Methods and systems for dissociating biological tissue into single cells using ultrasonic energy

Abstract

A method for dissociating a single cell from a biological tissue sample and a system for performing such a method are described herein. The method described herein 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 sample container holding the biological tissue sample through a coupling medium to dissociate a single cell from the biological tissue, the coupling medium coupling the one or more FASA elements to the sample container.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 909,484, filed on October 2, 2019, the contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] Described herein are methods and systems for dissociating single cells from biological tissue samples using ultrasonic energy. Background Art

[0004] In the field of medical and biological research, many current analyses are performed by techniques that treat each tumor sample as a homogenate, but it is known that tumor tissue is a heterogeneous body of cancer cells (e.g., infiltrating immune cells and tumor stroma). Such methods provide a large amount of molecular information, but do not retain the background of the cellular components in the original sample. These results are average values ​​for the entire tumor tissue, which excludes the identification of rare subpopulations of important cells. It has been shown that the information lost due to this practice may hide key revelations to tumor progression, metastasis, and drug resistance. In addition, the microenvironment of the tumor includes various arrays of cell types that maintain homeostasis and drive further development. This intratumoral cell heterogeneity has been identified as a key factor in the development of potential progression, metastasis, and drug resistance.

[0005] Flow sorting methods have been developed because they provide high-throughput, multiplexed information about each cell within a sample. Cell sorting can also be used to isolate rare cell types, such as cancer stem cells, metastatic precursors, and drug-resistant clones, for additional studies. However, for single-cell analysis, tissues must first be disaggregated into living single cells without compromising phenotypic and genotypic information, which requires considerable time and effort.

[0006] Previously, proteolytic enzymes have been used in combination with some form of physical force to decompose cell adhesion molecules and / or the underlying extracellular matrix, thereby dissociating 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 enzymes may be harmful to cells and negatively affect the ability of the resulting single cells to subsequently survive and / or divide. For example, enzymatic dissociation typically includes incubating tissue at an elevated temperature (e.g., 37° C.), which may result in the expression of undesirable proinflammatory and / or stress-induced genes. The expression of these genes can significantly change the state of the cell from its native state. Summary of the invention

[0007] Described herein are methods of dissociating single cells from a biological tissue sample using a transducer having one or more Fresnel annular sector actuator (FASA) elements that generate a large amount of lateral ultrasonic energy, and systems for dissociating cells from a biological tissue sample.

[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 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, wherein the coupling medium couples the one or more FASA elements to the sample container.

[0009] In some embodiments, a method for dissociating single cells from a biological tissue sample includes: 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 while applying the energy from the generated one or more ultrasonic pulses to the biological tissue sample.

[0010] 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 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.

[0011] In some embodiments of the above methods, controlling the temperature of the biological tissue sample comprises 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 to 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.

[0012] In some embodiments of the above methods, controlling the temperature of the biological tissue sample comprises using energy from the one or more ultrasonic pulses generated 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.

[0013] In some embodiments of the above methods, the biological tissue sample is heated by less than 15° C. or not heated during application of energy from the generated one or more ultrasound pulses to the sample container.

[0014] In some embodiments of the above methods, the coupling medium is a liquid, a solid, or a gel. In some embodiments, the coupling medium is a liquid, and the method further comprises circulating the coupling fluid through a temperature controller.

[0015] In some embodiments of the above methods, 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 living cells are dissociated from the biological tissue sample.

[0016] In some embodiments of the above methods, the biological tissue sample comprises cancer tissue.

[0017] 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 ultrasonic pulses generated. 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 suspension force applied to the sample.

[0018] In some embodiments, the plurality of ultrasound pulses are generated at a repetition rate of about 1 Hz to about 100,000 Hz.

[0019] In some embodiments, the one or more ultrasound pulses are generated at a duty cycle of about 0.1% to about 95%.

[0020] In some embodiments, energy from the one or more ultrasonic pulses is applied to the coupling medium for a duration of about 2 seconds to about 90 minutes.

[0021] In some embodiments of the above methods, the biological tissue is suspended in a liquid within the sample container.

[0022] In some embodiments of the above methods, the biological tissue is minced.

[0023] In some embodiments of the above methods, the biological tissue sample is substantially free of non-endogenous proteases.

[0024] In some embodiments of the above methods, the transducer includes four or more FASA elements.

[0025] In some embodiments of the above methods, the biological tissue sample is an unpreserved sample.

[0026] The present invention also describes a system for dissociating single cells from a biological tissue sample, the system comprising: a transducer comprising 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; and a coupling medium configured to couple the one or more FASA elements to the sample container and transmit energy from the multiple ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0027] 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 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 transmit energy from the multiple 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.

[0028] 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.

[0029] In some embodiments of the above system, the coupling medium is a liquid, and the system further comprises a pump configured to circulate the coupling medium between a location between the transducer and the sample container and the temperature controller.

[0030] In some embodiments of the above systems, the system is configured to control the temperature of the biological tissue sample by heating the biological tissue sample using energy from the generated one or more ultrasound pulses.

[0031] In some embodiments of the above systems, the transducer is configured to selectively generate: (1) one or more ultrasound pulses that heat the biological tissue sample, and (2) one or more ultrasound pulses that do not heat the sample.

[0032] In some embodiments of the above-mentioned system, the system further includes a control module, which is 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 multiple 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 multiple ultrasonic pulses at a repetition rate of 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 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 with a total duration of about 2 seconds to about 90 minutes. In some embodiments, the control module includes a radio frequency (RF) generator, which is 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 so 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 so that mixing forces or suspension forces are applied to the sample.

[0033] In some embodiments of the foregoing systems, the transducer includes four or more FASA elements.

[0034] In some embodiments of the above systems, the system is arranged in an array comprising a plurality of systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The foregoing summary and the following detailed description of the embodiments may be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, the accompanying drawings show example embodiments of the present disclosure; however, the present disclosure is not limited to the specific methods and instrumentalities disclosed. In the drawings:

[0036] Figure 1 An exemplary system for dissociating cells from a biological tissue sample is shown.

[0037] Figure 2 Shown is an electrode pattern of a FASA element that may be used with the transducers of the systems described herein.

[0038] Figure 3 Shown is a top view of an exemplary ultrasonic transducer having four 90 degree FASA elements that may be used with a system for dissociating single cells from a biological tissue sample as described herein.

[0039] Figure 4 An exemplary 8x12 transducer array is shown in which 96 transducer cells are arranged, each cell having four FASA elements.

[0040] Figure 5 An exemplary system is shown having a temperature controller and a pump configured to control the temperature of a coupling fluid when a transducer is operating.

[0041] Fig. 6A Shown are the results of flow cytometric analysis of cells dissociated from rabbit spleen tissue samples using an enzymatic dissociation method in order to distinguish between live and dead dissociated cells.

[0042] Figure 6B Shown are the results of flow cytometric analysis of cells dissociated from a rabbit spleen tissue sample using high-volume lateral ultrasound energy as described herein in order to distinguish between live and dead dissociated cells.

