system, the system comprising (a) a cassette on which one or more microfluidic cartridges are releasably coupled and supported, and (b) a plurality of components for facilitating fluid transport and process control; receiving user input for an execution protocol entered via the control panel; invoking the execution protocol on the system to process a sample by using one or more microfluidic cartridges to separate one or more target particles from the sample; and displaying progress or status substantially in real time while the system is processing the sample, the progress or status being depicted by graphic changes to the visual representation of the system. In certain embodiments, the plurality of components include flow channels, valves, pressure sensors, and pumps. In certain embodiments, the plurality of components further include one or more bubble sensors and at least one degassing unit. In certain embodiments, the graphic changes include the on / off state of one or more of the plurality of components. In certain embodiments, the graphic changes include the fluid flow of the sample or other medium through the cassette and one or more microfluidic cartridges. In certain embodiments, the method further includes the step of generating one or more notifications on a GUI indicating that the system is processing a sample according to an execution protocol. In certain embodiments, the method further includes the step of generating one or more notifications on a GUI indicating that the system is experiencing one or more deviations from the execution protocol while the sample is being processed. In certain embodiments, the method further includes the step of generating one or more options on a GUI for the user to correct one or more deviations. In certain embodiments, the method further includes the step of automatically reducing the pressure and flow rate of the sample when one or more deviations from the execution protocol are detected.In certain embodiments, the method further includes the step of generating a report containing multiple running metrics once the system has completed processing the sample. In certain embodiments, while the system is processing the sample, a GUI allows the user to observe the status and control the operation of one or more of the components in substantially real time.

[0015] The systems and methods described herein in specific embodiments relate to the use of cells in enrichment for cell therapies (e.g., CART cell therapy, cell-based immunotherapy, and stem cell therapy). The source of cells for enrichment, isolation, or separation is a blood-related product obtained from a patient under treatment or an HLA-matched donor. In specific embodiments, the sample is a human sample. In specific embodiments, the sample contains a blood-related product. In specific embodiments, the blood-related product contains an apheresis product. In specific embodiments, the apheresis product is a leukocyte apheresis product. In specific embodiments, one or more target particles isolated from the sample contain cells. In specific embodiments, the cells are human cells. In specific embodiments, the cells have a viability greater than about 90% at recovery. In specific embodiments, the cells contain peripheral blood mononuclear cells. In specific embodiments, the cells are greater than about 80%, 85%, 90%, or 95% of peripheral blood mononuclear cells recovered from the starting sample. In specific embodiments, the cells contain CD3+ T cells. In certain embodiments, the T cells exhibit a naive or central memory phenotype. In certain embodiments, the method further includes the step of culturing or growing the cells in vitro. In certain embodiments, the method further includes the step of transgenicizing the cells using an exogenous nucleic acid. In certain embodiments, the exogenous nucleic acid encodes a chimeric antigen receptor or a recombinant T cell receptor. In certain embodiments, the sample has a volume of at least about 300 mL. In certain embodiments, the sample has a volume of at least about 100 mL. In certain embodiments, the sample has a volume of at least about 100 mL. In certain embodiments, the sample has a volume of at least about 150 mL. In certain embodiments, the sample has a volume of at least about 200 mL.

[0016] In certain embodiments, the white blood cells are enriched to at least about 85% or more of the resulting product. In certain embodiments, the white blood cells are enriched to at least about 90% or more of the resulting product. In certain embodiments, the white blood cells are enriched to at least about 85% or more of the resulting product. In certain embodiments, at least about 2×10 9 white blood cells are obtained from at least about 200 mL of leukopak. In certain embodiments, at least about 2×10 9 white blood cells are obtained from at least about 300 mL of leukopak. In certain embodiments, at least about 2×10 9 white blood cells are obtained from 100 mL of leukopak. In certain embodiments, at least about 5×10 9 white blood cells are obtained from at least about 200 mL of leukopak. In certain embodiments, at least about 5×10 9 white blood cells are obtained from at least about 300 mL of leukopak. In certain embodiments, at least about 5×10 9 white blood cells are obtained from at least about 100 mL of leukopak.

[0017] In certain embodiments, the enriched white blood cells have a telomere length of at least about 2 kilobases. In certain embodiments, the enriched white blood cells have a telomere length of at least about 3 kilobases. In certain embodiments, the enriched white blood cells have a telomere length of at least about 5 kilobases.

[0018] In certain embodiments, at least about 4×10 8 naive T cells are obtained from at least about 200 mL of leukopak. In certain embodiments, at least about 4×10 8 naive T cells are obtained from at least about 300 mL of leukopak. In certain embodiments, at least about 4×10 8 naive T cells are obtained from at least about 100 mL of leukopak. In certain embodiments, at least about 5×10 8At least 5 × 10¹ central memory T cells can be obtained from at least about 200 mL of LeucoPac. In certain embodiments, at least about 5 × 10¹ 8 At least 5 × 10¹ central memory T cells can be obtained from at least approximately 300 mL of LeucoPac. In certain embodiments, at least approximately 5 × 10¹¹ central memory T cells can be obtained. 8 At least 100 mL of LeucoPac can be obtained from a single central memory T cell.

[0019] In certain embodiments, a product is obtained containing at least about 1.5 times, about 2.5 times, or about 5 times more leukocytes compared to the number of leukocytes obtained by density gradient centrifugation. In certain embodiments, a product is obtained containing CD3+ cells, CD45+ cells, CD4+ cells, CD4+ naive cells, CD4+ memory cells, CD4+ effector cells, CD8+ cells, CD8+ naive cells, CD8+ effector cells, CD8+ memory cells, memory cells, effector cells, naive cells, Temra cells, or any combination thereof. In certain embodiments, a product is obtained containing at least about 1.5 times, about 2.5 times, or about 5 times more CD45+ cells compared to that obtained by density gradient centrifugation. In certain embodiments, a product is obtained containing at least about 1.5 times, about 2.5 times, or about 5 times more CD3+ cells compared to that obtained by density gradient centrifugation.

[0020] In some embodiments, the system or method further includes the step of removing one or more target particles from a sample. In some embodiments, the target particles are cells. In some embodiments, the cells are red blood cells or platelets. In some embodiments, at least about 95% of the red blood cells are removed from the sample. In some embodiments, at least 95% of the platelets are removed from the sample.

[0021] In certain embodiments, a product is produced containing a mixture of red blood cells and white blood cells in a ratio of less than 2.5:1, 1.5:1, or 0.7:1. In certain embodiments, a product is produced containing a mixture of platelets and white blood cells in a ratio of less than 9:1, 5:1, 3:1, or 1.1:1. In certain embodiments, a product is produced containing at least about 1.5 times, about 2.5 times, or about 5 times more CD4+ cells compared to that obtained by density gradient centrifugation. In certain embodiments, a product is produced containing at least about 1.5 times, about 2.5 times, or about 5 times more CD8+ cells compared to that obtained by density gradient centrifugation. In certain embodiments, a product is produced containing at least about 1.5 times, about 2.5 times, or about 5 times more naive CD4+ cells compared to that obtained by density gradient centrifugation. In certain embodiments, a product containing at least approximately 1.5 times, approximately 2.5 times, or approximately 5 times more memory CD4+ cells is produced compared to that obtained by using density gradient centrifugation. In certain embodiments, a product containing at least approximately 1.5 times, approximately 2.5 times, or approximately 5 times more effector CD4+ cells is produced compared to that obtained by using density gradient centrifugation. In certain embodiments, a product containing at least approximately 1.5 times, approximately 2.5 times, or approximately 5 times more naive CD8+ cells is produced compared to that obtained by using density gradient centrifugation. In certain embodiments, a product containing at least approximately 1.5 times, approximately 2.5 times, or approximately 5 times more memory CD8+ cells is produced compared to that obtained by using density gradient centrifugation. In certain embodiments, a product containing at least approximately 1.5 times, approximately 2.5 times, or approximately 5 times more effector CD8+ cells is produced compared to that obtained by using density gradient centrifugation. In certain embodiments, a product is produced containing at least about 1.5 times, about 2.5 times, or about 5 times more leukocytes compared to the number of leukocytes obtained by using density gradient centrifugation.In certain embodiments, a product is produced containing at least about 1.5 times, about 2.5 times, or about 3 times fewer red blood cells than the product obtained by density gradient centrifugation. In certain embodiments, a product is produced containing at least about 3 times, or about 10 times, fewer platelets than the product obtained by density gradient centrifugation.

[0022] In some embodiments, the methods and systems disclosed herein produce a population of cells exhibiting an increase in one or more biological properties compared to a population of cells produced by density gradient centrifugation. The one or more biological properties are selected from a list consisting of: the ability to readily accept lentiviral vectors, the ability to grow in culture, the ability to retain T cell memory compositions during cell culture, receptivity to viral transduction, differentiation of mean telomere length, the ability to retain a relative population of poorly differentiated naive and central memory cells, functional killing ability, IFNγ expression, GM-CSF expression, TNF-α expression, and viability. In some embodiments, the cell population exhibits at least about a 25% increase in the ability to readily accept lentiviral vectors compared to a population of cells produced by density gradient centrifugation. In some embodiments, the cell population exhibits an increased ability to grow in culture, and the cells grow before or after genetic modification compared to a population of cells produced by density gradient centrifugation. In some embodiments, the cell population exhibits at least about a 1.5-fold increased ability in culture compared to a population of cells produced by density gradient centrifugation. In some embodiments, the cell population exhibits an increased ability to retain the T cell memory composition during cell culture compared to a cell population produced by density gradient centrifugation, including retaining the same T cell memory composition at least 9 days after culturing. In some embodiments, the cell population exhibits at least a 30% increase in receptivity to viral transduction compared to a cell population produced by density gradient centrifugation. In some embodiments, the cell population exhibits at least a 30% increase in telomere length compared to a cell population produced by density gradient centrifugation.

[0023] In some embodiments, the cell population exhibits an increased ability to retain a relative population of poorly differentiated naive and central memory cells during cell culture, compared to a cell population produced by density gradient centrifugation, and retains approximately the same relative population of poorly differentiated naive and central memory cells after at least 9 days of culture. In some embodiments, the cell population shows at least a 30% increase in functional killing capacity compared to a cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a twofold increase in IFNγ expression after 9 days of culture, compared to a cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 1.5-fold increase in GM-CSF expression after 9 days of culture, compared to a cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 10% increase in TNF-α expression after 9 days of culture, compared to a cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 10% increase in viability compared to a cell population produced by density gradient centrifugation.

[0024] In some embodiments, the methods and systems disclosed herein produce a population of cells that exhibit a reduction in one or more biological properties compared to a population of cells produced by density gradient centrifugation. Here, one or more biological properties are selected from a list consisting of the time required to grow in culture and produce a single therapeutic dose equivalent of cells, the time required to express the gene delivered by the vector, the relative population of effector or Temra cells, IL-1Ra expression, IL-6 expression, IL-13 expression, MCP-1 expression, PD1 and Tim3 co-expression, cellular senescence or depletion, tendency to induce cytokine release syndrome, and the culture time required before delivery to a patient. In some embodiments, the population of cells exhibits a reduction of at least 3 days in the time required to grow in culture and produce a single therapeutic dose equivalent of cells compared to a population of cells produced by density gradient centrifugation. In some embodiments, the population of cells exhibits a reduction of at least 1 day in the time required to express the gene delivered by the vector compared to a population of cells produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 10% decrease in the relative population of effector or Temra cells compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 40% decrease in IL-1Ra expression after 13 days of culture compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 60% decrease in IL-6 expression after 13 days of culture compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 10% decrease in IL-13 expression compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 20% decrease in MCP-1 expression compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 50% decrease in PD1 and Timp3 co-expression compared to the cell population produced by density gradient centrifugation.In some embodiments, the cell population shows at least a 50% reduction in cellular senescence or depletion compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 20% reduction in the tendency to induce cytokine release syndrome compared to the cell population produced by density gradient centrifugation. In some embodiments, the cell population shows at least a 3-day reduction in the culture time required before delivery to a patient. In some embodiments, compared to the cell population produced by density gradient centrifugation, the indicated increase or decrease in one or more biological properties becomes apparent at least about 0, 3, 6, 9, 13, or 16 days after production.

[0025] (Integration by reference) All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually incorporated by reference. Furthermore, U.S. applications 62 / 954478, 62 / 875942, PCT / US2020 / 066812, and PCT / US2020 / 042634 are each incorporated by reference as a whole. To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained herein, this Specified is intended to substitute and / or supersede any such conflicting material. [Brief explanation of the drawing]

[0026] Novel features of the present invention are described in particular with the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings (hereinafter referred to as “Figure” and “Fig.”) which describe exemplary embodiments in which the principles of the present invention are utilized.

[0027] [Figure 1] A schematic diagram illustrates a system for processing an inlet solution and generating an outlet solution enriched with one or more target particles, according to several embodiments. [Figure 2A] The rear perspective view of a particle separation unit according to several embodiments is shown. [Figure 2B] The image shows a perspective view of the front of a particle separation unit with a door in a closed position, according to several embodiments. [Figure 2C] The following is a perspective view of the front of a particle separation unit with a door in the open position, according to several embodiments. [Figure 2D] The upper perspective view of a particle separation unit according to several embodiments is shown. [Figure 2E] A perspective view of the side of a particle separation unit according to several embodiments is shown. [Figure 2F] Front and side views of particle separation units equipped with primary and secondary displays according to several embodiments are shown. [Figure 3A] A cassette with several components, according to several embodiments, is shown. [Figure 3B] A cassette with several components, according to several embodiments, is shown. [Figure 3C] A cassette with several components, according to several embodiments, is shown. [Figure 3D] Several embodiments of product bags and waste bags are shown. [Figure 3E] The following are perspective views of the top of the cassette according to several embodiments. [Figure 3F] A perspective view of the bottom of a cassette, including multiple valve gaskets and two pressure sensor gaskets according to several embodiments, is shown, which fluidly connect the cassette to a particle separation unit. [Figure 3G] Examples of color-coded connections to cassettes of samples, diluents, buffers, priming solutions, products, and waste bags, according to several embodiments, are shown. [Figure 4A] Perspective views of microfluidic cartridges according to several embodiments are shown. [Figure 4B]Perspective views of microfluidic cartridges according to several embodiments are shown. [Figure 4C] Perspective views of microfluidic cartridges according to several embodiments are shown. [Figure 5A] Several embodiments of the system's graphical user interface are shown. [Figure 5B] Several embodiments of the system's graphical user interface are shown. [Figure 5C] Several embodiments of the system's graphical user interface are shown. [Figure 5D] Several embodiments of the system's graphical user interface are shown. [Figure 5E] Several embodiments of the system's graphical user interface are shown. [Figure 5F] Several embodiments of the system's graphical user interface are shown. [Figure 5G] Several embodiments of the system's graphical user interface are shown. [Figure 5H] Several embodiments of the system's graphical user interface are shown. [Figure 5I] The following are exemplary data reports generated by the system according to several embodiments. [Figure 5J] The following are exemplary data reports generated by the system according to several embodiments. [Figure 5K] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5L] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5M] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5N]This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5O] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5P] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5Q] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5R] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 5S] This document illustrates a user interaction with a graphical user interface according to several embodiments. [Figure 6] This section describes sample processing that can be performed using the system according to several embodiments. [Figure 7] The following are exemplary digital processing devices programmed, or otherwise configured, to control various aspects of target particle separation according to several embodiments. [Figure 8] Compared to Ficol, this study demonstrates improved recovery of CD3+ T cells from leucopack enriched according to the system and methods described here. [Figure 9A] Compared to Ficol, this system and method demonstrates improved recovery of specific favorable cell subsets from leucopack enriched according to the methods described here. It shows greater recovery of central memory and naive T cells. [Figure 9B] Compared to Ficol, this system and method demonstrates improved recovery of specific favorable cell subsets from leucopack enriched according to the methods described here. It shows greater recovery of CD4+ T cell subsets. [Figure 10]Cells enriched according to the system and methods described here exhibit longer telomere lengths and greater proliferative capacity compared to Ficol cells. Analysis was performed using qPCR analysis for absolute telomere length (aTL). [Figure 11] This study demonstrates that cells enriched according to the system and methods described herein have fewer platelets (A) and red blood cells. [Figure 12] This study demonstrates that cells enriched according to the system and methods described here produce more cells after culture compared to Ficol. After treatment, T cells were activated and selected using magnetic CD3 / 28 beads. The cells were seeded at a density of 1 × 10⁶ / mL in TexMACS medium supplemented with 5 ng / mL each of IL-7 and IL-15. [Figure 13] Compared to Ficol, this system and method demonstrates improved recovery of all leukocytes (WBCs) / CD45+ cells from leucopack enriched according to this study. [Figure 14] The system and method described here demonstrate that, at the initiation of cell therapy, a greater number of CD4 poorly differentiated naive and central memory cell subsets can be recovered from LeucoPak, produced in zero days compared to Ficol. [Figure 15] Compared to Ficol, this study shows improved recovery of all leukocytes (WBCs) / CD45+ cells and CD3+ T cells from patient leucopacks enriched according to the system and methods described here. [Figure 16] Compared to Ficol, cells enriched according to the system and methods described here from cancer patient LeucoPacs have fewer platelets and red blood cells. [Figure 17] Compared to Ficol preparations, cells enriched according to the system and method described herein are shown to be more readily introduced with lentiviruses. [Figure 18]Compared to Ficol preparations, cells enriched according to the system and methods described here more readily accept lentiviruses and express reporter genes at an earlier time. [Figure 19] Compared to Ficol preparations, cells enriched according to the system and method described herein are more receptive to viral transduction. [Figure 20] Compared to Ficol preparations, cells enriched, lentivirally transduced, and proliferated according to the system and methods described herein produce a higher dose equivalent of therapeutic leukocytes at an earlier time point. [Figure 21] Compared to Ficol preparations, the population of cells enriched and treated with high-transition lentivirus according to the system and methods described herein has fewer terminally differentiated cells. [Figure 22] Compared to Ficol preparations, the system and method described here demonstrate that they recover a larger subset of poorly differentiated naive and central memory cells from a normal donor leucopack, resulting in fewer terminally differentiated cells. [Figure 23] Compared to the Ficol preparation, we demonstrate that a viable CD3+ memory cell population enriched and treated with high-transition lentivirus according to the system and methods described herein retains a relative population of T memory cells. [Figure 24] Compared to the Ficol preparation, the cell population enriched according to this system and method shows a 2-fold reduction in senescence and depletion, significantly lower PD1 / Tim3 co-expression at day 13 of culture, and fewer cells entering the TEMRA state (CD45RA-expressing T effector memory cells). [Figure 25] Compared to Ficol preparations, cell populations enriched according to the system and methods described herein are shown to have normal or increased killing capacity. [Figure 26]Compared to Ficol preparations, cell populations enriched according to the system and methods described herein exhibit better cytokine expression during proliferation and a better safety profile. [Figure 27] Compared to Ficol preparations, cell populations enriched according to the system and methods described herein exhibit better cytokine expression and a better safety profile during proliferation, as demonstrated using cytokine release with a CD19CAR-T structure (+ / - functional CD28 signaling domain). [Figure 28] Compared to Ficol preparations, cell populations enriched according to the system and methods described herein exhibit better cytokine expression during proliferation and a better safety profile, as demonstrated using cytokine release with TCR-T structure and lentiviral-GFP control. [Figure 29] Compared to Ficol preparations, the various advantages of cell populations enriched according to the system and methods described herein are summarized. [Modes for carrying out the invention]

[0028] The present invention relates primarily to systems and methods for separating target particles. The text herein describes and relates to the manufacture and use of systems and methods for performing separation of biological materials.

[0029] While various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Many variations, modifications, and substitutions can be conceived by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be adopted.

[0030] Whenever the terms “at least,” “greater than,” or “greater than or equal to” precede the first number in a set of two or more numbers, the terms “at least,” “greater than,” or “greater than or equal to” apply to each number in that set. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0031] Whenever the terms “no more than,” “less than,” or “less than or equal to” precede the first number in a set of two or more numbers, the terms “no more than,” “less than,” or “less than or equal to” apply to each number in that set. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0032] (definition) Apheresis: As used herein, this term refers to the procedure by which blood from a patient or donor is separated into its components, such as leukocytes, platelets, and erythrocytes. “Apheresis sample” is the final product of this procedure. More specific terms are “platelet apheresis” (referring to the separation of platelets) and “leukocyte apheresis” (referring to the separation of leukocytes). In this context, the term “separation” refers to the acquisition of a product enriched with specific components compared to whole blood or other starting material, and does not imply that absolute purity is achieved.

[0033] Sample: The term “sample” as used herein generally refers to any sample that contains or is suspected of containing nucleic acid molecules or nucleic acid cells. For example, a sample may be a biological sample containing one or more nucleic acid molecules or nucleic acid cells. A biological sample may be obtained (e.g., extracted or isolated) from or contain blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal discharge, sputum, feces, and tears. A sample may also contain blood, blood-related products (such as leukocyte apheresis products or apheresis products), and anticoagulants (e.g., EDTA, EGTA, heparin, citrate, ACD-A, or thrombin inhibitors). A sample may be a human-derived sample. A biological sample may be a fluid sample or a tissue sample (e.g., a skin sample). In some examples, a sample may be obtained from a cell-free fluid such as plasma. In some examples, a sample may contain circulating tumor cells. In some examples, the samples are environmental samples (e.g., soil, waste, outside air), industrial samples (e.g., samples from any industrial process), and food samples (e.g., dairy products, vegetable products, and meat products). The samples may be processed before being placed in the microfluidic device. The samples may, appropriately, be apheresis products or leukocyte apheresis products [e.g., leukopak].

