Kit for processing biological samples, device and method thereof

Abstract

Methods and systems for enhancing the separation of cells from solid biological tissue samples are described. In some embodiments, a stand-alone kit device includes a first chamber for receiving the tissue sample, enabling ultrasonic energy from a transducer assembly of a processing unit to separate cells from the sample in the first chamber and collect living cells of interest from an aqueous suspension in a second chamber fluidically connected to the first chamber via a channel. In some embodiments, to enhance the separation of living cells, a filter device includes a tubular body configured to be telescopically inserted into a container containing the tissue sample in an aqueous fluid. The filter device also includes a cell strainer covering a bottom opening of the tubular body and configured to compress the tissue sample to expel cells from the sample when the filter device is fully inserted into the container.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 909,476, filed on October 2, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates generally to systems and methods for processing solid biological tissue samples in a self-contained kit, and more particularly, for isolating single cells from a biological tissue sample in a self-contained kit using ultrasonic energy. Background Art

[0004] Traditional methods for isolating single cells from solid biological tissue samples require the use of proteolytic enzymes, incubation at 37°C, and mechanical disruption to digest or disrupt cell adhesion molecules and / or the underlying extracellular matrix. For example, mechanical disruption can include manual trituration (e.g., moving the minced tissue up and down in a pipette) or a mechanical mixer / disruptor (e.g., MiltenyigentleMACS). TM Separator), followed by centrifugation and pouring the processed tissue sample through a filter to remove the enzymes and recover the separated viable cells in a cell suspension (e.g., an aqueous suspension).

[0005] In some embodiments, the present invention relates to the method for separating the cell mass of interest from the living tissue sample. For example, the cell mass of interest of the living tissue sample is separated by enzyme and mechanical destruction. However, it is inefficient to use enzyme and mechanical destruction to carry out cell separation, and causes the high loss or degradation of the cell mass of interest. For example, due to the physical stress of pipetting, centrifugation and filtration treatment, and during the period when the tissue sample is transferred from one container to another container for treatment, many cells are lost or broken (i.e., no longer complete). In addition, due to the concentration of the enzyme used, the enzyme used, the treatment temperature and the long incubation time required for using these enzymes, the living cell mass can be lost. The stress caused by enzymolysis also causes the rise of proinflammatory and stress-induced genes that do not exist in the original tissue sample. Therefore, the gene expression profile of the separated cell of interest is usually different from the gene expression profile of the original tissue sample.

[0006] Current cell separation methods are also typically manual processes that require users to transfer tissue samples from one container to another to obtain viable, isolated cells. For example, users may need to mix enzymes and a biological tissue sample in a first container, transfer the mixture to a second container to perform mechanical disruption to obtain isolated cells, and then transfer the mixture from the second container to a third container to filter the mixture to obtain the viable cells of interest. This process of transferring the mixture exposes the tissue sample to environmental contaminants, which can make the experiment or test inaccurate. Summary of the Invention

[0007] As described above, the use of enzymes and mechanical disruptors to isolate single cells from biological tissue samples is suboptimal and results in high loss or degradation of the cell population of interest. Therefore, there is a need for systems, methods, and techniques for improving cell isolation while preserving the gene expression profiles of viable cells isolated from tissue samples.

[0008] In some embodiments, instead of using enzymes to perform cell separation, ultrasonic processing devices can be used to direct ultrasonic energy to a tissue sample to perform cell separation. This approach eliminates the drawbacks discussed above regarding the use of enzymes. While ultrasonic energy-based separation methods can improve the quality and quantity of isolated cells compared to enzymatic separation, the results achieved are limited by the need to perform necessary sample processing steps in other devices before and after applying ultrasonic energy to the tissue sample.

[0009] In particular, conventional liquid handling techniques need to be integrated to complete the workflow from solid tissue samples to suspended cells for safe transfer to downstream cell separation and analysis techniques. These conventional techniques include mincing tissue samples in open culture dishes, pipetting the minced tissue before separation, and pipetting (e.g., aspirating and dispensing) the separated samples along with enzymes or chemical reagents, as well as transporting samples between various containers, centrifuges, ice baths, instruments, and filtration devices to isolate cells of interest in a clean suspension. As described above, conventional liquid handling techniques are time-intensive and expose tissue samples to environmental contamination.

[0010] In some embodiments, a system for processing an improved solid biological tissue sample includes a stand-alone test kit device configured to load the tissue sample so that ultrasonic energy can separate single cells from the tissue sample in suspension and collect the living cells of interest from the suspension without the need for pipettes, centrifugation or enzymes, heating, and extended processing time. By performing the cell separation and living cell collection process within the stand-alone test kit device, the improved system achieves higher quality results because the separated cells are not exposed to the external environment. In addition, since enzymes are not used in the test kit device, the gene expression profile of the separated cells in the test kit device is closely consistent with the gene expression profile of the natural tissue (i.e., the tissue sample initially loaded into the test kit device).

[0011] In some embodiments, an apparatus for separating single cells from a solid biological tissue sample comprises: a housing unit configured to enable the apparatus to be loaded into an ultrasonic processing device; a first chamber configured to receive the solid biological tissue sample and an aqueous fluid, wherein when the apparatus is loaded into the processing device, the first chamber is aligned with a transducer assembly of the processing device so that ultrasonic energy applied by the processing device can separate cells from the biological tissue sample, thereby producing an aqueous suspension; a channel fluidically connecting the first chamber to a second chamber; and the second chamber configured to collect the separated cells in the aqueous suspension flowing from the first chamber through the channel.

[0012] In some embodiments, the second chamber is detachable from the device.

[0013] In some embodiments, the first chamber comprises a first inlet for receiving the solid tissue sample and the aqueous fluid from outside the device. In some embodiments, the first chamber comprises a lid to seal the received solid tissue sample and the aqueous fluid from the external environment.

[0014] In some embodiments, the first chamber comprises a bottom surface that is acoustically conductive to allow the ultrasonic energy to enter the first chamber to separate cells from the biological tissue. In some embodiments of the device, the bottom surface comprises a planar layer that is configured to couple to the transducer assembly when the device is loaded into the processing device.

[0015] In some embodiments, the aqueous fluid comprises a cell culture medium solution.

[0016] In some embodiments, the apparatus comprises a connector component coupled to the channel and configured to connect to an actuator of the processing device, wherein the actuator is configured to control the flow of a fluid through the channel. In some embodiments, the actuator comprises a pump or a plunger. In some embodiments, the connector component comprises a fitting or adapter configured to connect the channel and the actuator.

[0017] In some embodiments, the apparatus includes a valve positioned in the channel and configured to be controlled by an actuation device of the processing device to control fluid flow through the channel.

[0018] In some embodiments, the device includes a third chamber comprising a third inlet for receiving a second aqueous fluid into the interior of the device. In some embodiments, the device includes a channel fluidically coupling the third chamber to the first chamber to allow the second aqueous fluid to flow into the first chamber.

[0019] In some embodiments, the device comprises a series of two or more filtration chambers connecting the first chamber to the second chamber, wherein the channel comprises a plurality of sub-channels that fluidically connect successive chambers in the series of two or more filtration chambers so that the aqueous fluid flows from the first chamber to the second chamber. In some embodiments of the device, the two or more filtration chambers are configured to capture effluent material in the aqueous fluid as the aqueous fluid flows through the plurality of sub-channels so that target components can be collected in the second chamber. In some embodiments, the effluent includes non-target components, cellular debris, and extracellular debris.

[0020] In some embodiments, a filtration chamber from the two or more filtration chambers includes one or more marker beads configured to bind to the effluent material to prevent the effluent material from flowing through the filtration chamber.

[0021] In some embodiments, a filter chamber from the two or more filter chambers is configured to align with a second transducer assembly of the treatment device when the apparatus is loaded into the treatment device, and wherein the filter chamber includes a bottom surface that acoustically couples the filter chamber with the second transducer assembly to receive ultrasonic energy from the treatment device to promote selective flow of the target component.

[0022] In some embodiments, the second chamber is configured to collect the intracellular or extracellular target analyte, and wherein the device comprises a one-way channel fluidly connecting the second chamber to the first chamber to allow aqueous fluid to flow back to the first chamber.

[0023] In some embodiments, the plurality of subchannels are configured to allow the aqueous fluid to flow back through the series of two or more filtration chambers without flowing back into the first chamber.

[0024] In some embodiments, a method for isolating single cells from a solid biological tissue sample comprises: introducing the solid biological tissue sample and an aqueous liquid into a first chamber of a test kit apparatus; loading the test kit apparatus into an ultrasonic processing device, wherein the first chamber is aligned with a transducer assembly of the processing device; activating the ultrasonic processing device to apply ultrasonic energy from the transducer assembly to ultrasonically separate cells from the tissue sample, thereby generating an aqueous suspension in the first chamber; and controlling the flow of the aqueous suspension from the first chamber to the second chamber of the test kit apparatus, wherein the second chamber is configured to collect the separated cells in the aqueous suspension.

[0025] In some embodiments, the method comprises mincing the tissue sample within the first chamber. In some embodiments, mincing the tissue sample comprises inserting a pestle and mortar into the container; and mincing the tissue sample using the inserted pestle and mortar.

[0026] In some embodiments, the method comprises separating the second chamber from the kit device.

[0027] In some embodiments, the method includes receiving the solid tissue sample and the aqueous fluid from outside the device via a first inlet of the first chamber.

[0028] In some embodiments, the first chamber comprises a lid, and the method comprises sealing the received solid tissue sample and the aqueous fluid from the external environment by closing the lid.

[0029] In some embodiments, the first chamber comprises a bottom surface that is acoustically conductive to allow the ultrasonic energy to enter the first chamber to separate cells from the biological tissue. In some embodiments, the bottom surface comprises a planar layer, and the method comprises coupling the planar layer of the test kit apparatus to the transducer assembly when the test kit apparatus is loaded into the processing device.

[0030] In some embodiments, the aqueous fluid comprises a cell culture medium solution.

[0031] In some embodiments, the kit device includes a connector component coupled to the channel, and the method includes connecting the channel to an actuator of the processing device using the connector component, wherein the actuator is configured to control the flow of a fluid through the channel. In some embodiments, the actuator includes a pump or a plunger. In some embodiments, the connector component includes a fitting or adapter configured to connect the channel and the actuator.

[0032] In some embodiments, the kit apparatus includes a valve positioned in the channel, and the method includes operating the valve via an actuation device of the processing device to control fluid flow through the channel.

[0033] In some embodiments, the kit device comprises a third chamber comprising a third inlet, and the method comprises receiving a second aqueous fluid into the interior of the device via the third inlet.

[0034] In some embodiments, the method includes fluidly coupling the third chamber to the first chamber via a channel to allow the second aqueous fluid to flow into the first chamber.

[0035] In some embodiments, the kit device includes a series of two or more filtration chambers connecting the first chamber to the second chamber, and wherein the channel includes a plurality of sub-channels that fluidly connect successive chambers in the series of two or more filtration chambers to allow the aqueous fluid to flow from the first chamber to the second chamber.

[0036] In some embodiments, the method includes aligning a filter chamber from the two or more filter chambers with a second transducer assembly of the processing device when the test kit apparatus is loaded in the processing device, wherein the filter chamber includes a bottom surface that acoustically couples the filter chamber with the second transducer assembly to receive ultrasonic energy from the processing device to promote selective flow of the target component.

[0037] In some embodiments, a filter device for enhancing the separation of living cells from a solid biological tissue sample comprises: a tubular body configured to be telescopically inserted into a container containing a biological tissue sample in an aqueous fluid; and a cell strainer covering a bottom opening of the tubular body, wherein the cell strainer is configured to compress the biological tissue sample to expel cells from the tissue sample when the filter device is fully inserted into the container.

[0038] In some embodiments, the cells are expelled into a surrounding aqueous fluid that flows through the cell strainer into the center of the tubular body.

[0039] In some embodiments, the filter device comprises a transport channel that transports the draining cells that flow into the center of the tubular body to a collection chamber. In some embodiments, the transport channel comprises a tube within the interior of the tubular body and extending along the length of the tubular body.

[0040] In some embodiments, a lower portion of the tubular body is submerged in the container when the tubular body is fully inserted into the container, and wherein the lower portion comprises one or more openings covered by one or more corresponding cell strainers.

[0041] In some embodiments, the cells are expelled into a surrounding aqueous fluid that flows into the center of the tubular body through the one or more cell strainers corresponding to the one or more openings in the lower portion.

[0042] In some embodiments, the tubular body is configured to extend into the container at most a predetermined distance to enable the cell strainer to compress the biological tissue sample.

[0043] In some embodiments, the tubular body includes a raised portion that prevents the tubular body from extending into the container beyond the predetermined distance.

[0044] In some embodiments, the container comprises a stopper material at the bottom of the container, the stopper material preventing the tubular body from extending into the container beyond the predetermined distance. In some embodiments, the stopper material comprises sponge, rubber, or plastic.

[0045] In some embodiments, the biological tissue sample is ultrasonically disaggregated into cells prior to being compressed by the cell strainer.

[0046] In some embodiments, the biological tissue sample is enzymatically disrupted into cells before being compressed by the cell strainer.

[0047] In some embodiments, the container corresponds to a reaction chamber of a kit apparatus.

[0048] In some embodiments, a method for enhancing the separation of living cells from a solid biological tissue sample includes: telescopically inserting the tubular body of the filter device into a container containing the biological tissue sample in an aqueous fluid, wherein the filter device includes a cell strainer covering a bottom opening of the tubular body; fully inserting the tubular body of the filter device into the container to compress the biological tissue sample with the cell strainer, wherein the cell strainer compresses the biological tissue sample to expel cells from the tissue sample.

[0049] In some embodiments, the method includes: withdrawing the tubular body of the filter device from the container; and repeatedly performing full insertion and withdrawal of the tubular body of the filter device to repeatedly compress the biological tissue sample to increase the amount of cells discharged.

[0050] In some embodiments, the cells are expelled into a surrounding aqueous fluid that flows through the cell strainer into the center of the tubular body.

[0051] In some embodiments, the method comprises delivering the expelled cells that flow into the center of the tubular body to a collection chamber. In some embodiments, the delivery channel comprises a tube within the interior of the tubular body and extending along the length of the tubular body.

[0052] In some embodiments, the method comprises submerging a lower portion of the tubular body in a container when the tubular body is fully inserted into the container, wherein the lower portion comprises one or more openings covered by one or more corresponding cell strainers.

[0053] In some embodiments, the cells are expelled into a surrounding aqueous fluid that flows into the center of the tubular body through the one or more cell strainers corresponding to the one or more openings in the lower portion.