[0043] Figure 6C Shown are the results of flow cytometric analysis of cells dissociated from rabbit spleen tissue samples using an enzymatic dissociation method in order to distinguish nucleated live cells from other cellular debris.

[0044] Fig.6D Shown are the results of flow cytometric analysis of cells dissociated from a rabbit spleen tissue sample using high-level sideways ultrasound energy as described herein in order to distinguish nucleated, live cells from other cellular debris.

[0045] Fig. 7A Shown are the results of flow cytometric analysis of cells dissociated from clinical breast cancer tumor samples using an enzymatic dissociation method.

[0046] Figure 7B Shown are the results of flow cytometric analysis of cells dissociated from a clinical breast cancer tumor sample using high-volume lateral ultrasound energy as described herein.

[0047] Fig. 8A Shown are the results of flow cytometric analysis of cells dissociated from formalin-fixed paraffin-embedded (FFPE) human breast cancer xenograft (PDX) tumor biopsies obtained from a mouse model using an enzymatic dissociation method. The upper right quadrant indicates the substantial absence of dissociated CD326+ / CD24+ cells.

[0048] Figure 8B Shown are the results of flow cytometric analysis of cells dissociated from FFPE human breast cancer xenograft (PDX) tumor biopsies obtained from a mouse model using high-volume lateral ultrasound energy as described herein. The upper right quadrant indicates dissociated CD326+ / CD24+ cells.

[0049] Figure 8C Shown are the results of flow cytometric analysis of cells dissociated from residual FFPE human breast cancer xenograft (PDX) tumor tissue after enzymatic dissociation using a large amount of lateral ultrasound energy as described herein for further treatment. The upper right quadrant indicates dissociated CD326+ / CD24+ cells that were not successfully dissociated when using the enzymatic method.

[0050] Fig.9A Shown are changes in gene expression in cells dissociated from tissue by high-volume lateral ultrasound energy and in cells dissociated from solid tissue using an enzymatic process compared to a control sample (solid tissue) for all available genes.

[0051] Fig. 9B Shown are changes in gene expression for pro-inflammatory or stress-induced genes in cells dissociated from tissue by high-volume lateral ultrasound energy and in cells dissociated from solid tissue using an enzymatic process compared to a control sample (solid tissue). DETAILED DESCRIPTION

[0052] Methods for dissociating single cells from a biological tissue sample using ultrasonic energy emitted from an ultrasonic transducer and systems for performing such methods are described herein. 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.

[0053] FASA transducers can be used to apply energy to biological samples, providing a thorough but gentle application of energy that separates single cells while minimizing cell lysis (a common problem when applying traditional ultrasonic energy to tissues). As a result, the proportion of single cells separated from tissue that are alive and / or can express proteins is increased compared to traditional ultrasonic energy applications. In addition, the methods described herein allow for faster and more thorough cell dissociation than methods that include the use of enzymes (such as proteases) to separate cells from biological tissue samples, while also avoiding enzyme-induced changes in the in vivo state of the cells.

[0054] The large amount of lateral ultrasonic waves generated by the FASA element can be configured to apply one or more of mixing forces, suspension forces, shear forces, or heating energy (or configured not to apply one or more of them). The energy from the emitted ultrasonic waves used to dissociate single cells from biological tissue can, for example, apply mixing forces and / or suspension forces 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 shear forces, which may cause cell lysis.

[0055] The temperature of the sample can be controlled during single cell dissociation, which can limit the expression of proinflammatory and / or stress-induced genes. Heating the sample for an extended period of time may lead to the expression of proinflammatory and / or stress-induced genes, which may change the biological state of the cell. For example, heating the tissue to 37 ° C during enzymatic cell dissociation induces proinflammatory and stress-induced gene expression. However, a short burst of heat applied to the sample can help tissue dissociation while limiting the expression of proinflammatory or stress-induced genes. Therefore, in some embodiments, the ultrasonic energy applied to the sample is configured to apply thermal energy to the sample. Thermal energy can controllably warm the sample for a desired duration, which may be less than the total duration of the ultrasonic energy applied to the cell.

[0056] In some embodiments, a coupling medium is used to control the temperature of a sample, for example, to reduce the temperature of a sample heated by applied ultrasonic energy (or to counteract the temperature increase caused by heating by applied ultrasonic energy so that it does not increase as much as it would without using a 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.

[0057] Biological tissue samples often have heterogeneous cell populations, including two or more different cell types. Using the methods described herein, living single cells can be dissociated from biological tissue samples, wherein these single cells are within a heterogeneous living cell population. 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 method results in the dissociation of two or more different types of living cells from a sample (such as a cancer sample).

[0058] In an exemplary method, dissociating single cells from a biological tissue sample may include: generating a plurality of ultrasonic pulses using a transducer including one or more Fresnel annular sector actuator (FASA) elements; and applying energy from the generated plurality of ultrasonic pulses to a sample container holding the biological tissue sample through a coupling medium, wherein the coupling medium couples the one or more FASA elements to the container, thereby dissociating the single cells from the biological tissue.

[0059] In another exemplary method of dissociating a single cell from a biological tissue sample, the method 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 sample container holding the biological tissue sample through a coupling medium, the coupling medium coupling the one or more FASA elements to the sample container, to dissociate the single cell 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 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.

[0060] In an exemplary system for dissociating single cells from a biological sample, the system may 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.

[0061] In another exemplary system for dissociating single cells from a biological tissue sample, the system includes: a transducer comprising 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 transmit energy from the multiple ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0062] definition

[0063] As used herein, the singular form "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0064] Reference herein to "about" a value or parameter includes (and describes) variations with respect to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0065] A "viable" cell refers to a cell that is alive and capable of cell growth or expansion in culture.

[0066] It should be understood that the aspects and variations of the present invention described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0067] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other stated or intervening value within the stated range is included within the scope of the present disclosure. Where a stated range includes an upper or lower limit, ranges excluding any of those included limits are also included within the present disclosure.

[0068] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. The description is presented to enable one of ordinary skill in the art to make and use the invention, and is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles herein may be applied to other embodiments. Therefore, the present invention is not intended to be limited to the embodiments shown, but to conform to the widest scope consistent with the principles and features described herein.

[0069] 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.

[0070] Systems for dissociating cells from biological tissue

[0071] 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 an ultrasonic pulse, the coupling medium transfers energy from the ultrasonic wave to the container, which allows single cells to be dissociated from the biological tissue sample.

[0072] 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 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).

