Systems and methods for tissue analysis

Abstract

This document provides methods and systems for processing preserved and / or dissected tissue to release cells, nuclei, and / or other subcellular organelles. The instrument includes an interface connecting the cartridge via a fluid line for introducing or removing liquid into or from the cartridge's processing chamber. The system also includes a reagent subsystem configured to prevent liquid spillage and ensure a secure connection between the reagent container and the fluid line. The system also includes real-time feedback of the pressure exerted on the grinding rotor within the cartridge's processing chamber.

Description

[0001] Statement on Federally Funded Research none.

[0002] Citation of relevant applications This application claims the priority date of U.S. Provisional Application 63 / 556,364, filed February 21, 2024, the contents of which are incorporated herein by reference in their entirety.

[0003] background Isolating single cells, nuclei, and other subcellular organelles from fresh or fixed tissues is an area of ​​interest for life science researchers. Treatment of fixed tissues (e.g., FFPE tissues) may involve removing preservatives, such as by dissolving in an organic solvent, and rehydrating the released tissue. Treatment of fresh or rehydrated tissues may involve dissociating the tissue to disrupt the extracellular matrix, for example, in the presence of an enzyme solution that breaks chemical bonds in the extracellular matrix, or by 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, such as single-cell genomic analysis.

[0004] Methods for automating the separation of single cells, nuclei, and / or other subcellular organelles from fresh or preserved tissues have accelerated this process and made it more predictable. Brief description of the attached diagram The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate exemplary embodiments and, together with the specification, further serve to enable those skilled in the art to make and use these embodiments, as well as other embodiments that will be apparent to them. The invention will be described in more detail with reference to the following drawings, in which: Figure 1 A sample processing system is shown that processes samples into biological components such as single cells or cell nuclei for bioanalysis.

[0006] Figure 2 An overview of the tissue processing system and some exemplary modules are shown. Tissue samples or other samples are processed into single cells, cell nuclei, nucleic acids, single-cell libraries, and other biological products using one or more of a housing and one or more of a physical ionization system, an enzymatic and chemical ionization system, a measurement subsystem, a fluid subsystem, a control subsystem, and a magnetic module.

[0007] Figure 3 The overall design concept of the prototype is shown, along with examples of functional systems, several exemplary modes of mechanical fragmentation, and chemicals and enzymes used to dissociate FFPE tissue samples into single cells, nuclei, and other biomolecules.

[0008] Figure 4An example of a single-sample tissue processing system is shown, in which mechanical disruption is performed in a single chamber, and the instrument is equipped with enzymes and reagents to dissociate solid tissue samples into single cells, nuclei, and other biomolecules.

[0009] Figure 5 Another example of a single-sample tissue processing system is shown, in which mechanical disruption is performed in a single housing, and enzyme and reagent sets are set separately from the instrument in a reagent module.

[0010] Figure 6 A front view of an example single-sample tissue processing system is shown, which uses a housing to dissociate tissue samples into single-cell nucleus suspensions and other biomolecules.

[0011] Figure 7 A rear view of an example of a single-sample tissue processing system is shown.

[0012] Figure 8 A- Figure 8 C shows an example of a housing having a processing chamber, a post-processing chamber, and a vacuum trapping chamber for processing tissue samples into single-cell nuclei, single cells, and other biomolecules.

[0013] Figure 9 A- Figure 9 D illustrates an example of adding reagents to the container, mixing reagents, removing reagents, and mechanically breaking down tissue to process tissue samples into single cells, nuclei, and other biomolecules.

[0014] Figure 10 An exemplary computer system is shown.

[0015] Figure 11 The housing structure is shown, which uses a pinch valve to guide the flow of liquid.

[0016] Figure 12 An exemplary box-type fluid architecture is shown.

[0017] Figure 13 A shows a cross-sectional view of an exemplary box. Figure 13 B shows a housing containing a filter located in the post-processing chamber.

[0018] Figure 14 A top view of an exemplary box is shown.

[0019] Figure 15 An exemplary grinder component is shown.

[0020] Figure 16 A cross-sectional view of an exemplary housing is shown, in which the grinder assembly is positioned for insertion into the processing chamber.

[0021] Figure 17An exemplary housing is shown, in which the grinder assembly is inserted into the processing chamber.

[0022] Figure 18 A- Figure 18 B illustrates an exemplary housing having features designed to center the head of the rotor within the processing chamber and to set bottom and lateral clearances between the rotor and the processing chamber walls.

[0023] Figure 19 A- Figure 19 D shows a low-hardness port cap on the port, which is secured by a port cap retaining sleeve, or by crimping or heat fusion.

[0024] Figure 20 A- Figure 20 E illustrates the engagement of the cap with a rotary motor adapter and with a housing having a processing chamber, a post-processing chamber, and a vacuum trapping chamber for processing tissue samples into single cells, nuclei, and other organelles or biomolecules. Figure 20 A shows the actuator and plunger assembly before engagement. Figure 20 B and Figure 20 C shows a joint state with two different rotational orientations. Figure 20 D and Figure 20 E shows the plunger assembly in both partially depressed and fully depressed states.

[0025] Figure 21 An exemplary workflow is shown for dewaxing and rehydrating FFPE samples followed by nuclear separation.

[0026] Figure 22 An image of a box suitable for handling preserved tissue is shown.

[0027] Figure 23 A schematic diagram of a cassette suitable for processing preserved tissue is shown, including ports for connecting the cassette interface to instruments.

[0028] Figure 24 A cross-sectional view of a cassette suitable for handling preserved tissue is shown.

[0029] Figure 25 A cross-sectional view of a cassette suitable for handling preserved tissue is shown.

[0030] Figure 26 A top view of the box is shown.

[0031] Figure 27 An exemplary reagent module is shown, including a tray for holding reagent containers and waste containers.

[0032] Figure 28 A side view of an exemplary reagent module is shown.

[0033] Figure 29 An exemplary system is shown.

[0034] Figure 30 A and Figure 30 B shows a cross-sectional view of the processing chamber, including the plunger assembly (crusher head) translated along the Z-axis (1) and the impact stator (2).

[0035] Figure 31 A- Figure 31 C shows a mechanical assembly with a force sensor for monitoring the force on the rotor surface on the Z-axis.

[0036] Figure 32 A mechanical component for actuating the plunger assembly from another direction is shown.

[0037] Figure 33 The graph shows the force acting on the rotor as a function of the Z-axis position. The force increases slightly when the motor engages the plunger assembly, and reaches an inflection point where the force increases sharply when the rotor contacts the bottom of the processing chamber.

[0038] Figure 34 A schematic diagram of the mechanical components of an actuator for translating a plunger assembly along the Z-axis is shown.

[0039] Overview This article provides systems, kits, and methods for isolating cells, nuclei, and / or subcellular organelles from fresh, frozen, or preserved tissues.

[0040] Systems are provided that engage different cartridges adapted to process preserved tissue or for dissociating cells, nuclei, and / or other subcellular organelles from tissue. In some embodiments, both types of cartridges include a processing chamber in fluid communication with a post-processing chamber. The processing chamber of both types of cartridges is adapted to accommodate a plunger assembly including a rotor. For the preserved tissue cartridge, the rotor has a circumference such that, when the rotor is inserted into the processing chamber, the gap between the wall of the processing chamber and the rotor is small enough to prevent tissue fragments 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 wall of the processing chamber and the rotor is large enough to allow cells, nuclei, and / or other subcellular organelles to pass between the rotor and the chamber wall. More specifically, for tissue dissociation cassettes, some rotors may have peripheries that allow cells, nuclei, and other subcellular organelles to pass through, while other rotors may have peripheries that allow nuclei and / or other subcellular organelles to pass through but not intact cells.

[0041] Furthermore, in a cassette suitable for tissue dissociation, the processing chamber may include a stator comprising abrasive features, and the rotor may include a surface facing the stator, which also comprises abrasive features. Such abrasive features may not be present on the stator and rotor surfaces of the preserved tissue processing cassette.

[0042] A cassette for processing preserved tissue on one hand, and a cassette for releasing cells and / or subcellular organelles from the tissue on the other, has a geometry such that its respective features can engage the same element that the cassette can engage in the system. Thus, for example, after engagement, both cassettes can have the following elements for engaging ports in the instrument: (1) a plunger configured to enter the processing chamber through a top orifice and engage an actuator in the system, (2) a processing chamber port engaging a port in the cassette interface, (3) a post-processing chamber port engaging a port in the cassette interface, and (4) a vacuum port. Each of these elements can be positioned relatively in each of the preserved tissue processing cassette and the tissue dissociation cassette such that these ports engage the same port / actuator in the instrument. Furthermore, the cassette interface of the instrument can include alignment features to properly align the cassettes for engagement. Thus, both the preserved tissue processing cassette and the tissue dissociation cassette can include alignment, guiding, or registration features complementary to the alignment, guiding, or registration features in the same cassette interface, such that each cassette can be engaged. This could be, for example, a hole for a pin or vice versa, a groove for a guide rib or key or vice versa; or a dovetail joint.

[0043] The post-processing chambers of the two types of cartridges can be configured for different purposes. Both post-processing chambers can be connected to a vacuum source to draw liquid from the processing chamber into the post-processing chamber. In the tissue dissociation cartridge, the post-processing chamber can be configured to collect cells in the chamber without being drawn out of the cartridge by the vacuum source. In the preserved tissue processing cartridge, the post-processing chamber can be configured to draw liquid from the processing chamber into the post-processing chamber and then withdraw it. This can be achieved, for example, by including a port at the same location in the post-processing chamber. In the tissue dissociation cartridge, a vacuum chamber may also be present, communicating with the post-processing chamber and connected to a vacuum source in the instrument. In the preserved tissue processing cartridge, the port at the same location can be connected to the vacuum source and positioned in the post-processing cartridge as a drain port to collect liquid that moves into the post-processing cartridge when a vacuum is applied. In one embodiment, the port can be used as a drain port when the bottom plate of the post-processing cartridge is positioned at approximately the port liquid level.

[0044] A system for performing both preserved tissue processing and tissue dissociation may include the following features. For tissue dissociation, the instrument may include a housing interface for engaging a housing, the housing interface including a connector, such as a connector configured as a sleeve, positioned to engage a port in the housing that is in fluid communication with a processing chamber, a post-processing chamber, and / or a separate vacuum chamber. The system may also include a fluid subsystem comprising a reservoir configured to contain reagents (e.g., enzyme solutions) used for tissue dissociation. The system may also include one or more positive and / or negative pressure sources configured to move liquid from the fluid subsystem into and / or out of the housing. The system may also include a mechanical subsystem comprising an actuator configured to actuate a plunger assembly. Actuation may be either rotational (clockwise or counterclockwise) or linear, i.e., moving a rotor up and down within the processing chamber (i.e., along the Z-axis).

[0045] The system may also include a force sensor operatively connected to the rotor, configured to measure forces on the rotor in real time. Using feedback from the force sensor and a computer program configured to control the pressure on the rotor, the mechanical components can control the movement of the plunger assembly along the Z-axis. Thus, for example, when the force sensor senses a force on the rotor coinciding with the rotor impacting the stator, the software can instruct the mechanical components to stop the downward movement of the rotor. Similarly, when the force sensor senses a force on the rotor coinciding with the impacting of tissue in the processing chamber, the software can instruct the mechanical components to pause the downward movement and begin rotating the rotor. The system can store the position of the rotor along the Z-axis in a computer memory, the position corresponding to a force measurement coinciding with the rotor contacting the tissue sample and / or the chamber floor. Then, in subsequent grinding operations, whether in the same run in the currently used chamber or in a different chamber, the rotor can be pressed down to the relevant position stored in memory.

