Apparatus, systems, and methods of analyzing biological particles

Abstract

Described herein are inline flow cells having at least one inlet configured to receive a fluid comprising biological particles; a body in fluid communication with the at least one inlet, the body configured to form a plurality of flow channels containing the biological particles; and at least one energy source (e.g., a light source, laser, acoustic source, etc.) configured to traverse light through the plurality of flow channels, wherein, upon interaction with the traversed light, the biological particles generate at least one signal. Further described are systems containing at least one inline flow cell and methods of using inline flow cells to measure biological particles.

Description

OK Ref.11080007.01098 APPARATUS, SYSTEMS, AND METHODS OF ANALYZING BIOLOGICAL PARTICLES FIELD

[0001] The present disclosure relates to apparatus, systems, and methods of analyzing biological particles using, for example, an inline flow cell. BACKGROUND

[0002] In biopharmaceutical analysis, compositions of biological samples can be complex. Due to the diversity of biological matrices, the analysis of target substances in these samples presents significant challenges to sample processing. Typically, to measure biological particles such as cells within a sample, the biological particles are labelled with different mass tags, and then analyzed using, for example, high throughput cell labeling, mass cytometry, and / or tandem mass tagging. However, these techniques may destroy the cells during the process, are unable to sort cells for downstream analysis of specific cell populations, and generally have a low sample throughput. Moreover, methods such as mass cytometry utilize heavy metal reporters having high sensitivity, making it difficult to detect antigens or cellular components expressed at low levels within a cell, and these methods rely on pre-selected targets (e.g., antibodies against specific proteins) that limit analysis to particular markers. In addition, these methods are not conducive to on-line processing and automation.

[0003] What is needed therefore, are efficient devices, systems and methods for native detection and analysis of biological samples (i.e., the ability to detect and analyze biological particles in their original state, without the need for extensive processing or labeling) in real-time with one or more analytical instruments. Preferably such devices, systems and methods should be easy to implement, low cost, efficient, reliable and compatible with instruments such as laser force cytology instruments.OK Ref.11080007.01098 BRIEF SUMMARY

[0004] According to various embodiments, described herein are inline flow cells, comprising one or more of the following: at least one inlet configured to receive a fluid comprising biological particles; a body in fluid communication with the at least one inlet, the body configured to form a plurality of flow paths containing the biological particles; and at least one light source configured to traverse light through the plurality of flow paths, and / or at least one light capturing device configured to receive light from the inline flow cell, wherein, upon interaction with the traversed light, the biological particles generate at least one signal.

[0005] In one or more embodiments, described herein are inline flow cells for analyzing biological particles, comprising one or more of the following: a first inlet configured to receive a first fluid; a second inlet configured to receive a second fluid comprising biological particles; a body in fluid communication with the first inlet and the second inlet, wherein the body is configured to form a plurality of flow paths; a wall disposed within the body, the wall comprising a plurality of openings configured to receive small biological particles having a size of less than about 50 µm from the second fluid; and at least one source of energy configured to traverse energy through the body to measure and / or move the small biological particles, wherein the small biological particles are received in the plurality of flow paths.

[0006] In various embodiments, in inline flow cells, comprise one or more of the following: an inlet configured to receive a fluid comprising biological particles, optionally wherein the biological particles include, but are not limited to, T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof; a body including a pass-through flow path in fluid communication with an outlet; a plurality of flow paths or collectors, configured to receive biological particles of varying size; and at least one laser configured to apply a force to one or more of the biological particles entering the body through the inlet.

[0007] In embodiments, disclosed herein are in inline flow cells, comprising one or more of the following: at least one inlet configured to receive a fluid comprising biological particles; a body configured to form a plurality of flow paths or collectors containing the biological particles; andOK Ref.11080007.01098 at least one of an acoustic analyzer, laser source, light emitting diode source, brightfield source, darkfield source, Epi illumination source, or camera configured to traverse energy through the plurality of flow paths, wherein, upon interaction with the energy, the biological particles generate at least one signal.

[0008] Further disclosed herein are systems for analyzing biological particles, comprising one or more of the following: a supply reservoir configured to store a fluid comprising the biological particles; at least one inline flow cell according to the various embodiments described herein in fluid communication with the supply reservoir; at least one biological particle analyzer comprising a light source, acoustic analyzer, camera, or combinations the at least one biological particle analyzer configured to receive, analyze, and / or image an optical or acoustical signal from the biological particles; and a processor in communication with the at least one biological particle analyzer, wherein the biological particles include, but are not limited to, T-cells, engineered T- cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β- cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

[0009] Further described herein according to various embodiments are systems for analyzing biological particles, comprising one or more of the following: a supply reservoir configured to store a fluid comprising the biological particles; a first inline flow cell according to embodiments described herein in fluid communication with the supply reservoir; a second inline flow cell according to embodiments described herein in fluid communication with the supply reservoir; at least one biological particle analyzer comprising a light source, acoustic analyzer, camera, or combinations thereof, the at least one biological particle analyzer configured to receive, analyze, and / or image an optical or acoustical signal from the biological particles in the first inline flow cell, the second inline flow cell, or both; and a processor in communication with the at least one biological particle analyzer, wherein the biological particles include, but are not limited to, T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells,OK Ref.11080007.01098 endocrine cells, β-cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

[0010] In further embodiments, described herein are methods of analyzing biological particles in a sample, comprising one or more of the following: receiving a fluid in at least one inline flow cell according to embodiments described herein, wherein the fluid comprises a plurality of biological particles; introducing light into the flow cell, wherein the light interacts with the plurality of biological particles in the fluid and forms at least one signal; using the at least one signal to identify each of a plurality of flow paths containing one or more of the plurality of biological particles; and measuring the one or more of the plurality of biological particles within the identified flow paths using at least one biological particle analyzer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The foregoing summary, as well as the following detailed description of illustrative embodiments of the present application, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the present application, there is shown in the drawings illustrative embodiments of the disclosure. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown:

[0012] FIG.1 is a perspective view of an inline flow cell having a plurality of mechanical channels according to one or more embodiments of the disclosure;

[0013] FIG.2 is a perspective view of an inline flow cell having a plurality of fluidic flow paths according to one or more embodiments of the disclosure.

[0014] FIG.3 is a schematic of an inline flow cell having a plurality of flow channels each with a non-uniform geometry according to one or more embodiments of the disclosure.

[0015] FIG.4 is a schematic of an inline flow cell having an analysis region and a counting region according to one or more embodiments of the disclosure.

[0016] FIG.5A is a deconstructed view of an inline flow cell formed of three (3) layers according to one or more embodiments of the disclosure.

[0017] FIG. 5B is a perspective view of an inline flow cell constructed of three (3) layers according to one or more embodiments of the disclosure.OK Ref.11080007.01098

[0018] FIG.5C is a top view of an inline flow cell constructed of three (3) layers according to one or more embodiments of the disclosure.

[0019] FIG.6 is a schematic of an inline flow cell having an energy source configured to apply a force to biological particles received in the inline flow cell and separating the biological particles by size into flow paths and / or bins according to one or more embodiments.

[0020] FIG.7 is a schematic of a plurality of flow cells operating in parallel according to one or more embodiments.

[0021] FIG. 8 is a schematic of a flow cell constructed of multiple channels and a membrane bonded therebetween according to one or more embodiments.

[0022] FIG. 9 is a schematic of an inline flow cell having tangential flow side channels, a membrane, and a bottom channel according to one or more embodiments of the disclosure.

[0023] FIG.10 is a schematic of an inline flow cell having dean flow side channels according to one or more embodiments of the disclosure.

[0024] FIG. 11 is a schematic of an inline flow cell having a size-based particle sorting configuration according to one or more embodiments.

[0025] FIG. 12 is a schematic of an inline flow cell having a size-based particle sorting configuration according to one or more embodiments.

[0026] FIG.13 is a schematic of an inline flow cell according to one or more embodiments of the disclosure.

[0027] FIG.14 is a schematic of an inline flow cell according to one or more embodiments of the disclosure.

[0028] FIG. 15 is a schematic of a camera arrangement used for imaging biological particles in an inline flow cell according to one or more embodiments.

[0029] FIG. 16 is a schematic of a camera arrangement used for imaging biological particles in an inline flow cell according to one or more embodiments.

[0030] FIG. 17 is a schematic of a camera arrangement having illumination used for imaging biological particles in an inline flow cell according to one or more embodiments.

[0031] FIG.18 is a schematic of an inline flow cell having two analysis regions according to one or more embodiments.

[0032] FIG. 19 is a schematic of an inline flow cell having calibration targets and servo motors according to one or more embodiments of the disclosure.OK Ref.11080007.01098

[0033] FIG.20A is a schematic of an inline flow cell having a plurality of channels the images of which may be distorted based on flow according to one or more embodiments.

[0034] FIG.20B is a schematic of an inline flow cell having a plurality of channels, the images of which may be distorted based on flow according to one or more embodiments.

[0035] FIG.21A is an image of a first tracked population of biological particles entering a field of vision of an inline flow cell according to one or more embodiments.

[0036] FIG. 21B is an image of a second tracked population of biological particles flowing through an inline flow cell according to one or more embodiments.

[0037] FIG.21C is an image of a bounded region drawn between the first tracked population of biological particles and the second tracked population of biological particles of the inline flow cell according to one or more embodiments.

[0038] FIG. 21D is an image of a bounded region sent to an analyzer according to one or more embodiments.

[0039] FIG. 21E is an image of a third tracked population of biological particles and bounded region drawn between the second tracked population and the third tracked population of biological particles according to one or more embodiments.

[0040] FIG. 21F is an image of a bounded region sent to an analyzer according to one or more embodiments.

[0041] FIG.22 is a schematic of a system comprising an inline flow cell suitable to dispense fluid into a receptacle according to one or more embodiments.

[0042] FIG.23 is a schematic of a system comprising a plurality of inline flow cells according to one or more embodiments.

[0043] FIG.24 is a schematic of a system comprising an inline flow cell suitable to dispense fluid into a plurality of receptacles according to one or more embodiments.

[0044] FIG.25 is a schematic of a system comprising an inline flow cell suitable to dispense fluid into an implantable medical device according to one or more embodiments. DEFINITIONS

[0045] Before describing various embodiments of the present invention in detail, it is to be understood that the terminology used in the specification is for the purpose of describing particularOK Ref.11080007.01098 embodiments, and is not necessarily intended to be limiting. Although many methods, structures and materials similar, modified, or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, preferred methods, structures and materials are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0046] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a light source” includes a single light source as well two or more of the same or different light sources.

[0047] As used herein, the term “about” in connection with a measured quantity or time, refers to the normal variations in that measured quantity or time, as expected by one of ordinary skill in the art in making the measurement and exercising a level of care commensurate with the objective of measurement. In certain embodiments, the term “about” includes the recited number ±10%, such that “about 10” would include from 9 to 11, or “about 1 hour” would include from 54 minutes to 66 minutes.

[0048] The term “at least about” in connection with a measured quantity refers to the normal variations in the measured quantity, as expected by one of ordinary skill in the art in making the measurement and exercising a level of care commensurate with the objective of measurement and precisions of the measuring equipment and any quantities higher than that. In certain embodiments, the term “at least about” includes the recited number minus 10% and any quantity that is higher such that “at least about 10” would include 9 and anything greater than 9. This term can also be expressed as “about 10 or more.” Similarly, the term “less than about” typically includes the recited number plus 10% and any quantity that is lower such that “less than about 10” would include 11 and anything less than 11. This term can also be expressed as “about 10 or less.”

[0025] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to illustrate certain materials and methods and does not pose a limitation on scope.OK Ref.11080007.01098

[0049] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0050] The term “biological particles” as used herein refers to biologic structures, including T- cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof. Cell clusters may be comprised of more than one cell of one or more biological cell types or classes joined together. DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

[0051] Reference will now be made in detail to the various embodiments of the present disclosure illustrated in the accompanying drawings. Wherever possible, the same or like reference numbers will be used throughout the drawings to refer to the same or like features. It should be noted that the drawings are in simplified form and are not drawn to precise scale. In reference to the disclosure herein, for purposes of convenience and clarity only, directional terms such as top, bottom, left, right, above, below and diagonal, are used with respect to the accompanying drawings. Such directional terms used in conjunction with the following description of the drawings should not be construed to limit the scope of the present disclosure in any manner not explicitly set forth.

[0052] Disclosed herein according to various embodiments are inline flow cells suitable for analysis of biological particles. Further disclosed are related systems and methods for measuring biological particles. In some embodiments, described is a cell and cell cluster counting device, systems and methods of using the same. In some embodiments, the inline flow cells are configured to slow flow biological particles in a sample through parallel flow paths (e.g., fluidic or mechanical) into a field of view for analyzing (e.g., imaging) the biological particles. The parallel flow paths may be formed from a single input or multiple inputs to one or more inline flow cells. Inline flow cells according to embodiments herein alternatively may include non-parallel flow paths.OK Ref.11080007.01098

[0053] In various embodiments, an analyzer and / or analysis system is configured to enumerate the exact number of biological particles (e.g., cells, cell clusters, etc.) to fill the device, for example, when sample flow through the device is paused. The analyzer and corresponding system may include an energy source including, but not limited to, brightfield, darkfield, phase, laser, LED, camera imaging source, light scattering source, impedance source, line camera, dual cameras, or any combination thereof for measuring, enumerating, an acoustic wave generator, and / or analyzing the biological particles (e.g., counting) in the sample. In some embodiments, the system further comprises a computational system for real-time analysis. The computational system may utilize an algorithm for using imaging and light scatter for improved counting. Also described are methods for measuring the size and / or counting the number of cells or cell clusters in a sample.