[0073] Figure 1 A side view of an exemplary system 100 including a transducer 102 and a sample container 110 for an exemplary 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 a Fresnel annular sector actuator (FASA) that, when operated, moves in a target direction (e.g., in Figure 1 The control module 109 is configured to operate the transducer to generate ultrasonic waves. The sample container 110 contains a biological tissue sample 112 that can be suspended in a liquid (e.g., water or a buffer). The system 100 further includes a coupling medium 114, which is configured to couple the transducer 104 to surround the sample container 110. The coupling medium can be held within the side wall of the system. The biological tissue sample 112 in the sample container 110 is optionally below a surface 116 of the coupling medium 114. The coupling medium 114 provides acoustic coupling between the transducer 104 and the sample container 110, so that it transmits energy from the ultrasonic wave emitted by the transducer 102 to the sample container, which causes single cells to dissociate from the biological tissue sample 112 in the sample container 110.

[0074] One or more Fresnel annular sector actuator (FASA) elements of the transducer allow the transducer to emit ultrasonic waves having a high lateral acoustic potential energy distribution pointing in the target direction, which allows mixing of fluids in the target direction (e.g., liquids holding biological tissue samples). Exemplary FASA elements are described in US 6,682,214 or Vivek et al., Novel Acoustic-Wave Micromixer, Proceedings MEMS 2000, pp. 668-673 (2000). FASA is based on a self-focusing acoustic wave transducer that focuses acoustic waves by long-wave interference. Strong ultrasonic waves are generated on the electrode area, and the ultrasonic waves interfere with each other as they propagate. This results in 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 operate independently.

[0075] The electrodes of the FASA element can sandwich piezoelectric materials such as lead zirconate titanate (PZT), BaTiO 3 , KNbO 3 etc. The current passing through the electrode 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°, for example, 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°.

[0076] The electrodes of the FASA element are patterned by one annular segment, or by a plurality of concentric annular segments arranged in a Fresnel lens pattern. For example, the electrodes of the FASA element may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more concentric annular segments. The FASA element is operated by driving current through the electrodes via the electrode terminals, for example by operating a control module to pass the current. Figure 2 The electrode pattern of a FASA element that can be used with the transducer of the system described herein is shown. 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 electrodes clamp the piezoelectric material (see Figure 1 ), the piezoelectric material generates electric waves when current passes through the electrodes.

[0077] The transducer of the system can have one or more FASA elements that guide ultrasound 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 that are configured to transmit ultrasound waves in the direction of the sample container. The FASA elements can be arranged in a circular pattern, wherein the apex of the FASA element is positioned near the center of the circle. In some embodiments, the diameter of the circular pattern is about 5mm to about 20mm diameter (for example, about 5mm to about 7mm diameter to about 9mm diameter, about 9mm diameter to about 11mm diameter, about 11mm diameter to about 13mm diameter, about 13mm diameter to about 15mm diameter, about 15mm diameter to about 17mm diameter or about 17mm diameter to about 20mm diameter), and the FASA elements of the transducer can be separated by gaps, and the gaps can be consistent 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.

[0078] Multiple FASA elements (or a portion of FASA elements) on the same transducer can be activated simultaneously or non-simultaneously (e.g., two or more FASA elements are activated sequentially or alternately) to generate ultrasound. Random or controlled mixing is achieved through appropriate control of the activation of the FASA elements. In some embodiments, 50% or more of the FASA elements of the same transducer are activated simultaneously. In some embodiments, 75% or more of the FASA elements of the same transducer (e.g., 3 of 4 FASA elements) are activated simultaneously. In some embodiments, all FASA elements of the same transducer are activated simultaneously. When less than all FASA elements are activated simultaneously, one or more inactive FASA elements may alternate between all FASA elements of the transducer during operation.

[0079] Figure 3A top view of an ultrasonic transducer 300 with four 90 degree FASA elements labeled T1, T2, T3, and T4 is shown. The 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.

[0080] The transducers may be individual transducers or may be arranged in a transducer array. The transducer array may include a plurality of separate piezoelectric elements or may have a common (i.e., shared) piezoelectric element 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. An array with any number of transducers can be used, for example, between 1 and 1000 transducers within the array. The number of transducers within the array can match the number of wells in a multiwell plate, for example, 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).

[0081] The control module can be used to operate the FASA element of the transducer using one or more selected parameters to generate ultrasonic waves. The control module may include, for example, a radio frequency (RF) generator that can induce 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 may also include one or more additional electronic circuits that are configured to adjust one or more parameters of the ultrasonic wave emitted by the transducer, for example, by controlling the RF signal. The control module may 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 wave emitted from the transducer. The control module may include a user interface (which may include, for example, one or more buttons, switches, or digital interfaces) that may 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 wave generated by the transducer. The frequency of the drive current flowing through the electrodes of the FASA element can be based on the resonant frequency of the piezoelectric material to enhance the generation of ultrasonic waves (e.g., within 10%, within 20%, or within 30% or more of the resonant frequency). The shape of the drive current can be sinusoidal, square, or any other suitable shape.

[0082] The selected parameters for controlling the RF energy applied to the ultrasonic transducer can affect the type of ultrasonic wave generated by the ultrasonic transducer. The FASA element generates a large number of lateral ultrasonic waves, which can be configured to generate one or more of mixing force, suspension force, shear force and heating energy. Mixing force and suspension force are generally beneficial to the dissociation of single cells from tissue samples, while shear force may cause cell lysis and is preferably avoided. Optionally, a large number of lateral ultrasonic waves generated by the transducer can be 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 ultrasonic wave does not apply thermal energy to the biological tissue sample. The energy from the ultrasonic wave applied to the biological tissue sample can be uneven during the cell dissociation process. For example, the first part of the ultrasonic wave can apply thermal energy to the biological tissue sample, while the second part of the ultrasonic wave does not apply thermal energy to the biological tissue sample.

[0083] The control module can be configured to operate the transducer to generate ultrasound or ultrasound 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 ultrasound generated by the transducer are selected to exert mixing and suspension forces in the liquid held by the container so as to dissociate single cells from the tissue but minimize cell lysis.

[0084] In some embodiments, the control module operates the transducer to generate one or more ultrasonic pulses.The ultrasonic pulses may be generated at a selected repetition rate, which may be controlled, for example, by the repetition rate of RF energy applied to the transducer. For example, the repetition rate can be from about 1 Hz to about 100,000 Hz (such as from 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). Repetition rate refers to the frequency of ultrasonic pulses when the transducer is operated to transmit a plurality of ultrasonic pulses. In some embodiments, the ultrasonic pulses are generated with 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%.

[0085] If ultrasonic energy is applied to the sample container for too long a period of time, cells within the sample container may lyse. Therefore, the control module is configured to operate the transducer to generate ultrasonic waves (or wave pulses) for a selected duration, for example, 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).

[0086] In certain embodiments, the system is configured to control the temperature of the 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 used to apply energy to the biological tissue sample is configured to heat the sample. The temperature of the biological tissue sample can be controlled (alternatively or additionally) using a coupling medium. 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 reduce the temperature of the biological tissue sample. In some embodiments, the temperature of the biological tissue sample is heated using one or more ultrasonic pulses configured to heat the biological tissue sample for a period of time, and a coupling fluid can be used to reduce the temperature of the biological tissue sample after the biological tissue sample is heated. This allows heat to be controllably applied to the biological tissue sample in a relatively short period of time, which can help to dissociate a single cell from the tissue, but then the temperature of the biological tissue sample can be reduced using a coupling medium so that the cell limits the expression of pro-inflammatory and / or stress-induced genes.