[0054] In some embodiments, the method includes extending the tubular body into the container up to a predetermined distance to enable the cell strainer to compress the biological tissue sample.

[0055] In some embodiments, the tubular body includes a raised portion that prevents the tubular body from extending into the container beyond the predetermined distance.

[0056] In some embodiments, the container comprises a stopper material at the bottom of the container, the stopper material preventing the tubular body from extending into the container beyond the predetermined distance. In some embodiments, the stopper material comprises sponge, rubber, or plastic.

[0057] In some embodiments, the method includes ultrasonically separating the tissue sample into cells prior to telescopically inserting the tubular body of the filter to compress the tissue sample through the cell strainer.

[0058] In some embodiments, the method includes enzymatically dissociating the tissue sample into cells prior to telescopically inserting the tubular body of the filter to compress the tissue sample through the cell strainer.

[0059] In some embodiments, the container corresponds to a reaction chamber of a kit apparatus.

[0060] In some aspects, provided herein is a kit for isolating and obtaining separate single cells from a biological sample, the kit comprising: (a) a sample processing unit comprising: a sealable port configured to receive the biological sample for introduction into the sample processing unit; a sample chamber configured to collect the biological sample; and a grinder, wherein the grinder is configured to interface with the sample chamber; (b) a reaction unit comprising a reaction chamber configured to receive ultrasonic energy, wherein the sample processing unit is connected to the reaction unit via a first channel; (c) a filtration unit comprising: a filtration chamber; and a filtration device configured to enter the filtration chamber, wherein the reaction chamber is connected to the filtration unit via a second channel; and (d) a collection unit configured to receive separate single cells from the biological sample, wherein the filtration unit and the collection unit are connected via a third channel.

[0061] In some embodiments, the test kit further includes a first gate, which is configured to control the fluid connection between: a fluid connection between the sample processing unit and the reaction unit via the first channel; and / or a fluid connection between the reaction unit and the filtration unit via the second channel. In some embodiments, the first gate is configured to be positioned in a first position, and wherein when the first gate is in the first position, the sample processing unit and the reaction unit are fluidically connected, and the reaction unit and the filtration unit are not fluidically connected. In some embodiments, the first gate is configured to be positioned in a second position, and wherein when the first gate is in the second position, the reaction unit and the filtration unit are fluidically connected, and the sample processing unit and the reaction unit are not fluidically connected.

[0062] In some embodiments, the test kit further includes a second gate configured to control the fluid connection between the filtration unit and the collection unit via the third channel. In some embodiments, the second gate is configured to be positioned in a first position, wherein when the second gate is in the first position, the filtration unit and the collection unit are not fluidically connected. In some embodiments, the second gate is configured to be positioned in a second position, wherein when the second gate is in the second position, the filtration unit and the collection unit are fluidically connected.

[0063] In some embodiments, the first channel, the second channel, and the third channel are positioned and configured so that the flow between the sample processing unit, the reaction unit, the filtration unit, and the collection unit is controlled by tilting the reagent box. In some embodiments, the tilting of the reagent box occurs through a single axis. In some embodiments, the single axis is substantially perpendicular to any one or more of the following axes: an axis substantially parallel to the rod of the grinder and / or the direction of movement of the grinder; an axis substantially perpendicular to the bottom of the reaction chamber; and an axis substantially parallel to the movement of the filtration device. In some embodiments, the sample processing unit, the reaction unit, the filtration unit, and the collection unit are configured to be placed on a plane, wherein the single axis of the tilt of the reagent box is substantially perpendicular to the plane.

[0064] In some embodiments, the sealable port of the sample processing unit is further configured to receive a fluid for introduction into the sample processing unit. In some embodiments, the sealable port comprises a wall, wherein the sealable port of the sample processing unit is inclined toward the sample chamber.

[0065] In some embodiments, the kit further comprises a cap configured to seal the sealable port of the sample processing unit.

[0066] In some embodiments, the sample chamber comprises one or more walls that slope toward the center.

[0067] In some embodiments, the reaction chamber of the reaction unit comprises a sound-conducting bottom surface. In some embodiments, the bottom surface of the reaction chamber is substantially flat. In some embodiments, the reaction chamber of the reaction unit comprises cylindrical walls.

[0068] In some embodiments, the filtration device of the filtration unit comprises a tubular assembly, wherein an outer surface of the tubular assembly is configured to provide a seal with a sidewall of the filtration chamber, and wherein the tubular assembly comprises a cell strainer covering a bottom opening of the tubular assembly. In some embodiments, the sidewall of the filtration chamber is cylindrical.

[0069] In some embodiments, the filtration chamber of the filtration unit comprises a bottom surface having a raised structure. In some embodiments, the filtration unit comprises a vent.

[0070] In some embodiments, the collection unit is configured to receive a removable collection device for receiving a single cell from the biological sample. In some embodiments, the removable collection device is an Eppendorf tube.

[0071] In some embodiments, the kit is configured to be loaded into an ultrasonic processing device.

[0072] In some aspects, provided herein is an ultrasonic processing device comprising: (a) an ultrasonic transducer; (b) a temperature-controlled water bath; (c) a tilting assembly, wherein the tilting assembly is configured to hold the reagent kit according to any one of embodiments 65-86 so that (i) the reaction chamber of the reagent kit is positioned relative to the ultrasonic transducer, and (ii) the reaction chamber, sample chamber, and filtration chamber of the reagent kit, or portions thereof, are positioned relative to the water level of the temperature-controlled water bath, the tilting assembly comprising: a tilt actuator; a mincer actuator; and a filter actuator; and (d) a control unit.

[0073] In some embodiments, the ultrasonic processing apparatus further includes one or more gate actuators, each of which is configured to interface with the gate of the test kit. In some embodiments, the tilting assembly is configured to hold the test kit so that when a removable collecting device is placed in the collection unit of the test kit, the removable collecting device or a portion thereof is positioned relative to the water level of the temperature-controlled water bath. In some embodiments, the reaction chamber, the sample chamber, the filtration chamber, and the removable collecting device or a portion thereof are below the water level of the temperature-controlled water bath.

[0074] In some embodiments, the mincer actuator comprises a clamp that interfaces with a mincer of the kit. In some embodiments, the filter actuator comprises a clamp that interfaces with a filtration device of the kit.

[0075] In some embodiments, the control unit comprises: one or more processors; and a memory storing one or more programs, the one or more programs being configured to be executed by the one or more processors, the one or more programs comprising instructions for executing a method for isolating and obtaining separate single cells from a biological sample. In some embodiments, the instructions for executing the method for isolating and obtaining separate single cells from a biological sample comprise instructions for any one or more of: operating the ultrasonic transducer; operating the tilt actuator; operating the mincer actuator; operating the filter actuator; operating one or more gate actuators; and operating the temperature-controlled water bath.

[0076] In some aspects, provided herein is a method for obtaining separated single cells from a biological sample, the method comprising: loading the kit of any embodiment described herein into the ultrasonic processing device of any embodiment described herein; loading the biological sample into the sample processing unit of the kit via a sealable port; and initiating a programmed method of the ultrasonic processing device to separate and obtain separated single cells from the biological sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The foregoing summary and the following detailed description can be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, the accompanying drawings show example embodiments of the present disclosure; however, the present disclosure is not limited to the specific methods and tools disclosed. In the drawings:

[0078] Figure 1 shows a cross-sectional view of a system for processing a solid biological tissue sample in a kit device according to some embodiments;

[0079] Figure 2shows a schematic diagram of a kit device for processing a solid biological tissue sample according to some embodiments;

[0080] Figure 3 A kit device for processing a solid biological tissue sample according to some embodiments is shown;

[0081] Figure 4 A system for enhancing the separation of cells from a solid biological tissue sample is shown, according to some embodiments;

[0082] Figure 5 A method for operating a standalone kit device to separate a solid biological tissue sample into viable cells is shown according to some embodiments;

[0083] Figure 6 Methods for operating a filter device to enhance separation of viable cells from a solid biological tissue sample are shown according to some embodiments;

[0084] Figure 7A is a graph showing representative changes in the gene expression profile of an untreated pig liver tissue sample across all EnsemblS scrofa11.1 genes when the pig liver tissue sample is separated using ultrasonic energy and enzymes;

[0085] Figure 7B is a graph showing representative changes in the gene expression profile of untreated porcine liver tissue samples at selected pro-inflammatory and stress-induced genes of the Ensembl Scrofa11.1 gene set when the porcine liver tissue samples were separated using ultrasonic energy and enzymes;

[0086] Figure 8 is a table showing representative numbers of viable cells obtained when pig liver tissue was ultrasonically separated and ultrasonically separated and filter pressed;

[0087] Figure 9A is a graph showing representative numbers of viable cells per milligram obtained from various sized samples of pig liver tissue by applying ultrasonic separation and filtration in the kit apparatus; and

[0088] Figure 9B is a graph showing representative numbers of viable cells per milligram of various sizes obtained from pig liver tissue samples of various sizes by applying ultrasonic separation and filtration in the kit apparatus.

[0089] Figure 10A A schematic diagram of the kits described herein is shown. Figure 10B and 10C A photograph showing the reagent cartridge in a tilted assembly. DETAILED DESCRIPTION

[0090] In some aspects, provided herein is a kit for processing a biological sample including a plurality of cells to obtain a composition including a single cell separated from a plurality of cells. In some embodiments, the kit is a disposable kit for single use. In some embodiments, the kit is configured to subject a biological sample or a portion thereof to a separation force, such as ultrasonic energy. In some embodiments, the kit is configured to receive a biological sample, such as a solid biological tissue sample, perform preliminary processing of the biological sample, facilitate application of ultrasonic body wave energy to separate single cells in the biological sample, and filter and collect living cells of interest from the separated cells without substantially changing the separated single cells compared to their state in the biological sample, such as substantially changing the gene expression profile of the separated single cells. In some embodiments, ultrasonic body wave energy can be generated by an ultrasonic processing device as described in reference to U.S. Patents Nos. 8,319,398, 8,127,614, and 6,682,214, the contents of each of which are incorporated herein by reference in their entirety. In other aspects, described herein are systems, devices, and methods for processing biological samples, such as solid biological tissue samples, in a stand-alone kit as described herein.

[0091] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0092] Reference herein to "about" a value or parameter includes (and describes) variations with respect to the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0093] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof, are intended to be equivalent in meaning and to be open-ended in that the one or more items following any of these words are not intended to be an exhaustive list of such one or more items, or to be limited to only the listed one or more items. For example, an article that "comprising" components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may contain not only components A, B, and C, but may also contain one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and grammatical equivalents thereof include disclosure of embodiments that "consist essentially of" or "consist of."

[0094] It should be understood that aspects and variations of the present invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0095] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other stated or intervening value in that range is included within the scope of the present disclosure. Where the stated range includes an upper limit or a lower limit, ranges excluding any of those included limits are also included in the present disclosure.

[0096] The description of the present invention is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles herein may be applied to other embodiments. Therefore, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features described herein.

[0097] The disclosures of all publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. If any reference incorporated by reference conflicts with the present disclosure, the present disclosure controls.

[0098] In some aspects, the kits described herein are configured to perform any one or more of the following steps: (i) receiving a biological sample; (ii) receiving an additional substance input useful for processing the biological sample, such as an aqueous solution, e.g., a buffer; (iii) processing the biological sample, such as by mincing; (iv) subjecting the biological sample or a portion thereof to a separation force, such as ultrasonic energy; (v) filtering the biological sample or a portion thereof to separate individual cells of the biological sample; and (vi) collecting the separated individual cells of the biological sample. In some embodiments, the kit includes a module that can be used to perform functions for processing a biological sample. In some embodiments, the kit includes a module that is configured to perform one or more functions related to obtaining the separated individual cells of the biological sample. In some embodiments, the kit includes a plurality of modules, each of which is configured to perform a specific function or a set of functions involved in obtaining the separated individual cells of the biological sample. For example, in some embodiments, the kit includes a sample processing unit, a reaction unit, a filtration unit, and a collection unit. In some embodiments, the sample processing unit is configured to receive the biological sample and any additional substances input into the kit, such as an aqueous solution, e.g., a buffer. In some embodiments, the sample processing unit is configured to process the biological sample, for example, by grinding the biological sample through a grinder. In some embodiments, the reaction unit is configured to subject the biological sample or a part thereof to a separating force, for example, ultrasonic energy. In some embodiments, the filtration unit is configured to filter the biological sample or a part thereof to separate, for example, disperse, the single cells of the biological sample. In some embodiments, the collection unit is configured to collect the separated single cells of the biological sample. In some embodiments, the separated single cells are collected in a removable collection device, for example, an Eppendorf tube. In some embodiments, the collection unit includes a feature for engaging and forming a seal with the removable collection device. In some embodiments, the test kit includes a plurality of modules, and the test kit further includes a channel for fluidically connecting each module. In some embodiments, the channel is configured based on the desired material flow from one module to another module. In some embodiments, the test kit includes one or more gates for controlling the material flow from one module to another module. In some embodiments, the test kit is configured so that gravity can be used to manipulate (for example, move) the material in the test kit, for example, by pouring the test kit.

[0099] In some aspects, provided herein is a kit for processing a biological sample, for example by isolating and obtaining separate single cells from the biological sample, the kit comprising: (a) a sample processing unit comprising: a sealable port configured to receive the biological sample for introduction into the sample processing unit; a sample chamber configured to collect the biological sample; and a grinder, wherein the grinder is configured to interface with the sample chamber; (b) a reaction unit comprising a reaction chamber configured to receive ultrasonic energy, wherein the sample processing unit is connected to the reaction unit via a first channel; (c) a filtration unit comprising: a filtration chamber; and a filtration device configured to enter the filtration chamber, wherein the reaction chamber is connected to the filtration unit via a second channel; and (d) a collection unit configured to receive separate single cells from the biological sample, wherein the filtration unit and the collection unit are connected via a third channel.

[0100] In some embodiments, the test kit further includes a first gate configured to control the following fluid connections (e.g., material flow): a fluid connection between the sample processing unit and the reaction unit via the first channel; and / or a fluid connection between the reaction unit and the filtration unit via the second channel. In some embodiments, the gate described herein is used as a valve and can block (e.g., seal) a channel or allow a substance to flow through a channel. In some embodiments, the gate described herein can provide control over the flow of material through a channel, such as by partially opening / blocking a channel. In some embodiments, the first gate is configured to be positioned in a first position, wherein when the first gate is in the first position, the sample processing unit and the reaction unit are fluidically connected, and the reaction unit and the filtration unit are not fluidically connected. In some embodiments, the positioning of the first gate is controlled by pivoting on an axis.