[0054] According to various embodiments, inline flow cells and corresponding systems may include one or more additional components including, but not limited to, a cell cluster reservoir, a fillable device, a waste reservoir (or output stream), one or more fillable devices (e.g., sample containers, test tubes, vials, beakers, etc.), an intermediate holding area, an implantable device, one or more channel separators, a flat mirror that is optionally configured to rotate, a curved mirror (or microfluidic device), an illuminator, a calibration target, a large diameter channel for debris, a tangential flow channel, a porous membrane, a sheath fluid (e.g., water, buffer, etc.) to push the sample downwards towards the porous membrane or a bottom channel, an inertial flow side channel, a barrier, and / or combinations thereof. Methods and systems as described herein may include one or more flow cells including one or more of these additional components. Inline flow cells and corresponding systems and methods may be used to count, image and / or otherwise analyze biological particles according to embodiments herein.

[0055] Inline flow cells, systems and methods as described herein are suitable to measure biological particles such as pancreatic islet cells and / or pancreatic endocrine cells (e.g., β-cells, α- cells, δ-cells and FC cells) formed by the methods described in U.S. Patent Nos. 11,992,506, 12,006,516, 12,049,645, and 12,173,324 the contents of which are incorporated by reference herein in their entirety. These patents are directed to inter alia compositions and methods for differentiation of stem cells into pancreatic endocrine cells and / or pancreatic islet cells. In some aspects, the methods described relate to generation of pancreatic β-cells, α-cells, δ-cells and FC cells in vitro. In some aspects, the patents describe pharmaceutical compositions including the cells generated according to the methods disclosed, as well as methods of treatment making useOK Ref.11080007.01098 thereof. The cells and compositions as described in these patents may be analyzed and / or sorted using the inline flow cells, systems and methods according to embodiments herein.

[0056] Inline flow cells, systems and methods as described herein are further suitable to measure biological particles (e.g., β cells and precursors thereof) produced and used by the methods of U.S. Patent Nos. 11,466,256, 11,525,120, and 11,999,971 each of which is incorporated by reference herein in its entirety. These patents are directed to inter alia methods of producing β cells and precursors thereof utilizing a WNT signaling inhibitor or PKC activator, or both. These patents also describe in vitro cultures comprising such cells, methods of treating a subject with a disease characterized by high blood pressure levels over a prolonged period of time by administering these cells, and devices for encapsulating such cells. The cells and compositions as described in these patents may be analyzed and / or sorted using the inline flow cells, systems and methods according to embodiments herein.

[0057] Inline flow cells, systems and methods as described herein are further configured to measure biological particles (e.g., SC- β cells) produced and used by the methods of U.S. Patent No.11,945,795 the contents of which is incorporated by reference herein its entirety. This patent is directed to inter alia compositions and methods useful for manufacturing SC-β cell and isolated populations of SC-β cells for use in various applications, such as cell therapy. The cells and compositions as described in these patents may be analyzed and / or sorted using the inline flow cells, systems and methods according to embodiments herein.

[0058] Inline flow cells, systems and methods as described herein are further configured to utilize the principles and methods as described in U.S. Patent No.10,281,385, which is incorporated by reference herein in its entirety. This patent is directed to inter alia a device including a collimated light source operable to generate a collimated light source beam, which includes a beam direction. The device includes a first channel in a first plane and a second channel in a second plane different from the first plane. The second channel communicates with the first channel and includes a flow direction. The second channel is oriented to receive the collimated light source beam. The device includes a third channel in a third plane different from the second plane and communicates with the second channel. The collimated light source beam is orientated to enter a cross-section of the first channel, then to pass through the second channel, and then to enter a cross-section of the third channel such that the beam direction is opposite to the flow direction in the second channel. The device includes a focused particle stream nozzle operably connected to the first channel.OK Ref.11080007.01098

[0059] Inline flow cells, systems and methods as described herein are configured to implement the principles and methods as described in U.S. Patent No. 10,730,050 the contents of which are incorporated by reference herein in its entirety. This patent is directed to integrating optical forces and electrokinetics allows for the pooled separation vectors of each to be applied, providing for separation based on combinations of features such as size, shape, refractive index, charge, charge distribution, charge mobility, primitivity and deformability. The interplay of these separation vectors allows the selective manipulation of analytes with a finer degree of variation. Embodiments include methods of separating particles in a microfluidic channel using a device comprising a microfluidic channel, a source of laser light focused by an optic into the micro fluidic channel and a source of electrical field operationally connected to the microfluidic channel via electrodes so that the laser light and the electrical field to act jointly on the particles in the microfluidic channel.

[0060] Inline flow cells, systems and methods as described herein are further configured to implement the principles and methods as described in U.S. Patent No.11,913,870 the contents of which are incorporated by reference herein in its entirety. This patent is directed to methods and devices for assessing biological particles for use in cell immunotherapy. By utilizing a microfluidic chip device together with optical force measurement and cell imaging, the methods enable comprehensive assessment and characterization of biological particles with regard to morphology, motility, binding affinities, and susceptibility to external forces, including but not limited to, chemical, biochemical, biological, physical and temperature influences. The methods enable the selection and production of biological particles, such as engineered T-cells, for use in immunotherapy and biomanufacturing.

[0061] Inline flow cells, systems and methods as described herein are further configured to implement the principles and methods as described in U.S. Patent Application Serial No. 16 / 982,935, which is incorporated by reference herein in its entirety. This patent is directed to methods and devices for assessing biological particles for use in cell immunotherapy. By utilizing a microfluidic chip device together with optical force measurement and cell imaging, the methods enable comprehensive assessment and characterization of biological particles with regard to morphology, motility, binding affinities, and susceptibility to external forces, including but not limited to, chemical, biochemical, biological, physical and temperature influences. The methodsOK Ref.11080007.01098 enable the selection and production of biological particles, such as engineered T-cells, for use in immunotherapy and biomanufacturing.

[0062] Inline flow cells, systems and methods as described herein are further configured to implement the principles and methods as described in U.S. Patent Application Serial No. 17 / 470,639 the contents of which are incorporated by reference herein in their entirety. This patent is directed to devices for automated analysis of one or more samples in single or multi-well plates or vessels, wherein the process of automated analysis comprises automated flow, wherein the samples comprise liquid or particles in a sample vessel, and wherein the devices comprise an assembly of components that enable processing of a sample for analytical assessment by fluidic and / or particle based instruments. Automated flow may comprise systems for moving samples including vacuum systems, pressure-based systems, pneumatic systems, pumps, peristaltic pumps, diaphragms, or syringes. The devices may comprise an assembly of components that enable movement in X, Y, and Z dimensions, as well as switches, microfluidic tubing, well plate block, electronic pressure controllers, pneumatic or fluidic mixing devices, components for fluid handling, sampling vessels, and mechanical components for translating or transporting system components.

[0063] Inline flow cells, systems and methods as described herein are further configured to implement the principles and methods as described in U.S. Patent Application Serial Nos. 17 / 370,457 and 18 / 220,772 the contents of which are incorporated by reference herein in their entirety. These applications are directed to methods and devices for automated analysis of one or more samples in single or multi-well plates or vessels, wherein the process of automated analysis comprises flow and hydrodynamic, electrokinetic, and optical forces for the analysis and sorting of samples, wherein the samples comprise liquid or particles in microfluidic channels, and wherein the devices comprise an assembly of components that enable processing of a said samples for analytical assessment by fluidic and / or particle based instruments. Microfluidic structures (channels, “T's”, “Y's”, branched “Y's”, wells, and weirs) are described for facilitating sample interaction and observation, sample analysis, sorting, or isolation. Detection can be accomplished using spectroscopic methods including, but not limited to, Raman spectroscopy of single cells and bulk cellular samples (collections of cells; several individuals to hundreds or thousands of cells).

[0064] Inline flow cells, systems and methods as described herein are further configured to implement the principles and methods as described in U.S. Patent Application Serial No.OK Ref.11080007.01098 18 / 275,997, which is incorporated by reference herein in its entirety. This patent is directed to improved biomanufacturing devices, systems and methods for using the same for monitoring biologic products such as biologics, vaccines, cell and gene therapies for viral safety and identification of cytopathic effect. In certain embodiments, the embodiments described herein enable the objective analysis of adventitious agents including adventitious viruses, bacteria and mycoplasma. Inline Flow Cells for Sorting and / or Analyzing Biological Particles

[0065] According to various embodiments described herein are inline flow cells for sorting and / or analyzing biological particles. An embodiment of an inline flow cell 100 is shown in FIG. 1. Inline flow cell 100 includes a body a body in fluid communication with the at least one inlet 104. Inlet 104 and body 102 are configured to receive a fluid containing a plurality of biological particles 112. Suitable fluids include, but are not limited to, water, a buffer, blood, or combinations thereof. The biological particles 112 include, but are not limited to, biologic structures including T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

[0066] According to various embodiments, a sample comprised of a carrier fluid (e.g., water, buffer, blood, etc.) and biological particles is received by the inlet 104 and into body 102. The sample may be at a flow rate and pressure suitable to fill the entire internal space of body 102. In some embodiments, the sample flows through body 102 at a laminar flow rate. In various embodiments, each of the plurality of flow paths 108 is configured to provide about the same flow rate of the fluid when flowing therethrough. In one or more embodiments, each of the plurality of flow paths 108 is configured to provide about the same resistance to the fluid as each other’s flow path when flowing therethrough. According to various embodiments, the parallel flow paths 108 may be suitable to slow the sample flow after its entry into the inlet 104. The sample, once flowing at a slower rate, may be configured to fill a field of view 109 of an optional at least one energy source 110. When implemented, the at least one energy source 110 may traverse energy throughOK Ref.11080007.01098 the plurality of channels for detection by an analyzer to measure, image, and / or analyze the biological particles (e.g., small / large of cells or cell clusters). Inline flow cells according to embodiments herein may have varying cross-sections disposed upstream from the plurality of flow paths to hydrodynamically position biological particles in an equilibrium position before entering the plurality of flow paths. For example, the inlets may be tapered and / or the flow cell body may include one or more tapered sections.

[0067] Fields of view as described herein may refer to analysis regions within the inline flow cells. The analysis region may be a singular shape and / or channel in which the flow of flow paths of equal flow rate Q combine to normalize the velocity distribution of biological particles entering the analysis region. Before the analysis region, flow paths may be arranged such that each flow channel has about the same resistance and Q. Before the analysis region, particles may flow at 1 > Rp > 7 such that most particles share a singular focal plane.

[0068] The Reynolds number of the sample when flowing through the body 102 may be about 1 to about 2300, about 10 to about 2000, about 50 to about 1000, about 1 to about 10, less than 2300, less than 2000, less than 1000, less than 20, or any individual value or sub-range within these ranges. The Reynolds number may be determined using the following formula: ^^^^^^^^^^^^ ^^^^^^^^ =^^^^ρ is the fluid density; u is the fluid velocity; L is a characteristic length scale (e.g., diameter of the pipe or channel). μ is the dynamic viscosity of the fluid as measured using ASTM D2196-20 or D7042.

[0069] The inline flow cell 100 may be oriented vertically and configured to receive the sample from the inlet 104 at the top such that flow is in the direction of gravity toward the outlet 106. In one or more embodiments, inline flow cell 100 is free of horizontal flow paths for sample flow. In some embodiments, the inlet 104 is disposed at the center of the top wall of the body 102 as shown in FIG. 1. In some embodiments, the inlet 104 is disposed off-center (not shown) with respect to a center line of the body 102. The inlet 104 may be disposed on a top wall of the body 102, or a side wall of the body 102. In at least one embodiment, the inlet has a cross-section or orifice size substantially equivalent to a depth of the body 102 as shown in FIG.1. The length orOK Ref.11080007.01098 diameter of the body 102 may be about 2 to about 10 times the cross-section or orifice size of the inlet 104. In yet further embodiments, the inlet 204 is on the bottom wall of body 102 and the outlet 206 is on the top wall of body 102.

[0070] According to various embodiments, the inlet 106 is disposed at the center of the bottom wall of the body 102 as shown in FIG.1. In some embodiments, the outlet 106 is disposed off- center (not shown) with respect to a center line of the body 102. The outlet 106 may be disposed on a top wall of the body 102, or a side wall of the body 102. In at least one embodiment, the outlet 106 has a cross-section or orifice size substantially equivalent to a depth of the body 102 as shown in FIG.1. The length or diameter of the body 102 may be about 2 to about 10 times the cross-section or orifice size of the outlet 106. In one or more embodiments, the body has a greater internal volume than the inlet 104 or the outlet 106.

[0071] Body 102 is configured to form a plurality of flow paths 108 through which individual biological particles or clusters thereof can flow through to an outlet 106. The term “flow path” as used herein refers to a substantially linear path that one or more biological particles travels as it flows through body 102. The height of each flow path 108 is the dimension in the direction of sample flow (i.e., from inlet 104 to outlet 106). The width or diameter of each flow path is the dimension perpendicular to the height (i.e., from left to right, or right to left as shown in FIG.1). In some embodiments, body 102 comprises a plurality of mechanical flow paths 108, for example, baffles, plates, or other dividing components disposed within the body 102. The baffles may be arranged and spaced linearly in the direction of flow (i.e., from inlet 104 to outlet 106) within the body 102. In this configuration, a sample may enter body 102 via inlet 104 and divide between the mechanical flow paths 108 forming the plurality of flow paths.