[0087] The coupling medium of the system couples one or more FASA elements of the transducer to the sample container. Energy from the ultrasound waves generated by the transducer can be transmitted to the sample container through a coupling fluid, which then dissociates single cells from the biological tissue sample in 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, an oil, a water-based gel, propylene glycol, glycerin, or a glycol ether.

[0088] The system may include a temperature controller configured to control the temperature of the coupling medium. Since the coupling medium can be thermally coupled to the sample container, the temperature of the sample in 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 inviable. However, too cold a temperature may also make the cells inviable. In some embodiments, the coupling medium is controlled at a temperature of 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 about 2°C to about 80°C (such as about 4°C to about 10°C, about 10°C to about 15°C, about 15°C to about 25°C, about 25°C to about 35°C, about 35°C to about 45°C, about 45°C to about 55°C, about 55°C to about 65°C, or about 65°C to about 80°C). In some embodiments, during cell dissociation from the 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 the location between the transducer and the sample container and the temperature controller.

[0089] Figure 5 An exemplary system having a temperature controller and a pump configured to control the temperature of a coupling fluid when a transducer is operating is shown. Figure 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 via a coupling medium 510. The transducer is operated using a control module 504 that can selectively pass an electric current through the 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 the system 500, the coupling medium 510 can be circulated using the pump 512 to the temperature controller 514, which can operate to warm or cool the coupling medium 510. Because 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.

[0090] The sample container can be a sample tube, a hole or other suitable container configured to keep the liquid and / or biological tissue sample of the desired amount. 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 arranged in a sample tube array, for example, a hole in a porous plate (for example, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate). For example, a porous plate can be used together with a transducer array as discussed above.

[0091] The devices described above may be used with any of the methods described herein.

[0092] Methods for dissociating single cells from biological tissue samples

[0093] Single cells can be dissociated from a biological tissue sample by applying energy from one or more ultrasonic pulses to a sample container holding 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 guide lateral ultrasonic energy in the direction of the sample container through a coupling medium. The coupling medium couples the FASA element to the sample container and transmits energy from the ultrasonic waves generated by the transducer to the sample container. The energy applied to the sample container applies mixing forces and / or suspension forces to 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 when applying energy from the generated one or more ultrasonic pulses to the biological tissue sample in the sample container.

[0094] In some embodiments, the temperature of the biological tissue sample is controlled by using a coupling medium, energy from an ultrasonic pulse, or both. As discussed above, the ultrasound can be configured to apply thermal energy to the biological tissue sample, which can increase the temperature of the biological tissue sample in the container. The temperature increase for a controlled period of time can increase the dissociation of single cells from the biological tissue sample, although prolonged heating may cause induction of one or more pro-inflammatory or stress-related gene expression. Exemplary pro-inflammatory or stress-related genes that may be expressed under prolonged heat 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.

[0095] 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).

[0096] The biological tissue sample can be thermally coupled to the coupling medium so 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 reduce the temperature of the biological tissue sample. Cooling the biological tissue sample by the coupling medium can reduce the temperature of the sample, for example, after heating using ultrasound configured to apply thermal energy to the sample. In some embodiments, the coupling medium is heated to increase the temperature of the biological tissue sample. The temperature of the coupling medium can be controlled, for example, 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). For example, the coupling medium can be a liquid or gel maintained in a temperature-controlled compartment, or the coupling medium can be circulated by a temperature controller. The coupling medium can be thermally coupled to the sample container (and therefore thermally coupled to the biological tissue sample in the sample chamber). Therefore, 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 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 wave does not apply thermal energy to the biological tissue sample. The energy from the ultrasonic wave applied to the biological tissue sample can be uneven during the cell dissociation process. For example, the first part of the ultrasonic wave can apply thermal energy to the biological tissue sample, while the second part of the ultrasonic wave does not apply thermal energy to the biological tissue sample. The biological tissue sample can be heated using ultrasonic waves, and then cooled, for example, by a 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).

[0097] The ultrasound waves emitted by the transducer may be emitted in one or more ultrasound pulses. In some embodiments, a plurality of ultrasound pulses are emitted by the transducer. 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. Ultrasonic pulses have lateral forces that allow single cells to be dissociated from biological tissue.

[0098] In some embodiments, one or more ultrasonic pulses are generated with 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%.

[0099] In some embodiments, 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).

[0100] Single cell is effectively dissociated from biological tissue sample, and the substantial part in single cell keeps alive after dissociation.The method provided herein is enough to dissociate single cell thoroughly, but is gentle 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 in the single cell dissociated from tissue keeps alive after dissociation from 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%) in the cell dissociated from tissue keeps alive after dissociation from biological tissue sample.

[0101] The biological tissue sample can be a solid tissue sample, although the solid tissue sample can be minced into adherent cell clusters before single cells are isolated from the biological tissue sample. A blender, 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 5mm 3 (For example, about 0.25 mm 3 To about 0.5mm 3 , about 0.5mm 3 To about 1mm 3 , about 1mm 3 To about 2mm 3 , about 2mm 3 To about 3mm 3 , about 3mm 3 To about 4mm 3 , or about 4mm 3 To about 5mm 3 In some embodiments, the biological tissue sample is suspended in a liquid in a sample container, such as water, a buffer, or other suitable liquid culture medium (e.g., Rosewell Park Memorial Institute (RPMI) medium, Minimal Essential Medium (MEM), Iscov's Modified Dulbecco's Medium (IMDM), or Dulbecco's Modified Eagle's Medium (DMEM)).

[0102] Cells bind to each other in tissues through cell adhesion molecules (such as integrins, IgSF CAMs, cadherins, and selectins), which can form cell connections. Although cell adhesion molecules can be enzymatically degraded using, for example, proteases (such as trypsin, collagenase, and / or dispase). However, treating cells with such enzymes may reduce cell viability because proteases may cut unexpected proteins. The methods described herein can effectively dissociate single cells from biological tissue samples without using non-endogenous proteases (tissues may naturally include one or more proteases, but such endogenous proteases are generally ineffective when dissociating living single cells from tissues). In some embodiments, biological tissue samples are substantially free of or free of non-endogenous proteases. In this context, "substantially free of" non-endogenous proteases should be understood to mean that a small amount of non-endogenous proteases may be present, as long as such non-endogenous proteases do not affect the proportion of living single cells dissociated from biological tissue samples.

