Tissue dissociation using female-threaded tubes

Abstract

A laboratory processing tube assembly is mounted on the processing unit for dissociating an organic-derived sample. The tube assembly includes a tube and a cap, which form a tube chamber that contains the sample during dissociation. The tube includes at least one internal thread extending inward from and circumferentially around the inner surface of the tube in a helical arrangement, the internal helical thread including threads defining two impact surfaces facing generally opposite each other, such that during reciprocating axial tube assembly oscillation to dissociate the sample, the sample impacts a first of the impact surfaces when flowing in a first axial direction through the tube chamber and impacts a second of the impact surfaces when flowing in an opposite second axial direction through the tube chamber.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Non-provisional Patent Application No. 18 / 410,533, filed January 11, 2024, and U.S. Provisional Patent Application No. 63 / 438,854, filed January 13, 2023, which are incorporated herein by reference.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to laboratory instruments and methods for processing sample materials, and more particularly to tubes for holding samples during the process of tissue cell dissociation. [Background technology]

[0003] A common need in life sciences is for the output of tissue sample dissociation to be single cells preserved as intact cells for downstream applications, but these output intact single cells are typically, but not always, viable. Such samples may be, for example, human or other animal tissues (e.g., liver), fluids (e.g., blood), or bone, other organic matter, or other substances. Such output intact single cells are needed for a variety of downstream uses, including life science applications such as biomedical research, cell therapy, culture, identification, and diagnostics, and sorting for these and other downstream processes (e.g., DNA and RNA detection and analysis). Thus, sample dissociation is a critical step at the front end of almost all workflows that use intact single cells.

[0004] Current methods for dissociating samples into intact single cells include enzymatic dissociation and mechanical dissociation, which can be either manual (using handheld non-energized tools such as surgical instruments or dounce homogenizers) or automated (using powered instruments with blades and / or magnets).

[0005] However, both of these techniques have drawbacks. Manual mechanical dissociation is the traditional approach. In this approach, the surgical tools are typically scalpels, knives, or tweezers used to process samples held in Petri dishes. Dounce homogenizers are hand-held mortar and pestle-style tools. Both of these types of manual mechanical dissociation techniques are highly variable and dependent on the individual user / operator. This is due to the fact that the person must manually and vigorously apply input motion / force (e.g., stirring or grinding) for an extended period of time (typically about 20 minutes, but this varies depending on the individual, as some individuals are more efficient than others), different individuals use different stirring / grinding techniques, and even the same individual may change their stirring / grinding technique over time (e.g., due to fatigue). Thus, in practice, the results obtained with these manual tools are highly variable, highly inconsistent, and extremely unreliable. Furthermore, Dounce homogenizers are extremely ineffective on samples containing connective tissue, such as muscle.

[0006] Furthermore, the dissociation performed by automated / powered tools with blades and magnets is very violent, resulting in significantly fewer intact single cells. This is because cells are damaged during the violent dissociation process. Therefore, these automated tools are not suitable for use in dissociating samples when a large output of intact single cells is required. Finally, enzymes are also not suitable for use in dissociating samples when a large output of intact single cells is required, as enzymes tend to damage cells and alter the cellular transcriptome.

[0007] Thus, all current sample dissociation techniques tend to result in inconsistent, slow / time-consuming, and / or inefficient / ineffective yields of intact single cells from tissue samples. Summary of the Invention [Means for solving the problem]

[0008] Generally, the present disclosure relates to a laboratory tube assembly mounted on a processing unit used to dissociate organic-derived samples by tube assembly vibration including an axial reciprocating component. In an exemplary embodiment, the tube assembly includes a tube and a cap removably coupled to one another to form a tube chamber, the tube chamber having a longitudinal axis, the tube chamber containing the sample during dissociation, and the tube having a peripheral inner surface. Additionally, the tube assembly includes at least one internal thread extending inward from and circumferentially around the inner surface of the tube in a helical arrangement. The helical internal thread includes threads defining two impact surfaces facing generally opposite each other at a non-perpendicular angle relative to the longitudinal axis of the tube chamber and to the inner surface of the tube. In use, during axial reciprocating tube assembly vibration to dissociate the sample, the sample impacts a first of the impact surfaces as it flows through the tube chamber in a first axial direction, and impacts a second of the impact surfaces as it flows through the tube chamber in an opposite second axial direction.

[0009] In a typical embodiment, a sample impacts the impact surface of the internal helical thread during use for dissociation, separating the sample into intact single cells, where no enzymes or magnetic forces are used in the tube chamber (for completely mechanical dissociation) or only mild enzymes are used in the tube chamber (for enzyme-assisted / enhanced mechanical dissociation). The impact surface is angled from the longitudinal axis of the tube chamber, so that a blunt impact force and a shear force can be applied to the sample during use for dissociation. For example, the blunt impact force on the sample can be greater in a first direction than in a second direction, and the shear force on the sample can be greater in the second direction than in the first direction. This is because the internal helical thread generates a spiral angular flow of the sample.

[0010] In some embodiments, the internal helical thread forms a single continuous thread, for example, the internal helical thread can define multiple (at least two) complete revolutions around the inner peripheral surface of the tube.

[0011] In some embodiments, the internal helical thread extends along 80% to 90% of the total length of the inner peripheral surface of the pipe and / or has a thread pitch (threads per inch) of 15 to 25. In some embodiments, the internal helical thread has a profile with a thread angle of 25 degrees to 75 degrees (so that opposing striking faces are angled relative to each other) or a square profile with a thread angle of 0 degrees (so that opposing striking faces are parallel to each other).

[0012] In some embodiments, the internal helical thread is formed on a sleeve that is inserted into the pipe to form the inner peripheral surface of the pipe, and in some embodiments, the internal helical thread is formed by two threads in a double helix arrangement.

[0013] Another exemplary embodiment includes a laboratory dissociation system for dissociating a sample. In the exemplary embodiment, the system includes a processing unit that generates a tube assembly vibration that includes an axial reciprocating component. The system also includes a tube assembly mounted to the processing unit. The tube assembly includes a tube and a cap that are removably coupled to one another to form a tube chamber, the tube chamber having a longitudinal axis, the tube chamber containing the sample during use for dissociation. Additionally, the tube has a peripheral inner surface and at least one internal thread that extends circumferentially inwardly from and around the peripheral inner surface in a helical arrangement. The internal helical thread includes threads that define two impact surfaces that face generally opposite each other at a non-perpendicular angle relative to the longitudinal axis of the tube chamber and to the inner surface of the tube. In use, during axial reciprocating tube assembly vibration to dissociate a sample, the sample collides with a first one of the collision surfaces as it flows through the tube chamber in a first axial direction, and the sample collides with a second one of the collision surfaces as it flows through the tube chamber in an opposite second axial direction.

