Ultrasound Device and Method of Use

Abstract

The ultrasonic device includes a housing having an ultrasonic surface, an ultrasonic transducer, and an ultrasonic horn that provides and focuses energy from the ultrasonic transducer to the ultrasonic surface. A method for removing trapped air bubbles from a cell culture vessel includes positioning a cell culture vessel containing air bubbles trapped in liquid within a microcavity well of the cell culture vessel on the ultrasonic surface of the ultrasonic device. Mechanical agitation is generated by the ultrasonic device and applied to the cell culture vessel to remove the trapped air bubbles from the microcavity well. A method for releasing cell aggregates from a cell culture vessel includes positioning a cell culture vessel containing cell aggregates in the microcavity wells on the ultrasonic surface of the ultrasonic device. By applying mechanical agitation from the ultrasonic device to the surface of the cell culture vessel, the cell aggregates are released from the microcavity wells.

Description

Priority

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Patent Application No. 63 / 043877, filed June 25, 2020, the contents of which are relied upon and incorporated herein by reference in their entirety. [Technical Field]

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to ultrasonic devices and methods of using ultrasonic devices for cell culture. [Background technology]

[0003] The use of spheroids and organoids in three-dimensional (3D) cell culture continues to increase due to the physiological relevance of structured cells in living tissues. Spheroid and organoid 3D cell cultures are used in many applications, particularly in tissue engineering, regenerative medicine, and to better understand the pharmacokinetic and pharmacodynamic effects of drugs in preclinical trials.

[0004] Commercially available spheroid and organoid generation platforms seek to meet the growing need to generate large quantities of spheroids and organoids for research and other uses. These platforms often include substrates with many microcavity wells, with individual spheroids or organoids to be generated within each well. Summary of the Invention [Problem to be solved by the invention]

[0005] However, the generation of spheroids is problematic when air bubbles are introduced into the wells before seeding. If air bubbles are present in the wells before seeding, the cells cannot settle to the bottom of the wells, preventing the uniform distribution and aggregation of spheroids or organoids. [Means for solving the problem]

[0006] The ultrasonic device according to the present invention enables the mechanical removal of trapped air bubbles within the microcavity substrate of a cell culture vessel, thereby avoiding the manual agitation or chemical treatment techniques previously used to remove trapped air bubbles. Manual agitation can involve banging or hitting the substrate against a work surface to remove bubbles, which can lead to damage and loss of integrity of the vessel. Chemical treatment methods for removing bubbles may include functionalizing the surface of the substrate to increase wettability, pre-wetting the surface with a solvent with a low wetting angle, such as ethanol, flushing the system at a high flow rate, and coating the surface with a polysaccharide to allow dissolution across the surface of the substrate. However, chemical treatment methods require additional process steps and special packaging of the treated substrate.

[0007] The device of the present invention uses a piezoelectric ultrasonic transducer that vibrates at a consistent frequency. The ultrasonic vibrations create a cavitation event, exposing trapped gas bubbles to sonic frequencies, causing them to expand and contract, thereby removing them from the microcavity wells within the cell culture vessel. In this way, mechanical vibration can remove trapped gas bubbles within the microcavity wells without adding complex process steps, requiring special packaging, or causing damage to the cell culture vessel. By providing an ultrasonic device with a small footprint, the device can be used in a sterile cell culture hood, minimizing process steps and simplifying the user experience by saving time and increasing the efficiency of the spheroid generation process.

[0008] In accordance with one aspect of the present invention, an ultrasonic device includes a housing having an ultrasonic surface, an ultrasonic transducer disposed within the housing, and an ultrasonic horn for delivering and focusing energy from the ultrasonic transducer to the ultrasonic surface.

[0009] The ultrasonic transducer may be an ultrasonic piezoelectric transducer. The ultrasonic frequency of the ultrasonic device may be in the range of about 25 kHz to about 100 kHz. In some embodiments, the ultrasonic frequency may be about 40 kHz. The ultrasonic device may further include an ultrasonic power driver board. The ultrasonic device may provide continuous vibrations. The ultrasonic device may provide pulsed vibrations.

[0010] The ultrasonic surface may include an opening through which a face of the ultrasonic horn protrudes. The device may further include a power switch disposed on the surface of the housing. The device may further include an indicator light disposed on the surface of the housing. The device may further include an activation switch on the ultrasonic surface. The ultrasonic device may be portable.