[0103] In some embodiments, the biological tissue sample is an animal tissue, and can be, for example, a vertebrate or invertebrate tissue. For example, the tissue can be, but is not limited to, a mammal, a reptile, a bird, a fish, an insect or a nematode tissue. In some embodiments, the tissue is a plant tissue. In some embodiments, the tissue comprises a eukaryotic cell. In some embodiments, the cell is a multicellular prokaryotic organism (e.g., a biofilm). In some embodiments, the tissue is a tumor tissue or a 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 a diseased tissue or a healthy tissue, such as a cancerous tissue or a noncancerous tissue.

[0104] The cells dissociated from the tissue sample can include two or more different types of living 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 a variety of different cell types, such as macrophages, T cells, natural killer (NK) cells, and cancer cells, one or more of which can be dissociated from the tissue sample.

[0105] Exemplary embodiments

[0106] The following embodiments are exemplary and are not intended to limit the scope of the invention described herein. It will be appreciated by those skilled in the art that the exemplary embodiments may be modified by adding or excluding one or more features from the exemplary embodiments.

[0107] Embodiment 1. A method for dissociating single cells from a biological tissue sample, comprising:

[0108] generating one or more ultrasonic pulses using a transducer comprising one or more Fresnel annular sector actuator (FASA) elements; and

[0109] Energy from the generated one or more ultrasonic pulses is applied to a biological tissue sample contained by a sample container through a coupling medium that couples the one or more FASA elements to the sample container to dissociate single cells from the biological tissue.

[0110] Embodiment 2. A method for dissociating single cells from a biological tissue sample, comprising:

[0111] generating one or more ultrasonic pulses using a transducer including one or more Fresnel annular sector actuator (FASA) elements;

[0112] applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained by a sample container through a coupling medium coupling the one or more FASA elements to the sample container to dissociate single cells from the biological tissue; and

[0113] The temperature of the biological tissue sample is controlled while energy from the generated one or more ultrasound pulses is applied to the biological tissue sample.

[0114] Embodiment 3. A method according to embodiment 2, wherein controlling the temperature of the biological tissue sample includes controlling the temperature of the coupling medium.

[0115] Embodiment 4. A method according to embodiment 3, wherein the temperature of the coupling medium is controlled to cool the biological tissue sample or limit the temperature increase of the biological tissue sample.

[0116] Embodiment 5. The method of Embodiment 3 or 4, wherein the coupling medium is controlled to a temperature between about 2°C and about 70°C.

[0117] Embodiment 6. The method of any one of Embodiments 3-5, wherein the coupling medium is controlled to a temperature between about 2°C and about 25°C.

[0118] Embodiment 7. A method according to any one of embodiments 2-6, wherein controlling the temperature of the biological tissue sample includes using energy from the one or more generated ultrasonic pulses to heat the biological tissue sample.

[0119] Embodiment 8. A 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.

[0120] Embodiment 9. A 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.

[0121] Embodiment 10. A method according to any one of embodiments 1-6, wherein the biological tissue sample is heated by less than 15°C or not heated during the application of energy from the one or more generated ultrasonic pulses to the sample container.

[0122] Embodiment 11. A method according to any one of embodiments 2-10, wherein the temperature of the biological tissue sample is controlled to between about 2°C and about 70°C.

[0123] Embodiment 12. A 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 compared to not controlling the temperature of the biological tissue sample.

[0124] Embodiment 13. A 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.

[0125] Embodiment 14. The method of any one of Embodiments 1-13, wherein the coupling medium is a liquid, a solid, or a gel.

[0126] Embodiment 15. The method of embodiments 1-14, wherein the coupling medium is a liquid, the method further comprising circulating the coupling fluid through a temperature controller.

[0127] Embodiment 16. The method of any one of embodiments 1-15, wherein about 20% or more of the dissociated single cells are alive.

[0128] Embodiment 17. The method of embodiment 16, wherein about 20% to about 95% of the dissociated single cells are alive.

[0129] Embodiment 18. A method according to any one of embodiments 1-17, wherein two or more different types of living cells are dissociated from the biological tissue sample.

[0130] Embodiment 19. A method according to any one of Embodiments 1-18, wherein the tissue sample comprises cancer tissue.

[0131] Embodiment 20. A method according to any one of Embodiments 1-19, wherein the energy applied to the sample does not result in shear forces being applied to the sample.

[0132] Embodiment 21. A method according to any of embodiments 1-20, comprising 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 ultrasonic pulses generated.

[0133] Embodiment 22. A method according to any one of Embodiments 1-21, wherein the energy applied to the sample results in a mixing force or a suspension force applied to the sample.

[0134] Embodiment 23. A method according to any one of Embodiments 1-22, wherein multiple ultrasonic pulses are generated at a repetition rate of about 1 Hz to about 100,000 Hz.

[0135] Embodiment 24. A method according to any one of Embodiments 1-23, wherein the one or more ultrasonic pulses are generated with a duty cycle of about 0.1% to about 95%.

[0136] Embodiment 25. The method of any one of Embodiments 1-24, wherein energy from the one or more ultrasonic pulses is applied to the coupling medium for a duration of about 2 seconds to about 90 minutes.

[0137] Embodiment 26. A method according to any one of embodiments 1-25, wherein the biological tissue is suspended in a liquid within the sample container.

[0138] Embodiment 27. A method according to any one of embodiments 1-26, wherein the biological tissue is minced.

[0139] Embodiment 28. The method of any one of claims 1-27, wherein the biological tissue sample is substantially free of non-endogenous proteases.

[0140] Embodiment 29. A method according to any of embodiments 1-28, wherein the transducer comprises four or more FASA elements.

[0141] Embodiment 30. A method according to any one of embodiments 1-29, wherein the biological tissue sample is an unpreserved sample.

[0142] Embodiment 31. A system for dissociating single cells from a biological tissue sample, comprising:

[0143] a transducer comprising one or more Fresnel annular sector actuator (FASA) elements configured to generate one or more ultrasonic pulses;

[0144] a sample container configured to hold the biological tissue sample; and

[0145] A coupling medium is configured to couple the one or more FASA elements to the sample container and transmit energy from the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0146] Embodiment 32. A system for dissociating single cells from a biological tissue sample, comprising:

[0147] a transducer comprising one or more Fresnel annular sector actuator (FASA) elements configured to generate one or more ultrasonic pulses;

[0148] a sample container configured to hold the biological tissue sample, wherein the system is configured to control a temperature of the biological tissue sample; and

[0149] A coupling medium is configured to couple the one or more FASA elements to the sample container and transmit energy from the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

[0150] Embodiment 33. A 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.

[0151] Embodiment 34. The system of any one of Embodiments 31-33, further comprising a temperature controller configured to control the temperature of the coupling medium.

[0152] Embodiment 35. The system of Embodiment 34, wherein the temperature controller is configured to control the coupling medium to a temperature between about 2°C and about 70°C.

[0153] Embodiment 36. The system of Embodiment 34, wherein the temperature control is configured to cool the coupling medium to a temperature between about 2°C and about 25°C.

[0154] Embodiment 37. A system according to any 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.

[0155] Embodiment 38. A system according to any 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 energy from the one or more generated ultrasonic pulses.

