System and method for tissue analysis

Abstract

Provided herein are methods and systems for processing preserved tissue and / or dissociating tissue to release cells, nuclei and / or other subcellular organelles. Instruments comprise an interface for engaging cartridges through fluidic lines to introduce or remove liquids from a processing chamber of a cartridge. Systems also include reagent subsystems configured to prevent liquid spills and securely engage fittings between reagent containers and fluidic lines. Systems also include real-time feedback on the pressure on a grinding rotor in a processing chamber of a cartridge.

Description

SYSTEM AND METHOD FOR TISSUE ANALYSISSTATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] None.REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the priority date of United States provisional application 63 / 556,364, filed February 21 , 2024, the contents of which are incorporated herein in their entirety.BACKGROUND

[0003] 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, can involve removal of the preservative, for example by dissolving in an organic solvent, and rehydrating the released 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.

[0004] Automated methods of isolating single cells, nuclei and / or other subcellular organelles from fresh or preserved tissue speeds up this process and makes them more predictable.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

[0006] Figure 1 shows a Sample processing System that processes specimens into biocomponents such as single cells or nuclei for bioanalysis.

[0007] Figure 2 shows an overview of a Tissue Processing System and some exemplary modules. Tissue specimens or other specimens are processed into single cells, nuclei, nucleic acids, single-cell libraries, and other biologicals through the use of one or more cartridges and one or more of the Physical Dissociation Subsystem, the Enzymatic and Chemical DissociationSubsystem, the Measurement Subsystem, the Fluidic Subsystem, the Control Subsystem, and a Magnetic Module.

[0008] Figure 3 shows the overall design concept for a prototype showing functional system and a few example modalities of mechanical disruption and examples of chemicals and enzymes to dissociate FFPE tissue specimens into single cells, nuclei, and other biomolecules.

[0009] Figure 4 shows an example of a Single-Sample Tissue Processing System with mechanical disruption in a single cartridge with a bank of enzymes and reagents located in the instrument to dissociate solid tissue specimens into single cells, nuclei, and other biomolecules.

[0010] Figure 5 shows another example of a Single-Sample Tissue Processing System with mechanical disruption in a single cartridge with a bank of enzymes and reagents located separately from the instrument in a reagent module.

[0011] Figure 6 shows the front of an example of a Single-Sample Tissue Processing System used to dissociate tissue specimens into suspensions of single nuclei, and other biomolecules, using a cartridge.

[0012] Figure 7 shows the back of an example of the Single-Sample Tissue Processing System.

[0013] 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.

[0014] 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.

[0015] Figure 10 shows an exemplary computer system.

[0016] Figure 11 shows a cartridge architecture using pinch valves to direct liquid flows.

[0017] Figure 12 shows an exemplary cartridge fluidic architecture.

[0018] Figure 13A shows a cutaway view of an exemplary cartridge. Figure 13B shows a cartridge comprising a filter positioned in the post-processing chamber.

[0019] Figure 14 shows a top-down view of an exemplary cartridge.

[0020] Figure 15 shows an exemplary grinder assembly.

[0021] Figure 16 shows a cutaway view of an exemplary cartridge with a grinder assembly positioned for insertion into the processing chamber.

[0022] Figure 17 shows an exemplary cartridge with a grinder assembly inserted into the processing chamber.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Figure 21 shows an exemplary workflow to deparaffinize and rehydrate FFPE specimens followed by nuclei isolation.

[0027] Figure 22 shows a picture of cartridge adapted for processing preserved tissue.

[0028] Figure 23 shows a drawing of a cartridge adapted for processing preserved tissue, including ports for engaging a cartridge interface of an instrument.

[0029] Figure 24 shows a cut-away view of a cartridge adapted for processing preserved tissue.

[0030] Figure 25 shows a cut-away view of a cartridge adapted for processing preserved tissue.

[0031] Figure 26 shows a top-down view of a cartridge.

[0032] Figure 27 shows an exemplary reagent module including caddy holding reagent containers and a waste container.

[0033] Figure 28 shows a side view of an exemplary reagent module.

[0034] Figure 29 shows an exemplary system.

[0035] 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).

[0036] Figures 31A-C show a mechanical assembly with a force sensor to monitor force on the rotor face in the z-axis.

[0037] Figure 32 shows a mechanical assembly for actuating the plunger assembly from a different aspect.

[0038] 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.

[0039] Figure 34 shows a schematic of a mechanical assembly of an actuator for translating a plunger assembly along a z-axis.SUMMARY

[0040] Provided herein are systems, kits and methods for isolating cells, nuclei and / or subcellular organelles from fresh, frozen, or preserved tissue.

[0041] Systems are provided that engage different cartridges, adapted either for processing preserved tissue or for dissociating cells, nuclei and / or other subcellular organelles from tissue. In some embodiments, 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. In the case of the preserved tissue cartridge, 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. In contrast, for the dissociation cartridge 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.

[0042] Furthermore, in cartridges adapted for tissue dissociation, 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.

[0043] Cartridges for processing preserved tissue, on the one hand, and releasing cells and / or subcellular organelles from tissue, on the other hand, 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 cartridgeinterface and (4) a vacuum port. Each of these can be relatively positioned in each of the preserved tissued processing cartridge and the tissue dissociation cartridge such that these ports all engage the same ports / actuator in the instrument. Also, the cartridge interface of the instrument can comprise alignment features to properly align a cartridge for engagement. Accordingly, 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.

[0044] 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. In the tissue dissociation cartridge, 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. In the preserved tissue processing cartridge, 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. In the tissue dissociation cartridge, there also can be a vacuum chamber communicating with the post-processing chamber and connected to a source of vacuum in the instrument. In the preserved tissue processing cartridge, 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. In one embodiment, the port can function as a drain when a floor of the post-processing cartridge is positioned at about the level of the port.

[0045] A system for performing both processing of preserved tissue and dissociating tissue can comprise the following features. For dissociating tissue 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).

[0046] The system can also comprise a force sensor operatively linked to the rotor configured to measure, in real time, the force on the rotor. Using feedback from the force sensor, and a computer program configured to control pressure 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. Similarly, when the force sensor senses a force on the rotor consistent with hitting tissue in the processing chamber, 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.

[0047] For processing preserved tissue, 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.DETAILED DESCRIPTIONI. Introduction

[0048] 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.