[0034] Target Cells: As used herein, “target cells” refers to cells required by the various procedures described herein, or cells designed to be purified, harvested, manipulated, etc. What constitutes a specific cell varies depending on the context in which the term is used. For example, if the objective of a procedure is to isolate a particular type of stem cell, then that cell is the target cell for that procedure.

[0035] Isolation or Purification: Unless otherwise specified, these terms are synonymous as used herein and refer to the enrichment of an unwanted material with a desired product. This term does not necessarily mean that the product is completely isolated or completely pure. For example, if a starting sample has target cells that make up 2% of the cells in the sample, and the procedure is performed to produce a composition in which 60% of the present cells are target cells, then the procedure has succeeded in isolating or purifying the target cells.

[0036] Obstacle Array: As used herein, this term describes an array of obstacles arranged in a flow channel (channel) through which a fluid carrying cells or particles can pass. An obstacle array comprises a number of obstacles arranged in a column (along the channel of fluid flow). Gaps are formed between the obstacles (along the channel of fluid flow) that allow the passage of cells or other particles. Such arrays or columns are arranged in a repeating manner of one or more transverse rows (perpendicular to the channel of fluid flow).

[0037] As used herein, the term “Deterministic Lateral Displacement” or “DLD” refers to a process in which particles in a channel through an array are deterministically deflected based on their size in relation to some array parameters. This process can be used to separate cells, which is generally the context discussed herein. However, it is important to recognize that DLD can also be used to enrich cells and for buffer exchange. The process is described herein in general terms from the perspective of a continuous flow (DC) state (i.e., bulk fluid flow in only one direction). However, DLD can also function under oscillatory flow (AC) states (i.e., bulk fluid flow alternating between two directions).

[0038] Critical Size: The "critical size," "critical diameter," or "predetermined size" of particles passing through an obstacle array represents the limit of particle size that can follow the laminar flow of a fluid. Particles larger than the critical size may be "pushed" out of the fluid flow path, while particles smaller than the critical size (or predetermined size) are not displaced.

[0039] Fluid Flow: As used herein in relation to microfluidic cartridges, the terms “fluid flow” and “bulk fluid flow” refer to the macroscopic movement of fluid in the overall direction across an obstacle array. These terms do not consider the transient displacement of the fluid stream as the fluid moves around the obstacles in order for the fluid to continue moving in the overall direction.

[0040] In an obstacle array device, the tilt angle ε is the angle between the direction of the bulk fluid flow and the direction determined by the alignment of a series of obstacles in the array.

[0041] Array Direction: In an obstacle array device, the "array direction" is the direction determined by the alignment of a series of obstacles in the array. When a particle passes through a gap and encounters a downstream obstacle, it is "deflected" in the obstacle array (i.e., moves at an angle ε relative to the bulk fluid flow) if the overall trajectory of the particle follows the array direction of the obstacle array. The particle is not displaced if its overall trajectory follows the direction of the bulk fluid flow under such circumstances.

[0042] Approximately: As used herein, the term approximately refers to numbers that are ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% of that number.

[0043] Real-time: As used herein, “real-time” generally refers to a response time in which a graphical element is pressed by the user through a user interface and does not appear to the user as a substantial delay. In some embodiments, the response time can be associated with data processing by, for example, a computer processor, and may be as small as 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, 1 / 10 second, 1 / 100 second, 1 millisecond, or less. Real-time can also refer to the simultaneous or substantially simultaneous occurrence of a first event with the occurrence of a second event. The terms “real-time” or “real-time” generally refer to an event (e.g., an action, process, method, technique, computer calculation, computation, analysis, visualization, optimization, etc.) performed using recently obtained (e.g., collected or received) data. In some cases, a real-time event may occur almost immediately or within a sufficiently short time range, for example, within the range of at least 0.0001 milliseconds (ms), 0.0005 ms, 0.001 ms, 0.005 ms, 0.01 ms, 0.05 ms, 0.1 ms, 0.5 ms, 1 ms, 5 ms, 0.01 seconds, 0.05 seconds, 0.1 seconds, 0.5 seconds, 1 second or longer. In some cases, a real-time event may occur almost immediately or within a sufficiently short time range, for example, within the range of about 1 second, 0.5 seconds, 0.1 seconds, 0.05 seconds, 0.01 seconds, 5 ms, 1 ms, 0.5 ms, 0.1 ms, 0.05 ms, 0.01 ms, 0.005 ms, 0.001 ms, 0.0005 ms, 0.0001 ms or shorter.

[0044] Input: As used herein, the terms “input” or “inlet” are interchangeable herein and generally refer to the solution flowing into the cassette. For example, a sample solution. Such solutions may include a fluid or buffer containing cells, or a fluid or buffer not containing cells. These cells may be human cells.

[0045] Output: As used herein, the terms “output” or “outlet” are interchangeable herein and generally refer to the inflow solution from the cassette. For example, the product containing isolated or enriched cells and / or “non-target” cells and / or wastewater containing waste components. Isolated or enriched cells and non-target cells may be human cells.

[0046] (Sample processing system) Figure 1 schematically shows a sample processing system 100 for processing an input solution to produce an output solution enriched with one or more target particles. The sample processing system 100 may include a particle separation unit 110, an input unit 120, and an output unit 130.

[0047] The input unit 120 may include one or more containers or bags for storing an input solution that can contain a sample. The sample may contain or be suspected of containing nucleic acid molecules or cells. For example, the sample may be a biological sample containing one or more nucleic acid molecules or cells. Biological samples may be obtained (e.g., extracted or isolated) from blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, feces, or tears, and may include these. The sample may also contain blood, blood-related products (such as leukocyte apheresis products or apheresis products), and may also contain anticoagulants (e.g., EDTA, EGTA, heparin, citrate, ACD-A, or thrombin inhibitors). The sample may be a human-derived sample. The biological sample may be a fluid sample or a tissue sample (e.g., a skin sample). In some examples, the sample is obtained from a cell-free fluid such as plasma. In some examples, the sample may contain circulating tumor cells. In some examples, the samples are environmental samples (e.g., soil, waste, outside air), industrial samples (e.g., samples from any industrial process), and food samples (e.g., dairy products, vegetable products, and meat products). The samples may be processed before being placed in the microfluidic device. The samples may, appropriately, be apheresis products or leukocyte apheresis products [e.g., leukopak].

[0048] The particle separation unit 110 can be configured to process sample solutions of various volumes. For example, the particle separation unit 110 can be configured to process sample solutions of approximately 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or approximately 500 mL or more. The sample may contain blood products or apheresis products such as leukemia products. The particle separation unit 110 can efficiently and effectively separate target cells from the input sample solution. For example, the sample solution can be processed in approximately 120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or approximately 10 minutes or less, producing a product solution enriched with collected target particles. To achieve this, the particle separation unit 110 can operate at various flow rates. For example, the particle separation unit 110 can process the sample solution at flow rates of approximately 200 mL / hour, 250 mL / hour, 300 mL / hour, 350 mL / hour, 400 mL / hour, 450 mL / hour, or approximately 500 mL / hour or more.

[0049] The input unit 120 may also include a container for holding several other types of input solutions. For example, the input unit 120 may include a priming solution. The priming solution may include a low surface tension liquid such as alcohol. For example, the priming solution may include isopropyl alcohol in water at various concentrations. The isopropyl alcohol can be configured in a solution of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or about 99%, or more, with the remaining percentage of the solution being water. The priming solution may further include a surfactant added to the buffer. For example, the priming solution may include a surfactant (e.g., polyethylene glycol sorbitan monolaurate (TWEEN)) in a buffer of physiological saline at 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less. The priming solution may further contain poloxamers added to the buffer. For example, the priming solution may contain poloxamers (e.g., F68, F127, 407, L64, P65, P84, P85, F88, P103, P104, P105, F108, or P123) in the buffer or isotonic solution at a concentration of approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less. The priming solution may be the first input solution that enters the particle separation unit 110 before the separation process takes place. The priming solution may be configured to prime the particle separation unit 110 in preparation for receiving the sample. For example, the priming solution can ensure the removal of all air from the fluid pathways within the particle separation unit 110. The particle separation unit 110 is primed with a priming solution and is ready to accept a sample into the solution within approximately 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or approximately 10 minutes or less.

[0050] The input unit 120 may also include a media solution. The media solution may include a clean buffer solution configured to enter the particle separation unit 110 in parallel with the sample solution. The media solution may include a solution with or without added proteins. For example, the media solution may include physiological saline (e.g., 0.9% NaCl, phosphate buffer-physiological saline, Tris buffer-physiological saline, or Hanks equilibrium salt solution) with or without the presence of proteins such as bovine serum albumin (BSA) or human serum albumin (HSA). The media system solution may include plasmalyte, DMEM, TexMACS, OpTmers, RPMI, MEM, Hams F12, EMEM, F-12K, SteXVivo, Stemline, IMDM, or other commercially available cell culture media. The solution may be appropriately buffered at or near normal physiological pH (e.g., about 7.4).

[0051] The input unit 120 may contain a diluent. The diluent can also be configured to enter the particle separation unit 110 in parallel with the sample solution and the media solution. The diluent can be combined with the sample solution to achieve a desired concentration of the sample solution. The exposure time of the diluent to the sample solution can be short and limited to the time each solution is inside the cassette 112. The diluent may be the same as the sample solution. The diluent may be different. For example, the diluent may contain physiological saline with or without protein, such as BSA or HSA. The diluent may also contain reagents that are exposed to the sample solution and washed during the separation process.

[0052] The particle separation unit 110 may further comprise a cassette 112 and a microfluidic cartridge 114 subunit. The particle separation unit 110 may also comprise a housing for storing the cassette 112. The cassette 112 may be configured to accept any one of the input solutions described above. The cassette 112 may be configured to accept and hold the microfluidic cartridge 114 subunit. The cassette 112 may be configured to accept and hold one or more microfluidic cartridges 114, and such microfluidic cartridges may operate in series or in parallel, or there may be a combination of series and parallel microfluidic cartridges. The microfluidic cartridge 114 may be configured to process the sample solution to produce an outlet solution enriched with one or more target particles of the sample solution.

[0053] The input unit 120, particle separation unit 110, and output unit 130 can all be configured to be fluidically coupled to one another. For example, the input unit 120 may comprise multiple containers containing one or more input solutions as described herein. The particle separation unit 110 may comprise a cassette and one or more microfluidic cartridges configured to perform target particle separation. The cassette may comprise multiple fluid channels extending between the input, outlet, and microfluidic cartridges. The output unit 130 may comprise multiple containers containing one or more outlet solutions as described herein. The sample processing system 100 can be configured such that each of these components is releasable and fluidically coupled to one another. The components of the input unit 120, particle separation unit 110, and output unit 130 can be fluidically coupled to one another, so that the entire sample processing system 100 operates in a closed, end-to-end sterile environment. The closed environment provides the advantage of operating in a sterile environment. For example, manual control and / or contact without user intervention is required for the sample processing system 100 to perform a separation process in which one or more target particles are enriched and a contamination-free product is produced. The product can then be collected in one of the outlet containers that is not exposed to the external non-sterile environment. Furthermore, the sample from the input container can enter the cassette without being exposed to the external non-sterile environment. Moreover, the closed end-to-end environment is such that the sample solution does not need to come into contact with any non-reusable components of the sample processing system 100 to prevent contamination. Non-reusable components may include pumps, sensors, and valves, as described here. Furthermore, during the separation process, there are no intermediate reagents that need to be added externally or removed internally to the closed end-to-end environment to support the sterile in-line continuous processing of the incoming sample.

[0054] The sample processing system 100 can be configured to use multiple sterile coupling mechanisms to maintain the sterility of the sample processing system 100. For example, the coupling mechanism between the input container and the tube connecting the input container to the cassette may include a sterile spike and a sterile spike port. For example, the input container may be a bag having a sterile spike port, and the tube containing the sterile spike may be configured to fluidly connect the bag to the cassette to maintain the sterility of the sample processing system 100.

[0055] The output unit 130 may include one or more containers or bags for storing an output solution that may contain the product. For example, the output unit 130 may contain a product solution, a primary wastewater, and a secondary wastewater. The product solution may contain target particles that are to be separated from the input sample solution. The product solution may further contain a clean buffer from the input medium solution, or the product solution may contain the target particles alone. The primary and secondary wastewater may contain unwanted particles from the input sample solution. For example, the target particles may be separable by the particle separation unit 110 and can leave the particle separation unit 110 in the product solution, while unwanted particles may remain in the input sample solution stream and leave the particle separation unit 110 in the primary and secondary wastewater streams. The product outlet stream may have a higher percentage recovery rate of the target particles compared to the input sample stream. For example, the percentage recovery rate of target particles in the product solution can be approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or approximately 95% of the target particles from the sample input stream. The recovered target particles can be recovered in an intact and viable form in the product solution stream. For example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more of the target cells recovered in the product solution can be made viable after the separation process has occurred. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the target cells recovered in the product solution can be made viable after the separation process has occurred with respect to the input material or sample or input sample.

[0056] In certain embodiments, the enriched target particles may include living cells from blood or a blood-related sample input solution. In certain embodiments, the enriched target cells include leukocytes. In certain embodiments, the enriched target cells include stem cells. In certain embodiments, the enriched target cells include peripheral blood mononuclear cells. In certain embodiments, the peripheral blood mononuclear cells include CD3+ cells. The recovered target particles can be recovered in an intact and viable form in the product solution stream. To achieve this, the fluid content within the particle separation unit 110 can be configured to be subjected to a pressure of about 30 psi to about 60 psi or less. For example, the fluid content may generally be subjected to a pressure of about 5 psi to about 40 psi during the separation process. In further embodiments, the average pressure during particle / cell separation is approximately 10–30 psi, 10–20 psi, 10–15 psi, or 15–20 psi. The fluid pressure can be varied or adjusted depending on the volume and / or flow rate of the fluid moving through the channel. These forces allow for gentle separation of target particles from the sample solution, thereby improving cell viability in the product stream.

[0057] In certain embodiments, unwanted particles in the sample solution may include platelets. In certain embodiments, unwanted particles may also include red blood cells. In certain embodiments, target particles in the sample solution may include white blood cells. The particle separation unit 110 can separate unwanted particles from the product solution at a high percentage. For example, the particle separation unit 110 can separate about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% or more of the unwanted particles in the sample solution from the product solution.

[0058] The particle separation unit 110 can also be configured to recirculate either the input solution after it has passed through the cassette 112 and the microfluidic cartridge 114. Recirculation allows the cassette 112 to concentrate the target cells to a certain volume or concentration. For example, target cells can be recovered and fixed in approximately 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, or approximately 150 mL or more of buffer and target cell saturation. Recirculation can occur in parallel, in series, or a combination of series and parallel with respect to target cell separation, thereby enabling the recovery of product cells in a predetermined volume in clean buffer. When recirculation occurs in parallel, the recirculation process does not affect the overall execution time of target cell separation. Recirculation can be configured to start at a predetermined time in the separation process. Recirculation can also be configured to be initiated based on feedback from the sample processing system 100. For example, the input unit 120 and output unit 130 may be equipped with mass sensors configured to weigh each of the solutions contained in one or more containers or bags of the input unit 120 and output unit 130. For example, the mass sensors can measure the weight of the solution in the product solution bag, and once a threshold weight is reached in the product solution bag, the recirculation process can be initiated.

[0059] The particle separation unit 110 can be fully or partially automated. For example, a user can input desired process settings, and the particle separation unit 110 can perform separation without user intervention, as described here. While the particle separation unit 110 is performing the separation process, it is also possible for the user to manually adjust the process settings.

[0060] In certain embodiments, data from a pressure sensor, bubble sensor, or mass sensor is recorded over a time interval to track and periodically evaluate sensor drift. In some embodiments, sensor data may be tracked over time intervals that may consist of several days, weeks, weeks, months, or months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, or 72 months). In some embodiments, sensor data may be tracked over intervals of 1, 5, 10, 20, 50, 100, 1000, or 2000 over execution protocol instances. In some embodiments, sensor data is evaluated to identify drift once per hour, once per day, once per instance of the execution protocol, or in real time during a course of the execution protocol. In some embodiments, sensor data is evaluated to identify whether the drift is significant by comparing the measured sensor value to an expected sensor value. In some embodiments, the expected sensor value is determined at least partially on factory sensor calibration. In some embodiments, the expected sensor value is determined at least partially on stored sensor data from past time intervals of days, weeks, weeks, months, or months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, or 72 months) or from past execution protocol instances up to 1, 5, 10, 20, 50, 100, 1000, or 2000. In some embodiments, the expected sensor value is determined at least partially on information about the current execution protocol. In some embodiments, the expected sensor value is determined at least partially on a maximum error tolerance (e.g., about 1%, about 5%, about 10%, about 15%, or about 20%). In some embodiments, the maximum error tolerance defines the maximum allowable deviation of the sensor data from the expected value determined using factory sensor calibration and / or information about the current execution protocol. In some embodiments, sensor data is evaluated to identify whether drift is significant by comparing the measured sensor values ​​with expected sensor values.In some embodiments, the expected sensor value may include a range defined by the expected value ± a maximum error tolerance. In some embodiments, if drift outside the expected value range is detected and the user is warned, the execution protocol may be interrupted, an error condition may be set, and / or a service warning may be displayed.

[0061] In certain embodiments, multiple systems or particle separation units may be networked together to provide greater throughput.

[0062] (Particle separation unit) Figures 2A to 2E show several perspective views of the particle separation unit 210 that can be used in the embodiments described herein. Figure 2A shows a rear perspective view of the particle separation unit 210. The particle separation unit 210 can be housed in a housing 215 that can be sized and shaped to hold the particle separation unit 210. The housing 215 can be lightweight, compact, and portable. For example, the housing can house the particle separation unit 210 and can be placed on a desktop / tabletop such as a wet bench. The housing 215 can have height, width, or depth dimensions ranging from approximately 280 mm to approximately 1220 mm. The housing 215 can have dimensions of approximately 470,400 mm 2 ~Approximately 8,930,400 mm 2 It can have a surface area in the range of 21,952,000 mm². 3 ~Approximately 1,815,848,000 mm 3 The internal volume can be in the range of [range]. The particle separation unit 210, together with the housing 215, can have a total weight in the range of approximately 20 kg to approximately 120 kg, approximately 40 to approximately 100 kg, or approximately 50 to approximately 70 kg. In some embodiments, the particle separation unit can weigh less than approximately 100 kg, less than 80 kg, or less than approximately 70 kg.

[0063] The rear of the particle separation unit 210 may include a power switch 220, a power inlet 230, and a network connection unit 250. The power switch 220 can be toggled to turn the particle separation unit on and off. The network connection unit 250 may include Ethernet®, USB-C, and USB mini-B connections. The network connection unit 250 allows the particle separation unit 210 to be connected to a computer network, as discussed herein, so that the computer system can control the processing settings, and the user can monitor the separation process and change the processing settings in real time.

[0064] The rear of the particle separation unit 210 may also be equipped with a vent 240. The vent 240 can be configured to dissipate heat from inside the housing 215 while the particle separation unit is performing the separation process.

[0065] The rear of the particle separation unit 210 can also be configured to have one or more hangers 270 extending laterally from the rear of the housing 215. The hangers 270 can be releasably or permanently coupled to the housing 215 of the particle separation unit 210. The hangers 270 can be configured to receive and hold various inlet / outlet solution bags. For example, the hangers 270 can hold sample solution bags for loading into the particle separation unit 210. Each of the hangers 270 can also be configured to include a mass sensor. The mass sensor can help the system maintain real-time tracking of how much solution remains in the solution bags and monitor and manage the fluid movement throughout the particle separation unit 210 in real time.

[0066] Figure 2B shows a perspective view of the front of a particle separation unit 210 with a door 260 in the closed position. The front of the particle separation unit 210 may include a door 260. The door 260 may further include a microfluidic cartridge housing 280 and a handle 290. The door 260 can be opened and closed, allowing a user to access the interior of the particle separation unit 210.

[0067] Figure 2C shows a front perspective view of the particle separation unit 210 with a door 260 in the open position, and shows the cassette 300 inside the particle separation unit 210. The housing 215 may include a receptive section containing recesses, cavities, or slots. The cassette 300 can be releasably coupled to the particle separation unit 210 in this receptive section. The cassette 300 can be configured to be replaced and / or mounted on the particle separation unit 210 using a quick-release mechanism and / or without the use of tools. The receptive section of the housing 215 may be configured to releasably couple with various different types of cassettes. Other cassettes may be configured for different types of cell / target particle processing. For example, an exemplary cassette may be configured for blood sample processing, while another cassette may be configured for processing silica beads in a sample solution. The housing 215 can be configured to releasably couple with these various cassettes, enabling the particle separation unit 210 to perform various separation processes for desired target particle separation. Furthermore, various microfluidic cartridges can be removably coupled to the cassette, enabling the separation of target particles with various critical sizes.

[0068] Figure 2D shows a top perspective view of the particle separation unit 210. As shown in the figure, all of the hangers 270 are visible in this perspective view. The particle separation unit 210 may have two, three, four, five, six, seven, eight, nine, or more than ten hangers 270. As shown in Figure 2D, the particle separation unit has seven hangers 270. The hangers 270 may include a priming bag hanger 271, a medium bag hanger 272, a diluent bag hanger 273, a sample bag hanger 274, a secondary waste bag hanger 275, a primary waste bag hanger 276, and a product bag hanger 277. These hangers may be configured to hold bags containing solutions that are to be introduced into or discharged from the particle separation unit 210. For example, the sample bag hanger 274 may be configured to receive and hold a sample bag containing a sample solution to be separated within the particle separation unit 210. The solution bag can be removably coupled to the hanger 270.

