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WO2025179189A1 - Système et procédé d'analyse de tissus - Google Patents

Système et procédé d'analyse de tissus

Info

Publication number
WO2025179189A1
WO2025179189A1 PCT/US2025/016864 US2025016864W WO2025179189A1 WO 2025179189 A1 WO2025179189 A1 WO 2025179189A1 US 2025016864 W US2025016864 W US 2025016864W WO 2025179189 A1 WO2025179189 A1 WO 2025179189A1
Authority
WO
WIPO (PCT)
Prior art keywords
cartridge
processing chamber
rotor
tissue
port
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/016864
Other languages
English (en)
Inventor
Stevan Bogdan Jovanovich
Ezra Van Gelder
David Eberhart
Nathan PEREIRA
John BASHKIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
S2 Genomics Inc
Original Assignee
S2 Genomics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by S2 Genomics Inc filed Critical S2 Genomics Inc
Publication of WO2025179189A1 publication Critical patent/WO2025179189A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor

Definitions

  • the isolation of single cells, nuclei and other subcellular organelles from fresh or fixed tissue is an area of interest to investigators in the life sciences.
  • Processing of fixed tissue such as FFPE tissue
  • processing of fresh or rehydrated tissue can involve dissociating tissue to disrupt extracellular matrix, for example in the presence of enzymatic solutions that break chemical bonds in the extracellular matrix or with mechanical grinding to release cells and/or nuclei and other subcellular organelles.
  • the released cells, nuclei and/or other subcellular organelles can then be collected for analysis, for example, genomic analysis of single cells.
  • Figure 1 shows a Sample processing System that processes specimens into biocomponents such as single cells or nuclei for bioanalysis.
  • Figures 8 A-C shows an example of a cartridge with processing, post-processing, and vacuum trap chambers for processing tissue specimens into single nuclei, single cells, and other biomolecules.
  • Figures 9 A-D show an example of adding reagents to a cartridge mixing the reagents, removing the reagents, and mechanically disrupting the tissue for processing tissue specimens into single cells, nuclei, and other biomolecules.
  • Figure 10 shows an exemplary computer system.
  • Figure 11 shows a cartridge architecture using pinch valves to direct liquid flows.
  • Figure 13A shows a cutaway view of an exemplary cartridge.
  • Figure 13B shows a cartridge comprising a filter positioned in the post-processing chamber.
  • Figure 14 shows a top-down view of an exemplary cartridge.
  • Figure 15 shows an exemplary grinder assembly.
  • Figure 16 shows a cutaway view of an exemplary cartridge with a grinder assembly positioned for insertion into the processing chamber.
  • Figure 17 shows an exemplary cartridge with a grinder assembly inserted into the processing chamber.
  • Figures 18 A-B show an exemplary cartridge with a feature designed to center the head of the rotor in the processing chamber and set the bottom gap and side gaps between the rotor and the wall of the processing chamber.
  • Figures 19 A-D show a port cover with a low durometer over a port secured by a port cover retaining cylinder, or a crimp, or a heat staked port cover retaining cylinder.
  • Figures 20 A-E show a cap engaging with a rotary motor adapter and with a cartridge with processing, post-processing, and vacuum trap chambers for processing tissue specimens into single cells, nuclei, and other organelles or biomolecules.
  • Figure 20A shows actuator before engagement with the plunger assembly.
  • Figures 20B and 20C show the engaged in two different rotational orientations.
  • Figures 20D and 20E show the plunger assembly in partially and fully depressed states.
  • Figure 21 shows an exemplary workflow to deparaffinize and rehydrate FFPE specimens followed by nuclei isolation.
  • Figure 22 shows a picture of cartridge adapted for processing preserved tissue.
  • Figure 23 shows a drawing of a cartridge adapted for processing preserved tissue, including ports for engaging a cartridge interface of an instrument.
  • Figure 24 shows a cut-away view of a cartridge adapted for processing preserved tissue.
  • Figure 25 shows a cut-away view of a cartridge adapted for processing preserved tissue.
  • Figure 26 shows a top-down view of a cartridge.
  • Figure 27 shows an exemplary reagent module including caddy holding reagent containers and a waste container.
  • Figure 28 shows a side view of an exemplary reagent module.
  • Figure 29 shows an exemplary system.
  • Figures 30A and 30B show a cutaway view of a processing chamber including a plunger assembly (disruption head) being translated along the Z axis (1) and hitting the stator (2).
  • Figures 31A-C show a mechanical assembly with a force sensor to monitor force on the rotor face in the z-axis.
  • Figure 32 shows a mechanical assembly for actuating the plunger assembly from a different aspect.
  • Figure 33 shows a graph of force on the rotor as a function of position along the Z axis. Force increases slightly when the motor engages the plunger assembly and reaches an inflection point with dramatic increase in force when the rotor touches the bottom of the processing chamber.
  • Figure 34 shows a schematic of a mechanical assembly of an actuator for translating a plunger assembly along a z-axis.
  • kits and methods for isolating cells, nuclei and/or subcellular organelles from fresh, frozen, or preserved tissue are provided herein.
  • both types of cartridges comprise a processing chamber in fluidic communication with a post-processing chamber.
  • the processing chambers of both cartridges are adapted to accommodate a plunger assembly that comprises a rotor.
  • the rotor has a circumference such that when the rotor is inserted into the processing chamber the gap between the walls of the processing chamber and the rotor is small enough to prevent passage of pieces of tissue containing cells, nuclei and/or other subcellular organelles from passing between the rotor and the chamber wall.
  • the rotor has a circumference such that when the rotor is inserted into the processing chamber the gap between the walls of the processing chamber and the rotor is large enough to allow passage of cells, nuclei and/or other subcellular organelles between the rotor and the chamber wall. More particularly, for the tissue dissociation cartridge, certain rotors may have a circumference that allows passage of cells, nuclei, and other subcellular organelles, and other rotors may have a circumference that allows passage of nuclei and/or other subcellular organelles, but not whole cells.
  • the processing chamber can comprise a stator comprising grinding features, and, the rotor can comprise a face facing the stator that also comprises grinding features.
  • Such grinding features can be absent from the stator and the rotor face of the preserved tissue processing cartridge.