[0049] Referring to Figure 2 , in many embodiments, the Tissue Processing System 110 processing is performed in cartridges 200 in the system. Tissue samples, e.g., fresh tissue 101, or preserved tissue samples, such as FFPE tissue specimens 150 or OCT (optimal cuttingtemperature) tissue specimens 160, or other specimens 101 are converted to single nuclei 1050, single cells 1000, or other organelles, or biomolecules or single cell libraries 1200 or bulk libraries 1210 through the use of cartridge 200 with one or more of the Physical Dissociation Subsystem 300, the Enzymatic and Chemical Dissociation Subsystem 400, the Measurement Subsystem 500, the Fluidic Subsystem 600, the Control Subsystem 700, the Magnetic Module 900, and the Temperature Subsystem 1475.

[0050] The Physical Dissociation Subsystem 300 can perform mixing or perform physical disruption by one or more of passing the specimen through orifices, grinding, rotating a rotor with features to dissociate tissue, forcing tissue through screens or mesh, sonication, ultrasonics, blending, homogenization, bead beating, and other methods known to one skilled in the art to physically disrupt tissue to help produce single cells.

[0051] 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. The Enzymatic and Chemical Dissociation Subsystem 400 can perform crosslink reversal and / or enzymatic disruption by using heat or adding formulations of reagents or mixture of components comprised of but not limited to proteinase K, collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, trypsin, papain, hyaluronidase, chymotrypsin, neutral protease, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, RNase inhibitors, or other enzymes, biochemicals, or chemicals such as EDTA, protease inhibitors, buffers, acids, or base.

[0052] 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, IGEPAL™ CA630 octylphenyl polyethylene glycol, n-octyl-beta-Dglucopyranoside (betaOG), n-dodecyl-beta, Tween™. 20, polyethylene glycol sorbitan monolaurate, Tween™ 80 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.

[0053] In other embodiments, different reagents or mixtures of reagents are applied sequentially to dissociate the FFPE or OCT sample or specimen into single cells 1000 or nuclei. 1050. The physical and enzymatic / chemical dissociation systems can be separate from each other, or they can be co-located (e.g., acting upon the sample simultaneously or sequentially).

[0054] In some embodiments, the amount of dissociation is monitored at intervals during the dissociation or at the endpoint, and in some instances the viability is determined during processing using a Measurement Subsystem 500. The Measurement Subsystem 500 can be an optical imaging device to image cells or nuclei or tissue using brightfield, phase contrast, fluorescence, chemiluminescence, near-field, or other optical readouts, or an electrical measurement, such as an impedance measurement of the change in conductivity when a cell passes through a sensor, or other types of measurement.

[0055] 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.

[0056] 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. In some instances 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.

[0057] In another aspect provided herein is a device for the dissociation of a biological sample, the device 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.

[0058] In another aspect provided herein is a device for the dissociation of a biological sample and the production of single-cell 1000 or nuclei 1050 suspensions or matched bulk nucleic acids 1010 or single cell libraries 1200 or matched bulk libraries 1210, the device comprising: (i) a chamber or area to input a biological sample or specimen either directly or in a device;(ii) a cartridge capable of dissociating tissue or specimen; (iii) an instrument to operate the cartridge and provide fluids as needed (iv) a measurement module such as an optical imager to measure titer, clumping, and / or viability, necrosis, cytotoxicity, apoptosis, etc. (v) sources of positive and / or negative pressure for exchange of dissociation solution for buffer or growth media at the desired titer, and (vi) output vessels such as 8 well strip tubes, microtiterplates, Eppendorf tubes, a chamber in the cartridge, or other vessels capable of receiving cell suspensions.

[0059] Referring to Figure 4, 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. In some embodiments the beads have a surface chemistry that facilitates the purification of the biologicals in conjunction with the chemical conditions. In other embodiments 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.

[0060] 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.

[0061] 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.

[0062] In the instant invention, many embodiments are possible and are incorporated by reference from patent application PCT / US2017 / 063811 filed November 29, 2017 (Jovanovich, Chear, McIntosh, Pereira, and Zaugg, “Method and Apparatus for processing Tissue Samples”) and from patent application PCT / US19 / 35097 filed June 1, 2019 (Jovanovich, Chear, Leisz, Eberhart, and Bashkin, “Method and Apparatus for processing Tissue Samples”); the contents of all of which are incorporated herein in their entirety as well as the number system used therein except where there is conflict the numbering herein predominates.

[0063] 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.II. Cartridge designs

[0064] Cartridges 200 can be used to process tissue into single-cell 1000 suspensions or nuclei 1050 and can be for single-use.

[0065] Referring to Figure 3, cartridge 200 will input specimen 101 (e.g., fresh tissue, FFPE tissue specimen 150 or OCT tissue specimen 160) and output single cells 1000 or nuclei 1050. The Tissue Processing System 110 as shown conceptually in Figure 3 combines the mechanical disruption of specimen 101 on cartridge 200, adds reagents such as chemicals, detergents, enzymatic or chemical dissolution solutions 410 and other fluids according to the protocols, and controls sample movement, pressures, and temperature. The Tissue Processing System 110 can move or rotate mechanical tissue disruptor elements comprised of, without limitation, a syringe plunger, pestle, Dounce pestle, or grinder, using a z axis stepper 2110 with a rotary motor 2120 coupled through the cap 210.

[0066] 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. In some embodiments, flexible tubing 493 can connect chambers and create simple pinch valves 491 to direct flow. In other embodiments, channels are created in the cartridge 200 and valves can be incorporated such as pneumatic valves, or other valves.A. Tissue Dissociation Cartridge

[0067] Referring to Figure 8C, the term plunger, or plunger assembly, is at times used to refer to combination of shaft / piston 216 and rotor 218 with optional disruption features {e.g., teeth) 355 with spring 213 in sheath 212.

[0068] In an embodiment, 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. In some embodiments, the mechanical tissue disruptor elements does not have features 355 on the bottom of the rotor or grinder but can be flat andmechanically 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.

[0069] 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. Also shown are 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. Not shown here is 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. In certain embodiments, the teeth can have a height of about 500 microns and a width of about 1 mm to 2 mm.

[0070] Spin rates for the dissociation can be 10-200 rpm. Total revolutions of the grinding element can be 5-500. In an exemplary protocol, 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.B. Preserved Tissue Processing Cartridge

[0071] It is desirable that 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.

[0072] 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.

[0073] 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.

[0074] 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 thatspent 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.

[0075] In the cartridge for processing preserved tissue, the face of the rotor and the stator of the processing chamber can comprise or can not comprise grinding features.