[0072] In one or more embodiments, each of the plurality of flow paths 108 is uniform in size and / or shape. For example, in some embodiments, each of the plurality of flow paths has the same height and width (e.g., a rectangular cross-section) or the same height and diameter (e.g., a cylindrical cross-section) as each of the other flow paths. In some embodiments, at least two of the plurality of flow paths 108 is non-uniform in size and / or shape. For example, at least one of the plurality of flow paths 108 has a larger width or diameter than another of the plurality of flow paths. For channels 108 have a rectangular cross-section, each of the plurality of flow paths may have a hydraulic diameter defined by ^^^^ 2^^^^^ ℎ = ^^^ , wherein H is the height and W is the width ofeach flow path. In some embodiments, the plurality of flow paths has an aspect ratio ofOK Ref.11080007.01098 height to width, or of height to diameter of about 2 to about 12. In some embodiments, the aspect ratio of each flow path is determined by the expected concentration of a sample, in % v / v, in each channel. The aspect ratio may be defined as As= H / W or H / D. The formula for determining a io may be ^^^^100^^^^^^^^^^2maximum aspect rat^^^^^^= 6×% where % is the % v / v concentration of particles in the channel with a range of about 0.1 to about 9.4, or a range of concentrations.

[0073] In one or more embodiments, the width of each of the plurality of flow paths is about 1% to about 70% larger, or about 5% to about 60% larger, or about 10% to about 50% larger, or about 20% to about 40% larger, or any individual value or sub-range within these ranges, than the average length, width, or diameter of each of the biological particles. In some embodiments, each of the plurality of flow paths 108 has a width of about 1 µm to about 20000 µm, about 2 µm to about 5000 µm, about 10 µm to about 2000 µm, about 20 µm to about 500 µm, about 30 µm to about 100 µm, or any individual value or sub-range within these ranges. The width or diameter of each flow path may be determined by the particle diameter, a, and a width factor determined by the application, fw, which is defined as ^^^^^^^^= ^^^^ ^^^^; the range of may be about 0.4 to about 0.7.

[0074] In various embodiments, at least of energy 110 (e.g., a light source), or at leastone energy capturing device (e.g., a camera) 109, is arranged, for example, as shown in FIG.1, to traverse energy (e.g., light energy) through the plurality of flow paths 108 within the body 102, or to receive energy (e.g., light) emitting from the plurality of flow paths 108 within the body 102. The traversed energy may be in a direction perpendicular to the direction of sample flow as shown in FIG. 1. In one or more embodiments, the traversed energy may be in a direction inline (or parallel) with the direction of sample flow. In yet further embodiments, the traversed energy may be at an angle with respect to sample flow. Body 102 may include a transparent or translucent material at a first end of the body 102 that allows energy (e.g., light) from the at least one energy source to pass through the fluid in all of the flow paths and exit (or reflect) with a signal suitable for detection. Novel inline flow cells according to embodiments herein may have transparent or translucent flow path edges that enable the complete transmission of energy through the fluid sample. The body 102 may also include a transparent or translucent material at a second end of the body 102 that allows light from the at least one energy source to pass through the first end of the body to the second end of the body as shown in FIG.1.OK Ref.11080007.01098

[0075] In one or more embodiments, the at least one source of energy 110 comprises at least one light source. Suitable light sources and / or light capturing devices include (or are comprised in), but are not limited to, a laser, a fluorescence episcopic (Epi)-illumination microscope, a camera (e.g., a line scan camera, area scan camera, high framerate area scan camera, etc.), light scattering analyzer, brightfield microscope, or combinations thereof. The light source may be a laser configured to scatter light upon interaction with biological particles (e.g., cells, cell clusters, or combinations thereof). In some embodiments, the light source comprises a laser configured to cause fluorescence of the biological particles. Inline flow cells according to embodiments herein are suitable for use with Epi-illumination light scatter and fluorescence. In embodiments, this technique may implement an optics train containing at least one dichroic beam splitter that enables the light to travel completely through the plurality of channels of the inline flow cell. In embodiments, this technique uses laser light scattering 114 suitable for detection and data from biological particle size detection to normalize and estimate the light scattering as a function of biological particles in the composition. In embodiments, this technique also may use fluorescence for native detection (i.e., the ability to detect and analyze biological particles in their original state, without the need for extensive processing or labeling). Such analysis techniques may also address decreased fluorescence from cores of necrotic centers. In some embodiments, the at least one source of energy may additionally or alternatively include a through chip lamp, a light emitting diode (LED), a laser beam stripe (e.g., with a cylindrical lens) for measurement of absorption directly on a charge coupled device (CCD) with an neutral density (ND) filter to set normal intensity “high” for subsequent detection of low light when, for example, a cluster of biological particles (e.g., a cluster or cells) interacts with the incident energy (e.g., different wavelengths such as ultraviolet, blue light, red light, etc. may be used to measure biological particles).

[0076] A detector (not shown), also referred to herein as an energy capturing device, may be arranged proximate the body 102 to receive the at least one signal 114 generated upon interaction of the at least one energy source 110 with an individual biological particle or a cluster of biological particles. In some embodiments, the at least one signal 114 comprises scattered light, diffracted light, refracted light, reflected light, absorbed light, or combinations thereof. To analyze the at least one signal 114 (e.g., light), a Laser-Raman detector may be configured to receive light scattered when one or more biological particles interact with light from the light source. Suitable detectors according to embodiments herein include, but are not limited to, a photodiode arrayOK Ref.11080007.01098 (PDA), a charge coupled device (CCD), camera, line scan camera, area scan camera, photomultiplier tube, or combinations thereof.

[0077] Additional, or alternative detection techniques suitable for use with inline flow cells according to embodiments herein may include laser Raman detection including laser tweezers Raman spectroscopy (LTRS), which allows for label-free detection and quantification of biological particles. Such technique may involve optical trapping where a laser beam (e.g., the same laser that is used for Raman excitation) is focused on one or more biological particles to create a trap that physically holds a biological particle in place within a flow path. The laser beam may also excite Raman signals from the trapped particle. A spectrometer in combination with a confocal detection arrangement may be arranged to receive a scattered Raman light signal from the inline flow cell and analyze the scattered light to characterize the particle's molecular composition.

[0078] In one or more embodiments, body 102 may contain an inlet window (not shown) disposed on a sidewall thereof proximate the source of energy 110. The inlet window may be formed of a translucent or transparent material suitable to allow the energy to enter the body 102 and interact with the biological particles 112. For example, suitable materials may be chosen to be transparent at the operating wavelength of the energy source. Suitable translucent or transparent material include, but are not limited to, frosted glass, etched glass, tinted glass, optical glass (e.g., etched, frosted, amorphous, etc.), nylon, polyethylene, polypropylene, tinted acrylic, etched acrylic, polymethylmethacrylate, amorphous acrylic, clear glass, fused silica, optical glass (e.g., clear, polarized, etc.), silica, borosilicate, boron oxide, diboron trioxide, phosphorous pentoxide, phosphate, aluminosilicate, aluminum oxide, lead oxide, barium oxide, germanium oxide, Li2O- CaO-SiO2, sapphire, calcium fluoride, magnesium fluoride, zinc selenide, gallium arsenide, and / or combinations thereof.

[0079] Upon interaction of the biological particles 112 in the fluid with the traversed energy, the biological particles generate at least one signal 114 that is detectable by a suitable analyzer (not shown). Body 102 also may include an outlet window (not shown) configured to allow the energy to exit body 102 to be received by the detector of at least one analyzer. The outlet window may be arranged on the wall opposite from the inlet window, for example, as shown in FIG.1. The outlet window also may be formed of a translucent or transparent material as described herein. In some embodiments, the entire body 102 is formed of the translucent or transparent material. TheOK Ref.11080007.01098 inlet and outlet windows may be suitable as laser scatter detection windows and / or biological particle counting windows for use with corresponding lasers and particle analyzers.

[0080] In other embodiments, for example, as shown in FIG.2, body 202 is configured to form a plurality of fluidic flow paths 208. In this embodiment, body 202 does not include mechanical flow separators such as the baffles, plates, or other dividing components 102 as shown in FIG.1. Instead, the fluid flows through body 202 with fluid mechanics (e.g., a laminar flow rate) such that the sample fluid forms parallel layers creating flow lines of individual biological particles 212 and / or clusters of biological particles 212. In this embodiment, without mechanical flow paths, the velocity of the biological particles 112, 212 flowing through the fluidic flow paths 208 may be faster than the particles flowing through mechanical flow paths 108 such that the field of view 209 is relatively smaller than the field of view 109. Inline flow cell 200 further includes at least one source of energy 210. The at least one source of energy 210 may be defined as the at least one source of energy 110 described above.

[0081] Fluid containing the biological particles 212 flow into body 202 through inlet 204 disposed on a top wall of body 202 at the center. Although shown on the top wall and in the center of body 202, inlet 204 also may be positioned off center and / or on a side wall of body 202. The sample fluid may flow through body 102 and exit through outlet 206.

[0082] Referring to FIG. 3 (and FIG. 10), another embodiment of an inline flow cell 300 may include a plurality of flow channels 308A, 308B, 308C each being formed of a plurality of sections such that at least two sections have a different dimension (e.g., shape, volume, length, depth, diameter, etc.). Each of the plurality of flow channels 308A, 308B, 308C may include an inlet section 303A, 303B, 303C configured to receive a sample fluid containing biological particles (not shown) through inlet 304A, 304B, 304C, respectively. In some embodiments, the plurality of flow channels 308A, 308B, 308C may have a section of increased width / depth, which slows the velocity of the biological particles (e.g., cells) for imaging / counting. For example, inlet section 303A, 303B, 303C may have a first dimension through which biological particles may flow at a first mean velocity (v1). Body section 305A, 305B, 305C may have a second dimension through which the biological particles may flow at a second mean velocity (v2). In some embodiments, v1 is greater than v2such that the volumetric increase in the channel between the inlet section 303A, 303B, 303C and body section 305A, 305B, 305C result in a decrease in velocity. In one or more embodiments, the plurality of flow channels 308A, 308B, 308C may include an outlet sectionOK Ref.11080007.01098 307A, 307B, 307C having a third dimension, which may or may not be the same as the first dimension, through which the sample fluid exits each flow path 307A, 307B, 307C to the outlet 306A, 306B, 306C. The biological particles may flow through outlet section 307A, 307B, 307C at a third velocity (v3), which may or may not be the same as v1. The plurality of flow channels 308A, 308B, 308C are suitable for use in any of the inline flow cells according to embodiments herein.

[0083] With reference to FIG.4, an inline flow cell 400 as described herein may include a plurality of energy sources (e.g., light, acoustic, etc.) and / or energy capturing devices. In some embodiments, the inline flow cell 400 comprises a plurality of light sources or light capturing devices 410, 418, each light source or light capturing device being associated with a respective light scattering analyzer, brightfield microscope, line scan camera, area scan camera, high framerate area scan camera, or combinations of any two or more thereof. In one or more embodiments, inline flow cell 400 includes camera imaging 418 in combination with laser light scatter 410 for duplicative detection and counting. For example, inline flow cell 400 may include at least one energy source comprised in a respective line scan camera (not shown) having field of view 418. Inline flow cell 400 may include a second energy source 410, for example, a laser, configured to traverse light through the fluid in field of view 416 while in the plurality of flow paths 408. In some embodiments, the line scan camera 410 may be configured to image the biological particles 412 when they pass by the line scan field of view 418 at a known velocity determined by the second energy source 410. The laser may pass through a laser scatter detection window into the sample fluid upon which the laser light interacts with individual biological particles or clusters of biological particles in field of view 416 and thereby scatters light 414. The light scattering signals may be used to identify, track, and / or count the biological particles individually or as a group as they travel through individual channels (e.g., Channel Numbers 2, 7, 12, 16, 20) of the plurality of channels 408. In embodiments, when the biological particles enter field of view 418, only the biological particles in the identified lanes by laser scatter may be imaged with the line scan camera. In some embodiments, pattern of biological particles may be tracked as a group for tracking purposes. The combination of the laser scatter analysis and line scan imaging may be used to ensure that each biological particle is counted only once.

[0084] Inline flow cells according to the various embodiments herein may include at least one of an acoustic analyzer, laser source, light emitting diode source, brightfield source, darkfield source,OK Ref.11080007.01098 Epi illumination source, or camera configured to traverse energy through the plurality of flow paths. In some embodiments, the acoustic analyzer, laser source, light emitting diode source, brightfield source, darkfield source, Epi illumination source, or camera is configured to turn on or activate when a particle of interest is detected in a respective field of view. Upon interaction with the energy traverse through the sample from the at least one energy source, the biological particles may generate at least one signal.

[0085] In some embodiments, the acoustic analyzer is an acoustic particle analyzer, or an acoustic impedance detector. The at least one acoustic analyzer may be configured to measure an acoustic wave, acoustic impedance, resistance, dielectric properties, or combinations thereof of the biological particles. In some embodiments, the at least one acoustic analyzer is configured to modify a trajectory of the particle of interest (e.g., acoustic manipulation) to sort the particle of interest into a channel of the plurality of flow paths or collectors. The laser source of the inline flow cells may be comprised in a biological particle analyzer, light emitting diode source, brightfield source, darkfield source, Epi illumination source, or camera.

[0086] In some embodiments, the inline flow cells according to embodiments herein may include a device configured to apply a force to one or more of the biological particles. Such a device may be a laser that is simultaneously configured to traverse light through the sample containing the biological particles that generate a signal (e.g., scattered light) upon interaction with the light with the particles. The force applied may be a magnetic, optical, electrical, mechanical, and / or thermal force.

[0087] An embodiment of an inline flow cell 500 is shown in FIGs.5A-5C. Inline flow cell 500 may be constructed of a plurality of layers. In at least one embodiment, inline flow cell 500 is formed of three layers. A first layer 502 comprises through slots configured to form and separate the plurality of channels 508 through which the biological particles may flow and be imaged. First layer 502 having the channel separators, may extend down into a second layer 520 and attach to a third layer 501. In some embodiments, first layer 502 is bonded to second layer 520 and / or third layer 501.

