Method and system for dissociating biological tissue into single cells using ultrasonic energy

Abstract

Described herein is a method of dissociating single cells from a biological tissue sample and a system for performing such a method. A method described herein includes: generating one or more ultrasound pulses using a transducer including one or more Fresnel sector actuator (FASA) elements; and applying energy from the generated one or more ultrasound pulses to a sample container holding the biological tissue sample through a coupling medium coupling the one or more FASA elements to the sample container to dissociate single cells from the biological tissue.

Description

This application is a divisional application of the invention application with an application date of October 1, 2020, a Chinese application number of 202080082517.3, and an invention name of "Methods and Systems for Dissociating Biological Tissue into Single Cells Using Ultrasound Energy". Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 909,484, filed on October 2, 2019, the content of which is incorporated herein by reference for all purposes. Technical field

[0002] Described herein are methods and systems for dissociating single cells from a biological tissue sample using ultrasound energy. Background art

[0003] In the fields of medical and biological research, many current analyses are performed by techniques that treat each tumor sample as a homogeneous mass, but tumor tissue is known to be a heterogeneous body of cancer cells (e.g., infiltrating immune cells and tumor stroma). Such methods provide a wealth of molecular information, but do not preserve the context of the cellular components in the original sample. These results are averages of the entire tumor tissue, which exclude the identification of rare sub - populations of important cells. It has been shown that information lost due to this practice may hide key insights into tumor progression, metastasis, and drug resistance. Additionally, the tumor microenvironment includes a diverse array of cell types that maintain homeostasis and drive further development. This intra - tumor cell heterogeneity has been identified as a key factor in potential progression, metastasis, and the development of drug resistance.

[0004] Flow sorting methods have been developed because they provide high - throughput multiplex information about each cell within a sample. Cell sorting can also be used to isolate rare cell types, such as cancer stem cells, pre - metastatic precursors, and drug - resistant clones, for further study. However, for single - cell analysis, the tissue must first be broken down into viable single cells without affecting phenotypic and genotypic information, which requires considerable time and effort.

[0005] Previously, proteolytic enzymes in combination with some form of physical force have been used to break down cell adhesion molecules and / or the underlying extracellular matrix to dissociate tumor tissue into single cells. However, not all cell types can be easily dissociated using enzymes. For those cell types that are sensitive to enzymatic dissociation, it has been shown that the enzymes can be harmful to the cells and negatively affect the ability of the resulting single cells to subsequently survive and / or divide. For example, enzymatic dissociation typically involves incubating the tissue at an elevated temperature (e.g., 37°C), which can lead to the expression of unwanted pro - inflammatory and / or stress - induced genes. The expression of these genes can significantly alter the state of the cells from their native state. Summary of the invention

[0006] This disclosure describes a method for dissociating single cells from a biological tissue sample using a transducer having one or more Fresnel annular sector actuator (FASA) elements, and a system for dissociating cells from a biological tissue sample, wherein the one or more FASA elements generate a substantial amount of lateral ultrasonic energy.

[0007] In some embodiments, a method for dissociating single cells from a biological tissue sample includes: generating one or more ultrasonic pulses using a transducer including one or more Fresnel annular sector actuator (FASA) elements; and applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container.

[0008] In some embodiments, a method for dissociating single cells from a biological tissue sample includes: generating one or more ultrasonic pulses using a transducer including one or more Fresnel annular sector actuator (FASA) elements; applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container; and controlling the temperature of the biological tissue sample while applying energy from the generated one or more ultrasonic pulses to the biological tissue sample.

[0009] In some embodiments, the temperature of the biological tissue sample is controlled between about 2°C and about 70°C. In some embodiments, controlling the temperature of the biological tissue sample reduces the expression of one or more pro-inflammatory or stress-induced genes as compared to not controlling the temperature of the biological tissue sample. In some embodiments, the one or more pro-inflammatory or stress-induced genes are selected from CD69, LRRK2, TNF, FOS, PDE4B, NR4A3, S100A9, S100A8, THBD, IL-1β, IL-1α, HK2, CXCL8, CXCL1, AREG, ERG2, TIMP1, and CXCR4.

[0010] In some embodiments of the above method, controlling the temperature of the biological tissue sample includes controlling the temperature of the coupling medium. In some embodiments, the temperature of the coupling medium is controlled to cool the biological tissue sample or limit the temperature increase of the biological tissue sample. In some embodiments, the coupling medium is controlled to a temperature between about 2°C and about 70°C. In some embodiments, the coupling medium is controlled to a temperature between about 2°C and about 25°C.

[0011] In some embodiments of the above method, controlling the temperature of the biological tissue sample includes using the energy from the one or more generated ultrasonic pulses to heat the biological tissue sample. In some embodiments, a first portion of the one or more ultrasonic pulses generated by the transducer is configured to heat the biological tissue sample, and a second portion of the one or more ultrasonic pulses generated by the transducer is configured not to heat the biological tissue sample. In some embodiments, the biological tissue sample is heated to a peak temperature between about 20°C and about 70°C.

[0012] In some embodiments of the above method, during application of the energy from the one or more generated ultrasonic pulses to the sample container, the biological tissue sample is heated less than 15°C or not heated.

[0013] In some embodiments of the above method, the coupling medium is a liquid, a solid, or a gel. In some embodiments, the coupling medium is a liquid, and the method further includes circulating the coupling fluid through a temperature controller.

[0014] In some embodiments of the above method, about 20% or more of the dissociated single cells are alive. In some embodiments, about 20% to about 95% of the dissociated single cells are alive. In some embodiments, two or more different types of live cells are dissociated from the biological tissue sample.

[0015] In some embodiments of the above method, the biological tissue sample includes cancerous tissue.

[0016] In some embodiments of the above method, the method includes selecting one or more of a repetition rate, a pulse duration, a duty cycle, a peak power, or a total duration for the one or more generated ultrasonic pulses. In some embodiments, the energy applied to the sample does not result in a shear force applied to the sample. In some embodiments, the energy applied to the sample results in a mixing force or a suspending force applied to the sample.

[0017] In some embodiments, multiple ultrasonic pulses are generated at a repetition rate of about 1 Hz to about 100,000 Hz.

[0018] In some embodiments, the one or more ultrasonic pulses are generated at a duty cycle of about 0.1% to about 95%.

[0019] In some embodiments, the energy from the one or more ultrasonic pulses is applied to the coupling medium for about 2 seconds to about 90 minutes.

[0020] In some embodiments of the above method, the biological tissue is suspended in a liquid within the sample container.

[0021] In some embodiments of the above method, the biological tissue is minced.

[0022] In some embodiments of the above method, the biological tissue sample is substantially free of non-endogenous proteases.

[0023] In some embodiments of the above method, the transducer comprises four or more FASA elements.

[0024] In some embodiments of the above method, the biological tissue sample is an unpreserved sample.

[0025] Also described herein is a system for dissociating single cells from a biological tissue sample, the system comprising: a transducer comprising one or more Fresnel annular sector actuators (FASA) elements configured to generate one or more ultrasonic pulses; a sample container configured to hold the biological tissue sample; and a coupling medium configured to couple the one or more FASA elements to the sample container and transmit the energy of the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0026] Further described is a system for dissociating single cells from a biological tissue sample, the system comprising: a transducer comprising one or more Fresnel annular sector actuators (FASA) elements configured to generate one or more ultrasonic pulses; a sample container configured to hold the biological tissue sample, wherein the system is configured to control the temperature of the biological tissue sample; and a coupling medium configured to couple the one or more FASA elements to the sample container and transmit the energy of the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample. In some embodiments, the system is configured to control the temperature of the biological sample to a temperature between about 2°C and about 70°C.

[0027] In some embodiments of the above system, the system further comprises a temperature controller configured to control the temperature of the coupling medium. In some embodiments, the temperature controller is configured to control the coupling medium to a temperature between about 2°C and about 70°C. In some embodiments, the temperature control is configured to cool the coupling medium to a temperature between about 2°C and about 25°C.

[0028] In some embodiments of the above system, the coupling medium is a liquid, and the system further includes a pump configured to circulate the coupling medium between a position between the transducer and the sample container and the temperature controller.

[0029] In some embodiments of the above system, the system is configured to control the temperature of the biological tissue sample by heating the biological tissue sample using the energy from the one or more generated ultrasonic pulses.

[0030] In some embodiments of the above system, the transducer is configured to selectively generate: (1) one or more ultrasonic pulses that heat the biological tissue sample, and (2) one or more ultrasonic pulses that do not heat the sample.

[0031] In some embodiments of the above system, the system further includes a control module configured to operate the transducer to generate the one or more ultrasonic pulses. In some embodiments, the control module is configured to operate the transducer to generate the plurality of ultrasonic pulses at one or more of a selected repetition rate, a selected pulse duration, a selected duty cycle, a selected peak power, a selected average pulse power, or a selected total duration. In some embodiments, the control module is configured to operate the transducer to generate the plurality of ultrasonic pulses at a repetition rate of from about 1 Hz to about 100,000 Hz. In some embodiments, the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a duty cycle of from about 0.1% to about 95%. In some embodiments, the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a total duration of from about 2 seconds to about 90 minutes. In some embodiments, the control module includes a radio frequency (RF) generator configured to operate the transducer to generate the ultrasonic pulses. In some embodiments, the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that shear forces are not applied to the sample. In some embodiments, the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that mixing forces or levitation forces are applied to the sample.

[0032] In some embodiments of the above system, the transducer includes four or more FASA elements.

[0033] In some embodiments of the above system, the system is arranged in an array including a plurality of systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The foregoing summary, as well as the following detailed description of the embodiments, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, the drawings show exemplary embodiments of the present disclosure; however, the present disclosure is not limited to the specific methods and means disclosed. In the drawings:

[0035] Figure 1 An exemplary system for dissociating cells from a biological tissue sample is shown.