[0076] 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.

[0077] 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.

[0078] 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. In certain embodiments, floor 2580 may be slanted so that liquid pools at first post-processing port 2585.

[0079] In alternative embodiments, 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.

[0080] In operation, 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.

[0081] Similarly, in a tissue dissociation process, 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. After tissue dissociation, the rotor is depressed and liquid including released cells, nuclei and / or other subcellular organellescollect above the top of the rotor. Because 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. In contrast to the use of the preserved tissue processing cartridge, suction can be pulled from vacuum port 467 which is configured to apply vacuum to the post-processing chamber. However, because 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.III. Tissue Processing System

[0082] In one embodiment of the Sample processing System 50 as a Tissue Processing System 110, as shown in Figure 1, 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. As will be obvious to one skilled in the art, the biological materials produced such as single cells 1000, nuclei 1050, nucleic acids 1072, single cell libraries 1200, single nuclei libraries 1250, bulk libraries 1210, or other biocomponents comprised of subcellular components 1060, or biomolecules 1070 such as macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073 and RNA 1074, can also be used for many genomic, cell biology, proteomics, metabolomics, and other analytical methods.

[0083] 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 spectrometry, protein sequencing, and other detection modalities of proteins and peptides and their adducts and modifications with and without labels; carbohydrate characterization and profiling including sequencing, fluorescent, Raman, optical, mass spectrometry, and other detection modalities ofcarbohydrates and their adducts and other covalent polymers with and without labels; lipid characterization and profiling including sequencing, fluorescent, Raman, optical, mass spectrometry, and other detection modalities of lipids and their adducts and other covalent polymers with and without labels; flow cytometry; characterization of cells and profiling including fluorescent, Raman, optical, mass spectrometry, and other detection modalities of cells and their adducts and other covalent polymers with and without labels; metabolic profiling including sequencing, fluorescent, Raman, optical, mass spectrometry, and other detection modalities of metabolites and their adducts and other covalent polymers with and without labels; functional analysis including protein-protein interactions, protein-lipid interactions, protein-DNA interactions, RNA-DNA interactions, and other interactions between molecules derived from biological materials, with and without labels; bioinformatic analysis of cells, organelles, and biomolecules; and mass spectrometry and other analytical methods. In some embodiments the measurement system 500 can be physically integrated and fluids transferred by robotic pipetting, fluid flow through tubing or capillaries, centrifugal methods, or other methods.

[0084] Referring to Figure 4, in this embodiment 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. In some embodiments, 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.IV. Instrument and SystemA. Instrument

[0085] 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. As shown in Figure 4, 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.

[0086] 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.

[0087] Referring to Figure 4, 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.B. Fluidic Subsystem

[0088] 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. In one embodiment, cartridge 200 has two processing chambers 440 and a single post-processing chamber 460. In an embodiment, 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.

[0089] An embodiment of the Single-Sample Tissue processing System 2010 with a case on is shown in Figure 5. This embodiment has a reagent module 1430 which can be separate from Single Sample Tissue processing Instrument 2010 as shown in Figure 4 with power and control provided by Single Sample Tissue processing Instrument 2010 or a separate power source and processor can be used, or as shown in Figure 4 reagent module 1430 can be integrated inside a single instrument case.

[0090] Referring to Figures 27 and 28, the system can comprise a reagent block or reagent subsystem configured for handling reagents in the processing of preserved tissue. Such 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.

[0091] Certain reagents used for removing preservatives from preserved tissue may be considered hazardous materials. Accordingly, tracking and controlling these materials can provide a benefit to the user. In certain embodiments, 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.

[0092] 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.C. Force Sensor

[0093] Referring to Figure 6, in an embodiment, 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.

[0094] 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.

[0095] Referring to Figures 30-34, in some embodiments, 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 grinding routine can include a combination of rotating the rotor in one or both of clockwise or counterclockwise directions, and further depressing the rotor when the force measurement indicates to the operating software that more forceful grinding is necessary. A force measurement consistent with the rotor contacting the bottom of the chamber, can indicate to theoperating software that no further grinding is necessary and that liquid that is collected above the top of the rotor is ready for movement into the processing chamber.

[0096] The force sensor can be used to calibrate the plunger for operation in the same or different cartridges. In the first cartridge, position of the rotor and the z-axis consistent with tissue contact in or state or contact can be received into memory as calibrated positions. In the same cartridge, in the execution of a tissue dissociation or preserved tissue processing protocol, the plunger can be depressing the z-axis two positions in memory consistent with tissue contact or stator contact. Calibration can be performed on each cartridge engaged with the cartridge interface. Alternatively, a single calibration run can be performed in the calibrated positions used in a plurality of different cartridges engaged with the interface.D. Cartridge Interface

[0097] Cartridge 200 is placed into cartridge receiver tray 1510 on cartridge slide 1450 which is designed to hold cartridge 200 precisely, with the center of processing chamber 440 concentric with the center of rotary motor shaft 2121 of rotary motor 2120 within a distance or 1 or, 5, or 10, or 15, or 20, or 25, or 50, or 100, or 250 pm, or more when inserted by moving cartridge 200 in cartridge receiver tray 1510 on cartridge slide 1450 on cartridge slide rail 1480 until spring-loaded cartridge slide knob 1452 locks into place into a hole in cartridge slide 1450 with cartridge 200 held in place near or in contact with the thermal transfer plate 1470 and making fluidic connections with the pogo pins 1415 of cartridge interface 1510.E. Temperature Sensor

[0098] The temperature regulating subsystem 1475 can set the thermal transfer plate 1470 to a given temperature by cartridge Peltier 1440 or other temperature regulating device such as strip resistive heaters, circulating fluids, etc. to set the cartridge temperature in the processing chamber 440 and post-processing chamber 460 under control of board 2250. In some embodiments, the temperature of processing chamber 440 and post-processing chamber 460 can be set independently. In some embodiments the temperature regulating system can use a thermocouple, or thermistor, or IR camera to set the temperature of the thermal transfer plate 1470 or the outside of cartridge 200.