[0088] Inline flow cell 500 may include a plurality of inlets 504A, 504B. A fluid containing biological particles of different properties 512, 513 may be received through inlet 504A. The fluid may be divided between the plurality of flow paths 508 formed in at least the first layer 502. The plurality of parallel flow paths 508 may be configured to maintain a consistent fluid velocity profileOK Ref.11080007.01098 within each flow path. In some embodiments, the flow paths 508 are large enough to keep the parabolic velocity profile of the fluid flowing therein substantially flat in one or more dimension depending on the aspect ratio of the channel.

[0089] Inline flow cells according to the various embodiments described herein may implement cell sorting and / or sample enrichment through one (or a combination) of passive technologies to facilitate analysis. In one hydrodynamic implementation, tangential flow filtration includes channels / pores perpendicular to the flow of sample. In an inertial flow-based implementation, a combination of varying wall lift forces, shear gradient forces, and secondary forces (dean) are utilized to passively modify the trajectories of particles with differing geometric properties. Size- based sorting implementations may include on-chip filters, for example, involving a series of posts within the channel to prevent larger particles from entering the sequential channels (remove larger particles / debris). Another size-based sorting implementation may include deterministic lateral displacement that uses an array of posts that are offset to allow larger particles to be displaced in a direction away from original flow field (e.g., to sort remaining particles). In another implementation, particles may be positioned using hydrodynamic forces, which allows particles to reach an equilibrium position (with respect to the x, y, and / or z position in the channel cross section) to ease the analysis. For example, particles may reach equilibrium positions in sufficiently long channels, their position being dependent on the cross-section of the channel. A series of varying cross-sections can better focus particles at a specific equilibrium position. In another example, channel cross sections may be modified so that flow velocity is constant in part of channel where the particles reside.

[0090] Inline flow cells according to embodiments herein may be configured to separate smaller biological particles from larger biological particles so that each population may be detected, analyzed, and / or characterized separately. Sorting cells (e.g., smaller individual cells from larger clusters of cells, cells of different composition, refractive index, biochemistry, etc.), for example, in complex biological samples (such as tissue and blood) enables the downstream analysis of specific cell populations. Tissue and blood may be made up of different cell types having varying functions and characteristics. Analyzing a mixed biological particle population can average out these differences, masking unique behaviors of individual cells or rare cell populations. Specific cell populations of interest may be isolated based on characteristics such as cell surface markers (e.g., using fluorescence characteristics), morphology, size, chemical analysis, etc. which enablesOK Ref.11080007.01098 focused analysis of these biological particle populations and their particular properties. Moreover, separating and / or isolating a desired biological particle type may increase the sensitivity of downstream analytical methods by reducing noise (i.e., signals generated by non-target particles, too many particles in the samples, debris, aggregates, etc.) and / or enriching a sample with target particles in low abundance.

[0091] Separating chemically different particles according to embodiments offers new possibilities for analysis and possible purified collections of samples such as organic particulates, inorganic particles (e.g., glass and metal particles), and biological species such as cells, bacteria, and viruses. Differentiation of biological samples such as bacteria is based upon chemical differences in their capsules. Polysaccharides, lectins, lipoteichoic acids, and proteins are some of the biomolecules present in various bacterial species and strains. There exists a substantial range of refractive indices in bacterial and viral samples due to their different chemical compositions. The ability to separate biological species based upon physical and chemical properties using light interaction with samples in an inline flow is new and has potential benefits when applied to bio- warfare detection and biomedical analysis. Not only are samples physically separable using techniques described herein, but from their position in the separation field their refractive index can be determined. From a predicted location, one may identify specific entities in an unknown mixture.

[0092] In some embodiments of inline flow cell 500, the second layer 520 may be or may comprise a wall, partition, barrier, or membrane received between the first layer 502 and the third layer 501. The wall, partition, membrane, or barrier may contain openings 580 suitable to receive small biological particles, for example, having a mean size of less than about 100 µm, less than about 50 µm, less than about 40 µm, less than about 20 µm, less than about 10 µm, or about 0.1 µm to about 50 µm, about 10 µm to about 500 µm, about 20 µm to about 100 µm, about 40 µm to about 60 µm, or any individual value or sub-range within these ranges. Larger biological particles, for example, clusters of biological particles, are unable to pass through the wall, partition, membrane, or barrier. In some embodiments, a sheath fluid (such as water, buffer, etc.), for example, received in inlet 504B, may be used to push the smaller particles through the partition or membrane.

[0093] In one or more embodiments, inline flow cell 500 may include a source of energy configured to apply a force to one or more of the biological particles 512, 513. For example, a laser configured to direct energy to the sample while flowing through the inlet 504A, 504B orOK Ref.11080007.01098 upstream of the plurality of flow paths 508 may be configured to push the smaller biological particles 512 into the first section 502 or to push the larger biological particles 513 in or through the third section 501 thereby separating these populations from the bulk sample. Suitable lasers may be configured to provide additional power to separate the particle populations in the regions of the inlet 504A, 504B or upstream of the plurality of flow paths 508 as the velocity of the fluid / biological particles will be higher in these regions than in the plurality of flow paths 508.

[0094] In some embodiments, inline flow cell 500 may utilize laser optical force illumination to push larger biological particles 513 (e.g., cell clusters) in the sample fluid toward the third layer and through outlet 506B. The fluid in the first layer 502 may then become enriched with the smaller biological particles 512. In some embodiments, a laser optical force 516 may be implemented if needed to keep larger biological particles 513 (e.g., cell clusters) floating in the sample fluid, but smaller biological particles 512 (e.g., single cells) would experience less force and be overcome by gravity floating downward as shown in FIG.5C. Alternatively, optical or some other force as described herein could be used to push the single cells through the wall, partition, member, or barrier faster in order to have a higher flow rate not dependent upon gravity settling.

[0095] As shown in FIGs. 5B and 5C, outlet 506A may receive fluid containing the smaller biological particles 512 and outlet 506B may receive the remainder of the bulk fluid containing, for example, the larger biological particles. In some embodiments, the smaller biological particle 512 may be analyzed and sent to waste via outlet 506A whereas the remaining sample fluid may be collected for further analysis via outlet 506B.

[0096] In one or more embodiments, inline flow cell 500 may be comprised of a first inlet 504B configured to receive a first fluid. A second inlet 504A configured to receive a second fluid comprising biological particles. A body 501, 502, 520 in fluid communication with the first inlet 504A and the second inlet 504B. The body 501, 502, 520 may be configured to form a plurality of flow paths 508. A wall (e.g., a partition, membrane, barrier, filter, screen, etc.) may be disposed within the body and may constitute a second section 520. The wall may include a plurality of openings 580 configured to receive smaller biological particles (e.g., single biological particles, particles having a mean size of less than about 50 µm) and not larger biological particles (e.g., clusters of biological particles having a size of greater than about 50 µm) from the second fluid sample.OK Ref.11080007.01098

[0097] Inline flow cell 500 may include at least one source of energy (e.g., at least one light source) or energy capturing device (e.g., a camera) configured to traverse light through the body to measure and / or move the single biological particles, wherein the single biological particles are received in the plurality of flow paths. The at least one light source or light capturing device also may be configured to provide a force to small biological particles 512 in the second fluid. The force may be an optical force, gradient force, and / or scattering force. The force may be configured to move a population of biological particles (e.g., small, large, individual, cluster, etc.) through the openings in the wall 520. In some embodiments, the force is configured to move the population of biological particles through the openings faster to provide a higher flow rate of the small biological particles in the plurality of flow paths 508.

[0098] According to one or more embodiments, the plurality of flow paths 508 may be formed by fluidic (not shown), or may be formed by a plurality of dividers (e.g., the parallel flow dividers) configured to receive the first fluid or the second fluid. According to various embodiments, each of the plurality of flow paths 508 may have an aspect ratio of height to width, or height to diameter of about 2 to about 12. In various embodiments, each of the plurality of flow paths 508 is configured to provide a flat parabolic velocity profile of fluid flowing through a channel.

[0099] Inline flow cell further includes a first outlet 506A in fluid communication with first section 502. First outlet may be configured to receive a first discharge fluid enriched with a first population (e.g., small) biological particles. A second outlet may be in fluid communication with the second section 501. The second outlet may be configured to receive a second discharge fluid enriched with large (e.g., clusters of) biological particles.

[0100] With reference to FIG.6, another embodiment of an inline flow cell 600 configured to sort and analyze populations of biological particles is presented. Inline flow cell 600 includes an inlet 604 configured to receive a fluid comprising a plurality of biological particles. A body 602 may be configured to form a pass-through flow path 628, for example, comprising small biological particles 612 (e.g., with a mean size of less than about 10 µm) in fluid communication with an outlet (not shown). Body 602 is further configured to form a plurality of flow paths each configured to receive biological particles of varying size or composition 609, 611, 613. In some embodiments, the pass-through flow path has a larger diameter than the diameter of each of the plurality of flow paths or collectors.OK Ref.11080007.01098

[0101] The biological particles 609, 611, 613 may be received in collectors 634, 632, 630 (e.g., reservoirs, vials, containers, etc.) respectively. At least one device configured to apply a force 617 (e.g., a laser) to one or more of the biological particles may be positioned proximate inlet 604. Inline flow cell 600 may include a transparent or translucent window configured to allow the laser light to contact the particles as they enter body 602 through inlet 604. The amount of optical force applied by the laser may be indicative of the size of a particular biological particle and its flow path or collector. Although not shown, inline flow cell 600 may also include a plurality of mechanical flow paths.

[0102] Inline flow cell may further include at least one energy source (e.g., light or acoustical source) or energy capturing device (e.g., a camera) 615 configured to traverse energy through the plurality of flow paths or collectors 630, 632, 634 to contact particles that flow into the field of view 616. The at least one energy source or energy capturing device 615 may be comprised in a light scattering analyzer, brightfield microscope, line scan camera, area scan camera, or high framerate area scan camera. In some embodiments, the inline flow cell 600 includes a plurality of energy sources or energy capturing devices 615, each energy source being comprised in a respective light scattering analyzer, brightfield microscope, line scan camera, area scan camera, high framerate area scan camera, or combinations of any two or more thereof. In some embodiments, the inline flow cell 600 comprises a plurality of energy capturing devices 615, each energy capturing device being comprised in a respective line scan camera or area scan camera, wherein the line scan cameras or area scan cameras are positioned adjacent to one another and configured to produce parallel images of the biological particles that are combinable into one larger image. Refractive index of the populations of biological particles 609, 611, 612, 613 also may be determined. Increasing refractive index 626 is a function of size of the populations of biological particles.

[0103] FIG.7 is a schematic of a plurality of flow cells according to embodiments herein operating in parallel according to one or more embodiments. Implementing parallel inline flow cells may be suitable to reduce local flow rates and handle large volumes of samples. In this configuration, a fluid containing biological particles may be received in inlet 704 and divided between the two inline flow cells 700A, 700B. Flow cells 700A, 700B may contain a plurality of flow paths (not shown) as described herein through which the sample comprising the biological particles may flow. Flow cells 700A, 700B may be configured as shown in FIGs. 5A-5C such that eachOK Ref.11080007.01098 comprises two outlets 706A, 706B and 707A, 707B, respectively, suitable to discharge bulk fluid containing non-target particles (e.g., after sorting). A sample stream 711 enriched with target particles may flow through the field of view 710 of a particle detector and / or analyzer as described herein (e.g., an imaging, impedance, or optical force device) for measurement.

[0104] FIG.8 is a schematic of an embodiment of an inline flow cell 800 constructed of multiple channels 801, 802 and a membrane 820 as described herein arranged therebetween. Inline flow cell 800 may provide a tangential flow of a population of particles (e.g., small particles, large particles, single particles, clusters, etc.) to separate the population from biological particles in the sample stream. Membrane 820 is suitable for use as a particle filter and / or enricher to reduce the sample volume. Suitable membranes include, but are not limited to, a cell filter, cell strainer, mesh filter, sieve, tangential flow filter, on-chip filter, or combinations thereof. Membrane 820 may be porous with openings having a mean size of less than about 100 µm, less than about 50 µm, less than about 40 µm, less than about 20 µm, less than about 10 µm, or about 0.1 µm to about 50 µm, about 10 µm to about 500 µm, about 20 µm to about 100 µm, about 40 µm to about 60 µm, or any individual value or sub-range within these ranges.

[0105] A source of energy 815 (e.g., an imager, impedance, laser force cytology, etc.) may be disposed near outlet 806A and configured to measure biological particles in channel 801 as shown in FIG.8. Additionally or alternatively, a source of energy may also be disposed near outlet 806B and configured to measure biological particles in channel 802. Channels 801 and 802 may each be bonded 803A, 80B to membrane 820, for example, at an interface of the two channels. In one or more embodiments, inline flow cell 800 may receive a sheath fluid 810 (e.g., water, buffer, etc.) configured to push the sample received in channel 801 towards membrane 820 and into channel 802.

[0106] Referring to FIG.9, an embodiment of an inline flow cell 900 may include a membrane 920 similar to membrane 820 and a channel 923 similar to channel 802, but with a tangential flow channel 901, 921, 902 implemented instead of channel 801. In some embodiments, tangential flow channel 901, 921, 902 may be used without membrane 920 or channel 923.

[0107] In the embodiment shown in FIG.9, inline flow cell 900 has a tangential flow channel 901, 921, 902 disposed upstream from membrane 920. Inline flow cell 900 may be configured to receive a fluid comprising biological particles through inlet 904 and into sample channel 921. Sample channel 921 may be in fluid communication with tangential side channels 908. SideOK Ref.11080007.01098 channels 908 may be configured to remove a population of biological particles from the sample of biological particles flowing through sample channel 921. In some embodiments, tangential flow channels 908 may be configured to remove from sample channel 921 biological particles having a mean size of greater than about 10 µm, greater than about 20 µm, greater than about 30 µm, about 10 µm to about 1,000 µm, about 50 µm to about 500 µm, about 100 µm to about 250 µm, or any individual value or sub-range within these ranges.