[0101] In some embodiments, the test kit further includes a second gate, which is configured to control the fluid connection between the filtration unit and the collection unit via the third channel. In some embodiments, the second gate is configured to be positioned in a first position, wherein when the second gate is in the first position, the filtration unit and the collection unit are not fluidically connected. In some embodiments, the second gate is configured to be positioned in a second position, wherein when the second gate is in the second position, the filtration unit and the collection unit are fluidically connected. In some embodiments, the positioning of the second gate is controlled by pivoting on an axis.

[0102] In some embodiments, the first channel, the second channel, and the third channel are positioned and configured so that the flow of substances between the sample processing unit, the reaction unit, the filtration unit, and the collection unit is controlled by tilting the reagent cartridge. In some embodiments, the channels are tilted to facilitate the flow of substances from one module to another, for example, from a sample processing unit to a reaction unit.

[0103] In some embodiments, the sealable port of the sample processing unit is configured to receive a fluid for introduction into the sample processing unit. In some embodiments, the fluid is an aqueous fluid, such as a buffer or a cell culture medium solution.

[0104] In some embodiments, the sealable port of the sample processing unit comprises a wall, wherein the wall or a portion thereof is inclined toward the sample chamber, for example, toward the bottom of the sample chamber. In some embodiments, the kit further comprises a cap configured to seal the sealable port of the sample processing unit. In some embodiments, the sealable port has a circular shape and the cap is circular. In some embodiments, the cover is configured to be removed and replaced.

[0105] In some embodiments, the sample chamber is configured so that the biological sample and the added substance will be concentrated in a predetermined position of the sample chamber. In some embodiments, the sample chamber includes one or more walls inclined toward the center. In some embodiments, the sample chamber includes two walls inclined toward the center. In some embodiments, the sample chamber includes a tapered portion with a substantially flat bottom. In some embodiments, the sample chamber includes a substantially flat bottom.

[0106] In some embodiments, the sample processing unit includes a guide configured to position the mincer. In some embodiments, the guide enables the mincer to slide up and down, for example, to repeatedly enter the sample chamber.

[0107] In some embodiments, the sample processing unit is configured to control the extent to which the mincer can slide, such as depth and / or height. In some embodiments, the mincer includes a rod. In some embodiments, the mincer includes a cutting surface on the end of the mincer that interfaces with the sample chamber.

[0108] In some embodiments, the reaction chamber of the reaction unit includes a sound-conducting bottom surface. In some embodiments, the bottom surface of the reaction chamber is substantially flat. In some embodiments, the reaction chamber of the reaction unit includes cylindrical walls. In some embodiments, the volume of the reaction chamber is configured to accommodate a predetermined volume of material. In some embodiments, when the material is in the reaction chamber, the volume of the reaction chamber is configured so that the top surface of the material, such as a meniscus, is at a predetermined position or within the range of a predetermined position. In some embodiments, the predetermined position or the range of a predetermined position is relative to the ultrasonic transducer.

[0109] In some embodiments, the filtration device of the filtration unit includes a tubular assembly, wherein the outer surface of the tubular assembly is configured to provide a seal with the sidewall of the filtration chamber, and wherein the tubular assembly includes a cell strainer covering the bottom opening of the tubular assembly. In some embodiments, the tubular assembly comprises a cylindrical shape without top and bottom flat surfaces. In some embodiments, the tubular assembly includes one or more side holes, wherein the side holes are covered by the cell strainer. In some embodiments, the sidewall of the filtration chamber or a portion thereof is cylindrical. In some embodiments, the filtration chamber of the filtration unit includes a bottom surface having a raised structure. In some embodiments, the filtration unit includes a guide configured to position the tubular assembly in the filtration chamber, such as via a rod extending from the tubular assembly. In some embodiments, the guide enables the tubular assembly to slide up and down in the filtration chamber. In some embodiments, the filtration device is configured to compress the biological sample or a portion thereof in the filtration chamber, for example, to expel separated cells from the filtration chamber. In some embodiments, the filtration unit is configured to control the extent to which the tubular assembly can slide, such as its depth and / or height. In some embodiments, the filter unit includes a vent. In some embodiments, the vent is configured to allow air to flow into and out of the filter unit, such as when the tubular assembly moves up and down in the filter chamber. In some embodiments, the vent is configured to prevent fluid from leaving the filter unit.

[0110] In some embodiments, the cell strainer is configured to filter the particles in the aqueous solution based on particle size, for example, cells. In some embodiments, the cell strainer allows some particles, for example, separate individual cells, to pass through the cell strainer while preventing larger particles, for example, cell clusters and / or extracellular matrix from passing through the cell strainer. In some embodiments, the cell strainer has a mesh size (for example, pore size) of approximately 40 μm to approximately 125 μm, for example, approximately 60 μm to approximately 100 μm, approximately 60 μm to approximately 80 μm, or approximately 65 μm to approximately 75 μm. In some embodiments, the mesh size of the cell strainer is less than about 125 μm, for example, less than about 120 μm, 115 μm, 110 μm, 105 μm, 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, or any one of 40 μm. In some embodiments, the cell strainer has a mesh size greater than about 40 μm, such as greater than about any of 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, or 125 μm. In some embodiments, the cell strainer has a mesh size of about any of 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, or 125 μm. In some embodiments, the cell strainer has an average mesh size of about 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, or 125 μm. In some embodiments, the cell strainer comprises a PEEK mesh, a polyester mesh, a nylon mesh, a polypropylene mesh, a glass fiber mesh, or a stainless steel mesh.

[0111] In some embodiments, the collection unit is configured to receive and hold a removable collection device for receiving isolated cells from a biological sample. In some embodiments, the removable collection device is an Eppendorf tube.

[0112] In some embodiments, the test kit is configured to be loaded into an ultrasonic treatment apparatus. In some embodiments, some features of the test kit are configured to be attached to an ultrasonic treatment apparatus, such as functionally attached to an ultrasonic treatment apparatus. For example, in some embodiments, the grinder of the sample processing unit is configured to engage with a grinder actuator, such as via a clip of an ultrasonic treatment apparatus. In some embodiments, the rod of the filter device of the filtration unit is configured to engage with a filter actuator, such as via a clip, of the ultrasonic treatment apparatus.

[0113] In some embodiments, the kit includes one or more substances, wherein the one or more substances are compatible with the biological sample and any added substances (eg, buffers) and with the forces and temperatures applied thereto.

[0114] In some embodiments, the biological sample includes a plurality of cells. In some embodiments, the biological sample includes a plurality of cells, wherein at least a portion of the cells are, for example, interconnected via an extracellular matrix. In some embodiments, the biological sample is a solid tissue sample. In some embodiments, the biological sample is a sample from a vertebrate or an invertebrate. For example, the biological sample can be from a mammal, a reptile, a bird, a fish, an insect or a nematode sample. In some embodiments, the biological sample is from humans. In some embodiments, the biological sample is a plant sample, such as a plant tissue. In some embodiments, the biological sample comprises eukaryotic cells. In some embodiments, the biological sample comprises multicellular prokaryotes (for example, biofilm). In some embodiments, the biological sample is a cancer sample, such as a tumor tissue or a biopsy. In some embodiments, the biological sample is a healthy biological sample, such as a healthy non-lesioned tissue. In some embodiments, the biological sample is selected from lung, kidney, liver, pancreas, stomach, brain, skin, intestine, muscle, breast, spleen, bladder, uterus, ovary, prostate, heart and bone marrow sample.

[0115] In some aspects, provided herein is an ultrasonic processing device comprising: (a) an ultrasonic transducer; (b) a temperature-controlled water bath; (c) a tilting assembly, wherein the tilting assembly is configured to hold the reagent kit described herein so that (i) the reaction chamber of the reagent kit is positioned relative to the ultrasonic transducer, and (ii) the reaction chamber, sample chamber, and filtration chamber of the reagent kit, or portions thereof, are positioned relative to the water level of the temperature-controlled water bath, the tilting assembly comprising: a tilt actuator; a mincer actuator; and a filter actuator; and (d) a control unit.

[0116] In some embodiments, the ultrasonic treatment device further comprises one or more gate actuators, each of the gate actuators being configured to interface with a gate (eg, the first gate or the second gate) of the reagent cartridge.

[0117] In some embodiments, an actuator such as a tilt actuator, a mincer actuator, a filter actuator, or a gate actuator comprises a stepper motor or a servo motor.

[0118] In some embodiments, the tilt assembly is configured to hold the test kit so that when the removable collection device is placed in the collection unit of the test kit, the removable collection device or a portion thereof is positioned relative to the water level of the temperature-controlled water bath. In some embodiments, the reaction chamber, the sample chamber, and the filtration chamber and the removable collection device or a portion thereof are below the water level of the temperature-controlled water bath. For example, in some embodiments, the reaction chamber, the sample chamber, and the filtration chamber and the removable collection device or a portion thereof are below the water level of the temperature-controlled water bath so that when the biological sample or a portion thereof is present in the test kit, the biological sample is maintained at a desired temperature or temperature range.

[0119] In some embodiments, the ultrasonic transducer is located below the temperature-controlled water bath. In some embodiments, the temperature-controlled water bath is configured to maintain a fluid, such as water, at a predetermined temperature or range thereof. In some embodiments, the predetermined temperature or range thereof is configured so that the biological sample remains below the predetermined temperature for the duration of the method for separating and obtaining separate single cells from the biological sample. In some embodiments, the predetermined temperature or range thereof is configured so that the biological sample remains below about 20°C, for example, below any one of about 15°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, or 1°C. In some embodiments, the predetermined temperature or range thereof is about 4°C to about 20°C, for example, about 4°C to about 15°C, 4°C to about 10°C, or 4°C to about 8°C. In some embodiments, the temperature-controlled water bath is configured to hold a fluid, such as water, so that the fluid serves as a coupling medium from the sensor to the sample.

[0120] In some embodiments, the mincer actuator includes features, such as clamps, that interface with the mincer of the reagent cartridge.

[0121] In some embodiments, the filter actuator includes features, such as a clamp, to interface with the filtration device of the kit.

[0122] In some embodiments, the control unit includes: one or more processors; and a memory storing one or more programs, the one or more programs being configured to be executed by the one or more processors, the one or more programs including instructions for executing a method for separating and obtaining separate single cells from a biological sample. In some embodiments, the instructions for executing the method for separating and obtaining separate single cells from a biological sample include instructions for any one or more of the following: operating the ultrasonic transducer; operating the tilt actuator; operating the grinder actuator; operating the filter actuator; operating one or more gate actuators; and operating the temperature-controlled water bath. In some embodiments, the instructions for operating the ultrasonic transducer include instructions for controlling the duration and intensity of the ultrasonic transducer. In some embodiments, the instructions for operating the tilt actuator include instructions for controlling the positioning and movement of the test kit (e.g., the degree of tilt, the speed of movement, and the duration that the test kit is in a specific position). In some embodiments, the instructions for operating the grinder actuator include instructions for controlling the positioning of the grinder, the speed of the grinder, the depth of the grinder penetrating the sample chamber, and the number of times the grinder penetrates the sample chamber. In some embodiments, the instructions for operating the filter actuator include instructions for controlling the positioning of the filter device, the speed of the filter device, the depth to which the filter device penetrates the filter chamber, and the number of times the filter device penetrates the filter chamber. In some embodiments, the instructions for operating one or more gate actuators include instructions for controlling the positioning of the gate, the opening and closing of the gate, and the degree to which the gate blocks the passage. In some embodiments, the instructions for operating a temperature-controlled water bath include instructions for controlling the temperature and water level of the water in the temperature-controlled water bath.

[0123] In some embodiments, the ultrasonic treatment apparatus includes a user interface. In some embodiments, the user interface includes a screen with information about the system and the method for processing a biological system. In some embodiments, the user interface includes features for inputting information into the ultrasonic treatment apparatus, such as buttons or a keyboard. In some embodiments, the user interface transmits a state to the user, such as indicating the need to add additional substances or the state that the method has been completed to the test kit.

[0124] In some aspects, the present invention provides a method for obtaining separated single cells from a biological sample, the method comprising: loading a test kit as described herein into an ultrasonic processing device as described herein; loading the biological sample into a sample processing unit of the test kit via a sealable port; and activating a programming method of the ultrasonic processing device to separate and obtain separated single cells from the biological sample. In some embodiments, the method further comprises adding an additional substance, such as a buffer, to the test kit through the sealable port. In some embodiments, the method further comprises sealing the sealable port with a cap.

[0125] Figure 1 Shown is a cross-sectional view of a system 100 for processing a solid biological tissue sample 140 in a test kit device 102 according to some embodiments. According to some embodiments, the system 100 includes an ultrasonic treatment device 120 (also referred to as a treatment device) having an opening 120A for receiving the test kit device 102. In some embodiments, the test kit device 102 is configured to be handheld so that it is easy to use. For example, the test kit device 102 can have a width in 100mm, 85mm, 70mm, or 55mm so that the test kit device 102 can be held in one hand.

[0126] like Figure 1 As shown, the housing unit 102A of the test kit device 102 is configured to be placed within the opening 120A of the processing device 120. In some embodiments, the test kit device 102 includes a bottom surface 102B, which is a reference surface relative to the transducer assembly 130 of the ultrasonic processing device 120. In some embodiments, the test kit device 102 is configured to be inserted into the opening 120A until the bottom surface 102B is a predetermined distance from the transducer assembly 130. For example, the predetermined distance can be less than 10 mm, 8.5 mm, 7 mm, or 5.5 mm. In some embodiments, when the test kit device 102 is loaded into the ultrasonic processing device 120, the predetermined distance is maintained because changing the distance can change the manner in which the ultrasonic energy generated by the ultrasonic processing device 120 separates the solid tissue sample 140.

[0127] In some embodiments, the test kit device 102 may include a loading chamber 104 and a collecting chamber 106. In some embodiments, the loading chamber 104 may have a top opening (e.g., a circular top opening), a bottom surface 104B (e.g., a circular surface) and an inner surface (e.g., a tubular surface). In some embodiments, the top opening of the loading chamber 104 can be used as an inlet 104A for receiving a solid tissue sample. In some embodiments, the solid tissue sample 140 is animal tissue, and can be, for example, vertebrate or invertebrate tissue. For example, the tissue can be, but is not limited to, mammalian, reptilian, bird, fish, insect, or nematode tissue. In some embodiments, the tissue is plant tissue. In some embodiments, the tissue comprises eukaryotic cells. In some embodiments, the cell is a multicellular prokaryotic organism (e.g., a biofilm). In some embodiments, the tissue is tumor tissue or cancerous tissue, or a biopsy sample. The biological tissue sample 140 can be, for example, lung, kidney, liver, pancreas, stomach, brain, skin, intestine, muscle, breast, spleen, bladder, uterus, ovary, prostate, heart, bone marrow, or any other solid tissue, which can be diseased tissue or healthy tissue, such as cancerous tissue or non-cancerous tissue.