[0099] In an embodiment, fluidic ports on cartridge 200 dock with spring-loaded pogo pins 1415 to connect fluids, gases, or vacuum to cartridge 200 on cartridge insertion. In another embodiment, pogo pins 1415 or canula 1416 are moved to connect with cartridge 200 after cartridge insertion. In another embodiment, canula 1416 connected to fluidic lines from syringe pump 2130 are held rigidly attached to the thermal transfer plate 1470 or other part of instrument and cartridge 200 has flexible materials on cartridge ports that seal with the canula(s) 1416 after cartridge insertion, as described below. Cartridge ports are ports opening out of a cartridge. A cartridge port may communicate directly with a chamber by being a port inthe chamber, or indirectly, e.g., through another chamber comprising the port and communicating with the chamber in question.F. Magnetic Processing Module[000100] 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.[000101] Referring to Figure 7, in an embodiment, 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.[000102] Systems that process one or more cartridges simultaneously are within the scope of the present invention. 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.[000103] In one embodiment, illustrated in Figure 8, cap 210, alternatively referred to as a tissue disruptor, is placed on top of processing chamber 440 after specimen 101 is added into processing chamber 440 of cartridge 200. After cartridge 200 is inserted into the instrument, pogo pins 1415, canula 1416, or other fluidic connectors can connect with none, one, or more of cartridge ports 470 to supply reagents to processing chamber 440, first post-processing port 485 to supply reagents or vacuum to post-processing chamber 460, cartridge vacuum trap port 467 to supply vacuum to vacuum trap chamber 468, or cartridge waste port 2355 to supply vacuum or reagents to cartridge waste line 2351.[000104] An embodiment of cartridge 200 for processing tissue specimen 150 illustrated in Figure 8 fluidically connects processing chamber 440 to post-processing chamber 460 using fluidic line 453, which can be tubing, connecting from processing chamber nipple 471 to port 452 (connected through nipple 454) positioned over a strainer 2711 inserted into postprocessing chamber 460. In other embodiments, no strainer can be used or strainer 2711 can be incorporated as an in-line filter, for example in a Swinny filter holder attached to the output ofprocessing chamber 440 or in fluidic line 453 or attached to lid 462. In an embodiment, dual or triple or more filters are used in strainer 2711, for example, a 145 micron filter followed by a 40 micron filter followed by a 20 micron filter; other combinations are envisioned.[000105] 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.[000106] In some embodiments, cartridge 200 can have on-cartridge valves which can be pinch valves on fluidic lines such as fluidic line 453 which the instrument actuates to open and close lines, or by using a T junction and two lines, route fluids down different paths such as to on cartridge waste or to an a optics imaging system 520, or to multi’omics processing of another workflow or analysis method. In another embodiment, fluidic lines such as fluidic line 453 can be partially closed to create a variable orifice that can disrupt partially dissociated tissue. Actuators can open and pinch close tubing in the cartridge 200, or operate the variable orifice using variable orifice device when desired. In other embodiments, cartridge 200 can have on- cartridge valves which can be miniaturized pneumatic valves, or microvalves. In some embodiments, microfluidics or microchips are used for fluidic lines. In an embodiment there are no valves on the cartridge 200, with all fluidic control coming from the instrument.[000107] Referring to Figure 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.[000108] Referring to Figure 8B and Figure 8C, the cap coupler (also referred to as “drive head”) 211 is held inside cap sheath 212 which in one embodiment has cap sheath hole 214. Cap coupler 211 is attached to cap shaft 216 which passes through cap sheath hole 214 and is attached to the head 218 which can be a rotor 353 with grinding teeth 355. The assembly of cap coupler 211 attached to cap shaft 216 and head 218 are referred to as a plunger assembly which is a type of moveable mechanical tissue disruptor 345.[000109] Referring to Figure 18A, in a preferred embodiment, 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. When z-axis stepper motor 2110 lowers and cap coupler 211 is pushed down by rotary motor coupler 2125, 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. In addition, if 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. In addition, to allow passage of disrupted tissue when head 218 is seated on inwardly annular beveled preprocessor chamber feature 357, the inwardly annular beveled preprocessor chamber feature 357can 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. In a preferred embodiment, 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. In some embodiments, 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.[000110] Referring to Figure 19, 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. As illustrated in Figure 19A and in cutout Figure 19B, 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. In some configurations, 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.[000111] In a preferred embodiment 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. As shown in Figure 20 A, in a preferred embodiment, rotary motor coupler 2125 has a single blade to engage cap coupler 211 as stepper motor 2110 lowers, the rotary motor coupler 2125 attached to rotary motor shaft 2121 engages cap coupler 211 in cap 210 and if the rotary motor coupler 2125 is not lined up with cap coupler groove 217, the rotary motor coupler 2125 can not directly insert into the cap coupler groove 217. In a preferred embodiment, cap coupler 211 has two surfaces on either side of cap coupler groove 217 which slope in opposite directions across the cap coupler 211 such that each side has a higher and lower wall on either side of cap coupler groove 217. When rotary motor shaft 2121 turns in the clockwise direction (looking from above), rotary motor coupler 2125 blade spins in the clockwise direction and encounters the high side of the wall of cap coupler groove 217 and begins to rotate cap coupler 211 clockwise. As stepper motor 2110 lowers, the rotary motor coupler 2125 will engage the cap coupler groove 217, as shown in Figure 20 C. As shown in Figure 20 D, as stepper motor 2110 continues to lower, the rotary motor 2120 and rotary motor shaft 2121 attached to rotary motor coupler 2125 will lower, pushing on cap coupler groove 217 and the cap coupler 211 will compress cap spring 213 against the bottom of cap sheath 212 and lower head 218. As shown in Figure 20 E, head 218 can be lowered close to or in contact with the bottom of processing chamber 440, which can be a stator 354, and head 218 can be rotatedto disrupt tissue. When stepper motor 2110 raises, rotary motor 2120 and rotary motor coupler 2125 raise up and cap spring 213 decompresses to push cap coupler 211 against rotary motor coupler 2125 to continue engagement.[000112] In another embodiment of the Single Sample Singulator Instrument 2050, stepper motor 2110 controls the vertical position of rotary motor 2120 which is magnetically coupled to moveable disruptor 345 with a magnetic or paramagnetic element embedded with cap 210 as part of cap coupler 211 or as part of moveable disruptor 345 or head 218.[000113] When rotary motor coupler 2125 is engaged with cap coupler 211 by mechanical coupling, magnetic coupling, pneumatic, or fluidic coupling, or other coupling methods, and rotary motor 2120 rotates, moveable disruptor 325 and head 218 are rotated. 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.[000114] 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. In a preferred embodiment, 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.[000115] Moveable tissue disruptor 345 can be embodied in many different shapes with many different profiles. In one embodiment, moveable tissue disruptor 345 can have a head 218 which is a rotor 353 with optional features, for example, grinding teeth 355 on the bottom of rotor 353 and grinding teeth 355 on stator 354 which is on the top surface of the bottom of the processing chamber 440 to assist in disruption of large pieces of tissue specimens 120 into smaller pieces or assist in the dissociation into single cells 1000 or nuclei 1050 or biomolecules 1070. As shown in Figures 8 and 18, the sides of head 218 can be a cylinder to create an inside gap 221 with the inside wall over the length of the cylinder. By raising and lowering head 218 without turning head 218, thereby using it as a moveable disruptor 345, the system can process specimen 101 by trituration. In another embodiment 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. In other embodiments, 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. In other embodiments, the stator can be stationary or movable (e.g., rotating). In other embodiments, the stator is non-porous. In otherembodiments, either or both of the processing chamber or the rotor can have a circular or noncircular cross-section. In the case of a non-circular 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.V. Methods of Use[000116] Methods of isolating dissociated single cells, nuclei or other subcellular organelles from preserved tissue can proceed as follows. A first part of the process involves processing preserved tissue samples to remove preservatives and, typically, rehydrate the tissue, and optionally to further treat the deparaffinized, rehydrated sample with enzymatic processing, or heat processing or chemical processing. A second part of the process involves disrupting the rehydrated tissue to release individual cells, nuclei, and / or subcellular organelles. These two parts can be performed in different cartridges. The first part can be performed in a cartridge adapted for processing preserved tissue samples, as described herein. The second part can be performed in a cartridge adapted for dissociating tissue samples, also as described herein.A. Processing Preserved Tissue Samples1. Removal of Preservatives[000117] 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. In one embodiment 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. As discussed herein, 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. Using 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. In an alternative embodiment, liquidis 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.2. Rehydration of preserved tissue[000118] Preserved tissue from which preservatives have been removed can now be rehydrated. In this process, one or more concentrated alcohol solutions are sequentially exchanged with less concentrated alcohol solutions and then with buffer solutions until the tissue is sufficiently rehydrated. This process can involve moving alcohol-containing solutions in the same manner into the processing chamber through the second processing chamber port. Again, the rotor can be 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. After first incubation with an alcohol- containing solution, the rotor can be sufficiently depressed to push the spent solution to position above the top of the rotor.