[0014] In some embodiments, the laboratory processing unit is a bead mill homogenizer. In such embodiments, the tube assembly motion generated by the bead mill homogenizer can be a swash motion that includes an axial reciprocating component.

[0015] Another exemplary embodiment includes a laboratory dissociation method. In the exemplary embodiment, the method includes loading a sample and a buffer into a tube assembly, the tube assembly including a tube and a cap removably coupled to one another to form a tube chamber, the tube chamber having a longitudinal axis, the tube chamber containing the sample during use for dissociation. The tube has a peripheral inner surface and at least one internal thread extending inwardly from and circumferentially around the peripheral inner surface in a helical arrangement. The internal helical thread includes threads defining two impact surfaces facing generally opposite each other at a non-perpendicular angle relative to the longitudinal axis of the tube chamber and to the inner surface of the tube. The method further includes mounting the loaded tube assembly to a processing unit. The method further includes a step of operating the processing unit to generate tube assembly motion that includes an axial reciprocating component, such that during axially reciprocating tube assembly vibration to dissociate the sample, the sample collides with a first one of the collision surfaces when it flows in a first axial direction within the tube chamber, and with a second one of the collision surfaces when it flows in an opposite second axial direction within the tube chamber.

[0016] In some embodiments, the methods do not involve the use of any enzymes or magnetic forces within the tube chamber, and such methods involve mechanical (and only mechanical) dissociation of the sample within the tube.

[0017] In another embodiment, the method includes using a mild enzyme in the tubing chamber. Such a mild enzyme may be, for example, an enolase enzyme, a hydroxylase enzyme, and / or a neutral metalloproteinase enzyme. Such a method involves mechanical dissociation that is enhanced / augmented by the mild enzymatic dissociation.

[0018] During operation of the processing unit, the impact surface is angled from the longitudinal axis of the tube chamber, typically imparting a blunt impact force and a shear force to the sample during use for dissociation. Additionally, the helical arrangement of the internal threads typically induces a vortex-like angular flow of the sample, with the blunt impact force on the sample being greater in the first direction than in the second direction, and the shear force on the sample being greater in the second direction than in the first direction. Furthermore, the sample typically impacts the internal helical threads multiple times in the first direction and multiple times in the second direction (e.g., in embodiments in which the internal helical threads define multiple complete revolutions around the inner peripheral surface of the tube).

[0019] In typical embodiments, the step of loading the sample and buffer includes loading an organic sample and perfusion buffer into the tubing assembly, and the step of operating the processing unit includes dissociating the sample into dissociated intact single cells. In some such embodiments, the step of loading the sample and buffer further includes loading the sample and buffer into the tubing assembly so that the sample and buffer occupy 20% to 90% of the tubing chamber. Also, in some embodiments, the step of operating the processing unit includes operating at a speed of 0.8 m / s to 1.6 m / s, for a time period of 30 seconds to 60 seconds, or both.

[0020] The method may include at least one pre-step prior to the processing step, which may include, for example, red blood cells (RBC) lysis to remove all RBCs from the sample being processed.

[0021] Additionally, the method may include at least one post-processing step, such as further processing and / or downstream utilization of the dissociated sample. Further processing may include, for example, subjecting the cells of the dissociated sample to separation, sorting, enumeration, enrichment, purification, analysis, and / or other techniques prior to downstream utilization. And downstream utilization may include, for example, flow cytometry, genomic diagnostics, cell therapy, proteomic diagnostics, cell culture, cell imaging, NG2 sequencing, extraction, Western, ADME / Tox, and / or libraries, some of which may include additional / secondary dissociation processes (e.g., repeating the processing steps).

[0022] In some embodiments, the dissociated sample is transferred to another tube for further processing and / or downstream use. In other embodiments, the method further includes processing and / or downstream use of the dissociated sample while still in the same tube. For example, in the case of further processing using a centrifuge, after processing is completed, the threaded tube assembly containing the dissociated sample is transferred directly to and placed in a centrifuge, which operates to spin down the dissociated sample to separate intact single cells from extracellular debris.

[0023] As will be appreciated by those skilled in the art, all details described herein with respect to any of the tube assemblies, dissociation systems, and dissociation methods are applicable to all of the tube assemblies, dissociation systems, and dissociation methods. In exemplary embodiments, the tube assemblies may be used as components of a dissociation system to implement the dissociation methods. Accordingly, for the sake of brevity, some of these details will not be repeated unnecessarily throughout this disclosure.

[0024] The specific techniques and structures used to overcome the shortcomings of prior art devices and achieve the advantages described herein will become apparent from the following detailed description of exemplary embodiments and the accompanying drawings and claims. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is an exploded view of a process tube assembly according to an exemplary embodiment having a tube with internal helical threads shown in cross section and a cap shown in side view. [Figure 2] 2 is a top schematic view of the tube of FIG. 1 showing the internal helical threads. [Figure 3] 2 shows a section of the tube of FIG. 1 with the internal helical threads shown in detail. [Figure 4] 2 shows the tube of FIG. 1 in use for processing according to an exemplary method embodiment, with the circular angular flow path of the sample indicated by the circular direction arrow. [Figure 5] 5 shows a schematic representation of the lower portion of the tube of FIG. 4 in use for processing, with two successive portions of the sample flow indicated by directional arrow segments in both a first axial direction and a first angular direction. [Figure 6A] ~ [Figure 6B] 6 shows a top view of the tube section of FIG. 5 in use for processing, with two successive portions of the sample flow indicated by two respective angle-directional arrow segments. [Figure 7] 6 shows a schematic representation of the tube section of FIG. 5 in use for processing, with two further successive sections of sample flow indicated by directional arrow segments in both opposite second axial directions and the same first angular direction. [Figure 8A] ~ [Figure 8B] 8 shows a top view of the tubing section of FIG. 7 in use for processing, with two successive portions of the sample flow indicated by two respective angle-directional arrow segments. [Figure 9] 5 shows the sequence of operation of the tube of FIG. 4 in use for processing, with a plot of the axial displacement of the tube against time shown in the top diagram, the respective axial positions of the samples in the tube in the side view shown in the middle diagram, and the respective angular positions of the samples in the tube in the top view shown in the bottom diagram. [Figure 10] ~ [Figure 13] 10A-10C are side cross-sectional views of four tube assemblies having alternative configurations of helical threads with various thread pitches. [Figure 14] ~ [Figure 19] Figures 14, 16, and 18 are top views of three tube assemblies having alternative helical thread configurations with multiple helical threads, and Figures 15, 17, and 19 are cross-sectional views taken along lines 15-15, 17-17, and 19-19, respectively, of Figures 14, 16, and 18. [Figure 20] FIG. 1 includes side cross-sectional views of nine tube assemblies having alternative configurations of helical threads with various thread profiles. [Figure 21] FIG. 10 is a side cross-sectional view of a portion of a pipe assembly having an alternative configuration of helical threads, with recessed helical threads. [Figure 22] ~ [Figure 24] FIG. 22 is an exploded perspective view of a tube assembly having an alternative helical thread configuration in which the helical thread is molded separately on the insertion sleeve, FIG. 23 is a perspective view of the insertion sleeve, and FIG. 24 is a side cross-sectional view of the insertion sleeve. [Figure 25] ~ [Figure 32] 25, 27, 29, and 31 are perspective views of four insertion sleeves having alternative slotted configurations, and FIGS. 26, 28, 30, and 32 are respective cross-sectional views of the insertion sleeves. [Figure 33] ~ [Figure 34] 10 illustrates the results of tests conducted using exemplary method embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0026] Generally, the present disclosure relates to laboratory processing apparatus and methods for dissociating samples into output cells for further downstream utilization (e.g., processing, culturing, treatment, or analysis for life science and other applications). In exemplary embodiments, the processing apparatus and methods are operable to perform a relatively gentle, low-impact dissociation (also called disaggregation) of a sample to obtain output cells, most of which are preserved as intact single cells suitable for these further applications. In such embodiments, the processing apparatus and methods are capable of processing a variety of tissue samples in an automated, highly consistent manner, resulting in higher volume, higher throughput, and more consistent output. In another embodiment, the processing apparatus and methods are operable to apply relatively high forces to the sample to perform relatively vigorous, high-impact dissociation or other processing of the sample.