[0128] In some embodiments, entrance 104a can also receive aqueous fluid 142 to keep solid tissue sample 140.In some embodiments, aqueous fluid 142 comprises culture medium (also referred to as cell culture medium or growth medium), it is the liquid or gel of the selected component that supports and maintains cell (for example, the separation cell of solid tissue sample 140).In some embodiments, culture medium can comprise artificial culture medium, for example balanced salt solution, basal medium or compound culture medium.For example, conventional basal medium comprises minimum essential medium (MEM) or Dulbecco's modified Eagle culture medium (DMEM).For example, complicated culture medium may comprise the modified Dulbecco's medium (IMDM) of Roswell Park Memorial Institute (RPMI) 1640 or ISCOVE.In some embodiments, after solid tissue sample 140 is inserted, the entrance 104A of loading chamber can be sealed, so that test kit equipment 102 can process solid tissue sample 140, and solid tissue sample 140 is not exposed to external environment.For example, test kit equipment 102 can comprise the lid (for example, snap cap, screw cap etc.) that can be coupled to entrance 104A. After the user inserts the solid tissue sample 140 , the cover may be pressed and coupled to the inlet 104A to provide a tight seal.

[0129] In some embodiments, the loading chamber 104 includes a bottom surface 104B for acoustically coupling an aqueous fluid 142 and a solid tissue sample 140 to the ultrasonic processing device 120. By providing a sound-conducting bottom surface 104B, the loading chamber 104 is configured to enable the ultrasonic energy generated by the processing device 120 to separate single cells from the biological tissue sample 140, thereby producing an aqueous suspension. In some embodiments, the bottom surface 104B may include a sound-conducting layer such as a plastic layer, a glass layer, or a ceramic layer. In some embodiments, the sound-conducting layer may be a film (e.g., a plastic film) constituting the bottom surface 104B. In some embodiments, the bottom surface 104B is a flat layer that can be more evenly and tightly coupled to the transducer assembly 130 of the test kit device 102, as will be further described below.

[0130] In some embodiments, the collection chamber 106 can receive cells separated from a solid tissue sample 140, as will be described below with reference to Figure 2 Further described. In some embodiments, the collection chamber 106 can include a removable container (e.g., a test tube or plastic container) that can be separated from the test kit device 102 to enable its contents to be further processed in downstream analysis. For example, the contents of the collection chamber 106 can be processed in primary cell culture, single-cell fluorescence activated cell sorting (FACS) analysis, single-cell DNA and RNA analysis, or single-cell sequencing. In some embodiments, the collection chamber 106 includes a lid 108 (e.g., a snap cap, a screw cap, etc.) to seal the contents of the collection chamber 106 relative to the external environment.

[0131] In some embodiments, the kit apparatus 102 can include other chambers, such as a fluid source chamber 110 (also referred to as a reagent chamber or culture medium chamber) or one or more filtration chambers 112 and 114. In some embodiments, the fluid source chamber 110 can include an inlet 110A for receiving and holding an aqueous fluid (e.g., aqueous fluid 142 or another fluid) from outside the kit apparatus 102. In some embodiments, the kit apparatus 102 includes a channel that fluidically connects the fluid source chamber 110 to the loading chamber 104 to enable the aqueous fluid to flow from the fluid source chamber 110 to the loading chamber 104. In some embodiments, the kit apparatus 102 includes a plurality of channels that fluidically couple the loading chamber 104 to the collection chamber 106 via the filtration chambers 112 and 114. In some embodiments, the filtration chambers 112 and 114 can be configured to selectively capture effluent from an aqueous suspension flowing through the plurality of channels.

[0132] As will be described further below, once the solid tissue sample 140 is loaded with the aqueous fluid 142 in the loading chamber 104, the solid tissue sample 140 remains contained in the kit device 102 during processing until target components, such as cells, separated from the solid tissue sample 140 are received into the collection chamber 106. Thus, in contrast to conventional methods that require transferring the solid tissue sample and the associated aqueous suspension between different containers during processing, thereby exposing the solid tissue sample to the external environment, the kit device 102 provides a sealed, self-contained environment.

[0133] In some embodiments, the ultrasonic processing device 120 includes a power supply 132 that supplies power to various components to enable single cells to be separated from the solid tissue sample 140 and subsequently collected in the collection chamber 106. Figure 1 As shown, the ultrasonic processing device 120 includes at least one transducer assembly 130 that is configured to align with the reaction chamber of the reagent kit device 102 when the housing unit 102A of the reagent kit device 102 is loaded into the opening 120A of the ultrasonic processing device 120. In some embodiments, processing of the solid tissue sample 140 can be performed in the loading chamber 104. In these embodiments, the loading chamber 104 is also the reaction chamber.

[0134] In some embodiments, the transducer assembly 130 can include a transducer and an acoustic wave guide. For example, the acoustic wave guide can be a spherical lens (e.g., a Fresnel lens) fabricated on the transducer plate of the transducer assembly to focus the ultrasonic waves in the loading chamber 104. Various embodiments of the transducer assembly 130 are described in U.S. Patent Application No. 2013 / 0199298A1 and U.S. Patent Nos. 6,682,214 and 8,319,398, the entire contents of each of which are incorporated herein by reference.

[0135] In some embodiments, the ultrasonic treatment device 120 includes a control processor 124 configured to control the operation of the ultrasonic treatment device 120. For example, the control processor 124 can operate the transducer assembly 130 to generate ultrasonic body wave energy and direct the ultrasonic body wave energy to the reaction chamber containing the solid tissue sample 140. In some embodiments, to generate ultrasonic body wave energy, the transducer assembly 130 is configured to emit ultrasonic waves having a high transverse acoustic potential distribution directed in a target direction, which allows mixing of the fluid (e.g., the aqueous fluid 142 holding the solid biological tissue sample 140) in the target direction. In some embodiments, the control processor 124 is configured to control a radio frequency (RF) generator 126 and an RF amplifier 128 to drive the transducer assembly 130.

[0136] In some embodiments, ultrasonic body waves generated by the transducer assembly 130 exert mixing and suspension forces in the aqueous fluid 142 contained in the loading chamber 104 to separate single cells from the solid tissue sample 140. Compared to traditional cell separation methods, ultrasonic energy is used instead of enzymes. Therefore, in addition to maintaining the gene expression profile of cells separated from the solid tissue sample 140, faster processing times can be achieved.

[0137] In some embodiments, the ultrasonic treatment device 120 can include one or more additional transducer assemblies (similar to the transducer assembly 130) that are configured to align with one or more corresponding filtration chambers 112 and 114 when the reagent kit device 102 is loaded into the opening 120A of the ultrasonic treatment device 120. In some embodiments, the ultrasonic energy generated by these transducer assemblies can be directed toward the aqueous solution flowing through the filtration chambers 112 and 114 to achieve positive filtration and negative filtration, as will be further described below.

[0138] In some embodiments, the control processor 124 can be connected to an input device 122 that enables a user to interact with and control the ultrasonic treatment device 120. In some embodiments, the input device 122 includes a control panel or touch screen for receiving user input. For example, the input specified by the user can include activating the transducer assembly 130 to separate cells from the solid tissue sample 140 for a specific amount of time. In some embodiments, the input device 122 can be any suitable device for providing input, such as a touch screen, a keyboard or keypad, a mouse, or a voice recognition device. In some embodiments, the ultrasonic treatment device 120 includes an output device 136, which can be any suitable device for providing output, such as a touch screen, a tactile device, or a speaker.

[0139] In some embodiments, the ultrasonic treatment device 120 includes an actuation device 134 that is connected to one or more channels in the reagent kit apparatus 102. In some embodiments, the reagent kit apparatus 102 can include one or more connection components that are coupled to the one or more channels and configured to physically connect the reagent kit apparatus 102 to the actuation device 134. For example, the connection components can include fittings or adapters.

[0140] In some embodiments, the control processor 124 can be configured to operate the actuator 134 to control the flow of the aqueous solution generated by the ultrasonic treatment from the loading chamber 104 through the one or more filtration chambers 112 and 114 to the collection chamber 106. For example, the actuator 134 can include a vacuum pump that is connected to the reagent cartridge apparatus 102 via one or more connecting components and forces the aqueous solution through the one or more filtration chambers 112 and 114. In some embodiments, the pump can include a positive displacement pump, such as a diaphragm pump, a piston pump, a peristaltic pump, or a valveless pump.

[0141] In some embodiments, one or more reagent cartridge channels connecting the loading chamber 102 to the collection chamber 106 may include a plunger assembly, as will be described below with reference to Figure 2 Further described. In these embodiments, the reagent cartridge device 102, the plunger device may include a connector component (e.g., a latch, a handle, etc.) configured to connect to the actuation device 134. In these embodiments, the actuation device 134 may include a motor device configured to drive a pulling force and / or a pushing force to operate the plunger device.

[0142] Figure 2 A diagram illustrating a kit apparatus 200 for processing a solid biological tissue sample 212 according to some embodiments is shown. The kit apparatus 200 may be Figure 1 102 . In some embodiments, the kit device 200 may include one or more fluid source chambers 202 (also referred to as reagent chambers or culture medium chambers), a loading chamber 204, one or more filtration chambers 206, and a collection chamber 208. In some embodiments, the kit device 200 can be configured to include fluid channels 210A-H that fluidically couple two or more of the chambers 202-208, as further described below. In some embodiments, the one or more fluid channels can include tubes or etched or molded channels within the kit device 102.

[0143] In some embodiments, the fluid source chamber 202 may correspond to Figure 1 The fluid source chamber 110 is configured to receive and store an aqueous fluid 214 reservoir to accommodate single cells separated from the solid biological tissue sample 212. For example, the fluid source chamber 202 may include three fluid source chambers 202A-C having respective inlets for receiving the same or different aqueous fluids. In some embodiments, the test kit device 200 may include fluid channels 210A-C to fluidically connect the respective fluid source chambers 202A-C to the sample loading chamber 204. In some embodiments, once the test kit device 200 is loaded into an ultrasonic processing device (e.g., Figure 1Within the ultrasonic treatment device 120 (e.g., an ultrasonic treatment device 120), the flow of aqueous fluid from one or more fluid source chambers 202A-C can be controlled by the actuation device 134 to flow to the loading chamber 204. In some embodiments, the channels 210A-C include corresponding valves 212A-C, which can be controlled by the actuation device 134 to control which aqueous fluid (e.g., reagent) from the fluid source chambers 202A-C and in what amount can flow to the loading chamber 204. For example, one of the valves 212A-C can include a gate valve that can be opened or fully closed by the actuation device 134 to select an aqueous fluid from one or more of the corresponding fluid source chambers 202A-C. In another example, one of the valves 212A-C can include a shutoff valve to control the flow of aqueous fluid to the loading chamber 204.

[0144] In some embodiments, the loading chamber 204 can include an inlet for receiving a solid biological tissue sample 212. In some embodiments, the loading chamber 204 can be configured to receive an aqueous fluid 214 from the inlet or from one or more fluid source chambers 202. In some embodiments, the loading chamber 204 is configured to be positioned so that when the test kit apparatus 200 is loaded into the ultrasonic processing device 120, the loading chamber 204 is aligned with the transducer assembly 130 of the ultrasonic processing device 120. In some embodiments, the loading chamber 204 can be configured to hold the solid biological tissue sample 212 during a mincing operation and / or during ultrasonic separation of cells from the solid tissue sample 212. For example, when the loading chamber 204 is located on the transducer assembly 130, single cells from the biological tissue sample 212 can be separated by the transverse ultrasonic body wave energy generated by the transducer assembly 130. In some embodiments, the loading chamber 204 can be sealed from the external environment before activating the ultrasonic processing device 120 to perform ultrasonic cell separation. In some embodiments, the minced biological tissue sample 212 can be transported to a separate reaction chamber where cells are separated from the minced biological tissue sample 212 .

[0145] In some embodiments, after ultrasonic separation is completed, the loading chamber 204 contains an aqueous suspension comprising a mixture of the original aqueous fluid 214, the target component separated from the solid tissue sample 212, and the effluent material separated from the solid tissue sample 212. For example, the target component may include isolated cells of interest. For example, the effluent material may include non-targeted components separated from the tissue sample 212, such as cell debris and extracellular debris.

[0146] In some embodiments, the test kit device 200 includes one or more filter chambers 206A-C that selectively filter outflow materials from the aqueous solution as it flows from the loading chamber 204 to the collection chamber 208. In some embodiments, the filter chambers 206A-C include a series of two or more filter chambers 205A-C that connect the loading chamber 204 to the collection chamber 208. In some embodiments, the filter chamber 206 can include a positive filter chamber in which the analyte of interest is captured, or a negative filter chamber through which the analyte of interest passes. In some embodiments, the filter chamber (e.g., one of the filter chambers 206A-C) can include a filter that is configured to filter particles in the aqueous solution based on size (e.g., mesh size or membrane pore size). For example, the filter can be a mesh filter that captures the extracellular matrix of the sample tissue 212 and allows smaller particles in the aqueous solution to pass through a 70 μm mesh.

[0147] In some embodiments, the filtration chamber (e.g., one of the filtration chambers 206A-C) may include a marker (e.g., an antibody on a bead or microvesicle) configured to bind to a cell type in an aqueous solution. In positive filtration, these cell types may include an analyte of interest, while in negative filtration, these cell types may include effluent material (e.g., a non-target component). In some embodiments, the filtration chamber may include a filter (e.g., a mesh filter) that prevents the marker from passing to a subsequent chamber. In some embodiments, in addition to or instead of using a filter, the ultrasonic treatment device 120 may include one or more magnets that apply a pulling force to the marker to prevent it from passing through the subsequent chamber.

[0148] In some embodiments, the kit apparatus 200 includes a channel 210D configured to fluidically couple the loading chamber 204 to one or more of the filtration chambers 206A-C. In some embodiments, the kit apparatus 200 includes a plurality of channels 210E-G that fluidly connect successive chambers of the filtration chambers 206A-C to allow the aqueous suspension to flow from the loading chamber 204 to the collection chamber 208. In some embodiments, one or more of the channels 210D-G can include one or more corresponding valves (e.g., valves or shut-off valves) operated by the actuator 134 to control the flow of the aqueous solution.