[0108] Tangential flow channels 908 may be formed in an array and may be offset from each other to allow the population of biological particles have a size as described above (e.g., large biological particles greater than about 40 µm) to be displaced in a direction opposite fluid flow. In some embodiments, tangential side channels 908 provide at least one of wall lift forces, shear gradient forces, or Dean flow forces that modify trajectories of biological particles having differing geometric properties. Fluid received in channels 901, 902 may flow through outlets 906A, 906B to be collected in receptacles for further analysis or disposed in waste.

[0109] Membrane 920 may be arranged between the tangential flow channel 901, 921, 902 and flow channel 923. Membrane 920 may be configured to receive fluid from sample channel 921. A source of energy 915 (e.g., an imager, impedance, laser force cytology, etc.) disposed near outlet 907 is configured to measure biological particles in channel 921. In some embodiments, a source of energy may be arranged near outlet 913.

[0110] FIG. 10 is a schematic of an inline flow cell 1000 having Dean flow side channels and configured to sort and measure biological particles according to one or more embodiments of the disclosure. Inline flow cell 1000 receives a fluid containing biological particles through inlet 1004. The fluid may flow through inertial channel 1008A or inertial channel 1008B. The inertial flow channels 1008A, 1008B may be non-linear, for example, with a serpentine configuration. Dean flow refers to the generation of duplicate counter-rotating vortexes in a plane perpendicular to the main flow in a curved channel. Each inertial channel 1008A, 1008B contains both straight channel portions 1003 and curved channel portions 1005. The straight channels 1003 (e.g., with an aspect ratio of about 2 to about 12) are configured to reduce migration effects created in the curved channels 1005, for example, by focusing the smaller particles within the center of inertial channels 1008A, 1008B. The curved channels 1005 are configured to induce Dean flow and promote particle migration. As the fluid containing biological particles flows through inertial channels 1008A, 1008, relatively larger particles (e.g., greater than about 50 µm) flow to the outer regionsOK Ref.11080007.01098 whereas relatively smaller particles stay within the center of the channels (e.g., the inner channel, low velocity, low migration).

[0111] Fluid flows through inertial channels 1008A, 1008B into non-uniform flow channels 1009A, 1009B. Each non-uniform flow channel 1009A, 1009B is comprised of a first section 1019A, 1019B having a first configuration (e.g., shape, volume, length, depth, diameter, etc.) and a second section 1021A, 1021B having a second configuration (e.g., shape, volume, length, depth, diameter, etc.). For example, first section 1019A, 1019B may have a smaller dimension (e.g., width, diameter, volume, etc.) than that of second section 1021A, 1021B as shown in FIG.10. In the embodiment shown in FIG. 10, first section 1019A, 1019B has a tapered opening that transitions to a straight (e.g., cylindrical) section. Second section 1021A, 1021B may be entirely tapered from a narrower width / diameter to a wider width / diameter as shown in FIG. 10. The widening of the channels may be configured to reduce wall effects and promote migration to the internal walls of the channels 1019A, 1021A, 1019B, 1021B. Inline flow cell 1000 with its combination of serpentine channels flowing into widening channels is configured to passively move relatively larger biological particles to the internal walls of the channels thereby providing filtration / sorting / enrichment for ease of measurement / detection / analysis.

[0112] FIG. 11 is a schematic of an inline flow cell 1100 having a size-based particle sorting configuration according to one or more embodiments. In this embodiment, inline flow cell 1100 utilizes a deterministic lateral displacement to perform size-based biological particle sorting. Particles larger than a first mean size 1109 (e.g., having a diameter of greater than about 50 µm) may be displaced toward outlet 1106 (e.g., for measurement or waste) while smaller particles 1112 may flow in their original path towards outlet 1107 (e.g., for measurement or waste). While described in the context of size-based sorting, it should be understood that particles may be sorted based on other properties, additionally or alternatively, such as composition, refractive index, biochemistry, and / or other biological differences.

[0113] Fluid containing biological particles may be received in inlet 1104. Inlet 1104 may have a non-linear shape that flows into a uniform body 1102 (e.g., cylindrical). Disposed within body 1102 may be an array of posts 1113 to prevent larger particles from entering the sequential channels (e.g., to remove larger particles / debris). A sheath fluid may be received in inlet 1110 and configured to force fluid containing the biological particles through body 1102. For example, the relatively small particles 1112 may flow toward outlet 1107 whereas the relatively larger particlesOK Ref.11080007.01098 1109 may flow through outlet 1106. Disposed near outlet 1106 and / or outlet 1107 is a source of energy 1115 as described according to embodiments herein.

[0114] FIG. 12 is a schematic of an inline flow cell 1200 having a size-based particle sorting configuration according to one or more embodiments. A fluid containing biological particles 1212 is received in inlet 1204 and enters the field of view of an impedance analyzer 1215 (e.g., a laser, analyzer, detector, etc.). The impedance measurement determines the dielectric properties of the biological particles (e.g., cells). Particles of interest are identified and tracked (e.g., their flow path is identified and the they are tracked therethrough) based on their dielectric properties. At pre- determined (i.e., characterized) thresholds, when a particle of interest is detected, an acoustic analyzer 1238 turns on to modify trajectory of the particle of interest for sorting in the direction of 1234 or the direction of 1236 through outlet 1206. Sorting could also be accomplished using magnetic, optical, electrical, mechanical or thermal forces. A central processing unit (CPU) 1232 of a computer may be used to control and / or analyze the data representing the dielectric properties, particle tracking, and / or impedance and / or acoustic control. For example, the CPU may be used to make decisions concerning whether to activate an acoustic analyzer to measure and / or manipulate particles flowing through body 1202.

[0115] FIG. 13 is a schematic of an inline flow cell 1300 configured for sorting, enrichment, counting and imaging according to one or more embodiments of the disclosure. A fluid comprising biological particles is received by inlet 1304 and fills a top channel 1301. Outlet 1306A may be open while top channel 1301 is being filled and closed once channel 1301 is filled. Outlet 1306B may then be opened, for example, to apply negative pressure and draw the sample in top channel 1301 towards a porous membrane 1320. The pores may be much smaller (e.g., at least about 10%, at least about 20%, at least about 50% smaller) than the particles of interest. Fluid may flow from top channel 1301 through membrane 1320 to a bottom channel 1302 as described with respect to FIGs.8 and 9.

[0116] An imaging device (e.g., a camera) may be positioned above top channel 1301, the bottom of which forms a focal plane for the imaging device. Imaging may be done to determine the size and / or count the biological particles. Outlet 1306B may be closed to apply a positive pressure to remove fluid sample from membrane 1320 with inlet 1304 either closed or supplying buffer. Outlet 1306A may subsequently be opened to collect the enriched sample that has been imaged.OK Ref.11080007.01098 This process may be implemented in inline flow cell 1300 and repeated until the complete sample imaged / measured / analyzed.

[0117] With reference to FIGs. 14-17, a camera (e.g., a line scan camera or area scan camera) 1417, 1517, 1617, 1717 suitable for imaging biological particles within inline flow cells according to embodiments herein may be fixed or movable in a parallel or transverse direction to the flow of the fluid within the body of the inline flow cell. Inline flow cells according to embodiments herein may be configured to count cells using light scatter and / or brightfield and a camera (e.g., a line scan camera) to image biological particles.

[0118] In some embodiments, camera 1417, 1517, 1617, 1717 is a line scan camera. In embodiments, where the scan line is not fixed in position, the scan line may be oriented either parallel or transversely to the direction of sample velocity in the analysis region when it is moved across the field of vision to create an image. If mounted transversely to the direction of sample velocity, the scan line rate of acquisition may be increased by the average drift velocity of the cells. This velocity correction may minimize velocity related sampling errors. This correction can come from increasing the velocity of the scan line with fixed frequency or increasing the frequency with fixed velocity.

[0119] Known line scan cameras are very fast, but only have a one (1) pixel sensor height. Inline flow cells according to embodiments herein are suitable to create two dimensional objects as the biological particles (e.g., cells or clusters of cells) move through the field of view. According to embodiments, devices, systems and methods have been developed to take into account the parabolic velocity profile of sample fluid flowing through flow paths and the fluid mechanics may be adjusted, for example, based on mechanical design such that certain frames may be dropped (i.e., not used) for objects closer to the internal walls that move at a slower speed. For example, the relative velocity of a particle does not equal 0 px / s and some pixels are lost due to under- sampling. The output image of the particle appears skewed or squished. When the velocity of the particle is accounted for, the output image of the particle is properly scaled (e.g., the surrounding image of the fluid / flow cell is under-sampled) and appears without distortion.

[0120] Referring to FIG.14, in this arrangement 1400, the camera 1417 may be fixed in position proximate (e.g., above, near a side wall, near a viewing window, etc.) the inline flow cell 1444. The camera 1417 may be fixed in position above an analysis region. In some embodiments, a reflector 1442 (e.g., fixed or rotatable) is arranged to receive and reflect light from the analysisOK Ref.11080007.01098 region of the inline flow cell 1444 to the camera 1417. Reflector 1442 may include a mirror, prism, electro-optic reflector, digital electromechanical reflector, dichroic reflector, or combinations thereof. In some embodiments, the inline flow cell 1444 (e.g., the body) is flat (1544 as shown in FIG.15) or curved 1444 as shown in FIG.14. In some embodiments, the reflector 1442 may be arranged to maintain a focal point on the body of the inline flow cell 1444 and the sample fluid contained therein as the reflector 1442 rotates. For example, reflector 1442 may be mounted to a servo to move reflector 1442 as light from the analysis region is reflected to the scan line. The reflector 1442 may be rotated at a known rate to gather an image of the field of view.

[0121] As shown in FIG.15, in this arrangement 1500, the camera 1517 may be connected to a linear actuator the motion of which is indicated by the double arrow beneath camera 1517. According to various embodiments, the linear actuator is configured to move the camera 1517 relative to the reflector 1542 to keep a focal point on the inline flow cell 1544 as the reflector rotates. In some embodiments, a motion platform may be implemented to move camera 1517 three dimensionally.

[0122] As shown in FIG.16, in this arrangement 1600, the camera 1617 may be connected to a gantry 1642. The gantry 1642 in turn may be connected to a linear actuator (represented by the two way arrow above the gantry) or a motion platform to permit three dimensional movement. The linear actuator or motion platform may be configured to move the gantry 1642 over the inline flow cell 1644 (e.g., its body) to create an image of the biological particles flowing through the inline flow cell 1644.

[0123] Referring to FIG.17, in this arrangement 1700, the inline flow cell 1744 may be proximate an illuminator 1746 configured to illuminate the fluid containing biological particles as it flows through the plurality of flow paths (not shown). In some embodiments, as shown, the illuminator 1746 is positioned beneath the inline flow cell 1744 although the illuminator 1746 may be arranged near the sides or top in a manner that enables light to travel through the sample. As shown, light travels from the inline flow cell 1744 to reflector 1742, which may or may not be rotatable, and to camera 1717, which may be fixed or rotatable. The illuminator 1746 may be connected to a linear actuator or motion platform that moves the illuminator in two or three dimensional motion focusing the light traversing the sample within the inline flow 1744 to the camera 1717. at least one servo motor configured to move the body.OK Ref.11080007.01098

[0124] With reference to FIG. 18, an inline flow cell 1800 may include a plurality of energy sources (e.g., light or acoustic) or energy capturing devices (e.g., a camera) 1810, 1815. In some embodiments, each energy capturing device 1810, 1815 is comprised in a respective line scan camera. In other embodiments, the inline flow cell 1800 contains at least one line scan camera 1810 and at least one area scan camera 1815. The line scan camera 1810 or area scan camera 1815 may be configured to produce images of the biological particles 1811, 1812, 1813 that are combinable into one larger image.

[0125] In some embodiments, the plurality of light capturing devices include a line scan camera 1815 and a high framerate area scan camera 1810. The high framerate area scan camera 1810 may be configured to track a subset of particles of the biological particles 1811, 1812, 1813 to determine their respective velocities. The respective velocities of the scanned biological particles may be received by the line scan camera and used to scale the sampling rate or correct velocity for a line scan output image as shown in FIGs.20A and 20B.

[0126] Referring to FIG.19, the body of an inline flow cell 1952 according to embodiments herein may further include at least one reticle 1954. The at least one reticle 1954 may be configured for size calibration using a biological particle analyzer or camera. Built-in reticle for size calibration on a per flow cell basis. Device is calibrated with beads during QC but uses chip-based reticle to account for variations in device manufacturing.

[0127] Reference is made to FIGs. 20A and 20B, which show representations of a fluid comprising biological particles 2011, 2012, 2013 sorted by size within a plurality of flow paths 2008 disposed in the body 2002 of an inline flow cell 2000 according to various embodiments described herein. Inline flow cell 2000 may include a camera 2010 (e.g., a line scan camera) configured to image the biological particles 2011, 2012, 2013 as they flow through the flow cell. The camera 2010 may be implemented, for example, in a fixed position or movable in two or three dimensions. If fixed, when one or more biological particles 2011, 2012, 2013 pass by the scan line at a known rate an image is generated. If movable, the scan line passes over the one or more biological particles at a known rate to generate the image. The scan line camera may be configured to refresh at variable frequency to create a properly scaled image. Each refresh is configured to capture a line of pixels, one pixel thick. The frequency may be referred to as the “sampling rate.” If the pixel is 10 µm wide and the scan line is passed over biological particle of 100 µm long at a rate of 100 µm / s, then the desired frequency would be 10 Hz. If a biological particle 2011, 2012,OK Ref.11080007.01098 2013 is imaged too many times (i.e., over-sampling), then it will appear elongated in the generated image as shown in FIG. 20A. If a biological particle is imaged too few times (i.e., under- sampling), then it will appear squished (not shown).