  • Cartridges for processing preserved tissue, on the one hand, and releasing cells and/or subcellular organelles from tissue have geometries such that features of each can engage the same element in a system to which the cartridges can engage. So, for example, both cartridges can have the following elements for engaging ports in the instrument once engaged: (1) a plunger configured to fit through a top orifice into a processing chamber that engages an actuator in the system, (2) a processing chamber port that engages a port in the cartridge interface, (3) a post processing chamber port that engages a port in the cartridge interface and (4) a vacuum port.
  • both the preserved tissue processing cartridge and the tissue dissociation cartridge can comprise alignment, guide, or registration features complementary to the alignment, guide, or registration feature in the same cartridge interface such that each cartridge can be engaged. This might be, for example, a hole for a pin or vice versa, a groove for guide rib or key or vice versa, or a dovetail.
  • the post-processing chambers of both cartridges can be configured for different purposes. Both post-processing chambers can communicate with a source of vacuum to pull liquid from the processing chamber into the post-processing chamber.
  • the post-processing chamber can be configured to collect the cells in the chamber without being pulled out of the cartridge by the source of vacuum.
  • the post-processing chamber can be configured to pull liquid from the processing chamber into, and then out of, the post-processing chamber. This can be accomplished by, for example, including a same-positioned port in the post-processing chamber.
  • the same-positioned port can be connected to a source of vacuum, and positioned to function as a drain in the post-processing cartridge to collect liquid moved into the post-processing cartridge when vacuum is applied.
  • the port can function as a drain when a floor of the post-processing cartridge is positioned at about the level of the port.
  • a system for performing both processing of preserved tissue and dissociating tissue can comprise the following features.
  • the instrument can comprise a cartridge interface to engage the cartridge and that comprises connectors, e.g., configured as cannulas positioned to engage ports in the cartridge that are in fluid communication with the processing chamber, the post-processing chamber and/or a separate vacuum chamber.
  • the system can further comprise a fluidic subsystem comprising reservoirs adapted to contain reagents used in tissue dissociation, such as enzymatic solutions.
  • the system can further comprise one or more sources of positive and/or negative pressure configured to move liquids from the fluidic subsystem into and/or out of the cartridge.
  • the system can further comprise a mechanical subsystem comprising an actuator configured to actuate the plunger assembly.
  • Actuation can be either or both of rotational (in either clockwise or counterclockwise direction) and linear, which is to say, to move the rotor up and/or down within the processing chamber (/.e., along the Z axis).
  • the system can also comprise a force sensor operatively linked to the rotor configured to measure, in real time, the force on the rotor.
  • the mechanical assembly can control motion of the plunger assembly in the Z axis. So, for example, when the force sensor senses a force on the rotor consistent with the rotor hitting the stator, the software can instruct the mechanical assembly to cease downward motion of the rotor.
  • the software can instruct the mechanical assembly to pause downward motion and begin rotating the rotor.
  • the system can store in computer memory a position of the rotor along the Z axis that corresponds to a force measurement consistent with the rotor touching a tissue sample and/or touching the floor of the chamber. Then, in subsequent grinding operations, either in the currently used cartridge in the same run, or in different cartridges, the rotor can be depressed to the relevant position stored in memory.
  • the instrument can comprise a reversibly engageable tissue processing subsystem that comprises a caddy for holding a plurality of containers and one or more elements selected from (A) a containment barrier configured to contain liquids within the caddy, (B) a plurality of containers in the caddy including at least one container for containing a reagent liquid, and a waste container, (C) a scale configured to measure the weight of the waste container, (D) an optical sensor that senses liquid in the bottom of the caddy, and (E) a plurality of fluidic conduits that connect the containers with ports through couplings in a sensing mechanism that senses when a proper coupling has been made.
  • the instrument further comprises fluidic conduits that connect the couplings with ports in the cartridge interface and one or more sources of pressure for moving liquids in either direction between the containers in the caddy and chambers in the cartridge.
  • Figure 1 shows a Sample processing System 50 that can input specimen 101 and process them to produce biologicals such as single cells 1000 or nuclei 1050, microtissues 6001, organoids 6002, or other biocomponents comprised of subcellular components 1060, and biomolecules 1070 such as macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073 and RNA 1074; proteins 1075; carbohydrates 1076; lipids 1077; biomolecules 1070 with multiple types of macromolecules 1071 , metabolites 1078; and other biological components, including natural products 1079 for bioanalysis.
  • biologicals such as single cells 1000 or nuclei 1050, microtissues 6001, organoids 6002, or other biocomponents comprised of subcellular components 1060
  • biomolecules 1070 such as macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073 and RNA 1074; proteins 1075; carbohydrates 1076; lipids 1077; biomolecules 1070 with multiple types of macromolecules 1071 , metabolites 1078; and other biological
  • the Enzymatic and Chemical Dissociation Subsystem 400 can perform deparaffinization by adding organic solvents, such as xylene or xylene substitutes, to the cartridge and perform rehydration by adding mixtures of ethanol with increasing amounts of water or buffer.
  • organic solvents such as xylene or xylene substitutes
  • Another aspect of the Enzymatic and Chemical Dissociation Subsystem 400 is that it can perform chemical disruption or chemical and enzymatic disruption by adding formulations of chemicals that can disrupt tissue or cellular integrity, such as Triton X-100, Tween, Nonident P40, octyl glucoside, polyoxyethylene(9)dodecyl ether, digitonin, IGEPALTM CA630 octylphenyl polyethylene glycol, n-octyl-beta-Dglucopyranoside (betaOG), n-dodecyl-beta, TweenTM.
  • chemicals that can disrupt tissue or cellular integrity, such as Triton X-100, Tween, Nonident P40, octyl glucoside, polyoxyethylene(9)dodecyl ether, digitonin, IGEPALTM CA630 octylphenyl polyethylene glycol, n-octyl-beta-Dglu
  • polyethylene glycol sorbitan monolaurate polyethylene glycol sorbitan monooleate, polidocanol, n-dodecyl beta- D-maltoside (DDM), NEMO nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol ndodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether (C14E06), octyl-betathioglucopyranoside (octyl thioglucoside, OTG), Emulgen, and polyoxyethylene 10 lauryl ether other surfactants, or detergents or chemicals that can dissociate tissue into cells or produce nuclei or other organelles.
