Patent Description:
<CIT> purports to teach an apparatus for supplying multiple samples for rapid processing in flow cytometry. The apparatus requires a syringe to inject a sample into a sample loop, then requires a reciprocating valve to provide the sample from the sample loop to a flow cytometer. To attempt a throughput goal of approximately <NUM> samples/minute, the apparatus purports to use about <NUM> second of aspiration, <NUM> seconds of time delay, and <NUM> seconds for the valve to be switched. <CIT> purports to avoid the potential problem of air bubbles lodging in troublesome areas such as an analysis region of a flow cell, further noting that air bubbles can be broken up and dispersed at connector junctions, causing inaccuracies in the sample draw.

<CIT> purports to teach an apparatus for high throughput flow cytometry. The apparatus provides for separation of adjacent samples by using a separation gas, such as an air bubble, to prevent mixing of adjacent samples. As a probe, the apparatus preferably uses a stainless steel needle.

In the field of mass spectrometry and high performance liquid chromatography, <CIT> discloses a sampling probe having an open end. In the sampling probe, a liquid supply conduit delivers liquid to the open end of the probe at a first flow rate, and a liquid exhaust removes liquid at a second flow rate lower than the first flow rate. The sample material is received into the open end of the probe. The probe is useful for mass spectrometry and high performance liquid chromatography. <CIT> does not mention applications in flow cytometry, nor does it mention avoidance of air or other gas in the liquid provided from the probe, nor does it mention sorting or selection of cells, viruses, proteins, or other biological materials or of research reagents such as beads.

Also in the field of mass spectrometry and high performance liquid chromatography, <CIT> discloses a sampling probe having an open end. In the sampling probe, a liquid supply conduit delivers liquid to the open end of the probe at a first flow rate, and an exhaust conduit removes liquid at a second flow rate higher than the first flow rate. A gas flow containing the sample is received into the open end of the probe. The probe is useful for mass spectrometry and high performance liquid chromatography. <CIT> does not mention applications in flow cytometry, nor does it mention sorting or selection of cells, viruses, proteins, or other biological materials or of research reagents such as beads, and its method require introduction of air or another gas into the probe, thereby creating a gas/liquid interface in the exhaust conduit.

Additionally in the field of mass spectrometry, <CIT> discloses a sampling interface which is fluidly coupled to an ion source. This interface includes a sampling probe having an outer capillary tube for providing a fluid to a distal end of the probe and an inner capillary tube for removing a fluid from a distal end of the probe. A flow rate through the outer capillary tube can be temporarily increased relative to the flow rate through the inner capillary tube such that the fluid in the distal end of the probe overflows to clean any residual sample deposited by the withdrawn substrate and/or to prevent any airborne material from being transmitted into the inner capillary tube. <CIT> does not mention applications in flow cytometry, nor does it mention avoidance of air or other gas, as such, in the fluid provided from the probe, nor does it mention sorting or selection of cells, viruses, or proteins or of research reagents such as beads.

The above-discussed art lacks the ability to provide high sample throughput for flow cytometry or flow cytometry sorting without introducing air bubbles or using a complicated system of syringes, loops, and valves.

<NPL> informs about the state of microfluidic cell sorting devices, providing details on and alternatives to other types of flow cytometry cell sorting. It describes classification and current methods for active and passive sorting including fluorescent label-based, bead-based, and label-free sorting. It also describes the principles by which cells are being sorted for each methodology.

<NPL> discusses the great degree of potential in various cell-based therapy techniques for cancer treatment. It briefly mentions a high throughput investigation but does not discuss high throughput sampling or the sorting or selection of different components in one or more samples. As other references regarding cell-based therapies, there are <NPL> and <NPL>.

<NPL> notes the importance of high-throughput cell sorting for cell-based therapies, comparing the speeds of fluorescence sorting and magnetic sorting. However, it does not particularly mention the processing of large numbers of different and/or separate samples, nor does it mention problems with air or other gas bubbles in the intake of a plurality of samples to be sorted.

<CIT> discloses a sampling probe with an irrigation conduit having an outlet end positioned at the probe end. Analytes enter the irrigation fluid and are aspirated into a sampling tip for delivery to an analysis instrument.

"Flow cytometry" may refer to any process involving the detection of a property or properties of a collection of cells, particles, or molecules in a fluid suspension, and measurement and/or sorting based on the property or properties, using the fluidity of the suspension. The property or properties may be related to one or more labels provided on the collection of cells, particles, or molecules, such as a fluorescent, magnetic, isotopic, or chemical label. Additionally or alternatively, the property or properties may be unrelated to any label, for example wherein the property is the size, shape, density, binding capability, conductivity, or acoustic properties of a cell, particle, or molecule.

The present invention provides an analysis and/or sorting method, comprising: conveying a first fluid within a probe to an open end of a probe; collecting at least one sample, comprising at least one first component and at least one second component, into the open end of a probe; removing a fluid stream comprising the at least one sample and the first fluid from the open end of the probe; conveying the fluid stream to a flow cytometer and analyzing the fluid stream by flow cytometry, and/or separating the fluid stream into at least a first separated stream comprising the at least one first component and a second separated stream comprising the at least one second component by flow cytometry.

A sufficient flow rate of the first fluid is maintained so as to replace at least the removed fluid and/or a sufficiently low flow rate through the fluid exhaust is maintained so as to prevent intake of gas or air along with the sample and the first fluid supplied through the fluid supply to the open end such that the fluid stream is substantially free of both air bubbles and a gas/liquid interface.

Preferably, the at least one sample comprises a first sample, initially contained in a first container, and a second sample, initially contained in a second container, wherein the first container is a first well in a plate and the second container is a second well in the same plate or in a second plate. In a further embodiment, the at least one sample comprises a plurality of samples contained in a plurality of containers, and the plurality of containers are wells in one well plate or in a plurality of well plates. The containers can also be compartments within a compartmentalized container or array or collection of containers.

Preferably, the at least one sample comprises a plurality of samples, and wherein samples are collected at a rate of greater than <NUM> samples per minute, and optionally, wherein collecting the plurality of samples comprises at least partial insertion of the probe into a plurality of containers or transferring the Preferably, the first fluid is water or a sheath fluid or other fluid medium suitable for flow cytometry or flow cytometry sorting.