[000119] In one embodiment 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. As previously, liquid is pulled from the first processing chamber port out the reagent port, depending on configuration (and optionally through into the post-processing chamber), and into the waste container. This process is repeated with increasingly more dilute alcohol solutions until tissue is fully rehydrated.[000120] 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.3. Post-processing of deparaffinized and rehydrated tissues.[000121] It may be desirable to further process the deparaffinized and rehydrated tissue in the preserved tissue cartridge. In some embodiments, an enzymatic solution is used to loosen the cells and nuclei in the tissue. In one embodiment, Liberase TH (Roche) can be added and incubated for 45 min at a low temperature, such as 8 °C, to post-process the tissue while minimizing degradation of nucleic acids. In other embodiments, heat can be applied to assist in antigen presentation. In another embodiment, buffers can be applied to postprocess the deparaffinized and rehydrated tissue.B. Disruption of Tissue Samples to Release Cells, Nuclei, and / or Other Subcellular Organelles[000122] Rehydrated tissue can now be subjected to disruption to release individual cells, nuclei, and / or other subcellular organelles. The process involves moving the rehydrated tissue sample from the preserved tissue processing cartridge into the processing chamber of the cartridge adapted for tissue dissociation.[000123] Disruption of tissue can include a plurality of disruption steps, each involving positioning the head a different distance from floor of the chamber to produce gaps of different sizes. Typically, at each position, the head will rotate, further facilitating disruption or mixing. In certain embodiments, an organ can be auto-minced by the disruptor before tissue disruption into single cells 1000 or nuclei 1050 or other biological materials. Such a method can involve a first disruption step, which can include setting the head at a plurality of different distances from the floor of the chamber and rotating at each gap distance, to provide tissue with greater surface area and less distance for access by enzymes. A next step can involve incubating the autominced organ with enzymes or chemicals for tissue disruption into single cells 1000 or nuclei 1050. A next step can involve a second disruption step, which, in turn, can include setting the head at a plurality of different distances from the floor of the chamber and rotating the head.[000124] Processes described here can be performed using one or more computer systems that can be networked together. Calculations can be performed in a cloud computing system in which data on the host computer is communicated through the communications network to a cloud computer that performs computations and that communicates, or outputs, results to a user through a communications network. For example, nucleic acid sequencing can be performed on sequencing machines located at a user site. The resulting sequence data files can be transmitted to a cloud computing system where the sequence classification algorithm performs one or more operations of the methods described herein. At any step a cloud computing system can transmit results of calculations back to the computer operated by the user.[000125] 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. Alternatively, 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.VI. Computer Systems[000126] Methods provided herein can be executed by programmable digital computer.[000127] Figure 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. The computer readable memory 9910, storage unit 9915, interface 9920 and peripheral devices 9925 are in communication with the CPU 9905 through a communication bus (solid lines), such as a motherboard. The storage unit 9915 can be a data storage unit (or data repository) for storing data. The computer system 9901 can be operatively coupled to a computer network (“network”) 9930 with the aid of the communication interface 9920. The network 9930 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 9930 in some cases is a telecommunication and / or data network. The network 9930 can include one or more computer servers, which can enable distributed computing, such as cloud computing.[000128] The CPU 9905 can execute a sequence of machine-readable instructions, which can be embodied in a program or software (code). The instructions may be stored in a memory location, such as the computer readable memory 9910. The instructions can be directed to the CPU 9905, which can subsequently program or otherwise configure the CPU 9905 to implement methods of the present disclosure.[000129] The storage unit 9915 can store files, such as drivers, libraries, and saved programs. The storage unit 9915 can store user data, e.g., user preferences, log files, video or other images, and user programs. The computer system 9901 in some cases can include one or more additional data storage units that are external to the computer system 9901 , such as located on a remote server that is in communication with the computer system 9901 through an intranet or the Internet.[000130] The computer system 9901 can communicate with one or more remote computer systems through the network 9930.[000131] Methods as described herein can be implemented by way of machine {e.g., computer processor) executable code stored on an electronic storage location of the computer system 9901, such as, for example, on the computer readable memory 9910 or electronic storage unit 9915. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 9905. In somecases, the code can be retrieved from the storage unit 9915 and stored on the memory 9910 for ready access by the processor 9905. In some situations, the electronic storage unit 9915 can be precluded, and machine-executable instructions are stored on memory 9910. The code can be used to communicate and issue instructions to electronic devices, e.g., circuit boards 9940, modules, or subsystems, on the instrument ,for example, the rotary DC motor relay board 2134 or the heater relay board 2240 driving Peltier 1440 to accomplish tasks such as rotating a motor or controlling the temperature of the cartridge 200.[000132] Machine-executable code can be stored on an electronic storage unit, such as memory {e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks.[000133] The computer system 9901 can include or be in communication with an electronic display 9935 that comprises a user interface (III) 9940 for providing, for example, input parameters for methods described herein. Examples of Ills include, without limitation, a graphical user interface (GUI) and web-based user interface.EXEMPLARY EMBODIMENTS[000134] 1. A cartridge for processing preserved tissue, comprising:(a) 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(b) 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.[000135] 2. The cartridge of embodiment 1, wherein the plunger assembly comprises a plunger attached to the rotor.[000136] 3. The cartridge of embodiment 1, wherein the space between the rotor and the side wall is no more than any of 200 .m, 100 .m, 50 .m, and 25 .m.[000137] 4. The cartridge of embodiment 1, wherein:(i) the first processing chamber port is positioned in the side wall of the processing chamber, such that, when the rotor is fully depressed in the processing chamber, at least part of the first processing chamber port is positioned above a top of the rotor.[000138] 5. The cartridge of embodiment 4, further comprising:(c) a post-processing chamber comprising an inlet, a side wall, a floor, and a first post-processing port positioned in the side wall, and, optionally, wherein the inlet is positioned in a cover covering an orifice of the post-processing chamber; wherein:(i) the post-processing chamber is in fluidic communication with the first processing chamber port through a fluidic conduit; and(ii) the first post-processing port is positioned to drain fluid that collects in the post-processing chamber.[000139] 6. The cartridge of embodiment 5, wherein the floor of the post-processing chamber is slanted.[000140] 7. The cartridge of embodiment 4, further comprising:(c) a first post-processing port in fluidic communication with the first processing chamber port through a fluidic conduit, and configured to engage a cartridge interface connector (e.g., in the form of a cannula) of a cartridge interface of an instrument.[000141] 8. An instrument comprising:(i) 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;(ii) a reagent subsystem comprising at least a waste container; and(iii) a fluidic subsystem comprising:(1) one or more fluid lines connecting the waste container to the first cartridge interface connector (e.g., in the form of a cannula); and sources of pressure configured to move liquids from the processing chamber, through the first post-processing port and the first cartridge interface connector, and into the waste container.[000142] 9. The instrument of embodiment 8, further comprising:(iv) 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.