  • DDM n-dodecyl beta- D-maltoside
  • NEMO nonylphenyl polyethylene glycol C12E8 (octaethylene glycol ndodecyl monoether), hexaethyleneglycol
  • the addition and movement of fluids can be performed by a Fluidic Subsystem 600.
  • the Fluidic Subsystem 600 can use syringe pumps, piezopumps, on-cartridge pumps and valves, vacuum (negative pressure), pressure, pneumatics, or other components well known to one skilled in the art.
  • the Tissue Processing System 110 can be controlled by software in a Control Subsystem 700 which can be comprised of a user interface 740 through a monitor, embedded display, or a touch screen 730 to communicate with and control devices, modules, subsystems, instruments, and systems.
  • the Control Subsytem 700 can include interfaces to smart devices, laboratory information management systems, other instruments, analysis software, display software, databases, email, and other applications.
  • the Control Subsystem 700 can include control software 725 and scripts that control the operation and in some embodiments the scripts can be revised, created, or edited by the operator.
  • a device for the dissociation of a biological sample comprising: (i) a biological sample or specimen 101; (ii) a cartridge 200 capable of dissociating tissue; (iii) an instrument to operate the cartridge 200 and provide fluids as needed (iv) a measurement module 500 such as an optical imager to measure titer, clumping, and/or viability, (v) pumps and containers for exchange of dissociation solution for buffer or growth media at the desired titer, and (vi) output vessels such as a chamber in the cartridge, 8-well strip tubes, microtiter plates, Eppendorf tubes, conical centrifuge tubes, or other vessels capable of receiving cell suspensions.
  • a measurement module 500 such as an optical imager to measure titer, clumping, and/or viability
  • a measurement module 500 such as an optical imager to measure titer, clumping, and/or viability
  • pumps and containers for exchange of dissociation solution for buffer or growth media at the desired titer and
  • a Magnetic processing module 900 can use magnetic processing of magnetic and paramagnetic particles or beads or surfaces of other sizes and shapes, referred to as beads, to separate single cells 1000, or cell types, or nuclei 1050, or other biocomponents comprised of subcellular components 1060, and biomolecules 1070 such as macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073 and RNA 1074; proteins 1075; carbohydrates 1076; lipids 1077; biomolecules 1070 with multiple types of macromolecules 1071; metabolites 1078; and other biological components, including natural products 1079 for bioanalysis.
  • the beads have a surface chemistry that facilitates the purification of the biologicals in conjunction with the chemical conditions.
  • the beads have affinity molecules comprised of antibodies, aptamers, biomolecules, etc. that specifically purify certain biologicals such as cell types, nucleic acids, nuclei 1050, or other components of tissue or samples.
  • the basic elements of the Tissue Processing System 110 can be configured in multiple ways depending on the specimen(s) 101 or FFPE tissue specimens 150 or OCT tissue specimens 160 and analytes to be analyzed. In the following example, one of the numerous configurations is described in detail but in no way is the invention limited to these configurations as will be obvious to one skilled in the art.
  • the Tissue Processing System 110 can accommodate many different types of specimens 101, comprised of fresh tissue; snap-frozen tissue; microtome slices (cryo, laser or vibrating) of tissue; fixed tissue; bulk material obtained by surgical excision, biopsies, fine needle aspirates; samples from surfaces, and other matrices, or FFPE tissue specimens 150.
  • the instant disclosure teaches how to produce a system that processes FFPE tissue specimens 150 and OCT tissue specimens 160 and other samples into preferentially nuclei 1050 or into single-cells 1000.
  • the process may require adapting to the widely varying starting types of FFPE tissue specimens 150, with different requirements depending on the tissue, species, age, and state.
  • This disclosure describes how to automate, integrate, and, importantly, standardize the complete process to create single-nuclei 1050 in a single sample Tissue processing 110 system embodiment using a novel mechanism to retain the tissue and a novel cartridge design. It is clear to one skilled in the art that multi-sample embodiments can be accomplished with the same instant invention.
  • the Tissue Processing System 110 will greatly enable basic researchers, students, and translational researchers as well as clinicians and others with its ease of use and high performance.
  • Cartridges 200 can be used to process tissue into single-cell 1000 suspensions or nuclei 1050 and can be for single-use.
  • Cartridges 200 can be designed for 3D printing, injection molding in plastics with single or double pulls and low labor assembly, or layered assembly of fluidic and other layers, combinations of methods, and other methods well known to one skilled in the art. Fluids can be delivered to cartridge 200 by pumps such as a syringe pump 2130 or by vacuum or can be preloaded onto cartridge 200 or by any combination of the foregoing.
  • flexible tubing 493 can connect chambers and create simple pinch valves 491 to direct flow.
  • channels are created in the cartridge 200 and valves can be incorporated such as pneumatic valves, or other valves.
  • plunger or plunger assembly
  • shaft/piston 216 and rotor 218 with optional disruption features ⁇ e.g., teeth 355 with spring 213 in sheath 212.
  • the mechanical tissue disruptor elements have features 355 on the bottom of the rotor or grinder that can mechanically disrupt tissue at the bottom or floor of processing chamber 440 which in some embodiments may have complementary features 355 to aid in the disruption of the tissue.
  • the mechanical tissue disruptor elements does not have features 355 on the bottom of the rotor or grinder but can be flat and mechanically disrupt tissue against a flat surface at the bottom or floor of processing Chamber 440. Disruption also occurs in the ‘side gap’ between the rotor and the side wall of processing Chamber 440 in some embodiments.
  • Figures 13-17 show an exemplary cartridge of this disclosure.
  • Figure 13 shows a cutaway view of an exemplary cartridge.
  • the cartridge 200 includes a processing chamber 440 comprising a stator comprising teeth arranged in an annular array.
  • the processing chamber further comprises a first processing port from which a cell, nuclei organelle suspension can be removed from the processing chamber.
  • post-processing chamber 460 and a vacuum chamber.
  • the vacuum chamber 468 comprises a vacuum port 467.
  • Figures 15 and 16 show an exemplary grinder assembly 345 of this disclosure.
  • the grinder assembly 345 includes a plunger comprising a piston 216 and a rotor 218 positioned at an end of the piston.
  • the rotor comprises on a bottom surface, grinding elements, e.g., teeth 355, including a central tooth 356 and an annular array of three rings of teeth.
  • the teeth 355 can have a blunt or sharp shape. They may take the shape of a trapezoid in cross section.