Preferably, the first fluid is a buffered aqueous saline solution or a cell culture medium.

Preferably, the first fluid is a buffered aqueous saline solution comprising a phosphate buffer.

Preferably, the at least one sample comprises cells.

Preferably, at least a portion of the sample is labeled, for example with a fluorescent, magnetic, isotopic, or chemical label. In other embodiments, the sample is not labeled, and analysis and/or sorting occurs based on at least one other detected property.

Preferably, the at least one first component comprises a labeled component and wherein the at least one second component comprises a component which is not labeled.

Preferably, the at least one sample comprises leukocytes, stem cells, or circulating tumor cells.

Preferably, the at least one sample comprises T cells.

Preferably, at least some of the T cells comprise a T cell receptor or a chimeric antigen receptor.

Preferably, the at least one sample comprises tumor infiltrating lymphocytes.

Preferably, the at least one first component comprises T cells comprising a T cell receptor or a chimeric antigen receptor and wherein the at least one second component comprises T cells which are substantially free of T cell receptors and chimeric antigen receptors.

Preferably, the conveying the first fluid to the open end of the probe comprises pumping the first fluid with a first pump, and wherein removing the fluid stream from the open end of the probe comprises removing the fluid stream with a second pump.

Preferably, the method further comprises controlling a first flow rate of the first fluid into the open end of the probe, and simultaneously controlling a second flow rate of the fluid stream from the open end of the probe.

Preferably, the method further comprises adding a second fluid to the fluid stream, before, during, or after conveying the fluid stream to the flow cytometer, wherein the second fluid is the same as or different from the first fluid.

<FIG> show an embodiment of an analysis and/or sorting system <NUM>, shown in these figures in the presence of a well plate <NUM>. In this embodiment, analysis and/or sorting system <NUM> includes probe <NUM>, probe input line <NUM> configured to provide a fluid to probe <NUM>, and probe to flow cytometer line <NUM> configured to remove a fluid from probe <NUM> and/or provide a fluid to flow cytometer <NUM>. Also in this embodiment, probe input pump <NUM> is configured to move a fluid through probe input line <NUM> to probe <NUM>, and probe to flow cytometer pump <NUM> is configured to move a fluid through probe to flow cytometer line <NUM>, away from probe <NUM> and/or into flow cytometer <NUM>. Autosampler <NUM> in this embodiment may be configured to move probe <NUM> between wells 51a, 51b, and 51c of well plate <NUM>. In <FIG>, the embodiment of probe <NUM> is depicted in an orientation such that an open end thereof would be pointing downward during operation. However, in other embodiments, for example where the wells or other sample container or containers are sufficiently narrow so as to retain the sample by forces such as surface tension or adhesion even in other orientations, the open end of the probe may point in an upward direction, a sideways direction, or at a skew angle with respect to a vertical direction. In some such embodiments, an upward direction for the probe and an inverted orientation of the sample container or containers, wherein an open end of the sample container or containers faces downward, may be particularly preferred.

In some embodiments, analysis and/or sorting system <NUM> preferably orients probe <NUM> such that it can be directed into a sample container, such as a sample holder, tube, cartridge or microchip, and/or well plate, or compartments within a compartmentalized container or array or collection of containers. <FIG> shows probe <NUM> oriented to collect a sample from well 51c in well plate <NUM>. <FIG> shows probe <NUM> collecting a sample from well 51c in well plate <NUM>. In the embodiment shown in <FIG>, autosampler <NUM> is configured to reposition probe <NUM> for collection of samples in well 51a and well 51b in well plate <NUM>. In other embodiments, autosampler <NUM> may be configured to reposition the well plate <NUM> for collection of samples in well 51a and well 51b by probe <NUM>, and probe <NUM> may be provided in a stationary position.

<FIG>, and <FIG> show an embodiment of a probe <NUM>. In this embodiment, probe <NUM> comprises an outer wall <NUM>, an open end <NUM> at an end of the outer wall <NUM> which in these views is pointed upward, a fluid supply <NUM> configured for providing fluid to the open end <NUM>, and a fluid exhaust <NUM> configured for removing fluid from open end <NUM>.

In the embodiment of probe <NUM> shown in <FIG>, and <FIG>, the fluid supply <NUM> is an annular region immediately within outer wall <NUM>, and fluid exhaust <NUM> is a coaxial conduit positioned centrally in the probe <NUM>. In some embodiments, an opening of fluid exhaust <NUM> may not extend axially for the entire length of outer wall <NUM> extending to open end <NUM>, leaving some space at open end <NUM> for flow to more readily occur. In other embodiments, an opening of fluid exhaust <NUM> may extend axially for the entire length of the outer wall <NUM> extending to open end <NUM>, or may extend axially even further than the entire length of the outer wall <NUM> and out from the open end <NUM>.

In other embodiments, fluid supply <NUM> and fluid exhaust <NUM> can be configured differently. For example, in some other embodiments, the fluid exhaust <NUM> is an annular region immediately within outer wall <NUM>, and fluid supply <NUM> is a coaxial conduit positioned centrally in the probe <NUM>. In other embodiments, the fluid supply <NUM> may be provided as a plurality of fluid supply conduits and/or the fluid exhaust <NUM> may be provided as a plurality of fluid exhaust conduits. Conduits of the fluid supply <NUM> and/or the fluid exhaust <NUM> may be provided as shown in <FIG>, and <FIG> in a parallel direction, or in other embodiments may be provided at oblique angles from one another.

In the aforementioned embodiment of probe <NUM> as shown in <FIG> and <FIG>, probe <NUM> is configured to provide flow of a fluid along a flow path <NUM>. In such embodiment, fluid supply <NUM> can provide a fluid to open end <NUM> of probe <NUM> along incoming flow path 106a, then the fluid can flow through open end <NUM> along open-end flow path 106b, then the fluid can flow into outgoing flow path 106c through fluid exhaust <NUM>. In this or other embodiments, open-end flow path 106b can include paths of flow which are less direct than as depicted in <FIG> and <FIG>, extending into open end <NUM> which then forms fluid dome <NUM>. To measure or control the flow of a fluid inward through fluid supply <NUM>, fluid supply <NUM> can include a flow meter and/or a flow regulator. Similarly, but independently, to measure or control the flow of a fluid outward through fluid exhaust <NUM>, fluid exhaust <NUM> can include a flow meter and/or a flow regulator.