[0046] For processing preserved tissue, the instrument may include a reversibly engageable tissue processing subsystem comprising a holder for holding multiple containers and one or more elements selected from: (A) a leak-proof barrier configured to contain liquid within the holder, (B) multiple containers in the holder, including at least one container for containing reagent liquid and a waste container, (C) a scale configured to measure the weight of the waste container, (D) an optical sensor for sensing liquid at the bottom of the holder, and (E) multiple fluid conduits connecting the containers to ports via couplings located in a sensing mechanism for sensing whether a proper connection has been established. The instrument also includes fluid conduits connecting the couplings to ports in a housing interface, and one or more pressure sources for moving liquid in either direction between the containers in the holder and chambers in the housing.

[0047] Detailed description I. Introduction Figure 1 A sample processing system 50 is shown, which can take in a sample 101 and process it to produce biological products, such as single cells 1000 or cell nuclei 1050, microtissues 6001, organoids 6002, or other biological components consisting of subcellular components 1060, as well as biomolecules 1070, such as macromolecules 1071 and nucleic acids 1072 (containing DNA 1073 and RNA 1074); proteins 1075; carbohydrates 1076; lipids 1077; biomolecules 1070 comprising multiple types of macromolecules 1071; metabolites 1078; and other biological components, including natural products 1079 for bioanalysis.

[0048] Reference Figure 2 In many embodiments, the processing of the tissue processing system 110 is carried out in the housing 200 within the system. Tissue samples, such as fresh tissue 101, or preserved tissue samples, such as FFPE tissue sample 150 or OCT (optimal cutting temperature) tissue sample 160, or other samples 101, are converted into single-cell nuclei 1050, single cells 1000, or other organelles, or biomolecules, single-cell libraries 1200 or bulk libraries 1210, by using the housing 200 and one or more of the physical ionization system 300, the enzymatic and chemical ionization system 400, the measurement subsystem 500, the fluid subsystem 600, the control subsystem 700, the magnetic module 900, and the temperature subsystem 1475.

[0049] The physical deionization system 300 can mix or physically break down tissues in one or more of the following ways: passing a sample through an orifice, grinding, rotating a rotor with features for dissociating tissues, forcing tissues through a sieve or mesh, sonication, ultrasound, homogenization, bead milling, and other methods known to those skilled in the art for physically breaking down tissues to help generate single cells.

[0050] The enzymatic and chemical deionization system 400 can be dewaxed by adding an organic solvent (e.g., xylene or a xylene substitute) to the cartridge and rehydrated by adding a progressive mixture of ethanol and water or a buffer solution. The enzymatic and chemical deionization system 400 can be used for cross-linking reversal and / or enzymatic disruption by heating or adding reagent preparations or mixtures of components, including but not limited to proteinase K, collagenases (e.g., type I, II, III, IV, and other collagenases), elastase, trypsin, papain, hyaluronidase, chymotrypsin, neutral protease, clostridium protease, casein, neutral protease (Dispase®), DNase, type XIV protease, RNase inhibitors, or other enzymes, biochemicals or chemicals such as EDTA, protease inhibitors, buffer solutions, acids, or bases.

[0051] Another aspect of the enzymatic and chemical hydrolysis ionization system 400 is that it can be chemically broken down or chemically and enzymatically broken down by adding chemical agents that can disrupt the integrity of tissues or cells, such as Triton X-100, Tween, Nonident P40, octyl glucoside, polyoxyethylene (9) lauryl ether, digitalis saponins, IGEPA™ CA630 octylphenyl polyethylene glycol, n-octyl-β-D-glucopyranoside (betaOG), n-dodecyl-β, Tween™ 20 (polyethylene glycol sorbitan monolaurate), Tween™ 80 (polyethylene glycol sorbitan monooleate), poloxacillin, n-dodecyl-β-D-maltodextrin (DDM), NEMO nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol n-dodecyl monoether), hexaethylene glycol n-tetradecyl monoether (C14E06), octyl-β-thioglucopyranoside (octylthioglucoside, OTG), Emulgen, and polyoxyethylene (10) lauryl ether, other surfactants, or detergents or chemicals that can dissociate tissue into cells or produce cell nuclei or other organelles.

[0052] In other embodiments, different reagents or reagent mixtures are applied sequentially to dissociate FFPE or OCT samples or specimens into single cells 1000 or cell nuclei 1050. The physical dissociation system and the enzymatic / chemical dissociation system may be separate from each other or may be co-located (e.g., acting on the sample simultaneously or sequentially).

[0053] In some embodiments, the amount of dissociation is monitored at time intervals during dissociation or at the endpoint, and in some cases, the measurement subsystem 500 is used to determine activity during treatment. The measurement subsystem 500 may be an optical imaging device using brightfield, phase contrast, fluorescence, chemiluminescence, near-field, or other optical readouts, or an electrical measurement, such as impedance measurement of the change in conductivity as cells pass through a sensor, or other types of measurement.

[0054] The addition and movement of fluid can be performed by the fluid subsystem 600. The fluid subsystem 600 may use an injection pump, a piezoelectric pump, a pump and valve on a housing, a vacuum (negative pressure), a positive pressure, a pneumatic device, or other components known to those skilled in the art.

[0055] The tissue processing system 110 may be controlled by software in a control subsystem 700, which may include a user interface 740 implemented via a monitor, embedded display, or touchscreen 730 to communicate with and control devices, modules, subsystems, instruments, and systems. In some cases, the control subsystem 700 may include interfaces with smart devices, laboratory information management systems, other instruments, analytical software, display software, databases, email, and other applications. The control subsystem 700 may include control software 725 and scripts that control operations, and in some embodiments, an operator may modify, create, or edit the scripts.

[0056] This document also provides an apparatus for dissociating biological samples, the apparatus comprising: (i) a biological sample or specimen 101; (ii) a container 200 capable of dissociating tissue; (iii) instruments for operating the container 200 and providing fluid as needed; (iv) a measurement module 500, such as an optical imager for measuring titers, agglomeration, and / or activity; (v) a pump and container for exchanging the dissociated solution for a buffer or growth medium at the desired titer; and (vi) an output container, such as a chamber in the container, an 8-tube array, a microplate, an Eppendorf tube, a conical centrifuge tube, or other container capable of receiving a cell suspension.

[0057] This document, in another respect, provides an apparatus for dissociating biological samples and generating a single-cell 1000 or nucleus 1050 suspension or a matching batch of nucleic acids 1010 or a single-cell library 1200 or a matching batch of libraries 1210, the apparatus comprising: (i) a chamber or region for directly or incorporating a biological sample or specimen; (ii) a cassette capable of dissociating the tissue or specimen; (iii) an instrument for manipulating the cassette and providing fluid as needed; (iv) a measurement module, such as an optical imager for measuring titers, clumping and / or activity, necrosis, cytotoxicity, apoptosis, etc.; (v) a positive and / or negative pressure source for exchanging the dissociation solution for a buffer or growth medium at the desired titer; and (vi) an output container, such as an 8-tube array, a microplate, an Eppendorf tube, a chamber in the cassette, or other container capable of receiving the cell suspension.

[0058] Reference Figure 4The magnetic processing module 900 can use magnetic and paramagnetic particles or beads, or other surfaces of different sizes and shapes (referred to as beads), to separate single cells 1000, or cell types, or cell nuclei 1050, or other biological components composed of subcellular components 1060, as well as biomolecules 1070, such as macromolecules 1071 and nucleic acids 1072 (containing DNA 1073 and RNA 1074); proteins 1075; carbohydrates 1076; lipids 1077; biomolecules 1070 comprising multiple types of macromolecules 1071; metabolites 1078; and other biological components, including natural products 1079 for bioanalysis. In some embodiments, the beads have surface chemical properties that facilitate the purification of biological products under combined chemical conditions. In other embodiments, the beads have affinity molecules composed of antibodies, aptamers, biomolecules, etc., which specifically purify certain biological products, such as cell types, nucleic acids, cell nuclei 1050, or other components of tissues or samples.

[0059] The basic elements of the tissue processing system 110 can be configured in various ways according to sample 101, FFPE tissue sample 150, or OCT tissue sample 160, and the analytes to be analyzed. One of many configurations is described in detail in the following examples, but the invention is by no means limited to these configurations, as will be apparent to those skilled in the art. The tissue processing system 110 can accommodate many different types of samples 101, including fresh tissue; quick-frozen tissue; tissue sections (freezing, laser, or vibratory sections); fixed tissue; bulk material obtained by surgical excision, biopsy, or fine-needle aspiration; samples from surfaces and other matrices; or FFPE tissue sample 150.

[0060] This disclosure teaches how to produce a system that preferentially processes FFPE tissue samples 150 and OCT tissue samples 160, as well as other samples, into nuclei 1050 or single cells 1000. The process may need to be adapted to a wide range of starting types for FFPE tissue samples 150, with different requirements depending on the tissue, species, age, and state.

[0061] In this invention, numerous embodiments are possible, and the contents of PCT / US2017 / 063811 (Jovanovich, Chear, McIntosh, Pereira and Zaugg, "Method and Apparatus for processing Tissue Samples"), filed November 29, 2017, and PCT / US19 / 35097 (Jovanovich, Chear, Leisz, Eberhart and Bashkin, "Method and Apparatus for processing Tissue Samples"), filed June 1, 2019, are incorporated herein by reference in their entirety, along with the numbering system used therein, unless otherwise specified in this document.

[0062] This disclosure describes how a novel tissue preservation mechanism and a novel housing design automate, integrate, and, importantly, standardize the entire process of creating single-cell nuclei 1050 in a single-sample tissue processing 110 system embodiment. It will be apparent to those skilled in the art that multi-sample embodiments can be implemented using the same invention. The tissue processing system 110 will greatly empower basic researchers, students, translational researchers, clinicians, and others with its ease of use and high performance.

[0063] II. Box Design The box 200 can be used to process tissue into a single-cell suspension of 1000 cells or a nucleus of 1050, and is for single use only.

[0064] Reference Figure 3 The box 200 will input sample 101 (e.g., fresh tissue, FFPE tissue sample 150, or OCT tissue sample 160) and output single cells 1000 or cell nuclei 1050. For example... Figure 3 The tissue processing system 110, conceptually illustrated, combines mechanical fragmentation of sample 101 on a housing 200 with the addition of reagents such as chemicals, detergents, enzymatic or chemical dissolving solutions 410, and other fluids, and controls sample movement, pressure, and temperature. The tissue processing system 110 can move or rotate mechanical tissue fragmentation elements using a Z-axis stepper 2110 with a rotary motor 2120 connected via a cap 210. These mechanical tissue fragmentation elements include, but are not limited to, syringe plungers, pestles, Dounce pestles, or grinders.

[0065] The housing 200 can be designed using 3D printing, plastic injection molding (supporting single or double core-pulling structures with low assembly labor costs), layering of fluid layers with other functional layers, combinations of various methods, and other methods well known to those skilled in the art. Fluid can be delivered to the housing 200 via a pump (e.g., syringe pump 2130) or via vacuum, or pre-loaded onto the housing 200, or delivered via any combination of the foregoing. In some embodiments, a flexible tube 493 can connect to a chamber and form a simple clamp valve 491 to guide flow. In other embodiments, channels are formed in the housing 200, and valves, such as pneumatic valves or other valves, can be incorporated.

[0066] A. Tissue dissociation box Reference Figure 8 C. The term “plunger” or “plunger assembly” is sometimes used to refer to the combination of shaft / piston 216 and rotor 218, said rotor 218 having optional breaking features (e.g., teeth) 355, and spring 213 located in sleeve 212.