[0069] Figure 2E shows a perspective view of the side of the particle separation unit 210. The housing 215 of the particle separation unit 210 may have a recess 265 on the side of the particle separation unit 210. The recess may be on the left side, the right side, or both sides of the particle separation unit 210. The recess 265 can be configured to hold tubes that fluidly connect the inlet solution and outlet solution to the particle separation unit 210, as described herein.

[0070] Figure 2F shows a front view and a side view of an exemplary particle separation unit according to several embodiments. The particle separation unit includes a primary touchscreen display 275 and a secondary status display 285.

[0071] (cassette) In exemplary embodiments, Figures 3A–3F show a cassette 300 for sorting target particles. This disclosure assumes a wide range of target particles, including, for example, organic and inorganic particles, natural and synthetic particles, and combinations thereof. The target particles presented herein are exemplary embodiments and are not limited by such embodiments. In various embodiments, the target particles may include any particles having a critical size of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 microns. In some embodiments, the target particles may be fluorescently identifiable. In some embodiments, the target particles may include inorganic particles, such as metal beads and silica beads. For example, the metal beads may include beads containing alumina (e.g., gamma alumina). In some embodiments, the target particles may include silica beads. The target particles may include polymers, such as polystyrene, polyethylene, poly(vinylpyrrolidone), acrylamidopropyl-PEG, and derivatives thereof. For example, the polymer may be Merrifield resin, hydroxymethyl resin, Wang resin, aminomethyl resin, SASRIN resin, TentaGel S AC resin, TentaGel PHB resin, or TentaGel S NI-I2 resin. The target particles may also include carbon nanotubes and fullerenes. These target particles may include particles used in split-pool synthesis. Furthermore, in some embodiments, the target particles may include cellular material. The cellular material may include whole cells, lysed cells, cellular components, extracellular matrix, biological tissue, and parts thereof. In some embodiments, the target particles may also include biomolecules, including proteins, peptides, antibodies, carbohydrates, lipids, nucleic acids, nucleotides, primary metabolites, secondary metabolites, and natural products. The target particles may also include both small molecules, synthetic small molecules, and natural small molecules weighing less than 1000 daltons. In some embodiments, the target particles may include viruses. The cellular material may include cell suspensions.The cells may be any cells of biological origin, including human, animal, fungal, microorganism, insect, and modified cells thereof. The cell material mixture comprises cell material, opaque particles, and aqueous solutions. Examples of solutions include, for example, a media, buffer, and water. The devices of this disclosure may be used to isolate cells that specifically express or produce proteins, carbohydrates, enzymes, peptides, hormones, receptors, as well as cells that produce antibodies, genetically modified cells, and activated cells. In exemplary embodiments, according to Figures 3A to 3F, the cassette 300 can be configured to be operably coupled to the panel of a particle separation unit 210, which comprises a degassing unit 310, a bubble sensor 320, a pressure sensor 330, a valve 340, and a pump 350. The cassette 300 may comprise an inline mixer 360, a pressure sensor membrane 332, a pressure sensor gasket 335, a valve membrane 342, a valve gasket 345, a microfluidic cartridge housing 370 for receiving and holding one or more microfluidic cartridges, and a tube 380. Alternatively, the cassette 300 can be configured to comprise a degassing unit 310, a bubble sensor 320, a pressure sensor 330, a pressure sensor membrane 332, a valve 340, a valve membrane 342, a pump 350, an inline mixer 360, a microfluidic cartridge housing 370, and a tube 380.

[0072] The cassette 300 may be configured for single use, or it may be reusable for multiple uses. In one embodiment of the cassette 300 for sorting target particles, the cassette 300 may comprise a microfluidic cartridge housing 370 configured to receive and hold one or more microfluidic cartridges configured for sorting target particles, as described herein. One or more microfluidic cartridges may be releasably coupled to the cassette 300 at one or more microfluidic cartridge ports 372 of the cassette 300. The cassette may comprise a tube 380 configured to connect the cassette 300 to a sample bag which may contain a sample containing target particles to be separated. The tube 380 may also be configured to connect the cassette 300 to various other sources and may be configured to provide various outlets to the cassette. For example, the tube 380 may comprise an inlet tube including a priming solution tube 381, a medium tube 382, ​​a diluent tube 383, and a sample tube 384. Furthermore, tube 380 may include an outlet tube, which includes a product recirculation tube 385, a product recovery tube 386, and a waste tube 387. Tube 380 can be configured to allow the sample to flow freely through multiple paths within the cassette 300, enabling the separation process to be feasible. Tube 380 may be configured to enter and exit at the bottom of the cassette 300, as shown in Figure 3B. The inlet tube may be configured to enter on the left side of the bottom of the cassette 300, and the outlet tube may be configured to exit on the right side of the bottom of the cassette 300. Tube 380 may extend through the cassette 300, providing multiple paths within the cassette.

[0073] The product recovery tube 386 and waste tube 387 can be configured to fluidly connect the cassette 300 to the product bag 396 and waste bag 398 via the tube connector 392, as shown in Figure 3D. The product recovery tube 386 and waste tube 387 can enable fluid communication between the product bag 396 and waste bag 398, the cassette 300, and the microfluidic device. Similarly, the priming solution tube 381, medium tube 382, ​​diluent tube 383, and sample tube 384 may also be configured to enable fluid communication between the priming solution bag, medium bag, diluent bag, and sample bag, respectively, and the cassette 300 and the microfluidic device. The product recovery tube 386 and waste tube 387 can also be configured to enable fluid communication between the product recovery bag and waste bag, respectively, and the cassette 300 and the microfluidic device. The product recirculation tube 385 may be configured to take the outlet stream from the microfluidic device and recirculate it back to the microfluidic device for further processing.

[0074] The product recirculation tube 385 can be operably connected to at least one recirculation path within the cassette 300. The recirculation path can be configured to be isolated from any other path within the cassette 300, or it can be configured to join or connect to one or more of the other paths within the cassette 300. The recirculation path can be configured to extend from the inlet to the outlet of the cassette 300 and can be configured to manipulate and recirculate target particles in a clockwise or counterclockwise direction on the cassette 300. Furthermore, multiple recirculation paths can be configured to transfer samples into and out of one or more microfluidic cartridges. The microfluidic cartridges may be operably coupled to one or more cartridge ports 372 to establish fluid communication with the cassette, so that one or more microfluidic cartridges are configured to separate one or more target particles from the sample, and at least one recirculation path is configured to recirculate one or more target particles through the cassette 300 to concentrate one or more target particles into a predetermined volume of medium. The recirculation pathway can be configured to operate in this manner to achieve a desired concentration or volume of target particles in the product solution. The microfluidic cartridge and at least one recirculation pathway can be configured to operate in parallel, in series, or a combination of series and parallel. Parallel operation allows for optimized and minimized run time for the particle separation unit to complete the target particle separation process.

[0075] The microfluidic cartridge and recirculation pathway can be configured to operate independently of each other. For example, the microfluidic cartridge can be configured to separate target particles from the sample solution without affecting the recirculation process performed in the recirculation pathway. Furthermore, the recirculation process performed in the recirculation pathway can be performed without affecting the separation process performed by the microfluidic cartridge. The microfluidic cartridge and at least one recirculation pathway may also be individually controllable in real time to achieve a desired concentration of one or more target particles in the product.

[0076] Tube 380 may be releasably coupled to the inlet solution bag and the outlet solution bag. For example, the tube 380 and tube connector 392 of the solution bag may include weldable tubing, and the tube 380 and tube connector 392 may be joined together using a sterile tube welding machine.

[0077] The sample bag may be configured to hold a sample containing any of the target particles described herein that are to be separated. The priming tube 381 can connect to the cassette 300 a priming bag that can hold a priming solution. The priming solution may be configured to flow through the cassette before any other samples / solutions are introduced, ensuring that the cassette is ready to receive samples / solutions for processing. For example, the priming solution may be configured to flow through the cassette, ensuring that all air is removed from the fluid path. The medium tube 382 can connect to the cassette 300 a medium bag that can hold a medium solution. The medium solution may contain a clean buffer solution configured to be collected together with the target cells in the product recovery bag. The diluent tube 383 can connect to the cassette a diluent bag that can hold a diluent. The diluent tube may be configured to introduce the diluent into the cassette 300, which affects the concentration at which the target cells are collected in the product recovery bag.

[0078] Product recovery tube 386 can connect a product recovery bag to cassette 300, which can recover a product stream containing target cells from the microfluidic device. The product recovery stream may also contain buffer from the media bag. Waste tube 387 can connect a waste bag to cassette 300, which can recover a waste stream containing unseparated targets from the original sample from the microfluidic device. Product recirculation tube 385 can recirculate target cells to the microfluidic cartridge, as described herein. This allows the cassette to concentrate target cells to a certain volume. For example, target cells can be immobilized to be recovered in approximately 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, or approximately 150 mL or more of buffer and target cell fluid. A certain recovery volume can be achieved by using a dedicated bag designed with a compartment having the desired immobilization volume. For example, cells may enter the product bag on the side with the compartment, and the bag may be configured so that there is only a minimum amount of overflow to the other side of the bag, which is the recirculation side. The fluid on the recirculation side may remain relatively free of cells settling on the fixed-volume compartment side. For example, if the inflow into the bag is constant at 300 mL / hour, the product is a fixed percentage of the total flow rate. The outflow rate may be changed to be equal to the buffer flow rate. The buffer flow rate may be controlled using a mass trigger to determine when to initiate recirculation. For example, if the product bag mass is less than the high mass setpoint (e.g., 30 g), the product is supplied to the product bag at a constant rate. When the product bag reaches the high mass setpoint (i.e., 30 g in this example), the product may be drawn out from the recirculation side until the product bag mass reaches the low mass setpoint (e.g., 25 g). When the low mass setpoint is reached, recirculation (or drawing of product from the bag) stops, and the buffer is returned to the DLD, filling the bag with product until the high mass setpoint is reached.In some examples, the buffer flow rate may be 600 mL / hour, in which case the net flow rate to the bag may be 300 mL / hour or -300 mL / hour (300 inflow and 600 outflow). In some examples, the flow rate is adjusted to oscillate continuously between the upper and lower mass triggers until the separation run is complete, after which the product bag can be filled to the final dilution level, for example, to produce a final mass of 40 g. In some embodiments, the weir bag is configured to overflow at 20-25 g with a lower mass trigger of 25 g, so that a small amount of fluid is always present on the other side of the weir, reducing the risk of air being drawn into the system even when the lower mass trigger is reached. The mass triggers are user-selectable. The weir bag may also be user-selected based on the selected mass trigger to prevent air from entering the system. In some embodiments, recirculation can be performed without a dedicated weir bag, as long as the cell richness in the product bag is maintained at a level of 150 M / mL or less. Recirculation can be performed in parallel, in series, or a combination of parallel and series for target cell isolation, enabling the recovery of product cells in a specified volume within the clean buffer. When recirculation is performed in parallel, the recirculation process does not affect the overall execution time of target cell isolation.

[0079] As shown in Figure 3A, the flexible fluid channel 352 of the cassette 300 can be operably coupled to one or more pumps 350 on the panel of the particle separation unit 210. Alternatively, the pumps 350 can be configured to be part of the cassette 300 itself. The flexible fluid channel 352 may be configured to extend longitudinally on the cassette 300. The flexible fluid channel 352 may include a subset of fluid channels. Each subset of the flexible fluid channel 352 may include two or more fluid channels. The pumps 350 may comprise two or more pump heads configured to be operably coupled to two or more fluid channels within each subset of the flexible fluid channel 352. One or more pumps 350 can be configured to transport and control fluid contents of one or more inlet and outlet streams of the cassette 300 at a certain flow rate. For example, the pumps can transport streams of sample, medium, diluent, waste, product, priming, and recirculation solution throughout the cassette 300. Pump 350 can be configured to transport fluid contents without any moving parts that come into contact with the fluid contents. Pump 350 may include a peristaltic pump configured to peristaltically couple to a flexible fluid channel 352 and control the flow rate of the stream in the cassette 300. Pump 350 can be configured to be operably coupled to the flexible fluid channel 352 of the cassette 300 and to extend longitudinally on the flexible fluid channel 352. Pumps 350 may also be configured to be controllable independently of each other. Once the separation process is performed, pumps 350 can be controlled independently in real time. Pumps 350 can operate in opposite or in-phase relative to each other. For example, pump 350 can operate in reverse phase at approximately 180 degrees, 170 degrees, 160 degrees, 150 degrees, 140 degrees, 130 degrees, 120 degrees, 110 degrees, 100 degrees, 90 degrees, 80 degrees, 70 degrees, 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20 degrees, or approximately 10 degrees or less. Furthermore, pump 350 can be configured to achieve the same flow rate, different flow rates, the same flow direction, or different flow directions, respectively.

[0080] The panel of the particle separation unit 210 may include independent pumps operably coupled to each of the inlet and outlet streams of the cassette 300. For example, independent pumps may be included for each of the sample solution, diluent, medium solution, and priming solution. In addition, independent pumps can be used for each of the outlet streams, for example, the waste stream, product stream, and recirculation stream. These pumps can be individually controllable to adjust the flow rate of each of these streams. In an example where independent pumps are provided for the sample solution stream and the diluent stream, the pumps may also be independently controllable to adjust the dilution ratio of the target particles entering the cassette 300. The pumps controlling the flow rate of the sample solution and the pumps controlling the flow rate of the diluent can be independently controlled in real time to adjust or achieve the desired dilution ratio of the target particles once the target particles enter the cassette 300.

[0081] Furthermore, independent pumps may be included to control the flow rate of the outlet stream. For example, independent pumps can be configured to control the flow rates of the waste stream and the product stream. For example, independent pumps can be provided for each stream to achieve a desired flow rate ratio of the waste stream leaving the particle separation unit to the amount entering the cassette 300 from either of the inlet streams and the product stream leaving the cassette 300. For example, independent pumps can be used to control the flow rate of the waste stream leaving the cassette 300 to influence the ratio of the amount of waste to the amount of product leaving the cassette and / or the amount of sample supplied to the cassette 300. Furthermore, independent pumps can be used to control the flow rate of the sample stream to the flow rate of the diluent stream to influence the ratio of the amount of sample to the amount of diluent entering the cassette 300. Furthermore, independently controllable pumps for the recirculation stream and the sample stream can be used to achieve a desired concentration of target particles in the product solution.

[0082] As described above, the pump 350 may be equipped with a peristaltic pump. The peristaltic pump may be required to have the ability to gently promote fluid flow throughout the entire path of the cassette 300. This can increase the likelihood of recovering viable cells at the end of the separation process. For example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the target cells recovered in the product solution can be made viable after the separation process has been performed.

[0083] The pump 350 can be operably coupled to a flexible fluid channel 352 that extends longitudinally along the cassette 300 and configured to clamp the flexible fluid channel 352. In this way, the pump 350 can be configured to act longitudinally parallel to the flexible fluid channel 352. The pump 350 can also be configured to act transversely perpendicular to the flexible fluid channel 352.

[0084] The pump 350 may comprise a set of pump heads that can be operably coupled to each subset of the multiple flexible fluid channels 352. Each set of pump heads in each pump may comprise two or more pump heads. Two or more pump heads may comprise two or more sets of rollers. Two or more pump heads may be configured to operate in phase with each other. Two or more pump heads may be configured to operate in opposite phase with each other to transport fluid contents through the flexible fluid channels 352 toward or away from the microfluidic cartridge. By operating two or more pump heads in opposite phase with each other in this way, a flow of fluid contents with less pulsation can be obtained than when the pump has a single pump head. Two or more pump heads can be configured to operate in reverse phase at approximately 180 degrees, 170 degrees, 160 degrees, 150 degrees, 140 degrees, 130 degrees, 120 degrees, 110 degrees, 100 degrees, 90 degrees, 80 degrees, 70 degrees, 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20 degrees, or approximately 10 degrees or less.

[0085] Each of the two or more pump heads in each pump 350 can be configured to operably connect to and clamp two or more fluid channels within a subset of the flexible fluid channel 352. The fluid contents in the two or more fluid channels within each subset of the flexible fluid channel 352 can be transported by the two or more pump heads and can merge together into a single fluid path at the outlet of each subset. The two or more pump heads in each pump 350 can be configured to have fixed movements relative to each other. For example, the two or more pump heads in each pump 350 can move toward each other at the same speed and in the same direction. Alternatively, the two or more pump heads in each pump 350 can move toward each other at different speeds and in opposite directions.

[0086] Pump 350 can be configured so that the fluid contents flowing through cassette 300 are subjected to a pressure of approximately 30 psi to 60 psi. For example, the fluid contents may generally be subjected to a pressure of approximately 5 psi to 40 psi during the separation process. The pump can operate at any desired frequency. For example, the pump can operate at approximately 200 revolutions per minute (rpm), 190 rpm, 180 rpm, 170 rpm, 160 rpm, 150 rpm, 140 rpm, 130 rpm, 120 rpm, 110 rpm, 100 rpm, 90 rpm, 80 rpm, 70 rpm, 60 rpm, or approximately 50 rpm or less. The pressure of the fluid contents can be varied or adjusted depending on the volume and / or velocity at which the fluid moves through the channel. Pump 350 can thus slowly move the fluid along the path of cassette 300 before the inlet solutions meet and mix. Pump 350 operating in this manner can also ensure that a minimum amount of particle aggregation occurs within any given stream.

[0087] In addition to the pump 350, the panel of the particle separation unit 210 may also be configured to include one or more valves 340 that can be configured to help control the flow rate of the stream in the cassette 300. For example, a valve 340 can be operably coupled to a valve membrane 342 on the cassette 300. A pressure sensor 330 can be operably coupled to a pressure sensor membrane 332 that converts fluid pressure into a linear force on a load cell. The panel and cassette 300 may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more valves 340 and valve membranes 342 that are operably coupled to one another. The valve membranes 342 may be configured to be located in several places throughout the cassette 300. For example, a stream may contact a valve membrane 342 upstream of one or more bypass channels, immediately after contacting a pressure sensor membrane 332, before contacting a microfluidic device, after contacting a microfluidic device, or immediately after contacting a microfluidic device, before contacting a valve membrane 342. The valve membrane 342 may be located inline and configured to communicate fluidly with several streams of the cassette 300. Each valve 340 can be individually controlled to help achieve a desired flow rate for each of the individual streams. For example, each valve 340 can completely or partially prevent flow through a path in the cassette 300 by completely or partially closing the valve membrane 342 in a certain path. The valve 340 may have a pin or other suitable component that can completely or partially push the closed valve membrane 342 within a path in the cassette 300 to restrict the flow of fluid through the path. For example, a valve membrane 342 may be present in the line for the diluent stream, which can be closed or fully or partially opened by the valve 340, helping to achieve a desired flow rate of the diluent stream entering the cassette 300, thereby helping to achieve a desired dilution ratio of the target particles entering the cassette 300 in the sample solution.The valve membrane 342 for the diluent stream can be configured to be completely or partially closed by, for example, the valve 340, while the valve membrane 342 for the sample solution stream can remain closer to or further away from the closed position to help achieve a desired dilution ratio. The valve 340 can also close or open the valve membrane 342 in percentage values ​​compared to a completely closed or open state. For example, the valve membrane 342 can be open at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more to achieve a desired flow rate for the stream in which the valve membrane 342 is located.

[0088] In addition to the pump 350 and valve 340, the panel of the particle separation unit 210 may also be configured to include one or more pressure sensors 330 which can be configured to help control the flow rate of the stream in the cassette 300. The cassette 300 may be configured to include one or more flexible pressure sensor membranes 332 operably coupled to one or more pressure sensors 330. For example, the particle separation unit 210 and the panel of the cassette 300 may include one, two, three, four, five, six, seven, eight, nine, or ten or more pressure sensors 330 and pressure sensor membranes 332 operably coupled together. The stream may come into contact with the pressure sensor membranes 332 after coming into contact with the flexible pump tube 352 and before coming into contact with the microfluidic device. The pressure sensors 330 can measure the pressure of the fluid contents passing through the path in the cassette 300 based on the force applied to the pressure sensor membranes 332 by the fluid contents as the fluid contents pass through. The pressure sensor 330 may communicate with other devices on the panel of the particle separation unit 210. For example, if the pressure sensor reads a pressure higher than a desired pressure measurement, the pressure sensor may transmit information to one or more valves or one or more pumps, which may adjust to reduce the flow rate of the stream with the high-pressure measurement and lower the pressure.

[0089] The panel of the particle separation unit 210 may be configured to include a degassing unit 310. The degassing unit 310 may be configured to be operably coupled to a gas permeable tube in the cassette 300. The degassing unit 310 may be operably coupled to the gas permeable tube at the top of the cassette 300 and / or in close proximity to the microfluidic cartridge.

[0090] In cassette 300, it may be desirable that any inlet stream to the microfluidic cartridge be free of bubbles or dissolved gases. This may be desirable to ensure efficient and proper processing of the microfluidic cartridge. The degassing unit 310 can be configured to remove dissolved gases via a gas-permeable tube and to prevent bubble formation before the sample is circulated through one or more microfluidic cartridges. This allows cassette 300 to be ready to accept the sample solution and ensures that cassette 300 performs the separation process efficiently and properly throughout.