The material or materials constituting probe <NUM> are not particularly limited. Any of outer wall <NUM>, fluid supply <NUM>, and/or fluid exhaust <NUM> can comprise at least one metal, metal alloy, plastic, glass, ceramic, or any combination thereof, preferably comprising stainless steel and/or medical-grade plastic. Any part or entirety of outer wall <NUM>, fluid supply <NUM>, and/or fluid exhaust <NUM> can be coated or uncoated, preferably coated with a hydrophobic or hydrophilic coating. In some embodiments, at least a portion of outer wall <NUM>, fluid supply <NUM>, and/or fluid exhaust <NUM> can comprise a hydrophilic material or be provided with a hydrophilic coating, thereby providing an adhesive effect with a fluid flowing along flow path <NUM> and/or the fluid in fluid dome <NUM>. In the same or other embodiments, at least a portion of outer wall <NUM>, fluid supply <NUM>, and/or fluid exhaust <NUM> can comprise a hydrophobic material or be provided with a hydrophobic coating, thereby avoiding an adhesive effect with a fluid in order to better form a desired dome shape for fluid dome <NUM>.

In some embodiments, probe <NUM> can further include a fluid collection conduit, configured to collect excess or overflowing fluid from open end <NUM>. Such a fluid collection conduit may be positioned within or provided on outer wall <NUM>. The fluid collection conduit can comprise an opening or a plurality of openings, preferably located proximally to open end <NUM>. The opening or openings of the fluid collection conduit can be one or more annular openings or a plurality of openings in an annular arrangement.

In some embodiments as shown in <FIG>, probe <NUM> may be configured for at least partial insertion into a sample container or plurality of sample containers, preferably a well <NUM> or a plurality of such wells in a well plate <NUM> or plurality of such well plates. In these embodiments, an outer width w of probe <NUM> is preferably less than an inner width w' of the well <NUM>, thereby allowing for insertion of probe <NUM> into well <NUM>. The outer width w of probe <NUM> may be the width of the probe <NUM> at open end <NUM> or, more preferably, may be the width of the probe <NUM> at an axial distance from open end <NUM> which is less than or approximately equal to the depth of well <NUM>. In some embodiments, outer width w of probe <NUM> may be less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, or less than <NUM>. In some embodiments, outer width w of probe <NUM> may be a width of the probe at the open end <NUM> of probe <NUM>, or at an axial distance of from <NUM> to <NUM> from the open end <NUM> of probe <NUM>, or at an axial distance of from <NUM> to <NUM> from the open end <NUM> of probe <NUM>.

In other embodiments, probe <NUM> may not necessarily be configured for insertion into a sample container. For example some embodiments of probe <NUM> and/or analysis and/or sorting system <NUM> may be suitable for use with an apparatus for transmitting a sample from individual wells within a well plate to the open end <NUM> of probe <NUM>, for example with an automated pipetting system, or by directing acoustic energy into the samples within individual wells to eject droplets of the sample. In these embodiments, the open end of the probe may point in an upward direction, a downward direction, a sideways direction, or at a skew angle with respect to a vertical direction. In some such embodiments, an upward direction for the probe and an inverted orientation of the sample container or containers may be particularly preferred. In still other embodiments, probe <NUM> may be configured to receive a sample or more preferably a plurality of samples without direct sampling from wells in a well plate, for example in the provision of drops or other small quantities of a sample or plurality of samples directly to the open end <NUM> of the probe <NUM>.

In some embodiments, the analysis and/or sorting system <NUM> has only one probe <NUM> and no more. In other embodiments, analysis and/or sorting system <NUM> can comprise a plurality of probes. In cases where multiple probes <NUM> are utilized in parallel, the sampling rate may be multiplied accordingly, such as if autosampler <NUM> is configured to hold and/or move four probes simultaneously to collect samples and/or fluid from four different wells in parallel. In another embodiment, autosampler <NUM> may be configured to hold and/or move <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more probes <NUM> in parallel.

In certain embodiments, a flow cytometer <NUM> can include a flow cell <NUM>, a laser <NUM>, and at least two containers 61a and 61b, which can be, for example, wells in a plurality of well plates or wells in the same well plate, as shown in <FIG>. Flow cytometer <NUM> is not particularly limited, except in that it is configured to sort components of a fluid stream from probe <NUM>. In particular, flow cytometer <NUM> is configured to sort a sample into at least two containers 61a and 61b. A fluid stream provided to flow cytometer <NUM> can pass into flow cell <NUM>, which can regulate the fluid stream such that many or most cells or other particles of the sample in the fluid stream emerge individually in separate droplets. In certain embodiments, the fluid stream thus regulated then passes through a measuring system. The measuring system can be, but is not limited to, an impedance, conductive, size exclusion, filtration, acoustic, thermodynamic, electromagnetic, mass spectrometric, or optical system. In some embodiments, the measuring system can include a laser beam emitted from laser <NUM>, which can excite a fluorescent marker in a sample in the fluid stream. A detector can then detect the presence or absence of the fluorescent marker by the presence or absence of fluorescence. Some embodiments include more than one laser and/or more than one detector. In certain embodiments, droplets are charged, and droplets containing cells or particles marked with the fluorescent marker are sorted by their charges into one container 61a, while droplets containing cells or particles not marked with the fluorescent marker are sorted into a different container 61b, as shown in the embodiment in <FIG>. In other embodiments, droplet-based sorting is not used and instead, cells or particles marked with the fluorescent marker are sorted out of a fluid flow into one container 61a using a magnetic solenoid sorting valve, while unlabeled cells or particles are sorted into a different container. As further shown in <FIG>, these containers can be separate wells of a well plate, for example a microplate. In some configurations, flow cytometer <NUM> may sort samples into larger volume containers, tubes, cartridges or microchips.