  • the outermost ring of teeth 355 is positioned at the edge of the rotor.
  • the grinding assembly 345 further includes a sleeve or sheath 212 around the piston 216.
  • the grinding assembly 345 further includes a cap 210 to position the plunger in the processing chamber.
  • the cap 210 further comprises a slot or other mechanism configured to engage a key of an actuator to actuate the grinder.
  • a spring which biases the rotor 218 toward the cap 210 so that positive pressure must be asserted on the plunger by the actuator to press the rotor 218 against the stator.
  • the annular rings of teeth 355 in the rotor and the stator are positioned complementary to one another so that when the grinder is pressed against the stator the rings of the stator mesh with the rings of the rotor ⁇ e.g., are staggered against). That is, in an exemplary embodiment, teeth in the stator do not touch teeth in the rotor. This configuration facilitates rotation of the rotor against the stator so that teeth from one part do not collide with teeth from another part.
  • the number of rings of teeth in each of the rotor and the stator can be determined by a skilled artisan. Factors influencing the determination include the total surface area of the stator and the face of the rotor, as well as the size of the teeth. In certain embodiments the number of rings of teeth in the stator and/or the rotor 218 can be any of none, one, two, three, four, five, or six. In one embodiment teeth can have a trapezoidal cross-section.
  • the processing chamber can have a cylindrical shape.
  • the stator can have a radius between, for example, 5 mm and 25 mm, e.g., about 12 mm.
  • the processing chamber can have a volume less than 1 ml, or between about 1 mL and 50 mL, for example, between about 10 mL and about 30 mL, e.g., about 15 mL.
  • the rotor and the sidewalls of the processing chamber can be configured so that when the plunger is inserted into the processing chamber there is a gap between the sidewall of the processing chamber and an edge of the rotor.
  • the size of the gap can be optimized to allow passage of whole cells, nuclei or organelles between the sidewall and the rotor.
  • the teeth can have a height of about 500 microns and a width of about 1 mm to 2 mm. [00070]
  • Spin rates for the dissociation can be 10-200 rpm.
  • Total revolutions of the grinding element can be 5-500.
  • the spin rate is about 45 rpm (slow) or about 150 rm (fast), with about 4 seconds of revolution, about 1-2 second pause, then about another 4 seconds, then repeat (about 16 seconds total rotation time) at each vertical displacement step of the stepper motor, sequentially going lower towards the bottom of the cartridge, about 9 vertical displacements in all, and at the bottom-most step, there are about 3 repetitions of the rotation periods rather than 2.
  • disposable cartridge 200 process multiple types of preserved FFPE 150 or OCT 160 tissues with mechanical disruption and enzymatic or chemical dissociation that can be adjusted according to the tissue type and condition of the FFPE tissue, such as age, or chemical process.
  • the cartridge 200 can be designed to process tissue as quickly and as gently as possible, not expose the operator to the tissue being processed, and be manufacturable at low cost.
  • Multiple mechanical methods may be needed to accommodate the wide range of tissues and their individual requirements: designs are shown that can be readily adapted to multiple different mechanical disruption methods comprising variable orifice 490, grinding with rotating plungers 336, pestles 361 , and straining and filtering using a plunger 362 as well as other mechanical methods without limitation.
  • Figures 22-26 show an exemplary embodiment of a cartridge for preserved tissue processing.
  • the cartridge of this embodiment shares many similar elements with the tissue dissociation cartridge described above with adaptations for processing of preserved tissue.
  • Cartridge 2500 comprises processing chamber 2540 and post-processing chamber 2560.
  • Processing chamber 2540 comprises first processing chamber port 2504 including nipple 2571 that is fluidically connected to post-processing chamber 2560 through fluidic conduit 2553.
  • Processing chamber 2540 further comprises a second processing chamber port 2570 which can be used to introduce liquids into the processing chamber.
  • First processing chamber port 2504 and second processing chamber port 2570 can be placed in the same position as ports 1604 and 470 of exemplary tissue dissociation cartridge in order to meet with the same ports in the cartridge interface of the instrument.
  • Processing chamber 2540 can have inserted therein, plunger assembly 2510.
  • Plunger assembly 2510 can differ from a plunger assembly used in a tissue dissociation cartridge. More particularly, the rotor in a tissue dissociation cartridge has a circumference that provides a space between the outer wall of the rotor and the inner wall of the processing chamber. This space is selected to allow cells, nuclei, and/or subcellular organelles to pass around the rotor and collect on top of it when the rotor is fully depressed. In a cartridge configured for preserved tissue processing, the space is selected to be small enough so that spent liquid, but not pieces of tissue or in some embodiments released cells, nuclei and/or subcellular organelles (depending on what is to be collected) can pass. In certain embodiments, this gap can be no more than any of 200 .m, 100 .m, 50 .m, and 25 .m.
  • the face of the rotor and the stator of the processing chamber can comprise or can not comprise grinding features.
  • First processing chamber port 2504 can be positioned on a side wall of the cartridge such that when the rotor of plunger assembly 2510 is fully depressed at least part of the port is positioned to drain liquid collecting above the top of the rotor. In some embodiments, part of the port is below the top of the fully depressed rotor. In other embodiments, all of the port is positioned above the top of the rotor. In any case, the port can be positioned such that the expected volume of liquid displaced above the top of the rotor contacts the port and can be removed via the port.
  • Post-processing chamber 2560 comprises an inlet 2554.
  • Inlet 2554 can be positioned in a lid or cover 2562 that covers orifice 2566.
  • Inlet 2554 can be fluidically connected to the first processing chamber through fluidic conduit 2553 and port 2504.
  • a first postprocessing port 2585 can be placed in the same position as first post-processing port 485 of the exemplary tissue dissociation cartridge described herein.
  • Post-processing chamber 2560 can further comprise floor 2580 positioned such that liquid collecting in the post-processing chamber can drain to first post-processing port 2585.
  • floor 2580 may be slanted so that liquid pools at first post-processing port 2585.
  • fluidic conduit 2553 can connect directly with a port in the cartridge connectable to a source of pressure in the instrument, for example, either first post-processing port 2585, or a port corresponding in position to vacuum port 467.