[0091] In addition to the degassing unit 310, the panel of the particle separation unit 210 may include multiple bubble sensors 320. For example, the panel may have one, two, three, four, five, six, seven, eight, nine, or ten or more bubble sensors 320 configured to detect the presence of bubbles in any of several streams within the cassette 300. The paths within the cassette 300 may be configured to contact the bubble sensors 320 on the panel so that the bubble sensors can detect the presence or absence of bubbles in the paths. The paths can contact the bubble sensors as they enter the cassette 300, before and after contacting the microfluidic device, in front of the pump 350. For example, the bubble sensors 320 may be configured to detect the presence of bubbles in a sample stream before the sample stream enters the microfluidic cartridge. If the bubble sensors 320 detect the presence of bubbles, the system can provide a warning / notification to the user. Non-limiting examples of bubble sensors may include optical, ultrasonic, and capacitive bubble sensors.

[0092] The cassette 300 may be configured to include one or more bypass channels. The cassette 300 can be configured to use valves 340 and pumps 350 to direct the stream to the bypass channels, thereby directing the stream away from one of the sensors and / or microfluidic cartridges of the cassette 300.

[0093] The bubble sensor 320 may be configured to work in cooperation with the bypass channel of the cassette 300. For example, if the bubble sensor 320 detects the presence of bubbles in a sample stream, the bypass channel can be used to direct this sample stream away from the microfluidic cartridge to ensure that the stream does not enter the microfluidic cartridge with the bubbles. As an additional example, the bubble sensor 320 can help redirect other streams that have bubbles detected therein before they come into contact with the sample stream, thereby preventing contamination of the sample stream with bubbles.

[0094] The cassette 300 may be further configured to include an inline mixer 360. The inline mixer 360 may be configured to fluidize any of the streams in the cassette 300. The inline mixer 360 may be configured to come after the pump 350 and before the microfluidic device. The inline mixer 360 may be configured to mix any of the streams in the cassette to deliver a homogeneous mixed solution. For example, the inline mixer 360 may be configured to mix the product recirculation stream with the medium stream to reach a homogeneous mixture of product and buffer in the product recovery stream.

[0095] The inline mixer 360 may have channels extending in parallel with other paths and channels passing through the cassette 300. The inline mixer 360 may be configured to receive multiple different solution streams and may have one or more gates / obstacles that can loosely mix one or more streams together. The inline mixer 360 may also have multiple channels and the gates / obstacles may be configured to facilitate mixing. The components of the inline mixer may be configured so that no moving parts are required to perform the mixing of the fluid channels. This mixing can be performed loosely, which increases the likelihood of recovering viable cells at the end of the separation process. For example, the inline mixer may be configured to mix the incoming sample inline with the diluent on the cassette before the sample circulates through the microfluidic cartridge. The inline mixer 360 may receive the sample solution in a first fluid channel and the diluent in a second fluid channel, and the first and second channels may merge to allow mixing of the solutions. The inline mixer 360 may have two or more channels to provide mixing of two or more solutions.

[0096] (Microfluidic cartridge) Figures 4A to 4C show microfluidic cartridges 410 that can be used in the embodiments described herein. A microfluidic cartridge can be configured for single use, or it can be made reusable for multiple uses. As shown, two microfluidic cartridges 410 can be used stacked parallel to each other in a cassette 300 to perform a separation process. The separation process can also be configured to be performed using one microfluidic cartridge, or three or more microfluidic cartridges, all of which can be stacked to operate in series, parallel, or a combination of series and parallel. As shown, a microfluidic cartridge 410 may have two inlets 420 and two outlets 430. A microfluidic cartridge may also have one inlet and one outlet, or three or more inlets and outlets. The number of inlets 420 may differ from the number of outlets 430. For example, a microfluidic cartridge may have more or fewer inlets 420 than outlets 430.

[0097] As shown in Figures 4B and 4C, the microfluidic cartridge 410 may be equipped with vents 440. The vents 440 can be configured to allow bubbles to escape the stream while maintaining the fluid contents within the channels of the microfluidic cartridge 410 as the bubbles pass through the cartridge 410. For example, the vents 440 may be part of a dual filter system. The two filters may comprise a hydrophilic membrane and a hydrophobic or oleophobic membrane. The hydrophilic membrane can be configured to be closest to the fluid stream passing through the microfluidic cartridge 410, while the hydrophobic or oleophobic membrane can be closest to the surrounding environment surrounding the microfluidic cartridge 410. Each membrane can be of varying thicknesses. Each membrane may be the same thickness, or one membrane may be thicker or thinner than the others. For example, the hydrophilic membrane may be thicker than the hydrophobic membrane. The membranes may be approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, or approximately 350 microns or more in thickness. Either or both membranes can be selectively permeable to allow gases to escape from the system, while cells and fluids are retained within the system and contaminants (e.g., viruses, bacteria) are excluded. These selectively permeable membranes allow potentially harmful gases and bubbles to escape from the system while maintaining its sterility.The pore size of the membrane may be approximately 5.0, 4.0, 3.0, 2.0, 1.0, 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or approximately 0.01 microns or less. The membrane can contain different pore sizes; for example, a hydrophilic membrane may contain larger pore sizes than a hydrophobic membrane. By configuring the vent holes 440 in this way, bubbles can escape from the stream as they pass through the microfluidic cartridge, while maintaining the fluid in the pathway of the microfluidic cartridge 410.

[0098] As described here, the microfluidic cartridge 410 can be releasably coupled to the cassette 300. As shown in Figure 4C, the microfluidic cartridge 410 can be releasably coupled to the cassette 300 via tabs 374 on the cassette. Tabs 374 allow the microfluidic cartridge 410 to be snapped into and out of the cassette 300 without the need for tools.

[0099] The microfluidic cartridge 410 is typically rectangular and may have a planar support that can be fabricated from any material compatible with the separation method, including silicon, glass, hybrid materials, or (preferably) polymers. The support has a top and a bottom, one or both of which have at least one embedded channel extending from one or more sample inlets and one or more separate fluid inlets to one or more product outlets and one or more separate waste outlets. The fluid inlet (opposite the sample inlet) may sometimes be called a “buffer” or “wash” inlet and can be used to transport various fluids into the channel, depending on the purpose of separation. Unless otherwise stated in use or context, it will be understood that “fluid” may be a buffer, may contain reagents, may constitute a growth medium for cells, or may be any liquid in general, and may contain any components compatible with the operation of the device and the user’s purpose.

[0100] When a fluid is applied to the device through the sample inlet or fluid inlet, it passes through the channel toward the outlet, thereby defining the bulk fluid flow direction. To separate cells or particles of different sizes, the channel includes an array of obstacles organized into one or more columns extending longitudinally along the channel (from inlet to outlet) and rows extending laterally across the channel. Each subsequent obstacle is shifted laterally relative to the preceding row, thereby defining an array direction that deviates from the bulk fluid flow direction by an angle of inclination (ε). The obstacles are positioned to define a critical size, so when a sample is applied to the inlet of the microfluidic cartridge 410 and flows toward the outlet, particles or cells in the sample larger than the critical size follow the array direction, while particles smaller than the critical size flow in the bulk fluid flow direction, thereby achieving separation.

[0101] (Target cells and methods for producing target cells) The systems and devices described herein can be used to isolate, purify, or enrich one or more target cells or populations of target cells from a suitable sample. Target cells can be isolated based on critical size or critical diameter. Enriched cells may have a critical size greater than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 microns.

[0102] By setting an appropriate critical size, the isolation of specific cell types is possible, for example, hematopoietic stem cells, peripheral blood mononuclear cells (PBMCs), leukocytes (WBCs or CD45+ cells), or other immune cells. The enriched cell population may include one or more target cells selected from T cells (CD3+ cells), B cells (CD19+ cells), NK cells (CD56+ cells), or combinations thereof. The enriched T cell population may include CD4+ T cells, CD8+ T cells, or combinations thereof. The T cells may exhibit a naive, central memory, central memory effector or effector, or Temra phenotype. The enriched cell population can be enriched so that the products obtained from the system are approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% pure for a particular cell type. In some cases, the resulting product is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% leukocytes. Specifically, a leukapheresis product or leukopak may be applied to a system and / or device to obtain a population of PBMCs that are at least 90%, 95%, 96%, 97%, 98%, or 99% PBMC cells. This purity can be determined by examining CD45 (pan-leukocyte marker) expression on the resulting cells.

[0103] The systems and devices described herein can also produce particle or leukocyte populations in higher yield compared to the input population. For example, a sample can be applied to the device and a population of particles or cells that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the input particles or cells can be recovered. For example, a leukocyte-depleted transfusion product can be applied to the device and a population of PBMCs that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the PBMC input cells can be recovered. For example, a leukocyte-depleted transfusion product can be applied to the device and a population of T cells that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the T cell input cells can be recovered. One or more recovered cell or particle populations may contain enrichment products. The recovered cell population may have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% viability at the time of recovery.

[0104] Enriched target cells can be retrieved from the device in one or more step-posts, including culture, growth, cryopreservation, or genetic engineering. These processes may be carried out in vitro or ex vivo within a cell culture device, hood, or incubator. For example, cells may be further cultured or grown in vitro. These steps can be used to produce populations of cells expressing chimeric antigen receptors, recombinant T cell receptors, or other therapeutic proteins. For example, these steps can be used to transgenicize cells using exogenous nucleic acids configured to encode chimeric antigen receptors or recombinant T cell receptors. These cells can be genetically engineered by one or more steps, including viral transduction, electroporation, or the use of chemical transfection reagents (e.g., cationic lipids, calcium chloride precipitates). Viral transduction can be carried out using lentiviral vectors, retroviral vectors, or adenovirus vectors. The chimeric antigen receptor may include tisagenlecleucel, axicabtagene ciloleucel, or a combination thereof. The chimeric antigen receptor may also include one or a combination of sequences that can facilitate cell recognition events, such as MART-1, CD444v6, CAIX, CEA, CD133, c-Met, EGFR, EGFRvIII, Epcam, EphA2, FR alpha, GD2, GPC3, GUCY2C, HER1, HER2, ICAM-1, IL13R alpha 2, IL11R alpha, Kras, Kras G12D, L1CAM, MAGE, MET, Mesothelin, MUC1, MUC16 ecto, NKG2D, NY-ESO-1, PSCA, or WT-1.

[0105] The method described herein is for producing a product containing cells or particles by passing a sample through a system or device. In some cases, the system is a microfluidic system. In some embodiments, the microfluidic system comprises a cassette or cartridge for separating one or more particles from the system or device. In some cases, the system comprises a cassette to which one or more microfluidic cartridges are releasably coupled and supported. In some cases, the cassette is primed by passing a priming solution through the system. The method may also involve processing the sample by passing the sample through the system and using one or more microfluidic cartridges to separate one or more target particles from the system. The product containing one or more target particles separated from the system or device can be recovered. In some cases, the sample has a volume of at least about 40 mL. In some cases, the product is enriched with a recovery of at least about 70% of one or more target particles. In some cases, the method is achieved in a closed-system sterile setting that is continuously in-line. In some embodiments, the method is performed in less than one hour.

[0106] Described herein is a method for obtaining enriched cell products or populations of cells, comprising flowing or processing a blood-related sample through a microfluidic device to produce enriched cell products containing at least 90% of WBCs from the sample and less than 5% of red blood cells and less than 5% of platelets from the sample. In some embodiments, the method processes the blood-related sample at a flow rate of 300 mL / hour. In some embodiments, the method processes 30 × 10⁻⁶ 9 It has the highest cell throughput per cell / hour.

[0107] Described herein are methods for obtaining enriched cell products or populations of cells, which involve flowing or processing a blood-related sample through the device or system described herein. In some embodiments, the method includes generating a product comprising target cells, including leukocytes. In some methods, the leukocyte product is at least about 95% pure.

[0108] In some cases, the sample is processed at a rate of at least approximately 10 mL / hour, 30 mL / hour, 100 mL / hour, 150 mL / hour, 200 mL / hour, 300 mL / hour, 400 mL / hour, 500 mL / hour, 600 mL / hour, or 700 mL / hour. In some embodiments, the sample is processed at at least 1 × 10 8 Cells / hour, 10 × 10 8 Cells / hour, 30 x 10 8 Cells / hour, 10 × 10 9 Cells / hour, 30 x 10 9 cells / hour, or 100 × 10⁶ 9 The cells are processed at a cell throughput of cells / hour. In some cases, the product is enriched with particles having a diameter of at least approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 microns. In some cases, the washing efficiency of the method described herein is greater than at least 1 log, 2 logs, 3 logs, 3.3 logs, 4 logs, or 5 logs. In some cases, the method described herein produces a dose equivalent of therapeutic lymphocytes within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the method described herein produces a cell population from a single LeucoPak that can produce a greater dose equivalent of therapeutic lymphocytes than cells produced by ficoll or other methods. In some cases, the methods described here produce a cell population capable of producing at least approximately 2, 2.5, 3, 4, 5, 7, 10, or 20 times more dose-equivalent therapeutic lymphocytes than cells produced by Ficol or other methods. For example, the DLD method has been shown to produce a cell population that produces at least twice the dose-equivalent therapeutic lymphocytes than cells produced by Ficol or other methods. See Figure 20.

[0109] Other methods for operating the device described herein include applying a heterogeneous sample to one or more inlets and obtaining a target cell population enriched with one or more target cells from one or more outlets. In specific examples, this method includes applying a sample containing a heterogeneous cell population in a volume of 100 mL, 200 mL, 300 mL, 400 mL or more, and collecting a population enriched with one or more target cells. For example, at least 100 mL of leukocyte-depleting transfusion product may be applied to one or more inlets of the device, and a cell population larger than approximately 90% PBMCs may be obtained from one or more outlets of the device. In certain embodiments, the sample can be processed in one hour or less.

[0110] In some embodiments, this method can be completed in approximately 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, or less. In some cases, this method uses a cartridge or cassette configured for single or multiple uses.

[0111] In some cases, the sample is a human sample and contains human cells of a specific cell type, such as hematopoietic stem cells, red blood cells, platelets, peripheral blood mononuclear cells (PBMCs), white blood cells (WBC or CD45+ cells), or leukocytes, or other immune cells. In some cases, the sample volume is at least about 20 mL to about 500 mL. In some cases, the sample volume is at least about 20 mL to about 40 mL, about 20 mL to about 100 mL, about 20 mL to about 200 mL, about 20 mL to about 300 mL, about 20 mL to about 400 mL, about 20 mL to about 500 mL, about 40 mL to about 100 mL, about 40 mL to about 200 mL, about 40 mL to about 300 mL, about 40 mL to about 400 mL, about 40 mL to about The ranges are 500 mL, approximately 100 mL to 200 mL, approximately 100 mL to 300 mL, approximately 100 mL to 400 mL, approximately 100 mL to 500 mL, approximately 200 mL to 300 mL, approximately 200 mL to 400 mL, approximately 200 mL to 500 mL, approximately 300 mL to 400 mL, approximately 300 mL to 500 mL, or approximately 400 mL to 500 mL. In some cases, the sample volume is at least approximately 20 mL, approximately 40 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, or approximately 500 mL. In some cases, the sample volume is at least approximately 20 mL, approximately 40 mL, approximately 100 mL, approximately 150 mL, approximately 200 mL, approximately 300 mL, or approximately 400 mL. In some cases, the sample volume is at least about 40 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, or about 500 mL.

[0112] In some cases, the sample can be applied to the device, and enriched target cell products can be obtained by introducing blood-related products to remove target particles from the sample. In some cases, removing target particles involves simultaneously depleting red blood cells and platelets. The sample may also be applied to the device, and enriched target cell products can be obtained by introducing blood-related products to deplete platelets while maintaining red blood cells. Red blood cells can be maintained at red blood cell-to-target cell ratios of approximately 0.2:1, 0.5:1, 0.7:1, 1:1, 2:1, 2.5:1, 3:1, 5:1, 10:1, 25:1, 50:1, 100:1, 150:1, 200:1, 250:1, or 500:1 or smaller. See Figure 11. Platelet cells may be depleted to achieve platelet cell-to-target cell ratios of approximately 0.7:1, 1:1, 2:1, 2.5:1, 3:1, 5:1, 8:1, 9:1, 25:1, 50:1, 100:1, 150:1, 200:1, 250:1, or 500:1 or smaller. See Figure 11. Target cells may be peripheral blood monocular cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells. Such isolation can be achieved by configuring a system that allows enrichment of cells beyond a critical size of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 microns. In some cases, at least approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of red blood cells are removed from the sample. In some cases, at least approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of platelets are removed from the sample. In some cases, the product contains a smaller ratio of red blood cells to white blood cells, platelets to white blood cells, or a combination thereof, compared to the cell population obtained by using density gradient centrifugation.

[0113] The systems and methods described herein enable greater recovery of target cell populations and less waste, including cell populations contaminated with unwanted cell types such as platelets and / or red blood cells. Furthermore, because cells do not aggregate together due to rapid sedimentation, the system promotes potentially beneficial cell-cell interactions, such as lymphocyte-RBC interactions, while reducing potentially harmful interactions, such as platelet-lymphocyte interactions. This system also reduces platelet and leukocyte lysis and red blood cell rosette formation, thereby reducing potentially harmful soluble mediators (e.g., by promoting activation or differentiation) for target cell populations such as leukocytes, B cells, T cells, naive T cells, central memory T cells, and NK cells.

[0114] The systems and methods described herein can recover white blood cells (WBCs) to a purity of 80%, 85%, 90%, 95%, or higher. The systems and methods described herein can deplete platelets to 75%, 80%, 85%, 90%, 95%, or higher. The systems and methods described herein can deplete red blood cells to 75%, 80%, 85%, 90%, 95%, or higher. In certain embodiments, the systems and methods described herein produce cell populations having at least about 1.5, 2, 2.5, 3, or 4, 5, 10, or 20 times fewer red blood cells, platelets, or combinations thereof compared to Ficol or other methods. See Figure 11. Similarly, the methods described herein are superior to Ficol or other methods in producing fewer red blood cells or platelets when the sample is from a cancer patient. See Figure 16.

[0115] [Table 1]

[0116] The methods and systems described herein can produce target cell populations having equal or greater volumes for proliferation in vitro after enrichment. Cells isolated by the methods and systems described herein can yield doubling times and / or larger doubling volumes compared to current systems and methods using density gradient media and centrifugation (e.g., density gradient method, density gradient medium method, density gradient centrifugation, e.g., Ficol or Ficol, e.g., Ficol®, GE Healthcare) for isolation and cell separation.

[0117] In certain embodiments, the systems and methods described herein produce target cell populations with increased doubling / growth capacity compared to density gradient separation methods. Cells can undergo 1, 2, 3, 4, 5, 6, 7, or more doublings in vitro compared to separation by density gradient centrifugation (e.g., Ficol). In certain embodiments, doubling capacity is indicated by telomere length. In certain embodiments, cells enriched by the systems and methods described herein have an average telomere length of at least about 4 or at least about 5 kilobases. In certain embodiments, cells enriched by the systems and methods described herein have an average telomere length of 6 kilobases or more. In certain embodiments, cells enriched by the systems and methods described herein have an average telomere length of 7 kilobases or more. See 10.

[0118] Larger doubling and / or reduced doubling times allow for the production of target cell populations at an earlier time compared to other methods, which is useful for downstream therapeutic applications. In certain embodiments, the target cell population is at least approximately 2 × 10⁶ immediately after separation (e.g., day 0) from approximately 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL of whole blood samples, apheresis products, or a single LeucoPac. 9 , about 3×10 9 , about 4×10 9 , about 5×10 9 , about 9×10 9 , about 1×1010 , about 5×10 7 , or approximately 1 × 10 8 A total cell count of approximately 1 × 10¹⁶ cells can be achieved within approximately 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or less, from approximately 100 mL, 200 mL, 300 mL, or 400 mL of whole blood sample, apheresis product, or a single LeucoPac. 9 , about 5×10 9 , about 1×10 10 , about 2×10 10 , about 5×10 7 , or 1 × 10 8 The total number of cells can be achieved. See Figure 12. In certain embodiments, the target cells are peripheral blood mononuclear cells. In certain embodiments, the target cells are T cells. In some cases, the T cells are naive T cells, inactivated T cells (CD25-), or central memory T cells derived from leucopac or other apheresis products.

[0119] The methods and systems described herein can produce target cell populations that have a greater capacity to be transduced by the virus after enrichment. Cells isolated by the methods and systems described herein can result in greater efficiency of viral transduction compared to current systems and methods that use density gradient media (e.g., Ficol) for isolation.

[0120] In certain embodiments, the systems and methods described herein produce cell populations with greater efficiency of viral transduction compared to density gradient isolation. Cells can be transduced to have a transduction efficiency of at least 60%, 70%, 80%, or 90% or higher after isolation.

[0121] In certain embodiments, the systems and methods described herein produce a cell population having at least about 1.5 times, 2 times, 2.5 times, 3 times, or 4 times, 5 times, or 6 times more leukocytes compared to Ficol or other methods. In some cases, the leukocytes include CD3+ cells, CD45+ cells, CD4+ cells, CD4+ naive cells, CD4+ memory cells, CD4+ effector cells, CD8+ cells, CD8+ naive cells, CD8+ effector cells, CD8+ memory cells, memory cells, effector cells, naive cells, Temra cells, or any combination thereof. In certain embodiments, the systems and methods described herein produce a cell population having at least about 1.5 times, 2 times, 2.5 times, 3 times, or 4 times, 5 times, or 6 times more CD3+ T cells compared to Ficol or other methods. See Figure 8. In certain embodiments, the systems and methods described herein produce cell populations having at least 1.5, 2, 2.5, 3, or 4, 5, or 6 times more CD45+ T cells compared to Ficol or other methods. For example, the use of the DLD method has been shown to produce approximately 2.5 times more CD45+ cells or leukocytes than the Ficol method. See Figure 13.