[0036] Figure 2 An electrode pattern of a FASA element that can be used with a transducer of the system described herein is shown.

[0037] Figure 3 A top view of an exemplary ultrasonic transducer having four 90-degree FASA elements is shown, and the exemplary ultrasonic transducer can be used with a system for dissociating single cells from a biological tissue sample as described herein.

[0038] Figure 4 An exemplary 8x12 transducer array is shown, in which 96 transducer units are provided, and each unit has four FASA elements.

[0039] Figure 5 An exemplary system having a temperature controller and a pump is shown, and the temperature controller and the pump are configured to control the temperature of a coupling fluid during transducer operation.

[0040] Figure 6A Results of flow cytometry analysis of cells dissociated from a rabbit spleen tissue sample using an enzymatic dissociation method to distinguish between live and dead dissociated cells are shown.

[0041] Figure 6B Results of flow cytometry analysis of cells dissociated from a rabbit spleen tissue sample using a large amount of lateral ultrasonic energy as described herein to distinguish between live and dead dissociated cells are shown.

[0042] Figure 6C Results of flow cytometry analysis of cells dissociated from a rabbit spleen tissue sample using an enzymatic dissociation method to distinguish between nucleated live cells and other cell debris are shown.

[0043] Figure 6D Results of flow cytometry analysis of cells dissociated from a rabbit spleen tissue sample using a large amount of lateral ultrasonic energy as described herein to distinguish between nucleated live cells and other cell debris are shown.

[0044] Figure 7A Results of flow cytometry analysis of cells dissociated from a clinical breast cancer tumor sample using an enzymatic dissociation method are shown.

[0045] Figure 7B Shows the results of flow cytometry analysis of cells dissociated from a clinical breast cancer tumor sample using a large amount of lateral ultrasonic energy as described herein.

[0046] Figure 8A Shows the results of flow cytometry analysis of cells dissociated from a formalin-fixed paraffin-embedded (FFPE) human breast cancer xenograft (PDX) tumor biopsy obtained from a mouse model using an enzymatic dissociation method. The upper right quadrant indicates substantially no dissociated CD326+ / CD24+ cells.

[0047] Figure 8B Shows the results of flow cytometry analysis of cells dissociated from an FFPE human breast cancer xenograft (PDX) tumor biopsy obtained from a mouse model using a large amount of lateral ultrasonic energy as described herein. The upper right quadrant indicates dissociated CD326+ / CD24+ cells.

[0048] Figure 8C Shows the results of flow cytometry analysis of cells dissociated from residual FFPE human breast cancer xenograft (PDX) tumor tissue after further treatment with a large amount of lateral ultrasonic energy as described herein after enzymatic dissociation. The upper right quadrant indicates dissociated CD326+ / CD24+ cells that were not successfully dissociated using the enzymatic method.

[0049] Figure 9A Shows gene expression changes in cells dissociated from tissue by a large amount of lateral ultrasonic energy and in cells dissociated from solid tissue using an enzymatic process compared to a control sample (solid tissue) for all available genes.

[0050] Figure 9B Shows gene expression changes in cells dissociated from tissue by a large amount of lateral ultrasonic energy and in cells dissociated from solid tissue using an enzymatic process compared to a control sample (solid tissue) for pro-inflammatory or stress-induced genes. Detailed Description

[0051] Described herein are methods for dissociating single cells from a biological tissue sample using ultrasonic energy emitted from an ultrasonic transducer and systems for performing such methods. The ultrasonic transducer includes one or more Fresnel annular sector actuator (FASA) elements that allow a large amount of lateral ultrasonic energy to be applied to a biological tissue sample held in a sample container.

[0052] The FASA transducer can be used to apply energy to a biological sample, providing thorough yet gentle energy application that separates individual cells while minimizing cell lysis (a common problem when applying conventional ultrasonic energy to tissue). Thus, compared to conventional ultrasonic energy application, a greater proportion of single cells isolated from tissue are viable and / or capable of expressing proteins. In addition, the methods described herein allow for faster and more thorough cell dissociation than methods that include using enzymes (such as proteases) to isolate cells from a biological tissue sample, while also avoiding enzyme-induced changes in the in vivo state of the cells.

[0053] The substantial lateral ultrasonic waves generated by the FASA element can be configured to apply one or more of a mixing force, a suspending force, a shearing force, or heating energy (or can be configured not to apply one or more of these). The energy from the emitted ultrasonic waves used to dissociate single cells from biological tissue can, for example, apply a mixing force and / or a suspending force to the biological tissue sample, thereby dissociating single cells from the biological tissue sample. At the same time, the ultrasonic energy applied to the sample can be applied to avoid applying a shearing force that may cause cell lysis.

[0054] The temperature of the sample can be controlled during single cell dissociation, which can limit the expression of pro-inflammatory and / or stress-induced genes. Heating the sample for an extended period of time can lead to the expression of pro-inflammatory and / or stress-induced genes, which can alter the biological state of the cells. For example, heating tissue to 37°C during enzymatic cell dissociation induces the expression of pro-inflammatory and stress-induced genes. However, a brief heat burst applied to the sample can aid in tissue dissociation while limiting the expression of pro-inflammatory or stress-induced genes. Thus, in some embodiments, the ultrasonic energy applied to the sample is configured to apply thermal energy to the sample. The thermal energy can controllably warm the sample for a desired duration, which can be less than the total duration of the ultrasonic energy applied to the cells.

[0055] In some embodiments, a coupling medium is used to control the temperature of the sample, for example to reduce the temperature of the sample heated by the applied ultrasonic energy (or to counteract the temperature increase caused by the applied ultrasonic energy heating so that it does not increase as much as it would without using the coupling medium to control the temperature). For example, the coupling medium can absorb heat from the sample, and a temperature controller can be used to control the temperature of the coupling medium. In some embodiments, the sample is warmed by a coupling medium that can be heated by a temperature controller.

[0056] Biological tissue samples often have heterogeneous cell populations, including two or more different cell types. Using the methods described herein, viable single cells can be dissociated from a biological tissue sample, where these single cells are within a heterogeneous population of live cells. Previously known methods of dissociating cells typically result in preferential cell lysis, which may destroy certain cell types from a heterogeneous biological tissue sample. In some embodiments, the methods result in the dissociation of two or more different types of viable cells from a sample, such as a cancer sample.

[0057] In an exemplary method, dissociating single cells from a biological tissue sample can include: using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements to generate a plurality of ultrasonic pulses; and applying energy from the generated plurality of ultrasonic pulses to a sample container holding the biological tissue sample through a coupling medium, thereby dissociating single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the container.

[0058] In another exemplary method of dissociating single cells from a biological tissue sample, the method includes: using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements to generate one or more ultrasonic pulses; applying energy from the generated one or more ultrasonic pulses to a sample container holding the biological tissue sample through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container; and controlling the temperature of the biological tissue sample when applying energy from the generated one or more ultrasonic pulses to the sample container. Controlling the temperature of the biological tissue sample can reduce the expression of one or more pro-inflammatory or stress-induced genes compared to not controlling the temperature of the biological tissue sample.

[0059] In an exemplary system for dissociating single cells from a biological sample, the system can include: a transducer comprising one or more Fresnel annular sector actuator (FASA) elements configured to generate a plurality of ultrasonic pulses; a sample container configured to hold a biological tissue sample; and a coupling medium configured to couple the one or more FASA elements to the container and transmit energy from the plurality of ultrasonic pulses generated by the transducer to the container, thereby dissociating single cells from the biological tissue sample.

[0060] In another exemplary system for dissociating single cells from a biological tissue sample, the system includes: a transducer including one or more Fresnel annular sector actuator (FASA) elements configured to generate one or more ultrasonic pulses; a sample container configured to hold the biological tissue sample, wherein the system is configured to control the temperature of the biological tissue sample; and a coupling medium configured to couple the one or more FASA elements to the sample container and transfer the energy of a plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample. Definition

[0061] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" include plural referents.

[0062] References herein to "about" a value or parameter include (and describe) variations that are directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0063] A "live" cell refers to a cell that is alive and capable of cell growth or expansion in a culture medium.

[0064] It should be understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" multiple aspects and variations.

[0065] Where a range of values is provided, it is understood that each intermediate value between the upper and lower limits of the range and any other stated value or intermediate value within the stated range is included within the scope of the present disclosure. Ranges that exclude any of the included limits of those stated ranges are also included within the scope of the present disclosure when the stated range includes the upper or lower limit.

[0066] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. The description is provided to enable a person of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its claims. Various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles herein can be applied to other embodiments. Thus, the invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features described herein.

[0067] The disclosures of all publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. If any reference incorporated by reference conflicts with the present disclosure, the present disclosure shall control. System for dissociating cells from biological tissue

[0068] A system for dissociating cells from a biological tissue sample includes a transducer, a sample container, and a coupling medium. The transducer includes one or more Fresnel annular sector actuator (FASA) elements configured to generate ultrasonic pulses. The sample container can hold the biological tissue sample, and the coupling medium is positioned to couple the one or more FASA elements to the container at a defined distance. When the transducer emits ultrasonic pulses, the coupling medium transfers energy from the ultrasonic waves to the container, which allows for the dissociation of single cells from the biological tissue sample.

[0069] In some embodiments of the system for dissociating single cells from a biological tissue sample, the system is configured to control the temperature of the biological tissue sample. For example, the system can be configured to control the temperature of the biological tissue sample to be between about 2°C and about 70°C (such as between about 2°C and about 5°C, between about 5°C and about 10°C, between about 10°C and about 15°C, between about 15°C and about 25°C, between about 25°C and about 35°C, between about 35°C and about 45°C, between about 45°C and about 55°C, or between about 55°C and about 70°C).