  • both the tissue dissociation cartridge and the preserved tissue processing cartridge can have ports positioned to engage the same cartridge interface connector in the instrument. So, for example, in a preserved tissue processing step, processing reagents can be introduced into processing chamber 2540 through second processing chamber port 2570. After the rotor is fully depressed in the processing chamber, spent reagents collect above the rotor and can be pulled by a vacuum exerted at first post-processing port 2585 through fluid line 2553 and into the post-processing chamber. From here, the spent reagents can be pulled from the post-processing chamber.
  • dissociation reagents can be introduced into processing chamber 440 through second processing chamber port 470, positioned to engage the same connector in the cartridge interface as port 2570.
  • the rotor is depressed and liquid including released cells, nuclei and/or other subcellular organelles collect above the top of the rotor.
  • first processing chamber port 1604 is positioned in fluid communication with the region above the top of the fully depressed rotor, the cells, nuclei, in or other subcellular organelles can be pulled through fluidic line 453 and into the postprocessing chamber.
  • suction can be pulled from vacuum port 467 which is configured to apply vacuum to the post-processing chamber.
  • vacuum port 467 is positioned well above the floor of the postprocessing chamber and can be separated by a wall almost reaching the top of the chamber, liquid comprising cells, nuclei, and/or other subcellular organelles is not pulled through vacuum port 467.
  • the Tissue Processing System 110 can perform powerful integrated tissue-to-genomics or sample-to-other answer (genomic, proteomic, metabolomic, or epigenetic, multi-omics, etc.) analysis functionality for scientists to simply and standardize the production and or analysis of single-cell 1000 or nuclei 1050 suspensions, affinity purified single cells 1100, affinity purified nuclei 1105, nucleic acids 1072, and bulk libraries 1210 from solid or liquid tissues.
  • the Tissue Processing System 110 can integrate the preparation of biological materials from preserved tissue samples, e.g., FFPE tissue specimens 150 or OCT tissue specimen 160, with measurement subsystems 500 that perform an analysis selected from one or more of: DNA or RNA sequencing, next generation DNA or RNA sequencing, next next generation DNA or RNA sequencing of nucleic acids and their adducts (such as epigenetic modifications); nanopore sequencing of nucleic acids and their adducts; single cell DNA sequencing of nucleic acids and their adducts; single nuclei RNA sequencing of nucleic acids and their adducts; PCR, digital droplet PCR, qPCR, RT-qPCR; genomic analysis, gene expression analysis, gene mapping, DNA fragment mapping; imaging including optical and mass spectrometry imaging; DNA or RNA microarray analysis; fluorescent, Raman, optical, mass spectrometry and other detection modalities of nucleic acids and their adducts with and without labels; proteomic analysis including fluorescent, Raman, optical, mass
  • mechanical and enzymatic dissociation is performed in single-use cartridges 200 in one or more processing chambers 440 to produce nuclei suspensions 1050, single-cell suspension 1000 or, nucleic acids 1072, biomolecules 1070, subcellular components 1060, or other products.
  • the samples can then be processed in the one or more post-processing chamber(s) 460 by optional bead-based affinity purification of cell types by surface antigens to produce affinity purified single-cell suspensions 1000 or nuclear suspensions 1050 by nuclear antigens or nucleic acids 1072, biomolecules 1070, subcellular components 1060 can be further processed into purified mRNA, NGS libraries, or other sample types.
  • one or more of the processing 440 and postprocessing chambers 460 and strain chambers 450 and vacuum trap chambers 468 and waste chambers 430 or other chambers can be combined.
  • the Tissue Processing System 110 can mechanically disrupt tissue and enzymatically dissociate and reverse crosslinks of the disrupted tissue in a cartridge 200 into single cells 1000 or nuclei 1050.
  • a Single Sample Tissue Processing System 2010 can combine the Physical Dissociation Subsystem 300 and the Enzymatic and Chemical Dissociation Subsystem 400 to produce single-cell 1000 or nuclei 1050 suspensions.
  • the instrument provides the mechanical motion and fluidics to the cartridge which in turn mechanically and enzymatically or chemically process the FFPE tissue specimen 150 into single cells 1000 or nuclei 1050.
  • Multiple reagents 411 can be stored on the instrument or reagent module 1430 with cooling as needed.
  • a 3D CAD representation of one embodiment of a Single-Sample Tissue processing 2010 design packaged with a ‘skin’ is shown in Figure 4 and another embodiment is shown in Figures 6 and 7. Both embodiments have a two axis mechanical motion (Z axis stepper 2110 and rotary motor 2120) integrated with fluidics based on a syringe pump ,for example, with 1.6 pL resolution with a six-way valve (C2400MP, TriContinent) controlled by control software 725.
  • Z axis stepper 2110 and rotary motor 2120 integrated with fluidics based on a syringe pump ,for example, with 1.6 pL resolution with a six-way valve (C2400MP, TriContinent) controlled by control software 725.
  • a computer 720 with an operating system for example, such as Windows 10 and 85 Gbytes HD (Beelink, AP42) can run control software 725 to control the system with display on a 10” touchscreen 730 (eleduino, Raspberry Pi 10) or on a tablet 750 such as a Windows Surface Pro 6.
  • Chassis 1010 provides the framework to mount components and the exterior case of the system.
  • the embodiment of the Single-Sample Tissue Processing System 2010 shown in Figure 4 has a fluidic subsystem 600 with a single syringe pump 2130 with a single six-way valve 2140 to supply liquids, pressure, or vacuum to cartridge 200 from reagent block 415.
  • cartridge 200 has two processing chambers 440 and a single post-processing chamber 460.
  • magnetic processing module 900 can apply magnetic force to cartridge 200 under software control to enable the use of paramagnetic beads, paramagnetic surfaces, paramagnetic nanoparticles, and other magnetic or paramagnetic particles to purify and analyze single cells 1000, nuclei 1050, nucleic acids 1072, biomolecules 1070, subcellular components 1060, or other products.
  • the system can comprise a reagent block or reagent subsystem configured for handling reagents in the processing of preserved tissue.
  • reagents typically include organic solvents, such as xylene, for removing preservatives from preserved tissue, such as paraffin, as well as alcohols in aqueous solutions for rehydrating tissues from which preservatives have been removed.
  • the reagent block can comprise a caddy configured to hold one or a plurality of containers, including reagent containers and a waste container.
  • the caddy can include a containment barrier to contain any liquid spilled into the caddy.
  • the caddy also can contain optical sensors that sense the presence of liquids at the bottom of the caddy.