[0122] In certain embodiments, the systems and methods described herein produce cell populations having at least two, three, or four times more central memory T lymphocytes (CD45RO+CD62L+ or CD45RO+CCR7+) compared to Ficol or other methods. See Table 2 and Figure 9A.

[0123] In certain embodiments, the systems and methods described herein produce cell populations having at least two, three, or four times more naive T lymphocytes (CD25RA+ or CD25-) compared to Ficol or other methods. See Table 2.

[0124] In certain embodiments, the systems and methods described herein produce cell populations having at least about 1.5 times, 2 times, 2.5 times, 3 times, or 4 times, or 5 times or more, CD4+, CD8+T, or combinations thereof, compared to Ficol or other methods. See Table 2 and Figure 9B. In some cases, the CD4+ or CD8+T lymphocytes are subtypes of inactive, naive, central memory or memory, effector, or Temra. In some further embodiments, the leukocytes or lymphocytes are derived or obtained from samples taken from healthy patients. In other examples, they are taken from cancer patients. In some cases, the cancer patients have breast cancer, lymphoma, leukemia, Hodgkin lymphoma, colorectal cancer, skin cancer, or other cancers. In some cases, the samples taken from cancer patients have a reduced leukocyte count. In some embodiments, the methods described herein generate cell populations from cancer patients with reduced WBC counts that have at least 1.5, 2, 2.5, 3, or 4, 5, or 10 times more CD3+, CD4+, CD8+, CD45+, or combinations thereof lymphocytes or leukocytes compared to Ficol or other methods. For example, the DLD method has been shown to produce more than 5 times more CD45+ cells and more than 2.5 times more CD3+ cells than Ficol or other methods when using the same volume from the same cancer patient with reduced WBC counts. See Figure 15.

[0125] [Table 2]

[0126] In certain embodiments, the systems and methods described herein recover high percentages of naive, central memory, or CD4+ T cells. The recovery of naive, central memory, or CD4+ T cells is increased compared to other systems, such as density gradient separation. In some embodiments, the methods described herein produce at least approximately 2, 2.5, 3, 4, 5, or 10 times more naive or fewer differentiated cells than those produced by other systems, such as concentration gradient separation. For example, the DLD system and methods have been shown to produce at least approximately 3 times more naive and central memory T cells compared to the Ficol method. See Table 2. This includes cases where the sample is taken from a healthy donor and the method is performed on the same day. In certain embodiments, the systems and methods described herein recover at least approximately 50%, 60%, 70%, 80%, 95%, 97%, 98%, or 99% of naive T cells compared to, for example, approximately 100 mL, 200 mL, 300 mL, or 400 mL of whole blood samples, apheresis products, or samples not enriched from LeucoPac. In some examples, the methods described herein produce more naive and central memory T cells for effector and Tera cells than cells produced using other methods such as Ficol. For example, the DLD method has been shown to produce slightly more naive and central memory T cells for effector and Tera cells than cells produced using other methods such as Ficol. See Figure 14. This is also true when using samples taken from healthy donors. See Figure 22.

[0127] In certain embodiments, the method described herein produces a population of cells exhibiting an increase in one or more biological properties compared to a population of cells produced by density gradient centrifugation. In certain embodiments, the method described herein produces a population of cells exhibiting a decrease in one or more biological properties compared to a population of cells produced by density gradient centrifugation. Biological characteristics include the ability to readily introduce lentiviral vectors, the ability to grow in culture, the ability to retain T cell memory compositions during cell culture, receptivity to viral transduction, differentiation of mean telomere length, the ability to retain relative populations of poorly differentiated naive and central memory cells, cytotoxic killing ability, IFNγ expression / secretion, GM-CSF expression / secretion, TNF-α expression / secretion, viability, time required to grow in culture and produce a single therapeutic dose equivalent of cells, time required to express the genes delivered by the vector, relative population of effector or Temra cells, IL-IRa expression / secretion, IL-6 expression / secretion, IL-13 expression / secretion, MCP-1 expression / secretion, PD1 and / or Tim3 expression, cellular senescence or depletion, the possibility of causing cytokine release syndrome, and the culture time required before delivery to the patient. For example, it is shown here that the DLD method produces a cell population with a favorable cytokine expression profile and a reduced possibility of causing cytokine release syndrome compared to cells produced using the Ficol method. See Figures 26-28. In general, DLD methods and the methods described herein exhibit many favorable increases or decreases in biological properties regarding the production, delivery, efficacy, and safety of therapeutic lymphocytes. See Figure 29.

[0128] In some cases, the method described herein produces a population of cells that exhibits an increased ability to grow in culture, and these cells proliferate before or after genetic engineering compared to a population of cells produced by density gradient centrifugation. In other cases, the population of cells shows at least a 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in the ability to grow in culture, and these cells proliferate before or after genetic engineering compared to a population of cells produced by density gradient centrifugation.

[0129] In some cases, the method described herein produces a population of cells that exhibits increased ability to readily accept lentiviral vectors (i.e., at least a portion of the lentiviral nucleic acid is inserted into the cell's genome or exosome) compared to a population of cells produced by density gradient centrifugation. In other cases, the cell population exhibits an increased ability to readily accept lentiviral vectors by at least approximately 5%, 10%, 15%, 25%, 30%, 50%, 100%, 200%, 300%, 400%, or 500% compared to a population of cells produced by density gradient centrifugation. In some examples, the method described herein shows that it produces a population of cells with a 30% increased ability to readily accept lentiviruses. See Figure 17.

[0130] In some cases, the method described herein produces a population of cells that exhibits an increased ability to retain the T cell memory composition during cell culture compared to the population of cells produced by density gradient centrifugation. In some cases, the cell population retains its relative T cell memory composition for at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days longer than the population of cells produced by density gradient centrifugation. For example, DLD has been shown to produce a cell population that retains its relative memory T cell population longer than other methods, including Ficol. See Figure 23.

[0131] In some cases, the method described here produces a population of cells that exhibits increased receptivity to viral transduction compared to a population of cells produced by density gradient centrifugation. In other cases, the cell population shows increased receptivity to viral transduction by at least approximately 5%, 10%, 15%, 25%, 30%, 50%, 100%, 200%, 300%, 400%, or 500% compared to a population of cells produced by density gradient centrifugation. For example, DLD has been shown to produce a population of cells that exhibits approximately 20–40% greater receptivity to viral transduction than cells produced by Ficol or other methods. See Figure 19.

[0132] In some cases, the method described here produces a population of cells that exhibit increased telomere length compared to the population of cells produced by density gradient centrifugation. In some cases, the population of cells exhibits telomere length increased by at least approximately 5%, 10%, 15%, 25%, 30%, 50%, 100%, 200%, 300%, 400%, or 500% compared to the population of cells produced by density gradient centrifugation.

[0133] In some cases, the method described here produces a population of cells that, during cell culture, exhibits an increased ability to retain a relative population of poorly differentiated naive and central memory cells compared to the population of cells produced by density gradient centrifugation. In some cases, the cell population retains a relative population of poorly differentiated naive and central memory cells for at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days longer than the population of cells produced by density gradient centrifugation.

[0134] In some cases, the method described herein produces a population of cells that exhibits increased functional lethality compared to the population of cells produced by density gradient centrifugation. In some cases, the population of cells exhibits increased functional lethality of at least approximately 5%, 10%, 15%, 25%, 30%, 50%, 100%, 200%, 300%, 400%, or 500% compared to the population of cells produced by density gradient centrifugation. For example, the DLD method has been shown to produce cells with at least 30% greater lethality compared to cells produced using the Ficol method, when seeded at two cells per target cell for killing. See Figure 25.

[0135] In some cases, the method described herein produces a population of cells showing increased IFNγ expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows at least approximately 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in IFNγ expression compared to a population of cells produced by density gradient centrifugation. In some cases, the increase in IFNγ expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0136] In some cases, the method described herein produces a population of cells showing increased GM-CSF expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows at least a 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in GM-CSF expression compared to a population of cells produced by density gradient centrifugation. In some cases, the increase in GM-CSF expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0137] In some cases, the method described herein produces a population of cells showing increased TNFα expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows an increase of at least approximately 5%, 10%, 15%, 25%, 30%, 50%, 100%, 200%, 300%, 400%, or 500% in TNFα expression compared to a population of cells produced by density gradient centrifugation. In some cases, the increase in TNFα expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0138] In some cases, the method described here produces a population of cells that exhibits increased viability compared to the population of cells produced by density gradient centrifugation. In some cases, the population of cells shows an increase in viability of at least approximately 5%, 10%, 15%, 25%, 30%, 50%, 100%, 200%, 300%, 400%, or 500% compared to the population of cells produced by density gradient centrifugation.

[0139] In some cases, the method described here produces a population of cells that exhibits an increased ability to readily accept lentiviral vectors compared to the population of cells produced by density gradient centrifugation.

[0140] In some cases, the method described here produces a population of cells that exhibits a reduced time required to grow in culture and produce a single therapeutic dose equivalent of cells, compared to the population of cells produced by density gradient centrifugation. In some cases, the population of cells requires fewer than approximately 1, 2, 3, 4, 5, 6, 7, or 8 days to grow in culture and produce a single therapeutic dose equivalent of cells, compared to the population of cells produced by density gradient centrifugation.

[0141] In some cases, the method described here produces a population of cells that exhibits a reduced time required to express the vector-delivered gene compared to the population of cells produced by density gradient centrifugation. In other cases, the cell population requires fewer than approximately 1, 2, 3, 4, 5, 6, 7, or 8 days to express the vector-delivered gene compared to the population of cells produced by density gradient centrifugation. For example, DLD has been shown to produce a faster vector-delivered gene-expressing cell population than Ficol or other methods. See Figure 18.

[0142] The therapeutic dose equivalent varies depending on the exact type of treatment, but in some cases, it is at least approximately 1 × 10⁻⁶. 7 ,2×10 7 ,3×10 7 ,4×10 7 ,5×10 7 ,1×10 8 ,2×10 8 ,3×10 8 ,4×10 8 ,5×10 8 ,1×10 9 ,2×10 9 ,3×10 9 ,4×10 9 , or 5 × 10 9 The total number of cells may also be used. The therapeutic dose is approximately 1 × 10⁻⁶. 7 ,2×10 7 ,3×10 7 ,4×10 7 ,5×10 7 ,1×10 8 ,2×10 8 ,3×10 8 ,4×10 8 ,5×10 8 ,1×10 9 ,2×10 9 ,3×10 9 ,4×10 9 , or 5 × 10 9 Even individual transfected cells are acceptable.

[0143] The therapeutically relevant amount varies depending on the exact type of treatment, but in some cases is at least about 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , or 5×10 9 total cells. The therapeutic dose is about 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , ..... 9 , or 5×10 9 transfected cells.

[0144] In some cases, the methods described herein produce a population of cells that shows a decrease in the relative population of effector or Temra cells when compared to a population of cells produced by density gradient centrifugation. In some cases, the population of cells shows at least about a 5%, 10%, 15%, 25%, 30%, 40%, 50%, 75%, or 90% decrease in the relative population of effector or Temra cells when compared to a population of cells produced by density gradient centrifugation. For example, the DLD method has been shown to produce a cell population with about 40% fewer Temra cells than cells produced using Ficoll or other methods after treatment with high transduction lentivirus. See Figure 21.

[0145] In some cases, the method described herein produces a population of cells exhibiting reduced IL-1Ra expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows a reduction of at least approximately 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in IL-1Ra expression compared to a population of cells produced by density gradient centrifugation. In some cases, the reduction in IL-1Ra expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0146] In some cases, the method described herein produces a population of cells exhibiting reduced IL-6 expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows a reduction of at least approximately 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in IL-6 expression compared to a population of cells produced by density gradient centrifugation. In some cases, the reduction in IL-6 expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0147] In some cases, the method described herein produces a population of cells exhibiting reduced IL-13 expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows a reduction of at least approximately 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in IL-13 expression compared to a population of cells produced by density gradient centrifugation. In some cases, the reduction in IL-13 expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0148] In some cases, the method described herein produces a population of cells exhibiting reduced MCP-1 expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows a reduction of at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in MCP-1 expression compared to a population of cells produced by density gradient centrifugation. In some cases, the reduction in MCP-1 expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0149] In some cases, the method described herein produces a population of cells exhibiting reduced PD1 and Timp3 co-expression compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population shows a reduction of at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in PD1 and Timp3 co-expression compared to a population of cells produced by density gradient centrifugation. In some cases, the reduction in PD1 and Timp3 co-expression becomes apparent at 0, 3, 6, 9, 13, or 16 days after production by the method and system described herein.

[0150] In some cases, the method described here produces a population of cells that exhibit reduced cellular senescence or depletion compared to the population of cells produced by density gradient centrifugation. In other cases, the population of cells shows a reduction of at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in cellular senescence or depletion compared to the population of cells produced by density gradient centrifugation. For example, the DLD method has been shown to produce cells showing less than 50% co-expression of PD11 and Timp3 compared to cells produced from Ficol, suggesting lower senescence and depletion than cells produced using Ficol. See Figure 24.

[0151] In some cases, the method described here produces a population of cells that exhibits a reduced tendency to cause cytokine release syndrome compared to the population of cells produced by density gradient centrifugation. In some cases, the population of cells exhibits a reduction of at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 90% in the cell tendency to cause cytokine release syndrome compared to the population of cells produced by density gradient centrifugation.

[0152] In some cases, the method described here produces a population of cells that exhibits a reduced culture time before delivery to the patient compared to a population of cells produced by density gradient centrifugation. In some cases, the cell population requires approximately 1, 2, 3, 4, 5, 6, 7, or 8 fewer days of culture before delivery to the patient compared to a population of cells produced by density gradient centrifugation.

[0153] In some cases, an increase or decrease in one or more biological properties is evident at 0, 3, 6, 9, 13, or 16 days after production by the methods and systems described herein, compared to a population of cells produced by density gradient centrifugation.

[0154] In some cases, the method described here produces a population of cells that exhibits a reduced time required to grow in culture and produce a single therapeutic dose equivalent of cells, compared to the population of cells produced by density gradient centrifugation.

[0155] In some cases, the method described here produces a population of cells that exhibits a reduced time required to grow in culture and produce a single therapeutic dose equivalent of cells, compared to the population of cells produced by density gradient centrifugation.

[0156] (User Interface) Figures 5A–5H show graphical user interfaces (GUIs) that can be used in the methods and systems described herein. The GUI may include a control panel and a visual representation of the processing system. The processing system may comprise a particle separation unit, a cassette on which one or more microfluidic cartridges are releasably coupled and supported, and a plurality of components configured to become part of the particle separation unit, the cassette, and / or the microfluidic cartridges. The GUI may be configured to receive user input, for example, to input preliminary data before the separation process starts, or to adjust one or more of the plurality of components while the separation process is in progress. The GUI may include various execution protocols that the user can select to achieve the desired target particle separation. The GUI may include a calibration protocol that allows the user to check or adjust the calibration of the mass sensors. For calibration adjustment, the GUI may prompt the user to sequentially place a plurality of calibration weights, each containing distinct fixed mass values ​​at each mass sensor. The sequence of weights may be from low mass to high mass, high mass to low mass, or randomized. In a weight sequence, there may be at least 3, 4, 5, 6, 7, 8, 9, or 10 calibration weight values. For each calibration weight, the user may be prompted to place a specific weight value on the mass sensor being calibrated to confirm that the weight is present on the sensor. The user may be allowed to input custom calibration weight values ​​using an input device, for example, to change the default calibration mass used to one of the available calibration weights.

[0157] For example, the user is sequentially prompted to place a calibration weight of 100g on the mass sensor to be calibrated. The user can then confirm that the weight is placed on the mass sensor, prompting the system to save the 100g calibration value, and the GUI can repeat the process by prompting the user to replace the 100g calibration weight with a 200g calibration weight, followed by the other calibration weights in the sequence. If the user does not have a specific calibration weight, they can change the calibration point using a custom calibration value corresponding to a different calibration weight with a mass similar to the missing weight by selecting an option on the GUI. For example, if the user has lost the 400g calibration weight required by the default calibration sequence but has a 500g calibration weight, they can enter a custom calibration value of 500g into the GUI and proceed with the calibration. The processing system can verify the new calibration weight by ensuring that the new calibration falls within the expected drift range before allowing the user to replace the previous calibration. For example, if a new calibration slope differs from the factory calibration slope by at least 5%, 10%, 15%, 20%, or 25%, the new calibration may fall outside the expected drift range. If the processing system is unable to validate the new calibration due to a slope outside the expected drift range, the user may be prompted to repeat the calibration procedure. After a series of at least two, three, four, or five consecutive failed calibrations, a service warning may be displayed prompting the user to request repair of the mass sensor.

[0158] As shown in Figure 5A, the control panel may be included on the left side of the GUI, and the visual representation of the processing system may be included on the right side of the GUI. The control panel may include several options for the user to select, for example, a scripting execution option that the user can select to achieve the desired target particle separation. The visual representation of the system may include a visual depiction of each component within the processing system. For example, there may be one or more visual representations for the degassing unit, bubble sensor, pressure sensor, pump, valve, pathway, flow sensor, and individual tubes entering and leaving the cassette. Each of these components may be assigned its own color to help the user distinguish which visual representation corresponds to which component. The user can click on each individual representation to see what a particular component is measuring or to manually adjust a particular component. The system can display readings from the components in real time, allowing the user to see the status of the separation process as it progresses. The user can see the status of the components in real time or manually control the operation of any of the components while the separation process is in progress.

[0159] The particle separation unit may be configured to include multiple lights that can change color depending on the mode the particle separation unit is in. For example, the lights may be white before the separation process begins and may change to yellow to indicate that user input is required before the separation process begins. The lights may also be red while the separation process is in progress and change to green when the process is complete. These lights may also be repeatedly turned on or off, or gradually dimmed or brightened to indicate the status to the user. For example, a blue light may slowly fade from dim to bright to indicate that the separation is proceeding normally, while a rapidly flashing red light may indicate that an error condition has occurred.

[0160] Figures 5B to 5F show various notifications / commands that can pop up for the user on the GUI. Commands can include requests for information about various inputs the system needs before the separation process can begin. For example, the system may ask the user to input the sample ID, execution ID, cell richness of the sample (e.g., number of cells / mL), and sample volume in the inlet sample bag. Notifications can be shown to the user to inform them of the system status during the separation process. For example, a notification may be shown to inform the user that the system is running according to the execution protocol selected before the separation process began. A notification may also be shown to the user to inform them that the system is experiencing one or more deviations from the execution protocol selected by the user. The notification may show the user that one or more deviations have occurred in the system and that one or more options are available to the user to correct the deviations and return the system to conform to the execution protocol. Alternatively, the system may automatically make the necessary changes itself to return the system to conform to the execution protocol. For example, if a bubble sensor detects bubbles in the fluid in the path, the path may be redirected to avoid entry into the microfluidic device. Furthermore, the speed of one or more pumps can be increased or decreased depending on whether high / low pressure is detected in the fluid path, or whether a higher / lower flow rate is desired to return the system to conform to the execution protocol. Also, the mass readings in each of the inlet or outlet solution bags can be changed if the mass sensors in each hanger detect a loss or gain in fluid content. Thus, the system can operate completely autonomously throughout the entire separation process, or it can receive user input once the user sees a fit.

[0161] As an addition to or alternative to notifications, the state of the isolation process can also be made visible to the user by implementing graphic changes to the system's visual representation. For example, a graphic change could include switching the color of the system's visual representation of a valve to indicate to the user that the valve has switched from the "on" to the "off" position, or from the "off" to the "on" position. A graphic change could also include changing the text area next to the visual representation of a valve as it opens and closes, changing from "on" to "off" or from "off" to "on". A graphic change could also include the text area next to the visual representation of one or more inlet streams, outlet streams, or paths within a cassette to indicate the flow rate of fluid passing through them. The text area can change as the flow rate through these areas changes.

[0162] As shown in Figures 5G and 5H, the system can also provide notification of when the separation process is complete. Once the execution is complete, the system can also generate a report that provides the user with an overview of the system's components measured during the separation process. An example of a system-generated execution report for the separation of leukocytes from red blood cells and platelets using leukocyte washing into a new buffer medium is shown in Figure 5I. In the exemplary report, the information is divided into system information, general information, results, and results and estimates for the specific execution, actual measurements (tracking sensor data as a function of time throughout the execution). The system and general information sections include information about the execution, the device (e.g., serial number SN), the protocol used, the sample, the organization running the device, the device location, user identification information, timing information, the version of the software used, and the execution status (e.g., completed, aborted, or other specific error state).