[0149] In some embodiments, one or more of the filtration chambers 206A-C can be positioned to align with one or more corresponding transducer assemblies of the ultrasonic treatment device 120 when the test kit apparatus 200 is loaded within the ultrasonic treatment device 120. These transducer assemblies can be separate from the transducer assembly 130. In some embodiments, the bottom surface of one or more filtration chambers 206A-C is configured to acoustically couple to one or more corresponding transducer assemblies to receive ultrasonic body wave energy to promote the selective flow of target components. In some embodiments, a filtration chamber located on a transducer assembly (separate from the transducer assembly 130) can achieve higher filtration efficiency. For example, the ultrasonic energy (e.g., transverse ultrasonic body wave energy) applied by the transducer assembly can exert mixing forces on the contents of the filtration chamber (i.e., the marker and the aqueous solution) to increase the likelihood of binding.

[0150] In some embodiments, when the test kit device 200 is loaded into the ultrasonic treatment device 120, the actuator 134 of the ultrasonic treatment device 120 can control the flow of the aqueous suspension including the separated cells through the fluid channels 210D-210G. Figure 1 As discussed, the actuating device 134 may include a pump or a mechanical motor.

[0151] In some embodiments, the collection chamber 208 can be configured to receive and collect target components of interest from isolated cells of the solid biological tissue sample 212 that have not been filtered by the filtration chambers 206A-C. In some embodiments, the collection chamber 208 is removable and can be configured to be separated from the kit device 200.

[0152] In some embodiments, the kit device 200 includes a channel 210H that fluidically couples the collection chamber 208 to the loading chamber 204. In some embodiments, the channel 210H is configured as a one-way fluid channel that allows aqueous fluid from the collection chamber 208 to flow back into the loading chamber 204, but prevents fluid from the loading chamber 204 from flowing back into the collection chamber 208. For example, the channel 210H can include a check valve 210D to prevent fluid from flowing back into the collection chamber 208. One advantage provided by the channel 210H is that the aqueous fluid can be reused for future ultrasonic separation processes.

[0153] Figure 3 A kit device 300 for processing a solid biological tissue sample 330 is shown according to some embodiments. In some embodiments, the kit device 300 may be Figure 1 Kit device 102 or Figure 2 An example implementation of the kit device 200 is configured to be placed in Figure 1 The ultrasonic treatment device 120 is placed in the opening 120A.

[0154] like Figure 3 As shown, the base 302 of the reagent kit device 300 includes a plurality of chambers, such as a loading chamber 306 and a collection chamber 308. Figure 1 In the loading chamber 104, the loading chamber 306 may include an inlet 306A for receiving a solid biological tissue sample 330. In some embodiments, the inlet 306A of the loading chamber 306 may be configured to also receive an aqueous fluid to accommodate the solid tissue sample 330. For example, as described above with respect to Figure 1 In some embodiments, the kit device 300 may include one or more fluid source chambers having corresponding inlets for receiving one or more aqueous fluids and controlling one or more of these aqueous fluids to flow to the loading chamber 306.

[0155] In some embodiments, the loading chamber 306 is configured to allow the contained solid tissue sample 330 to be minced by the mincing device 312. Figure 3 Shown, grinding device 312 can comprise pestle tool 312A and mortar tool 312B.In some embodiments, mortar tool 312B comprises the handle being connected to mortar bottom, and this mortar bottom can be inserted in loading chamber 306, and pestle tool 312A comprises pestle tip, and this pestle tip can be inserted in loading chamber 306, so that solid tissue sample 330 is ground on the mortar bottom of the mortar tool 312B of insertion.Therefore, grinding operation can be directly performed at test kit equipment 300 place, and does not need biological sample 330 to be ground before being loaded in loading chamber 306.In some embodiments, grinding operation can be performed automatically.For example, ultrasonic treatment apparatus 120 can comprise one or more actuating devices (for example, motor, piston etc.), and described actuating device is configured to mechanically operate grinding device 312 when test kit equipment 300 is loaded in ultrasonic treatment apparatus 120.

[0156] In some embodiments, the kit device 300 can be loaded into the solid tissue sample 330 before or after it is minced. Figure 1 In the ultrasonic treatment device 120. Figure 1 As described above, when the test kit device 300 is loaded into the ultrasonic processing device 120, the loading chamber 306 can be positioned to align with the transducer assembly 130. In some embodiments, the minced sample tissue 330 contained in the loading chamber can be separated into single cells by the ultrasonic body wave energy generated by the ultrasonic processing device 120. In some embodiments, the resulting fluid contained in the loading chamber 306 is referred to as an aqueous suspension, which includes an aqueous fluid, a portion of the solid tissue sample 330, ultrasonically separated cells of the solid tissue sample 330, and an effluent. For example, the effluent can include cell debris or extracellular debris generated by mincing and ultrasonic separation.

[0157] In some embodiments, the base 302 of the test kit device 300 includes an isolation compartment 304 having an opening 304A for receiving and holding the filtration device 310 in place. In some embodiments, after the solid tissue sample 330 has been minced by the mincing device 312 and ultrasonically separated by the ultrasonic processing device 120, the aqueous suspension obtained in the loading chamber 306 can be filtered by the filtration device 310. For example, the filtration device 310 can be inserted into the loading device 306 to compress the remaining portion of the solid tissue sample 330 to expel additional cells separated from the solid tissue sample 330, as will be described below. Figure 4 Further description. In some embodiments, the filter device 310 is configured to engage with the loading device 306 in a sliding fit so that a small gap exists between the outer surface of the filter device 310 and the inner wall of the loading device 306. Therefore, the solid tissue sample 330 can be minced and filtered without the need to transfer the solid tissue sample 330 from the reagent kit device 202 to other devices, which increases the risk of environmental contamination. In some embodiments, the filtering operation can be performed automatically. For example, the ultrasonic treatment device 120 may include one or more actuators (e.g., motors, pistons, etc.) that are configured to mechanically operate the filter device 310 when the reagent kit device 300 is loaded into the ultrasonic treatment device 120. For example,

[0158] In some embodiments, the kit apparatus 300 includes a collection chamber 308 configured to receive and collect separated cells of interest from the aqueous suspension held in the loading chamber 306. In some embodiments, the collection chamber 308 can be a container that is detachable from the kit apparatus 300. Before separating the collection chamber 308, a cap 314 can seal the entrance to the collection chamber 308 to prevent the collected target components of interest from leaking or contacting the external environment.

[0159] In some embodiments, the kit apparatus 300 can include one or more channels configured to fluidically connect multiple chambers. For example, the fluid channel 320 can be a plastic tube that, when the filtration apparatus 322 is inserted into the loading chamber 306, fluidically connects the loading chamber 306 to the collection chamber 308. In some embodiments, the aqueous solution contained in the loading chamber 306 can be drawn into the collection chamber 308 through the channel 322. In some embodiments, the kit apparatus 300 includes a lid 324 that is configured to fluidically connect the channel 322 and the channel 320 and can be coupled to the collection chamber 308. In some embodiments, the channel 320 can be coupled to one or more barrels 318 of a plunger device 316, which can be actuated to cause the aqueous solution to flow from the loading chamber 306 through the channel 322 to the collection chamber. For example, the plunger device 316 can be actuated by pulling on a handle, causing air to be drawn from the channel 322 into the collection chamber 308 and through the channel 320 toward the barrel 318, thereby causing the aqueous solution to follow the channel 322 into the collection chamber 308.

[0160] In some embodiments, the plunger device 316 can be pulled or pushed by the user. In other embodiments, the plunger device 316 can be mechanically actuated by the ultrasonic treatment device 120 when the test kit device 300 is loaded into the ultrasonic treatment device 120. For example, the ultrasonic treatment device 120 may include an actuating device configured to apply a pulling force on the plunger device (e.g., a motor or hydraulic pulling device that drives a pulley, chain, or cable). In some embodiments, the actuating device may include a vacuum pump, such as Figure 1 As described above, the vacuum pump induces a pressure change within the channel 322 to control the flow of the aqueous solution from the loading chamber 306 to the collection chamber 308 .

[0161] Figure 4 Shown is a system 400 for enhancing separation of cells from a solid biological tissue sample 412, according to some embodiments. In some embodiments, the system 400 includes a filter device 402 operable to increase the recovery of viable cells separated from the tissue sample 412, as compared to conventional cell strainers.

[0162] In traditional filtration techniques, an aqueous suspension containing cells separated from a solid tissue sample is poured over a cell strainer to retrieve viable cells. However, residual viable cells are often trapped within the cell strainer itself, rather than being retrieved. This may be due to the separated viable cells being physically located within the fibrous network of the solid tissue sample's cellular matrix. Other traditional filtration techniques require transferring the aqueous suspension to a separate centrifuge apparatus to filter the cells, which also leaves residual tissue containing viable cells in the centrifuge.

[0163] In some embodiments, to improve existing filtration technology, the filter device 402 includes a tubular body 402B that can be telescopically inserted into a container 410 containing a solid biological tissue sample 412 in an aqueous fluid 414. Therefore, the circumference of the tubular body 402B is slightly smaller than the circumference of the container 410 so that the filter device 402 can be inserted into the container 410. Figure 1-3 As mentioned, the aqueous fluid 414 can be a cell culture medium such as MEM or RPMI 1640.

[0164] In some embodiments, filter device 402 can be made up of materials compatible with solid biological tissue sample 412. For example, the tubular body 402B of filter device 402 can be made of plastics (for example, polystyrene) that do not react with biological tissue. In some embodiments, tubular body 402B is configured to rigidity, to slide with container 410 when filter device 402 is inserted in container 410, thereby do not form tight seal. During insertion, slide fit produces gap between the outer surface of tubular body 402B and the inner surface of container 402, to allow filter device 402 to easily insert and remove from container 410, to repeatedly compress solid biological tissue sample 412, to discharge the cell of separation, as will be discussed further below. In some embodiments, in order to reduce the pressure caused in the top of tubular body 402B when being inserted into container 410, filter device 402 can be configured to include one or more holes along top. For example, tubular body 402B can comprise one or more holes 402D positioned in the top of tubular body 402B. In some embodiments, the holes 402D can be positioned on the top surface of the filter device 402. In some embodiments, the holes 402D can have a diameter between 0.5-3 mm, 1-2.5 mm, or 1-2 mm.

[0165] In some embodiments, the filter device 402 includes a cell strainer 404 covering the bottom opening 402A of the tubular body 402B. In some embodiments, the cell strainer 404 can include a PEEK mesh, a polyester mesh, a nylon mesh, a polypropylene mesh, a glass fiber mesh, or a stainless steel mesh. In some embodiments, the cell strainer 404 can have a mesh size of 40 μm, 70 μm, or 100 μm.

[0166] In some embodiments, when the filter device 402 is fully inserted into the container 410, the cell strainer 404 is configured to compress the tissue sample 412 to expel cells from the extracellular matrix of the tissue sample 412. In some embodiments, full insertion occurs when the tubular body 402B is inserted to the bottom of the container 410 and / or cannot be inserted further. In some embodiments, the filter device 402 is configured to extend into the container 410 approximately a predefined distance 403 sufficient to enable the cell strainer 404 to squeeze and compress the tissue sample 412. In some embodiments, the predetermined distance 403 enables the tissue sample 412 to be compressed by at least 50%, 60%, 70%, 80%, or 90%.

[0167] In some embodiments, the tubular body 402B includes a raised portion 402E that prevents the tubular body 402B from extending beyond the predetermined distance 403. For example, the raised portion 402E can be a tab or ring attached to the tubular body 402B (as shown). The raised portion 402E can be any rigid material compatible with the container 410, such as plastic, rubber, ceramic, etc.

[0168] In some embodiments, in addition to or in lieu of the raised portion 402E, the container 410 includes a stopper material 418 positioned at the bottom of the container 410 to prevent the tubular body 402E from extending beyond the predetermined distance 403. In some embodiments, the stopper material 418 can be one or more tabs or rings (as shown) composed of sponge, rubber, or plastic material. In some embodiments, the stopper material 418 can have a height of at least 0.1 mm, 0.2 mm, 0.25 mm, or 0.5 mm.

[0169] In some embodiments, the stop material 418 can have a height of less than 0.7 mm, 0.5 mm, 0.25 mm, or 0.2 mm.

[0170] In some embodiments, when the filter device 402 is fully inserted into the container, the lower portion 402C of the tubular body 402B is positioned within the container 410. In some embodiments, the lower portion 402C includes one or more openings that are covered by one or more corresponding cell strainers 406A-B, which can be identical to the cell strainer 402A.

[0171] In some embodiments, when the tissue sample 412 is compressed, the aqueous fluid is expelled from the compressed tissue sample 412 to expel the separated cells, and the expelled cells are passed through the cell strainer 404 to the interior, for example, the center, of the tubular body 402B. Thus, the separated living cells physically located within the extracellular matrix of the solid tissue sample 412 can be released to increase the recovery of living cells. In some embodiments, the aqueous fluid 414 also flows through the cell strainer pores 406A-B within the tubular body 402B.

[0172] In some embodiments, the aqueous fluid collected within the tubular body 402B can be delivered to a collection chamber, e.g. Figure 1 Collection chamber 106 or Figure 3 In some embodiments, the filter device 402 includes a channel 416 configured to draw fluid (including dispersed cells) collected within the tubular body 402B into the collection chamber. For example, the channel 416 can be a plastic tube. In some embodiments, the channel 416 extends along the length of the tubular body 402B and within the tubular body 402B to a position within the lower portion 402C.

[0173] In some embodiments, the filter device 402 can be operated on a solid tissue sample 412 that has undergone enzymatic hydrolysis. However, as explained in the background above, the use of enzymes requires long incubation and processing times and may alter the gene expression profile of the isolated cells. Therefore, in some embodiments, it may be more efficient to operate the filter device 402 on a solid tissue sample 412 that has undergone ultrasonic separation, as described above with respect to Figure 1-3 In these embodiments, the container 410 may correspond to a Figure 1 The reaction chamber or loading chamber (e.g., loading chamber 306) of the reagent kit apparatus 102 or the reagent kit apparatus 300.

[0174] Figure 5 A method 500 for operating a stand-alone kit device to separate a solid biological tissue sample into viable cells is shown in accordance with some embodiments. In some embodiments, the kit device may be Figure 1 Reagent kit equipment 102, Figure 2 Kit device 200 or Figure 3 The kit device 300.

[0175] In step 502, a solid biological tissue sample and an aqueous liquid are introduced into a first chamber of a kit device. In some embodiments, the first chamber includes a first inlet that allows the tissue sample to be placed in the first chamber. In some embodiments, the first chamber can be, for example, Figure 2 Loading chamber 204 or Figure 3In some embodiments, aqueous liquids can be introduced into the kit device through separate inlets of one or more fluid supply chambers, as described above with respect to Figure 1-2 As stated.