In other embodiments, a flow cytometer may be configured for analysis of the sample and collection of data about the presence, content, frequency, and/or distribution of components within the sample having different properties, or satisfying various parameters. In particular, the flow cytometer may be configured to measure and collect data as to the presence and/or frequency of a labeled component, such as a labeled biological material, a labeled analysis reagent, or labeled particulate component. In some embodiments, flow cytometer <NUM> may be configured for both analysis and sorting, as both described above. In other embodiments, the flow cytometer may be configured for analysis alone without sorting, and the fluid stream comprising the sample may be discarded after analysis.

In still other embodiments, the flow cytometer <NUM> is a microfluidic sorting device configured for the sorting of samples into at least two containers 61a and 61b or other receiving points as directed by the microfluidic sorting device, for example, wells in a plurality of well plates or wells in the same well plate, as shown in <FIG>. The microfluidic sorting device is not particularly limited, except in that it is configured to sort components of a fluid stream from probe <NUM> based on any fluorescent, magnetic, isotopic, or chemical label or labels provided on the components, and/or based on a property or properties of the components unrelated to any label such as size, shape, density, binding capability, conductivity, or acoustic properties. A fluid stream provided to the microfluidic sorting device can be regulated by the microfluidic sorting device such that many or most cells or other particles of the sample in the fluid stream are separated into distinct components based on the labels and/or properties mentioned above. In certain embodiments, components containing a given set of labels and/or properties are separated and sorted into one container 61a, while components lacking the given set of labels and/or properties are sorted into a different container 61b, as shown in the embodiment in <FIG>. These containers can be separate wells of a well plate, for example a microplate. In some embodiments, for example in embodiments that do not involve analysis or sorting based on fluorescence or any other light- or radiation-based phenomenon, a microfluidic sorting device as flow cytometer <NUM> may not comprise a laser, in contrast with the embodiment of <FIG>, showing laser <NUM>.

In some embodiments, for example as shown in <FIG>, analysis and/or sorting system <NUM> can include a probe input line <NUM>, configured to supply a fluid to probe <NUM>. Probe input line <NUM> is not particularly limited beyond its configuration for conveying a fluid stream to the probe <NUM>. Probe input line <NUM> can be rigid, partially rigid, or flexible. Probe input line <NUM> is preferably a closed and sealed line to prevent introduction of air or gas into the fluid which it conveys. It can include a tube, hose, pipe, conduit through another element, or any combination thereof. It can be made of metal, glass, plastic, rubber, or any combination thereof, or any other material or combination of materials which can convey the fluid to the probe <NUM>. Preferably, probe input line <NUM> comprises silicone or PVC tubing. In some embodiments, probe input line <NUM> can include or exclude valves, joints, and/or junctions. Preferably, probe input line <NUM> excludes any reciprocating valve. In some embodiments, probe input line <NUM> can include or exclude sample loops, preferably excluding sample loops. Probe input line <NUM> is joined to or integral with fluid supply <NUM> to provide fluid to fluid supply <NUM>.

In some embodiments, for example as shown in <FIG>, probe input line <NUM> can include or be interrupted by a probe input pump <NUM>, configured to supply a fluid to probe <NUM>. Probe input pump <NUM> is not particularly limited, except in that it provides a fluid to probe <NUM>. Probe input pump <NUM> can be any of various types of laboratory pumps including peristaltic pumps, diaphragm pumps, syringe pumps, gear pumps, or microfluidic pumps. Preferably, probe input pump <NUM> is a microfluidic pump. In other embodiments, a fluid can be conveyed through probe input line <NUM> to probe <NUM> in other ways. For example, a vacuum in or near probe <NUM> can reduce pressure at or near the end of probe input line <NUM>, thereby causing the pressure (for example, ambient pressure) at a source of the fluid to push the fluid through probe input line <NUM>. Force can also be applied to the fluid to convey it through probe input line <NUM>, for example, by siphoning, or by gravity, from an elevated fluid source. The skilled artisan will understand that other methods of conveying fluid through a line are known and may be suitable for conveying the fluid to probe <NUM> through probe input line <NUM>.

Analysis and/or sorting system <NUM> includes a probe to flow cytometer line <NUM>. Probe to flow cytometer line <NUM> is not particularly limited beyond its configuration for conveying a fluid stream from the probe <NUM> to the flow cytometer <NUM>. Probe to flow cytometer line <NUM> is joined to or integral with fluid exhaust <NUM> to provide fluid to fluid exhaust <NUM>. Probe to flow cytometer line <NUM> can be rigid, partially rigid, or flexible. Probe to flow cytometer line <NUM> is preferably a closed and sealed line to prevent introduction of air or gas into the fluid stream which it conveys. It can include a tube, hose, pipe, conduit through another element, or any combination thereof. It can be made of metal, glass, plastic, rubber, or any combination thereof, or any other material or combination of materials which can convey the fluid stream to the flow cytometer <NUM>. The material or materials of flow cytometer line <NUM> can be the same as or different from the materials of probe input line <NUM>. Preferably, probe to flow cytometer line <NUM> comprises silicone or PVC tubing. In some embodiments, probe to flow cytometer line <NUM> can include or exclude valves, joints, and/or junctions. Preferably, probe to flow cytometer line <NUM> excludes any reciprocating valve. In some embodiments, probe to flow cytometer <NUM> can include or exclude sample loops, preferably excluding sample loops.

In some embodiments, for example as shown in <FIG>, probe to flow cytometer line <NUM> can include or be interrupted by a probe to flow cytometer pump <NUM>, configured to draw a fluid stream from probe <NUM>. Probe to flow cytometer pump <NUM> is not particularly limited, except in that it draws a fluid from probe <NUM> to provide it to flow cytometer <NUM>. Probe to flow cytometer pump <NUM> can be any of various types of laboratory pumps including peristaltic pumps, diaphragm pumps, syringe pumps, gear pumps, or microfluidic pumps. Preferably, probe to flow cytometer pump <NUM> is a microfluidic pump. Probe to flow cytometer pump <NUM> can be the same type as or a different type from probe input pump <NUM>. In other embodiments, a fluid can be conveyed through probe to flow cytometer line <NUM>, from probe <NUM> to flow cytometer <NUM>, in other ways. For example, a vacuum in or near flow cytometer <NUM> can reduce pressure at or near the end of probe to flow cytometer line <NUM>, thereby causing the pressure (for example, ambient pressure) at the probe <NUM> to push the fluid through probe to flow cytometer line <NUM>. Force can also be applied to the fluid to convey it through probe to flow cytometer line <NUM>, for example, by siphoning or gravitational force from a difference in elevation between probe <NUM> and flow cytometer <NUM>. The skilled artisan will understand that other methods of conveying fluid through a line are known and may be suitable for conveying the fluid from probe <NUM> to flow cytometer <NUM> through probe to flow cytometer line <NUM>.