[0163] The results and estimation section includes the sample input volume (130 mL in this case), the estimated weight of the processed sample, the final product weight, and the actual run time. In this case, the actual weighing section includes time-course data measurements from pressure sensor 1, pressure sensor 2, product bag scale (mass sensor), and sample bag scale. For example, the actual weighing in the run report of Figure 5I shows three color codes for the displayed time-course data. When the solution is introduced into the system, the yellow trace (0 to 20 minutes across the graph) indicates the priming phase, where the system is primed to remove any bubbles. The red trace (>20 minutes to approximately 53 minutes across the graph) indicates the enrichment phase, when the sample is being converted into the enriched product by the DLD array, causing a corresponding pressure increase at both pressure sensors. During the red trace, an increase in the mass of the product bag is observed throughout the run, and as the sample is converted into the enriched product, a depletion of mass in the sample is observed. The slopes of these graphs indicate the flow rate to the product bag and the flow rate from the sample bag, respectively. The blue trace (from approximately 53 minutes into the graph to the end) indicates the shutdown phase after the target product mass has been reached.

[0164] Other parameters that can be included in the actual weighing are time-course data series from the diluent bag scale, buffer bag scale, first and / or second waste bag scale, and / or any of the bubble sensors 1-6, and combinations thereof. Exemplary graphs of each of these parameters are shown in Figure 5J. The example in Figure 5J shows an example of recirculation after the target product amount has been obtained. Mass sensor data indicates the amount of solution in each bag corresponding to the individual mass sensor, while the slope of these data indicates the mass flow rate into or out of the bag. Pressure sensor data is used to monitor the pressure experienced by target particles or cells as they progress from the sample bag through the DLD to the product bag. Pressure data is monitored in real time to ensure that the pressure does not exceed the maximum pressure threshold that the target cells or particles can tolerate without damage. Pressure data also corresponds to, or may be used to estimate, the viscosity and / or cell richness of the product. This data summary allows the user to inspect the separation process and determine whether or not modifications are possible or should be made to achieve different desired results. In some embodiments, the system can respond to pressure data and adjust the separation in real time or near real time during the separation process, for example, by adjusting the flow rate. The summary may be exported via a network connection or saved to a USB drive connected to a USB port on the system.

[0165] Figures 5K to 5S illustrate exemplary embodiments of user interaction with the GUI according to some embodiments described herein. In Figure 5K(i), the user is prompted by the primary touchscreen display to enter their login credentials. Once the user is authenticated, the primary touchscreen display presents them with a default splash screen similar to the example shown in Figure 5K(ii). When the user selects the run button as in Figure 5L(i), several run mode options are presented that can be selected as shown in Figure 5L(ii). Once a mode is selected, the user may be prompted to enter several run parameter values ​​using an on-screen keypad or another input device, as in the example in Figure 5M(i). Once all the necessary run parameters have been entered, the user can proceed with the run. Before they proceed, the system can calculate the required reagent volume, predicted output, and estimated run time, which are displayed to the user as shown in Figure 5M(ii). Once the user instructs the system to proceed, the GUI can prompt the user to measure the tare weight of the mass sensor by removing all bags or weights from the mass sensor, as shown in Figure 5N(i).

[0166] As shown in Figure 5N(i), if all mass sensors are zero, the tare weight is enabled and the user is allowed to proceed. On the other hand, as shown in Figure 5N(ii), if the sensors detect a non-zero mass, an error message is displayed and the user cannot proceed until the weight is removed from the mass sensors (in case of user error) or until the sensors are repaired or recalibrated (in case of hardware or software error). Once the user is allowed to proceed, the GUI can provide instructions for loading cassettes, samples, reagents, waste bags, and priming the system. For example, the GUI can prompt the user to ensure all bag clips are closed, as shown in Figure 5O(i). The GUI then uses a secondary status display, as seen in Figure 5O(ii), to instruct the user to load cartridges, and subsequently prompt the user to scan their barcodes and load each color-coded bag into its corresponding bag hanger. Figures 5O(ii-iv) show the yellow-coded priming solution.

[0167] Once all necessary bags are loaded, the GUI can use a secondary status display, as shown in Figure 5P(i-iii), to prompt the user to scan the cartridge barcode, lock the cartridge in place, and close the instrument door. The GUI can then display the data coded by each scanned barcode (which may include the serial number of each loaded bag and the serial number of the cartridge) on the main screen, and further display the loaded mass of each bag, as shown in the example in Figure 5P(iv). The GUI can also display a preview of the execution parameters, as shown in Figure 5Q(i), giving the user one last chance to go back and reconfigure before starting the execution procedure. As the user proceeds, the system can verify that the sensors and other system components are functioning correctly and that the correct amount of required reagents has been loaded. During this progress, a progress bar for prerun verification may be displayed by the GUI, as shown in Figure 5Q(ii). If the verification has passed all system checks, the user may be instructed to remove all bag clips, as shown in Figure 5R(i), before they proceed and start execution. Once execution begins, progress can be tracked on either or both the main and secondary status displays. An example of a main screen status readout is shown in Figure 5R(ii). Once execution is complete, the GUI can prompt the user to seal the bag clip and retrieve the product bag, as shown in Figure 5S(i). Once the user has done this, the GUI can prompt the user to print or export an execution report, such as the report described above, as shown in Figure 5S(ii).

[0168] Figure 6 shows process 600 which can follow the separation process. Process 600 can be started in step 610, at which point the system displays a GUI to the user. The GUI may include a control panel and visual representation of the processing system. The processing system may include a particle separation unit, a cassette to which one or more microfluidic cartridges are releasably coupled and supported, and a plurality of components configured to be part of the particle separation unit, the cassette, and / or the microfluidic cartridges.

[0169] In step 620, the system can receive user input for the execution protocol used in the separation process. User input may be received via a GUI. The GUI may include various execution protocols that the user can select to achieve the desired target particle separation. The GUI can also be configured to receive user input, for example, to input preliminary data before the separation process starts, or to adjust one or more of several components while the separation process is in progress. Based on the user input, the GUI can calculate one or more predictions of the volume of reagents required to complete the separation, and the volume and concentration of products and wastes produced by the execution, and can further prompt the user to load at least the minimum predicted required volume. The GUI can sequentially prompt the user to scan barcodes on samples, reagents, or waste bags using a barcode scanner and place the bags on designated bag hooks. Designated hooks may be indicated to the user by color codes. For example, a red marking or light on the hook for a product bag may correspond to a red tag on the corresponding product bag. When the barcode on the red-tagged product bag is scanned, the GUI can prompt the user to load the product bag onto the red-marked bag hook (for example, by illuminating the bag hook or its vicinity with red light or other visible indicator through visual representation). Once the bag is loaded, the system can detect its presence as a weight on the mass sensor corresponding to the loaded bag hook. The user is similarly prompted by the GUI to load the priming solution bag (which may be color-coded yellow), the buffer bag (which may be color-coded blue), and / or the waste bag (which may be color-coded black). For each loaded bag, the system can verify that the correct weight is loaded onto the hook before prompting the user to load the next bag.In some embodiments, the bags are color-coded and can be connected to a cassette, as illustrated in Figure 3G, with sample bags coded in red, diluent bags in purple, buffer bags in blue, priming solution bags in yellow, product bags in green, and waste bags in black.

[0170] In step 630, the system can invoke execution protocol 630. The execution protocol may be based on the user input received in step 620.

[0171] In step 640, the system can display the progress of sample processing in real time or near real time. For example, the control panel may be included on the left side of the GUI, and the visual representation of the processing system may be included on the right side of the GUI. The control panel may include several user-selectable options, including script execution protocols that the user can choose to achieve the desired target particle separation. The visual representation of the system may include a visual depiction of each component within the processing system. For example, visual representations may be provided for one or more of the degassing unit, bubble sensor, pressure sensor, pump, valve, pathway, flow sensor, and individual tubes entering and leaving the cassette. Each of these components may be assigned its own color to help the user distinguish which visual representation corresponds to which component. The user can click on each individual representation to see what a particular component is reading or to manually adjust a particular component. The system can display readings from the components in real time, allowing the user to see the status of the separation process as it progresses. The user can manually adjust any of the components in real time while the separation process is in progress.

[0172] Step 640 may also include displaying various notifications / commands that can pop up to the user on the GUI. Commands may include requests for information about various inputs the system needs before the separation process can begin. For example, the system may request the user to enter a user authentication certificate, sample ID, execution ID, cell richness of the sample (e.g., number of cells / mL), and sample volume in the inlet sample bag. Requests for information may be implemented, for example, as a series of prompts, requiring the user to scan the barcode on the sample bag and enter the sample ID and execution ID. Notifications may be shown to the user to inform them of the system's status during the separation process. For example, a notification may be shown to the user to inform them that the system is running according to the execution protocol selected before the separation process began. The system may also show a notification to the user to inform them that it is experiencing one or more deviations from the execution protocol selected by the user. The notification may show the user where the one or more deviations are occurring in the system and may provide the user with one or more options to correct the deviations and restore the system to conform to the execution protocol. Alternatively, the system may automatically make the necessary changes itself to restore the system to conform to the execution protocol. For example, if a bubble sensor detects bubbles in the fluid within a path, the path can be redirected to avoid entry into the microfluidic device. Furthermore, the speed of one or more pumps can be increased or decreased depending on whether the fluid in the path is detected to have high / low pressure or whether higher / lower flow rates are desired, allowing the system to recover to conform to the execution protocol. In this way, the system can operate completely autonomously throughout the entire separation process, or, if the user has made a conformance, the system can also receive user input.

[0173] As an addition to or alternative to notifications, the status of the isolation process can be visualized to the user by implementing graphic changes in the system's visual representation in step 640. For example, a graphic change could include switching the color of the system's visual representation of a valve to indicate to the user that the valve has switched from the on to the off position, or from the off to the on position. A graphic change could also include changing a text area next to the valve's visual representation as the valve opens and closes, changing from "on" to "off," or from "off" to "on." A graphic change could include a text area next to the visual representation of one or more inlet streams, outlet streams, or paths within a cassette, indicating the flow rate of fluid passing through them. The text area can change as the flow rate through these areas changes.

[0174] The system may provide an additional notification in step 640 if the isolation process is complete. Upon completion of the execution, the system may also generate a report that provides the user with a summary of the system components' metrics during the isolation process. This data summary allows the user to examine the isolation process and determine whether or not it is possible to modify it to achieve different desired results.

[0175] (Digital processing devices) In some embodiments, the platforms, systems, media, and methods described herein utilize digital processing devices. In further embodiments, the digital processing device includes one or more hardware central processing units (CPUs) or general-purpose graphics processing units (GPGPUs) or field-programmable gate arrays (FPGAs) that perform the functions of the device. In further embodiments, the digital processing device further includes an operating system configured to execute executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Internet to access the World Wide Web. In further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.

[0176] As described herein, suitable digital processing devices include, in non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, subnotebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, internet devices, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those skilled in the art will recognize that many smartphones are suitable for use in the systems described herein. Those skilled in the art will also recognize and select any television, video player, or digital music player with computer network connectivity as suitable for use in the systems described herein. Suitable tablet computers include those with booklets, slates, and compatible configurations known to those skilled in the art.

[0177] In some embodiments, a digital processing device includes an operating system configured to execute executable instructions. The operating system is software, including programs and data, that manage the device's hardware and provide services for running applications. Those skilled in the art will recognize that suitable server operating systems include, in non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux®, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those skilled in the art will also recognize that suitable personal computer operating systems include, in non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided through cloud computing. Those skilled in the art will recognize that suitable mobile smartphone operating systems include, but are not limited to, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.A person skilled in the art will also recognize that suitable media streaming device operating systems include, but are not limited to, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. A person skilled in the art will also recognize that suitable video game console operating systems include, but are not limited to, Sony PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U, and Ouya®.

[0178] In some embodiments, the device includes a storage and / or memory device. A storage and / or memory device is one or more physical devices used to store data or programs temporarily or permanently. In some embodiments, the device is volatile memory and requires power to maintain the stored information. In some embodiments, the device is non-volatile memory and retains the stored information when the digital processing device is not powered. In further embodiments, non-volatile memory includes flash memory. In some embodiments, non-volatile memory includes dynamic random access memory (DRAM). In some embodiments, non-volatile memory includes ferroelectric random access memory (FRAM®). In some embodiments, non-volatile memory includes phase-change random access memory (PRAM). In other embodiments, the device is, in non-limiting examples, a storage device including CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tape drives, optical disk drives, and cloud computing-based storage. In further embodiments, the storage and / or memory device is a combination of devices such as those disclosed herein.

[0179] In some embodiments, the digital processing device includes a display for sending visible information to the user. In some embodiments, the display is a cathode ray tube (CRT). In some embodiments, the display is a liquid crystal display (LCD). In further embodiments, the display is a thin-film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light-emitting diode (OLED) display. In various further embodiments, the OLED display is a passive-matrix OLED (PMOLED) or an active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In yet another embodiment, the display is a combination of devices such as those disclosed herein. In other embodiments, the digital processing device includes a plurality of displays for sending visual information to the user. In some embodiments, the plurality of displays include a status display for sending visual state information to the user and a control display for sending visual control information to the user.

[0180] In some embodiments, the digital processing device includes an input device that receives information from the user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device, including, in non-limiting examples, a mouse, trackball, trackpad, joystick, game controller, or stylus. In some embodiments, the input device is a touchscreen or multitouchscreen. In other embodiments, the input device is a microphone that captures voice or other sound input. In other embodiments, the input device is a video camera or other sensor that captures motion or visual input. For example, the input device may include a barcode scanner. In some embodiments, the input device may include a haptic feedback device. In further embodiments, the input device is Kinect, Leap Motion, or similar. In further embodiments, the input device is a combination of devices such as those disclosed herein.

[0181] Referring to Figure 7, in a particular embodiment, the exemplary digital processing device 701 is programmed or otherwise configured to operate a particle separation system. The device 701 can control various aspects of the target particle separation of this disclosure, such as performing processing steps. In this embodiment, the digital processing device 701 includes a central processing unit (CPU, also referred to herein as “processor” and “computer processor”) 705, which can be a single-core or multi-core processor, or multiple processors for parallel processing. The digital processing device 701 also includes memory or memory locations 710 (e.g., random-access memory, read-only memory, flash memory), electronic storage devices 715 (e.g., hard disks), a communication interface 720 for communicating with one or more other systems (e.g., a network adapter), and peripheral devices 725 such as a cache, other memory, data storage and / or an electronic display adapter. The memory 710, storage devices 715, interface 720 and peripheral devices 725 communicate with the CPU 705 through a communication bus (real line), such as a motherboard. The storage device 715 may be a data storage device (or data repository) for storing data. The digital processing device 701 can be operationally connected to a computer network ("network") 730 with the help of the communication interface 720. The network 730 may be the Internet, the Internet and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, the network 730 is a telecommunications and / or data network. The network 730 may include one or more computer servers, which can operate distributed computing such as cloud computing. In some cases, the network 730 can operate a peer-to-peer network with the help of device 701, which can operate devices connected to device 701 that act as clients or servers.

[0182] Continuing with the reference to Figure 7, the CPU 705 is capable of executing 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 memory 710. The instructions can be directed to the CPU 705, thereby allowing the CPU 705 to be programmed or otherwise configured to subsequently perform the methods of this disclosure. Examples of operations performed by the CPU 705 may include retrieving, decoding, executing, and writing back. The CPU 705 may be part of a circuit such as an integrated circuit. One or more other components of device 701 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC) or a field-programmable gate array (FPGA).

[0183] Continuing with the reference to Figure 7, the storage device 715 is capable of storing files such as drivers, libraries, and saved programs. The storage device 715 is also capable of storing user data, such as user preferences and user programs. The digital processing device 701 may include one or more additional external data storage devices, such as those located on a remote server communicating via an intranet or the internet.

[0184] Continuing with the reference to Figure 7, the digital processing device 701 is capable of communicating with one or more remote computer systems via the network 730. For example, device 701 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., Apple® iPad®, Samsung® Galaxy Tab), telephones, smartphones (e.g., Apple® iPhone® or Android-enabled devices, BlackBerry®), or personal digital assistants.

[0185] The methods described herein can be executed by machine-executable code (e.g., a computer processor) stored on an electronic storage location of a digital processing device 701, such as on memory 710 or electronic storage device 715. 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 705. In some cases, the code can be retrieved from storage device 715 and stored on memory 710 for easy access by the processor 705. In some situations, electronic storage device 715 can be excluded, and the machine-executable instructions are stored on memory 710.

[0186] (Non-temporary computer-readable storage medium) In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-temporary computer-readable storage media programmed with instructions executable by the operating system of a digital processing device, which is optionally connected to a network. In further embodiments, the computer-readable storage medium is a tangible component of the digital processing device. In further embodiments, the computer-readable storage medium is optionally removable from the digital processing device. In some embodiments, the computer-readable storage medium includes, in non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid-state memory, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and similar devices. In some cases, the programs and instructions are coded on the medium permanently, substantially permanently, semi-permanently, or non-temporarily.

[0187] (Computer program) In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program or the use thereof. For example, a computer program includes a sequence of instructions written to perform a specific task, executable on the CPU of a digital processing device. Computer-readable instructions may be executed as program modules such as functions, objects, application programming interfaces (APIs), data structures, and the like, which perform a specific task or execute a specific abstract data type. In light of the disclosures provided herein, it will be recognized by those skilled in the art that computer programs may be written in various versions of various languages.

[0188] The functionality of computer-readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program includes a single sequence of instructions. In some embodiments, a computer program includes multiple sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from multiple locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plugins, extensions, add-ins, or add-ons, or a combination thereof.

[0189] (Web application) In some embodiments, a computer program includes a web application. Those skilled in the art will recognize, in light of the disclosures provided herein, that a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, the web application is built on a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, the web application utilizes one or more database systems, including, in non-limiting examples, relational, non-relational, object-oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, in non-limiting examples, Microsoft® SQL Server, MySQL®, and Oracle®. Those skilled in the art will also recognize that, in various embodiments, the web application is written in one or more versions of one or more languages. The web application may be written in one or more markup languages, presentation-definition languages, client-side scripting languages, server-side coding languages, database query languages, or a combination thereof. In some embodiments, the web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). In some embodiments, the web application is written to some extent in a presentation-definition language such as Cascading Style Sheets (CSS).In some embodiments, the web application is written to some extent in a client-side scripting language such as Asynchronous Java® Script and XML (AJAX), Flash® ActionScript, Javascript, or Silverlight®. In some embodiments, the web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java®, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python®, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In some embodiments, the web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, the web application integrates enterprise server products such as IBM® Lotus Domino®. In some embodiments, the web application includes a media player element. In various further embodiments, the media player element may utilize one or more of many suitable multimedia technologies, including, but not limited to, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java®, and Unity®.

[0190] (Mobile application) In some embodiments, the computer program includes a mobile application provided to a mobile digital processing device. In some embodiments, the mobile application is provided to the mobile digital processing device at the time of its manufacture. In other embodiments, the mobile application is provided to the mobile digital processing device via a computer network as described herein.

[0191] As provided herein, mobile applications are created using hardware, languages, and development environments known in the art, and techniques known to those skilled in the art. Those skilled in the art will recognize that mobile applications are written in several languages. Suitable programming languages ​​include, but are not limited to, C, C-Hk, C4, Objective-C, Java®, Javascript, Pascal, Object Pascal, Python®, Ruby, VB.NET, WML, and XHTML / HTML with or without CSS, or a combination thereof.

[0192] Suitable mobile application development environments are available from several sources. Commercial development environments include, but are not limited to, AirplaySDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments, but are not limited to, Lazarus, MobiFlex, MoSync, and Phonegap, are available free of charge. In addition, mobile device manufacturers sell software developer kits, but are not limited to, iPhone and iPad (iOS) SDK, Android® SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.

[0193] Those skilled in the art will recognize that several commercial forums are available for the distribution of mobile applications, including, but not limited to, the Apple® App Store, Google® Play, Chrome WebStore, BlackBerry® App World, App Store for Palm devices, App Catalog for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, and Nintendo® DSi Shop.

[0194] (Standalone application) In some embodiments, a computer program includes a standalone application, which is a program that runs as an independent computer process, not as an add-on to an existing process, for example, as a plug-in. Those skilled in the art will recognize that standalone applications are often compiled. A compiler is a computer program(s) that translates source code written in a programming language into binary object code, such as assembly language or machine code. Suitable compiled programming languages, in non-limiting examples, include C, C++, Objective-C, COBOL, Delphi, Eiffel, Java®, Lisp, Python®, Visual Basic, and VB.NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable compiled applications.

[0195] (Web browser plugin) In some embodiments, a computer program includes web browser plugins (e.g., extensions). In computer operation, a plugin is one or more software components that add specific functionality to a larger software application. Software application creators support plugins, which generate the ability of third-party developers to extend the application, facilitate the addition of new functionality, and reduce the size of the application. Where supported, plugins allow customization of the functionality of the software application. For example, plugins are commonly used in web browsers to play videos, generate interactivity, scan for viruses, and display specific file types. Those skilled in the art will be familiar with several web browser plugins, including Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. In some embodiments, a toolbar includes one or more web browser extensions, add-ins, or add-ons. In some embodiments, a toolbar includes one or more explorer bars, toolbands, or deskbands.

[0196] In terms of the disclosures provided herein, those skilled in the art will recognize that, as non-limiting examples, several plugin frameworks are available that enable the development of plugins in a variety of programming languages, including C++, Delphi, Java™, PHP, Python™, and VB.NET, or combinations thereof.