[0067] In one embodiment, the mechanical tissue-breaking element has a feature 355 at the bottom of the rotor or grinder, which can mechanically break up tissue at the bottom or base plate of the processing chamber 440. In some embodiments, the processing chamber 440 may have complementary features 355 to assist in tissue breaking. In some embodiments, the mechanical tissue-breaking element does not have feature 355 at the bottom of the rotor or grinder, but may be flat and mechanically break up tissue against a flat surface at the bottom or base plate of the processing chamber 440. In some embodiments, breaking also occurs in a “lateral gap” between the rotor and the sidewall of the processing chamber 440.

[0068] Figures 13-17 An exemplary box is shown to illustrate the contents of this disclosure. Figure 13 A cross-sectional view of an exemplary housing is shown. Housing 200 includes a processing chamber 440, which includes a stator containing teeth arranged in a circular array. The processing chamber also includes a first processing port through which a suspension of cells, nuclei, and organelles can be removed from the processing chamber. A post-processing chamber 460 and a vacuum chamber are also shown. The vacuum chamber 468 includes a vacuum port 467. Figure 15 and Figure 16An exemplary grinder assembly 345 of this disclosure is shown. The grinder assembly 345 includes a plunger comprising a piston 216 and a rotor 218 located at the end of the piston. The rotor includes grinding elements, such as teeth 355, on its bottom surface, including a central tooth 356 and an annular array of three toothed rings. The teeth 355 may have a blunt or sharp shape. Their cross-section may be trapezoidal. The outermost teeth 355 are located at the edge of the rotor. The grinder assembly 345 also includes a sleeve or sheath 212 surrounding the piston 216. The grinder assembly 345 also includes a cap 210 for positioning the plunger in a processing chamber. The cap 210 also includes a slot or other mechanism configured to engage an actuator to actuate the grinder. A spring, not shown here, biases the rotor 218 toward the cap 210 such that the actuator must apply positive pressure to the plunger to press the rotor 218 against the stator. The annular rings of the rotor teeth 355 and the annular rings of the stator teeth 355 are positioned to be complementary to each other, such that when the grinder is pressed against the stator, the stator rings mesh with the rotor rings (e.g., staggered arrangement). That is, in an exemplary embodiment, the teeth in the stator do not contact the teeth in the rotor. This configuration facilitates rotation of the rotor relative to the stator, ensuring that the teeth of one component do not collide with the teeth of another component. Those skilled in the art can determine the number of tooth rings in each of the rotor and stator. Factors influencing this determination include the total surface area of ​​the stator and rotor surfaces, and the size of the teeth. In some embodiments, the number of tooth rings in the stator and / or rotor 218 can be any of none, one, two, three, four, five, or six. In one embodiment, the teeth may have a trapezoidal cross-section. The processing chamber may have a cylindrical shape. The radius of the stator may be, for example, between 5 mm and 25 mm, such as about 12 mm. The volume of the processing chamber may be less than 1 ml, or between about 1 mL and 50 mL, such as between about 10 mL and about 30 mL, such as about 15 mL. The rotor and the sidewalls of the processing chamber can be configured such that a gap exists between the sidewalls of the processing chamber and the edge of the rotor when the plunger is inserted into the processing chamber. The size of the gap can be optimized to allow intact cells, nuclei, or organelles to pass between the sidewalls and the rotor. In some embodiments, the tooth height can be about 500 micrometers and the width can be about 1 mm to 2 mm.

[0069] The rotational speed for dissociation can be 10-200 rpm. The total number of revolutions of the grinding element can be 5-500. In one exemplary embodiment, the rotational speed is approximately 45 rpm (slow) or approximately 150 rpm (fast), rotating for approximately 4 seconds, pausing for approximately 1-2 seconds, then rotating again for approximately 4 seconds, and then repeating (total rotation time approximately 16 seconds). In each vertical displacement step of the stepper motor, the rotation proceeds sequentially towards the bottom of the housing, for a total of approximately 9 vertical displacements, and in the bottommost step, the rotation cycle is repeated approximately 3 times instead of 2 times.

[0070] B. Preserved tissue processing cassette The disposable container 200 is expected to be able to process various types of preserved FFPE 150 or OCT 160 tissues via mechanical fragmentation and enzymatic or chemical dissociation, which can be tailored to the tissue type and condition of the FFPE tissue (e.g., preservation time or chemical treatment process). The container 200 can be designed to process tissues as quickly and gently as possible, without exposing the operator to the tissue being processed, and can be manufactured at low cost. A variety of mechanical methods may be required to accommodate a wide range of tissue types and their respective requirements: the design shown can be readily adapted to a variety of different mechanical fragmentation methods, including, but not limited to, variable orifice 490, grinding using a rotating plunger 336, pestle 361, and filtration and sieving using a plunger 362, as well as other mechanical methods.

[0071] Figures 22-26 An exemplary embodiment of a cartridge for processing preserved tissue is shown. This embodiment shares many similar elements with the tissue dissociation cartridge described above, and has modifications suitable for processing preserved tissue.

[0072] The housing 2500 includes a processing chamber 2540 and a post-processing chamber 2560. The processing chamber 2540 includes a first processing chamber port 2504, which includes a connecting pipe 2571 that is fluidly connected to the post-processing chamber 2560 via a fluid conduit 2553. The processing chamber 2540 also includes a second processing chamber port 2570 for introducing liquid into the processing chamber. The first processing chamber port 2504 and the second processing chamber port 2570 can be positioned at the same location as ports 1604 and 470 of the exemplary tissue dissociation housing to mate with the same ports in the instrument's housing interface.

[0073] The processing chamber 2540 may have a plunger assembly 2510 inserted therein. The plunger assembly 2510 may differ from the plunger assembly used in a tissue dissociation cassette. More specifically, the rotor in the tissue dissociation cassette has a periphery that provides 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 at the top of the rotor when it is fully depressed. In a cassette configured for processing preserved tissue, this space is selected to be small enough that used fluid can pass through, but tissue debris or (in some embodiments) released cells, nuclei, and / or subcellular organelles (depending on the material to be collected) cannot pass through. In some embodiments, this gap may not exceed any one of 200 μm, 100 μm, 50 μm, and 25 μm.

[0074] In a cassette used for processing preserved tissue, the rotor face and the stator of the processing chamber may or may not include abrasive features.

[0075] The first processing chamber port 2504 may be located on a side wall of the housing such that, when the rotor of the plunger assembly 2510 is fully depressed, at least a portion of the port is positioned to discharge liquid collected above the top of the rotor. In some embodiments, a portion of the port is below the top of the fully depressed rotor. In other embodiments, the entire port is located above the top of the rotor. In any case, the port may be positioned such that the intended volume of liquid pushed above the top of the rotor contacts and can be removed through the port.

[0076] The post-processing chamber 2560 includes an inlet 2554. The inlet 2554 may be located in a cover or cap 2562 that covers the orifice 2566. The inlet 2554 may be fluidly connected to the first processing chamber via a fluid conduit 2553 and a port 2504. A first post-processing port 2585 may be placed in the same location as the first post-processing port 485 of the exemplary tissue dissociation cassette described herein.

[0077] The post-processing chamber 2560 may also include a base plate 2580 positioned such that liquid collected in the post-processing chamber can be drained to a first post-processing port 2585. In some embodiments, the base plate 2580 may be inclined such that liquid collects at the first post-processing port 2585.

[0078] In an alternative embodiment, the fluid conduit 2553 may be directly connected to a port in the housing that can be connected to a pressure source in the instrument, such as a first post-processing port 2585, or a port located corresponding to the vacuum port 467.

[0079] During operation, both the tissue dissociation cassette and the preserved tissue processing cassette can have ports positioned to engage with the same cassette interface connector in the instrument. Thus, for example, in the preserved tissue processing step, processing reagents can be introduced into processing chamber 2540 through the second processing chamber port 2570. After the rotor is fully depressed in the processing chamber, used reagents are collected above the rotor and can be drawn into the post-processing chamber via fluid line 2553 by a vacuum applied at the first post-processing port 2585. From here, the used reagents can be extracted from the post-processing chamber.

[0080] Similarly, during tissue dissociation, dissociation reagents can be introduced into processing chamber 440 through a second processing chamber port 470, which, like port 2570, is positioned to engage with the same connector in the cassette interface. After tissue dissociation, the rotor is depressurized, and liquid containing the released cells, nuclei, and / or other subcellular organelles collects above the top of the rotor. Because the first processing chamber port 1604 is positioned in fluid communication with the area above the top of the fully depressurized rotor, cells, nuclei, or other subcellular organelles can be drawn into the post-processing chamber through fluid line 453. Aspiration can be applied from the vacuum port 467, configured to apply a vacuum to the post-processing chamber, in contrast to the use of a preserved tissue processing cassette. However, because the vacuum port 467 is positioned significantly above the bottom plate of the post-processing chamber and is separated by a wall that extends almost to the top of the chamber, liquid containing cells, nuclei, and / or other subcellular organelles is not drawn through the vacuum port 467.

[0081] III. Organization Processing System In one embodiment of the sample processing system 50 as the tissue processing system 110, such as Figure 1 As shown, the tissue processing system 110 can provide scientists with powerful integrated "tissue-to-genomics" or "sample-to-other-analysis-results" (genomics, proteomics, metabolomics or epigenetics, multi-omics, etc.) analysis functions to simplify and standardize the production and / or analysis of single-cell 1000 or cell nucleus 1050 suspensions, affinity-purified single-cell 1100, affinity-purified cell nuclei 1105, nucleic acids 1072, and batch libraries 1210 from solid or liquid tissues. It will be apparent to those skilled in the art that the resulting biological materials, such as single cells 1000, cell nuclei 1050, nucleic acids 1072, single-cell libraries 1200, single-cell nucleus libraries 1250, bulk libraries 1210, or other biological components consisting of subcellular components 1060, or biomolecules 1070 such as macromolecules 1071 and nucleic acids 1072 (containing DNA 1073 and RNA 1074), can also be used in many genomics, cell biology, proteomics, metabolomics and other analytical methods.

[0082] The tissue processing system 110 integrates the preparation and measurement subsystem 500 of biological material from preserved tissue samples (e.g., FFPE tissue sample 150 or OCT tissue sample 160), wherein the measurement subsystem 500 performs analyses selected from one or more of the following: DNA or RNA sequencing, next-generation DNA or RNA sequencing, next-generation DNA or RNA sequencing of nucleic acids and their adducts (e.g., epigenetic modifications); nanopore sequencing of nucleic acids and their adducts; single-cell DNA sequencing of nucleic acids and their adducts; single-cell nuclear 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; fluorescence, Raman, optical, mass spectrometry, and other detection methods for nucleic acids and their adducts, with or without labels; proteomics analysis, including fluorescence, Raman, optical, mass spectrometry, protein sequencing, and other detection methods for proteins and peptides and their adducts and modifications, with or without labels. Labeled; Characterization and spectral analysis of carbohydrates, including sequencing, fluorescence, Raman, optical, mass spectrometry and other detection methods for carbohydrates and their adducts and other covalent polymers, with or without labels; Characterization and spectral analysis of lipids, including sequencing, fluorescence, Raman, optical, mass spectrometry and other detection methods for lipids and their adducts and other covalent polymers, with or without labels; Flow cytometry; Characterization and spectral analysis of cells, including fluorescence, Raman, optical, mass spectrometry and other detection methods for cells and their adducts and other covalent polymers, with or without labels; Metabolic spectral analysis, including sequencing, fluorescence, Raman, optical, mass spectrometry and other detection methods for metabolites and their adducts and other covalent polymers, with or without labels; Functional analysis, including protein-protein interactions, protein-lipid interactions, protein-DNA interactions, RNA-DNA interactions, and other interactions of molecules derived from biological materials, with or without labels; Bioinformatics analysis of cells, organelles and biomolecules; and mass spectrometry and other analytical methods. In some embodiments, the measurement system 500 may be physically integrated, and fluid may be transferred via automated pipetting, fluid flow through tubes or capillaries, centrifugation, or other methods.