[0070] Figure 1 A side view of an exemplary system 100 including a transducer 102 and a sample container 110 for an example embodiment is shown. The transducer 102 includes a piezoelectric material 104 sandwiched between a top electrode 106 and a bottom electrode 108. As discussed below, the electrodes 106 and 108 are configured as Fresnel annular sector actuators (FASA) that direct ultrasonic waves in a target direction (e.g., upward in Figure 1 ). A control module 109 is configured to operate the transducer to generate ultrasonic waves. The sample container 110 houses a biological tissue sample 112 that can be suspended in a liquid (e.g., water or buffer). The system 100 further includes a coupling medium 114 configured to couple the transducer 104 to surround the sample container 110. The coupling medium can be held within the sidewalls of the system. The biological tissue sample 112 within the sample container 110 is optionally below the surface 116 of the coupling medium 114. The coupling medium 114 provides acoustic coupling between the transducer 104 and the sample container 110 such that it transfers the energy from the ultrasonic waves emitted by the transducer 102 to the sample container, which causes single cells to dissociate from the biological tissue sample 112 within the sample container 110.

[0071] One or more Fresnel annular sector actuators (FASA) elements of the transducer allow the transducer to emit ultrasonic waves having a high lateral acoustic potential energy distribution directed towards a target direction, which allows for the mixing of fluids within the target direction (e.g., a liquid holding a biological tissue sample). Exemplary FASA elements are described in US 6,682,214 or Vivek et al., Novel Acoustic-Wave Micromixer, Proceedings MEMS 2000, pages 668-673 (2000). FASA is a self-focusing acoustic wave transducer that focuses acoustic waves through constructive wave interference. Strong ultrasonic waves are generated over the electrode regions, and the ultrasonic waves interfere with each other as they propagate. This results in the mixing of the fluid present in the target direction. Mixing can be enhanced by providing selective activation of different FASA elements. That is, one or more FASA elements can be operated independently.

[0072] The electrodes of the FASA element can hold a piezoelectric material such as lead zirconate titanate (PZT), BaTiO 3 , KNbO 3 etc. A current passing through the electrodes activates the piezoelectric material, causing the transducer to generate ultrasonic waves. The electrodes of the FASA element are configured in one or more annular ring segments. When the annular ring is broken into angled segments, there is a proportional change in the vertical acoustic potential energy distribution and the lateral acoustic potential energy distribution. More specifically, as the angle of the sector distribution becomes smaller, the gradient of the lateral acoustic potential energy becomes larger, and the vertical potential energy distribution becomes more dispersed. The angle of the FASA element can be between greater than 0° and less than 360°, e.g., up to about 10°, about 10° to about 20°, about 20° to about 45°, about 45° to about 60°, about 60° to about 75°, about 75° to about 90°, about 90° to about 105°, about 105° to about 120°, about 120° to about 135°, about 135° to about 150°, about 150° to about 165°, about 165° to about 180°, about 180° to about 210°, about 210° to about 240°, about 240° to about 270°, about 270° to about 300°, about 300° to about 330°, or about 330° to less than 360°. In some embodiments, the angle of the FASA element is about 75° to about 105°. In some embodiments, the angle of the FASA element is about 90°.

[0073] The electrodes of the FASA element are patterned by one annular segment or by multiple concentric annular segment patterns arranged in a Fresnel lens pattern. For example, the electrodes of the FASA element can include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more concentric annular segments. The FASA element operates by driving a current through the electrodes via the electrode terminals, e.g., by operating a control module to deliver the current. Figure 2Shows the electrode pattern of the FASA element that can be used with the transducer of the system described herein. In this exemplary embodiment, the angle of the FASA element 200 is 90°, as defined by the edges of the FASA elements 202 and 206. The FASA element includes an electrode configured with a plurality of concentric annular segments (e.g., 204 and 208). The electrode includes terminals 210 and 212 through which the control module can drive current. The electrode holds the piezoelectric material (see Figure 1 ), and the piezoelectric material generates an electric wave when current passes through the electrode.

[0074] The transducer of the system can have one or more FASA elements that direct ultrasonic waves in the direction of the sample container. For example, the transducer can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more FASA elements configured to emit ultrasonic waves in the direction of the sample container. The FASA elements can be arranged in a circular pattern, where the vertices of the FASA elements are positioned near the center of the circle. In some embodiments, the diameter of the circular pattern is from about 5 mm to about 20 mm in diameter (e.g., from about 5 mm to about 7 mm in diameter to about 9 mm in diameter, from about 9 mm in diameter to about 11 mm in diameter, from about 11 mm in diameter to about 13 mm in diameter, from about 13 mm in diameter to about 15 mm in diameter, from about 15 mm in diameter to about 17 mm in diameter, or from about 17 mm in diameter to about 20 mm in diameter), and the FASA elements of the transducer can be separated by gaps that can be uniform along the radius of the FASA or can vary depending on the configuration of the FASA elements on the transducer. In some embodiments, two or more of the FASA elements overlap.

[0075] Multiple FASA elements (or a portion of the FASA elements) on the same transducer can be activated simultaneously or non-simultaneously (e.g., two or more FASA elements can be activated sequentially or alternately) to generate ultrasound. Through appropriate control of the activation of the FASA elements, random or controlled mixing is achieved. In some embodiments, 50% or more than 50% of the FASA elements of the same transducer are activated simultaneously. In some embodiments, 75% or more than 75% of the FASA elements of the same transducer (e.g., 3 out of 4 FASA elements) are activated simultaneously. In some embodiments, all of the FASA elements of the same transducer are activated simultaneously. When less than all of the FASA elements are activated simultaneously, one or more inactive FASA elements can alternate among all of the FASA elements of the transducer during operation.

[0076] Figure 3A top view of an ultrasonic transducer 300 having four 90-degree FASA elements labeled T1, T2, T3, and T4 is shown. Small gaps between the FASA elements allow the FASA elements to effectively combine mechanical effects to generate lateral mixing forces. The individual FASA elements can operate independently.

[0077] The transducer can be a single transducer or can be arranged in a transducer array. The transducer array can include a plurality of discrete piezoelectric elements or can have a common (i.e., shared) piezoelectric element that is configured with one or more FASA elements to form individual transducer units. An exemplary transducer array is described in U.S. Patent No. 8,319,398. Figure 4 An exemplary 8x12 transducer array is shown, in which 96 transducer units are provided, each unit having four FASA elements. Arrays with any number of transducers can be used, e.g., between 1 and 1000 transducers within the array. The number of transducers within the array can match the number of wells, for example, in a microplate used for sample containers (e.g., a 96-transducer array can be used with a 96-well plate, or a 384-transducer array can be used with a 384-well plate).

[0078] A control module can be used to operate the FASA elements of the transducer using one or more selected parameters to generate ultrasonic waves. The control module can include, for example, a radio frequency (RF) generator that can induce a current through the FASA element electrodes. In some embodiments, the control module includes an amplifier that can amplify the RF signal generated by the RF generator to operate the transducer. The control module can also include one or more additional electronic circuits that are configured to adjust one or more parameters of the ultrasonic waves emitted by the transducer, for example, by controlling the RF signal. The control module can control, for example, the peak power, pulse repetition rate, RF frequency, duty cycle, pulse duration, total duration, etc. of the RF energy applied to the ultrasonic transducer, which controls the ultrasonic waves emitted from the transducer. The control module can include a user interface (which can include, for example, one or more buttons, switches, or digital interfaces) that can be used to select one or more control module parameters. The parameters of the electrical signal flowing through the FASA element electrodes control the resulting ultrasonic waves generated by the transducer. The frequency of the drive current flowing through the electrodes of the FASA element can be based on the resonance frequency of the piezoelectric material to enhance the generation of ultrasonic waves (e.g., within 10%, 20%, or 30% or more of the resonance frequency). The shape of the drive current can be sinusoidal, square, or any other suitable shape.

[0079] Selected parameters for controlling the RF energy applied to an ultrasonic transducer can affect the type of ultrasonic waves generated by the ultrasonic transducer. The FASA element generates a large number of lateral ultrasonic waves, and the large number of lateral ultrasonic waves can be configured to generate one or more of a mixing force, a levitation force, a shear force, and heating energy. The mixing force and the levitation force are generally beneficial for dissociating single cells from a tissue sample, while the shear force may cause cell lysis and is preferably avoided. Optionally, the large number of lateral ultrasonic waves generated by the transducer can be configured to apply thermal energy to a biological tissue sample, and the thermal energy can be applied to control the temperature of the biological tissue sample. In some embodiments, the energy from the ultrasonic waves does not apply thermal energy to the biological tissue sample. The energy from the ultrasonic waves applied to the biological tissue sample can be non-uniform during the cell dissociation process. For example, a first portion of the ultrasonic waves can apply thermal energy to the biological tissue sample, while a second portion of the ultrasonic waves does not apply thermal energy to the biological tissue sample.

[0080] The control module can be configured to operate the transducer to generate ultrasonic waves or ultrasonic pulses (or multiple such pulses) at one or more of a selected repetition rate, a selected pulse duration, a selected duty cycle, a selected peak power, a selected average pulse power, or a selected total duration. The parameters of the ultrasonic waves generated by the transducer are selected to apply a mixing force and a levitation force in the liquid held by the container such that single cells are dissociated from the tissue while minimizing cell lysis.