[0156] Embodiment 39. A system according to any 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.

[0157] Embodiment 40. A system according to any one of Embodiments 31-39, further comprising a control module, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses.

[0158] Embodiment 41. A 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.

[0159] Embodiment 42. A system according to embodiment 40 or 41, wherein the control module is configured to operate the transducer to generate multiple ultrasonic pulses at a repetition rate of about 1 Hz to about 100,000 Hz.

[0160] Embodiment 43. A system according to any 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%.

[0161] Embodiment 44. A system according to any of Embodiments 40-43, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses with a total duration of about 2 seconds to about 90 minutes.

[0162] Embodiment 45. A system according to any of Embodiments 40-44, wherein the control module includes a radio frequency (RF) generator, and the RF generator is configured to operate the transducer to generate the ultrasonic pulses.

[0163] Embodiment 46. A system according to any 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.

[0164] Embodiment 47. A system according to any of Embodiments 40-46, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses so that mixing forces or levitation forces are applied to the sample.

[0165] Embodiment 48. A system according to any of Embodiments 30-47, wherein the transducer comprises four or more FASA elements.

[0166] Embodiment 49. A system according to any one of Embodiments 30-48, wherein the system is arranged in an array comprising a plurality of systems.

[0167] Example

[0168] The application can be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following embodiments are proposed to more fully illustrate the embodiments, but should not be interpreted as limiting the broad scope of the application in any way. Although some embodiments of the application have been shown and described herein, it will be apparent that such embodiments are provided only by way of example. Without departing from the spirit and scope of the present invention, it will be appreciated by those skilled in the art that many variations, changes and replacements are possible. It should be understood that the various alternatives of the embodiments described herein can be used to practice the methods described herein.

[0169] Example 1

[0170] Three tissue samples were prepared by adding approximately 100 mg of liver tissue to a culture dish supplemented with 250 μL of RPMI medium. The liver tissue samples were minced into approximately 1 mm 3 To about 3mm 3of tissue block. The tissue sample is transferred into a sample tube. The sample tube is placed on a transducer having 4 independently operable FASA elements, the elements being angled at 90°, arranged in a circular pattern having a diameter of approximately 9 mm. The sample temperature is controlled by a water bath (i.e., coupling fluid) set to 25°C with a chiller. The FASA elements are activated by applying RF energy to the FASA elements such that a large amount of lateral ultrasound (BLU) energy is applied to the sample. At any given point in time, three of the four FASA elements are activated within the inactive FASA element rotating clockwise.

[0171] After applying ultrasound 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 ultrasound pulse frequencies and extended durations of applying ultrasound resulted in cell lysis. However, lower pulse frequencies at shorter durations resulted in live cells.

[0172] Example 2

[0173] Liver tissue samples were prepared by adding about 100 mg of liver tissue to a culture dish supplemented with 250 μL of RPMI medium. The liver tissue samples were minced into pieces of about 1 mm in size. 3 To about 3mm 3 of tissue blocks. The tissue sample is transferred into a sample tube. The sample tube is then placed on a transducer having 4 independently operable FASA elements, the elements being angled at 90°, arranged in a circular pattern having a diameter of approximately 9 mm. The sample temperature is controlled by a water bath (i.e., coupling fluid) set to 25°C with a chiller. The FASA elements are activated by applying RF energy to the FASA elements such that a large amount of lateral ultrasound (BLU) energy is applied to the sample. At any given point in time, three of the four FASA elements are activated within the clockwise rotating inactive FASA element.

[0174] Control samples (from liver tissue) were also prepared by enzymatic dissociation method at 37°C.

[0175] Sample is transferred to the centrifuge tube of 15mL separately, and pours 5mL washing buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)).At 4 ℃, centrifuge tube is centrifuged for 5 minutes with 300x g, and pours out supernatant.Use large orifice 1mL pipette tip that sediment is resuspended in 1mL washing buffer, then add 5mL ACK (potassium ammonium chloride) lysis buffer and make cell at room temperature incubate 5-7 minute.By 5mL stop reaction buffer (Dulbecco's phosphate buffered saline (DPBS) (Ca-free) 4% bovine serum albumin (BSA) in phosphate buffered saline (PBS ... 2+ and Mg2+ ) in 1% BSA) is added to the sample, which is then centrifuged at 4 ° C for 5 minutes at 300xg. The supernatant is then removed, and the resulting precipitate is suspended in 5mL DPBS. Suspended cells are filtered using a 70 μm cell strainer, and the filtrate is poured to 5mL using other DPBS. Suspended cells are mixed, and 600 μL samples are used to determine cell viability using a Vi-CELL cell viability analyzer. The results are shown in Table 1.

[0176] Table 1

[0177]

[0178] About 77.5% (test sample) of cells dissociated from liver tissue using ultrasound were viable at a yield of about 9,800 viable cells per mg of liver tissue.

[0179] Example 3

[0180] Seven liver tissue samples (sample numbers 1-7) and seven kidney samples (samples 8-14) were prepared by adding about 100 mg of liver tissue or kidney tissue to a culture dish supplemented with 200 μL of RPMI medium. The liver tissue samples were minced into pieces of about 1 mm in size using scissors or a scalpel. 3 To about 3mm 3 of tissue blocks. 200 μL of 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 a 90° angle, arranged in a circular pattern with a diameter of approximately 9 mm. The water bath (i.e., 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 so that a large amount of applied lateral ultrasonic (BLU) energy was applied to the sample. At any given point in time, three of the four FASA elements were activated within the inactive FASA element rotating clockwise. The sample temperature peak was measured during the application of ultrasonic energy, as shown in Table 2.

[0181] Table 2

[0182]

[0183] Sample is transferred to the centrifuge tube of 15mL separately, and pours 5mL washing buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)).At 4 ℃, centrifuge tube is centrifuged for 5 minutes with 300x g, and pours out supernatant.Use large orifice 1mL pipette tip that sediment is resuspended in 1mL washing buffer, then add 5mL ACK (potassium ammonium chloride) lysis buffer and make cell at room temperature incubate 5-7 minute.By 5mL stop reaction buffer (Dulbecco's phosphate buffered saline (DPBS) (Ca-free) 4% bovine serum albumin (BSA) in phosphate buffered saline (PBS ... 2+ and Mg 2+ ) in 1% BSA) is added to the sample, which is then centrifuged at 4 ° C for 5 minutes at 300xg. The supernatant is then removed, and the resulting precipitate is suspended in 5mL DPBS. Suspended cells are filtered using a 70 μm cell strainer, and the filtrate is poured to 5mL using other DPBS. Suspended cells are mixed, and 600 μL samples are used to determine cell viability using a Vi-CELL cell viability analyzer. The results are shown in Table 2.