[0027] Some preliminary definitions are provided below: All of these definitions are in the context of laboratory processing equipment and methods, and the term "laboratory" should be interpreted broadly to mean any type of controlled, sterile environment used for processing samples, including clinical diagnostic and pathology laboratories, academic and industrial research laboratories, and teaching laboratories.

[0028] As used herein, "processing" should be broadly construed to mean high shear disaggregation in which components of the sample are reduced (and optionally mixed and / or resuspended) to significant micron-level particle sizes by vibration of a tube containing the sample (which may optionally contain another medium, such as beads). Disaggregation is a general, but not exclusive, example of such processing.

[0029] As used herein, "dissociating" should be broadly construed to mean a type of sample processing that results in tissue separation into a single-cell state. This means that there are no extracellular attachments. The cells are free-standing, not sessile. This should not be confused with anoikis, a form of programmed cell death that occurs in anchorage-dependent cells when they become detached from the surrounding extracellular matrix. Dissociation refers to the physical separation, not destruction, of cells found within a solid tissue sample, to produce / output a dissociated sample of intact (i.e., whole (no cell wall)) and single (i.e., solid and therefore not layered (or otherwise attached) to any other cells) cells, which are typically, but not always, viable (e.g., alive or otherwise usable for downstream applications) (these are also referred to as "intact single cells").

[0030] As used herein, the term "processing unit" should be broadly construed to mean a laboratory instrument operable to drive a tube holding a sample with a vibration that includes an axial reciprocating component in order to process the sample. A common, but not exclusive, example of a processing unit is a laboratory homogenizer, such as a bead mill homogenizer such as a BEADRUPTER bead mill homogenizer (Omni International, Inc., Kennesaw, Georgia), or another conventional bead mill homogenizer or other type of laboratory homogenizer. The processing unit generates vibration of the tube holding the sample. This vibration can be (a) a simple axial reciprocating motion (e.g., many conventional laboratory homogenizers), (b) a "swash" motion that includes an axial reciprocating component and an offset spinning motion about the axis of reciprocation at the same speed as the vibration (e.g., a BEADRUPTER bead mill homogenizer), or (c) any other vibration that includes an axial reciprocating component.

[0031] As used herein, the term "sample" should be broadly construed to encompass any type of organically derived material (i.e., organic matter and / or organically derived material) that can be processed and for which dissociation or other processing may be useful in life science applications, including, but not limited to, human and / or non-human bodily fluids and / or tissues (e.g., blood, bone marrow cells, coronary artery sections, or organ sections), other organic matter (e.g., plants or food), and / or other chemicals or substances.

[0032] "Tube" should be interpreted broadly to encompass any closable / sealable vessel or container capable of holding a sample during processing and is not limited to conventional cylindrical hard plastic vials with screw-on caps, but therefore also encompasses well plates, polygonal or other shaped vials, and other types of vessels and / or closure devices.

[0033] And "downstream applications" should be interpreted broadly to encompass the processing, culturing, treatment, and / or analysis of intact single cells for life science and other applications. A comprehensive list of common downstream applications includes extraction, Western, ADME / Tox, flow cytometry, genomic diagnostics, cell therapy, proteomic diagnostics, cell culture, libraries, cell imaging, and NG2 sequencing.

[0034] 1-9 illustrate a processing tube assembly 10 according to an exemplary embodiment. The tube assembly 10 may be used to dissociate a sample 2, for example, to generate output cells, most of which are intact single cells. This is done using a processing unit (not shown) operable to generate vibrations having an axial reciprocating component. The processing unit with which the tube assembly 10 may be used to generate the intended intact single cells may be conventional as defined herein.

[0035] It will be noted that the current protocol does not distinguish between recovered cells (all intact single cells, whether viable or not) and the total number of cells (which additionally includes damaged cell debris), and therefore there is no standard of measurement. In some embodiments, the majority of the output is not intact single cells, but at any rate, compared to conventional methods and devices, a significant portion of the output is intact single cells. For example, in some preliminary tests, only about 10% (e.g., about 10% to about 15%) of the output, relative to the total number, were intact single cells. Nevertheless, in most embodiments, if output is defined as recovered cells (excluding damaged cell debris), then the majority of the output cells are single and intact.