[0081] In some embodiments, the control module operates the transducer to generate one or more ultrasonic pulses. The ultrasonic pulses can be generated at a selected repetition rate, which can be controlled, for example, by the repetition rate of the RF energy applied to the transducer. For example, the repetition rate can be from about 1 Hz to about 100,000 Hz (such as about 1 Hz to about 5 Hz, about 5 Hz to about 10 Hz, about 10 Hz to about 25 Hz, about 25 Hz to about 50 Hz, about 50 Hz to about 100 Hz, about 100 Hz to about 150 Hz, about 150 Hz to about 200 Hz, about 200 Hz to about 300 Hz, about 300 Hz to about 400 Hz, about 400 Hz to about 500 Hz, about 500 Hz to about 750 Hz, or about 750 Hz to about 1000 Hz, about 1000 Hz to about 2500 Hz, about 2500 Hz to about 5000 Hz, about 5000 Hz to about 10,000 Hz, about 10,000 Hz to about 25,000 Hz, about 25,000 Hz to about 50,000 Hz, or about 50,000 Hz to about 100,000 Hz). The repetition rate refers to the frequency of the ultrasonic pulses when the transducer is operated to emit multiple ultrasonic pulses. In some embodiments, the ultrasonic pulses are generated with a duty cycle of from about 0.1% to about 95% (such as about 0.1% to about 0.25%, about 0.25% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 3%, about 3% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 95%).

[0082] If ultrasonic energy is applied to the sample container for too long a period of time, the cells within the sample container may lyse. Accordingly, the control module is configured to operate the transducer to generate ultrasonic waves (or wave pulses) for a selected duration, e.g., between about 2 seconds and about 90 minutes (e.g., about 2 seconds to about 5 seconds, about 5 seconds to about 10 seconds, about 10 seconds to about 15 seconds, about 15 seconds to about 30 seconds, about 30 seconds to about 1 minute, about 1 minute to about 2 minutes, about 2 minutes to about 4 minutes, about 4 minutes to about 6 minutes, about 6 minutes to about 8 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 30 minutes, about 30 minutes to about 45 minutes, about 45 minutes to about 60 minutes, about 60 minutes to about 75 minutes, or about 75 minutes to about 90 minutes).

[0083] In some embodiments, the system is configured to control the temperature of a biological tissue sample using a coupling medium, energy from an ultrasonic pulse, or both. As discussed above, an ultrasonic pulse can be configured to apply energy to the biological sample to heat the sample, or a portion of the ultrasonic wave that is configured to apply energy to the biological tissue sample is configured to heat the sample. A coupling medium can be (alternatively or additionally) used to control the temperature of the biological tissue sample. The coupling medium is thermally coupled to the biological tissue sample, and controlling the temperature of the coupling medium can control the temperature of the biological tissue sample. For example, the coupling medium can be cooled, which can be used to lower the temperature of the biological tissue sample. In some embodiments, one or more ultrasonic pulses configured to heat the biological tissue sample for a period of time are used to heat the temperature of the biological tissue sample, and a coupling fluid can be used to lower the temperature of the biological tissue sample after the biological tissue sample has been heated. This allows heat to be controllably applied to the biological tissue sample over a relatively short period of time, which can help dissociate single cells from the tissue, but then the coupling medium can be used to lower the temperature of the biological tissue sample so that the cells limit the expression of pro-inflammatory and / or stress-induced genes.

[0084] The coupling medium of the system couples one or more FASA elements of the transducer to the sample container. Energy from the ultrasonic waves generated by the transducer can be transmitted through the coupling fluid into the sample container, which then dissociates single cells from the biological tissue sample within the sample container. The coupling medium can be a fluid (e.g., a liquid), a solid, or a gel. Exemplary coupling media can include, but are not limited to, water, a buffer, oil, a water-based gel, propylene glycol, glycerol, or an ethylene glycol ether.

[0085] The system can include a temperature controller configured to control the temperature of a coupling medium. Since the coupling medium can be thermally coupled to the sample container, the temperature of the sample within the sample container can be controlled by controlling the temperature of the coupling fluid. If the temperature of the sample container becomes too hot, the cells may lyse or otherwise become non-viable. However, being too cold can also render the cells non-viable. In some embodiments, the coupling medium is controlled at a temperature of from about 2°C to about 70°C (such as between about 2°C and about 5°C, between about 5°C and about 10°C, between about 10°C and about 15°C, between about 15°C and about 25°C, between about 25°C and about 35°C, between about 35°C and about 45°C, between about 45°C and about 55°C, or between about 55°C and about 70°C). In some embodiments, during cell dissociation, the peak temperature of the sample is from about 2°C to about 80°C (such as from about 4°C to about 10°C, from about 10°C to about 15°C, from about 15°C to about 25°C, from about 25°C to about 35°C, from about 35°C to about 45°C, from about 45°C to about 55°C, from about 55°C to about 65°C, or from about 65°C to about 80°C). In some embodiments, during dissociation of cells from a tissue sample, the temperature of the sample increases by less than about 50°C, less than about 40°C, less than about 30°C, less than about 20°C, less than about 15°C, less than about 10°C, or less than about 5°C. The system can be further configured with a pump that can circulate the coupling medium between a location between the transducer and the sample container and the temperature controller.

[0086] Figure 5 An exemplary system with a temperature controller and a pump is shown, the system being configured to control the temperature of the coupling fluid during transducer operation. In Figure 5 FIG. 5, system 500 includes a transducer 502 having one or more FASA elements configured to generate ultrasonic waves that are directed to a sample container 506 coupled to the transducer through a coupling medium 510. The transducer is operated using a control module 504 that can selectively pass current through one or more FASA elements to generate ultrasonic waves. The sample container 506 is configured to hold a biological tissue sample 508. The coupling medium 510 is fluidly connected to a pump 512 and a temperature controller 514. During operation of system 500, the coupling medium 510 can be circulated to the temperature controller 514 using the pump 512, which can operate to warm or cool the coupling medium 510. Since the coupling medium 510 is thermally coupled to the sample container 506, the temperature controller can indirectly regulate the temperature of the biological tissue sample within the sample container.

[0087] The sample container can be a sample tube, well, or other suitable container configured to hold a desired amount of a liquid and / or biological tissue sample. For example, the sample container can be a standard laboratory tube, which can be made of plastic (such as polystyrene or polypropylene) or any other suitable material. In some embodiments, the sample container is disposed within an array of sample tubes, e.g., wells within a multi-well plate (e.g., a 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, or 384-well plate). For example, the multi-well plate can be used with the transducer array discussed above.

[0088] The devices described above can be used with any of the methods described herein. Method for dissociating single cells from a biological tissue sample

[0089] By applying the energy from one or more ultrasonic pulses to a sample container holding a biological tissue sample, single cells can be dissociated from the biological tissue sample. The ultrasonic pulses can be generated using a transducer configured with one or more Fresnel annular sector actuator (FASA) elements, which can direct lateral ultrasonic energy along the direction of the sample container through a coupling medium. The coupling medium couples the FASA element to the sample container and transmits the energy of the ultrasonic waves generated by the transducer to the sample container. The energy applied to the sample container exerts a mixing force and / or a suspending force on the sample, which causes single cells to dissociate from the biological tissue sample. In some embodiments, the method further includes controlling the temperature of the biological tissue sample while applying the energy from the generated one or more ultrasonic pulses to the biological tissue sample in the sample container.

[0090] In some embodiments, the temperature of the biological tissue sample is controlled by using a coupling medium, the energy from the ultrasonic pulses, or both. As discussed above, the ultrasonic waves can be configured to apply thermal energy to the biological tissue sample, which can raise the temperature of the biological tissue sample in the container. A temperature increase for a controlled period of time can increase the dissociation of single cells from the biological tissue sample, although prolonged heating may lead to the induction of the expression of one or more pro-inflammatory or stress-related genes. Exemplary pro-inflammatory or stress-related genes that may be expressed under prolonged thermal exposure include, but are not limited to, CD69, LRRK2, TNF, FOS, PDE4B, NR4A3, S100A9, S100A8, THBD, IL-1β, IL-1α, HK2, CXCL8, CXCL1, AREG, ERG2, TIMP1, and CXCR4.

[0091] In some embodiments, the temperature of the biological tissue sample is controlled between about 2°C and about 70°C (such as between about 2°C and about 5°C, between about 5°C and about 10°C, between about 10°C and about 15°C, between about 15°C and about 25°C, between about 25°C and about 35°C, between about 35°C and about 45°C, between about 45°C and about 55°C, or between about 55°C and about 70°C).

[0092] The biological tissue sample can be thermally coupled to a coupling medium such that controlling the temperature of the coupling medium can control the temperature of the biological tissue sample. For example, the coupling medium can be cooled to lower the temperature of the biological tissue sample. Cooling the biological tissue sample via the coupling medium can, for example, lower the temperature of the sample after heating using ultrasound configured to apply thermal energy to the sample. In some embodiments, the coupling medium is warmed to raise the temperature of the biological tissue sample. The temperature of the coupling medium can be controlled between, for example, about 2°C and about 70°C (such as between about 2°C and about 5°C, between about 5°C and about 10°C, between about 10°C and about 15°C, between about 15°C and about 25°C, between about 25°C and about 35°C, between about 35°C and about 45°C, between about 45°C and about 55°C, or between about 55°C and about 70°C). For example, the coupling medium can be a liquid or gel maintained in a temperature-controlled compartment, or the coupling medium can be circulated through a temperature controller. The coupling medium can be thermally coupled to the sample container (and thus to the biological tissue sample within the sample chamber). Thus, by controlling the temperature of the coupling medium, the temperature of the biological tissue sample itself can be controlled. In some embodiments, one or more ultrasonic pulses are configured to apply thermal energy to the biological tissue sample, and the thermal energy can be applied to control the temperature of the biological tissue sample. In some embodiments, the energy from the ultrasound does not apply thermal energy to the biological tissue sample. The energy from the ultrasound applied to the biological tissue sample can be non-uniform during the cell dissociation process. For example, a first portion of the ultrasound can apply thermal energy to the biological tissue sample while a second portion of the ultrasound does not apply thermal energy to the biological tissue sample. The biological tissue sample can be heated using ultrasound and then cooled, for example, via the coupling medium to limit the expression of pro-inflammatory and / or stress-induced genes. In some embodiments, the biological tissue sample is heated to a peak temperature between about 20°C and about 70°C (such as between about 20°C and about 25°C, between about 25°C and about 35°C, between about 35°C and about 45°C, between about 45°C and about 55°C, or between about 55°C and about 70°C).