[0083] Reference Figure 4In this embodiment, mechanical and enzymatic dissociation are performed in one or more processing chambers 440 of the disposable cartridge 200 to produce a nuclear suspension 1050, a single-cell suspension 1000, or nucleic acids 1072, biomolecules 1070, subcellular components 1060, or other products. The sample can then be processed in one or more post-processing chambers 460 by optional bead-based affinity purification, affinity purification of cell types via surface antigens to produce affinity-purified single-cell suspensions 1000, or affinity purification of nuclear suspensions 1050 via nuclear antigens. Alternatively, nucleic acids 1072, biomolecules 1070, and subcellular components 1060 can be further processed into purified mRNA, NGS libraries, or other sample types. In some embodiments, processing chambers 440 and post-processing chambers 460, as well as filtration chambers 450 and vacuum trapping chambers 468 and waste chambers 430, or other chambers, can be combined.

[0084] IV. Instruments and Systems A. Instruments The tissue processing system 110 can mechanically break down tissue within the housing 200, perform enzymatic hydrolysis and dissociation, and reverse the cross-linking of the broken tissue to produce single cells 1000 or cell nuclei 1050. For example... Figure 4 As shown, the single-sample tissue processing system 2010 combines a physical ionization system 300 and an enzymatic and chemical ionization system 400 to produce a suspension of single cells 1000 or cell nuclei 1050. The instrument provides mechanical motion and fluid to the housing, which then mechanically and enzymatically or chemically processes the FFPE tissue sample 150 into single cells 1000 or cell nuclei 1050. Various reagents 411 can be stored on the instrument or in the reagent module 1430 and are provided with cooling as needed.

[0085] Figure 4 A 3D CAD representation of one embodiment of a 2010 design for single-sample tissue processing encapsulated with "skin" is shown, and Figure 6 and Figure 7 Another embodiment is shown. Both embodiments feature a dual-axis mechanical motion mechanism (Z-axis stepper 2110 and rotary motor 2120) integrated with a syringe pump-based fluid system, for example, with a six-way valve (C2400MP, TriContinent) controlled by control software 725, with a resolution of 1.6 μL.

[0086] Reference Figure 4A computer 720 with an operating system (such as Windows 10 and an 85 GB hard drive (Beelink, AP42)) can run control software 725 to control the system, displayed on a 10-inch touchscreen 730 (eleduino, Raspberry Pi 10) or a tablet 750 (such as a Windows Surface Pro 6). A rack 1010 provides a frame for mounting components and the system enclosure.

[0087] B. Fluid Subsystem Figure 4 An embodiment of the single-sample tissue processing system 2010 shown has a fluid subsystem 600 with a single syringe pump 2130 and a single six-way valve 2140 to supply liquid, pressure, or vacuum from reagent block 415 to cartridge 200. In one embodiment, cartridge 200 has two processing chambers 440 and a single post-processing chamber 460. In one embodiment, a magnetic processing module 900 can apply magnetic force to cartridge 200 under software control, enabling the purification and analysis of single cells 1000, cell nuclei 1050, nucleic acids 1072, biomolecules 1070, subcellular components 1060, or other products using paramagnetic beads, paramagnetic surfaces, paramagnetic nanoparticles, and other magnetic or paramagnetic particles.

[0088] Figure 5 An embodiment of a single-sample tissue processing system 2010 with a housing is shown. This embodiment includes a reagent module 1430, which is detachable from the single-sample tissue processing instrument 2010, such as... Figure 4 As shown, power and control are provided by the single-sample tissue processing instrument 2010, or a separate power supply and processor can be used, or as... Figure 4 As shown, the reagent module 1430 can be integrated inside a single instrument housing.

[0089] Reference Figure 27 and Figure 28 The system may include reagent blocks or reagent subsystems configured to process reagents in the treatment of preserved tissue. Such reagents typically include organic solvents, such as xylene, for removing preservatives (e.g., paraffin) from preserved tissue, and alcohols in aqueous solutions for rehydrating the tissue after preservative removal. The reagent block may include a holder configured to hold one or more containers, including reagent containers and waste containers. The holder may include a leak-proof barrier to contain any liquid that leaks into the holder. The holder may also include an optical sensor for sensing the presence of liquid at the bottom of the holder.

[0090] Certain reagents used to remove preservatives from preserved tissue can be considered hazardous materials. Therefore, tracking and controlling these materials can benefit the user. In some embodiments, the rack includes a scale on which a waste container rests. The scale measures the weight of the waste container. Software in the system receives such measurements and can determine when the waste container is about to fill. In this case, the system can be configured to warn the user and / or stop the transfer of liquid into the waste container.

[0091] Fluid conduits from reagent containers in waste containers and / or racks can be connected to couplings in the instrument's fluid subsystem. Couplings may include sensors, such as those using the Hall effect, to confirm that the connector is correctly positioned within the coupling.

[0092] C. Force sensor Reference Figure 6 In one embodiment, the single-sample tissue processing instrument 1020 has a linear drive motor, such as a Z-axis stepper motor 2110, which may optionally be equipped with an encoder to control the vertical position of a rotary motor 2120 mounted on a Z-axis stepper slider 2111, which is attached to an inverted "U"-shaped structural frame 1020 mounted on a frame 1010.

[0093] A force sensor may be incorporated into the Z-axis stepper 2110 to provide force feedback control of the mechanical forces acting on the sample 101 or the cantilevered cassette slider 1450; this helps ensure very gentle mechanical handling steps and prevents the rotor 218 from applying high forces to the bottom of the processing chamber 440. An injection pump 2130 is fluidly connected to six-way valves 2141 and 2140 via tubing, capillary, microchip, or other fluid connectors to supply reagents, pressure, or vacuum from the reagent module 1430 to the cassette 200 (not shown).

[0094] Reference Figures 30-34 In some embodiments, the force feedback mechanism can be configured to determine the position of the rotor face within the processing chamber. More specifically, when the actuator engages the plunger assembly, the force on the rotor increases slightly ( Figure 33 The first inflection point in the process). Furthermore, when the rotor encounters the stator or base plate of the processing chamber, the force on the rotor increases significantly ( Figure 33 (The steep inflection point in the process). When the force sensor measures a force consistent with the rotor encountering tissue, the operating software can initiate a grinding program. The grinding program may include a combination of rotating the rotor in one or both clockwise or counterclockwise directions, and further depressing the rotor when the force measurement indicates to the operating software that more forceful grinding is needed. A force measurement consistent with the rotor contacting the bottom of the chamber indicates to the operating software that no further grinding is needed, and the fluid collected above the top of the rotor is ready to be moved into the processing chamber.

[0095] Force sensors can be used to calibrate the plunger for operation in the same or different housings. In the first housing, the rotor position and Z-axis position coinciding with tissue contact or a contact state can be stored as calibrated positions in memory. In the same housing, during tissue dissociation or preserved tissue processing, the plunger can be pressed down to two Z-axis positions coinciding with tissue contact or stator contact in memory. Calibration can be performed on each housing that engages with the housing interface. Alternatively, a single calibration run can be performed at the calibrated positions in multiple different housings for engagement with the interface.

[0096] D. Box Interface The housing 200 is placed in the housing receiving tray 1510 on the housing slider 1450, which is designed to precisely hold the housing 200 so that when the housing 200 is moved along the housing slide rail 1480 in the housing receiving tray 1510 on the housing slider 1450 until the spring-loaded housing slider knob 1452 locks into the hole in the housing slider 1450, the center of the processing chamber 440 is concentric with the center of the rotary motor shaft 2121 of the rotary motor 2120 by a distance of 1, or 5, or 10, or 15, or 20, or 25, or 50, or 100, or 250 μm or less, wherein the housing 200 is held near or in contact with the heat conduction plate 1470 and establishes a fluid connection with the spring probe 1415 of the housing interface 1510.

[0097] E. Temperature sensor The temperature control subsystem 1475 can set the heat transfer plate 1470 to a given temperature via the housing Peltier device 1440 or other temperature control devices (e.g., strip resistance heater, circulating fluid, etc.) to set the housing temperature in the processing chamber 440 and the post-processing chamber 460 under the control of the plate 2250. In some embodiments, the temperatures of the processing chamber 440 and the post-processing chamber 460 can be set independently. In some embodiments, the temperature control system can use thermocouples, thermistors, or IR cameras to set the temperature of the heat transfer plate 1470 or the exterior of the housing 200.

[0098] In one embodiment, a fluid port on housing 200 abuts a spring-loaded probe 1415 upon housing insertion to establish a fluid, gas, or vacuum connection with housing 200. In another embodiment, the spring probe 1415 or sleeve 1416 is moved to connect with housing 200 after housing insertion. In yet another embodiment, sleeve 1416, connected to a fluid line from syringe pump 2130, is rigidly attached to heat transfer plate 1470 or other parts of the instrument, and housing 200 has a flexible material at the housing port that seals against sleeve 1416 after housing insertion, as described below. The housing port is an outwardly opening into the housing. The housing port may communicate directly with a chamber as a port within a chamber, or indirectly, for example, through another chamber containing the port and communicating with said chamber.

[0099] F. Magnetic Processing Module Figure 6 An embodiment of the single-sample tissue processing system 2010 shown has a magnetic processing module 900, and a magnet 910 is moved by a magnetic actuator 935 mounted on an inverted "U"-shaped structural frame 1020 under the control of control software 725 using a controller 2122. Figure 9 As shown, magnet 910 can be located away from housing 200 and does not interact with any magnetic beads 685 in housing 200, or in an extended position, magnet 910 can be moved close to housing 200 to magnetically capture and process magnetic beads 685. Numerous embodiments of the geometric configuration of the magnetic processing module 900 and magnet 910 relative to housing 200 are possible.

[0100] Reference Figure 7 In one embodiment, the single-sample Singulator system 2000 has a rear structural frame 1021 on a structural frame 1020, which houses electronics 710 including a rotary motor controller 2122, a Z-axis stepper controller 2112, a 24V to 5V step-down power supply 2230, and a 24V to 12V step-down power supply 2225. The single-sample tissue processing system 2010 can be powered by plugging a 24V power supply into a connector 762 connected to a fuse 761 and a power switch 760. Six-way valves 2140 and 2141 are controlled by boards 2210 and 2212. A reagent Peltier relay board 2240 controls the reagent Peltier device.

[0101] Systems that process one or more cassettes simultaneously are within the scope of this invention. Cassette 200 may have one or more processing chambers 440 and zero, one or more post-processing chambers 460, as well as zero, one or more other chambers, such as cassette waste chamber 435 or vacuum collection chamber 468.

[0102] exist Figure 8In one embodiment shown, after sample 101 is added to the processing chamber 440 of cartridge 200, cap 210 (also referred to as a tissue disruptor) is placed on top of processing chamber 440. After the instrument is inserted into cartridge 200, spring probe 1415, sleeve 1416, or other fluid connectors can be connected to zero, one, or more cartridge ports 470 to supply reagents to processing chamber 440, to a first post-processing port 485 to supply reagents or vacuum to post-processing chamber 460, to a cartridge vacuum trapping port 467 to supply vacuum to vacuum trapping chamber 468, or to a cartridge waste port 2355 to supply vacuum or reagents to cartridge waste line 2351.

[0103] Figure 8 The embodiment of the housing 200 shown for processing tissue sample 150 uses a fluid line 453 to fluidly connect the processing chamber 440 to the post-processing chamber 460. This fluid line may be a tube connecting from the processing chamber nozzle 471 to a port 452 (connected via nozzle 454) positioned above a filter 2711 inserted into the post-processing chamber 460. In other embodiments, a filter may not be used, or the filter 2711 may be incorporated as an in-line filter, for example, in a Swinny filter holder attached to the output of the processing chamber 440, or in the fluid line 453, or attached to the cap 462. In one embodiment, dual, triple, or more filters are used in the filter 2711, such as a 145-micron filter followed by a 40-micron filter, and then a 20-micron filter; other combinations are also contemplated.