[0036] Tube assembly 10 includes a processing tube 20 and a cap 22, which are removably coupled to one another to form a tube interior chamber 24 having a longitudinal axis 26 and containing a sample 2 undergoing dissociation. Except as described herein, processing tube 20 and cap 22 may be of conventional types commercially available in the laboratory sample processing industry. For example, tube 20 may include a peripheral (e.g., cylindrical) side wall 28, a bottom wall 32, and an open top end 34, where peripheral side wall 28 defines a peripheral (e.g., cylindrical) inner surface 30 that circumferentially bounds inner chamber 24 (tube longitudinal axis 26 is parallel to inner tube surface 30), bottom wall 32 defines and bounds (closes) the bottom of inner chamber 24, and open top end 34 is for adding or removing sample 2 when cap 22 is removed. Also, cap 22 may be, for example, a screw-top design, with internal threads that mate with external threads on the upper portion of tube sidewall 24. The volume of interior chamber 24 of exemplary tube 20 may be 2 mL, 7 mL, or another standard or specialized size that may be required for a given application (e.g., 7 mL or other relatively larger tubes work better primarily for samples requiring a relatively large physical sample size, such as lung or liver samples, while 2 mL or other relatively smaller tubes work better primarily for samples not requiring a relatively large physical sample size, such as spleen samples).

[0037] In an exemplary embodiment, the tube assembly 10 holding the sample 2 does not additionally hold a grinding media (e.g., beads or active grinding media) during use for processing. In another embodiment, the tube assembly 10 holds the sample 2 and also holds a grinding media during processing.

[0038] To provide for the desired sample dissociation, tube 20 includes at least one internal thread extending from the inner surface 30 of peripheral sidewall 28 into interior chamber 24. Internal thread 40 extends circumferentially around (e.g., around) the peripheral inner surface 30 of tube 20 in a helical arrangement (i.e., a threaded arrangement). Internal helical thread 40 includes threads 42 that define two impact surfaces 46 and 48, which face in generally opposite directions and are angled relative to one another. Additionally, each of the two impact surfaces 46 and 48 is angled non-perpendicularly with respect to the inner surface 30 of tube 20 (see FIG. 3 ) and with respect to the longitudinal axis 26 of tube chamber 24 (which is parallel to and coaxial with inner surface 28).

[0039] Thus, the impact surfaces 46 and 48 of the helical thread 40 form angled surface areas that are configured / arranged (e.g., positioned / oriented) such that during use for processing (when subjected to axial reciprocating motion), the sample 2 impacts and causes a swirling flow path 50 of the sample 2 to form a vortex within the tube chamber 24. In this manner, the vortex-like angular flow 50 of the sample 2 causes the sample 2 to swirl around and collide with the internal thread 40 and the inner tube sidewall surface 30 (and, after actuation, already-dissociated sample particles), thereby dissociating the sample 2 into output cells.

[0040] Furthermore, as a result of the vortex flow 50 of the sample 2, the distance traveled by the sample 2 within the tube chamber 24 during each oscillation (compared to simply axial / linear reciprocating flow) is increased, and therefore the number of times the sample 2 impacts the inner tube sidewall surface 30 is greater than in conventional processing tubes. And the inclusion of the internal threads 40, which induces the vortex flow 50 of the sample 2, further increases the number of collisions of the sample 2. Because of this increased collision of the sample 2 (against the impact surfaces 46 and 48 of the peripheral internal threads 40 and against the inner tube surface 30) during use for processing, the sample 2 can be separated into dissociated, intact single cells at oscillation speeds low enough to prevent cell damage.

[0041] The motion of the tube 20 imparted by the processing unit creates a swirling flow path 50. In an exemplary embodiment, the helical thread 40 is oriented to enhance and / or optimize the impact of the thread 40 on the sample 2. It should be understood that because the tube 20 imparts energy to the sample 2, technically the helical thread 40 impacts the sample 2, not the sample 2 impacting the helical thread 40, and impact as referred to herein refers to impact due to relative motion between the helical thread 40 and the sample 2. It should also be understood that while the swirling vortex flow 50 is due to the tube 20 having the helical thread 40, the force and effect of the swirling vortex flow 50 can be enhanced by using a processing unit that imparts a swash motion to the tube 20.

[0042] Specifically, as a result of the vortex flow 50 of the sample 2, the sample 2 travels a longer distance within the tube chamber 24 during each oscillation, and therefore the sample 2 impacts the inner tube sidewall surface 30 more frequently than in conventional processing tubes. And, by including the internal threads 40 that induce a vortex flow of the sample 2, the sample 50 impacts the impact surfaces 46 and 48 of the internal threads 40 even more frequently. As a result, in the exemplary embodiment, during use for processing, the vortex angular flow of the sample 2 impacting the impact surfaces 46 and 48 of the peripheral internal threads 40 and the inner tube surface 30 separates the sample 2 into dissociated, intact single cells within the tube chamber 24 without the need for the use of enzymes or magnetic forces.

[0043] In some embodiments, the internal helical thread 40 extends along only a portion of the total length 52 of the inner surface 30 of the tube 20. This portion of the inner surface having the helical thread 40 may be an upper portion (adjacent the open top end 34), a lower portion (adjacent the bottom wall 32), a middle portion (between the upper and lower portions), or a combination thereof. For example, the internal helical thread 40 may extend along only a partial length 54 of the total length 52 of the inner surface 30 of the tube 20, as shown, where the partial length 52 may include the upper and middle portions of the inner surface 30, and the thread 40 may not extend to the lower portion of the inner surface 30. In testing, good results have been obtained in embodiments where the partial length 54 is greater than about 50% of the total length 52 of the inner surface 30, such as about 80% to about 90% (e.g., about 90%). In another embodiment, the internal thread extends along the entire length of the inner surface 30 of the tube 20.

[0044] Additionally, the threads 42 of the internal helical thread 40 typically extend a relatively short radial distance into the tube chamber 24, and the diameter 56 of the tube chamber 24 is significantly greater than the height 58 of the threads 42. The thread height (depth) 58 is selected to be large enough to induce a vortex flow 50 of the sample 2 and increase the surface area against which the sample impinges, but not so large as to significantly restrict or disrupt the sample flow. The thread height 58 is typically at least about 5% of the tube diameter 56, e.g., about 10% to about 30% (e.g., about 14%). In alternative embodiments, the thread height is relatively large or small relative to the tube radius.

[0045] The thread pitch (threads per inch) of the internal helical thread 40 is typically about 3 to about 32, e.g., about 15 to about 25 (e.g., about 20). In addition, the profile of the helical thread 40 typically has a thread angle of about 40 degrees to about 90 degrees, e.g., about 25 degrees to about 75 degrees (e.g., about 60 degrees), such that the opposing strike surfaces 46 and 48 are angled relative to each other and to the inner surface 30. In other embodiments, the helical thread has a different thread pitch and / or thread angle.

[0046] Also, the internal helical thread 40 in some embodiments forms a single continuous helical thread 42 (e.g., as shown). In addition, the helical thread 40 typically defines multiple (e.g., at least two) complete revolutions around the inner surface 30 of the tube 20. In other embodiments, the helical thread includes multiple thread portions in a helical arrangement, less than two complete revolutions, or a combination thereof.