[0128] The generated image may be corrected by taking into account the velocity of the biological particles as measured, for example, by laser scattering. In embodiments the scan line may be fixed in position relative to the inline flow cell 2000. A reflector (not shown) as described herein, such as a flat mirror, prism, electro-optic, digital electromechanical device, or a dichroic reflector, may be used to maintain the focal point of the camera 2010 on the sample within the inline flow cell 2010. In some embodiments, a size selective process is implemented upstream from the plurality of flow paths and prior to the field of view of the camera 2010. Size-based velocity differences may be determined and accounted for in the different channels 2008. Smaller particles 2012 will have a higher velocity than relatively larger particles 2013 and 2011. The scan rate of the camera 2010 may be based off the fastest moving particles (i.e., the smaller particles 2012). Slower moving particles (i.e., larger particles 2011), which become oversampled may be transformed (i.e., shrunk) in the corrected image, shown in FIG.20B, back to their estimated correct size based off the expected velocity of the average particles in that channel 2008.

[0129] FIGs.21A-21F are a series of images 2100 demonstrating the use of devices, systems, and methods of measuring biological particles according to the present disclosure. As shown in FIG. 21A, biological particles (e.g., cells) enter the field of view of a camera 2110 (e.g., a line scan camera). A first population 2102 of biological particles is imaged. As flow progresses through the inline flow cell, a second population 2104 of biological particles is identified as shown in FIG. 21B. A bounded region 2106 is drawn between the first and second populations 2102, 2104 of tracked biological particles as shown in FIG.21C. The bounded region 2106 may be sent to an analysis node for counting as shown in FIG.21D. As flow progresses through the inline flow cell, a third population 2112 of biological particles is imaged as shown in FIG.21E. A bounded region 2114 is drawn between the second and third populations 2104, 2112 and sent to an analysis node for counting as shown in FIG.21F.

[0130] Biological particles (e.g., cells) may be counted using a light scattering analyzer. The data generated by the light scattering technique may be used to identify the biological particles to be imaged and to track the particles using a brightfield method as they move through the inline flow cell. The downstream analyzer may be activated (i.e., turned on) to image the flow paths withOK Ref.11080007.01098 biological particles (e.g., cells) identified using light scatter. In some embodiments, not all of the flow paths are filled with biological particles in each frame, which would reduce imaging and tracking capabilities of the those lanes containing particles. The methods demonstrated with respect to FIGs.21A-21F enable tracking of the biological particles and identification of a pattern of a plurality of biological particles (e.g., cells, cell clusters), for example, more than one hundred particles in 250-500 channels that are tracked as a group, but only analyzed once. Scattered light and intensity may be measured on a CCD. The analyzer(s) may be calibrated for size and possibly composition. Image size may be used to normalize scatter data, for example, by backing out compositional changes.

[0131] Methods described herein may take into account parabolic velocity profiles of the fluid flowing through the plurality of channels, differences in drag force due to particle size, and wall effects. While not every particle can be counted in each frame without double counting some particles, to prevent double counting of particles, some or all particles are tracked. In a high- throughput application, the computational demands of tracking every particle limits throughput. Methods described with respect to FIGs. 21A-21F, minimize the number of tracked biological particles, account for differences in particle velocities, and minimize double counting if counted particles share a focal plane, and flow prevents particles from passing above and below each other. The images displayed in FIGs. 21A-21F do not display the differences in velocity because it is assumed that the tracked populations of biological particles would shift relative to each other between frames. Systems for Analyzing and / or Sorting Biological Particles

[0132] In further embodiments herein are systems comprising at least one inline flow cell as described herein. An embodiment of a system 2200 is shown in FIG.22. System 2200 includes a sample reservoir 2201 suitable to store a fluid comprising biological particles as defined herein. Suitable reservoirs include, but are not limited to, a receptacle, vial, beaker, jar, container, or combinations thereof. Reservoir 2201 is in fluid communication with at least one inline flow cell 2202 via an inlet 2204. Fluid flow through system 2200 may be by a positive or negative force or an electrophoretic force. Suitable devices for providing positive or negative forces include, but are not limited to, peristaltic pumps, centrifugal pumps, vacuum pumps, or combinations thereof.OK Ref.11080007.01098

[0133] The at least one inline flow cell 2202 may be of any embodiment as described herein. The at least one inline flow cell 2202 may be suitable for sizing counting, imaging, sorting, and / or otherwise analyzing biological particles (e.g., cells and cell clusters) received from reservoir 2201.

[0134] An analyzer 2210 may be arranged proximate the at least one inline flow cell 2202. Analyzer 2210 may be configured to focus energy (e.g., light, impedance, acoustic waves, etc.) through a window or transparent / translucent wall of inline flow cell 2202 and into the fluid sample flowing therethrough. The analyzer 2210 may be configured to enumerate the number of biological particles (e.g., clusters) required to fill the at least one inline flow cell 2202 and may send a signal to a control system or a processor to stop fluid flow once the flow cell is filled. The analyzer 2210 may provide real-time analysis of the biological particles.

[0135] In some embodiments, the at least one inline flow cell 2202 may be illuminated using brightfield, darkfield, phase, laser, or LED light sources, or any combination thereof to improve detection by the analyzer 2210. In at least one embodiment, the analyzer may be suitable for imaging (e.g., by camera, line scan camera, area scan cameras, etc.), light scatter analysis, impedance, or combinations thereof. In one or more embodiments, the at least one biological particle analyzer 2210 may include an energy source (e.g., a light source, impedance, or acoustic analyzer) or energy capturing device (e.g., a line scan camera, area scan camera, high framerate area scan camera, etc.) and may be configured to receive and analyze a signal (e.g., optical or acoustical) from the biological particles. Analyzed fluid exiting inline flow cell 2202 may flow through outlet 2206 into a reservoir, fillable device, implantable device, or a waste 2260. To fill a reservoir, fillable device, or implantable device, a minimum pressure may be required to transport fluid from reservoir 2201 to fillable device 2260.

[0136] An embodiment of a system 2300 having two inline flow cells is shown in FIG.23. System 2300 includes a sample reservoir 2301 suitable to store a fluid comprising biological particles as defined herein. Suitable reservoirs include, but are not limited to, a receptacle, vial, beaker, jar, container, or combinations thereof. Reservoir 2301 is in fluid communication with a first inline flow cell 2302 via an inlet 2304A and a second inline flow cell 2303 via inlet 2304B. In some embodiments, a portion of the sample flow is diverted from reservoir 2301 to an inline flow cell 2303 configured for counting. The ratio between the flow rates to the first and second inline flow cells 2302, 2303, enable ensuring that the particle / cluster count and distribution remains known in both paths.OK Ref.11080007.01098

[0137] Fluid flow through system 2300 may be by a positive or negative force or an electrophoretic force. Suitable devices for providing positive or negative forces include, but are not limited to, peristaltic pumps, centrifugal pumps, vacuum pumps, or combinations thereof. The first and second inline flow cells 2302, 2303 may be of any embodiment as described herein. Inline flow cells 2302, 2303 may be suitable for sizing counting, imaging, sorting, and / or otherwise analyzing biological particles (e.g., cells and cell clusters) received from reservoir 2301.

[0138] An analyzer 2310 may be arranged proximate inline flow cells 2302, 2303. Analyzer 2310 may be configured to focus energy (e.g., light, impedance, acoustic waves, etc.) through a window or transparent / translucent wall of inline flow cell 2302 or 2303 and into the fluid sample flowing therethrough. The analyzer 2310 may be configured to enumerate the number of biological particles (e.g., clusters) required to fill the at least one inline flow cell 2302, 2303 and may send a signal to a control system or a processor to stop fluid flow once the flow cell is filled. The analyzer 2310 may provide real-time analysis of the biological particles. In some embodiments, the at least one inline flow cell 2302, 2303 may be illuminated using brightfield, darkfield, phase, laser, or LED light sources, or any combination thereof to improve detection by the analyzer 2310. In some embodiments, the analyzer may be suitable for imaging (e.g., by camera, line scan camera, area scan cameras, etc.), light scatter analysis, impedance, or combinations thereof. In one or more embodiments, the at least one biological particle analyzer 2310 may include an energy source (e.g., a light source, acoustic analyzer, etc.), or energy capturing devices (e.g., a camera) and may be configured to receive and analyze a signal (e.g., optical or acoustical) from the biological particles.

[0139] Analyzed fluid exiting inline flow cell 2302 may flow through outlet 2306A into a reservoir, fillable device, implantable device, or a waste 2360. Analyzed fluid exiting flow cell 2303 may flow through outlet 2306B into a reservoir, fillable device, implantable device, or a waste 2362. To fill a reservoir, fillable device, or implantable device, a minimum pressure may be required to transport fluid from reservoir 2301 to fillable device 2360, 2362.

[0140] An embodiment of a system 2400 is shown in FIG. 24. System 2400 includes a sample reservoir 2401 suitable to store a fluid comprising biological particles as defined herein. Suitable reservoirs include, but are not limited to, a receptacle, vial, beaker, jar, container, or combinations thereof. Reservoir 2401 is in fluid communication with at least one inline flow cell 2402 via an inlet 2404. Fluid flow through system 2400 may be by a positive or negative force or anOK Ref.11080007.01098 electrophoretic force. Suitable devices for providing positive or negative forces include, but are not limited to, peristaltic pumps, centrifugal pumps, vacuum pumps, or combinations thereof.

[0141] The at least one inline flow cell 2202 may be of any embodiment as described herein. The at least one inline flow cell 2402 may be suitable for sizing counting, imaging, sorting, and / or otherwise analyzing biological particles (e.g., cells and cell clusters) received from reservoir 2401.

[0142] An analyzer 2410 may be arranged proximate the at least one inline flow cell 2402. Analyzer 2410 may be configured to focus energy (e.g., light, impedance, acoustic waves, etc.) through a window or transparent / translucent wall of inline flow cell 2402 and into the fluid sample flowing therethrough. The analyzer 2410 may be configured to enumerate the number of biological particles (e.g., clusters) required to fill the at least one inline flow cell 2402 and may send a signal to a control system or a processor to stop fluid flow once the flow cell is filled. The analyzer 2410 may provide real-time analysis of the biological particles.

[0143] In some embodiments, the at least one inline flow cell 2402 may be illuminated using brightfield, darkfield, phase, laser, or LED light sources, or any combination thereof to improve detection by the analyzer 2410. In at least one embodiment, the analyzer may be suitable for imaging (e.g., by camera, line scan camera, area scan cameras, etc.), light scatter analysis, impedance, or combinations thereof. In one or more embodiments, the at least one biological particle analyzer 2410 may include an energy source (e.g., a light source, impedance, or acoustic analyzer) and / or energy capturing device and may be configured to receive and analyze a signal (e.g., optical or acoustical) from the biological particles.

[0144] System 2400 may further include a flush fluid inlet 2462. The flush fluid may be used to flush the fluidic components of system 2400 following analysis of a sample in the inline flow cell 2402 and prior to re-filling the flow cell with the next sample.

[0145] Analyzed fluid exiting inline flow cell 2402 may flow through outlet 2406 into a multi- way valve 2466 configured to divert flow between a plurality of reservoirs, fillable devices, implantable devices, or a waste 2460A, 2460B, 2460C. To fill a reservoir, fillable device, or implantable device, a minimum pressure may be required to transport fluid from reservoir 2401 to fillable device 2460A, 2460B, 2460C. Multi-way valve 2466 may allow for dead volume to be pumped to waste 2462 while the system is being flushed. In some embodiments, multiple devices 2460A, 2460B may be filled without going to waste.OK Ref.11080007.01098

[0146] Another embodiment of a system 2500 is shown in FIG. 25. System 2500 includes a sample reservoir 2501 suitable to store a fluid comprising biological particles as defined herein. Suitable reservoirs include, but are not limited to, a receptacle, vial, beaker, jar, container, or combinations thereof. Reservoir 2501 is in fluid communication with at least one inline flow cell 2502. Fluid flow through system 2500 may be by a positive or negative force or an electrophoretic force. Suitable devices for providing positive or negative forces include, but are not limited to, peristaltic pumps, centrifugal pumps, vacuum pumps, or combinations thereof.

[0147] The at least one inline flow cell 2502 may be of any embodiment as described herein. The at least one inline flow cell 2502 may be suitable for sizing counting, imaging, sorting, and / or otherwise analyzing biological particles (e.g., cells and cell clusters) received from reservoir 2501.

[0148] An analyzer 2510 may be arranged proximate the at least one inline flow cell 2502. Analyzer 2510 may be configured to focus energy (e.g., light, impedance, acoustic waves, etc.) through a window or transparent / translucent wall of inline flow cell 2202 and into the fluid sample flowing therethrough. The analyzer 2510 may be configured to enumerate the number of biological particles (e.g., clusters) required to fill the at least one inline flow cell 2502 and may send a signal to a control system or a processor to stop fluid flow once the flow cell is filled. The analyzer 2510 may provide real-time analysis of the biological particles.

[0149] In some embodiments, the at least one inline flow cell 2502 may be illuminated using brightfield, darkfield, phase, laser, or LED light sources, or any combination thereof to improve detection by the analyzer 2210. In at least one embodiment, the analyzer may be suitable for imaging (e.g., by camera, line scan camera, area scan cameras, etc.), light scatter analysis, impedance, or combinations thereof. In one or more embodiments, the at least one biological particle analyzer 2510 may include an energy source (e.g., a light source, impedance, or acoustic analyzer) and / or energy capturing device (e.g., a camera) and may be configured to receive and analyze a signal (e.g., optical or acoustical) from the biological particles.