  • the caddy includes a scale on which the waste container rests.
  • the scale can measure the weight of the waste container.
  • Software in the system receives such measurements and can determine when the waste container is becoming full. In this case, the system can be configured to alert the user and/or stop moving liquids into the waste container.
  • Fluidic conduits from the waste container and/or reagent containers in the caddy can connect with couplings in the fluidic subsystem of the instrument.
  • the couplings can include sensors, for example, that use the Hall effect, to confirm that a connector is properly seated within the coupling.
  • Single Sample Tissue processing Instrument 1020 has a linear driver motor, such as a z-axis stepper motor 2110, which may have an optional encoder, that controls the vertical position of rotary motor 2120 mounted on z- axis stepper slide 2111 attached to the inverted ‘II’ shaped structural frame 1020 mounted on chassis 1010.
  • a linear driver motor such as a z-axis stepper motor 2110, which may have an optional encoder, that controls the vertical position of rotary motor 2120 mounted on z- axis stepper slide 2111 attached to the inverted ‘II’ shaped structural frame 1020 mounted on chassis 1010.
  • a force gauge can be incorporated into the z-stage stepper 2110 to provide forcefeedback control of the mechanical force on the specimen 101 or below cantilevered cartridge slide 1450; this can help ensure very gentle mechanical processing steps and prevent application of high force by the rotor 218 onto the bottom of processing chamber 440.
  • Syringe pump 2130 connects fluidically with tubing or capillaries or microchips or other fluidic connectors with six-way valve 2141 and six-way valve 2140 to supply reagents, pressure, or vacuum to cartridge 200 (not shown) from reagent module 1430.
  • the force feedback mechanism can be configured to determine the position of the face of the rotor in the processing chamber. More specifically, force on the rotor increases somewhat when the actuator engages the plunger assembly (first inflection point in Figure 33). Also, the force on the rotor increases significantly when the rotor encounters the stator, or floor, of the processing chamber (steep inflection point in Figure 33). When the force gauge measures a force consistent with the rotor encountering tissue, the operating software can commence a grinding routine.
  • the embodiment of the single-sample Tissue Processing System 2010 shown in Figure 6 has a Magnetic processing Module 900 and magnet 910 is moved by magnetic actuator 935 mounted on inverted ‘II’ shaped structural frame 1020 under control of control software 725 using controller 2122.
  • Magnet 910 can be far from cartridge 200 as shown in Figure 9 and not interact with any magnetic beads 685 in cartridge 200 or in an extended position magnet 910 is moved to be near cartridge 200 for magnetic capture and processing of magnetic beads 685.
  • Many embodiments of configurations of the geometric relationship of the Magnetic processing Module 900 and magnet 910 and cartridge 200 are possible.
  • the Single-Sample Singulator System 2000 has a back structural frame 1021 on structural frame 1020 that mounts electronics 710 comprising rotary motor controller 2122, z-axis stepper controller 2112, 24 V to 5 V step down power supply 2230 and 24 V to 12 V step down power supply 2225.
  • Power can be supplied to single-sample Tissue processing System 2010 by plugging a 24 V power supply into plug 762 connecting to fuse 761 and power switch 760.
  • Six way valves 2140 and 2141 are controlled by boards 2210 and 2212.
  • Reagent Peltier relay board 2240 can control reagent Peltier.
  • the cartridge 200 can have one or more processing Chamber(s) 440 and none, one, or more Post-Processing Chamber(s) 460 as well as none, one or more other chambers such as cartridge waste chamber 435 or vacuum trap chamber 468.
  • Lid 462 produces a vacuum tight seal of post-processing chamber 460 and vacuum trap chamber 468 when cap 465 is sealed on lid 462.
  • Lid 462 can be attached to cartridge body 201 by ultrasonic welding, glue, epoxy, adhesives, and other methods to produce a vacuum tight seal .
  • the permanent attachment of lid 462 ensures single usage of cartridge 200 to eliminate cross sample contamination by preventing changing of strainer 2711.
  • FIG. 8A Figure 13 and Figure 14, when vacuum is applied to vacuum trap port 467 or to first post-processing port 485, liquids including single cell suspensions 1000, nuclei 1050, and other subcellular components 1060, and biomolecules 1070 are pulled from processing chamber 440 through fluidic line 453 and strainer 2711 into strain drain 451 and into output collector region 461 of post-processing chamber 460.
  • Strainer 2711 can have pore sizes no more than any of 2, 5, 10, 15, 20, 25, 30, 40, 50, 70, 100, 125, 200 pm, or larger to filter the suspension of biological material. Multiple in-line or stacked strainers 2711 can be employed to successively remove different sized components of the dissociated tissue specimen 110.
  • Cap 210 with cap coupler 211, and head 218 (also referred to as “rotor” 253) is shown ready to be inserted into sample inlet port 425.
  • Head 218 can have a surface for disrupting tissue that can comprise raised features 355 that aid in mechanically disrupting a tissue, organ, microtissue 6001 , organoid 6002 or other biological material.
  • head 218 attached to cap shaft 216 has a outwardly annular beveled head feature 356 designed to improve centricity of head 218 inside processing chamber 440 and thereby the uniformity of side gap 221 at the bottom of travel.
  • head 218 will lower until outwardly annular beveled feature 356 engages with inwardly annular beveled preprocessor chamber feature 357 on the inside wall of processing chamber 440 to produce a centered head 218 as shown in Figure 18B.
  • the centering of head 218 will produce a uniform side gap 221 between head 218 and the inner wall of processing chamber 440.
  • the height of head 211 is less than the height of the processing chamber 440 below inwardly beveled feature 357, the engagement of outwardly annular beveled head feature 356 with inwardly annular beveled preprocessor chamber feature 357 will set a uniform bottom gap 222.
  • the size of the side gap and the bottom gap can be optimized for different cell types or for different sized nuclei or subcellular organelles, or multicellular structures such as intestinal crypts.
  • the inwardly annular beveled preprocessor chamber feature 357 can be fluted to have sections with the same or different depths.
  • the side gap 221 between the head 218 of moveable mechanical disruptor 345 and the inside wall is preferably greater than or equal to any of 1 pm, or 2 pm, or 5 pm, or 10 pm, or 15 pm, or 20 pm, or 25 pm, or 30 pm, or 40 pm, or 50 pm, or 75 pm, or 100 pm, or 150 pm, or 200 pm, or 250 pm, or 500 pm, and 1000 pm or more, as well as any size in between.