[0047] In typical embodiments, the internal helical thread 40 is molded integrally with the tube 20 as a single piece (e.g., as shown). The internal helical thread 40 is also typically made of a rigid material, which may be the same hard plastic or other material that the tube 20 is made of (e.g., as shown). Furthermore, the internal helical thread 40 is typically fixed in position relative to the tube 20 and does not move relative to other threads or other elements (mating or not) of the tube 20 during use (e.g., as shown). In other embodiments, the helical thread is formed on a separate piece, which may be inserted into a standard conventional tube (e.g., as described below) and / or may be made of a relatively flexible material with high resilience to provide a spring effect that accelerates processing of the sample 2 within the tube.

[0048] 4-9, a method of dissociating a sample 2 using a tube assembly 10 is shown. This method may be performed using the tube assembly 10 of FIGS. 1-3, or any other tube assembly described herein, and the method of dissociating a sample 2 is another exemplary embodiment.

[0049] The method includes loading sample 2 and a buffer solution into tubing assembly 10. The buffer solution may include, for example, a chemical reagent, such as a conventional perfusion buffer material. In a typical embodiment, sample 2 and buffer solution occupy about 10% to about 90%, e.g., about 10% to about 30% (e.g., about 20%) of tubing chamber 24. Thus, if the volume of tubing chamber 24 of tubing assembly 10 is about 2 mL, the volume of sample 2 and buffer solution loaded into tubing assembly 10 may be about 200 μL to about 1800 μL to correspond to a range of about 10% to about 90%, about 200 μL to about 600 μL to correspond to a range of about 10% to about 30%, or about 400 μL to correspond to a value of about 20%. In general, a relatively small amount of sample 2 and buffer solution provides sufficient headspace (for the intended dissociation) but not too much (to maintain a high yield of output cells). After the sample 2 and buffer are loaded into the tube 20, the tube is sealed (eg, by a cap 22).

[0050] The sample-loaded tube assembly 10 is then mounted in a processing unit, which may be of a conventional type as described herein.

[0051] The processing unit then operates to generate a tube assembly vibration that includes an axial reciprocating component. Due to the angular flow caused by the helical thread 40, the oscillatory reciprocating motion of the tube assembly 10 can be relatively slower than speeds conventionally used for processing applications. This speed is selected so that the sample 2 is caused to flow along / across the helical thread 40 of the tube 20 without being completely interrupted by other internal forces. It is believed that when some intact single cells are dissociated, at relatively low processing speeds, the intact single cells tend to cluster together and "hide" in the recesses between the threads of the helical thread 40, providing some protection from damage; whereas at relatively high processing speeds, more of the intact single cells tend to be pulled out of these cavities and become damaged or broken.

[0052] For example, to dissociate a sample into a dissociation product / output of intact single cells, the processing unit can operate at a speed of about 0.8 m / s to about 8.0 m / s, e.g., about 0.8 m / s to about 2.4 m / s, about 0.8 m / s to about 2.8 m / s, or about 0.8 m / s to about 3.2 m / s (e.g., about 1.6 m / s), and / or for a time period of about 30 seconds to about 150 seconds, e.g., about 30 seconds to about 60 seconds (e.g., about 45 seconds). The operation time can be in cycles, e.g., one 60-second cycle, or four 15-second cycles. These operation speeds and operation times are typically appropriate for vibration stroke lengths of, e.g., about 0.8 inches to about 3.0 inches of displacement (e.g., about 1.2 inches). Of course, the processing unit is capable of other operating speeds and other operating times for other vibration stroke lengths to produce the intended output dissociated, intact single cells.

[0053] During use for processing, when the tube assembly 10 is driven by vibrations that include an axial reciprocating component, the helical thread 40 causes a circulating flow 50 of the sample 2 within the tube chamber 24, as shown in FIG. 4. Thus, as shown in FIGS. 5 and 6A, the sample 2 impinges on the first impact surface 46 when the sample flow 50 is in a first axial direction within the tube chamber 24, and then, as shown in FIGS. 5 and 6B and 7 and 8A, the processing unit operates to reverse the axial direction of the vibrations, thereby reversing the sample flow 50. Thereafter, as shown in FIGS. 7 and 8B, the sample 2 impinges on the second impact surface 48 when the sample flow 50 is in an opposite second axial direction within the tube chamber 24.

[0054] In this manner, the helical thread 40 induces a circulating (e.g., vortex-like) angular flow 50 of the sample 2, as shown in Figures 5 and 6A-B and Figures 7 and 8A-B, with the angular direction of the vortex flow 50 being the same in both axial directions. Thus, when the sample 2 moves in a first axial direction within the tube 20, the angle of attack relative to the helical thread 40 is relatively large (the angular flow 50 is more directly opposed to the helical thread 40 of the tube 20 at this stage of the vibration stroke), and when it moves in an opposite, second axial direction, the angle of attack is relatively small. Typically, the first axial direction is the same direction as the first stroke of the vibration, because the helical thread 40 generates the vortex flow 50 in one angular direction during that first stroke (the vortex flow becomes stronger when the processing unit is used to impart a swash motion to the tube). The helical thread 40 is oriented in the opposite direction to the angular direction of the second axial vortex flow 50, but the height of the helical thread 40 is small enough (and therefore the surface area with which the sample comes into contact is small) that it does not exert a force on the sample 2 sufficient to reverse the angular flow direction of the second axial direction.

[0055] Additionally, because the impact surfaces 46 and 48 are angled from the longitudinal axis of the tube chamber 24, they impart a blunt impact force and a shear force to the sample 2 during use for the dissociation process. The blunt impact force on the sample 2 is greater in the first direction than in the second direction, and the shear force on the sample 2 is greater in the second direction than in the first direction. This is because the vortex sample flow 50 is at the same angular orientation during both axial vibrations. This is because the sample flow 50 impinges on the first impact surface 46 at a first angle in the first direction (see FIGS. 5 and 6A) and on the second impact surface 48 at a second angle in the second direction (see FIGS. 7 and 8A), with the second impact angle being closer to perpendicular than the first impact angle. In some embodiments, the second impact angle is perpendicular, while the first impact angle is always non-perpendicular.

[0056] FIG. 9 further illustrates this angular flow 50 of sample 2 within a threaded tube assembly 10 (threads not shown for simplicity). The top portion of the figure shows a plot of tube axial displacement (oscillatory reciprocating motion) versus time. Below that is shown a series of tube assemblies, each of which contains sample 2 at a respective axial position corresponding to the axial displacement shown immediately above it. Below that is shown a series of tube assemblies, each of which contains sample 2 at a respective angular position corresponding to the axial position shown immediately above it. The figure generally illustrates cyclical angular sample flow over one oscillation of tube assembly 10.