[0093] The ultrasonic waves emitted by the transducer can be emitted as one or more ultrasonic pulses. In some embodiments, multiple ultrasonic pulses are emitted by the transducer. The multiple ultrasonic pulses can be generated, for example, at a repetition rate of 1 Hz to about 100,000 Hz (such as about 1 Hz to about 5 Hz, about 5 Hz to about 10 Hz, about 10 Hz to about 25 Hz, about 25 Hz to about 50 Hz, about 50 Hz to about 100 Hz, about 100 Hz to about 150 Hz, about 150 Hz to about 200 Hz, about 200 Hz to about 300 Hz, about 300 Hz to about 400 Hz, about 400 Hz to about 500 Hz, about 500 Hz to about 750 Hz, or about 750 Hz to about 1000 Hz, about 1000 Hz to about 2500 Hz, about 2500 Hz to about 5000 Hz, about 5000 Hz to about 10,000 Hz, about 10,000 Hz to about 25,000 Hz, about 25,000 Hz to about 50,000 Hz, or about 50,000 Hz to about 100,000 Hz). The ultrasonic pulses have a lateral force that allows single cells to dissociate from biological tissue.

[0094] In some embodiments, one or more ultrasonic pulses are generated at a duty cycle of about 0.1% to about 95% (such as about 0.1% to about 0.25%, about 0.25% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 3%, about 3% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 95%).

[0095] In some embodiments, the energy from one or more ultrasonic pulses is applied to the coupling medium for about 2 seconds to about 90 minutes (e.g., about 2 seconds to about 5 seconds, about 5 seconds to about 10 seconds, about 10 seconds to about 15 seconds, about 15 seconds to about 30 seconds, about 30 seconds to about 1 minute, about 1 minute to about 2 minutes, about 2 minutes to about 4 minutes, about 4 minutes to about 6 minutes, about 6 minutes to about 8 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 30 minutes, about 30 minutes to about 45 minutes, about 45 minutes to about 60 minutes, about 60 minutes to about 75 minutes, or about 75 minutes to about 90 minutes).

[0096] Single cells are effectively dissociated from a biological tissue sample, and a substantial portion of the single cells remain viable after dissociation. The methods provided herein dissociate single cells thoroughly enough but gently enough to limit cell lysis. For example, in some embodiments, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more of the single cells dissociated from the tissue remain viable after dissociation from the biological tissue sample. In some embodiments, about 20% to about 95% (such as about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 95%) of the cells dissociated from the tissue remain viable after dissociation from the biological tissue sample.

[0097] The biological tissue sample can be a solid tissue sample, although the solid tissue sample can be minced into adherent cell clusters before the single cells are separated from the biological tissue sample. A chopper, scalpel, scissors, or other suitable device can be used to mince the cells, although care should be taken to avoid cell lysis. The tissue can be minced into, for example, about 0.25 mm 3 to about 5 mm 3 (e.g., about 0.25 mm 3 to about 0.5 mm 3 , about 0.5 mm 3 to about 1 mm 3 , about 1 mm 3 to about 2 mm 3 , about 2 mm 3 to about 3 mm 3 , about 3 mm 3 to about 4 mm 3 , or about 4 mm 3 to about 5 mm 3 ) in size. In some embodiments, the biological tissue sample is suspended in a liquid within a sample container, such as water, a buffer, or other suitable liquid medium (e.g., Rosewell Park Memorial Institute (RPMI) medium, Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), or Dulbecco's Modified Eagle Medium (DMEM)).

[0098] Cells bind to each other in tissues through cell adhesion molecules such as integrins, IgSF CAMs, cadherins, and selectins, which can form cell junctions. Although cell adhesion molecules can be enzymatically degraded, for example, using proteases such as trypsin, collagenase, and / or dispase. However, treating cells with such enzymes may reduce cell viability because proteases may cleave unintended proteins. The methods described herein can effectively dissociate single cells from a biological tissue sample without using non-endogenous proteases (tissues may naturally include one or more proteases, but such endogenous proteases are generally ineffective in dissociating live single cells from tissues). In some embodiments, the biological tissue sample is substantially free or free of non-endogenous proteases. In this context, "substantially free" of non-endogenous proteases is understood to mean that a small amount of non-endogenous proteases may be present, provided that such non-endogenous proteases do not affect the proportion of live single cells dissociated from the biological tissue sample).

[0099] In some embodiments, the biological tissue sample 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, avian, 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 cells are multicellular prokaryotes (e.g., biofilms). In some embodiments, the tissue is tumor tissue or cancerous tissue, or a biopsy sample. The biological tissue sample 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 or healthy tissue, such as cancerous or non-cancerous tissue). The cells dissociated from the tissue sample can include two or more different types of live cells. The tissue sample can be a heterogeneous tissue sample, and different cell types can be dissociated from the tissue sample. For example, a cancerous tissue sample can include multiple different cell types such as macrophages, T cells, natural killer (NK) cells, and cancer cells, and one or more of them can be dissociated from the tissue sample). Exemplary Embodiments

[0100] The following embodiments are exemplary and are not intended to limit the scope of the invention described herein. Those skilled in the art will understand that the exemplary embodiments can be modified by adding or excluding one or more features from the exemplary embodiments).

[0101] Embodiment 1. A method for dissociating single cells from a biological tissue sample, comprising: generating one or more ultrasonic pulses using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements; and Applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container.

[0102] Embodiment 2. A method for dissociating single cells from a biological tissue sample, comprising: Generating one or more ultrasonic pulses using a transducer including one or more Fresnel annular sector actuators (FASA) elements; Applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container; and Controlling the temperature of the biological tissue sample when applying energy from the generated one or more ultrasonic pulses to the biological tissue sample.

[0103] Embodiment 3. The method according to Embodiment 2, wherein controlling the temperature of the biological tissue sample includes controlling the temperature of the coupling medium.

[0104] Embodiment 4. The method according to Embodiment 3, wherein controlling the temperature of the coupling medium cools the biological tissue sample or limits the temperature rise of the biological tissue sample.

[0105] Embodiment 5. The method according to Embodiment 3 or 4, wherein the coupling medium is controlled to a temperature between about 2°C and about 70°C.

[0106] Embodiment 6. The method according to any one of Embodiments 3-5, wherein the coupling medium is controlled to a temperature between about 2°C and about 25°C.

[0107] Embodiment 7. The method according to any one of Embodiments 2-6, wherein controlling the temperature of the biological tissue sample includes heating the biological tissue sample using energy from the generated one or more ultrasonic pulses.

[0108] Embodiment 8. The method according to Embodiment 7, wherein a first portion of the one or more ultrasonic pulses generated by the transducer is configured to heat the biological tissue sample, and a second portion of the one or more ultrasonic pulses generated by the transducer is configured not to heat the biological tissue sample.

[0109] Embodiment 9. The method according to Embodiment 7 or 8, wherein the biological tissue sample is heated to a peak temperature between about 20°C and about 70°C.

[0110] Embodiment 10. The method according to any one of Embodiments 1-6, wherein during application of the energy from the one or more generated ultrasonic pulses to the sample container, the biological tissue sample is heated by less than 15 °C or not heated.

[0111] Embodiment 11. The method according to any one of Embodiments 2-10, wherein the temperature of the biological tissue sample is controlled to be between about 2 °C and about 70 °C.

[0112] Embodiment 12. The method according to any one of Embodiments 2-11, wherein controlling the temperature of the biological tissue sample reduces the expression of one or more pro-inflammatory or stress-induced genes as compared to not controlling the temperature of the biological tissue sample.

[0113] Embodiment 13. The method according to Embodiment 12, wherein the one or more pro-inflammatory or stress-induced genes are selected from CD69, LRRK2, TNF, FOS, PDE4B, NR4A3, S100A9, S100A8, THBD, IL-1β, IL-1α, HK2, CXCL8, CXCL1, AREG, ERG2, TIMP1, and CXCR4.

[0114] Embodiment 14. The method according to any one of Embodiments 1-13, wherein the coupling medium is a liquid, a solid, or a gel.

[0115] Embodiment 15. The method according to Embodiments 1-14, wherein the coupling medium is a liquid, and the method further comprises circulating the coupling fluid through a temperature controller.

[0116] Embodiment 16. The method according to any one of Embodiments 1-15, wherein about 20% or more of the dissociated single cells are alive.

[0117] Embodiment 17. The method according to Embodiment 16, wherein about 20% to about 95% of the dissociated single cells are alive.

[0118] Embodiment 18. The method according to any one of Embodiments 1-17, wherein two or more different types of live cells are dissociated from the biological tissue sample.

[0119] Embodiment 19. The method according to any one of Embodiments 1-18, wherein the tissue sample comprises cancer tissue.

[0120] Embodiment 20. The method according to any one of Embodiments 1-19, wherein the energy applied to the sample does not result in shear forces applied to the sample.

[0121] Embodiment 21. The method according to any one of embodiments 1-20, which includes selecting one or more of a repetition rate, a pulse duration, a duty cycle, a peak power, or a total duration for the generated one or more ultrasonic pulses.