[0104] When cap 465 is sealed onto lid 462, lid 462 creates a vacuum seal for post-processing chamber 460 and vacuum trapping chamber 468. Lid 462 can be attached to the housing body 201 by ultrasonic welding, glue, epoxy resin, adhesives, or other methods to create a vacuum seal. The permanent attachment of lid 462 ensures the single-use nature of housing 200 by preventing filter 2711 replacement, thus eliminating sample cross-contamination.

[0105] In some embodiments, housing 200 may have an on-housing valve, which may be a clamp valve on a fluid line (e.g., fluid line 453), actuated by the instrument to open and close the line, or to direct fluid along different paths, such as to waste on the housing or to the optical imaging system 520, or to multi-omics processing of another workflow or analytical method, by using a "T" joint and two lines. In another embodiment, the fluid line (e.g., fluid line 453) may be partially closed to create a variable orifice that can break up partially dissociated tissue. An actuator may open and clamp the tube in housing 200 as needed, or the variable orifice may be operated using a variable orifice device. In other embodiments, housing 200 may have an on-housing valve, which may be a miniaturized pneumatic valve or a micro-valve. In some embodiments, microfluidics or microchips are used for the fluid lines. In one embodiment, there is no valve on housing 200, and all fluid control is from the instrument.

[0106] Reference Figure 8 A, Figure 13 and Figure 14 When a vacuum is applied to the vacuum trapping port 467 or the first post-processing port 485, a liquid containing a single-cell suspension 1000, cell nuclei 1050 and other subcellular components 1060, and biomolecules 1070 is drawn from the processing chamber 440 through the fluid line 453 and filter 2711 into the filter drain port 451 and into the output collection area 461 of the post-processing chamber 460. The pore size of the filter 2711 may be no larger than any of 2, 5, 10, 15, 20, 25, 30, 40, 50, 70, 100, 125, 200 μm or larger to filter the biological material suspension. Multiple in-line or stacked filters 2711 may be used to sequentially remove different sized components of the dissociated tissue sample 110. A cap 210 with a cap connector 211 and a head 218 (also referred to as a “rotor” 253) is shown ready to be inserted into the sample inlet port 425. The head 218 may have a surface for breaking up tissue, and may include protrusions 355 that facilitate mechanical breaking up tissue, organs, micro-tissues 6001, organoids 6002 or other biological materials.

[0107] Reference Figure 8 B and Figure 8 C. The cap connector (also referred to as the "drive head") 211 is held inside the cap sheath 212, which in one embodiment has a cap sheath bore 214. The cap connector 211 is attached to a cap shaft 216, which passes through the cap sheath bore 214 and is attached to a head 218, which may be a rotor 353 with grinding teeth 355. The assembly to which the cap connector 211 is attached to the cap shaft 216 and the head 218 is referred to as a plunger assembly, which is a movable mechanical tissue disruptor 345.

[0108] Reference Figure 18 A. In a preferred embodiment, the head 218 attached to the cap shaft 216 has an outwardly annular bevel head feature 356, which is designed to improve the concentricity of the head 218 within the processing chamber 440, thereby improving the uniformity of the lateral clearance 221 at the bottom of the stroke. As the Z-axis stepper motor 2110 descends and the cap connector 211 is pushed downwards by the rotary motor connector 2125, the head 218 descends until the outwardly annular bevel feature 356 engages with the inwardly annular bevel pre-processing chamber feature 357 on the inner wall of the processing chamber 440, to produce, as Figure 18The centered head 218 is shown in Figure B. The centering of the head 218 will create a uniform lateral gap 221 between the head 218 and the inner wall of the processing chamber 440. Furthermore, if the height of the head 211 is less than the height of the processing chamber 440 below the inward annular slope feature 357, the engagement of the head feature 356 of the outward annular slope and the pre-processing chamber feature 357 of the inward annular slope will set a uniform bottom gap 222. The size of the lateral and bottom gaps can be optimized for different cell types or for different sizes of nuclei or subcellular organelles, or for multicellular structures (e.g., intestinal crypts). Additionally, to allow fragmented tissue to pass through when the head 218 is seated on the pre-processing chamber feature 357 of the inward annular slope, the pre-processing chamber feature 357 of the inward annular slope may have grooves with segments of the same or different depths. The lateral clearance 221 between the head 218 of the movable mechanical crusher 345 and the inner wall is preferably greater than or equal to any one of 1 μm, or 2 μm, or 5 μm, or 10 μm, or 15 μm, or 20 μm, or 25 μm, or 30 μm, or 40 μm, or 50 μm, or 75 μm, or 100 μm, or 150 μm, or 200 μm, or 250 μm, or 500 μm, and 1000 μm or greater, and any size in between. In a preferred embodiment, for the cell nucleus and other subcellular organelles, the lateral clearance 221 is greater than 50 μm and less than 150 μm, and for cells, the lateral clearance 221 is greater than 75 μm and less than 250 μm. The clearance size for separating the cell nucleus can be, for example, between about 30 μm and about 200 μm, for example, between about 40 μm and about 150 μm, or between about 100 µm and about 125 µm. The size of the gap used for cell separation can be, for example, between about 50 µm and about 400 µm, for example, between about 200 µm and about 300 µm, or about 250 µm. The bottom gap 222 between the bottom of the head 218 of the movable mechanical crusher 345 and the bottom of the processing chamber 440 is preferably greater than or equal to any one of 1 µm, or 2 µm, or 5 µm, or 10 µm, or 15 µm, or 20 µm, or 25 µm, or 30 µm, or 40 µm, or 50 µm, or 75 µm, or 100 µm, or 150 µm, or 200 µm, or 250 µm, or 500 µm, and 1000 µm or greater, and any size in between. In some embodiments, different heads can be selected for use with processing chambers 440 of the same diameter to produce different lateral gaps 221 or bottom gaps 222, thereby simplifying manufacturing and inventory management requirements. The bottom clearance between the flat surface of the head and the flat bottom surface of the processing chamber can also be defined by the location of a groove, a hemispherical dome, or other structures that prevent the clearance from forming or define the clearance between the flat surface of the head and the flat bottom surface of the processing chamber.

[0109] Reference Figure 19One or more ports leading to the housing 200 may have flexible or low-hardness port covers 442, for example, but not limited to, hardness of 40 to 100. Figure 19 A and Figure 19 As shown in cross-sectional view B, the port cap 442 can be inserted into the space between the port and the port cap retaining sleeve 441 to secure the port cap 442 in place, for example, to cover the reagent addition port 470 as shown. A fluid sleeve 1416 or a fluid spring probe 1415 with an outer diameter larger than the center hole 446 of the port cap can engage the port covered by the port cap 442, and due to its relatively low rigidity, the port cap 442 will be deformed by the fluid sleeve 1416 or the fluid spring probe 1415 to create a seal around the fluid sleeve 1416 or the fluid spring probe 1415. In some configurations, deformation can be used to eliminate the need for a spring, and the use of the fluid spring probe 1415 can be replaced by a non-removable fluid sleeve 1416. Figure 19 C shows the port cover 442 held in place by a press-fit seal 443. Figure 19 D shows a port cover 442 held in place by forming a port cover retaining cylinder 441 above the port cover 442 and melting the port cover retaining cylinder 441 to form a heat-fused flange 444 that holds the port cover 442.

[0110] In a preferred embodiment, the single-sample Singulator instrument 2050 has an actuator for mechanical processing, comprising a stepper motor 2110 that controls the vertical position of a rotary motor 2120, and a rotary motor shaft 2121 attached to a rotary motor connector 2125, which is mechanically coupled to a cap connector 211 of a cap 210 when inserted into the housing 200. The connector may have a drive head, which may take any suitable form, such as a slotted, cross-shaped, square, three-winged, pentagonal, or hexagonal head. The rotary motor connector 2125 has one or more faces that reversibly engage the cap connector 211 by actions such as downward movement and slow rotation. Figure 20As shown in Figure A, in a preferred embodiment, the rotary motor connector 2125 has a single blade to engage the cap connector 211. When the stepper motor 2110 descends, the rotary motor connector 2125, attached to the rotary motor shaft 2121, engages the cap connector 211 in the cap 210, and if the rotary motor connector 2125 is not aligned with the cap connector recess 217, the rotary motor connector 2125 cannot be directly inserted into the cap connector recess 217. In a preferred embodiment, the cap connector 211 has two surfaces on both sides of the cap connector recess 217, which are inclined in opposite directions on the cap connector 211, such that each side has a higher wall and a lower wall on both sides of the cap connector recess 217. When the rotary motor shaft 2121 rotates clockwise (viewed from above), the blade of the rotary motor connector 2125 rotates clockwise and encounters the higher side of the wall of the cap connector recess 217 and begins to rotate the cap connector 211 clockwise. When the stepper motor 2110 descends, the rotary motor connector 2125 will engage the cap connector groove 217, as... Figure 20 As shown in C. Figure 20 As shown in Figure D, as the stepper motor 2110 continues to descend, the rotary motor 2120 and the rotary motor shaft 2121 attached to the rotary motor connector 2125 will descend, pushing the cap connector groove 217, and the cap connector 211 will compress the cap spring 213 against the bottom of the cap sleeve 212 and lower the head 218. Figure 20 As shown in E, the head 218 can be lowered to near or in contact with the bottom of the processing chamber 440 (which may be the stator 354), and the head 218 can rotate to break up tissue. As the stepper motor 2110 rises, the rotary motor 2120 and the rotary motor connector 2125 rise, and the cap spring 213 decompresses to push the cap connector 211 against the rotary motor connector 2125 to continue engagement.

[0111] In another embodiment of the single-sample Singulator instrument 2050, a stepper motor 2110 controls the vertical position of a rotary motor 2120, which is magnetically coupled to the movable crusher 345 via a magnetic or paramagnetic element embedded in the cap 210 as part of the cap connector 211 or as part of the movable crusher 345 or the head 218.

[0112] When the rotary motor connector 2125 is engaged with the cap connector 211 via a mechanical, magnetic, pneumatic, or fluid connection or other connection method, and the rotary motor 2120 rotates, the movable crusher 325 and the head 218 are rotated. The stepper motor 2110 controls the vertical position of the rotary motor 2120, thereby controlling the vertical position of the rotary motor connector 2125 to raise or lower the movable crusher 345 and the head 218 within the processing chamber 440. The combination of the rotation of the rotary motor 2120 and the movement of the stepper motor 2110 enables the movable tissue crusher 345 and the head 218 to achieve multiple motion modes.

[0113] The inner wall of the processing chamber 440 can take on a variety of different shapes. The inner wall of the processing chamber 440 may have grooves in sections of varying depths. In a preferred embodiment, the inner wall may have a circular profile, and the maximum gap between the head 218 of the movable mechanical tissue disruptor 345 and the inner wall is preferably greater than or equal to any one of 1 μm, or 2 μm, or 5 μm, or 10 μm, or 15 μm, or 20 μm, or 25 μm, or 30 μm, or 40 μm, or 50 μm, or 75 μm, or 100 μm, or 150 μm, or 200 μm, or 250 μm, or 500 μm, and 1000 μm or greater, and any size in between.