[0057] As a result of the oscillatory reciprocating motion of the helically threaded tubing assembly, the sample 2 is dissociated into intact single cells, which are the output dissociated product. Furthermore, the method is able to achieve this result without the use of enzymes, magnetic forces, or moving mechanical parts within the tubing assembly 10. Thus, the method provides a completely mechanical dissociation.

[0058] It should be noted that the present methods typically include at least one pre-step prior to the processing step. The pre-step may include, for example, red blood cell (RBC) lysis, which removes all RBCs from the sample to be processed. Such a pre-step is typically performed before the sample is inserted into a tube used in the processing step, although in some embodiments the pre-step may be performed with the sample in the same tube used in the processing step.

[0059] Other method embodiments differ in that the tubes are performed with the sample additionally containing an enzyme, and therefore, these embodiments perform enzyme-assisted / enhanced mechanical dissociation, which may be included in the buffer used in the method. The enzymes may be of a mild (soft) type (class) of enzyme (i.e., milder (softer / less aggressive) than the aggressive enzymes traditionally used in enzymatic dissociation, such as serine proteases and hydrolases). That is, the enzymes are selected to generate biochemical reactions in the presence of the sample that do not degrade and / or destroy the cell walls of the sample, producing intact single cells while causing negligible transcriptomic abnormalities. For example, the enzymes may be enolase enzymes, hydroxylases, and neutral metalloproteinase enzymes (e.g., collagenase enzymes and anionic enzymes), or other enzymes that do not degrade and / or destroy the cell walls of the sample during use in the present mechanical dissociation methods. In such methods, the sample is incubated with a preparation of mild enzymes selected to assist in the release of single cells from within the sample tissue. The mild enzymes cleave the triple-helical protein chains of collagen (which are found in the extracellular matrix within the collagenous structure of all mammals), resulting in the isolation of the cells.

[0060] All of the methods disclosed herein involve mechanically (and optionally additionally enzymatically) dissociating a sample (into output dissociated intact single cells) using a threaded tube assembly and processing unit (e.g., as described herein). Another exemplary embodiment is a method of in-tube processing of a sample according to any of the processing methods disclosed herein, including a post-processing step such as further treatment and / or downstream application of the dissociated sample (in some methods, the intact single cells are used directly in the downstream application, in other methods, they are further processed prior to the downstream application).

[0061] Typically, the output dissociated sample is a heterogeneous cell population containing the desired intact single cells, plus some debris (e.g., cells that are non-viable (dead and / or damaged) and therefore unsuitable for downstream use, connective tissue, etc.). Thus, further processing may include subjecting the cells of the dissociated sample to separation, sorting, enumeration, enrichment, purification, analysis, and / or other techniques prior to downstream use. For example, further processing may include using a magnetic cell separation system (e.g., the MOJO SORT system from BioLegend, Inc.), a conventional laboratory centrifuge, or another conventional or new system for isolating, purifying, and sorting intact single cells from the heterogeneous cell population of the dissociated sample. Further processing may also include using a dual fluorescence cell counter (e.g., the CELLOMETER K2 system from Nexcelom Bioscience LLC), a high performance integrated virtual environment (HIVE) sequencing system, or another conventional or new system for counting and optionally analyzing intact single cells in a heterogeneous cell population of the dissociated sample. Downstream applications may then be of the type described herein, including flow cytometry, genomic diagnostics, cell therapy, proteomic diagnostics, cell culture, cell imaging, NG2 sequencing, extraction, Western, ADME / Tox, and / or library. Some of these downstream application steps may involve further processing using the mechanical dissociation methods disclosed herein (i.e., additional / secondary processing of the dissociated sample in the same or another threaded tube assembly using the same or another processing unit, with or without any additional steps / functions, such as filtration).

[0062] In some embodiments, the dissociated sample is transferred to another tube for further processing and / or downstream use. In another embodiment, the method further includes processing and / or downstream use of the dissociated sample while still in the same tube (which improves efficiency / yield by minimizing wasted / lost intact single cells during tube transfer). For such further processing, after processing is complete, the threaded tube assembly containing the dissociated sample is transferred and placed directly into the further processing system / instrument (e.g., centrifuge, MOJO SORT instrument, CELLOMETER K2 instrument, etc.). The dissociated sample does not need to be removed from the threaded tube assembly in which it was processed and transferred to another tube assembly until it is placed into the further processing system / instrument.

[0063] For example, in the case of further processing using a centrifuge, after processing is completed, the threaded tube assembly containing the dissociated sample is transferred directly to and placed in the centrifuge.

[0064] The centrifuge then operates to spin down the dissociated sample to separate intact single cells. These intact single cells are still in the tube chamber where the sample was loaded prior to processing, and are now separated as the supernatant from which the debris has been pushed down. This is important to remove debris from the intact single cells of the dissociated sample. The intact single cells may then be removed from the threaded tube assembly (the same one into which the sample was originally loaded). These intact single cells may then be further processed and / or used in downstream applications for various life science applications.

[0065] The pipe assembly 10 may include a helical thread in a variety of alternative configurations. Figures 10-32 illustrate just a few of the alternative helical thread configurations. Of course, various aspects of these various configurations can be combined into additional exemplary embodiments, which are not shown or described for the sake of brevity.

[0066] For example, Figures 10-13 show pipe 20 with alternative helical thread configurations (thread pitches). In Figure 10, helical thread 40a has the same high thread pitch as threaded pipe assembly 10 of Figures 1-9, except that thread 40a extends along the entire length of the pipe. In Figure 11, helical thread 40b has a slightly higher thread pitch, in Figure 12, helical thread 40c has a slightly lower thread pitch, and in Figure 13, helical thread 40d has a lower thread pitch. In alternative embodiments, the helical threads have different thread pitches. Also, the helical threads are right-handed in Figures 12-13, while left-handed in the other figures (the helical direction may be selected to enhance impact based on the motion generated by the processing unit).

[0067] Additionally, Figures 14-19 illustrate tube 20 with alternative helical thread configurations (multiple helices). Tube 20 has a double helix thread 40e (two helical threads in a double helix arrangement) in Figures 14-15, a triple helix thread 40f in Figures 16-17, and an eight-start helical thread 40g in Figures 18-19. The helical threads in these figures are multi-start or multiple-start threads / flutes intertwined to increase the number of active features while maintaining a reasonable thread count per inch. In other embodiments, the helical thread has a different helical arrangement.