[0122] Embodiment 22. The method according to any one of embodiments 1-21, wherein the energy applied to the sample results in a mixing force or a suspending force applied to the sample.

[0123] Embodiment 23. The method according to any one of embodiments 1-22, wherein the plurality of ultrasonic pulses are generated at a repetition rate of about 1 Hz to about 100,000 Hz.

[0124] Embodiment 24. The method according to any one of embodiments 1-23, wherein the one or more ultrasonic pulses are generated at a duty cycle of about 0.1% to about 95%.

[0125] Embodiment 25. The method according to any one of embodiments 1-24, wherein the energy from the one or more ultrasonic pulses is applied to the coupling medium for about 2 seconds to about 90 minutes.

[0126] Embodiment 26. The method according to any one of embodiments 1-25, wherein the biological tissue is suspended in a liquid within the sample container.

[0127] Embodiment 27. The method according to any one of embodiments 1-26, wherein the biological tissue is minced.

[0128] Embodiment 28. The method according to any one of claims 1-27, wherein the biological tissue sample is substantially free of non-endogenous proteases.

[0129] Embodiment 29. The method according to any one of embodiments 1-28, wherein the transducer comprises four or more FASA elements.

[0130] Embodiment 30. The method according to any one of embodiments 1-29, wherein the biological tissue sample is an unpreserved sample.

[0131] Embodiment 31. A system for dissociating single cells from a biological tissue sample, comprising: A transducer comprising one or more Fresnel annular sector actuators (FASA) elements configured to generate one or more ultrasonic pulses; A sample container configured to hold the biological tissue sample; and A coupling medium configured to couple the one or more FASA elements to the sample container and transfer the energy of the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0132] Embodiment 32. A system for dissociating single cells from a biological tissue sample, comprising: A transducer comprising one or more Fresnel annular sector actuators (FASA) elements configured to generate one or more ultrasonic pulses; A sample container configured to hold the biological tissue sample, wherein the system is configured to control the temperature of the biological tissue sample; and A coupling medium configured to couple the one or more FASA elements to the sample container and transfer the energy of the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0133] Embodiment 33. The system according to Embodiment 32, wherein the system is configured to control the temperature of the biological sample to a temperature between about 2°C and about 70°C.

[0134] Embodiment 34. The system according to any one of Embodiments 31-33, further comprising a temperature controller configured to control the temperature of the coupling medium.

[0135] Embodiment 35. The system according to Embodiment 34, wherein the temperature controller is configured to control the coupling medium to a temperature between about 2°C and about 70°C.

[0136] Embodiment 36. The system according to Embodiment 34, wherein the temperature control is configured to cool the coupling medium to a temperature between about 2°C and about 25°C.

[0137] Embodiment 37. The system according to any one of Embodiments 31-36, wherein the coupling medium is a liquid, and the system further comprises a pump configured to circulate the coupling medium between a position between the transducer and the sample container and the temperature controller.

[0138] Embodiment 38. The system according to any one of Embodiments 32-37, wherein the system is configured to control the temperature of the biological tissue sample by heating the biological tissue sample using the energy from the generated one or more ultrasonic pulses.

[0139] Embodiment 39. The system according to any one of embodiments 32-38, wherein the transducer is configured to selectively generate: (1) one or more ultrasonic pulses that heat the biological tissue sample, and (2) one or more ultrasonic pulses that do not heat the sample.

[0140] Embodiment 40. The system according to any one of embodiments 31-39, further comprising a control module configured to operate the transducer to generate the one or more ultrasonic pulses.

[0141] Embodiment 41. The system according to embodiment 40, wherein the control module is configured to operate the transducer to generate the plurality of ultrasonic pulses at one or more of a selected repetition rate, a selected pulse duration, a selected duty cycle, a selected peak power, a selected average pulse power, or a selected total duration.

[0142] Embodiment 42. The system according to embodiment 40 or 41, wherein the control module is configured to operate the transducer to generate a plurality of ultrasonic pulses at a repetition rate of about 1 Hz to about 100,000 Hz.

[0143] Embodiment 43. The system according to any one of embodiments 40-42, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a duty cycle of about 0.1% to about 95%.

[0144] Embodiment 44. The system according to any one of embodiments 40-43, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a total duration of about 2 seconds to about 90 minutes.

[0145] Embodiment 45. The system according to any one of embodiments 40-44, wherein the control module includes a radio frequency (RF) generator configured to operate the transducer to generate the ultrasonic pulses.

[0146] Embodiment 46. The system according to any one of embodiments 40-45, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that shear forces are not applied to the sample.

[0147] Embodiment 47. The system according to any one of embodiments 40-46, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that mixing forces or levitation forces are applied to the sample.

[0148] Embodiment 48. The system according to any one of embodiments 30-47, wherein the transducer comprises four or more FASA elements.

[0149] Embodiment 49. The system according to any one of embodiments 30-48, wherein the system is arranged in an array comprising a plurality of systems. Examples

[0150] The present application can be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the present application. The following embodiments are presented to more fully illustrate the embodiments, but should not be construed in any way as limiting the broad scope of the present application. Although certain embodiments of the present application have been shown and described herein, it will be apparent that such embodiments are provided by way of example only. Many variations, changes and substitutions can be envisioned by those skilled in the art without departing from the spirit and scope of the present invention. It should be understood that various alternatives of the embodiments described herein can be used to practice the methods described herein. Example 1

[0151] Three tissue samples were prepared by adding approximately 100 mg of liver tissue to a culture dish containing 250 μL of Roswell Park Memorial Institute (RPMI) medium. The liver tissue samples were minced into tissue pieces sized from approximately 1 mm 3 to approximately 3 mm 3 . The tissue samples were transferred to sample tubes. The sample tubes were placed on a transducer having 4 independently operable FASA elements, the elements having an angle of 90° and arranged in a circular pattern having an approximate diameter of 9 mm. The sample temperature was controlled by a water bath (i.e., coupling fluid) by setting the cooler to 25°C. The FASA elements were activated by applying RF energy to the FASA elements such that a large amount of lateral ultrasound (BLU) energy was applied to the sample. At any given time point, three of the four FASA elements were activated within the non-active FASA elements rotating clockwise.

[0152] After applying the ultrasonic waves to the sample, the sample was passed through a 70 μm cell strainer, and 10 μL of the filtrate was visualized under a microscope. High ultrasonic pulse frequencies and extended durations of applying the ultrasonic waves resulted in cell lysis. However, live cells were obtained at lower pulse frequencies for shorter durations. Example 2

[0153] A liver tissue sample was prepared by adding approximately 100 mg of liver tissue to a culture dish containing 250 μL of Roswell Park Memorial Institute (RPMI) medium. The liver tissue sample was minced into tissue pieces sized approximately 1 mm 3 to approximately 3 mm 3 . The tissue sample was transferred to a sample tube. The sample tube was then placed on a transducer having 4 independently operable FASA elements, the elements having an angle of 90° and arranged in a circular pattern having an approximate diameter of 9 mm. The sample temperature was controlled by a water bath (i.e., coupling fluid) by setting a cooler to 25°C. The FASA elements were activated by applying RF energy to the FASA elements such that a substantial amount of lateral ultrasound (BLU) energy was applied to the sample. At any given time point, three of the four FASA elements were activated within the non-active FASA elements that rotated clockwise.

[0154] Control samples (from liver tissue) were also prepared by an enzymatic dissociation method at 37°C.

[0155] The samples were individually transferred to 15 mL centrifuge tubes and rinsed with 5 mL of wash buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)). The centrifuge tubes were centrifuged at 300 x g for 5 minutes at 4°C and the supernatant was decanted. The pellet was resuspended in 1 mL of wash buffer using a large orifice 1 mL pipette tip, then 5 mL of ACK (ammonium chloride potassium) lysis buffer was added and the cells were incubated at room temperature for 5 - 7 minutes. 5 mL of stop reaction buffer (1% BSA in Dulbecco's phosphate buffered saline (DPBS) (without Ca 2+ and Mg 2+ ) was added to the samples, which were then centrifuged at 300 x g for 5 minutes at 4°C. The supernatant was then removed and the resulting pellet was resuspended in 5 mL of DPBS. The suspended cells were filtered through a 70 μm cell strainer and the filtrate was rinsed to 5 mL with additional DPBS. The suspended cells were mixed and cell viability was determined using a Vi-CELL cell viability analyzer using 600 μL of sample. The results are shown in Table 1. Table 1

[0156] Approximately 77.5% (test samples) of the cells dissociated from liver tissue using ultrasound survived at a yield of approximately 9,800 viable cells per mg of liver tissue. Example 3

[0157] Seven liver tissue samples (sample numbers 1 - 7) and seven kidney samples (sample numbers 8 - 14) were prepared by adding approximately 100 mg of liver tissue or kidney tissue to a culture dish containing 200 μL of Roswell Park Memorial Institute (RPMI) medium. The liver tissue samples were minced into tissue blocks approximately 1 mm 3 to approximately 3 mm 3 in size using scissors or a scalpel. 200 μL of the minced tissue sample was transferred to a sterile sample tube. The sample tube was then placed on a transducer with 4 independently operable FASA elements, the elements having an angle of 90° and arranged in a circular pattern with an approximate diameter of 9 mm. The water bath (i.e., the coupling fluid) controlled the sample temperature by setting the cooler to 22 °C (resulting in a 25 °C water bath). The FASA elements were activated by applying RF energy to the FASA elements such that a substantial amount of applied lateral ultrasound (BLU) energy was applied to the sample. At any given time point, three of the four FASA elements were activated within the non - active FASA elements that rotated clockwise. The peak sample temperature was measured during the application of ultrasound energy, as shown in Table 2. Table 2