In some embodiments, for example as shown in <FIG>, analysis and/or sorting system <NUM> can further include a control system for controlling probe input pump <NUM> and probe to flow cytometer pump <NUM>. The control system can monitor the flow through probe input line <NUM> and through probe to flow cytometer line <NUM> and can further monitor these two flows relative to one another. The control system can optionally include one or more flow meters for monitoring one or both of the flow through probe input line <NUM> and through probe to flow cytometer line <NUM>. The control system can adjust the pumping power or other determinative parameters for probe input pump <NUM> and/or probe to flow cytometer <NUM>, preferably both. The control system can include a graphical user interface. The control system can be integrated with or separate from a control system for flow cytometer <NUM>.

In some embodiments, for example as shown in <FIG>, analysis and/or sorting system <NUM> can include an autosampler <NUM>. In some embodiments, autosampler <NUM> comprises a holder for controllably holding and releasing probe <NUM>, and for moving probe <NUM> between multiple sample containers and wells. In other embodiments, autosampler <NUM> is fixedly attached or integral with probe <NUM>, and is configured to move probe <NUM> between multiple sample containers and/or wells. In still other embodiments, probe <NUM> may be configured to maintain a substantially fixed position during operation, while autosampler <NUM> comprises a holder for controllably holding and releasing a sample container, such as a sample holder, tube, and/or well plate, and for moving such a sample container to provide multiple samples for sampling by probe <NUM> in its fixed position. Autosampler <NUM> may be configured to collect separate samples from different wells or other containers at a rate of greater than <NUM> per minute, preferably at least <NUM> per minute, preferably at least <NUM> per minute, more preferably at least <NUM> per minute, even more preferably at least <NUM> per minute, and still further preferably at about <NUM> per minute. In some embodiments, autosampler <NUM> may be configured to collect separate samples from different wells or other containers at a rate of as high as <NUM> per minute or less. Embodiments involving these sample collection rates may particularly be embodiments in which probe <NUM> may be configured for at least partial insertion into a sample container or plurality of sample containers, preferably a well <NUM> or a plurality of such wells in a well plate <NUM> or plurality of such well plates.

The above-described system and substantially similar systems can be used in a method for selection and separation of different components within a sample.

A method of analyzing, sorting, separating, and/or selecting components in one or more samples can include conveying a fluid to an open end <NUM> of a probe <NUM>, collecting at least one sample into the open end <NUM> of the probe <NUM>, removing a fluid stream comprising the at least one sample and the fluid from the open end <NUM> of the probe <NUM>, conveying the fluid stream to a flow cytometer <NUM>, and analyzing and/or separating components of the sample with flow cytometer <NUM>.

The sample is not particularly limited. In some embodiments, at least a portion of it may be labeled, preferably prior to collection with probe <NUM>. In some preferred embodiments involving labeling, the sample comprises a labeled biological material or materials and also an unlabeled biological material or materials. The labeled and unlabeled biological material or materials may each independently comprise one or more cells, cell fragments, viruses, virus fragments, organelles, exosomes or extracellular vesicles and fragments thereof, proteins, nucleic acids, and/or carbohydrates. Proteins, if present, can include a protein complex, a multiprotein complex, a single polypeptide, an oligopeptide, or any combination thereof. Nucleic acids, if present, can include chromosomes, polynucleotides, oligonucleotides, a nucleic acid complex, or any combination thereof. Carbohydrates can include, for example, sugars, oligosaccharides, polysaccharides, a carbohydrate complex, or any combination thereof. The sample preferably comprises one or more cells, which in some embodiments may be immune cells, stem cells, or circulating tumor cells (CTCs). Examples of immune cells that may be included in the sample include leukocytes such as T cells, B cells, natural killer (NK) cells, dendritic cells, monocytes, and macrophages. Native or engineered T cells are preferred as leukocytes in certain embodiments, and may include tumor infiltrating lymphocytes, T cells including one or more chimeric antigen receptor proteins, or T cells including one or more T cell receptor proteins suitable for the diagnosis or treatment of cancer, infectious diseases, or autoimmune diseases. In other embodiments, the leukocytes may include native or engineered B cells, in particular engineered B cells suitable for the diagnosis or treatment of cancer, autoimmune diseases, infectious diseases, or protein deficiency diseases. In still other embodiments, the leukocytes may be native or engineered NK cells, for example NK cells including a chimeric antigen receptor protein, in particular those suitable for treatment of cancer, infectious diseases, or autoimmune diseases. In further embodiments, the leukocytes may be native or engineered dendritic cells, for example engineered dendritic cells suitable for the diagnosis or treatment of cancer, infectious diseases, inflammatory diseases, degenerative diseases, autoimmune diseases, and organ transplantation. In still further embodiments, the leukocytes may be native or engineered monocytes, for example engineered monocytes suitable for the diagnosis or treatment of cancer, infectious diseases, inflammatory diseases, degenerative diseases, autoimmune diseases, and organ transplantation. In other embodiments, the leukocytes may be native or engineered macrophages, for example engineered macrophages suitable for the diagnosis or treatment of cancer, infectious diseases, inflammatory diseases, degenerative diseases, autoimmune diseases, and organ transplantation. Stem cells may include, for example, embryonic stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, or induced pluripotent stem cells (iPSCs). Embodiments with a sample comprising stem cells may include stem cells suitable for genetically engineered correction of a disease or condition, or for transplantation. The stem cells, if present, may be suitable for the treatment of neurodegeneration, diabetes, multiple sclerosis, cerebral palsy, macular degeneration, cardiovascular diseases, or musculoskeletal diseases. CTCs may include, without limitation, tumor cells released from a solid or primary tumor into the surrounding vasculature or lymphatic system to then circulate in the bloodstream. CTCs may be suitable for the detection and diagnosis of cancer. Where the sample includes cells, the cells may be bacterial cells, plant cells, yeast or fungal cells, or animal cells. In certain embodiments, the cells are preferably engineered or native animal cells, and in particular embodiments, the cells are more preferably engineered or native human cells. The labeled and unlabeled biological material or materials may be naturally-occurring, or may be modified from their native states for example by mutation or genetic engineering, or may be a combination of both types of materials. In some preferred embodiments, the sample includes at least one modified and labeled biological material, preferably including one or more modified and labeled cells.