[0176] In some embodiments, the tissue sample can be minced in a first chamber of the test kit apparatus. In some embodiments, the first chamber is configured to receive a mincing apparatus comprising a pestle and a mortar to mince the tissue sample. In some embodiments, the tissue sample can be transferred from the first chamber to another chamber in the test kit apparatus for mincing.

[0177] In step 504, the reagent kit apparatus is loaded into the ultrasonic processing device such that the first chamber is aligned with the transducer assembly of the processing device. In some embodiments, after the reagent kit apparatus is loaded into the ultrasonic processing device, the tissue sample can be minced within the first chamber.

[0178] In step 506, the ultrasonic treatment device is activated to apply ultrasonic energy from the transducer assembly to ultrasonically separate the cells in the tissue sample in the first chamber to produce an aqueous suspension. In some embodiments, the ultrasonic transducer array is configured to generate and guide ultrasonic waves with a high level of transverse ultrasonic thrust toward the first chamber. These ultrasonic waves generate transverse body wave energy that applies mixing and levitation forces in the fluid in the first chamber to separate the cells from the tissue sample. In some embodiments, the aqueous suspension includes the aqueous fluid of step 502, the residual portion of the tissue sample, the separated cells, and effluent such as cell debris and extracellular debris.

[0179] In some embodiments, the ultrasonic treatment device can be activated for a predetermined period of time selected by the user. In some embodiments, the ultrasonic treatment device can be controlled based on user control to start and stop the generation of ultrasonic waves. For example, the processing unit can include a user interface that allows the user to control its operation, as described above with respect to Figure 1 The ultrasonic treatment device 120 is described.

[0180] In step 508, the aqueous suspension is controlled to flow from the first chamber of the kit device to the second chamber, wherein the second chamber is configured to collect the separated cells in the aqueous suspension. In some embodiments, the aqueous suspension can be controlled to flow through a series of two or more filtration chambers to filter the effluent from the aqueous suspension, as described above with respect to Figure 2 As stated.

[0181] Figure 6 A method 600 for operating a filter device to enhance separation of living cells from a solid biological tissue sample is shown according to some embodiments. In some embodiments, the filtration device can be the filtration device 310, as described with reference to FIG. Figure 3Said, or filtering device 402, as referenced Figure 4 As stated.

[0182] In step 602, the tubular body of the filter device is telescopically inserted into a container containing a biological tissue sample in an aqueous fluid. In some embodiments, the filter device includes a cell strainer covering a bottom opening of the tubular body. In some embodiments, the tubular body forms a sliding fit with the container during insertion.

[0183] In step 604, the tubular body of the filter device is fully inserted into the container to compress the biological tissue sample with the cell strainer. The action of the cell strainer compressing the biological tissue sample discharges free cells physically located in the fiber mesh of the extracellular matrix of the biological tissue sample. In some embodiments, when the tubular body is inserted, the filter device is fully inserted until it reaches the bottom of the container and / or cannot be further inserted. For example, the tubular body may include a raised portion that prevents the tubular body from extending beyond a predetermined distance. In another example, the container may include a stopper material attached to the bottom of the container, the stopper material having a preselected height that prevents the tubular body from extending into the container beyond a predetermined distance.

[0184] In some embodiments, the tubular body can be repeatedly lifted (e.g., removed) and reinserted to repeatedly compress the biological tissue sample in the container to increase the amount of cells discharged. In some embodiments, the filter device can be operated to repeatedly press the biological tissue a predetermined number of times before the cells discharged in the aqueous solution are transported to the collection chamber. In some embodiments, the biological tissue sample can be ultrasonically separated by the ultrasonic processing device 102 before one or more pressure filtrations.

[0185] In some embodiments, after the aqueous solution is delivered to the collection chamber, as described above in Figure 1-3 As described in , additional aqueous fluid may be introduced into the container. The filter device may then be configured to perform methods 602-604 and / or repeatedly extract and reinsert the filter device into the container to enable further cells to be expelled from the extracellular matrix of the biological tissue sample.

[0186] Exemplary embodiments

[0187] Embodiment 1. A device for separating single cells from a solid biological tissue sample, the device comprising: a housing unit configured to enable the device to be loaded into an ultrasonic processing device; a first chamber configured to receive the solid biological tissue sample and an aqueous fluid, wherein when the device is loaded into the processing device, the first chamber is aligned with the transducer assembly of the processing device so that the ultrasonic energy applied by the processing device can separate cells from the biological tissue sample, thereby producing an aqueous suspension; a channel fluidically connecting the first chamber to a second chamber; and the second chamber configured to collect the separated cells in the aqueous suspension flowing from the first chamber through the channel.

[0188] Embodiment 2. The apparatus of embodiment 1, wherein the second chamber is detachable from the apparatus.

[0189] Embodiment 3. The device of embodiment 1 or 2, wherein the first chamber comprises a first inlet for receiving the solid tissue sample and the aqueous fluid from outside the device.

[0190] Embodiment 4. The apparatus of embodiment 3, wherein the first chamber comprises a lid to seal the received solid tissue sample and the aqueous fluid from the external environment.

[0191] Embodiment 5. The apparatus of any one of embodiments 1-4, wherein the first chamber comprises a bottom surface that is acoustically conductive to allow the ultrasonic energy to enter the first chamber to separate cells from the biological tissue.

[0192] Embodiment 6. The apparatus of embodiment 5, wherein the bottom surface comprises a planar layer configured to couple to the transducer assembly when the apparatus is loaded into the processing device.

[0193] Embodiment 7. The apparatus of any one of embodiments 1-6, wherein the aqueous fluid comprises a cell culture medium solution.

[0194] Embodiment 8. The apparatus according to any one of embodiments 1-7, further comprising: a connector component coupled to the channel and configured to connect to an actuator of the processing device, wherein the actuator is configured to control the flow of fluid through the channel.

[0195] Embodiment 9. The apparatus of embodiment 8, wherein the actuating device comprises a pump or a plunger.

[0196] Embodiment 10. The apparatus of embodiment 8 or 9, wherein the connector component comprises a fitting or adapter configured to connect the channel and the actuation device.

[0197] Embodiment 11. The apparatus of any one of embodiments 8-10, further comprising a valve positioned in the channel and configured to be controlled by an actuating device of the processing device to control fluid flow through the channel.

[0198] Embodiment 12. The apparatus of any one of embodiments 1-11, further comprising a third chamber comprising a third inlet for receiving a second aqueous fluid into the interior of the apparatus.

[0199] Embodiment 13. The apparatus of embodiment 12, further comprising a channel fluidly coupling the third chamber to the first chamber to allow the second aqueous fluid to flow into the first chamber.

[0200] Embodiment 14. The apparatus according to any one of embodiments 1-13, further comprising: a series of two or more filter chambers connecting the first chamber to the second chamber, wherein the channel comprises a plurality of sub-channels, and the plurality of sub-channels fluidically connect successive chambers in the series of two or more filter chambers to allow the aqueous fluid to flow from the first chamber to the second chamber.

[0201] Embodiment 15. The apparatus of embodiment 14, wherein the two or more filtration chambers are configured to capture effluent material in the aqueous fluid as the aqueous fluid flows through the plurality of subchannels so that target components can be collected in the second chamber.

[0202] Embodiment 16. The apparatus of embodiment 15, wherein the effluent comprises non-target components, cellular debris, and extracellular debris.

[0203] Embodiment 17. The apparatus of embodiment 15 or 16, wherein the filtration chamber comprises one or more marker beads configured to bind to the effluent material to prevent the effluent material from flowing through the filtration chamber.

[0204] Embodiment 18. An apparatus according to any of embodiments 14-17, wherein a filter chamber from the two or more filter chambers is configured to align with a second transducer assembly of the processing device when the apparatus is loaded into the processing device, and wherein the filter chamber includes a bottom surface that acoustically couples the filter chamber with the second transducer assembly to receive ultrasonic energy from the processing device to promote selective flow of the target component.

[0205] Embodiment 19. A device according to any of embodiments 15-18, wherein the second chamber is configured to collect the intracellular or extracellular target analyte, and wherein the device further comprises: a one-way channel that fluidically connects the second chamber to the first chamber to allow the aqueous fluid to flow back to the first chamber.

[0206] Embodiment 20. The apparatus of any one of embodiments 14-19, wherein the plurality of subchannels are configured to allow the aqueous fluid to flow back through the series of two or more filtration chambers without flowing back into the first chamber.

[0207] Embodiment 21. A method for separating single cells from a solid biological tissue sample, the method comprising: introducing the solid biological tissue sample and an aqueous liquid into a first chamber of a test kit apparatus; loading the test kit apparatus into an ultrasonic processing device, wherein the first chamber is aligned with a transducer assembly of the processing device; activating the ultrasonic processing device to apply ultrasonic energy from the transducer assembly to ultrasonically separate cells from the tissue sample, thereby generating an aqueous suspension in the first chamber; and controlling the aqueous suspension to flow from the first chamber to the second chamber of the test kit apparatus, wherein the second chamber is configured to collect the separated cells in the aqueous suspension.

[0208] Embodiment 22. The method of embodiment 21, further comprising: mincing the tissue sample within the first chamber.

[0209] Embodiment 23. The method of embodiment 22, wherein mincing the tissue sample comprises: inserting a pestle and mortar into the container; and mincing the tissue sample using the inserted pestle and mortar.

[0210] Embodiment 24. The method according to any one of embodiments 21-23, further comprising: separating the second chamber from the kit device.

[0211] Embodiment 25. The method of any one of embodiments 21-24, further comprising: receiving the solid tissue sample and the aqueous fluid from outside the device via a first inlet of the first chamber.

[0212] Embodiment 26. The method of any one of embodiments 21-25, wherein the first chamber comprises a lid, and wherein the method further comprises: sealing the received solid tissue sample and the aqueous fluid from the external environment by closing the lid.

[0213] Embodiment 27. A method according to any one of embodiments 21-26, wherein the first chamber includes a bottom surface that is acoustically conductive to allow the ultrasonic energy to enter the first chamber to separate cells from the biological tissue.

[0214] Embodiment 28. The method of embodiment 27, wherein the bottom surface comprises a planar layer, and wherein the method further comprises coupling the planar layer of the test kit apparatus to the transducer assembly when the test kit apparatus is loaded in the processing device.

[0215] Embodiment 29. The method of any one of embodiments 21-28, wherein the aqueous fluid comprises a cell culture medium solution.

[0216] Embodiment 30. A method according to any of embodiments 21-29, wherein the test kit device further includes a connector component coupled to the channel, and wherein the method further includes: using the connector component to connect the channel to an actuator of the processing device, wherein the actuator is configured to control the flow of fluid through the channel.

[0217] Embodiment 31. A method according to embodiment 30, wherein the actuating device comprises a pump or a plunger.

[0218] Embodiment 32. The method of embodiment 30 or 31, wherein the connector component comprises a fitting or adapter configured to connect to the channel and the actuation device.

[0219] Embodiment 33. A method according to any of embodiments 30-32, wherein the test kit device further includes a valve positioned in the channel, and wherein the method further includes: operating the valve by an actuator of the processing device to control the flow of fluid through the channel.

[0220] Embodiment 34. The method of any one of embodiments 21-33, wherein the kit device further comprises a third chamber comprising a third inlet, and wherein the method further comprises: receiving a second aqueous fluid into the interior of the device via the third inlet.

[0221] Embodiment 35. The method of embodiment 34, further comprising fluidly coupling the third chamber to the first chamber via a channel to allow the second aqueous fluid to flow into the first chamber.

[0222] Embodiment 36. A method according to any of embodiments 21-35, wherein the kit device further includes a series of two or more filtration chambers connecting the first chamber to the second chamber, and wherein the channel includes a plurality of sub-channels, and the plurality of sub-channels fluidically connect successive chambers in the series of two or more filtration chambers to allow the aqueous fluid to flow from the first chamber to the second chamber.

[0223] Embodiment 37. The method of embodiment 36 further comprising: aligning a filter chamber from the two or more filter chambers with a second transducer assembly of the processing device when the test kit apparatus is loaded in the processing device, wherein the filter chamber includes a bottom surface that acoustically couples the filter chamber with the second transducer assembly to receive ultrasonic energy from the processing device to promote selective flow of the target component.

[0224] Embodiment 38. A filter device for enhancing the separation of living cells from a solid biological tissue sample, the filter device comprising: a tubular body configured to be telescopically inserted into a container containing a biological tissue sample in an aqueous fluid; and a cell filter covering a bottom opening of the tubular body, wherein the cell filter is configured to compress the biological tissue sample to expel cells from the tissue sample when the filter device is fully inserted into the container.

[0225] Embodiment 39. The filter device of embodiment 38, wherein the cells are expelled into a surrounding aqueous fluid that flows through the cell strainer into the center of the tubular body.

[0226] Embodiment 40. The filter device according to embodiment 38 or 39 further comprises: a transport channel that transports the discharged cells flowing into the center of the tubular body to the collection chamber.

[0227] Embodiment 41. The filter device of Embodiment 40, wherein the delivery channel comprises a tube within the interior of the tubular body and extending along the length of the tubular body.

[0228] Embodiment 42. A filter device according to any one of embodiments 38-41, wherein when the tubular body is fully inserted into the container, the lower portion of the tubular body is immersed in the container, and wherein the lower portion includes one or more openings covered by one or more corresponding cell strainers.

[0229] Embodiment 43. A filter device according to embodiment 42, wherein the cells are expelled into a surrounding aqueous fluid, which flows into the center of the tubular body through the one or more cell strainers corresponding to the one or more openings in the lower portion.

[0230] Embodiment 44. The filter device of any one of embodiments 38-43, wherein the tubular body is configured to extend into the container at most a predetermined distance to enable the cell strainer to compress the biological tissue sample.

[0231] Embodiment 45. The filter device of Embodiment 44, wherein the tubular body includes a raised portion that prevents the tubular body from extending into the container beyond the predetermined distance.

[0232] Embodiment 46. The filter device of Embodiment 44 or 45, wherein the container further comprises a stopper material at a bottom of the container, the stopper material preventing the tubular body from extending into the container beyond the predetermined distance.

[0233] Embodiment 47. The filter device of Embodiment 46, wherein the stop material comprises sponge, rubber, or plastic.

[0234] Embodiment 48. The filter device of any one of embodiments 38-47, wherein the biological tissue sample is ultrasonically broken down into cells before being compressed by the cell strainer.

[0235] Embodiment 49. The filter device of any one of embodiments 38-48, wherein the biological tissue sample is enzymatically disrupted into cells before being compressed by the cell strainer.

[0236] Embodiment 50. The filter device of any one of embodiments 38-49, wherein the container corresponds to a reaction chamber of a kit apparatus.