[000143] 10. The instrument of embodiment 9, further comprising:(v) a 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.[000144] 11. A kit comprising:(I) a first cartridge for processing preserved tissue comprising:(a) 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(b) 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:(i) the first processing chamber port is positioned in the side wall of the processing chamber, such that, when the rotor is fully depressed in the processing chamber, at least part of the first processing chamber port is positioned above a top of the rotor; and(c) a first post-processing port fluidically connected to the first processing chamber port, e.g., through a fluidic conduit;(d) 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; wherein:(i) the post-processing chamber is in fluidic communication with the first processing chamber port through the fluidic conduit; and(ii) the first post-processing port is positioned to drain fluid that collects in the post-processing chamber; and(II) 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(b) 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 face comprises grinding features, optionally wherein the rotor has a circumference such that when the rotor is inserted into the processing chamber a 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;(c) a post-processing chamber comprising (i) a floor, (ii) a side wall, (iii) an inlet, and (iv) a first post-processing port positioned in the side wall, and (v) an optional second post-processing chamber port; and(d) 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.[000145] 12. The kit of embodiment 11 , further comprising:(a) an instrument comprising:(i) at least one cartridge interface configured to engage the first cartridge and, separately, the second cartridge, wherein the cartridge interface comprises at least a first cartridge interface connector (e.g., in the form of a cannula) configured to engage the first post-processing port of the first cartridge, and the first post-processing port of the second cartridge;(ii) a reagent subsystem comprising at least one reagent container containing a reagent and at least one waste container;(iii) a fluidic subsystem comprising:(1) at least one fluid line connecting the reagent container with the first cartridge interface connector (e.g., in the form of a cannula), and atleast one fluid line connecting the waste container to the first cartridge interface connector (e.g., in the form of a cannula); and(2) one or more pressure sources configured to apply positive or negative pressure to move liquids and / or gasses through the one or more fluid lines;(iv) a mechanical subsystem comprising an actuator, a linear driver (e.g., a stepper motor or a pneumatic driver) that drives the actuator in an up-down (Z axis) direction, and a rotary motor that rotates the actuator around the Z axis; and (v) a 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; and wherein, when the first or second cartridge is engaged with the cartridge interface:(A) the first post-processing port engages the same first cartridge interface connector; and(B) optionally, the second processing chamber port engages the same second cartridge interface connector.[000146] 13. A method of isolating cells and / or subcellular organelles from preserved tissue comprising:(a) providing a kit of embodiment 11 or 12, wherein the first cartridge is engaged with the cartridge interface, wherein the processing chamber of the first cartridge comprises a preserved tissue sample;(b) processing the preserved tissue sample by:(i) using the actuator to depress the rotor to push liquid to the level of the first processing chamber port;(ii) using the fluidic subsystem, moving processing reagents into the processing chamber to remove preservative compounds from the preserved tissue ;(iii) using the fluidic subsystem to move the liquid out of the processing chamber (e.g., through the first post-processing port (optionally through the postprocessing chamber) and the first cartridge interface connector into a waste container fluidically connected therewith);(iv) optionally, using the fluidic subsystem, moving rehydrating reagents into the processing chamber to rehydrate the preserved tissue; and(v) using the fluidic subsystem, moving rehydrating reagents out of the processing chamber (e.g., through the first post-processing port (optionally through the post-processing chamber) and the first cartridge interface connector into a waste container fluidically connected therewith);(c) moving the processed tissue from the first cartridge into the processing chamber of the second cartridge, and, engaging the second cartridge with one of the cartridge interfaces;(d) dissociating cells and / or subcellular organelles from the processed tissue by:(i) using the fluidic subsystem, moving tissue dissociation reagents into the processing chamber;(ii) using the actuator to depress and rotate the rotor to release cells and / or nuclei from the processed tissue and to push liquid to move the released cells and / or subcellular organelles to the level of the first processing chamber port;(iii) using the fluidic subsystem to move the cells and / or subcellular organelles from the processing chamber into the post-processing chamber; and(iv) collecting the released cells and / or subcellular organelles from the post-processing chamber.[000147] 14. A system for processing tissue comprising:(a) an instrument comprising:(i) at least one cartridge interface for engaging a cartridge, wherein the cartridge interface comprises at least first, second and third cartridge interface connectors configured to engage ports in an engaged cartridge;(ii) a reversibly engageable processing reagent subsystem comprising one or more of:(A) a caddy for holding a plurality of containers, wherein the carriage comprises a containment barrier configured to contain liquids inside the carriage,(B) a plurality of containers held by the carriage, wherein the containers include at least one container containing a reagent for processing paraffinized tissue, and at least one waste container,(C) a scale configured to measure the weight of the waste container;(D) one or more optical sensors that senses liquid in the bottom of the caddy;(E) a plurality of fluidic conduits connecting the containers with one or more couplings of a fluidic subsystem of the instrument, wherein the instrument comprises a sensing mechanism that senses when the fluidic conduits and couplings are properly connected; and properly seated;(F) optionally a sensor that e.g., uses the Hall effect to sense the connection between the caddy and the rest of the instrument(iii) a fluidics subsystem comprising:(A) the one or more couplings connected to a plurality of fluidic conduits, wherein the fluidic conduits communicate with the cartridge interface connectors; and(B) one or more pressure sources for moving reagents from the one or more containers through one or more of the cartridge interface connectors, and for moving liquids from cartridge interface connectors into the waste container.[000148] 15. A method for releasing cells and / or subcellular organelles from tissue comprising:(a) providing a cartridge comprising:(1) 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;(2) a plunger assembly positioned in the processing chamber through the top orifice, wherein the plunger assembly comprises a plunger comprising a rotor, the rotor comprising a face facing the stator, wherein the face comprises grinding features; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the plunger assembly is operatively connected to a pressure sensor configured to measure pressure against the face of the rotor;(b) providing a tissue sample in the processing chamber;(c) depressing the rotor in the processing chamber until the pressure sensor measures a target pressure consistent with contact between the face of the rotor and the tissue sample;(d) rotating the rotor against the tissue sample while maintaining the target pressure, to dissociate cells and / or subcellular organelles from the tissue sample;(e) stopping depression of the rotor when the pressure sensor measures a pressure consistent with the face of the rotor contacting the stator.[000149] 16. A method of calibrating a plunger for cartridge comprising:(a) while depressing a plunger comprising a shaft attached to a rotor, within a chamber of a cartridge, measuring and recording force on the plunger at different positions along the Z axis of the plunger and, determining a calibrated fully depressed position consistent with the rotor contacting a floor of the chamber.[000150] 17. The method of embodiment 16, further comprising:(b) while depressing the plunger within a chamber of the cartridge, determining a calibrated tissue engagement position consistent with the rotor contacting tissue in the chamber.[000151] 18. The method of embodiment 16 or 17, further comprising:(c) performing a dissociation protocol on tissue in the chamber wherein the protocol comprises depressing the plunger to the determined fully depressed position and / or the determined contacting tissue position.[000152] 19. The method of embodiment 16 or 17, comprising using the calibrated fully depressed position and / or the determined contacting tissue position with a second, different cartridge.[000153] As used herein, the following meanings apply unless otherwise specified. The words “can” and “may” are used in a permissive sense (i.e. , meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The phrase “at least one” includes “one”, “one or more”, “one or a plurality”, and, therefore, contemplates the use of the term “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1 , at least 2 or at least 3”. The term “about” refers to a range that is 5% plus or minus from a stated numerical value within the context of the particular usage. The term "consisting essentially of' refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination.[000154] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.[000155] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A cartridge for processing preserved tissue, comprising:(a) 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(b) 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.