[0114] The movable tissue disruptor 345 can take on a variety of different shapes and profiles. In one embodiment, the movable tissue disruptor 345 may have a head 218, which is a rotor 353, with optional features such as grinding teeth 355 at the bottom of the rotor 353 and grinding teeth 355 on a stator 354 located on the top surface of the bottom of the processing chamber 440, to assist in breaking large tissue samples 120 into smaller pieces or to assist in dissociation into single cells 1000, cell nuclei 1050, or biomolecules 1070. Figure 8 and Figure 18As shown, the sides of the head 218 may be cylindrical to create an inner gap 221 with the inner wall along the length of the cylinder. By raising and lowering the head 218 without rotating it, thus using it as a movable tissue disruptor 345, the system can process the sample 101 by grinding. In another embodiment, the sides of the head 218 may be formed into a spherical structure to create a gap with the inner wall in a small area, and the bottom of the processing chamber 440 may be rounded to match the spherical structure, thereby creating a Dounce-like mechanical tissue disruptor 345. In other embodiments, multiple areas with the same or different sized gaps can be created by changing the side profile of the movable tissue disruptor 345 and the inner wall of the processing chamber 440. In other embodiments, the stator may be stationary or movable (e.g., rotating). In other embodiments, the stator is non-porous. In other embodiments, either or both of the processing chamber or the rotor may have a circular or non-circular cross-section. In the case of a non-circular cross-section, the rotor is configured to rotate within the processing chamber. During its rotation, a portion of the rotor wall will have a gap between the rotor and the processing chamber, which is between approximately 150 micrometers and 250 micrometers.

[0115] V. Usage Method The method for isolating dissociated single cells, nuclei, or other subcellular organelles from preserved tissue can be performed as follows. The first part of the process involves treating the preserved tissue sample to remove the preservative and typically rehydrating the tissue, and optionally further enzymatically, thermally, or chemically treating the dewaxed, rehydrated sample. The second part of the process involves disrupting the rehydrated tissue to release single cells, nuclei, and / or subcellular organelles. These two parts can be performed in different containers. The first part can be performed in a container suitable for processing preserved tissue samples, as described herein. The second part can be performed in a container suitable for dissociating tissue samples, as described herein.

[0116] A. Processing preserved tissue samples 1. Remove preservative The preserved tissue sample, such as an FFPE or OTC sample, is placed at the bottom of the processing chamber of the preserved tissue processing cassette. The cassette engages with the instrument's cassette interface. The fluid connection from the reagent container is checked to confirm proper placement. Xylene or other suitable organic solvent is transferred from the reagent container through the fluid line and cassette interface connector via the second processing chamber port 2570 into the processing chamber. The rotor is moved up and down to facilitate the dissolution of the preservative. After sufficient time, the actuation system presses the rotor down until it encounters the tissue sample. This pushes the used liquid reagent above the top of the rotor. In one embodiment, the rotor is lowered to a position where the top of the rotor is below the first processing chamber port 2504 and rotated to mix the solvent, or moved to a fixed position above the chamber floor and below the first processing chamber port 2504. As described herein, the gap between the rotor side and the inner wall of the processing chamber has been selected to allow liquid to pass between the rotor and the wall, but not tissue fragments or cells, nuclei, and / or subcellular organelles. If the purpose is to isolate cells, the gap size may be set too small to allow cells to pass through, even if the gap is large enough to allow the nucleus and other subcellular organelles to pass through. Using a vacuum applied at the first post-processing port 2585, liquid is drawn from the first processing chamber port 2504 into the processing chamber. The liquid collected at the first post-processing port 2585 is then typically moved further by the vacuum, through the housing interface connector, and via a fluid line into a waste container, for example, located in a holder. In an alternative embodiment, the liquid is drawn out through the first processing chamber port 2504, through a fluid conduit 2553, and discharged through a reagent port (e.g., located at the first post-processing port 2585). This process can be repeated until sufficient preservative is removed from the preserved tissue.

[0117] 2. Rehydration of preserved tissues The preserved tissue, now free of preservatives, is ready for rehydration. In this process, one or more concentrated alcohol solutions are sequentially exchanged for dilute alcohol solutions, then for buffer solutions, until the tissue is fully rehydrated. This process may involve transferring the alcohol-containing solution into the processing chamber through a second processing chamber port in the same manner. Similarly, the rotor can be pumped up and down to facilitate the dissolution of the preservatives. After a sufficient time, the actuation system presses the rotor down until it encounters the tissue sample. This pushes the used liquid reagent to the top of the rotor. After the first incubation with the alcohol-containing solution, the rotor can be pressed down sufficiently to push the used solution to the top of the rotor.

[0118] In one embodiment, the rotor is lowered to a position where the top of the rotor is below the first processing chamber port 2504 and rotated to mix the solvent, or moved to a fixed position above the chamber floor and below the first processing chamber port 2504. As previously described, depending on the configuration, liquid is drawn from the first processing chamber port through the reagent port (and optionally through into the post-processing chamber) and into the waste container. This process is repeated with gradually diluting alcohol solutions until the tissue is completely rehydrated.

[0119] The process can be further rehydrated with a buffer solution (e.g., PBS) using the same treatment method, with the rotor positioned to prevent tissue fragments from being extracted from the treatment chamber during mixing.

[0120] 3. Post-treatment of dewaxed and rehydrated tissues Further processing of the dewaxed and rehydrated tissue may be required within the preserved tissue cassette. In some embodiments, an enzyme solution is used to loosen the cells and nuclei in the tissue. In one embodiment, Liberase TH (Roche) may be added and incubated at a low temperature (e.g., 8°C) for 45 minutes for post-processing of the tissue while minimizing nucleic acid degradation. In other embodiments, heat may be applied to assist antigen presentation. In yet another embodiment, a buffer solution may be applied for post-processing of the dewaxed and rehydrated tissue.

[0121] B. Disrupt tissue samples to release cells, nuclei, and / or other subcellular organelles. Rehydrated tissue can now be broken down to release individual cells, nuclei, and / or other subcellular organelles. This process involves transferring a rehydrated tissue sample from a preserved tissue processing cassette into a processing chamber of a cassette suitable for tissue dissociation.

[0122] Tissue fragmentation may include multiple fragmentation steps, each involving positioning the head at different distances from the chamber floor to create gaps of varying sizes. Typically, at each location, the head will rotate to further facilitate fragmentation or mixing. In some embodiments, the organ may be automatically shredded by the fragmenter before being fragmented into single cells 1000 or nuclei 1050 or other biological material. Such a method may involve a first fragmentation step that may include positioning the head at multiple different distances from the chamber floor and rotating it at each gap distance, thereby providing a larger surface area for the tissue and reducing the distance for enzyme entry. A next step may involve incubating the automatically shredded organ with enzymes or chemicals for fragmenting the tissue into single cells 1000 or nuclei 1050. A next step may involve a second fragmentation step that may again include positioning the head at multiple different distances from the chamber floor and rotating the head.

[0123] The processes described herein can be performed using one or more networked computer systems. Computations can be performed in a cloud computing system, where data from the host computer is transmitted to a cloud computer via a communication network, and the cloud computer performs the computations and transmits or outputs the results to the user via the communication network. For example, nucleic acid sequencing can be performed on a sequencer located at the user's site. The resulting sequence data files can be transmitted to the cloud computing system, where a sequence classification algorithm performs one or more operations of the methods described herein. At any step, the cloud computing system can transmit the computation results back to the computer operated by the user.

[0124] Data can be transmitted electronically, such as via the Internet. Electronic communication can take place over any communication network, including, for example, high-speed transmission networks, including but not limited to Digital Subscriber Line (DSL), cable modems, fiber optics, wireless, satellite, and powerline broadband (BPL). Information can be transmitted to a modem for transmission, such as wireless or wired transmission, to a computer, such as a desktop computer. Alternatively, reports can be transmitted to mobile devices. Reports can be accessed through a subscription program, whereby a user visits a website displaying the report. Reports can be transmitted to a user-accessible user interface device. The user interface device can be, for example, a personal computer, laptop, smartphone, or wearable device, such as a watch, like a wristwatch.

[0125] VI. Computer Systems The method presented in this paper can be executed by a programmable digital computer.

[0126] Figure 10An exemplary computer system is illustrated. Computer system 9901 includes a central processing unit (CPU, also referred to herein as a “processor” and “computer processor”) 9905, which may be a single-core or multi-core processor or more than one processor for parallel processing. 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 for communicating with one or more other systems (e.g., network adapter), and peripheral devices 9925, such as cache memory, other memory, data storage, and / or electronic display adapters. Computer-readable storage 9910, storage unit 9915, interface 9920, and peripheral devices 9925 communicate with CPU 9905 via a communication bus (solid line) such as a motherboard. Storage unit 9915 may be a data storage unit (or data repository) for storing data. Computer system 9901 can be operatively coupled to computer network (“network”) 9930 by means of communication interface 9920. Network 9930 can be the Internet, the Internet and / or an extranet, or an intranet and / or an extranet communicating with the Internet. In some cases, Network 9930 is a telecommunications and / or data network. Network 9930 may include one or more computer servers that can enable distributed computing, such as cloud computing.

[0127] CPU 9905 can execute a series of machine-readable instructions, which can be embodied in a program or software (code). The instructions can be stored in a memory location such as computer-readable storage 9910. The instructions can be directed to CPU 9905, which can then program or otherwise configure CPU 9905 to implement the methods of this disclosure.

[0128] Storage unit 9915 can store files, such as drivers, libraries, and saved programs. Storage unit 9915 can store user data (e.g., user preferences, log files, videos, or other images) and user programs. In some cases, computer system 9901 may include one or more additional data storage units located outside of computer system 9901, such as on a remote server that communicates with computer system 9901 via an intranet or the Internet.

[0129] Computer system 9901 can communicate with one or more remote computer systems via network 9930.

[0130] The method described herein can be implemented in the form of machine-executable code (e.g., a computer processor), which is stored in an electronic storage location of computer system 9901 (e.g., computer-readable storage 9910 or electronic storage unit 9915). The machine-executable or machine-readable code can be provided in software form. During use, the code can be executed by processor 9905. In some cases, the code can be retrieved from storage unit 9915 and stored in memory 9910 for later access by processor 9905. In some cases, electronic storage unit 9915 can be excluded, and machine-executable instructions are stored in memory 9910. The code can be used to communicate with and issue instructions to electronic devices on an instrument (e.g., circuit board 9940, module, or subsystem), such as rotating DC motor relay board 2134 or heater relay board 2240 driving Peltier device 1440, to perform tasks such as rotating a motor or controlling the temperature of housing 200.

[0131] "Storage" media can include any or all tangible memory, or related modules, of computers, processors, etc., such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software may sometimes communicate via the Internet or various other telecommunications networks.

[0132] Computer system 9901 may include or communicate with an electronic display 9935, the electronic display 9935 including a user interface (UI) 9940 for providing input parameters, such as those described herein. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0133] Exemplary embodiments 1. A cassette for processing preserved tissue, comprising: (a) A processing chamber, comprising: (i) a stator, (ii) a sidewall, (iii) a top opening, (iv) a first processing chamber port located in the sidewall, and (v) an optional second processing chamber port located in the sidewall; and (b) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a rotor, the rotor optionally attached to the plunger, the rotor including a face facing the stator; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the rotor has a periphery such that the space between the rotor and the sidewall prevents tissue debris, cells, cell nuclei and other subcellular organelles from passing between the rotor and the sidewall.

[0134] 2. The housing according to embodiment 1, wherein the plunger assembly includes a plunger attached to the rotor.

[0135] 3. The housing according to Embodiment 1, wherein the space between the rotor and the sidewall does not exceed any one of 200μm, 100μm, 50μm and 25μm.

[0136] 4. The box body according to Embodiment 1, wherein: (i) The first processing chamber port is located in the side wall of the processing chamber such that when the rotor is fully depressed in the processing chamber, at least a portion of the first processing chamber port is located above the top of the rotor.

[0137] 5. The housing according to Embodiment 4 further includes: (c) A post-processing chamber, comprising an inlet, sidewalls, a bottom plate, and a first post-processing port located in the sidewalls, and optionally, wherein the inlet is located in a cover covering an opening of the post-processing chamber; in: (i) The post-processing chamber is in fluid communication with the port of the first processing chamber via a fluid conduit; and (ii) The first post-processing port is positioned to discharge the fluid collected in the post-processing chamber.