[0158] The samples were transferred individually to 15 mL centrifuge tubes and rinsed with 5 mL of wash buffer (1% bovine serum albumin (BSA) in phosphate - buffered saline (PBS)). The centrifuge tubes were centrifuged at 300 x g for 5 minutes at 4 °C and the supernatant was decanted. The pellet was resuspended in 1 mL of wash buffer using a large - bore 1 mL pipette tip, then 5 mL of ACK (ammonium chloride potassium) lysis buffer was added and the cells were incubated at room temperature for 5 - 7 minutes. 5 mL of stop - reaction buffer (1% BSA in Dulbecco's phosphate - buffered saline (DPBS) (without Ca 2+ and Mg 2+ ) was added to the samples, which were then centrifuged at 300 x g for 5 minutes at 4 °C. The supernatant was then removed and the resulting pellet was resuspended in 5 mL of DPBS. The suspended cells were filtered through a 70 μm cell strainer and the filtrate was rinsed to 5 mL with additional DPBS. The suspended cells were mixed and 600 μL of the sample was used to determine cell viability using a Vi - CELL cell viability analyzer. The results are shown in Table 2. Example 4

[0159] Approximately 100 mg of rabbit spleen tissue was placed in a culture dish containing Roswell Park Memorial Institute (RPMI) medium. The spleen tissue sample was minced into tissue blocks approximately 1 mm 3 to approximately 3 mm 3Tissue blocks. Transfer the minced tissue sample to a sterile sample tube. Then place the sample tube on a transducer with 4 independently operable FASA elements, the elements having an angle of 90° and arranged in a circular pattern with an approximate diameter of 9 mm. Apply a large amount of lateral ultrasonic energy to the sample to dissociate cells from the minced spleen tissue.

[0160] Individually, dissociate cells from rabbit spleen tissue using enzymatic dissociation for 45 minutes.

[0161] Transfer the samples individually to 15 mL centrifuge tubes and wash with 5 mL of wash buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)). Centrifuge the centrifuge tubes at 300 x g for 5 minutes at 4 °C and decant the supernatant. Resuspend the pellet in 1 mL of wash buffer using a large orifice 1 mL pipette tip, then add 5 mL of ACK (ammonium chloride potassium) lysis buffer and incubate the cells at room temperature for 5 - 7 minutes. Add 5 mL of stop reaction buffer (1% BSA in Dulbecco's phosphate buffered saline (DPBS) (without Ca 2+ and Mg 2+ )) to the samples, then centrifuge them at 300 x g for 5 minutes at 4 °C. Then remove the supernatant and resuspend the resulting pellet in 5 mL of DPBS. Filter the suspended cells through a 70 μm cell strainer and wash the filtrate to 5 mL with additional DPBS.

[0162] Use flow cytometry (FACS) to compare cells from rabbit spleen tissue dissociated using a large amount of lateral ultrasound and cells dissociated using an enzymatic method. The results are shown in Figures 6A to 6D below. Figure 6A and Figure 6B compare the cell population diversity, showing greater cell diversity using a large amount of lateral ultrasound to dissociate cells ( Figure 6A ) compared to enzymatic dissociation ( Figure 6B ) (compare the cell populations within the circles). Figures 6C to 6D Compare the cell viability of cells dissociated using enzymatic dissociation ( Figure 6C ) and a large amount of lateral ultrasound ( Figure 6D ), as indicated by viable (y - axis) nucleated cells (as determined by nuclear staining, x - axis). Enzymatic dissociation yielded approximately 7% viable cells, while a large amount of lateral ultrasound dissociation yielded approximately 31% viable cells. Example 5

[0163] The dissociation of cells from clinical breast cancer tumors extracted from patients by a large amount of lateral ultrasonic energy was compared with enzymatic dissociation. Approximately 100 mg of breast cancer tissue was placed in a culture dish supplemented with Roswell Park Memorial Institute (RPMI) medium. The breast cancer tissue sample was minced into tissue pieces sized from about 1 mm 3 to about 3 mm 3 . The minced tissue sample was transferred to a sterile sample tube. Then the sample tube was placed on a transducer with 4 independently operable FASA elements, which had an angle of 90° and were arranged in a circular pattern with a diameter of approximately 9 mm. A large amount of lateral ultrasonic energy was applied to the sample to dissociate cells from the minced spleen tissue.

[0164] Individually, cells were dissociated from the clinical breast cancer tissue sample using enzymatic dissociation for 45 minutes.

[0165] The sample was individually transferred to a 15 mL centrifuge tube and rinsed with 5 mL of wash buffer (1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)). The centrifuge tube was centrifuged at 300 x g for 5 minutes at 4 °C, and the supernatant was decanted. The pellet was resuspended in 1 mL of wash buffer using a large-orifice 1 mL pipette tip, then 5 mL of ACK (ammonium chloride potassium) lysis buffer was added and the cells were incubated at room temperature for 5 - 7 minutes. 5 mL of stop reaction buffer (1% BSA in Dulbecco's phosphate-buffered saline (DPBS) (without Ca 2+ and Mg 2+ ) was added to the sample, which was then centrifuged at 300 x g for 5 minutes at 4 °C. Then the supernatant was removed, and the resulting pellet was resuspended in 5 mL of DPBS. The suspended cells were filtered through a 70 μm cell strainer, and the filtrate was rinsed to 5 mL with additional DPBS.

[0166] Cells from the two samples were compared using flow cytometry (FACS), and the results are shown in Figure 7A (enzymatic dissociation) and Figure 7B (dissociation by a large amount of lateral ultrasonic energy). As Figure 7B shown, a large amount of lateral ultrasonic energy successfully dissociated CD45-cells, CD45+ cells, CD326- cells, CD326+ cells, CD326+CD49f+ cells, CD326+CD166+ cells, CD3- cells, CD3+ cells, CD8+ T cells, CD4+ T cells, monocytes, natural killer (NK cells), and B cells from cancer tissue. Example 6

[0167] The dissociation of cells in formalin-fixed paraffin-embedded (FFPE) human breast cancer xenograft (PDX) tumor biopsies obtained from a mouse model by a large amount of lateral ultrasonic energy was compared with enzymatic dissociation. Approximately 100 mg of PDX tissue was placed in a culture dish supplemented with Roswell Park Memorial Institute (RPMI) medium. The breast cancer tissue sample was minced into tissue pieces sized approximately 1 mm 3 to approximately 3 mm 3 . The minced tissue sample was transferred to a sterile sample tube. The sample tube was then placed on a transducer with 4 independently operable FASA elements, which had an angle of 90° and were arranged in a circular pattern with an approximate diameter of 9 mm. A large amount of lateral ultrasonic energy was applied to the sample to dissociate cells from the minced spleen tissue.

[0168] Separately, cells were dissociated from similar FFPE human breast cancer xenograft tumor biopsies using enzymatic dissociation for 45 minutes.

[0169] The samples were separately transferred to 15 mL centrifuge tubes and rinsed with 5 mL of wash buffer (1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)). The centrifuge tubes were centrifuged at 300 x g for 5 minutes at 4 °C, and the supernatant was decanted. The pellet was resuspended in 1 mL of wash buffer using a large-orifice 1 mL pipette tip, then 5 mL of ACK (ammonium chloride potassium) lysis buffer was added and the cells were incubated at room temperature for 5 - 7 minutes. 5 mL of stop reaction buffer (1% BSA in Dulbecco's phosphate-buffered saline (DPBS) (without Ca 2+ and Mg 2+ ) was added to the samples, which were then centrifuged at 300 x g for 5 minutes at 4 °C. The supernatant was then removed, and the resulting pellet was resuspended in 5 mL of DPBS. The suspended cells were filtered through a 70 μm cell strainer, and the filtrate was rinsed to 5 mL with additional DPBS.

[0170] Flow cytometry (FACS) was used to compare the cells from the two samples. Few CD326+, CD24+ cells were dissociated from the tissue samples by enzymatic dissociation ( Figure 8A ). In contrast, a large number of CD326+, CD24+ cells were dissociated from the tissue samples using a large amount of lateral ultrasonic energy. Additionally, when the residual tissue from the enzymatically dissociated samples was treated with a large amount of lateral ultrasonic energy, CD326+, CD24+ cells were successfully dissociated out ( Figure 8C ). Example 7

[0171] Four 45 mg tissue samples of porcine liver were obtained. Two of the samples were dissociated into cell suspensions using Bulk Lateral Ultrasound (BLU) waves. One sample was dissociated into a cell suspension using a standard Miltenyi enzymatic tissue dissociation protocol. One sample was retained as a control sample in its solid tissue state.

[0172] The cell suspensions and solid tissue samples were frozen, after which RNA was extracted and the minimum RIN value for valid sequencing data was evaluated. mRNA was prepared for sequencing using the TruSeq TM RNA Library Preparation Kit (Illumina), and then sequenced using the NextSeq TM Sequencer (Illumina). The resulting data was sequenced using standard next-generation sequencing data analysis tools.

[0173] Figure 9A The fold changes of all available genes are shown compared to the control sample (solid tissue). For most genes, the gene expression profiles of those dissociated using Bulk Lateral Ultrasound (BLU) energy closely matched the control sample tissue, while the gene expression of enzymatically dissociated (ENZYME) cells did not match as closely. Figure 9B The fold changes of pro-inflammatory and stress-induced genes (CD69, LRRK2, TNF, FOS, PDE4B, NR4A3, S100A9, S100A8, THBD, IL-1β, IL-1α, HK2, CXCL8, CXCL1, AREG, EGR2, TIMP1, and CXCR4) are shown compared to the control sample (solid tissue). For each measured pro-inflammatory and stress-induced gene, enzymatically dissociated cells showed a greater increase in gene expression compared to cells dissociated using Bulk Lateral Ultrasound.