[0237] Embodiment 51. A method for enhancing the separation of living cells from solid biological tissue samples, the method comprising: telescopically inserting the tubular body of the filter device into a container containing a biological tissue sample in an aqueous fluid, wherein the filter device includes a cell filter covering the bottom opening of the tubular body; and fully inserting the tubular body of the filter device into the container to compress the biological tissue sample with the cell filter, wherein the cell filter compresses the biological tissue sample to expel cells from the tissue sample.

[0238] Embodiment 52. The method of embodiment 51 further comprises: withdrawing the tubular body of the filter device from the container; and repeatedly performing full insertion and withdrawal of the tubular body of the filter device to repeatedly compress the biological tissue sample to increase the amount of cells discharged.

[0239] Embodiment 53. A method according to embodiments 51 and 52, wherein the cells are expelled into a surrounding aqueous fluid which flows through the cell strainer into the center of the tubular body.

[0240] Embodiment 54. The method according to any one of embodiments 51-53, further comprising: transporting the discharged cells flowing into the center of the tubular body to a collection chamber.

[0241] Embodiment 55. A method according to Embodiment 54, wherein the delivery channel comprises a tube within the tubular body and extending along the length of the tubular body.

[0242] Embodiment 56. The method according to any one of embodiments 51-55 further comprises: when the tubular body is fully inserted into the container, immersing the lower portion of the tubular body in the container, wherein the lower portion comprises one or more openings covered by one or more corresponding cell filters.

[0243] Embodiment 57. A method according to embodiment 56, wherein the cells are expelled into a surrounding aqueous fluid, which flows into the center of the tubular body through the one or more cell strainers corresponding to the one or more openings in the lower portion.

[0244] Embodiment 58. The method according to any one of embodiments 51-57, comprising: extending the tubular body into the container up to a predetermined distance to enable the cell strainer to compress the biological tissue sample.

[0245] Embodiment 59. The method of embodiment 58, wherein the tubular body further comprises a raised portion that prevents the tubular body from extending into the container beyond the predetermined distance.

[0246] Embodiment 60. The method of embodiment 58 or 59, wherein the container further comprises a stopper material located at the bottom of the container, the stopper material preventing the tubular body from extending into the container beyond the predetermined distance.

[0247] Embodiment 61. A method according to embodiment 60, wherein the stop material comprises sponge, rubber, or plastic.

[0248] Embodiment 62. The method according to any one of embodiments 51-61, further comprising: ultrasonically separating the tissue sample into cells before telescopically inserting the tubular body of the filter to compress the tissue sample through the cell strainer.

[0249] Embodiment 63. The method according to any one of embodiments 51-62, further comprising: enzymatically dissociating the tissue sample into cells before telescopically inserting the tubular body of the filter to compress the tissue sample through the cell strainer.

[0250] Embodiment 64. The method of any one of embodiments 51-63, wherein the container corresponds to a reaction chamber of a kit device.

[0251] Embodiment 65. A kit for separating and obtaining separate single cells from a biological sample, the kit comprising: (a) a sample processing unit comprising: a sealable port configured to receive the biological sample for introduction into the sample processing unit; a sample chamber configured to collect the biological sample; and a grinder, wherein the grinder is configured to interface with the sample chamber; (b) a reaction unit comprising a reaction chamber configured to receive ultrasonic energy, wherein the sample processing unit is connected to the reaction unit via a first channel; (c) a filtration unit comprising: a filtration chamber; and a filtration device configured to enter the filtration chamber, wherein the reaction chamber is connected to the filtration unit via a second channel; and (d) a collection unit configured to receive separate single cells from the biological sample, wherein the filtration unit and the collection unit are connected via a third channel.

[0252] Embodiment 66. The kit according to embodiment 65 further comprises: a first gate, which is configured to control the fluid connection between: the fluid connection between the sample processing unit and the reaction unit via the first channel; and / or the fluid connection between the reaction unit and the filtration unit via the second channel.

[0253] Embodiment 67. A kit according to embodiment 66, wherein the first gate is configured to be positioned in a first position, and wherein when the first gate is in the first position, the sample processing unit and the reaction unit are fluidically connected, and the reaction unit and the filtration unit are not fluidically connected.

[0254] Embodiment 68. A kit according to embodiment 66 or 67, wherein the first gate is configured to be positioned in a second position, and wherein when the first gate is in the second position, the reaction unit and the filtration unit are fluidically connected, and the sample processing unit and the reaction unit are not fluidically connected.

[0255] Embodiment 69. The kit according to any one of embodiments 65-68, further comprising a second gate configured to control the fluid connection between the filtration unit and the collection unit via the third channel.

[0256] Embodiment 70. The kit of embodiment 69, wherein the second gate is configured to be positioned in a first position, wherein the filtration unit and the collection unit are not fluidically connected when the second gate is in the first position.

[0257] Embodiment 71. The kit according to embodiment 69 or 70, wherein the second gate is configured to be positioned in a second position, wherein when the second gate is in the second position, the filtration unit and the collection unit are fluidly connected.

[0258] Embodiment 72. A test kit according to any one of embodiments 65-71, wherein the first channel, the second channel and the third channel are positioned and configured so that the flow between the sample processing unit, the reaction unit, the filtration unit and the collection unit is controlled by tilting the test kit.

[0259] Embodiment 73. The kit of any of embodiments 65-72, wherein the sealable port of the sample processing unit is further configured to receive a fluid for introduction into the sample processing unit.

[0260] Embodiment 74. The kit of any one of embodiments 65-72, wherein the wall of the sealable port of the sample processing unit is inclined toward the sample chamber.

[0261] Embodiment 75. The kit of any one of embodiments 65-74, further comprising a cap configured to seal the sealable port of the sample processing unit.

[0262] Embodiment 76. The kit of any one of embodiments 65-75, wherein the sample chamber comprises one or more walls that are inclined toward the center.

[0263] Embodiment 77. The kit according to any one of embodiments 65-76, wherein the reaction chamber of the reaction unit comprises a sound-conductive bottom surface.

[0264] Embodiment 78. A kit according to embodiment 77, wherein the bottom surface of the reaction chamber is substantially flat.

[0265] Embodiment 79. The kit according to any one of embodiments 65-78, wherein the reaction chamber of the reaction unit comprises a cylindrical wall.

[0266] Embodiment 80. A kit according to any one of embodiments 65-79, wherein the filtration device of the filtration unit comprises a tubular assembly, wherein the outer surface of the tubular assembly is configured to provide a seal with the side wall of the filtration chamber, and wherein the tubular assembly comprises a cell filter covering a bottom opening of the tubular assembly.

[0267] Embodiment 81. The kit of embodiment 80, wherein the sidewall of the filtration chamber is cylindrical.

[0268] Embodiment 82. The kit according to any one of embodiments 65 to 81, wherein the filtration chamber of the filtration unit comprises a bottom surface having a raised structure.

[0269] Embodiment 83. The kit of any one of embodiments 65-82, wherein the filtration unit comprises a vent.

[0270] Embodiment 84. The kit according to any one of embodiments 65-83, wherein the collection unit is configured to receive a removable collection device for receiving a single cell from the biological sample.

[0271] Embodiment 85. The kit of embodiment 84, wherein the removable collection device is an Eppendorf tube.

[0272] Embodiment 86. The kit of embodiment 85, wherein the kit is configured to be loaded into an ultrasonic processing device.

[0273] Embodiment 87. An ultrasonic treatment device comprising: (a) an ultrasonic transducer; (b) a temperature-controlled water bath; (c) a tilting assembly, wherein the tilting assembly is configured to hold the reagent kit according to any one of embodiments 65-86 so that (i) the reaction chamber of the reagent kit is positioned relative to the ultrasonic transducer, and (ii) the reaction chamber, sample chamber, and filtration chamber of the reagent kit, or portions thereof, are positioned relative to the water level of the temperature-controlled water bath, the tilting assembly comprising: a tilting actuator; a mincer actuator; and a filter actuator; and (d) a control unit.

[0274] Embodiment 88. The ultrasonic treatment device of embodiment 87, further comprising one or more gate actuators, each gate actuator being configured to interface with a gate of the reagent cartridge.

[0275] Embodiment 89. An ultrasonic treatment device according to embodiment 87 or 88, wherein the tilt assembly is configured to hold the reagent cartridge so that when the removable collection device is placed in the collection unit of the reagent cartridge, the removable collection device or a portion thereof is positioned relative to the water level of the temperature-controlled water bath.

[0276] Embodiment 90. The ultrasonic treatment device of embodiment 89, wherein the reaction chamber, the sample chamber, the filtration chamber, and the removable collection device or portion thereof are below the water level of the temperature-controlled water bath.

[0277] Embodiment 91. The ultrasonic treatment device of any one of embodiments 87-90, wherein the mincer actuator comprises a clamp that interfaces with the mincer of the reagent cartridge.

[0278] Embodiment 92. The ultrasonic treatment device of any one of embodiments 87-91, wherein the filter actuator comprises a clamp that interfaces with the filter device of the reagent cartridge.

[0279] Embodiment 93. An ultrasonic processing device according to any one of embodiments 87-92, wherein the control unit includes: one or more processors; and a memory storing one or more programs, wherein the one or more programs are configured to be executed by the one or more processors, and the one or more programs include instructions for executing a method for isolating and obtaining separate single cells from a biological sample.

[0280] Embodiment 94. An ultrasonic processing device according to embodiment 93, wherein the instructions for performing the method for isolating and obtaining separate single cells from a biological sample include instructions for any one or more of: operating the ultrasonic transducer; operating the tilt actuator; operating the mincer actuator; operating the filter actuator; operating one or more gate actuators; and operating the temperature-controlled water bath.

[0281] Embodiment 95. A method for obtaining separated single cells from a biological sample, the method comprising: loading the reagent kit according to any of embodiments 65-86 into the ultrasonic processing device according to any of embodiments 87-94; loading the biological sample into the sample processing unit of the reagent kit via a sealable port; and initiating a programmed method of the ultrasonic processing device to separate and obtain separated single cells from the biological sample.

[0282] Example

[0283] Example 1

[0284] Gene expression profiling of processed tissue samples

[0285] As explained in the background above, the use of enzymes to isolate cells from tissue samples may significantly alter the gene expression profiles of genes in the isolated cells due to enzymatically induced stress. Figure 3 The kit device 300) to apply ultrasonic separation (e.g., applying ultrasonic body wave energy) is unlikely to change the gene expression profile of the gene.

[0286] In order to observe the improvement of ultrasonic energy compared with enzyme in cell separation, four 45mg tissue samples excised from the pig liver of same commercial acquisition were experimented.The first sample remains on its solid, untreated tissue state as a control sample, and its gene expression spectrum can be compared with the gene expression spectrum of the tissue samples of other processes.The second sample is separated into a cell suspension using the Miltenyi enzymatic tissue separation scheme (provided by Mitenyi) of standard, which represents the method currently used to separate cells using enzyme.By applying ultrasonic energy to the 3rd sample in the reaction chamber placed on test kit equipment 300, the 3rd sample is separated into a cell suspension.The 4th sample is carried out ultrasonic separation with a method similar to the 3rd sample.

[0287] All four samples were frozen prior to RNA extraction and assessment of the minimum RIN value for valid sequencing data. RNA extraction and mRNA sequencing were then performed on all four samples using the Illumina TruSeq Stranded mRNA Library Preparation Kit. RNA was extracted from all four samples and sequenced on the NextSeq 500. Finally, sequencing data from the four samples were analyzed using the Strand NGS Data Analysis Tool to assess the effects of ultrasonic and enzymatic separation on the gene expression profiles of the tissue samples.

[0288] Figure 7A700A is a graph showing representative changes in the gene expression profile of untreated pig liver tissue samples across all Ensembl Sscrofa11.1 genes when using ultrasonic energy and enzymes to separate pig liver tissue samples. Graph 700A was generated by applying the experimental setup described above. The x-axis of graph 700A represents genes in the pig genome SSCROFA11.1 assembly. The y-axis of graph 700A shows the log2 fold changes of the SSCROFA11.1 gene compared to a control with a value of 0. The control sample corresponds to the gene expression profile of the Sscrofa11.1 gene calculated for the first sample (i.e., control and untreated sample). Line 702 represents the log2 fold change of the Sscrofa11.1 gene of the second sample after enzymatic hydrolysis. Lines 704 and 706 represent the log2 fold changes of the Sscrofa11.1 gene of the third and fourth samples after ultrasonic separation, respectively. As shown in graph 700A, line 702 shows much greater log2 fold changes for many Sscrofa11.1 genes compared to lines 704 and 706. In other words, the gene expression profiles of the sonicated third and fourth samples more closely resemble the gene expression profile of the native, unprocessed tissue sample (i.e., the first sample).

[0289] Figure 7B 700B is a diagram showing representative changes in the gene expression profile of untreated pig liver tissue samples on the proinflammatory and stress-induced genes 710 selected from the Ensembl Sscrofa11.1 gene when using ultrasonic energy and enzyme separation of pig liver tissue samples. 18 genes were selected from the Ensembl Sscrofa11.1 gene on the x-axis of chart 700A to generate chart 700B. The x-axis of chart 700B represents the gene 710 selected from the Sscrofa11.1 assembly of the pig genome. The y-axis of chart 700B shows the log2 fold change of the selected genes compared to the control with a set value of 0, and is represented by the gene expression profile calculated for the first sample (i.e., control and untreated sample). Line 712 represents the log2 fold change of the selected genes 710 of the second sample through enzymatic hydrolysis. Lines 714 and 716 represent the log2 fold change of the selected genes 710 of the third and fourth samples, respectively, which are separated by ultrasound. As shown in graph 700B, line 712 exhibits a much greater log2 fold change across all selected genes 710 compared to lines 714 and 716. In other words, the enzymatically digested cells of the second sample exhibited signs of stress in their gene expression, as evidenced by upregulation of selected pro-inflammatory and stress-induced genes 710. In contrast, the third and fourth samples exhibited much smaller log2 changes, meaning their gene expression profiles more closely resemble those of the natural, untreated first sample.

[0290] Example 2

[0291] Assessment of the number of viable cells obtained using the filter device

[0292] As mentioned above in the background and Figure 4 As explained, after ultrasonic separation, many separated living cells can be captured in the cell matrix of the solid tissue sample. The filter device 402 can be used according to Figure 6 The method 600 operates to compact and compress the solid tissue sample in the container to expel captured living cells to increase the number of living cells obtained. In order to observe the improvement achieved by using the filter device 402, one basic solution experiment and two filter pressing experiments were performed.