In some preferred embodiments, the sample has been originally or initially obtained from a subject. The subject may be a human subject, another organism, or a tissue sample, preferably a human subject or a human tissue sample. The subject may preferably be a patient in need of treatment, or in other embodiments, the subject is a different individual from a patient in need of treatment. The sample may be initially obtained from the subject as a blood sample or as a tissue sample, preferably as a blood sample and/or a sample obtained from a tumor, particularly a tumor stroma, or other tissue subject to a condition in need of treatment. After the sample is initially obtained from the subject, at least one component of the blood and/or tissue may be removed or isolated as the sample. The component to be removed or isolated as the sample may comprise one or more cells, cell fragments, viruses, virus fragments, exosomes or extracellular vesicles and fragments thereof, proteins, nucleic acids, or carbohydrates. The component to be removed or isolated as the sample is preferably one or more cells, more preferably leukocytes, and in particular, T cells are preferred in certain embodiments.

In preferred embodiments, once the sample has been removed or isolated, it is modified, or it is used to modify another biological material. Modification may include genetic engineering, including the introduction of DNA and/or RNA into a cell, in order to express a desired trait and/or produce a desired structure, preferably a protein; alternatively, the modifications may have the goal of removing or inhibiting the expression of a desired trait and/or production of a desired structure. In certain preferred embodiments, the removed or isolated sample comprises T cells, and the modification comprises providing the T cells with DNA which encodes for one or more receptor proteins. In these embodiments, the receptor protein can be a T cell receptor or chimeric antigen receptor configured to selectively bind to one or more tumor cell antigens. In some embodiments, the receptor protein is an anti-CD19 chimeric antigen receptor.

In some embodiments related to tumor-infiltrating lymphocyte (TIL) adoptive cell therapy, the sample may be initially obtained from blood or a tumor from a patient in need of treatment. The sample may be sorted to separate TILs, such as T cells, and tumor cells. In some embodiments, separated T cells may be further isolated as individual single cells. T cells are expanded ex vivo and then exposed to the tumor cells to identify T cells that react against the tumor cells. In some embodiments, the reactive T cells may be identified and selected for reactivity by the presence of cell surface proteins, such as but not limited to CD137/<NUM>-1BB, CD134/0X40, and/or CD107a/LAMP-<NUM>. T cells that indicate as reactive to the tumor cells may be selected and further expanded to be infused back into the patient with IL-<NUM> treatment to promote expansion and engraftment.

In other embodiments, the sample includes reagents or particulate components, which may be suitable for sorting or analysis, or for facilitating a sorting or analysis method. Reagents or particulate components can include beads. Such reagents or particulate components may be present in the sample instead of, or in addition to, a biological material. In some embodiments, the reagents or particulate components are provided with one or more biological materials. For example, a surface of a bead may be provided with an antibody, in particular an antibody configured to capture cells or other biological materials. The reagents or particulate components, for example beads, may include a label, for example a fluorescent or magnetic label. Such a label may be provided within the bead and/or on a surface of the bead, either directly or via a linking group.

To facilitate analysis, separation, selection, and/or sorting, the biological material may be labeled, preferably in a selective manner, and preferably prior to collection with probe <NUM>. The label may be a chemical, isotopic, magnetic, or fluorescent type label. In some embodiments, the label preferably includes a label which is suitable for detection in an optical manner, in particular a fluorescent label, for example phycoerythrin (PE) or carboxyfluorescein succinimidyl ester (CFSE), or fluorescent proteins such as enhanced GFP (eGFP), or nanoparticles such as quantum dots. The label can also include a magnetic label, instead of an optical label, or in some preferable embodiments in addition to an optical label. If the labeling is selective, then the selective labeling may be selective for the presence of a target biological material, such as a for a protein or receptor marker on the surface of a cell in the sample, for example a T cell receptor or chimeric antigen receptor capable of binding to one or more tumor cell antigens, such as an anti-CD19 chimeric antigen receptor. In other embodiments, the labeling may be selective for the presence of surface protein markers that indicate an activated or reactive T cell, for example, CD137/<NUM>-1BB, CD134/OX40, and/or CD107a/LAMP-<NUM>. A label may include one or more labels for different characteristics and/or markers, such as for the presence of different proteins, or for the presence of a protein and another characteristic. In such a plurality of labels, the labels may be the same or different.

To obtain the sample in the open end <NUM> of the probe <NUM>, a fluid may be provided from fluid supply <NUM>. The fluid is preferably a liquid, more preferably a saline solution, even more preferably a buffered saline solution. The buffer, if present, may be a phosphate buffer. In some embodiments, the fluid is a fluid suitable as a sheath fluid for flow cytometry, or a fluid which can be combined with one or more other substances to provide a sheath fluid for flow cytometry. Preferably, the fluid itself as provided through fluid supply <NUM> to open end <NUM> is suitable as a sheath fluid for flow cytometry.