[0150] An intermediate holding reservoir 2568 may be in fluid communication with inline flow cell 2502 and configured to receive analyzed fluid therefrom. Intermediate holding reservoir 2568 may have a sufficient volume to fill a downstream reservoir, fillable device, or implantable device. In some embodiments, a pair of valves may be positioned on either side of the intermediate holding reservoir 2568 to decouple the flow rates between the counting and filling sides, that is, suchOK Ref.11080007.01098 arrangement may enable slow flow for counting, but allow for quick filling of the downstream reservoir, fillable device, or implantable device. Methods of Using Inline Flow Cells for Analyzing and / or Sorting Biological Particles

[0151] Further described herein are methods of using inline flow cells according to various embodiments described herein for analyzing and / or sorting biological particles. An embodiment of a method of analyzing biological particles in a sample includes receiving a fluid in at least one inline flow cell according to embodiments herein. The fluid may comprise a plurality of biological particles as defined herein. In embodiments, methods as described herein include introducing energy (e.g., light, impedance or an acoustic wave) into the flow cell. The energy is configured to interact with the plurality of biological particles in the fluid and forms at least one signal. In some embodiments, the at least one signal comprises scattered light. In various embodiments, the energy source is a laser comprised in a light scattering analyzer. The at least one signal may comprise light scattered by a biological particle, wherein the scattered light and the intensity of the scattered light is measured by a charge coupled device.

[0152] In various embodiments, the methods include using the at least one signal to identify each of a plurality of flow paths (fluidic or mechanical) containing one or more of the plurality of biological particles. In various embodiments, the inline flow cell comprises about 100 to about 750 flow paths, or about 250 to about 500 flow paths. In some embodiments, for a given measurement frame by the biological particle analyzer, at least some of the plurality of flow paths do not contain biological particles. Interaction of the energy (e.g., light) with each of the biological particles enables each of the biological particles to be counted and / or tracked through the plurality of flow paths within the inline flow cell. Each of the identified biological particles may share a focal plane, and flow rate of the fluid through the inline flow cell prevents each of the biological particles from passing above or below another of the biological particles as it travels through a flow path of the plurality of flow paths.

[0153] In some embodiments, the energy capturing device is comprised in a line scan camera, and the method further includes imaging the biological particles using the line scan camera. The line scan camera may be fixed or movable in a parallel or transverse direction in relation to the direction of fluid flow through the inline cell. In embodiments, the line scan camera is mounted in aOK Ref.11080007.01098 transverse direction to the fluid flow and a line scan rate of acquisition is adjusted based on an average drift velocity of the biological particles. According to embodiments, the average drift velocity adjustment minimizes velocity relating to sampling errors. The line scan rate of acquisition may be adjusted by increasing or decreasing the velocity of the line scan with fixed line scan frequency, or increasing or decreasing the line scan frequency with fixed velocity of the line scan.

[0154] Methods may further include determining the size of individual biological particles upstream from the plurality of flow paths, and moving each of the biological particles into a particular flow path based on its size and / or velocity. In some embodiments, a line scan rate is determined based on the fastest moving particles within the particular flow path. Methods may further include adjusting size data for particles having a velocity of less than or greater than the average expected velocity.

[0155] In embodiments, the size of the plurality of biological particles varies and the drag force of each biological particle of the plurality of biological particles is a function of its size. Walls of the inline flow cell may be configured to produce drag on the fluid.

[0156] In some embodiments, methods include calibrating an analyzer or camera comprising at least one energy source or energy capturing device using at least one reticle as described herein comprised in the inline flow cell. In some embodiments, calibrating the analyzer or camera comprises imaging the at least one reticle. Imaging the at least one reticle may provide data used for position adjustment of the analyzer, camera, inline flow cell, and / or body of the inline flow cell.

[0157] Methods according to embodiments herein further include using the at least one signal to normalize background noise in the fluid. In some embodiments of the methods, the number of biological particles identified in the plurality of flow paths is minimized, velocity of each of the biological particles is determined, and double counting of each of the biological particles is minimized.

[0158] Methods as described herein may include receiving the fluid in a first frame, wherein the light interacts with the biological particles in the fluid to form a plurality of light scattering signals that are detected by a light scattering analyzer forming a first tracked population of biological particles. A second tracked population of biological particles downstream from the first tracked population of biological particles may be identified. Methods may further include forming a firstOK Ref.11080007.01098 bounded region between the first and second tracked populations. The first bounded region may be analyzed to count the number of biological particles in the first bounded region. Methods may further include forming a third tracked population of biological particles downstream from the second tracked population. A second bounded region may be formed between the second and third tracked populations. Methods may include analyzing the second bounded region to count the number of biological particles in the second bounded region.

[0159] Methods as described herein may further include illuminating the fluid in the inline fluid cell with an illuminator while imaging with the at least one energy capturing device. The illuminator may be moved synchronously with the energy capturing device as it interacts with the biological particles.

[0160] The foregoing written description is considered to be sufficient to enable one skilled in the art to practice the invention. The present disclosure is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

OK Ref.11080007.01098 IN THE CLAIMS 1. An inline flow cell, comprising: at least one inlet configured to receive a fluid comprising biological particles; a body in fluid communication with the at least one inlet, the body configured to form a plurality of flow paths containing the biological particles; and at least one light source configured to traverse light through the plurality of flow paths, and / or at least one light capturing device configured to receive light from the inline flow cell, wherein, upon interaction with the traversed light, the biological particles generate at least one signal.

2. The inline flow cell of claim 1, comprising a plurality of inlets configured to divide the fluid into the plurality of flow paths.

3. The inline flow cell of claim 1 or 2, wherein the body is configured to divide the biological particles between the plurality of flow paths.

4. The inline flow cell of any preceding claim, wherein the body comprises at least one partition configured to divide the fluid between the plurality of flow paths.

5. The inline flow cell of any preceding claim, wherein each of the plurality of flow paths is uniform in size and shape, or wherein each of the plurality of flow paths has the same height and width, or wherein at least two of the plurality of flow paths is non-uniform in size, shape, or both.

6. The inline flow cell of any preceding claim, wherein each of the plurality of flow paths has a hydraulic diameter defined by ^^^^ 2^^^^^^^^ ℎ = ^^^^+^^^^ , wherein H is the height and W is the width of each flow path.OK Ref.11080007.01098 7. The inline flow cell of any preceding claim, wherein each of the plurality of flow paths comprises a rectangular cross-section and has an aspect ratio of height to width, or of height to diameter of about 2 to about 12.

8. The inline flow cell of any preceding claim, wherein each of the plurality of flow paths is configured to provide about the same flow rate of the fluid when flowing therethrough, and / or wherein each of the plurality of flow paths is configured to provide about the same resistance to the fluid when flowing therethrough.

9. The inline flow cell of any preceding claim, wherein each of the biological particles has a Reynold’s Number of about 1 to about 10 when flowing through a flow path of the plurality of flow paths.

10. The inline flow cell of any preceding claim, wherein the width of each of the plurality of flow paths is about 1% to about 70% larger than the average length, width, or diameter of each of the biological particles, and / or wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α- cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

11. The inline flow cell of any preceding claim, wherein the inlet is disposed at the center of the body, or wherein the inlet is disposed off-center with respect to a center line of the body, and / or wherein the inlet is disposed on a top wall of the body, or a side wall of the body.

12. The inline flow cell of any preceding claim, wherein the inlet comprises a cross-section or orifice size substantially equivalent to a depth of the body.OK Ref.11080007.01098 13. The inline flow cell of any preceding claim, wherein a length or diameter of the body is about 2 to about 10 times the cross-section or orifice size of the inlet.

14. The inline flow cell of any preceding claim, further comprising an outlet in fluid communication with the body.

15. The inline flow cell of any preceding claim, wherein the outlet is disposed at the center of the body.

16. The inline flow cell of any preceding claim, wherein the outlet comprises a cross-section or orifice size equivalent to a depth of the body.

17. The inline flow cell of any preceding claim, wherein a length or diameter of the body is about 2 to about 10 times the cross-section or orifice size of the outlet.

18. The inline flow cell of any preceding claim, wherein the body comprises a transparent or translucent material at a first end of the body, wherein the transparent or translucent material is configured to allow light from the at least one light source to pass through the fluid while within the body, optionally wherein the body comprises the transparent or translucent material at a second end of the body, wherein the transparent or translucent material is configured to allow light from the at least one light source to pass through the first end of the body to the second end of the body, and / or to allow the at least one light capturing device to receive light.

19. The inline flow cell of any preceding claim, wherein the body is formed of a transparent or translucent material.

20. The inline flow cell of claim 18 or 19, further comprising a laser scatter detection window formed of the transparent or translucent material and a biological particle counting window formed of the transparent or translucent material.OK Ref.11080007.01098 21. The inline flow cell of any preceding claim, wherein the at least one light source comprises a laser.

22. The inline flow cell of any preceding claim, further comprising a detector configured to receive the at least one signal.

23. The inline flow cell of any preceding claim, further comprising a Laser-Raman detector configured to receive light scattered when the biological particles interact with light from the light source.

24. The inline flow cell of claim 22 or 23, wherein the detector comprises a photodiode array (PDA), a charge coupled device (CCD), or combinations thereof.

25. The inline flow cell of any preceding claim, wherein the at least one signal comprises diffracted light, refracted light, reflected light, absorbed light, or combinations thereof.

26. The inline flow cell of any preceding claim, wherein the light source is comprised in a fluorescence episcopic (Epi)-illumination microscope.

27. The inline flow cell of claim 26, wherein the fluorescence Epi-illumination microscope comprises an optics train comprising at least one dichroic beam splitter.

28. The inline flow cell of any preceding claim, wherein the light source comprises a laser configured to scatter light upon interaction with the biological particles.

29. The inline flow cell of any preceding claim, wherein the light source comprises a laser configured to cause ultraviolet fluorescence of the biological particles.

30. The inline flow cell of any preceding claim, wherein the body comprises a first portion having a first length, depth, or diameter, and a second portion having a second length, depth, or diameter different than the first length, depth, or diameter, respectively.OK Ref.11080007.01098 31. The inline flow cell of any preceding claim, wherein the body further comprises at least one reticle.

32. The inline flow cell of claim 31, wherein the at least one reticle is configured for size calibration using a biological particle analyzer or camera.

33. The inline flow cell of any preceding claim, wherein the body has a greater internal volume than the inlet or the outlet.

34. The inline flow cell of any preceding claim, wherein the at least one light capturing device is a line scan camera, or wherein the inline flow cell comprises a plurality of light capturing devices, each light capturing device being comprised in a respective line scan camera, or wherein the inline flow cell comprises a plurality of light capturing devices, wherein at least one light capturing device is comprised in a respective line scan camera, wherein the line scan camera is configured to image the biological particles when the biological particles pass by a line scan at a known velocity.

35. The inline flow cell of claim 34, wherein the line scan camera is fixed or movable in a parallel or transverse direction to the flow of the fluid.

36. The inline flow cell of claim 34 or 35, wherein the line scan camera has a line scan frequency of about 1 Hz to about 300 kHz, or about 10 Hz to about 100 kHz.

37. The inline flow cell of any preceding claim, further comprising a line scan camera in fixed position above the body.

38. The inline flow cell of claim 37, further comprising a rotating reflector positioned above the body and configured to receive light from the body and reflect light to the line scan camera.OK Ref.11080007.01098 39. The inline flow cell of claim 38, wherein the reflector comprises a mirror, prism, electro- optic reflector, electromechanical reflector, dichroic reflector, or combinations thereof.

40. The inline flow cell of claim 38 or 39, wherein the body is flat or curved to maintain a focal point on the body as the reflector rotates.

41. The inline flow cell of any one of claims 38 to 40, wherein the line scan camera is connected to a linear actuator, wherein the linear actuator is configured to move the line scan camera relative to the reflector to keep a focal point on the body as the reflector rotates.

42. The inline flow cell of any one of claims 34 to 41, wherein the line scan camera is connected to a gantry, wherein the gantry is connected to a linear actuator configured to move the gantry over the body to create an image of the biological particles.

43. The inline flow cell of any preceding claim, wherein the at least one light source or light capturing device is comprised in a light scattering analyzer, brightfield microscope, line scan camera, area scan camera, or high framerate area scan camera, or wherein the inline flow cell comprises a plurality of light sources or a plurality of light capturing devices, each light source or light capturing device being a respective light scattering analyzer, brightfield microscope, line scan camera, area scan camera, high framerate area scan camera, or combinations of any two or more thereof, or wherein the inline flow cell comprises a plurality of light sources or a plurality of light capturing devices, each light source being a respective line scan camera or area scan camera, wherein the line scan camera or area scan camera are configured to produce images of the biological particles that are combinable into one larger image.

44. The inline flow cell according to any preceding claim, further comprising an illuminator configured to illuminate the fluid as it flows through the plurality of flow paths.

45. The inline flow cell of claim 44, wherein the illuminator is connected to a linear actuator.OK Ref.11080007.01098 46. The inline flow cell according to any preceding claim, further comprising at least one servo motor configured to move the body.

47. The inline flow cell according to any preceding claim, comprising a plurality of light capturing devices, wherein a first light capturing device is comprised in a line scan camera and a second light capturing device is comprised in a high framerate area scan camera.

48. The inline flow cell of claim 47, wherein the high framerate area scan camera tracks a subset of particles of the biological particles to determine their respective velocities.

49. The inline flow cell of claim 48, wherein the respective velocities are received by the line scan camera and used to scale the sampling rate or correct velocity for a line scan output image.

50. The inline flow cell of any preceding claim, further comprising a filter disposed upstream of the plurality of flow paths, wherein the filter is configured to remove biological particles having a size of greater than about 20 µm to about 1,000 µm, or about 50 µm to about 500 µm, about 100 µm to about 250 µm.

51. The inline flow cell of claim 50, wherein the filter is a tangential flow filter or an on-chip filter.

52. The inline flow cell of claim 50 or 51, wherein the inlet, body, or both comprise internal protrusions disposed upstream of the plurality of flow paths to prevent large biological particles of greater than about 500 µm, greater than about 750 µm, or greater than about 1000 µm from entering the plurality of flow paths.