[0068] FIG. 20 further illustrates nine alternative configurations (profiles) of the helical thread of the tube 20, which may be selected and used to adjust the aggressiveness of the breakaway. These may include metric, American (Unified), 60-degree stub, square, ACME, stub ACME, buttress, knuckle, and Whitworth. For internal helical threads with a square profile, the thread angle is 0 degrees, so that opposing impact surfaces are parallel to each other. In other embodiments, the helical thread has other profiles, such as serrations, castellations / notches, or undulations. In FIG. 20, D is thread height and P is thread pitch, and the illustrated dimensions are representative (but not limiting). FIG. 21 also illustrates a tube 20 with an alternative configuration of recessed helical thread 40h.

[0069] Additionally, Figures 22-32 illustrate tubes 20 having alternative helical thread (separately molded) configurations. In these embodiments, the internal helical thread is formed on a sleeve 60, which is inserted into the tube 20 to form the tube's peripheral inner surface. In Figures 22-24, the insertion sleeve 60 has the same helical thread 40i as the embodiment of Figure 10. In Figures 25-26, the insertion sleeve 60 has the same helical thread 40i but includes two additional upper slots 62; in Figures 27-28, the insertion sleeve 60 has the same helical thread 40i but includes four additional upper slots 62; in Figures 29-30, the insertion sleeve 60 has the same helical thread 40i but includes four additional upper slots 62 and four additional lower slots 62; and in Figures 31-32, the insertion sleeve 60 has the same helical thread 40i but includes two additional upper slots 62 and two additional lower slots 62. The upper slots 62 may be provided to provide a tighter fit by preventing rotation or promoting ventilation within the tube 20, and are therefore optional. In other embodiments, the insertion sleeve has slots of other configurations.

[0070] As noted above, exemplary embodiments include a processing tube assembly including an internal helical thread for use with a separately provided processing unit, as well as a method of using such a tube assembly with such a processing unit to dissociate a sample. Another embodiment includes a processing system including such a tube assembly and such a processing unit (e.g., a bead mill homogenizer that generates a swash motion of the tube assembly, including an axial reciprocating component).

[0071] Tests were conducted to demonstrate the effectiveness of selected embodiments (referred to as "Omni") and to clarify processing parameters in comparison to conventional manual mechanical dissection / dissociation (referred to as "Manual"). The test results are shown in Figure 33. Tests were conducted using intact left liver lobes (top two graphs) and whole spleens (bottom two graphs) obtained from fresh (not frozen or fixed) mouse tissue samples. The reported / illustrated test results are the average of five Omni tests and three manual tests (N), with a standard deviation / variance (P) less than 0.05. Total cell counts were recorded and presented in the figures as cells per mL of sample.

[0072] The selected embodiment used in the Omni test included a BEADRUPTOR ELITE bead mill homogenizer (Omni International Inc.) as the processing unit and an L-20 2 mL processing tube (Omni International Inc.) adapted to include the internal helical thread shown and described with respect to Figures 1-9. Specifically, the processing tube used in the Omni test included an internal helical thread forming a continuous helical thread extending along the entire length of the interior chamber of the tube, the helical thread being made of the same polymeric material as the tube, and the helical thread having 20 threads per inch and a 60-degree thread angle. The homogenizer processing unit was operated at a speed of 1.6 m / s for 35 seconds.

[0073] Additionally, the Omni test treatment tube contained the sample and PBS buffer containing collagenase IV enzyme and DNASE I enzyme. No perfusion was performed on the sample.

[0074] In contrast, dissection / dissociation in manual studies was performed in a conventional manner: without the use of power tools, samples were dissociated manually in Petri dishes by vigorous and forceful agitation with tweezers for 20 minutes (agitation fast enough and forceful enough to be reasonably sustained by a graduate student with average adult physical ability). No enzymes, no buffers, and no perfusion of samples were used.

[0075] As can be seen in Figure 33, the total number of cells generated was greater with the Omni method than with the manual method. This is shown in the upper left graph for hepatocytes and the lower left graph for splenocytes. Furthermore, as shown, of those total number of cells generated, the Omni method generated more intact single cells (labeled intact / viable) than the manual method. This is shown in the upper right graph for hepatocytes and the lower right graph for splenocytes. These test results demonstrate that the Omni method achieves significantly higher throughput than the manual method, generating significantly more intact single cells in a much shorter time.

[0076] These intact single cells are now ready for downstream use (e.g., tissue culture or analysis, or other downstream uses disclosed herein), or further processing prior to such downstream use. As noted above, further processing may include cell sorting (e.g., using the MOJO SORT system). Additional testing was performed to demonstrate the effectiveness of combining the Omni processing method with a post-processing sorting step. The results are shown in Figure 34. The left graph shows the post-processing results for hepatocytes generated using the Omni method of Figure 33, and the right graph shows the post-processing results for splenocytes generated using the Omni method of Figure 33. The sorting step was performed using the MOJO SORT system, specifically, using MOJO SORT Human CD4 Nanobeads (#480013) and MOJO SORT Human CD19 Sorting Kit (#480105).

[0077] As can be seen in Figure 34, the Omni method produced a large number of whole cells, of which a high percentage were intact single cells (labeled intact / viable), as confirmed by post-processing sorting steps. In addition, the results showed a relatively high percentage of CD3 and CD19 (T and B cells), demonstrating the accuracy of the results.

[0078] There is always the possibility of variability between biological samples from different regions of a given tissue, and there is always the possibility of differences between different organisms. A gross anatomical evaluation of each animal was performed to rule out obvious abdominal lesions. However, without histopathological and molecular analysis of all tissues utilized in these experiments, it is not possible to guarantee the absence of lesions in all animals utilized in these experiments. Prior to tissue dissociation experiments, to reduce variability in sample preparation, all samples from mouse models for each included experiment were prepared in the same manner by the same scientist.

[0079] It should be understood that the present disclosure is not limited to the particular apparatus, methods, conditions, or parameters of the exemplary embodiments described and / or illustrated herein, and that the terminology used herein is intended solely for the purpose of describing particular embodiments by way of example. Accordingly, such terms are intended to be broadly interpreted and are not intended to unnecessarily limit the claimed subject matter. For example, in this specification, including the appended claims, the singular forms "a," "an," and "the" include the plural, the term "or" means "and / or," and references to specific numerical values ​​include at least that particular value, unless the context clearly contradicts otherwise. Furthermore, all uses of the terms "about," "substantially," and / or "generally" are intended to refer to the exact value or characteristic specified, as well as to an approximation that one of ordinary skill in the art would understand to be sufficiently close to the exact value or characteristic, given the context of the intended use and application. Additionally, unless expressly stated otherwise herein, all methods described herein are not limited to the order of steps described and may be performed in other orders.