2. The cartridge of claim 1 , wherein the plunger assembly comprises a plunger attached to the rotor.

3. The cartridge of claim 1 , wherein the space between the rotor and the side wall is no more than any of 200 .m, 100 .m, 50 .m, and 25 .m.

4. The cartridge of claim 1 , wherein:(i) the first processing chamber port is positioned in the side wall of the processing chamber, such that, when the rotor is fully depressed in the processing chamber, at least part of the first processing chamber port is positioned above a top of the rotor.

5. The cartridge of claim 4, further comprising:(c) a post-processing chamber comprising an inlet, a side wall, a floor, and a first postprocessing port positioned in the side wall, and, optionally, wherein the inlet is positioned in a cover covering an orifice of the post-processing chamber; wherein:(i) the post-processing chamber is in fluidic communication with the first processing chamber port through a fluidic conduit; and(ii) the first post-processing port is positioned to drain fluid that collects in the postprocessing chamber.

6. The cartridge of claim 5, wherein the floor of the post-processing chamber is slanted.

7. The cartridge of claim 4, further comprising:(c) a first post-processing port in fluidic communication with the first processing chamber port through a fluidic conduit, and configured to engage a cartridge interface connector (e.g., in the form of a cannula) of a cartridge interface of an instrument.

8. An instrument comprising:(i) at least one cartridge interface configured to engage a cartridge of any of claims 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;(ii) a reagent subsystem comprising at least a waste container; and(iii) a fluidic subsystem comprising:(1) one or more fluid lines connecting the waste container to the first cartridge interface connector (e.g., in the form of a cannula); and sources of pressure configured to move liquids from the processing chamber, through the first postprocessing port and the first cartridge interface connector, and into the waste container.

9. The instrument of claim 8, further comprising:(iv) 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.

10. The instrument of claim 9, further comprising:(v) a 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.

11. A kit comprising:(I) a first cartridge for processing preserved tissue comprising:(a) 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(b) 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:(i) the first processing chamber port is positioned in the side wall of the processing chamber, such that, when the rotor is fully depressed in theprocessing chamber, at least part of the first processing chamber port is positioned above a top of the rotor; and(c) a first post-processing port fluidically connected to the first processing chamber port, e.g., through a fluidic conduit;(d) 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; wherein:(i) the post-processing chamber is in fluidic communication with the first processing chamber port through the fluidic conduit; and(ii) the first post-processing port is positioned to drain fluid that collects in the post-processing chamber; and(II) 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(b) 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 face comprises grinding features, optionally wherein the rotor has a circumference such that when the rotor is inserted into the processing chamber a 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;(c) a post-processing chamber comprising (i) a floor, (ii) a side wall, (iii) an inlet, and (iv) a first post-processing port positioned in the side wall, and (v) an optional second post-processing chamber port; and(d) 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 post-processing 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 toengage separately with the same second cartridge interface connector of a cartridge interface of an instrument.