The fluid may be provided to fluid supply <NUM> by pumping, for example with a probe input pump <NUM>. In preferred embodiments, the flow rate of the fluid through fluid supply <NUM> to open end <NUM> is regulated, which may include measuring with a flow meter and/or regulating the flow rate with a flow regulator. The flow rate of the fluid through fluid supply <NUM> to open end <NUM> is, in some embodiments, also a volumetric flow rate suitable for flow cytometry. In such embodiments, the flow rate may be from <NUM> to <NUM>,<NUM>µL/min, in particular from <NUM> to <NUM>µL/min or from <NUM> to <NUM>,<NUM>µL/min. The flow rate of the fluid through fluid supply <NUM> to open end <NUM> may be greater than, substantially the same as, or identical to a flow rate for removing a fluid stream from the open end <NUM> of probe <NUM> through the fluid exhaust <NUM>. In some embodiments, the flow rate of the fluid through fluid supply <NUM> to open end <NUM> is from <NUM>% to <NUM>% greater than a flow rate for removing a fluid stream from the open end <NUM> of probe <NUM> through the fluid exhaust <NUM>, more preferably about <NUM>% greater. By maintaining a sufficient flow rate through fluid supply <NUM> to open end <NUM> so as to replace at least the fluid removed through fluid exhaust <NUM>, the probe <NUM> in the analysis and/or sorting system <NUM> and the method of using it can avoid intake of air or other gas into fluid exhaust <NUM>, thereby avoiding the undesired presence of gas into a flow cytometer <NUM>.

Upon arrival at open end <NUM> of probe <NUM>, the fluid may form a fluid dome <NUM> as shown in <FIG>. A sample may enter open end <NUM> by contact with fluid dome <NUM>. In some embodiments, the sample may enter the fluid dome <NUM> when the fluid dome <NUM> contacts a sample, for example a sample as such or an aqueous suspension or dispersion of the sample, in a sample container or containers such as tubes, flasks, or a well <NUM> of a well plate <NUM>. In other embodiments, the sample could be provided to open end <NUM>, preferably having dome <NUM>, by provision of drops or sprays of the sample to open end <NUM>. For example, some embodiments of the method may convey sample to open end <NUM> of probe <NUM> with an automated pipetting system, or by directing acoustic energy into the samples within individual wells to eject droplets of the sample toward open end <NUM> of probe <NUM>. In some such embodiments, for example where the wells or other sample container or containers are sufficiently narrow so as to retain the sample by forces such as surface tension or adhesion even in other orientations, the open end of the probe may point in an upward direction, a downwards direction, a sideways direction, or at a skew angle with respect to a vertical direction. In some such embodiments, an upward direction for the probe and an inverted orientation of the sample container or containers may be particularly preferred. In some cases, sample and/or fluid may be drawn into the probe without formation of a dome.

A fluid stream comprising the fluid and the sample may be removed from open end <NUM> through fluid exhaust <NUM> by pumping, for example with a probe to flow cytometer pump <NUM>. In preferred embodiments, the flow rate of the fluid stream through fluid exhaust <NUM> out from open end <NUM> is regulated, which may include measuring with a flow meter and/or regulating the flow rate with a flow regulator. In addition or in the alternative, a sensor may be used to directly or indirectly measure the flow rate. For example, a sensor, such as a light-based sensor, may measure the size and/or shape of dome <NUM> in order to maintain a balance between the flow rate through fluid supply <NUM> and the flow rate through fluid exhaust <NUM>. In some embodiments, a processor may be used to monitor and/or control the flow rate through fluid supply <NUM> and the flow rate through fluid exhaust <NUM>. Such a processor, if present, may receive data from one or more flow meters and/or one or more sensors such as light-based sensors. Additionally or alternatively, such a processor, if present, may provide data to control one or more flow regulators. The flow rate of the fluid stream through fluid exhaust <NUM> away from open end <NUM> is, in some embodiments, also a volumetric flow rate suitable for flow cytometry. In such embodiments, the flow rate may be from <NUM> to <NUM>,<NUM>µL/min, in particular from <NUM> to <NUM>µL/min or from <NUM> to <NUM>,<NUM>µL/min. The flow rate of the fluid stream through fluid exhaust <NUM> out from open end <NUM> may be less than, substantially the same as, or identical to a flow rate of the fluid through fluid supply <NUM> to open end <NUM>. In some embodiments, the flow rate through fluid exhaust <NUM> out from open end <NUM> is from <NUM>% to <NUM>% less than a flow rate through fluid supply <NUM> to open end <NUM>, more preferably about <NUM>% less. By maintaining a sufficiently low flow rate through fluid exhaust <NUM> so as to prevent intake of gas or air along with the sample and the fluid supplied through fluid supply <NUM> to open end <NUM>, the probe <NUM> in the analysis and/or sorting system <NUM> and the method of using it can avoid the undesired presence of gas into a flow cytometer <NUM>.

The fluid stream removed from open end <NUM> of the probe <NUM> through fluid exhaust <NUM> of the probe <NUM> is then conveyed to the flow cytometer <NUM>. In some embodiments, probe to flow cytometer line <NUM> transports the fluid stream. Probe to flow cytometer line <NUM> can be free of joints, junctures, or valves in some embodiments; in other embodiments, probe to flow cytometer line <NUM> includes at least a joint, juncture, or valve. The implementation of probe <NUM> to avoid the undesired presence of gas in the fluid stream may mean that, in some embodiments, the presence of a joint, juncture, or valve may cause fewer problems, as no air or gas bubbles would be provided into probe to flow cytometer line <NUM> to be retained deleteriously in the joint, juncture, or valve, thereby disrupting the flow of the fluid stream through probe to flow cytometer line <NUM>.

As the fluid stream is conveyed from open end <NUM> of the probe <NUM> through fluid exhaust <NUM> of the probe <NUM> to the flow cytometer <NUM>, the fluid stream may or may not receive or be joined by an additional component or components. In some embodiments, the fluid stream travels without added components to the flow cytometer <NUM>. In other embodiments, the fluid stream may be diluted with water, or an optionally-buffered saline solution may be added.