[0184] Example 4

[0185] About 100 mg of rabbit spleen tissue was placed in a culture dish supplemented with RPMI medium. The spleen tissue sample was minced into pieces of about 1 mm in size. 3 To about 3mm 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 at 90° angles arranged in a circular pattern with a diameter of approximately 9 mm. A large amount of lateral ultrasound energy was applied to the sample to dissociate cells from the minced spleen tissue.

[0186] Separately, cells were dissociated from rabbit spleen tissue using enzymatic dissociation for 45 minutes.

[0187] Sample is transferred to the centrifuge tube of 15mL separately, and pours 5mL washing buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)).At 4 ℃, centrifuge tube is centrifuged for 5 minutes with 300x g, and pours out supernatant.Use large orifice 1mL pipette tip that sediment is resuspended in 1mL washing buffer, then add 5mL ACK (potassium ammonium chloride) lysis buffer and make cell at room temperature incubate 5-7 minute.By 5mL stop reaction buffer (Dulbecco's phosphate buffered saline (DPBS) (Ca-free) 4% bovine serum albumin (BSA) in phosphate buffered saline (PBS ... 2+ and Mg 2+) was added to the sample, which was then centrifuged at 300 x g for 5 minutes at 4°C. The supernatant was then removed and the resulting precipitate was suspended in 5 mL DPBS. The suspended cells were filtered using a 70 μm cell strainer and the filtrate was poured to 5 mL using additional DPBS.

[0188] Flow cytometry (FACS) was used to compare cells from rabbit spleen tissue dissociated using extensive lateral ultrasound and cells dissociated using an enzymatic method. The results are shown in FIG. 6A to FIG. 6D middle. Fig. 6A and Figure 6B Cell population diversity was compared, showing the relationship with enzymatic dissociation ( Fig. 6A ) compared to the use of large amounts of lateral sonication to dissociate cells ( Figure 6B ) to obtain greater cell diversity (compare the cell populations in the circles). FIG. 6C to FIG. 6D Comparison of enzymatic dissociation ( Figure 6C ) and a large number of lateral ultrasound ( Fig.6D ) Cell viability of dissociated cells as indicated by live (y-axis) nucleated cells (as determined by nuclear staining, x-axis). Enzymatic dissociation yielded approximately 7% live cells, while extensive lateral sonication dissociation yielded approximately 31% live cells.

[0189] Example 5

[0190] The dissociation of cells in clinical breast cancer tumors extracted from patients by a large amount of lateral ultrasonic energy was compared with enzymatic dissociation. About 100 mg of breast cancer tissue was placed in a culture dish supplemented with Losvi Parker Memorial Institute (RPMI) culture medium. The breast cancer tissue sample was chopped into pieces of about 1 mm in size. 3 To about 3mm 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 at 90° angles arranged in a circular pattern with a diameter of approximately 9 mm. A large amount of lateral ultrasound energy was applied to the sample to dissociate cells from the minced spleen tissue.

[0191] Separately, cells were dissociated from clinical breast cancer tissue samples using enzymatic dissociation for 45 minutes.

[0192] Sample is transferred to the centrifuge tube of 15mL separately, and pours 5mL washing buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)).At 4 ℃, centrifuge tube is centrifuged for 5 minutes with 300x g, and pours out supernatant.Use large orifice 1mL pipette tip that sediment is resuspended in 1mL washing buffer, then add 5mL ACK (potassium ammonium chloride) lysis buffer and make cell at room temperature incubate 5-7 minute.By 5mL stop reaction buffer (Dulbecco's phosphate buffered saline (DPBS) (Ca-free) 4% bovine serum albumin (BSA) in phosphate buffered saline (PBS ... 2+ and Mg 2+ ) was added to the sample, which was then centrifuged at 300 x g for 5 minutes at 4°C. The supernatant was then removed and the resulting precipitate was suspended in 5 mL DPBS. The suspended cells were filtered using a 70 μm cell strainer and the filtrate was poured to 5 mL using additional DPBS.

[0193] Cells from the two samples were compared using flow cytometry (FACS) and the results are shown in Fig. 7A (enzymatic dissociation) and Figure 7B (a lot of lateral ultrasonic energy dissociation). Figure 7B As shown, a large amount of lateral ultrasound 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.

[0194] Example 6

[0195] The dissociation of cells from formalin-fixed paraffin-embedded (FFPE) human breast cancer xenograft (PDX) tumor biopsies obtained from a mouse model by high-volume lateral ultrasound energy was compared to enzymatic dissociation. Approximately 100 mg of PDX tissue was placed in a culture dish supplemented with RPMI medium. The breast cancer tissue sample was minced into approximately 1 mm 3 To about 3mm 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 at 90° angles arranged in a circular pattern with a diameter of approximately 9 mm. A large amount of lateral ultrasound energy was applied to the sample to dissociate cells from the minced spleen tissue.

[0196] Separately, cells were dissociated from similar FFPE human breast cancer xenograft tumor biopsies using enzymatic dissociation for 45 minutes.

[0197] Sample is transferred to the centrifuge tube of 15mL separately, and pours 5mL washing buffer (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)).At 4 ℃, centrifuge tube is centrifuged for 5 minutes with 300x g, and pours out supernatant.Use large orifice 1mL pipette tip that sediment is resuspended in 1mL washing buffer, then add 5mL ACK (potassium ammonium chloride) lysis buffer and make cell at room temperature incubate 5-7 minute.By 5mL stop reaction buffer (Dulbecco's phosphate buffered saline (DPBS) (Ca-free) 4% bovine serum albumin (BSA) in phosphate buffered saline (PBS ... 2+ and Mg 2+ ) was added to the sample, which was then centrifuged at 300 x g for 5 minutes at 4°C. The supernatant was then removed and the resulting precipitate was suspended in 5 mL DPBS. The suspended cells were filtered using a 70 μm cell strainer and the filtrate was poured to 5 mL using additional DPBS.

[0198] Flow cytometry (FACS) was used to compare cells from both samples. Very few CD326+, CD24+ cells ( Fig. 8A In contrast, the use of a large amount of lateral ultrasound energy dissociated a large number of CD326+, CD24+ cells from the tissue sample. In addition, when the residual tissue from the enzymatically dissociated sample was treated with a large amount of lateral ultrasound energy, CD326+, CD24+ cells were successfully dissociated ( Figure 8C ).

[0199] Example 7

[0200] Four 45 mg tissue samples of pig liver were obtained. Two of the samples were dissociated into a cell suspension using a large number of 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.

[0201] Cell suspensions and solid tissue samples were frozen before RNA extraction and the minimum RIN value for valid sequencing data was estimated. TM RNA library preparation kit (Illumina) was used for sequencing and then NextSeq TM The sequencing was performed using an Illumina sequencer and the obtained data were analyzed using standard next generation sequencing data analysis tools.