[0138] 6. The housing according to Embodiment 5, wherein the bottom plate of the post-processing chamber is inclined.

[0139] 7. The housing according to Embodiment 4 further includes: (c) A first post-processing port, which is in fluid communication with the first processing chamber port via a fluid conduit, and is configured to engage a housing interface connector (e.g., in the form of a sleeve) of the housing interface of the instrument.

[0140] 8. An instrument comprising: (i) At least one housing interface configured to engage a housing as described in any one of embodiments 1 to 7, wherein the housing interface includes at least a first housing interface connector configured to engage a first post-processing port in the housing that is in direct or indirect fluid communication with the first processing chamber port. (ii) A reagent subsystem, which includes at least a waste container; and (iii) A fluid subsystem comprising: - (1) One or more fluid lines connecting the waste container to the first housing interface connector (e.g., in the form of a sleeve); and a pressure source configured to move liquid from the processing chamber through the first post-processing port and the first housing interface connector and into the waste container.

[0141] 9. The instrument according to Embodiment 8 further includes: (iv) A mechanical subsystem comprising an actuator engaging the plunger assembly, and one or more motors that move the actuator along the vertical (Z-axis) direction of the processing chamber and rotate the actuator about the Z-axis.

[0142] 10. The instrument according to Embodiment 9 further includes: (v) A control subsystem comprising a digital computer containing a processor and a memory, wherein the memory contains code that, when executed by the processor, instructs the system to perform one or more operations.

[0143] 11. A reagent kit comprising: (I) A first container for processing preserved tissue, the first container comprising: - (a) a processing chamber comprising: (i) a stator, (ii) a sidewall, (iii) a top opening, (iv) a first processing chamber port located in the sidewall, and (v) an optional second processing chamber port located in the sidewall; and - (b) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a rotor optionally attached to the plunger, the rotor including a face facing the stator; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the rotor has a periphery such that the space between the rotor and the sidewall prevents tissue debris, cells, cell nuclei, and other subcellular organelles from passing between the rotor and the sidewall, and optionally, wherein the face of the rotor does not include abrasive features; wherein: -- (i) The first processing chamber port is located in the sidewall of the processing chamber such that when the rotor is fully depressed in the processing chamber, at least a portion of the first processing chamber port is located above the top of the rotor; and - (c) A first post-processing port, which is fluidly connected to the first processing chamber port, for example, via a fluid conduit; - (d) wherein the housing optionally includes a post-processing chamber, the post-processing chamber including an inlet, sidewalls, and a bottom plate, wherein the first post-processing port is located in the sidewalls, and optionally the post-processing chamber includes a cover covering an opening in the post-processing chamber and including the inlet; wherein: -- (i) The post-processing chamber is in fluid communication with the port of the first processing chamber via the fluid conduit; and -- (ii) The first post-processing port is positioned to discharge the fluid collected in the post-processing chamber; and (II) A second housing for dissociating cells, nuclei, and / or other subcellular organelles from tissue, the second housing comprising: - (a) a processing chamber comprising: (i) a stator including grinding features, (ii) sidewalls, (iii) a top orifice, (iv) a first processing chamber port located in the sidewalls; and (v) an optional second processing chamber port located in the sidewalls; and - (b) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a rotor, the rotor optionally attached to the plunger, the rotor including a face facing the stator, wherein the face includes abrasive features, and optionally wherein the rotor has a periphery such that when the rotor is inserted into the processing chamber, the gap between the wall of the processing chamber and the rotor is large enough to allow cells, nuclei and / or other subcellular organelles to pass between the rotor and the chamber wall; - (c) a post-processing chamber comprising (i) a base plate, (ii) sidewalls, (iii) an inlet, and (iv) a first post-processing port located in said sidewalls, and (v) an optional second post-processing chamber port; and - (d) A housing pressure port configured to apply negative pressure to the processing chamber directly or via a vacuum chamber; in: The first post-processing port of the first housing and the first post-processing port of the second housing are configured to engage with the same first housing interface connector of the instrument's housing interface, and optionally, the second processing chamber port of the first housing and the second processing chamber port of the second housing are configured to engage with the same second housing interface connector of the instrument's housing interface.

[0144] 12. The kit according to Example 11 further includes: (a) An instrument, said instrument comprising: - (i) At least one housing interface configured to engage the first housing and separately engage the second housing, wherein the housing interface includes at least a first housing interface connector (e.g., in the form of a sleeve), the first housing interface connector being configured to engage the first post-processing port of the first housing and the first post-processing port of the second housing. - (ii) A reagent subsystem comprising at least one reagent container containing reagents and at least one waste container; - (iii) A fluid subsystem comprising: -- (1) At least one fluid line connecting the reagent container to the first housing interface connector (e.g., in the form of a sleeve), and at least one fluid line connecting the waste container to the first housing interface connector (e.g., in the form of a sleeve); and -- (2) One or more pressure sources configured to apply positive or negative pressure to move liquid and / or gas through the one or more fluid lines; - (iv) A mechanical subsystem comprising an actuator, a linear driver (e.g., a stepper motor or pneumatic driver) driving the actuator in a vertical (Z-axis) direction, and a rotary motor for rotating the actuator about the Z-axis; and - (v) A control subsystem comprising a digital computer, the digital computer including a processor and a memory, wherein the memory contains code that, when executed by the processor, instructs the system to perform one or more operations; and Wherein, when the first box or the second box is engaged with the box interface: (A) The first post-processing port engages with the same first housing interface connector; and (B) Optionally, the second processing chamber port engages with the same second housing interface connector.

[0145] 13. A method for isolating cells and / or subcellular organelles from preserved tissue, comprising: (a) Provide the kit of Example 11 or 12, wherein the first housing is engaged with the housing interface, wherein the processing chamber of the first housing contains a preserved tissue sample; (b) The preserved tissue sample is processed in the following manner: - (i) Using the actuator to press down the rotor to push the liquid to the liquid level at the port of the first processing chamber; - (ii) Using the fluid subsystem, a treatment reagent is moved into the treatment chamber to remove the preservation compound from the preserved tissue; - (iii) Using the fluid subsystem to remove liquid from the processing chamber (e.g., through the first post-processing port (optionally through the post-processing chamber) and the first housing interface connector into a waste container in fluid connection with it). - (iv) Optionally, using the fluid subsystem, a rehydration agent is transferred into the processing chamber to rehydrate the preserved tissue; and - (v) Using the fluid subsystem, remove the rehydration reagent from the treatment chamber (e.g., through the first post-treatment port (optionally through the post-treatment chamber) and the first cartridge interface connector into a waste container in fluid connection with it). (c) The processed tissue is transferred from the first box to the processing chamber of the second box, and the second box is engaged with one of the box interfaces; (d) Dissociating cells and / or subcellular organelles from the treated tissue by means of: - (i) Using the fluid subsystem, tissue dissociation reagents are transferred into the processing chamber; - (ii) Using the actuator to press down and rotate the rotor to release cells and / or nuclei from the treated tissue and to push liquid to move the released cells and / or subcellular organelles to the liquid level at the port of the first treatment chamber; - (iii) Using the fluid subsystem, moving the cells and / or subcellular organelles from the processing chamber into the post-processing chamber; and - (iv) Collect the released cells and / or subcellular organelles from the post-processing chamber.

[0146] 14. A system for processing an organization, comprising: (a) An instrument, said instrument comprising: - (i) At least one housing interface for engaging a housing, wherein the housing interface includes at least a first housing interface connector, a second housing interface connector and a third housing interface connector configured to engage ports in the engaged housing. - (ii) A reversibly connectable processing reagent subsystem, comprising one or more of the following: -- (A) A support for holding multiple containers, wherein the support includes a leak-proof barrier configured to contain liquid within the support. -- (B) A plurality of containers held by the support frame, wherein the containers include at least one container containing reagents for treating 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 sense liquid in the bottom of the bracket; -- (E) A plurality of fluid conduits connect the container to one or more couplings of the fluid subsystem of the instrument, wherein the instrument includes a sensing mechanism that senses whether the fluid conduits and the couplings are properly connected and correctly positioned; -- (F) Optionally, a sensor, such as a sensor that uses the Hall effect to sense the connection between the bracket and the rest of the instrument; (iii) A fluid subsystem comprising: -- (A) One or more couplings connected to a plurality of fluid conduits, wherein the fluid conduits are in communication with the housing interface connector; and -- (B) One or more pressure sources for moving reagents from the one or more containers through one or more of the housing interface connectors, and for moving liquid from the housing interface connectors into the waste container.

[0147] 15. A method for releasing cells and / or subcellular organelles from a tissue, comprising: (a) Providing a housing, the housing comprising: - (1) A processing chamber, the processing chamber comprising: (i) a stator, (ii) a sidewall, (iii) a top opening, and (iv) a first processing chamber port located in the sidewall; and a second processing chamber port located in the sidewall; - (2) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a plunger containing a rotor, the rotor including a surface facing the stator, wherein the surface includes abrasive 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 acting on the surface of the rotor; (b) Providing a tissue sample in the processing chamber; (c) Press the rotor down in the processing chamber until the pressure sensor measures a target pressure consistent with the contact between the surface of the rotor and the tissue sample; (d) While maintaining the target pressure, the rotor is rotated against the tissue sample to dissociate cells and / or subcellular organelles from the tissue sample; (e) When the pressure sensor measures a pressure consistent with that of the surface of the rotor in contact with the stator, the pressure on the rotor is stopped.

[0148] 16. A method for calibrating a plunger in a housing, comprising: (a) While pressing a plunger, including a shaft attached to the rotor, into the chamber of the housing, measure and record the force of the plunger at different positions along the Z-axis of the plunger, and determine a calibrated full-press position that coincides with the bottom plate of the chamber where the rotor contacts the housing.

[0149] 17. The method according to embodiment 16 further includes: (b) While pressing the plunger into the chamber of the housing, determine a calibrated tissue engagement position that corresponds to the contact of the rotor with the tissue in the chamber.

[0150] 18. The method according to embodiment 16 or 17 further includes: (c) Perform a dissociation procedure on the tissue in the chamber, wherein the procedure includes pressing the plunger down to a determined fully depressed position and / or a determined contact tissue position.

[0151] 19. The method according to Example 16 or 17, comprising using the calibrated full compression position and / or the determined contact tissue position for a second different housing.

[0152] As used herein, unless otherwise stated, the following meanings apply. The words “may” and “possibly” are used in a permissive sense (i.e., meaning possible) rather than a mandatory sense (i.e., meaning must). The words “include,” “including,” and “includes,” etc., mean including but not limited to. The singular forms “a,” “one,” and “the” include plural indicators. Thus, for example, a reference to “one element” includes a combination of two or more elements, although other terms and phrases such as “one or more” are used for one or more elements. The phrase “at least one” includes “one,” “one or more,” “one or more,” and therefore also covers situations where the term “multiple” is used. Unless otherwise stated, the term “or” is non-exclusive, i.e., it covers “and” and “or.” The term “any” between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase “at least one of 1, 2, or 3” means “at least 1, at least 2, or at least 3.” The term “about” refers to a range of 5% plus or minus the numerical value stated in the context of its particular use. The term "consistent with essentially" means including the listed elements and other elements that do not substantially affect the essential and novel features of the claimed combination.

[0153] It should be understood that the specification and drawings are not intended to limit the invention to the specific forms disclosed; rather, the invention is intended to cover all modifications, equivalents, and substitutions falling within the spirit and scope of the 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 light of this specification. Therefore, this specification and drawings are to be interpreted only as illustrative and to teach those skilled in the art the general manner of practicing the invention. It should be understood that the forms of the invention shown and described herein will be considered as examples of embodiments. Elements and materials illustrated and described herein may be substituted, parts and steps may be reversed or omitted, and certain features of the invention may be used independently, all of which will be apparent to those skilled in the art after benefiting from the description of the invention. Changes may be made to the elements described herein without departing from the spirit and scope of the invention as set forth in the appended claims.