[0080] While the claimed embodiments have been shown and described in an illustrative manner, it will be apparent to those skilled in the art that various modifications, additions, and deletions may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.

Claims

1. 1. A laboratory processing tube assembly mounted to a processing unit for dissociating organic-derived samples by tube assembly vibration including an axial reciprocating component, said tube assembly comprising: a tube and a cap removably coupled to one another to form a tube chamber, the tube chamber having a longitudinal axis, the tube chamber containing the sample during dissociation, the tube having a peripheral inner surface; at least one internal thread extending inward from and circumferentially around the inner surface of the tube in a helical arrangement, the helical internal thread including threads defining two impact surfaces facing generally opposite each other at a non-perpendicular angle relative to the longitudinal axis of the tube chamber and to the inner surface of the tube, wherein the sample impacts a first one of the impact surfaces when it flows through the tube chamber in a first axial direction and a second one of the impact surfaces when it flows through the tube chamber in an opposite second axial direction during the axial reciprocating tube assembly oscillation to dissociate the sample; a laboratory processing tube assembly,

2. 10. The laboratory processing tube assembly of claim 1, wherein the sample impacts the impact surface of the helical internal thread during use for dissociation, separating the sample into dissociated intact single cells, wherein no enzymes or magnetic forces are used in the tube chamber, or wherein mild enzymes are used in the tube chamber.

3. 10. The laboratory processing tube assembly of claim 1, wherein the impact surface is angled from the longitudinal axis of the tube chamber to impart a blunt impact force to the sample and a shear force to the sample during use for dissociation.

4. 4. The laboratory process tube assembly of claim 3, wherein the internal helical thread induces an angular flow of the sample, the blunt impact force imparted by the internal helical thread to the angular flow of the sample is greater in a first direction than in a second direction, and the shear force imparted by the internal helical thread to the sample is greater in the second direction than in the first direction.

5. 10. The laboratory process tubing assembly of claim 1, wherein the internal helical thread forms a single continuous thread.

6. 10. The laboratory process tube assembly of claim 1, wherein the internal helical threads extend along 80% to 90% of the total length of the inner peripheral surface of the tube.

7. 2. The laboratory process tube assembly of claim 1, wherein the internal helical thread defines at least two complete revolutions around the inner peripheral surface of the tube.

8. 10. The laboratory process tubing assembly of claim 1, wherein the internal helical thread has a thread pitch (threads per inch) of 15 to 25.

9. 2. The laboratory process tube assembly of claim 1, wherein the internal helical thread has a square profile with a 0 degree thread angle so that the opposing impact surfaces are parallel to one another.

10. 10. The laboratory process tube assembly of claim 1, wherein the internal helical thread has a profile with a thread angle between 25 degrees and 75 degrees such that the opposing impact surfaces are angled relative to one another.

11. 2. The laboratory process tube assembly of claim 1, wherein the internal helical threads are formed on a sleeve, the sleeve being inserted into the tube to form the inner peripheral surface of the tube.

12. 10. The laboratory process tubing assembly of claim 1, wherein the internal helical thread is formed by two threads in a double helix arrangement.

13. 1. A laboratory dissociation system for dissociating a sample, comprising: a processing unit for generating a tube assembly vibration including an axial reciprocating component; a tube assembly mounted to the processing unit, the tube assembly comprising: a tube and a cap removably coupled to one another to form a tube chamber, the tube chamber having a longitudinal axis, the tube chamber containing the sample during use for dissociation, the tube having a peripheral inner surface; and at least one internal thread extending inwardly from and circumferentially around the peripheral inner surface in a helical arrangement, the helical internal thread including threads defining two impact surfaces facing generally opposite one another at an angle non-perpendicular to the longitudinal axis of the tube chamber and to the inner surface of the tube, wherein the sample impacts a first one of the impact surfaces when flowing through the tube chamber in a first axial direction and the sample impacts a second one of the impact surfaces when flowing through the tube chamber in an opposite second axial direction during the axial reciprocating tube assembly oscillation to dissociate the sample; Laboratory dissociation system including:

14. 14. The laboratory dissociation system of claim 13, wherein the laboratory processing unit is a bead mill homogenizer.

15. 15. The laboratory dissociation system of claim 14, wherein the tube assembly motion generated by the bead mill homogenizer is a swash motion that includes an axial reciprocating component.

16. 1. A laboratory sample dissociation method comprising: loading a sample and a buffer into a tube assembly, the tube assembly including a tube and a cap removably coupled to one another to form a tube chamber, the tube chamber having a longitudinal axis, the tube chamber containing the sample during use for dissociation, the tube having a peripheral inner surface, and at least one internal thread extending inwardly from and circumferentially around the inner surface in a helical arrangement, the helical internal thread including threads defining two impact surfaces facing generally opposite one another at a non-perpendicular angle to the longitudinal axis of the tube chamber and to the inner surface of the tube; mounting the loaded tube assembly in a processing unit; operating the processing unit to generate a tube assembly motion that includes an axial reciprocating component, such that during the axially reciprocating tube assembly vibration to dissociate the sample, the sample impacts a first one of the impact surfaces when flowing in a first axial direction within the tube chamber and impacts a second one of the impact surfaces when flowing in an opposite second axial direction within the tube chamber, wherein no enzyme or magnetic force is used within the tube chamber, or a mild enzyme is used within the tube chamber; A laboratory sample dissociation method comprising:

17. 17. The laboratory sample dissociation method of claim 16, wherein the step of loading a sample and a buffer comprises loading an organic-derived sample and a perfusion buffer into a tubing assembly, and wherein the step of operating the processing unit comprises dissociating the sample into dissociated intact single cells.

18. 17. The laboratory sample dissociation method of claim 16, wherein the step of loading a sample and a buffer comprises loading the sample and the buffer into a tube assembly such that the sample and the buffer occupy between 20% and 90% of the tube chamber.

19. 17. The laboratory sample dissociation method of claim 16, wherein the step of operating the processing unit comprises operating at a speed of between 0.8 m / s and 1.6 m / s, or for a time period of between 30 seconds and 60 seconds, or both.

20. 17. The laboratory sample dissociation method of claim 16, further comprising placing the processed tube assembly directly into a centrifuge without transferring the dissociated sample to another tube assembly, and using the centrifuge to spin down the dissociated sample to separate intact single cells from extracellular debris.

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