[0202] Fig.9AFold changes for all available genes compared to control samples (solid tissue) are shown. For most genes, gene expression profiles dissociated using bulk lateral ultrasound (BLU) energy closely matched control sample tissue, while gene expression for enzymatically dissociated (ENZYME) cells was less closely matched. Fig. 9B 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) compared to control samples (solid tissue) are shown. For each of the pro-inflammatory and stress-induced genes measured, enzymatically dissociated cells showed a higher increase in gene expression compared to cells dissociated using extensive lateral ultrasound.

[0203] For the purpose of explanation, the foregoing description has been described with reference to specific embodiments. However, the above illustrative discussion is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. In view of the above teachings, many modifications and variations are possible. The embodiments are selected and described in order to best explain the principles of the technology and its practical application. Thus, others skilled in the art can best utilize the technology and various embodiments with various modifications suitable for the intended specific use.

[0204] Although the present disclosure and embodiments have been fully described with reference to the accompanying drawings, it should be noted that various changes and modifications will become apparent to those skilled in the art. These changes and modifications should be understood to be included within the scope of the present disclosure and embodiments defined by the claims.

Claims

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 elements; applying energy from the generated one or more ultrasonic pulses to a biological tissue sample contained by a sample container through a coupling medium coupling the one or more Fresnel annular sector actuator elements to the sample container to dissociate single cells from the biological tissue, wherein the biological tissue sample is substantially free of non-endogenous proteases; as well as The temperature of the biological tissue sample is controlled while energy from the generated one or more ultrasonic pulses is applied to the biological tissue sample, wherein controlling the temperature of the biological tissue sample reduces expression of one or more pro-inflammatory or stress-induced genes compared to not controlling the temperature of the biological tissue sample. 2 . The method of claim 1 , wherein controlling the temperature of the biological tissue sample comprises controlling the temperature of the coupling medium. 3 . The method according to claim 2 , wherein the temperature of the coupling medium is controlled to cool the biological tissue sample or to limit a temperature increase of the biological tissue sample.

4. The method of claim 2, wherein the coupling medium is controlled to a temperature between 2°C and 70°C.

5. The method of claim 2, wherein the coupling medium is controlled to a temperature between 2°C and 25°C.

6. The method of claim 1, wherein controlling the temperature of the biological tissue sample comprises using energy from the generated one or more ultrasound pulses to heat the biological tissue sample.

7. A method according to claim 6, 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.

8. The method of claim 6, wherein the biological tissue sample is heated to a peak temperature between 20°C and 70°C.

9. The method of claim 1, wherein the biological tissue sample is heated by less than 15°C or not during application of energy from the generated one or more ultrasound pulses to the sample container.

10. The method of claim 1, wherein the temperature of the biological tissue sample is controlled between 2°C and 70°C.

11. The method of any one of claims 1-10, 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.

12. The method according to any one of claims 1 to 10, wherein the coupling medium is a liquid, a solid or a gel.

13. The method of any one of claims 1-10, wherein the coupling medium is a liquid, the method further comprising circulating the coupling fluid through a temperature controller.

14. The method of any one of claims 1-10, wherein 20% or more of the dissociated single cells are alive.

15. The method of claim 14, wherein 20% to 95% of the dissociated single cells are alive.

16. The method of any one of claims 1-10, wherein two or more different types of living cells are dissociated from the biological tissue sample.

17. The method of any one of claims 1-10, wherein the biological tissue sample comprises cancerous tissue.

18. The method of any one of claims 1-10, wherein the energy applied to the biological tissue sample does not result in shear forces being applied to the sample.

19. The method of any one of claims 1-10, comprising 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 ultrasonic pulses generated.

20. The method of any one of claims 1-10, wherein energy applied to the biological tissue sample results in mixing or suspension forces applied to the sample.

21. The method of any one of claims 1-10, wherein the plurality of ultrasonic pulses are generated at a repetition rate of 1 Hz to 100,000 Hz.

22. The method of any one of claims 1-10, wherein the one or more ultrasonic pulses are generated with a duty cycle of 0.1% to 95%.

23. The method of any one of claims 1-10, wherein energy from the one or more ultrasonic pulses is applied to the coupling medium for a duration of from 2 seconds to 90 minutes.

24. The method of any one of claims 1-10, wherein the biological tissue is suspended in a liquid within the sample container.

25. The method of any one of claims 1-10, wherein the biological tissue is minced.

26. The method of any of claims 1-10, wherein the transducer comprises four or more Fresnel ring sector actuator elements.

27. The method of any one of claims 1-10, wherein the biological tissue sample is an unpreserved sample.

28. A system for dissociating single cells from a biological tissue sample, comprising: a transducer comprising one or more Fresnel annular sector actuator elements configured to generate one or more ultrasonic pulses; a sample container configured to hold the biological tissue sample, wherein the biological tissue sample is substantially free of non-endogenous proteases, and wherein the system is configured to control a temperature of the biological tissue sample such that the temperature of the biological tissue sample reduces expression of one or more pro-inflammatory or stress-induced genes compared to a system that does not control the temperature of the biological tissue sample; as well as A coupling medium is configured to couple the one or more Fresnel annular sector actuator elements to the sample container and transmit energy from the plurality of ultrasonic pulses generated by the transducer to the sample container to dissociate single cells from the biological tissue sample.

29. The system of claim 28, wherein the system is configured to control the temperature of the biological tissue sample to a temperature between 2°C and 70°C.

30. The system of claim 28, further comprising a temperature controller configured to control a temperature of the coupling medium.

31. The system of claim 30, wherein the temperature controller is configured to control the coupling medium to a temperature between 2°C and 70°C.

32. The system of claim 30, wherein the temperature control is configured to cool the coupling medium to a temperature between 2°C and 25°C.

33. The system of any one of claims 30-32, wherein the coupling medium is a liquid, the system further comprising a pump configured to circulate the coupling medium between a location between the transducer and the sample container and the temperature controller.

34. The system of any one of claims 28-32, wherein the system is configured to control the temperature of the biological tissue sample by heating the biological tissue sample using energy from the generated one or more ultrasound pulses.

35. The system of any one of claims 28-32, 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.

36. The system of any one of claims 28-32, further comprising a control module configured to operate the transducer to generate the one or more ultrasonic pulses.

37. The system of claim 36, 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.

38. The system of claim 36, wherein the control module is configured to operate the transducer to generate a plurality of ultrasonic pulses at a repetition rate of 1 Hz to 100,000 Hz.

39. The system of claim 36, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses at a duty cycle of 0.1% to 95%.

40. The system of claim 36, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses with a total duration of 2 seconds to 90 minutes.

41. The system of claim 36, wherein the control module comprises a radio frequency generator configured to operate the transducer to generate the ultrasonic pulses.

42. The system of claim 36, 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.

43. The system of claim 36, wherein the control module is configured to operate the transducer to generate the one or more ultrasonic pulses such that mixing or levitation forces are applied to the sample.

44. The system of any of claims 28-32, wherein the transducer comprises four or more Fresnel ring sector actuator elements.

45. The system of any one of claims 28-32, wherein the system is arranged in an array comprising a plurality of systems.

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