  • the side gap 221 is greater than 50 microns and less than 150 microns for nuclei and other subcellular organelles and is greater than 75 microns and less than 250 microns for cells.
  • a gap size for isolation of nuclei can be, for example, between about 30 microns and about 200 microns, e.g., about 40 microns and about 150 microns, or about 100 pm to about 125 pm.
  • a gap size for isolation of cells can be, for example, between about 50 pm and about 400 pm, e.g., about 200 pm to about 300 pm, or about 250 pm.
  • the bottom gap 222 between the bottom of head 218 of moveable mechanical disruptor 345 and the bottom of processing chamber 440 is preferably greater than or equal to 1 pm, or 2 pm, or 5 pm, or 10 pm, or 15 pm, or 20 pm, or 25 pm, or 30 pm, or 40 pm, or 50 pm, or 75 pm, or 100 pm, or 150 pm, or 200 pm, or 250 pm, or 500 pm, and 1000 pm or more, as well as any size in between.
  • different heads can be selected to be used with the same diameter processing chamber 440 to produce different side gaps 221 or bottom gaps 222 to simplify manufacturing and inventory management requirements.
  • a bottom gap between a flat surface of the head and the flat bottom surface of the processing chamber can also be limited by the position of the flutes, or half domes, or other structures that prevent or define gaps between a flat surface of the head and the flat bottom surface of the processing chamber.
  • none, one, or more of the ports to cartridge 200 can have flexible or low durometer port covers 442, for example without limitation 40 to 100 durometer.
  • port cover 442 can be inserted into the space between the port and port cover retaining cylinder 441 to secure the port cover 442 in place over, for example as shown, reagent addition port 470.
  • a fluidic canula 1416 or fluidic pogo pin 1415 with an outside diameter larger than port cover center hole 446 can engage the port covered by port cover 442 and, because of the relatively low durometer, the port cover 442 will be deformed by fluidic canula 1416 or fluidic pogo pin 1415 to create a seal around the fluidic canula 1416 or fluidic pogo pin 1415.
  • the deformation can be used to eliminate the need for springs and the use of the fluidic pogo pin 1415 can be replaced by a non-movable fluidic canula 1416.
  • Figure 19C shows port cover 442 retained by crimp seal 443.
  • Figure 19D shows port cover 442 retained by forming port cover retaining cylinder 441 higher than the port cover 442 and melting the port cover retaining cylinder 441 to form a heat stake lip 444 that retains port cover 442.
  • the Single Sample Singulator Instrument 2050 has an actuator for mechanical processing that has a stepper motor 2110 that controls the vertical position of rotary motor 2120 and rotary motor shaft 2121 attached to rotary motor coupler 2125 that in turn can mechanically couples with cap coupler 211 of the cap 210 when inserted into cartridge 200.
  • the coupler can have a drive head that takes any appropriate form, such as a slot, a Phillips head, a quadrex, atri-wing, a spanner or a hex.
  • Rotary motor coupler 2125 has one or more facets that reversibly engage cap coupler 211 by actions such as moving downward and slowly rotating.
  • Stepper motor 2110 controls the vertical position of the rotary motor 2120 and thereby the vertical position of rotary motor coupler 2125, to raise or lower moveable disruptor 345 and head 218 in processing chamber 440. Combining rotation of rotary motor 2120 and movement of stepper motor 2110 enables many patterns of motion of moveable tissue disruptor 345 and head 218.
  • the inside walls of processing chamber 440 can be embodied in many different shapes.
  • the inside walls of processing chamber 440 can be fluted to have sections with different depths.
  • the inside wall can have a circular profile with the largest gap between the head 218 of moveable mechanical tissue disruptor 345 and the inside wall of preferably greater than or equal to any of 1 pm, or 2 pm, or 5 pm, or 10 pm, or 15 pm, or 20 pm, or 25 pm, or 30 pm, or 40 pm, or 50 pm, or 75 pm, or 100 pm, or 150 pm, or 200 pm , or 250 pm, or 500 pm, and 1000 pm or more, as well as any size in between.
  • the system can process specimen 101 by trituration.
  • the sides of the head 218 can form a ball-like structure to create a gap with the inside wall in a small area and the bottom of processing chamber 440 can be rounded to match the ball-like structure to create a Dounce-like mechanical tissue disruptor 345.
  • multiple regions with gaps of the same or different sizes can be created by varying the side profile of moveable tissue disruptor 345 and the inner wall of processing chamber 440.
  • the stator can be stationary or movable (e.g., rotating). In other embodiments, the stator is non-porous.
  • either or both of the processing chamber or the rotor can have a circular or noncircular cross-section.
  • the rotor is configured to rotate within the processing chamber. In its rotation, a portion of a wall of the rotor will have a gap between the rotor and the processing chamber of between about 150 and 250 microns.
  • a preserved tissue sample such as an FFPE sample or an OTC sample is placed into the bottom of the processing chamber of a preserved tissue processing cartridge.
  • the cartridge is engaged with the cartridge interface of the instrument. Fluidic connections from reagent containers are checked to confirm proper seating.
  • Xylene, or another appropriate organic solvent, is moved from a reagent container, through fluidic lines and a cartridge interface connector, into the processing chamber through the second processing chamber port 2570.
  • the rotor is pumped up and down to facilitate dissolving of preservatives. After sufficient time, the actuation system presses the rotor until it encounters the tissue sample. This pushes spent liquid reagents above the top of the rotor.
  • the rotor is lowered to a position such that the top of the rotor is below first processing chamber port 2504 and is rotated to mix the solvent, or moves to a fixed position above the bottom of the chamber and below the first processing chamber port 2504.
  • the gap between the side of the rotor and the internal wall of the processing chamber has been selected so that liquid can pass between the rotor and the wall but, tissue pieces or cells, nuclei, and/or subcellular organelles cannot. If the intention is to isolate cells, the gap can be of a size too small to pass cells, even if it is large enough to pass nuclei and other subcellular organelles.
  • vacuum, applied at first post-processing port 2585 liquid is pulled from first processing chamber port 2504 into the processing chamber.
  • Liquids collecting at first post-processing port 2585 are then further moved, typically by vacuum, through the cartridge interface connector, and through fluidic lines into the waste container located, for example, in the caddy.