53. The inline flow cell of any one of claims 50 to 52, wherein the inlet, body, or both comprises an array of internal protrusions, wherein the array of internal protrusions are offset from each other to allow the large particles to be displaced in a direction opposite fluid flow.OK Ref.11080007.01098 54. The inline flow cell of any preceding claim, wherein internal surfaces of the inlet, the body, or both are configured to provide at least one of wall lift forces, shear gradient forces, or Dean flow forces that modify trajectories of biological particles having differing geometric properties.

55. The inline flow cell of any preceding claim, wherein the inlet, the body, or both comprise varying cross-sections disposed upstream from the plurality of flow paths to hydrodynamically position biological particles in an equilibrium position before entering the plurality of flow paths.

56. An inline flow cell for analyzing biological particles, comprising: a first inlet configured to receive a first fluid; a second inlet configured to receive a second fluid comprising biological particles; a body in fluid communication with the first inlet and the second inlet, wherein the body is configured to form a plurality of flow paths; a wall disposed within the body, the wall comprising a plurality of openings configured to receive small biological particles having a size of less than about 50 µm from the second fluid; and at least one source of energy configured to traverse energy through the body to measure and / or move the small biological particles, wherein the small biological particles are received in the plurality of flow paths.

57. The inline flow cell of claim 56, wherein the plurality of flow paths are formed by laminar flow of the first fluid or second fluid, or are formed by a plurality of dividers configured to receive the first fluid or the second fluid.

58. The inline flow cell of claim 56 or 57, wherein each of the plurality of flow paths has an aspect ratio of height to width, or height to diameter of about 2 to about 12, and / or wherein each of the plurality of flow paths is configured to provide a flat parabolic velocity profile of fluid flowing through each channel.OK Ref.11080007.01098 59. The inline flow cell of any one of claims 56 to 58, wherein the at least one energy source comprises at least one light source configured to provide a force to small biological particles in the second fluid.

60. The inline flow cell of claim 59, wherein the force comprises an optical force, gradient force, or scattering force.

61. The inline flow cell of claim 59 or 60, wherein the force is configured to move individual biological particles through the openings in the wall, and / or wherein the force is configured to move the small biological particles through the openings faster to provide a higher flow rate of the small biological particles in the plurality of flow paths, and / or wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α- cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

62. The inline flow cell of any one of claims 56 to 61, further comprising a first outlet in fluid communication with the first body.

63. The inline flow cell of claim 62, wherein the first outlet is configured to receive a first discharge fluid enriched with small biological particles.

64. The inline flow cell of claim 62 or 63, further comprising a second outlet in fluid communication with the second body.

65. The inline flow cell of claim 64, wherein the second outlet is configured to receive a second discharge fluid enriched with clusters of biological particles.OK Ref.11080007.01098 66. The inline flow cell of any one of claims 56 to 65, wherein the openings are about 10 µm to about 500 µm, or about 20 µm to about 100 µm, or about 40 µm to about 60 µm.

67. An inline flow cell, comprising: an inlet configured to receive a fluid comprising biological particles, optionally wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof; a body comprising: a pass-through flow path in fluid communication with an outlet; a plurality of flow paths or collectors, configured to receive biological particles of varying size; and at least one laser configured to apply a force to one or more of the biological particles entering the body through the inlet.

68. The inline flow cell of claim 67, wherein the pass-through flow path has a larger diameter than the diameter of each of the plurality of flow paths or collectors.

69. The inline flow cell of claim 67 or 68, wherein the amount of optical force applied is indicative of the size of a particular biological particle and its flow path or collector.

70. The inline flow cell of any one of claims 67 to 69, further comprising at least one light source configured to traverse light through the plurality of flow paths or collectors, and / or at least one light capturing device configured to receive light from the inline flow cell.

71. The inline flow cell of claim 70, wherein the at least one light source or light capturing device is comprised in a light scattering analyzer, brightfield microscope, line scan camera, area scan camera, high framerate area scan camera, orOK Ref.11080007.01098 wherein the inline flow cell comprises a plurality of light sources or a plurality of light capturing devices, each light source being comprised in a respective light scattering analyzer, brightfield microscope, line scan camera, area scan camera, high framerate area scan camera, or combinations of any two or more thereof, or wherein the inline flow cell comprises a plurality of light sources or a plurality of light capturing devices, each light source being comprised in a respective line scan camera or area scan camera, wherein the line scan cameras or area scan cameras are positioned adjacent to one another and configured to produce parallel images of the biological particles that are combinable into one larger image.

72. An inline flow cell, comprising: at least one inlet configured to receive a fluid comprising biological particles; a body configured to form a plurality of flow paths or collectors containing the biological particles; and at least one of an acoustic analyzer, laser source, light emitting diode source, brightfield source, darkfield source, Epi illumination source, or camera configured to traverse energy through the plurality of flow paths, wherein, upon interaction with the energy, the biological particles generate at least one signal.

73. The inline flow cell of claim 72, wherein the acoustic analyzer is comprised in an acoustic particle analyzer, or an acoustic impedance detector, and / or wherein the laser source is comprised in a biological particle analyzer, light emitting diode source, brightfield source, darkfield source, Epi illumination source, or camera.

74. The inline flow cell of claim 72 or 73, wherein the at least one acoustic analyzer is configured to measure an acoustic wave, acoustic impedance, dielectric properties, or combinations thereof of the biological particles.

75. The inline flow cell of any one of claims 72 to 74, wherein the acoustic analyzer, laser source, light emitting diode source, brightfield source, darkfield source, Epi illumination source, or camera is configured to turn on when a particle of interest is detected.OK Ref.11080007.01098 76. The inline flow cell of claim 75, wherein the at least one acoustic analyzer is configured to modify a trajectory of the particle of interest to sort the particle of interest into a channel of the plurality of flow paths or collectors.

77. The inline flow cell of any one of claims 72 to 76, further comprising a device configured to apply a force to one or more of the biological particles.

78. The inline flow cell of claim 77, wherein the force comprises a magnetic, optical, electrical, mechanical, or thermal force.

79. The inline flow cell of any one of claims 72 to 78wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

80. A system for analyzing biological particles, comprising: a supply reservoir configured to store a fluid comprising the biological particles; at least one inline flow cell according to any preceding claim in fluid communication with the supply reservoir; at least one biological particle analyzer comprising a light source, acoustic analyzer, camera, or combinations the at least one biological particle analyzer configured to receive, analyze, and / or image an optical or acoustical signal from the biological particles; and a processor in communication with the at least one biological particle analyzer, optionally, wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor- infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-OK Ref.11080007.01098 cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

81. The system of claim 79, further comprising at least one discharge stream in fluid communication with the inline flow cell.

82. The system of claim 80, further comprising a multi-way valve configured to direct fluid flow between the at least one inline flow cell and the at least one discharge steam or a waste stream.

83. The system of any one of claims 79 to 81, further comprising a plurality of inline flow cells, wherein a first inline flow cell is in fluid communication with the a discharge stream, and a second inline flow cell is in fluid communication with a waste stream.

84. The system of any one of claims 79 to 82, further comprising at least one receptacle in fluid communication with the inline flow cell and the at least one discharge stream.

85. The system of any one of claims 79 to 83, further comprising at least one implantable device in fluid communication with the inline flow cell.

86. The system of claim 84, further comprising an intermediate reservoir in fluid communication with the at least one inline flow cell and the at least one implantable device.

87. A system for analyzing biological particles, comprising: a supply reservoir configured to store a fluid comprising the biological particles; a first inline flow cell according to any one of claims 1 to 78 in fluid communication with the supply reservoir; a second inline flow cell according to any one of claims 1 to 78 in fluid communication with the supply reservoir; at least one biological particle analyzer comprising a light source, acoustic analyzer, camera, or combinations thereof, the at least one biological particle analyzer configured toOK Ref.11080007.01098 receive, analyze, and / or image an optical or acoustical signal from the biological particles in the first inline flow cell, the second inline flow cell, or both; and a processor in communication with the at least one biological particle analyzer, optionally, wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor- infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β- cells, α-cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

88. The system of claim 86, further comprising at least one discharge stream in fluid communication with the at least one first inline flow cell, the at least one second inline flow cell, or both.

89. The system of claim 86 or 87, further comprising a waste reservoir in fluid communication with the at least one first inline flow cell, the at least one second inline flow cell, or both.

90. A method of analyzing biological particles in a sample, comprising: receiving a fluid in at least one inline flow cell according to any one of claims 1 to 78, wherein the fluid comprises a plurality of biological particles; introducing light into the flow cell, wherein the light interacts with the plurality of biological particles in the fluid and forms at least one signal; using the at least one signal to identify each of a plurality of flow paths containing one or more of the plurality of biological particles; and measuring the one or more of the plurality of biological particles within the identified flow paths using at least one biological particle analyzer.

91. The method of claim 90, wherein the size of the plurality of biological particles varies and the drag force of each biological particle of the plurality of biological particles is a function of its size, and / orOK Ref.11080007.01098 wherein the biological particles comprise T-cells, engineered T-cells, chimeric antigen receptor (CAR) T or CAR-T cells, CAR macrophage (M) or CAR-M cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, other engineered or effector cells, stem cells, nuclei, organelles, organoids, chloroplasts, mitochondria, islet cells, endocrine cells, β-cells, α- cells, δ-cells of the pancreas or other organs and / or FC cells, viruses, bacteria and mycoplasma, clusters thereof, cell clusters thereof, or combinations thereof.

92. The method of claim 90 or 91, wherein walls of the inline flow cell produce drag on the fluid.

93. The method of any one of claims 90 to 92, wherein interaction of each biological particle of the plurality of biological particles with the light causes the at least one signal.

94. The method of claim 93, wherein the at least one signal comprises scattered light.

95. The method of any one of claims 90 to 94, wherein interaction of the light with each of the biological particles enables each of the biological particles to be counted and tracked through the plurality of flow paths within the inline flow cell.

96. The method of any one of claims 90 to 95, wherein for a given measurement frame by the biological particle analyzer, at least some of the plurality of flow paths do not contain biological particles.

97. The method of any one of claims 90 to 96, wherein the inline flow cell comprises about 100 to about 750 flow paths, or about 250 to about 500 flow paths.

98. The method of any one of claims 90 to 97, wherein the light source is a laser comprised in a light scattering analyzer.OK Ref.11080007.01098 99. The method of claim 98, wherein the at least one signal comprises light scattered by a biological particle, wherein the scattered light and the intensity of the scattered light is measured by a charge coupled device.

100. The method of any one of claims 90 to 99, further comprising calibrating the at least one biological particle analyzer using at least one reticle comprised in the inline flow cell, optionally wherein calibrating the biological particle analyzer comprises imaging the at least one reticle.

101. The method of claim 100, wherein imaging the at least one reticle provides data used for position adjustment of the at least one biological particle analyzer, inline flow cell, and / or body of the inline flow cell.

102. The method of any one of claims 90 to 101, further comprising using the at least one signal to normalize background noise in the fluid.

103. The method of any one of claims 90 to 102, wherein the number of biological particles identified in the plurality of flow paths is minimized, velocity of each of the biological particles is determined, and double counting of each of the biological particles is minimized.

104. The method of any one of claims 90 to 103, wherein each of the identified biological particles shares a focal plane, and flow rate of the fluid through the inline flow cell prevents each of the biological particles from passing above or below another of the biological particles as it travels through a flow path of the plurality of flow paths.

105. The method of any one of claims 90 to 104, further comprising receiving the fluid in a first frame, wherein the light interacts with the biological particles in the fluid to form a plurality of light scattering signals that are detected by a light scattering analyzer forming a first tracked population of biological particles.

106. The method of claim 105, further comprising forming a second tracked population of biological particles downstream from the first tracked population of biological particles.OK Ref.11080007.01098 107. The method of claim 106, further comprising forming a first bounded region between the first and second tracked populations.

108. The method of claim 107, further comprising analyzing the first bounded region to count the number of biological particles in the first bounded region.

109. The method of any one of claims 106 to 108, further comprising forming a third tracked population of biological particles downstream from the second tracked population.

110. The method of claim 109, further comprising forming a second bounded region between the second and third tracked populations.

111. The method of claim 110, further comprising analyzing the second bounded region to count the number of biological particles in the second bounded region.

112. The method of any one of claims 90 to 111, wherein the at least one biological particle analyzer is a line scan camera, the method further comprising imaging the biological particles using the line scan camera.

113. The method of claim 112, wherein the line scan camera is fixed or movable in a parallel or transverse direction in relation to the direction of fluid flow through the inline cell.

114. The method of claim 113, wherein the line scan camera is mounted in a transverse direction to the fluid flow and a line scan rate of acquisition is adjusted based on an average drift velocity of the biological particles.

115. The method of claim 114, wherein the average drift velocity adjustment minimizes velocity relating to sampling errors.OK Ref.11080007.01098 116. The method of claim 114 or 115, wherein the line scan rate of acquisition is adjusted by increasing or decreasing the velocity of the line scan with fixed line scan frequency, or increasing or decreasing the line scan frequency with fixed velocity of the line scan.

117. The method of any one of claims 114 to 116, further comprising determining the size of individual biological particles upstream from the plurality of flow paths, and moving each of the biological particles into a particular flow path based on its size and / or velocity.

118. The method of claim 117, further comprising determining a line scan rate based on the fastest moving particles within the particular flow path.

119. The method of claim 117 or 118, further comprising adjusting size data for particles having a velocity of less than or greater than the average expected velocity.

120. The method of any one of claims 90 to 119, further comprising illuminating the fluid in the inline flow cell with an illuminator while imaging with the at least one biological particle analyzer.

121. The method of claim 120, wherein the illuminator is moved synchronously with the at least one biological particle analyzer as it interacts with the biological particles. 4898-5479-5341, v.1

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