[0174] For purposes of explanation, the foregoing description has been made 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. Many modifications and variations are possible in light of the above teachings. The embodiments were chosen and described in order to best explain the principles of the technology and its practical application. Thereby, others skilled in the art may best utilize the technology with various modifications suitable for the particular uses contemplated.

[0175] Although the disclosure and examples have been described fully with reference to the accompanying drawings, it should be noted that various changes and modifications will become apparent to those skilled in the art. These changes and modifications should be understood to be included within the scope of the disclosure and examples as defined by the claims.

[0176] The present disclosure relates to the following embodiments. 1. A method for dissociating single cells from a biological tissue sample, comprising: generating one or more ultrasonic pulses using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements; and applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container. 2. A method for dissociating single cells from a biological tissue sample, comprising: generating one or more ultrasonic pulses using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements; applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container; and controlling the temperature of the biological tissue sample when applying energy from the generated one or more ultrasonic pulses to the biological tissue sample. 3. The method according to item 2, wherein controlling the temperature of the biological tissue sample comprises controlling the temperature of the coupling medium. 4. The method according to item 3, wherein controlling the temperature of the coupling medium cools the biological tissue sample or limits the temperature rise of the biological tissue sample. 5. The method according to item 3 or 4, wherein the coupling medium is controlled to a temperature between about 2°C and about 70°C. 6. The method according to any one of items 3 - 5, wherein the coupling medium is controlled to a temperature between about 2°C and about 25°C. 7. The method according to any one of items 2 - 6, wherein controlling the temperature of the biological tissue sample comprises heating the biological tissue sample using energy from the generated one or more ultrasonic pulses. 8. The method according to item 7, wherein a first portion of the one or more ultrasonic pulses generated by the transducer is configured to heat the biological tissue sample, and a second portion of the one or more ultrasonic pulses generated by the transducer is configured not to heat the biological tissue sample. 9. The method according to item 7 or 8, wherein the biological tissue sample is heated to a peak temperature between about 20°C and about 70°C. 10. The method according to any one of items 1-6, wherein during applying the energy from the generated one or more ultrasonic pulses to the sample container, the biological tissue sample is heated less than 15 °C or not heated. 11. The method according to any one of items 2-10, wherein the temperature of the biological tissue sample is controlled between about 2 °C and about 70 °C. 12. The method according to any one of items 2-11, wherein controlling the temperature of the biological tissue sample reduces the expression of one or more pro-inflammatory or stress-induced genes as compared to not controlling the temperature of the biological tissue sample. 13. The method according to item 12, wherein the one or more pro-inflammatory or stress-induced genes are selected from CD69, LRRK2, TNF, FOS, PDE4B, NR4A3, S100A9, S100A8, THBD, IL-1β, IL-1α, HK2, CXCL8, CXCL1, AREG, ERG2, TIMP1, and CXCR4. 14. The method according to any one of items 1-13, wherein the coupling medium is a liquid, a solid, or a gel. 15. The method according to items 1-14, wherein the coupling medium is a liquid, and the method further comprises circulating the coupling fluid through a temperature controller. 16. The method according to any one of items 1-15, wherein about 20% or more of the dissociated single cells are alive. 17. The method according to item 16, wherein about 20% to about 95% of the dissociated single cells are alive. 18. The method according to any one of items 1-17, wherein two or more different types of live cells are dissociated from the biological tissue sample. 19. The method according to any one of items 1-18, wherein the biological tissue sample comprises cancer tissue. 20. The method according to any one of items 1-19, wherein the energy applied to the sample does not result in a shear force applied to the sample. 21. The method according to any one of items 1-20, which comprises selecting one or more of a repetition rate, a pulse duration, a duty cycle, a peak power, or a total duration for the generated one or more ultrasonic pulses. 22. The method according to any one of items 1-21, wherein the energy applied to the sample results in a mixing force or a suspension force applied to the sample. 23. The method according to any one of items 1-22, wherein a plurality of ultrasonic pulses are generated at a repetition rate of about 1 Hz to about 100,000 Hz. 24. The method according to any one of items 1 - 23, wherein the one or more ultrasonic pulses are generated with a duty cycle of from about 0.1% to about 95%. 25. The method according to any one of items 1 - 24, wherein the energy from the one or more ultrasonic pulses is applied to the coupling medium for about 2 seconds to about 90 minutes. 26. The method according to any one of items 1 - 25, wherein the biological tissue is suspended in a liquid within the sample container. 27. The method according to any one of items 1 - 26, wherein the biological tissue is minced. 28. The method according to any one of items 1 - 27, wherein the biological tissue sample is substantially free of non - endogenous proteases. 29. The method according to any one of items 1 - 28, wherein the transducer comprises four or more FASA elements. 30. The method according to any one of items 1 - 29, wherein the biological tissue sample is an unpreserved sample. 31. A system for dissociating single cells from a biological tissue sample, comprising: A transducer comprising one or more Fresnel annular sector actuators (FASA) elements configured to generate one or more ultrasonic pulses; A sample container configured to hold the biological tissue sample; and A coupling medium configured to couple the one or more FASA elements to the sample container and transmit the energy of the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample. 32. A system for dissociating single cells from a biological tissue sample, comprising: A transducer comprising one or more Fresnel annular sector actuators (FASA) elements configured to generate one or more ultrasonic pulses; A sample container configured to hold the biological tissue sample, wherein the system is configured to control the temperature of the biological tissue sample; and A coupling medium configured to couple the one or more FASA elements to the sample container and transmit the energy of the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample. 33. The system according to item 32, wherein the system is configured to control the temperature of the biological sample to a temperature between about 2°C and about 70°C. 34. The system according to any one of items 31 - 33, further comprising a temperature controller configured to control the temperature of the coupling medium. 35. The system according to item 34, wherein the temperature controller is configured to control the coupling medium to a temperature between about 2 °C and about 70 °C. 36. The system according to item 34, wherein the temperature control is configured to cool the coupling medium to a temperature between about 2 °C and about 25 °C. 37. The system according to any one of items 31 - 36, wherein the coupling medium is a liquid, and the system further comprises a pump configured to circulate the coupling medium between a position between the transducer and the sample container and the temperature controller. 38. The system according to any one of items 32 - 37, wherein the system is configured to control the temperature of the biological tissue sample by heating the biological tissue sample using the energy from the generated one or more ultrasonic pulses. 39. The system according to any one of items 32 - 38, wherein the transducer is configured to selectively generate: (1) one or more ultrasonic pulses that heat the biological tissue sample, and (2) one or more ultrasonic pulses that do not heat the sample. 40. The system according to any one of items 31 - 39, further comprising a control module configured to operate the transducer to generate the one or more ultrasonic pulses. 41. The system according to item 40, wherein the control module is configured to operate the transducer to generate the plurality of ultrasonic pulses at one or more of a selected repetition rate, a selected pulse duration, a selected duty cycle, a selected peak power, a selected average pulse power, or a selected total duration. 42. The system according to item 40 or 41, wherein the control module is configured to operate the transducer to generate a plurality of ultrasonic pulses at a repetition rate of about 1 Hz to about 100,000 Hz. 43. The system according to any one of items 40 - 42, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a duty cycle of about 0.1% to about 95%. 44. The system according to any one of items 40 - 43, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a total duration of about 2 seconds to about 90 minutes. 45. The system according to any one of items 40 - 44, wherein the control module includes a radio frequency (RF) generator configured to operate the transducer to generate the ultrasonic pulses. 46. The system according to any one of items 40 - 45, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that shear forces are not applied to the sample. 47. The system according to any one of items 40 - 46, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that mixing forces or levitation forces are applied to the sample. 48. The system according to any one of items 30 - 47, wherein the transducer includes four or more FASA elements. 49. The system according to any one of items 30 - 48, wherein the system is arranged in an array including a plurality of systems.

Claims

1. A method for dissociating single cells from a biological tissue sample, which comprises: generating one or more ultrasonic pulses using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements; and applying the energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container.

2. A method for dissociating single cells from a biological tissue sample, which comprises: generating one or more ultrasonic pulses using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements; applying the energy from the generated one or more ultrasonic pulses to a biological tissue sample contained in a sample container through a coupling medium to dissociate single cells from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container; and controlling the temperature of the biological tissue sample when applying the energy from the generated one or more ultrasonic pulses to the biological tissue sample.

3. The method according to claim 2, wherein controlling the temperature of the biological tissue sample comprises controlling the temperature of the coupling medium.

4. The method according to claim 3, wherein controlling the temperature of the coupling medium cools the biological tissue sample or limits the temperature rise of the biological tissue sample.

5. The method according to claim 3 or 4, wherein the coupling medium is controlled to a temperature between about 2°C and about 70°C.

6. The method according to any one of claims 3 - 5, wherein the coupling medium is controlled to a temperature between about 2°C and about 25°C.

7. The method according to any one of claims 2 - 6, wherein controlling the temperature of the biological tissue sample comprises heating the biological tissue sample using the energy from the generated one or more ultrasonic pulses.

8. The method according to claim 7, wherein a first portion of the one or more ultrasonic pulses generated by the transducer is configured to heat the biological tissue sample, and a second portion of the one or more ultrasonic pulses generated by the transducer is configured not to heat the biological tissue sample.

9. The method according to claim 7 or 8, wherein the biological tissue sample is heated to a peak temperature between about 20°C and about 70°C.

10. The method according to any one of claims 1 - 6, wherein during the application of the energy from the generated one or more ultrasonic pulses to the sample container, the biological tissue sample is heated less than 15°C or not heated.

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