12. The kit of claim 11 , further comprising:(a) an instrument comprising:(i) at least one cartridge interface configured to engage the first cartridge and, separately, the second cartridge, wherein the cartridge interface comprises at least a first cartridge interface connector (e.g., in the form of a cannula) configured to engage the first post-processing port of the first cartridge, and the first post-processing port of the second cartridge;(ii) a reagent subsystem comprising at least one reagent container containing a reagent and at least one waste container;(iii) a fluidic subsystem comprising:(1) at least one fluid line connecting the reagent container with the first cartridge interface connector (e.g., in the form of a cannula), and at least one fluid line connecting the waste container to the first cartridge interface connector (e.g., in the form of a cannula); and(2) one or more pressure sources configured to apply positive or negative pressure to move liquids and / or gasses through the one or more fluid lines;(iv) a mechanical subsystem comprising an actuator, a linear driver (e.g., a stepper motor or a pneumatic driver) that drives the actuator in an up-down (Z axis) direction, and a rotary motor that rotates the actuator around the Z axis; and(v) a 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; and wherein, when the first or second cartridge is engaged with the cartridge interface:(A) the first post-processing port engages the same first cartridge interface connector; and(B) optionally, the second processing chamber port engages the same second cartridge interface connector.

13. A method of isolating cells and / or subcellular organelles from preserved tissue comprising:(a) providing a kit of claim 11 or 12, wherein the first cartridge is engaged with the cartridge interface, wherein the processing chamber of the first cartridge comprises a preserved tissue sample;(b) processing the preserved tissue sample by:(i) using the actuator to depress the rotor to push liquid to the level of the first processing chamber port;(ii) using the fluidic subsystem, moving processing reagents into the processing chamber to remove preservative compounds from the preserved tissue;(iii) using the fluidic subsystem to move the liquid out of the processing chamber (e.g., through the first post-processing port (optionally through the postprocessing chamber) and the first cartridge interface connector into a waste container fluidically connected therewith);(iv) optionally, using the fluidic subsystem, moving rehydrating reagents into the processing chamber to rehydrate the preserved tissue; and(v) using the fluidic subsystem, moving rehydrating reagents out of the processing chamber (e.g., through the first post-processing port (optionally through the postprocessing chamber) and the first cartridge interface connector into a waste container fluidically connected therewith);(c) moving the processed tissue from the first cartridge into the processing chamber of the second cartridge, and, engaging the second cartridge with one of the cartridge interfaces;(d) dissociating cells and / or subcellular organelles from the processed tissue by:(i) using the fluidic subsystem, moving tissue dissociation reagents into the processing chamber;(ii) using the actuator to depress and rotate the rotor to release cells and / or nuclei from the processed tissue and to push liquid to move the released cells and / or subcellular organelles to the level of the first processing chamber port;(iii) using the fluidic subsystem to move the cells and / or subcellular organelles from the processing chamber into the post-processing chamber; and(iv) collecting the released cells and / or subcellular organelles from the postprocessing chamber.

14. A system for processing tissue comprising:(a) an instrument comprising:(i) at least one cartridge interface for engaging a cartridge, wherein the cartridge interface comprises at least first, second and third cartridge interface connectors configured to engage ports in an engaged cartridge;(ii) a reversibly engageable processing reagent subsystem comprising one or more of:(A) a caddy for holding a plurality of containers, wherein the carriage comprises a containment barrier configured to contain liquids inside the carriage,(B) a plurality of containers held by the carriage, wherein the containers include at least one container containing a reagent for processing paraffinized tissue, and at least one waste container,(C) a scale configured to measure the weight of the waste container;(D) one or more optical sensors that senses liquid in the bottom of the caddy;(E) a plurality of fluidic conduits connecting the containers with one or more couplings of a fluidic subsystem of the instrument, wherein the instrument comprises a sensing mechanism that senses when the fluidic conduits and couplings are properly connected; and properly seated(F) optionally a sensor that e.g., uses the Hall effect to sense the connection between the caddy and the rest of the instrument(iii) a fluidics subsystem comprising:(A) the one or more couplings connected to a plurality of fluidic conduits, wherein the fluidic conduits communicate with the cartridge interface connectors; and(B) one or more pressure sources for moving reagents from the one or more containers through one or more of the cartridge interface connectors, and for moving liquids from cartridge interface connectors into the waste container.

15. A method for releasing cells and / or subcellular organelles from tissue comprising:(a) providing a cartridge comprising:(1) 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;(2) a plunger assembly positioned in the processing chamber through the top orifice, wherein the plunger assembly comprises a plunger comprising a rotor, the rotor comprising a face facing the stator, wherein the face comprises grinding features; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the plunger assembly is operatively connected to a pressure sensor configured to measure pressure against the face of the rotor;(b) providing a tissue sample in the processing chamber;(c) depressing the rotor in the processing chamber until the pressure sensor measures a target pressure consistent with contact between the face of the rotor and the tissue sample;(d) rotating the rotor against the tissue sample while maintaining the target pressure, to dissociate cells and / or subcellular organelles from the tissue sample;(e) stopping depression of the rotor when the pressure sensor measures a pressure consistent with the face of the rotor contacting the stator.

16. A method of calibrating a plunger for cartridge comprising:(a) while depressing a plunger comprising a shaft attached to a rotor, within a chamber of a cartridge, measuring and recording force on the plunger at different positions along the Z axis of the plunger and, determining a calibrated fully depressed position consistent with the rotor contacting a floor of the chamber.

17. The method of claim 16, further comprising:(b) while depressing the plunger within a chamber of the cartridge, determining a calibrated tissue engagement position consistent with the rotor contacting tissue in the chamber.

18. The method of claim 16 or 17, further comprising:(c) performing a dissociation protocol on tissue in the chamber wherein the protocol comprises depressing the plunger to the determined fully depressed position and / or the determined contacting tissue position.

19. The method of claim 16 or 17, comprising using the calibrated fully depressed position and / or the determined contacting tissue position with a second, different cartridge.

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