[0197] A web browser (also called an internet browser) is a software application designed for use with a networked digital processing device to search, present, and traverse information resources on the World Wide Web. Suitable web browsers include, but are not limited to, Microsoft Internet Explorer®, Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software®, and KDE Konkeror. In some embodiments, a web browser is a mobile web browser. Mobile web browsers (also called microbrowsers, minibrowsers, and wireless browsers) are designed for use on mobile digital processing devices, including, but are not limited to, handheld computers, tablet computers, netbooks, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, but are not limited to, the Google® Android® browser, the RIM BlackBerry® browser, Apple® Safari®, Palm® Blazer, Palm WebOS browser, Mozilla Firefox for mobile, Microsoft® Internet Explorer Mobile, Amazon Kindle Basic Web, Nokia browser, Opera Software® Opera, and Sony PSP™ browser.

[0198] (Software module) In some embodiments, the platforms, systems, media, and methods disclosed herein include software, servers, and / or database modules, or uses thereof. From the standpoint of the disclosures provided herein, software modules are created using machines, software, and languages ​​known in the art, and techniques known to those skilled in the art. Software modules disclosed herein are executed in numerous ways. In various embodiments, a software module includes files, sections of code, programming objects, programming structures, or combinations thereof. In further various embodiments, a software module includes multiple files, multiple sections of code, multiple programming objects, multiple programming structures, or combinations thereof. In various embodiments, one or more software modules include, in non-limiting examples, web applications, mobile applications, and standalone applications. In some embodiments, a software module resides in one computer program or application. In other embodiments, a software module resides in more than one computer program or application. In some embodiments, a software module is provided on one machine. In other embodiments, a software module is provided on more than one machine. In further embodiments, a software module is provided on a cloud computing platform. In some embodiments, a software module is provided on one or more machines at a single location. In other embodiments, the software module is provided on one or more machines at more than one location.

[0199] (Database) In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases or uses thereof. Those skilled in the art will recognize from the disclosures provided herein that many databases are suitable for storing and retrieving information. In various embodiments, suitable databases include, in non-limiting examples, relational databases, non-relational databases, object-oriented databases, object databases, entity-relational model databases, associative databases, and XML databases. Further non-limiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, and Sybase. In some embodiments, the database is internet-based. In further embodiments, the database is web-based. In even further embodiments, the database is cloud computing-based. In other embodiments, the database is based on one or more local computer storage devices.

[0200] (Examples) Example 1 Compared to density gradient centrifugation, a larger number of CD45+ and CD3+ cells are recovered on day 0 after DLD isolation.

[0201] The performance of the DLD system was investigated against density gradient centrifugation (e.g., Ficoll®, GE Healthcare) based on the total number of leukocytes and T cells. Compared to Ficoll, LeucoPak enriched using the DLD system showed an increased overall amount of viable (DARQ7-)CD45+ cells (pan leukocyte markers), as determined by flow cytometry. As shown in Figure 13, when normalized to a 1200 mL input, 5 × 10⁶ cells were obtained. 9 An average of 10¹ CD45+ cells were isolated from LeucoPak, and 2 × 10¹⁶ cells were isolated using Ficol. 9This represents a 2.5-fold increase compared to [another method]. As shown in Figure 15, when processing patient samples with lower WBC counts, an increase in viable total CD45+ and CD3+ cells (pan-T cell markers) obtained using the DLD system is also observed compared to Ficol. This advantage is important for obtaining lymphocytes from patients with NHL, lymphoma, AML, breast cancer, colorectal cancer, or other cancers. Similarly, the increased lymphocyte and T cell retrieval performance of DLD can be expressed as a measure of tumor debulking efficiency, converted to a WBC ratio to the cells that are desirable to be depleted from the input sample. As shown in Figure 16, DLD products result in lower ratios of both RBC / WBC and PLT / WBC. This was determined by flow cytometry using CD41 as a marker for platelets, CD235a for red blood cells, and CD45 for white blood cells. The DLD protocol results in a population of white blood cells with significantly fewer RBCs (red blood cells) and PLTs (platelets).

[0202] Example 2 Compared to density gradient centrifugation, a greater number of beneficial T cell subtypes and a smaller number of undesirable or harmful T cell subtypes are recovered on day 0 after isolation.

[0203] The efficacy and safety of T cell therapy depend on the T cell subtype used to manufacture the treatment. Therefore, T cell subtypes isolated using the DLD system were compared to those purified by Ficol, a common method for isolating T cells from blood and leukocyte-depleted transfusion samples. Compared to Ficol, LeucoPak enriched using the DLD system showed a higher percentage composition of T central memory cells and fully dedifferentiated T effector cells. CD4+ and CD8+ T cell populations were isolated using the DLD and Ficol methods, respectively, as shown in Figure 14. On average, the populations isolated by the DLD method consisted of 30% T naive cells (CD3+ / CD45RA+ / CCR7+), 25% T central memory cells (CD3+ / CD45RA- / CCR7+), 29% T effector memory cells (CD3+ / CD45RA+ / CCR7-), and 17% Temra cells (effector memory differentiated) (CD3+ / CD45+ / CCR7-). On average, the populations isolated by the Ficoll method consisted of 32% T naive cells (CD3+ / CD45RA+ / CCR7+), 19% T central memory cells (CD3+ / CD45RA- / CCR7+), 28% T effector memory cells (CD3+ / CD45RA+ / CCR7-), and 21% Temra cells (effector memory differentiated) (CD3+ / CD45+ / CCR7-).

[0204] Example 3 T cell populations isolated by DLD are more receptive to lentiviral transduction, are transduced more efficiently by lentiviruses, express lentivirally transduced genes more rapidly, and retain more beneficial T cell subtypes compared to density gradient centrifugation.

[0205] The timely administration, efficacy, and safety of T cell therapy depend on how obediently isolated T cells are genetically engineered and how rapidly and efficiently they can express heterologous genetic material during subsequent proliferation. Therefore, we compared the T cell response to lentiviral transduction using T cells isolated with the DLD system to that obtained from Ficol purification. Leucopack cells from three different donors were treated, isolated / activated with CD3 / CD28 beads, and transduced with GFP-lentivirus. The cells were then grown in cell culture with IL-7 / IL-15 for 9 days. As shown in Figure 17, cells were analyzed by flow cytometry at the corresponding days. We monitored GFP-lentivirus uptake and transduction, showing that DLD-prepared cell populations transduced lentivirus more readily compared to untransduced cells at days 0, 3, 6, 9, and 12. At day 6, we showed a 30% increase in the number of transduced cells compared to Ficol-prepared cells. Figure 19 shows the average percentage of transduced cells in DLD and Ficol-prepared cell populations, indicating that the DLD population was more compliant to lentiviral transduction, in some cases ranging from 20% to 100%.

[0206] These findings were confirmed using immunofluorescence microscopy over nine days. T cells from the DLD and Ficol methods were isolated / activated from the same donor, transduced with GFP-lentivirus, and proliferated in cell culture. At the indicated time, cells were examined by microscopy monitoring GFP-lentivirus uptake and GFP expression, showing that DLD cells were transduced more readily than Ficol cells, as indicated by a greater GFP signal in the DLD-derived cell population shown in Figure 18. Cells prepared by DLD and the system were transduced more readily compared to the other system methods (see Figures 17–19). DLD-producing cells were consistently transduced more readily, with approximately 87.5% showing significant improvement compared to Ficol. The average improvement on day 3 was approximately twofold, and a 30% advantage was maintained on days 6 and 9. At all times, DLD-producing cells had, on average, higher transduction levels.

[0207] These findings are particularly prominent when translating these separation processes into clinical applications. A higher lentiviral transduction efficiency leads to a shorter time to administration, that is, a sufficient number of cells can be obtained for a therapeutic cell dose or multiple doses. As shown in Figure 20, the DLD method can produce sufficient lentivirally transduced cells corresponding to 10 doses of therapeutic cells after 3 days in culture, normalized to an initial input of 200 mL of leukopak material, and can produce a total dose more than Ficoll over 9 days. In some cases, the cell population of the DLD preparation produced more than twice the number of transduced cells over a 9-day period.

[0208] In addition to the total number of cells produced after separation and lentiviral transduction, it is important that the therapeutic cell dose includes effectively activated T cell types, such as T central memory and T effector memory cells. The T cell subset composition of GFP-Lv+ cells on days 3 and 6 after activation was compared between the cell populations of DLD and Ficoll preparations. Determination of T cell subtypes was performed by flow cytometry within GFP-Lv+ T cells as shown in Figure 21. On day 6, the DLD method resulted in a population of T cells containing 4% naive T (CD3+ / CD45RA+ / CCR7+), 19% T central memory (CD3+ / CD45RA- / CCR7+), 74% T effector memory (CDCD3+ / CD45RA+ / CCR7-), and 3% Temra (CD3+ / CD45+ / CCR7-). On day 6, the Ficoll method resulted in a population of T cells containing 28% naive T (CD3+ / CD45RA+ / CCR7+), 16% T central memory (CD3+ / CD45RA- / CCR7+), 51% T effector memory (CD3+ / CD45RA+ / CCR7-), and 5% Temra (CD3+ / CD45+ / CCR7-). Thus, compared to Ficoll GFP=Lv+ T cells, DLD cells have a larger pool of Tcm and result in a more robust conversion to Tem cells.

[0209] These findings were confirmed in additional experiments comparing the cell population T cell compositions and ficoll preparation compositions (from healthy donors) from DLD before lentiviral transduction (Figure 22) and 3, 6, and 9 days after transduction during culture (Figure 23). DLD cells on day 0 (before transduction) showed a larger number of CD4+ cells and less differentiated Tcm cells than ficoll cells, as determined by flow cytometry. The progression of T cell subtypes with GFP-Lv is shown in Figure 23. Viable CD3+ cells from the DLD protocol showed a bias towards Tcm over time compared to cells from the ficoll protocol. GFP-Lv+ and T cell subsets were determined by flow cytometry. For example, on day 9, the DLD method yielded a population of T cells containing 5% T naive (CD3+ / CD45RA+ / CCR7+), 29% T central memory (CD3+ / CD45RA- / CCR7+), 59% T effector memory (CD3+ / CD45RA+ / CCR7-), and 7% Temra (CD3+ / CD45+ / CCR7-). On day 9, the ficoll method yielded a population of T cells containing 9% T naive (CD3+ / CD45RA+ / CCR7+), 20% T central memory (CD3+ / CD45RA- / CCR7+), 59% T effector memory (CD3+ / CD45RA+ / CCR7-), and 12% Temra (CD3+ / CD45+ / CCR7-).

[0210] Example 4 The T cell population separated by DLD had lower expression of cell senescence and exhaustion markers after activation, lentiviral transduction, and proliferation compared to the T cell population prepared using the ficoll method.

[0211] The efficacy and production of therapeutic cells depend in part on having a viable and proliferating population of cells, i.e., non-senescent or non-depleted T cells. Activated, transduced, and proliferated T cells were examined for senescence (CD57+ / KLRG1+) and depletion (CD57 / KLRG1+ / PD1+ / Tim3+) on day 13 and expressed as a Ficol / DLD ratio. Ficol cells transduced with a complete CAR19 signaling domain had more pronounced expression of senescence and depletion markers (CD57+ / KLRG1+ and CD57- / KLRG1+ with PD1 and Timp3 co-expression) than DLD cells, as shown in Figure 24. In contrast, the difference was very small in cells transduced by control of no CAR or an inactive CAR (CAR19-Sig domain).

[0212] Example 5 T cell populations isolated by DLD have equivalent or increased killing capacity compared to those prepared using the Ficoll method.

[0213] The efficacy of therapeutic T cell preparations depends on cells containing the target peptide sequence that are effective in cell killing. T cells were isolated from DLD or Ficol, activated, and transduced using a TCRT lentivirus specific to the MART-1 antigen. Cells were harvested on day 6 and co-cultured with T2 target cells (Luc+) containing the MART-1 peptide in different ratios. During culture, T2 cell death was assessed by the loss of chemiluminescence in the co-culture. Both DLD and Ficol cells were capable of dose-dependently killing their target cells. As shown in Figure 25, cells prepared using the DLD method showed higher killing capacity than those prepared using Ficol at T cell:target cell ratios of 2:1, 1:1, and 0.5:1, and in some cases showed a 30% increase in killing capacity.

[0214] Example 6 T cell populations isolated by DLD exhibit higher desirable cytokine expression and lower undesirable cytokine expression compared to T cell populations isolated by Ficol.

[0215] The safety of therapeutic T cell preparation partially depends on isolated cells that exhibit greater cytotoxic activity, in contrast to inflammatory responses, in order to avoid adverse effects on patients. Therefore, it is desirable that T cell populations prepared using various isolation methods express more cytotoxic cytokines and have lower inflammatory cytokine expression. For this reason, cytokine expression was compared between T cell populations isolated using DLD and those isolated using the Ficol method.

[0216] Supernatants from DLD and Ficol cells were collected 0, 6, and 13 days after T cell isolation / activation, proliferation (IL7 / IL-15), and lentiviral transduction (CAR-T-CD19 or TCRT-MART-1). Fifteen different cytokines were analyzed in all supernatants using a Luminex multiplex assay. The results are expressed as Ficol / DLD (pg / ml) ratios, as shown in Figure 26. Figure 27 shows the time course of cytokine expression for IFNg, GM-CSF, IL-1Ra, and IL-6 in CAR-T-CD19 transduction cells. Figure 28 shows the time course of cytokine expression for IFNg, GM-CSF, IL-1Ra, and IL-6 in TCRT-MART-1 transduction cells. Ficol cells secreted more IL-6, MCP-1, and IL-1Ra, which are involved in the inflammatory response, while DLD cells expressed more IFNg and GM-CSF, typical markers of cytotoxic activity. Thus, DLD-prepared T cell populations exhibit a more desirable cytokine expression profile.

[0217] Preferred embodiments of the present invention have been illustrated and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. The present invention is not intended to be limited by any particular example provided herein. The present invention has been described with reference to the preceding specification, but the descriptions and examples of embodiments herein are not intended to be restrictive. Hereinafter, numerous variations, modifications, and substitutions will come to mind for those skilled in the art without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to any particular description, configuration, or relative proportion described herein, which will vary depending on various conditions and variables. It will be understood that various alternatives to the embodiments of the present invention described herein can be adopted in the practice of the present invention. Accordingly, the present invention is intended to encompass any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and the methods and structures within the scope of these claims and their equivalents are intended to be encompassed thereby.

[0218] Any reference listed herein is incorporated entirely by reference. Having described the present invention in detail, it will be understood by those skilled in the art that the invention can be practiced within a broad and equivalent range of conditions, parameters, etc., without affecting the spirit or scope of the invention or any embodiment thereof.

[0219] (Example embodiment) 1. A system for generating enriched products in one or more target particles, A cassette comprising (i) one or more cartridge ports, (ii) at least one inlet for receiving a sample, (iii) at least one outlet for outputting a product, and (iv) at least one recirculation path, A microfluidic cartridge comprising one or more cartridge ports operably coupled to establish fluid communication with a cassette, One or more microfluidic cartridges are used to separate one or more target particles from a sample. At least one recirculation pathway is used to recirculate one or more target particles through a cassette to concentrate one or more target particles to a predetermined volume of medium or a predetermined concentration. A system comprising one or more microfluidic cartridges and at least one recirculation path, operating in parallel to optimize execution time for product generation.

[0220] 2. The system according to Embodiment 1, wherein one or more microfluidic cartridges and at least one recirculation path operate independently of each other.

[0221] 3. The system according to Embodiment 1, wherein one or more microfluidic cartridges are configured to separate one or more target particles from a sample without affecting the recirculation process by at least one recirculation pathway.

[0222] 4. The system according to Embodiment 1, wherein at least one recirculation pathway is configured to recirculate one or more target particles through a cassette without affecting the separation process by one or more microfluidic cartridges.

[0223] 5. The system according to Embodiment 2, wherein one or more microfluidic cartridges and at least one recirculation pathway are individually controllable in real time to achieve a desired concentration of one or more target particles in the product.

[0224] 6. The system of Embodiment 1, wherein at least one recirculation path extends between at least one inlet and at least one outlet.

[0225] 7. The system of Embodiment 1, wherein at least one recirculation pathway is configured to recirculate one or more target particles in a clockwise direction on the cassette.

[0226] 8. The system of Embodiment 1, wherein at least one recirculation path is configured to recirculate one or more target particles counterclockwise on the cassette.

[0227] 9. The system of Embodiment 1, wherein the cassette further comprises a plurality of paths for loading and unloading samples to and from one or more microfluidic cartridges.

[0228] 10. The system of Embodiment 9, wherein at least one recirculation path is provided separately from the plurality of paths.

[0229] 11. The system of Embodiment 9, wherein at least one recirculation path is adjacent to or connected to one or more of the plurality of paths.

[0230] 12. A system for generating a product enriched in one or more target particles, comprising: a cassette to which one or more microfluidic cartridges are releasably coupled and supported, the cassette including a plurality of fluid channels that extend longitudinally and are spaced apart on the cassette; and a plurality of pumps peristaltically coupled to the plurality of fluid channels and configured to control the flow of a fluidic content through a plurality of fluid channels downstream of the one or more microfluidic cartridges for separation of one or more target particles from a sample. The system, wherein no moving part from the pumps directly contacts the fluidic content during its flow.

[0231] 13. The system according to Embodiment 12, wherein the plurality of fluid channels includes flexible tubes.

[0232] 14. The system according to Embodiment 12, wherein each of the plurality of pumps includes a set of pump heads peristaltically coupled to each subset of the plurality of fluid channels.

[0233] 15. The system according to Embodiment 14, wherein the set of pump heads in each pump includes two or more pump heads.

[0234] 16. The system according to embodiment 15, wherein two or more pump heads include two or more roller sets.

[0235] 17. The system according to Embodiment 16, wherein each subset of the multiple fluid channels comprises two or more fluid channels.

[0236] 18. The system according to Embodiment 12, wherein the fluid-containing material includes a sample, a medium solution, a diluent, and waste generated by one or more microfluidic cartridges after one or more target particles have been separated from the sample.

[0237] 19. The system according to embodiment 18, wherein the fluid-containing material further comprises a priming solution.

[0238] 20. The system according to Embodiment 18, wherein the fluid-containing material further comprises a recirculating solution containing a concentrated amount of one or more target particles.

[0239] 21. The system according to Embodiment 18, wherein the multiple pumps include a first pump for controlling the flow of a sample, a second pump for controlling the flow of a medium solution, a third pump for controlling the flow of a diluent, and a fourth pump for controlling the flow of waste.

[0240] 22. The system according to Embodiment 18, wherein multiple pumps are individually controllable to adjust the relative flow rates between the sample, medium solution, diluent, and waste.

[0241] 23. The system according to embodiment 14, wherein the set of pump heads in each pump are configured to operate in opposite phases to each other.

[0242] 24. The system according to embodiment 23, wherein the pump head set in each pump operates in reverse phase only for a period of approximately 180 degrees or less.

[0243] 25. The system according to Embodiment 14, wherein the set of pump heads in each pump have fixed movement relative to one another.

[0244] 26. The system according to embodiment 25, wherein the fixed motion includes the movement of a set of pump heads in each pump in the same direction at the same speed relative to one another.

[0245] 27. The system according to Embodiment 14, wherein for each subset of pumps and fluid channels, the fluid contents transported by the set of pump heads merge together into a single fluid path at the outlet of each subset of fluid channels.

[0246] 28. The system according to embodiment 14, wherein the pulsation of each pump is reduced by moving a set of pump heads in reverse phase.

[0247] 29. The system according to Embodiment 22, wherein multiple pumps are individually controllable to (a) control the ratio of waste volume to sample volume, (b) control the ratio of sample volume to diluent volume, or (c) adjust the dilution ratio.

[0248] 30. The system according to embodiment 12, wherein the multiple pumps can be individually controlled to be in phase or out of phase with respect to each other.

[0249] 31. The system according to Embodiment 12, wherein multiple pumps can be individually controlled to achieve the same flow rate, different flow rates, the same flow direction, or different flow directions.

[0250] 32. The system according to Embodiment 12, wherein multiple pumps are individually controllable to adjust the flow of fluid-containing material in real time as one or more microfluidic cartridges separate one or more target particles from a sample.

[0251] 33. The system according to Embodiment 12, wherein multiple pumps are individually controllable to enable a desired concentration of one or more target particles in the product.

[0252] 34. The system according to Embodiment 12, wherein the multiple pumps include peristaltic pumps.

[0253] 35. A system for generating enriched products in one or more target particles, One or more microfluidic cartridges configured to separate one or more target particles from a sample, One or more sensors for detecting the presence of bubbles in a sample or other solution, One or more controllable valves, A system comprising a cassette in which one or more microfluidic cartridges are releasably coupled and supported, the cassette including one or more bypass channels downstream of one or more controllable valves for redirecting a portion of a bubble-containing sample or other solution away from one or more microfluidic cartridges based on the detection of the presence of air by one or more sensors.

[0254] 36. The system according to embodiment 35, wherein one or more sensors are used to detect the presence of bubbles before the sample is circulated in one or more microfluidic cartridges.

[0255] 37. The system according to embodiment 35, wherein the system is configured to generate one or more alerts when one or more sensors detect the presence of bubbles in a sample or other solution.

[0256] 38. The system according to embodiment 35, wherein one or more sensors and one or more bypass channels work together to prevent air bubbles from entering and reducing the efficiency of one or more microfluidic cartridges.

[0257] 39. The system according to embodiment 35, wherein one or more sensors and one or more bypass channels work together to reduce or eliminate contamination in the product.

[0258] 40. A system for generating enriched products in one or more target particles, One or more microfluidic cartridges configured to separate one or more target particles from a sample, A cassette in which one or more microfluidic cartridges are releasably coupled and supported, A system comprising at least one degassing unit for removing dissolved gases and preventing bubble formation before a sample circulates through one or more microfluidic cartridges.