Once in the flow cytometer <NUM>, the fluid stream may in some embodiments be separated, for example though fluorescence-activated cell sorting. In some embodiments, the separation process includes regulation of flow of the fluid stream, preferably by hydrodynamic focusing, including generation of a sheath flow with the fluid stream comprising the sample in a middle portion thereof. In other preferred embodiments, the fluid flow can be regulated by acoustic focusing with ultrasonic waves to enhance generation of a sheath flow with the fluid stream including the sample in a middle portion thereof. The fluid stream can then be separated into droplets, most or all of which preferably contain one cell or other unit of the biological material, and the droplets may each be provided with a charge. A label, when present in a droplet, is then detected. Detection in some embodiments includes provision of electromagnetic energy, preferably in the form of a laser beam, to the droplets. The energy may then excite the label, preferably through fluorescent excitation. A camera or other light-detecting apparatus can then detect the presence or absence of fluorescence, thereby detecting the presence or absence of a labeled sample in the droplet. Droplets of the fluid stream can then be sorted based on the detected presence or absence of the labeled sample, including directing of the droplets to at least two containers or other receiving points such as wells in a well plate, tubes, or flasks through the charge provided to the droplets. In other embodiments, labeled sample is sorted out of a fluid flow using a magnetic solenoid sorting valve into at least two containers based on the detected presence or absence of the label. By these or other methods of flow cytometry-based sorting, the sample can be separated into at least one labeled component and at least one unlabeled or differently-labeled component. In certain embodiments, labeled components may be sorted as single or individual components into individual containers. For example if the labeled components are cells single or individual cells may be sorted into individual containers as the only labeled component within that container.

In still other embodiments, sorting by flow cytometry may comprise sorting the sample by microfluidic sorting into at least two containers 61a and 61b or other receiving points as directed by the microfluidic sorting device, for example, wells in a plurality of well plates or wells in the same well plate, as shown in <FIG>. Microfluidic sorting is not particularly limited, except in that it sorts components of a fluid stream from probe <NUM> based on any fluorescent, magnetic, isotopic, or chemical label or labels provided on the components, and/or based on a property or properties of the components unrelated to any label such as size, shape, density, binding capability, conductivity, or acoustic properties. Microfluidic sorting may involve regulating the fluid stream such that many or most cells or other particles of the sample in the fluid stream are separated into distinct components based on the labels and/or properties mentioned above. In certain embodiments, components containing a given set of labels and/or properties are separated and sorted into one container 61a, while components lacking the given set of labels and/or properties are sorted into a different container 61b, as shown in the embodiment in <FIG>. These containers can be separate wells of a well plate, for example a microplate, or separate tubes. In some embodiments, for example in embodiments that do not involve analysis or sorting based on fluorescence or any other light- or radiation-based phenomenon, a microfluidic sorting device as flow cytometer <NUM> may not comprise a laser, in contrast with the embodiment of <FIG>, showing laser <NUM>.

In some embodiments, after sorting by flow cytometry, one or more resultant fluid streams, fractions, and/or containers comprising sorted components may be enriched for labeled components of the sample, meaning labeled components are present in a greater number and/or amount relative to other fractions collected (e.g., the one or more second fluid streams, fractions, and/or containers). In some cases, being enriched for labeled components may refer to a measurable or detectable number or amount of labeled components. For example, an enriched fraction may contain at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>%, or more of the labeled components relative to unlabeled components in the fraction, or relative to the amount of total labeled components within the original sample. In some configurations, the unlabeled components may be collected in one or more second fractions. These may be discarded or analyzed. The one or more second fractions containing the unlabeled components are substantially devoid or depleted of labeled components. For example, these fractions may contain less than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>'%, or none of the labeled component relative to the fractions enriched for labeled components. In some cases, this relative amount may be based upon a particle count, an amount by mass, a weight %, target label intensity, etc. A determination of amount may be made using techniques well known in the art such as by absorption, mass spectrometry, a chromatograph, a cell counter, an enzymatic assay (e.g., a horseradish peroxidase-based assay, ELISA), a colorimetric assay, a fluorescent assay, or the like.

In some embodiments, after separation in the flow cytometer <NUM>, one or more components obtained from the sample may be prepared for administration to a subject in need of treatment. In some embodiments, where the one or more components obtained from the sample comprise cells, the cells may be cultivated. In addition, pharmaceutical adjuvants may be added, and certain components including the label may be removed, if desired.

In some embodiments, after separation in the flow cytometer <NUM> and in some embodiments after further preparation, one or more components obtained from the sample may be administered to a patient in need of treatment. The patient may be in need of treatment, for example, for cancer, in particular melanoma, acute lymphocytic leukemia, ovarian cancer, colon cancer, prostate cancer, brain cancer, or breast cancer.

The system and method may be further useful with respect to cell-based therapies, including other aspects of T cell receptor (TCR) therapy, chimeric antibody receptor T cell (CAR-T) therapy, tumor-infiltrating lymphocyte (TIL) therapy, or any combination thereof, for example as discussed in <NPL>.

In other embodiments, the sample may be analyzed in the flow cytometer and data may be collected about the presence, content, frequency, and/or distribution of components within the sample having different properties, or satisfying various parameters. In particular, the sample may be measured and data may be collected as to the presence and/or frequency of a labeled component, such as a labeled biological material, a labeled analysis reagent, or labeled particulate component. In some embodiments, analysis by flow cytometry may be combined with sorting by flow cytometry, as both described above. In other embodiments, analysis by flow cytometry may occur without sorting, and the fluid stream comprising the sample may be discarded after analysis.

Claim 1:
An analysis and/or sorting method, comprising:
conveying a first fluid within a probe from a fluid supply (<NUM>) to an open end (<NUM>) of the probe,
collecting at least one sample, comprising at least one first component and at least one second component, into the open end of a probe,
removing a fluid stream comprising the at least one sample and the first fluid through a fluid exhaust (<NUM>) from the open end of the probe,
conveying the fluid stream to a flow cytometer (<NUM>), and
analyzing the fluid stream by flow cytometry, and/or separating the fluid stream into at least a first separated stream comprising the at least one first component and a second separated stream comprising the at least one second component by flow cytometry,
characterized in that a sufficient flow rate of the first fluid is maintained so as to replace at least the removed fluid
and/or a sufficiently low flow rate through the fluid exhaust (<NUM>) with respect to the first fluid flow rate is maintained so as to prevent intake of gas or air along with the sample and the first fluid supplied through the fluid supply (<NUM>) to the open end (<NUM>),
such that the fluid stream is substantially free of air bubbles when the fluid stream is conveyed to the flow cytometer (<NUM>), and wherein the fluid stream is substantially free of a gas/liquid interface when the fluid stream is conveyed to the flow cytometer (<NUM>).