[0293] In the basic protocol experiment, ultrasonic separation was repeatedly applied to a 41.3 mg pig liver tissue sample without any filtration. Specifically, the following steps were performed:

[0294] 1. Loading the tissue sample into the 9 mm diameter reaction chamber (i.e., tube) of the kit device 300 without any mincing operation and without any enzyme;

[0295] 2. The ultrasonic processing device 120 applies ultrasonic energy for two minutes to separate living cells from the tissue sample;

[0296] 3. Evaluate the number of total recovered cells and total recovered viable cells from sonicated tissue samples; and

[0297] 4. Repeat steps 2-3 six times.

[0298] In the filter press experiments, two 45 mg portions of porcine liver tissue were ultrasonically separated and then filtered using a 402 filter unit. Specifically, the following steps were performed for each filter press experiment:

[0299] 1. Loading the tissue sample into the 9 mm diameter reaction chamber (i.e., tube) of the kit device 300 without any mincing operation and without any enzyme;

[0300] 2. The ultrasonic processing device 120 applies ultrasonic energy for two minutes to separate living cells from the tissue sample;

[0301] 3. Apply six pressure filtration passes to the tissue sample using filter device 402; and

[0302] 4. Evaluate the number of total recovered cells and total recovered viable cells from sonicated tissue samples.

[0303] Figure 8800 is a table showing representative numbers of viable cells obtained when pig liver tissue was ultrasonically isolated and ultrasonically isolated and filter pressed. Table 800 was generated using the experimental setup described above. Specifically, table 800 includes base case results 802 and filter press results 804 derived from the base case experiment and filter press experiment, respectively, as described above.

[0304] The basic scheme result 802 shows the total number of cells and the total number of viable cells per milligram obtained at the end of each round of six rounds of ultrasonic separation. At the end of six rounds of ultrasonic separation, the basic scheme experiment obtained a cumulative total of 39795 viable cells / mg. On the contrary, the filter press result 804 shows the total number of cells and the total number of viable cells per milligram obtained at the end of two filter press experiments (that is, corresponding to samples 3-1 and 4-1). As shown in Table 800, the two filter press experiments obtained a cumulative total of 76,220 and 73,260 viable cells respectively, which resulted in an average of 74,740 viable cells per milligram. Therefore, the results in Table 800 show that, compared with only using ultrasonic separation to obtain viable cells, using filter press 400 to discharge cells has achieved a nearly two-fold increase in viable cell recovery.

[0305] Example 3

[0306] Evaluating the performance of the disclosed kit device

[0307] To evaluate the performance of ultrasound separation of tissue samples using the disclosed method and kit apparatus, the following experimental protocol was performed on various porcine liver tissue sample sizes (i.e., 10 mg to 200 mg):

[0308] 1. Load the tissue sample of the selected size into the loading chamber 306 of the kit device 300 along with 370 μL of DMEM aqueous solution;

[0309] 2. The mincing device 312 is used to apply 100 mincing strokes to the tissue sample in the reagent kit device 300;

[0310] 3. Add another 370 uL of DMEM aqueous solution to the loading chamber 306 of the kit device 300;

[0311] 4. The ultrasonic processing device 120 applies ultrasonic energy for two minutes to separate living cells from the tissue sample;

[0312] 5. Apply six pressure filtrations to the tissue sample in the kit device 300 using the pre-wet filter device 310;

[0313] 6. The aqueous suspension resulting from the filter press is transferred to a recovery tube (e.g., collection chamber 308) via the filter apparatus 310; and

[0314] 7. A portion of the tissue sample ultrasonically separated in the aqueous solution in the recovery tube was evaluated to determine the number of viable cells recovered.

[0315] Figure 9A Graph 900A shows representative numbers of viable cells per milligram obtained from various sized porcine liver tissue samples using ultrasonic separation and filtration in the kit apparatus 300. Graph 900A was generated by applying the experimental setup described above to the following sized porcine liver tissue samples: 10 mg, 25 mg, 50 mg, 100 mg, and 200 mg. Graph 900A includes results shown in a bar graph portion 902 and a data table portion 904 corresponding to the bars in bar graph portion 902. As shown in graph 900A, a consistent total number of viable cells (i.e., at least 135,000 viable cells per milligram) was isolated from the porcine liver tissue samples, regardless of tissue sample size.

[0316] Figure 9B 900B is a graph showing the representative number of viable cells of various sizes per milligram obtained from pig liver tissue samples of various sizes by applying ultrasonic separation and filter pressing in the test kit device 300. Graph 900B is generated by evaluating the cell size of the viable cells obtained per milligram shown in graph 900A. Graph 900B includes a bar graph portion 906 and a data table portion 908 corresponding to the bars in bar graph portion 902. In particular, bar graph portion 906 graphically shows the viable cells recovered per milligram for four types of cell sizes (i.e., 11-20 μm, 21-30 μm, 31-40 μm, and >40 μm) under five different tissue sample specifications (i.e., 10 mg, 25 mg, 50 mg, 100 mg, and 200 mg). Data table portion 908 shows the numerical values ​​corresponding to the bars shown in bar graph portion 906. As shown in graph 900B, different cell populations with different cell sizes were successfully separated from tissue samples of different sizes.

[0317] Example 4

[0318] Exemplary kits, devices, and methods for obtaining isolated single cells from biological samples

[0319] This example demonstrates exemplary kits, devices, and methods of use of the present application for obtaining isolated single cells from a biological sample.

[0320] The kit was designed in Figure 10A1000 is shown in the schematic diagram of . As described therein, test kit 1000 is configured to have a sample processing unit, which includes a sealable port 1002 and an inclined wall 1004 to guide substances such as biological samples to a sample chamber 1006. The sample chamber 1006 is configured to have a wall inclined toward the center to ensure that the biological sample and any added substances are concentrated in a predetermined portion of the sample chamber, i.e., a grinder 1008 and the sample chamber interface. The sample processing unit and grinder 1008 are configured so that when the biological sample repeatedly descends and rises to the sample chamber, the grinder will process (such as crush) the biological sample. Test kit 1000 is configured to have an inclined first channel 1010 to guide substances from the sample processing unit to a reaction chamber 1012 when the test kit 1000 is tilted. The test kit 1000 is configured to have a first gate 1014 to control the flow of substances between the sample chamber 1006 and the reaction chamber 1012 and the reaction chamber 1012 and the filtration chamber 1016. The reagent kit 1000 is configured to have a second channel 1018 connecting the reaction chamber 1012 and the filtration unit. The filtration unit is configured to have: a filtration chamber 1016 having a convex bottom surface, a filtration device 1020, and a vent 1022. The reagent kit is configured to have a third channel connecting the filtration unit and the collection unit, wherein a second gate 1024 controls the flow of substances in the third channel. The reagent kit 1000 is configured to maintain and create a seal with an Eppendorf tube 1026 to collect separated single cells.

[0321] like Figure 10B and Figure 10C As shown in , the designed reagent kit is loaded into the tilting assembly 1050 of the ultrasonic processing device for processing. The tilting assembly is shown on the outside of the ultrasonic processing device to facilitate the display of the tilting reagent kit. In a method for obtaining separated single cells from a biological sample, the biological sample and cell culture medium buffer are loaded into the reagent kit through a sealable port of a sample processing unit. A cap is placed on the sealable port of the sample processing unit. The grinder is repeatedly placed into the sample chamber to process the biological sample. When the first gate is positioned to allow material to flow from the sample chamber to the reaction chamber, but not from the reaction chamber to the filtration chamber, the tilting actuator of the tilting assembly 1050 is activated, tilting the reagent kit to move the material in the sample chamber to the reaction chamber. The tilting actuator 1050 of the tilting assembly is then activated so that the reagent kit is positioned in the original position (the original position is such that when the tilting assembly is in the ultrasonic processing device, the reaction chamber will be positioned relative to the ultrasonic transducer). The first gate is positioned to allow material to flow from the reaction chamber to the filtration chamber, and the filtration device is in a raised position ( Figure 10B), the tilt actuator of the tilt assembly 1050 is activated to tilt the test kit to move the substance in the reaction chamber to the filtration chamber. The filtration device is repeatedly lowered into the filtration chamber to separate single cells by allowing single cells to flow through the cell filter. With the second gate positioned to allow the substance to flow from the filtration chamber to the collection unit through the third channel, the tilt actuator of the tilt assembly 1050 is activated to tilt the test kit to move the filtered material from the filtration chamber to the collection unit. The filtration device can receive the final filtrate when the test kit is tilted in this position to allow any final substance (e.g., separated single cells and cell culture medium) from the processed biological sample or a portion thereof to move to the collection unit.

[0322] The method can be repeated to further process the biological sample to obtain isolated single cells therefrom.In such an embodiment, additional cell culture medium is added to the kit through the sealable port.

[0323] For purposes of explanation, the foregoing description has been described with reference to specific embodiments. However, the above illustrative discussion is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. In light of the above teachings, many modifications and variations are possible. These embodiments were selected and described in order to best explain the principles of these techniques and their practical applications. Thus, others skilled in the art will be able to best utilize these techniques and various embodiments with various modifications to suit the specific application contemplated.

[0324] Although the present disclosure and examples have been fully described with reference to the accompanying drawings, it should be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications should be understood to be included within the scope of the disclosure and examples defined by the claims.

Claims

1. An ultrasonic treatment system comprising: (a) A kit for isolating and obtaining separate single cells from a biological sample, the kit comprising: (i) a sample processing unit comprising: a sealable port having a removable cap configured to receive the biological sample for introduction into the sample processing unit; a sample chamber configured to collect the biological sample; and mincer, wherein the mincer is configured to interface with the sample chamber; (ii) a reaction unit comprising a reaction chamber configured to receive ultrasonic energy, wherein the sample processing unit is connected to the reaction unit via a first channel; (iii) a filtration unit comprising: filter chamber; and a filtration device configured to enter the filtration chamber, wherein the reaction chamber is connected to the filtration unit via a second channel; and (iv) a collection unit configured to receive separated single cells from the biological sample, wherein the filtering unit and the collecting unit are connected via a third channel; (v) one or more gates, wherein each gate is configured to control fluid connection between any one or more of the following: a fluid connection between the sample processing unit and the reaction unit via the first channel; a fluid connection between the reaction unit and the filtration unit via the second channel; and a fluid connection between the filtration unit and the collection unit via the third channel; and (b) an ultrasonic treatment device comprising: (i) ultrasonic transducer device; (ii) a temperature-controlled water bath; (iii) tilting assembly, wherein the tilt assembly is configured to hold the reagent cartridge such that (i) the reaction chamber of the reagent cartridge is positioned relative to the ultrasonic transducer, and (ii) the reaction chamber, the sample chamber, positioning the filtration chamber, the removable collection device, or a portion thereof relative to the water level of said temperature-controlled water bath, The tilting assembly comprises: Tilt actuator; a mincer actuator; and a filter actuator; and (iv) Control unit.

2. The ultrasonic processing system according to claim 1, wherein the first channel, the second channel, and the third channel are positioned and configured so that the flow between the sample processing unit, the reaction unit, the filtration unit, and the collection unit is controlled by tilting the reagent cartridge.

3. The ultrasonic treatment system of claim 1 , wherein the one or more gates include a first gate configured to be positioned in a first position and a second position, wherein when the first gate is in a first position, the sample processing unit and the reaction unit are fluidically connected via the first channel, and When the first gate is located at the second position, the reaction unit and the filtration unit are fluidically connected via the second channel.

4. The ultrasonic treatment system of claim 1 , wherein the one or more gates include a second gate configured to be positioned in a first position and a second position, wherein when the second gate is in the first position, the filtration unit and the collection unit are not fluidically connected, and When the second gate is located at the second position, the filtering unit and the collecting unit are fluidically connected.

5. The ultrasonic processing system according to any one of claims 1 to 4, wherein the reaction chamber of the reaction unit comprises a sound-conductive bottom surface.

6. The ultrasonic processing system of claim 5, wherein the bottom surface of the reaction chamber is substantially flat.

7. The ultrasonic processing system according to any one of claims 1 to 4, wherein the filtration device of the filtration unit comprises a tubular component, wherein the outer surface of the tubular component is configured to provide a seal with the side wall of the filtration chamber, wherein the tubular component comprises a cell strainer covering the bottom opening of the tubular component, wherein the tubular component is configured to be telescopically inserted into the filtration chamber containing the biological sample or a portion thereof, and wherein the cell strainer is configured to compress the biological sample or a portion thereof to expel the single cells from the biological sample when the filtration device is fully inserted into the filtration chamber.

8. The ultrasonic treatment system of claim 7, wherein the tubular assembly is configured such that the cells are expelled into a surrounding aqueous fluid that flows through the cell strainer into the center of the tubular assembly.

9. The ultrasonic processing system of claim 7, wherein the side wall of the filtration chamber is cylindrical.

10. The ultrasonic processing system according to any one of claims 1 to 4, wherein the filter chamber of the filter unit includes a bottom surface having a convex structure.

11. The ultrasonic treatment system according to any one of claims 1 to 4, wherein the filter unit comprises a vent.

12. The ultrasonic processing system according to any one of claims 1 to 4, wherein the collection unit is configured to receive a removable collection device for receiving a single cell from the biological sample.

13. The ultrasonic treatment system according to any one of claims 1 to 4, wherein the reagent cartridge is configured to be loadable into the ultrasonic treatment device.

14. The ultrasonic processing system of any one of claims 1-4, further comprising one or more gate actuators, each gate actuator configured to interface with a gate of the reagent cartridge, wherein the mincer actuator comprises a clamp that interfaces with a mincer of the reagent cartridge, and wherein the filter actuator comprises a clamp that interfaces with a filter device of the reagent cartridge.

15. The ultrasonic treatment system according to any one of claims 1 to 4, wherein the control unit comprises: one or more processors; and A memory storing one or more programs configured to be executed by the one or more processors, the one or more programs including instructions for executing a method for isolating and obtaining separate single cells from a biological sample.

16. The ultrasonic processing system of claim 15, wherein the instructions for performing the method for isolating and obtaining separate single cells from a biological sample include instructions for any one or more of: operating the ultrasonic transducer; operating the tilt actuator; operating the mincer actuator; operating the filter actuator; operating one or more gate actuators; and The temperature-controlled water bath was operated.

17. A method for obtaining isolated single cells from a biological sample, the method comprising: loading the reagent kit in the ultrasonic treatment system according to any one of claims 1 to 16 into the ultrasonic treatment device in the ultrasonic treatment system according to any one of claims 1 to 16; loading a biological sample into the sample processing unit of the kit via a sealable port; and initiating a programmed method of the ultrasonic processing device to separate and obtain isolated single cells from the biological sample.

Images