[0259] 41. The system according to embodiment 40, wherein at least one degassing unit is integrated onto a cassette.

[0260] 42. The system according to embodiment 40, wherein at least one degassing unit is manufactured as part of the cassette.

[0261] 43. The system according to embodiment 40, wherein at least one degassing unit is installed on the cassette in close proximity to one or more microfluidic cartridges.

[0262] 44. The system according to embodiment 40, wherein at least one degassing unit is in fluid communication with multiple pathways that introduce fluid into one or more microfluidic cartridges.

[0263] 45. A system for generating enriched products in one or more target particles, A cassette on which one or more microfluidic cartridges are releasably coupled and supported, comprising: (i) a plurality of inlets having a plurality of input vessels releasably and fluidly coupled thereto, at least one of the plurality of input vessels containing an incoming sample; (ii) a plurality of outlets having a plurality of output vessels releasably and fluidly coupled thereto; (iii) one or more microfluidic cartridges for separating one or more target particles from the sample; and (iv) a plurality of fluid channels extending between the plurality of inlets, the plurality of outlets and the one or more microfluidic cartridges, A cassette having multiple input containers, multiple output containers, and one or more microfluidic cartridges coupled thereto, as a whole, provides a closed, end-to-end sterile environment, enabling in-line continuous processing of incoming samples without external manual operation or intervention, enriching one or more target particles and producing a contamination-free product.

[0264] 46. ​​The system according to embodiment 45, wherein multiple inlets and multiple input vessels are releasable and fluidly coupled using multiple sterile coupling mechanisms.

[0265] 47. The system according to embodiment 46, wherein the multiple sterilization bonding mechanisms include at least one sterilization spike and at least one spike port.

[0266] 48. The system according to embodiment 45, wherein the product is collected in at least one of a plurality of output containers.

[0267] 49. The system according to embodiment 45, wherein the product is collected in at least one of a plurality of output containers without exposing the product to an external non-sterile environment.

[0268] 50. The system according to Embodiment 45, wherein the product is collected in at least one of a plurality of output containers without exposing the product to an external non-sterile environment.

[0269] 51. The system according to Embodiment 45, wherein the sample is entered into the cassette from at least one of a plurality of input containers without exposing the sample to an external non-sterile environment.

[0270] 52. The system according to Embodiment 45, wherein the system does not require any intermediate reagents to be added externally from outside the closed end-to-end sterile environment during inline continuous processing of incoming samples.

[0271] 53. The system according to Embodiment 45, wherein the system does not require by-products to be removed outside the closed end-to-end sterile environment during in-line continuous processing of incoming samples.

[0272] 54. The sample has a volume of at least approximately 200 mL. The system according to embodiment 45, configured to process a sample to produce a product enriched with at least about 70% of one or more target particles in less than one hour.

[0273] 55. The system according to Embodiment 45, configured to process samples at a rate of approximately 300 mL / hour or more.

[0274] 56. A system for generating a product enriched in one or more target particles, One or more microfluidic cartridges configured to separate one or more target particles from a sample, A system comprising a cassette, the cassette on which one or more microfluidic cartridges are releasably coupled and supported, and the cassette including a mixer configured to mix the sample inline on the cassette with a diluent without using any moving parts before the sample circulates through the one or more microfluidic cartridges.

[0275] 57. The system according to embodiment 56, wherein the mixer includes a first fluid channel for the sample and a second fluid channel for the diluent.

[0276] 58. The system according to embodiment 57, wherein the first fluid channel and the second fluid channel merge to allow mixing of the sample and the diluent.

[0277] 59. The system according to embodiment 57, wherein the first fluid channel and the second fluid channel include a plurality of structural elements to facilitate in-line mixing of the sample and the diluent.

[0278] 60. (a) A step of preparing a system including a cassette in which one or more microfluidic cartridges are releasably coupled and supported, (b) A step of priming the cassette by passing a priming solution through the system, (c) A step of processing the sample by passing the sample through the system and separating one or more target particles from the system using one or more microfluidic cartridges, (d) a step of recovering a product containing one or more target particles separated from the sample, The sample has a volume of at least approximately 40 mL. The product is enriched by the recovery of at least approximately 70% of one or more target particles. Steps (b) to (d) are a method that is completed continuously in-line in a closed sterile environment for approximately one hour or less.

[0279] 61. The method according to embodiment 60, wherein step (b) is completed in approximately 20 minutes or less.

[0280] 62. The method according to Embodiment 60, wherein steps (c) and (d) are completed in approximately 40 minutes or less.

[0281] 63. The method according to embodiment 60, wherein the cassette and one or more microfluidic cartridges are configured for single use.

[0282] 64. Step (b) is the method of embodiment 60, which allows the cassette to be reused for multiple uses.

[0283] 65. The method according to embodiment 60, wherein step (b) allows one or more microfluidic cartridges to be reusable for multiple uses.

[0284] 66. The method according to any one of embodiments 60 to 65, wherein the sample is a human sample.

[0285] 67. The method according to any one of embodiments 60 to 66, wherein the sample comprises a blood-related product.

[0286] 68. The method according to embodiment 67, wherein the blood-related product includes an apheresis product.

[0287] 69. The method according to embodiment 68, wherein the apheresis product is a leukocyte apheresis product.

[0288] 70. The method according to any one of embodiments 60 to 69, wherein one or more target particles isolated from the sample include cells.

[0289] 71. The method according to embodiment 70, wherein the cells are human cells.

[0290] 72. The method according to Embodiment 70 or 71, wherein the cells have a viability of approximately 90% or more upon harvesting.

[0291] 73. The method according to any one of embodiments 70 to 72, wherein the cells include peripheral blood mononuclear cells.

[0292] 74. The method according to any one of embodiments 70 to 73, wherein the cells include CD3+ T cells.

[0293] 75. The method according to embodiment 74, wherein the T cells exhibit a naive or central memory phenotype.

[0294] 76. The method according to any one of embodiments 70 to 75, further comprising the step of culturing or growing cells in vitro.

[0295] 77. The method according to any one of embodiments 70 to 76, further comprising the step of transgenicizing cells using exogenous nucleic acids.

[0296] 78. The method according to embodiment 77, wherein the exogenous nucleic acid encodes a chimeric antigen receptor or a recombinant T cell receptor.

[0297] 79. The method according to Embodiment 60, wherein the sample has a volume of at least about 300 mL.

[0298] 80. The method according to Embodiment 60, wherein the sample has a volume of at least about 100 mL.

[0299] 81. The method according to Embodiment 60, wherein the product is enriched with the recovery of at least about 80% of one or more target particles.

[0300] 82. The method according to Embodiment 60, wherein the product is enriched with the recovery of at least about 90% of one or more target particles.

[0301] 83. The method according to Embodiment 60, wherein the product is enriched with the recovery of at least about 95% of one or more target particles.

[0302] 84. A step of displaying a graphical user interface (GUI) on a computer, wherein the GUI includes (i) a control panel and (ii) a visual representation of a system, the system comprising (a) a cassette on which one or more microfluidic cartridges are releasably coupled and supported, and (b) a plurality of components for facilitating fluid transport and process control. The steps include receiving user input for the execution protocol entered via the control panel, The system processes the sample by activating an execution protocol and separating one or more target particles from the sample using one or more microfluidic cartridges. A method comprising the steps of: displaying progress or status in substantially real time while the system is processing a sample, wherein the progress or status is depicted by graphical changes to the system's visual representation.

[0303] 85. The method according to embodiment 84, wherein the multiple components include a flow channel, a valve, a pressure sensor, and a pump.

[0304] 86. The method according to embodiment 85, wherein the multiple components further include one or more bubble sensors and at least one degassing unit.

[0305] 87. The method according to embodiment 86, wherein the graphic change includes an on / off state of one or more of a plurality of components.

[0306] 88. The method according to embodiment 86, wherein the graphic change includes the fluid flow of a sample or other medium through a cassette and one or more microfluidic cartridges.

[0307] 89. The method according to embodiment 84, further comprising the step of generating one or more notifications on a GUI indicating that the system is processing a sample in accordance with an execution protocol.

[0308] 90. The method according to embodiment 84, further comprising the step of generating one or more notifications on a GUI indicating that the system is experiencing one or more deviations from the execution protocol while a sample is being processed.

[0309] 91. The method according to embodiment 90, further comprising the step of generating one or more options on a GUI for a user to correct one or more deviations.

[0310] 92. The method according to Embodiment 90, further comprising the step of automatically reducing the pressure and flow rate of a sample when one or more deviations from the execution protocol are detected.

[0311] 93. The method according to embodiment 84, further comprising the step of generating a report including multiple run-by measurements when the system has completed processing the sample.

[0312] 94. The method according to Embodiment 84, wherein, when the system processes a sample, a GUI allows the user to observe the status and control the operation of one or more of several components in substantially real time.

[0313] 95. The system further includes one or more mass sensors, The system according to Embodiment 1, wherein the recirculation of one or more target particles through a cassette is controlled based on one or more readings obtained from one or more mass sensors.

[0314] 96. The system further includes a panel operably coupled to the cassette, The system according to embodiment 40, wherein at least one degassing unit is integrated onto a panel.

[0315] 97. The method according to Embodiment 60, wherein the sample has a volume of at least about 100 mL.

[0316] 98. The method according to Embodiment 60, wherein the sample has a volume of at least about 150 mL.

[0317] 99. The method according to Embodiment 60, wherein the sample has a volume of at least about 200 mL.

[0318] 100. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein leukocytes are enriched in at least about 85% or more of the resulting product.

[0319] 101. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein leukocytes are enriched in at least about 90% or more of the resulting product.

[0320] 102. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein leukocytes are enriched in at least about 85% or more of the resulting product.

[0321] 103. At least approximately 2 × 10 9 A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein 100 leukocytes are obtained from at least about 200 mL of LeucoPac.

[0322] 104. At least approximately 2 × 10 9 A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein 100 leukocytes are obtained from at least about 300 mL of LeucoPac.

[0323] 105. At least approximately 2 × 10 9 A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein 100 mL of leukocytes are obtained from 100 mL of LeucoPac.

[0324] 106. At least approximately 5 × 10 9 A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein 100 leukocytes are obtained from at least about 200 mL of LeucoPac.

[0325] 107. At least approximately 5 × 10 9 A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein 100 leukocytes are obtained from at least about 300 mL of LeucoPac.

[0326] 108. At least approximately 5 × 109 A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein 100 leukocytes are obtained from at least about 100 mL of LeucoPac.

[0327] 109. The enriched leukocytes have a telomere length of at least about 2 kilobases, according to the system described in any of Embodiments 1 to 59 or the method described in any of Embodiments 60 to 99.

[0328] 110. The enriched leukocytes have a telomere length of at least about 3 kilobases, according to the system described in any of Embodiments 1 to 59 or the method described in any of Embodiments 60 to 99.

[0329] 111. The enriched leukocytes have a telomere length of at least about 5 kilobases, according to the system described in any of Embodiments 1 to 59 or the method described in any of Embodiments 60 to 99.

[0330] 112. At least approximately 4 × 10 8 A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein naive T cells are obtained from at least about 200 mL of LeucoPac.

[0331] 113. At least approximately 4 × 10 8 A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein naive T cells are obtained from at least about 300 mL of LeucoPac.

[0332] 114. At least approximately 4 × 10 8 A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, wherein naive T cells are obtained from at least about 100 mL of LeucoPac.

[0333] 115. At least approximately 5 × 10 8A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein at least 200 mL of LeucoPac is obtained from a central memory T cell.

[0334] 116. At least approximately 5 × 10 8 A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein at least 300 mL of LeucoPac is obtained from a central memory T cell.

[0335] 117. At least approximately 5 × 10 8 A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, wherein at least 100 mL of LeucoPac is obtained from LeucoPac.

[0336] 118. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which yields a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more leukocytes compared to the number of leukocytes obtained by density gradient centrifugation.

[0337] A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, which yields a product comprising CD3+ cells, CD45+ cells, CD4+ cells, CD4+ naive cells, CD4+ naive cells, CD8+ effector cells, CD8+ memory cells, memory cells, effector cells, naive cells, Temra cells, or any combination thereof.

[0338] 120. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which yields a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more CD45+ cells compared to that obtained by using density gradient centrifugation.

[0339] 121. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which yields a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more CD3+ cells compared to that obtained by using density gradient centrifugation.

[0340] 122. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, further comprising the step of removing one or more target particles from a sample.

[0341] 123. The method or system of Embodiment 122, wherein the target particle is a cell.

[0342] 124. The method or system according to Embodiment 123, wherein the target particles are red blood cells or platelets.

[0343] 125. The method or system according to Embodiment 124, wherein at least about 95% of red blood cells or platelets are removed from the sample.

[0344] 126. The method or system according to Embodiment 124, wherein at least 95% of platelets are removed from the sample.

[0345] 127. A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, which produces a product containing a mixture of red blood cells and white blood cells in a ratio of 2.5:1, 1.5:1, or less than 0.7:1.

[0346] 128. A system according to any one of Embodiments 1 to 59 or a method according to any one of Embodiments 60 to 99, which produces a product containing a mixture of platelets and leukocytes in a ratio of less than 9:1, 5:1, 3:1, or 1.1:1.

[0347] 129. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more CD4+ cells compared to that obtained by using density gradient centrifugation.

[0348] 130. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more CD8+ cells compared to that obtained by using density gradient centrifugation.

[0349] 131. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more naive CD4+ cells compared to that obtained by using density gradient centrifugation.

[0350] 132. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more memory CD4+ cells compared to that obtained by using density gradient centrifugation.

[0351] 133. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more effector CD4+ cells compared to that obtained by using density gradient centrifugation.

[0352] 134. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more naive CD8+ cells compared to that obtained by using density gradient centrifugation.

[0353] 135. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more memory CD8+ cells compared to that obtained by using density gradient centrifugation.

[0354] 136. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more effector CD8+ cells compared to that obtained by using density gradient centrifugation.

[0355] 137. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 5 times or more leukocytes compared to the number of leukocytes obtained by using density gradient centrifugation.

[0356] 138. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about 1.5 times, about 2.5 times, or about 3 times fewer red blood cells than the product obtained by using density gradient centrifugation.

[0357] 139. A system according to any of Embodiments 1 to 59 or a method according to any of Embodiments 60 to 99, which produces a product containing at least about three times, or about ten times, fewer platelets than the product obtained by using density gradient centrifugation.

[0358] 140. A population of cells produced by any system of Embodiments 1 to 59 or by any method of Embodiments 60 to 99, wherein the population of cells exhibits an increase in one or more biological properties compared to a population of cells produced by density gradient centrifugation. Here, one or more biological properties are selected from the list consisting of the ability to readily introduce lentiviral vectors, the ability to proliferate in culture, the ability to retain T cell memory compositions during cell culture, receptivity to viral transduction, differentiation of mean telomere length, the ability to retain a relative population of poorly differentiated naive and central memory cells, functional killing ability, IFNγ expression, GM-CSF expression, TNF-α expression, and viability.

[0359] 141. A population of cells according to Embodiment 140, the population of cells exhibits at least about 25% increased ability to facilitate lentiviral vectors compared to a population of cells produced by density gradient centrifugation.

[0360] 142. A population of cells according to Embodiment 140, wherein the population of cells exhibits an increased ability to grow during culture, and the cells grow before or after genetic modification compared to a population of cells produced by density gradient centrifugation.

[0361] 143. A population of cells according to Embodiment 142, wherein the population of cells exhibits at least approximately 1.5 times increased capacity during culture compared to a population of cells produced by density gradient centrifugation.

[0362] 144. A population of cells according to Embodiment 140, the population of cells exhibits an increased ability to retain a T cell memory composition during cell culture compared to a population of cells produced by density gradient centrifugation, and includes retaining the same T cell memory composition at least 9 days after culturing.

[0363] 145. A population of cells according to Embodiment 140, the population of cells exhibits at least a 30% increase in receptivity to viral transduction compared to a population of cells produced by density gradient centrifugation.

[0364] 146. A population of cells according to Embodiment 140, wherein the population of cells shows at least a 30% increase in telomere length compared to a population of cells produced by density gradient centrifugation.

[0365] 147. A population of cells according to Embodiment 140, wherein the population of cells exhibits an increased ability to retain a relative population of poorly differentiated naive and central memory cells during cell culture, compared to a population of cells produced by density gradient centrifugation, and retains substantially the same relative population of poorly differentiated naive and central memory cells after at least 9 days of culture.

[0366] 148. A population of cells according to Embodiment 140, the population of cells exhibits at least a 30% increase in functional lethality compared to a population of cells produced by density gradient centrifugation.

[0367] 149. A population of cells according to Embodiment 140, the population of cells shows at least a twofold increase in IFNγ expression after 9 days of culture compared to a population of cells produced by density gradient centrifugation.

[0368] 150. The cell population of Embodiment 140 shows a 1.5-fold increase in GM-CSF expression after 9 days of culture, even if not, compared to the cell population produced by density gradient centrifugation.

[0369] 151. A population of cells according to Embodiment 140, the population of cells shows at least a 10% increase in TNF-α expression after 9 days of culture compared to a population of cells produced by density gradient centrifugation.

[0370] 152. A population of cells according to Embodiment 140, wherein the population of cells exhibits at least a 10% increase in viability compared to a population of cells produced by density gradient centrifugation.

[0371] 153. A population of cells produced by any system of Embodiments 1 to 59 or by any method of Embodiments 60 to 99, wherein the population of cells exhibits a reduction in one or more biological properties compared to a population of cells produced by density gradient centrifugation. Here, one or more biological properties are selected from the list consisting of the time required to grow in culture and produce a single therapeutic dose equivalent of cells, the time required to express the gene delivered by the vector, the relative population of effector or Temra cells, IL-1Ra expression, IL-6 expression, IL-13 expression, MCP-1 expression, PD1 and Tim3 co-expression, cellular senescence or depletion, tendency to induce cytokine release syndrome, and the culture time required before delivery to a patient.

[0372] 154. A population of cells according to Embodiment 153, the population of cells exhibits a reduction of at least 3 days in the time required to grow in culture and produce a single therapeutic dose equivalent of cells, compared to a population of cells produced by density gradient centrifugation.

[0373] 155. A population of cells according to Embodiment 153, the population of cells exhibits a reduction of at least one day in the time required to express the vector-delivered gene compared to a population of cells produced by density gradient centrifugation.

[0374] 156. A population of cells according to Embodiment 153, wherein the population of cells shows at least a 10% reduction in the relative population of effector or Temra cells compared to a population of cells produced by density gradient centrifugation.

[0375] 157. A population of cells according to Embodiment 153, the population of cells shows at least a 40% reduction in IL-1Ra expression after 13 days of culture compared to a population of cells produced by density gradient centrifugation.

[0376] 158. A population of cells according to Embodiment 153, the population of cells shows at least a 60% reduction in IL-6 expression after 13 days of culture compared to a population of cells produced by density gradient centrifugation.

[0377] 159. A population of cells according to Embodiment 153, the population of cells exhibits at least a 10% reduction in IL-13 expression compared to a population of cells produced by density gradient centrifugation.

[0378] 160. A population of cells according to Embodiment 153, the population of cells exhibits at least a 20% reduction in MCP-1 expression compared to a population of cells produced by density gradient centrifugation.

[0379] 161. A population of cells according to Embodiment 153, the population of cells exhibits at least a 50% reduction in PD1 and Timp3 co-expression compared to a population of cells produced by density gradient centrifugation.

[0380] 162. A population of cells according to Embodiment 153, wherein the population of cells exhibits at least a 50% reduction in cellular senescence or depletion compared to a population of cells produced by density gradient centrifugation.

[0381] 163. A population of cells according to Embodiment 153, the population of cells exhibits at least a 20% reduction in the tendency to cause cytokine release syndrome compared to a population of cells produced by density gradient centrifugation.

[0382] 164. A population of cells according to Embodiment 153, wherein the population of cells exhibits a reduction of at least 3 days in the culture time required before delivery to a patient.

[0383] 165. In any one of the cell populations of Embodiments 140 to 164, compared to a population of cells produced by density gradient centrifugation, one or more indicated increases or decreases in biological properties become apparent at least about 0, 3, 6, 9, 13, or 16 days after production.

Claims

1. A system for generating enriched products in one or more target particles, A cassette on which one or more microfluidic cartridges are releasably coupled and supported, comprising: (i) a plurality of inlets having a plurality of input vessels releasably and fluidly coupled thereto, at least one of the plurality of input vessels containing an incoming sample; (ii) a plurality of outlets having a plurality of output vessels releasably and fluidly coupled thereto; (iii) one or more microfluidic cartridges for separating one or more target particles from the sample; and (iv) a plurality of fluid channels extending between the plurality of inlets, the plurality of outlets and the one or more microfluidic cartridges, A cassette having multiple input containers, multiple output containers, and one or more microfluidic cartridges coupled thereto provides a closed, end-to-end sterile environment that enables in-line continuous processing of incoming samples without external manual operation or intervention. Multiple inlets and multiple input vessels are releasable and fluidly coupled using multiple sterilization coupling mechanisms. A system in which one or more target particles are enriched, producing a contamination-free product.

2. The system according to claim 1, wherein the product is collected in at least one of a plurality of output containers without exposing the product to an external non-sterile environment.

3. The system according to claim 1, wherein the sample is input into the cassette from at least one of a plurality of input containers without exposing the sample to an external non-sterile environment.

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