[0154] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. A cassette for processing preserved tissue, comprising: (a) A processing chamber, comprising: (i) a stator, (ii) a sidewall, (iii) a top opening, (iv) a first processing chamber port located in the sidewall, and (v) an optional second processing chamber port located in the sidewall; and (b) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a rotor, the rotor optionally attached to the plunger, the rotor including a face facing the stator; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the rotor has a periphery such that the space between the rotor and the sidewall prevents tissue debris, cells, cell nuclei and other subcellular organelles from passing between the rotor and the sidewall.

2. The box body according to claim 1, wherein, The plunger assembly includes a plunger attached to the rotor.

3. The box body according to claim 1, wherein, The space between the rotor and the sidewall does not exceed any one of 200μm, 100μm, 50μm and 25μm.

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

5. The housing according to claim 4, further comprising: (c) A post-processing chamber, comprising an inlet, sidewalls, a bottom plate, and a first post-processing port located in the sidewalls, and optionally, wherein the inlet is located in a cover covering an opening of the post-processing chamber; in: (i) The post-processing chamber is in fluid communication with the port of the first processing chamber via a fluid conduit; and (ii) The first post-processing port is positioned to discharge the fluid collected in the post-processing chamber.

6. The box body according to claim 5, wherein, The bottom plate of the post-processing chamber is inclined.

7. The housing according to claim 4, further comprising: (c) A first post-processing port, which is in fluid communication with the first processing chamber port via a fluid conduit, and is configured to engage a housing interface connector (e.g., in the form of a sleeve) of the housing interface of the instrument.

8. An instrument comprising: (i) At least one housing interface configured to engage a housing according to any one of claims 1 to 7, wherein the housing interface includes at least a first housing interface connector configured to engage a first post-processing port in the housing that is in direct or indirect fluid communication with the first processing chamber port. (ii) A reagent subsystem, which includes at least a waste container; as well as (iii) A fluid subsystem comprising: - (1) Connecting the waste container to one or more fluid lines (e.g., in the form of a sleeve) of the first housing interface connector; And a pressure source configured to move liquid from the processing chamber through the first post-processing port and the first housing interface connector, and into the waste container.

9. The instrument according to claim 8, further comprising: (iv) A mechanical subsystem comprising an actuator engaging the plunger assembly, and one or more motors that move the actuator along the vertical (Z-axis) direction of the processing chamber and rotate the actuator about the Z-axis.

10. The instrument according to claim 9, further comprising: (v) A control subsystem comprising a digital computer including a processor and a memory, wherein the memory contains code that, when executed by the processor, instructs the system to perform one or more operations.

11. A reagent kit, comprising: (I) A first container for processing preserved tissue, the first container comprising: - (a) a processing chamber comprising: (i) a stator, (ii) a sidewall, (iii) a top opening, (iv) a first processing chamber port located in the sidewall, and (v) an optional second processing chamber port located in the sidewall; and - (b) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a rotor optionally attached to the plunger, the rotor including a face facing the stator; wherein the rotor is rotatable and movable toward and away from the stator; and wherein the rotor has a periphery such that the space between the rotor and the sidewall prevents tissue debris, cells, cell nuclei, and other subcellular organelles from passing between the rotor and the sidewall, and optionally, wherein the face of the rotor does not include abrasive features; wherein: -- (i) The first processing chamber port is located in the sidewall of the processing chamber such that when the rotor is fully depressed in the processing chamber, at least a portion of the first processing chamber port is located above the top of the rotor; and - (c) A first post-processing port, which is fluidly connected to the first processing chamber port, for example, via a fluid conduit; - (d) wherein the housing optionally includes a post-processing chamber, the post-processing chamber including an inlet, sidewalls, and a bottom plate, wherein the first post-processing port is located in the sidewalls, and optionally the post-processing chamber includes a cover covering an opening in the post-processing chamber and including the inlet; wherein: -- (i) The post-processing chamber is in fluid communication with the port of the first processing chamber via the fluid conduit; and -- (ii) The first post-processing port is positioned to discharge the fluid collected in the post-processing chamber; and (II) A second housing for dissociating cells, nuclei, and / or other subcellular organelles from tissue, the second housing comprising: - (a) a processing chamber comprising: (i) a stator including grinding features, (ii) sidewalls, (iii) a top orifice, (iv) a first processing chamber port located in the sidewalls; and (v) an optional second processing chamber port located in the sidewalls; and - (b) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a rotor, the rotor optionally attached to the plunger, the rotor including a face facing the stator, wherein the face includes abrasive features, and optionally wherein the rotor has a periphery such that when the rotor is inserted into the processing chamber, the gap between the wall of the processing chamber and the rotor is large enough to allow cells, nuclei and / or other subcellular organelles to pass between the rotor and the chamber wall; - (c) a post-processing chamber comprising (i) a base plate, (ii) sidewalls, (iii) an inlet, and (iv) a first post-processing port located in said sidewalls, and (v) an optional second post-processing chamber port; and - (d) A housing pressure port configured to apply negative pressure to the processing chamber directly or via a vacuum chamber; in: The first post-processing port of the first housing and the first post-processing port of the second housing are configured to engage with the same first housing interface connector of the instrument's housing interface, and optionally, the second processing chamber port of the first housing and the second processing chamber port of the second housing are configured to engage with the same second housing interface connector of the instrument's housing interface.

12. The kit according to claim 11, further comprising: (a) An instrument, said instrument comprising: - (i) At least one housing interface configured to engage the first housing and separately engage the second housing, wherein the housing interface includes at least a first housing interface connector (e.g., in the form of a sleeve), the first housing interface connector being configured to engage the first post-processing port of the first housing and the first post-processing port of the second housing. - (ii) A reagent subsystem comprising at least one reagent container containing reagents and at least one waste container; - (iii) A fluid subsystem comprising: -- (1) At least one fluid line connecting the reagent container to the first housing interface connector (e.g., in the form of a sleeve), and at least one fluid line connecting the waste container to the first housing interface connector (e.g., in the form of a sleeve); and -- (2) One or more pressure sources configured to apply positive or negative pressure to move liquid and / or gas through the one or more fluid lines; - (iv) A mechanical subsystem comprising an actuator, a linear driver (e.g., a stepper motor or pneumatic driver) driving the actuator in a vertical (Z-axis) direction, and a rotary motor for rotating the actuator about the Z-axis; and - (v) A control subsystem comprising a digital computer, the digital computer including a processor and a memory, wherein the memory contains code that, when executed by the processor, instructs the system to perform one or more operations; and Wherein, when the first box or the second box is engaged with the box interface: (A) The first post-processing port engages with the same first housing interface connector; and (B) Optionally, the second processing chamber port engages with the same second housing interface connector.

13. A method for isolating cells and / or subcellular organelles from preserved tissue, comprising: (a) A kit according to claim 11 or 12, wherein the first housing is engaged with the housing interface, wherein the processing chamber of the first housing contains a preserved tissue sample; (b) The preserved tissue sample is processed in the following manner: - (i) Using the actuator to press down the rotor to push the liquid to the liquid level at the port of the first processing chamber; - (ii) Using the fluid subsystem, a treatment reagent is moved into the treatment chamber to remove the preservation compound from the preserved tissue; - (iii) Using the fluid subsystem to remove liquid from the processing chamber (e.g., through the first post-processing port (optionally through the post-processing chamber) and the first housing interface connector into a waste container in fluid connection with it). - (iv) Optionally, using the fluid subsystem, a rehydration agent is transferred into the processing chamber to rehydrate the preserved tissue; as well as - (v) Using the fluid subsystem, remove the rehydration reagent from the treatment chamber (e.g., through the first post-treatment port (optionally through the post-treatment chamber) and the first cartridge interface connector into a waste container in fluid connection with it). (c) The processed tissue is transferred from the first box to the processing chamber of the second box, and the second box is engaged with one of the box interfaces; (d) Dissociating cells and / or subcellular organelles from the treated tissue by means of: - (i) Using the fluid subsystem, tissue dissociation reagents are transferred into the processing chamber; - (ii) Using the actuator to press down and rotate the rotor to release cells and / or nuclei from the treated tissue and to push liquid to move the released cells and / or subcellular organelles to the liquid level at the port of the first treatment chamber; - (iii) Using the fluid subsystem, the cells and / or subcellular organelles are moved from the processing chamber into the post-processing chamber; as well as - (iv) Collect the released cells and / or subcellular organelles from the post-processing chamber.

14. A system for processing an organization, comprising: (a) An instrument, said instrument comprising: - (i) At least one housing interface for engaging a housing, wherein the housing interface includes at least a first housing interface connector, a second housing interface connector and a third housing interface connector configured to engage ports in the engaged housing. - (ii) A reversibly connectable processing reagent subsystem, comprising one or more of the following: -- (A) A support for holding multiple containers, wherein the support includes a leak-proof barrier configured to contain liquid within the support. -- (B) A plurality of containers held by the support frame, wherein the containers include at least one container containing reagents for treating 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 sense liquid in the bottom of the bracket; -- (E) A plurality of fluid conduits connecting the container to one or more couplings of the fluid subsystem of the instrument, wherein the instrument includes a sensing mechanism that senses whether the fluid conduits and the couplings are properly connected and correctly positioned; -- (F) Optionally, a sensor, such as a sensor that uses the Hall effect to sense the connection between the bracket and the rest of the instrument; - (iii) A fluid subsystem comprising: -- (A) One or more couplings connected to a plurality of fluid conduits, wherein the fluid conduits are in communication with the housing interface connector; and -- (B) One or more pressure sources for moving reagents from the one or more containers through one or more of the housing interface connectors, and for moving liquid from the housing interface connectors into the waste container.

15. A method for releasing cells and / or subcellular organelles from a tissue, comprising: (a) Providing a housing, the housing comprising: - (1) A processing chamber, the processing chamber comprising: (i) a stator, (ii) a sidewall, (iii) a top opening, and (iv) a first processing chamber port located in the sidewall; and a second processing chamber port located in the sidewall; - (2) A plunger assembly positioned in the processing chamber via the top orifice, wherein the plunger assembly includes a plunger containing a rotor, the rotor including a surface facing the stator, wherein the surface includes abrasive 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 acting on the surface of the rotor; (b) Providing a tissue sample in the processing chamber; (c) Press the rotor down in the processing chamber until the pressure sensor measures a target pressure consistent with the contact between the surface of the rotor and the tissue sample; (d) While maintaining the target pressure, the rotor is rotated against the tissue sample to dissociate cells and / or subcellular organelles from the tissue sample; (e) When the pressure sensor measures a pressure consistent with that of the surface of the rotor in contact with the stator, the pressure on the rotor is stopped.

16. A method for calibrating a plunger in a housing, comprising: (a) While pressing a plunger, including a shaft attached to the rotor, into the chamber of the housing, measure and record the force of the plunger at different positions along the Z-axis of the plunger, and determine a calibrated full-press position that coincides with the bottom plate of the chamber where the rotor contacts the housing.

17. The method of claim 16, further comprising: (b) While pressing the plunger into the chamber of the housing, determine a calibrated tissue engagement position that corresponds to the contact of the rotor with the tissue in the chamber.

18. The method according to claim 16 or 17, further comprising: (c) Perform a dissociation procedure on the tissue in the chamber, wherein the procedure includes pressing the plunger down to a determined fully depressed position and / or a determined contact tissue position.

19. The method of claim 16 or 17, comprising using the calibrated full depress position and / or the determined contact tissue position for a second different housing.

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