  • liquid is pulled through first processing chamber port 2504, through fluidic conduit 2553, and out a reagent port, e.g., positioned at first post-processing port 2585. This process can be repeated until sufficient preservatives are removed from the preserved tissue.
  • the process can be continued with rehydration in a buffer, e.g., PBS, using the same processing methods of mixing with the rotor in a position that prevents pieces of tissue from being pulled out of the processing chamber.
  • a buffer e.g., PBS
  • Data can be transmitted electronically, e.g., over the Internet.
  • Electronic communication can be, for example, over any communications network include, for example, a high-speed transmission network including, without limitation, Digital Subscriber Line (DSL), Cable Modem, Fiber, Wireless, Satellite and, Broadband over Powerlines (BPL).
  • Information can be transmitted to a modem for transmission, e.g., wireless or wired transmission, to a computer such as a desktop computer.
  • reports can be transmitted to a mobile device. Reports may be accessible through a subscription program in which a user accesses a website which displays the report. Reports can be transmitted to a user interface device accessible by the user.
  • the user interface device could be, for example, a personal computer, a laptop, a smart phone or a wearable device, e.g., a watch, for example worn on the wrist.
  • FIG. 10 shows an exemplary computer system.
  • the computer system 9901 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 9905, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 9901 also includes memory or memory location 9910 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 9915 (e.g., hard disk), communication interface 9920 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 9925, such as cache, other memory, data storage and/or electronic display adapters.
  • CPU central processing unit
  • computer processor computer processor
  • the post-processing chamber is in fluidic communication with the first processing chamber port through a fluidic conduit;
  • An instrument comprising:
  • At least one cartridge interface configured to engage a cartridge of any of embodiments 1-7, wherein the cartridge interface comprises at least a first cartridge interface connector configured to engage a first post-processing port in the cartridge that is fluidically connected, directly or indirectly, to the first processing chamber port;
  • a mechanical subsystem comprising an actuator that engages the plunger assembly, and one or more motors that move the actuator in an up-down (Z axis) direction of the processing chamber, and rotates the actuator around the Z axis.
  • control subsystem comprising a digital computer comprising a processor and memory, wherein the memory comprises code that, when executed by the processor, instructs the system to perform one or more operations.
  • a kit comprising:
  • a processing chamber comprising: (i) a stator, (ii) a side wall, (iii) a top orifice, (iv) a first processing chamber port positioned in the side wall, and (v) an optional second processing chamber port positioned in the side wall; and
  • a plunger assembly positioned in the processing chamber through the top orifice, wherein the plunger assembly comprises a rotor, optionally attached to a plunger, the rotor comprising a face facing the stator; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the rotor has a circumference such that a space between the rotor and the side wall prevents passage of pieces of tissue, cells, nuclei, and other sub-cellular organelles between the rotor and the side wall and, optionally, wherein a face of the rotor does not comprise grinding features; wherein:
  • the cartridge optionally comprises a post-processing chamber comprising an inlet, a side wall, a floor, wherein the first post-processing port is positioned in the side wall, and, optionally, a cover covering an orifice in the postprocessing chamber and comprising the inlet;
  • a second cartridge for dissociating cells, nuclei and/or other sub-cellular organelles from tissue comprising: (a) a processing chamber comprising: (i) a stator comprising grinding features, (ii) a side wall, (iii) a top orifice, (iv) a first processing chamber port positioned in the side wall; and (v) an optional second processing chamber port positioned in the side wall; and
  • a cartridge pressure port configured to apply negative pressure to the processing chamber through the post-processing chamber, directly or through a vacuum chamber; wherein: the first post-processing port of the first cartridge, and the first postprocessing port of the second cartridge are configured to engage, separately, with the same first cartridge interface connector of a cartridge interface of an instrument, and, optionally, the second processing chamber port of the first cartridge in the second processing chamber port of the second cartridge are configured to engage separately with the same second cartridge interface connector of a cartridge interface of an instrument.
  • a reagent subsystem comprising at least one reagent container containing a reagent and at least one waste container;
  • a method of isolating cells and/or subcellular organelles from preserved tissue comprising:
  • a system for processing tissue comprising:
  • cartridge interface for engaging a cartridge
  • the cartridge interface comprises at least first, second and third cartridge interface connectors configured to engage ports in an engaged cartridge
  • a method for releasing cells and/or subcellular organelles from tissue comprising:
  • a processing chamber comprising: (i) a stator, (ii) a side wall, (iii) a top orifice, and (iv) a first processing chamber port positioned in the side wall; and a second processing chamber port positioned in the side wall;

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Abstract

L'invention concerne des procédés et systèmes pour traiter un tissu conservé et/ou dissocier un tissu pour libérer des cellules, des noyaux et/ou d'autres organites subcellulaires. Les instruments comprennent une interface servant à mettre en prise des cartouches par l'intermédiaire de conduites fluidiques pour introduire ou retirer des liquides d'une chambre de traitement d'une cartouche. Les systèmes comprennent également des sous-systèmes de réactif conçus pour empêcher des déversements de liquide et mettre en prise de manière sûre des raccords entre des récipients de réactif et des conduites fluidiques. Les systèmes comprennent également une rétroaction en temps réel de la pression sur un rotor de broyage dans une chambre de traitement d'une cartouche.
PCT/US2025/016864 2024-02-21 2025-02-21 Système et procédé d'analyse de tissus Pending WO2025179189A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160003791A1 (en) * 2013-02-27 2016-01-07 Artem Lebedev System and method for radiosynthesis, quality control and dose dispensing
WO2023167974A2 (fr) * 2022-03-02 2023-09-07 S2 Genomics, Inc. Procédé et appareil pour traiter des échantillons de tissu
US20230407232A1 (en) * 2018-06-01 2023-12-21 S2 Genomics, Inc. Method and apparatus for processing tissue samples

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160003791A1 (en) * 2013-02-27 2016-01-07 Artem Lebedev System and method for radiosynthesis, quality control and dose dispensing
US20230407232A1 (en) * 2018-06-01 2023-12-21 S2 Genomics, Inc. Method and apparatus for processing tissue samples
WO2023167974A2 (fr) * 2022-03-02 2023-09-07 S2 Genomics, Inc. Procédé et appareil pour traiter des échantillons de tissu

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