[0011] The ultrasonic surface may be capable of accepting a cell culture vessel. The cell culture vessel may include a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. In some embodiments, the cell culture vessel is a microcavity flask, including a T-25 flask, a T-75 flask, a T-175 flask, or a T-225 flask. In some embodiments, the cell culture vessel is a microcavity plate, including a 96-well spheroid microplate, a 384-well spheroid microplate, or a 1536-well spheroid microplate. In some embodiments, the cell culture vessel is a multi-layer microcavity cell culture vessel, including a CellSTACK® vessel (manufactured by Corning Incorporated, Corning, NY), a HYPERFlask® vessel (manufactured by Corning Incorporated, Corning, NY), or a HYPERStack® vessel (manufactured by Corning Incorporated, Corning, NY).

[0012] According to one aspect of the present invention, a method for removing trapped air bubbles from a cell culture vessel includes positioning the cell culture vessel on an ultrasonic surface of an ultrasonic device, the cell culture vessel containing trapped air bubbles in a liquid within a microcavity well of the cell culture vessel, and performing mechanical agitation on the ultrasonic surface to remove the trapped air bubbles from the microcavity well.

[0013] The mechanical agitation may be pulsed vibration. The mechanical agitation may be continuous vibration. The applied mechanical agitation may include using an ultrasonic horn to provide and focus ultrasonic energy from an ultrasonic transducer. The mechanical agitation may be applied to the cell culture vessel in increments of about 15 seconds or less.

[0014] Mechanical agitation may be applied to a cell culture vessel by holding the cell culture vessel against an ultrasonic horn on an ultrasonic surface. The cell culture vessel may be a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. The cell culture vessel may include a microcavity substrate for generating spheroids or organoids.

[0015] According to one aspect of the present invention, a method for releasing cell aggregates from a cell culture vessel includes positioning a cell culture vessel containing cell aggregates in microcavity wells on an ultrasonic surface of an ultrasonic device, and releasing the cell aggregates from the microcavity wells by applying mechanical agitation from the ultrasonic device to the surface of the cell culture vessel.

[0016] The mechanical agitation may be applied to the cell culture vessel in increments of 15 seconds or less. The mechanical agitation may be applied to the cell culture vessel continuously. The mechanical agitation may be applied to the cell culture vessel in pulsed fashion. The cell culture vessel may include a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. The cell aggregate may be a spheroid or an organoid. The surface of the cell culture vessel may be a bottom surface.

[0017] According to one aspect of the invention, a kit may include an ultrasound device and a cell culture vessel including a microcavity substrate for generating spheroids or organoids.

[0018] Further scope of applicability of the described devices, methods, and kits will become apparent from the following detailed description, claims, and drawings. The detailed description and specific examples are given for purposes of illustration only, as various changes and modifications within the spirit and scope of the description will become apparent to those skilled in the art. [Brief explanation of the drawings]

[0019] A further understanding of the nature and features of the present disclosure may be obtained by reference to the following drawings, in which like components or features may have the same reference labels. [Figure 1] 1 is a perspective view of an apparatus according to an embodiment of the present invention; [Figure 2] 1 is a perspective view of an apparatus according to an embodiment of the present invention; [Figure 3] 1 is a perspective view of an apparatus according to an embodiment of the present invention; [Figure 4] 1 is an exploded view of an apparatus according to an embodiment of the present invention; [Figure 5] FIG. 1 is an explanatory diagram illustrating a method according to an embodiment of the present invention. [Figure 6] Schematic diagram showing a cell culture vessel before (A) and after (B) application of a method according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] Due to the inherent hydrophobicity of the bulk spheroid microcavity substrate geometry, high surface tension is generated by hydrogen bonding of water molecules in aqueous solutions. This surface tension causes the liquid to spread over the microcavities rather than enter them, thereby preventing wetting of the microcavity wells. Conventional techniques to address the surface tension issue include chemical treatments to pre-wet or coat the wells. For example, wells can be pre-wetted with a gradient of solvents ranging from a nonpolar solvent, such as ethanol, to a more polar solvent, such as water, or a mixture of solvents in between. As another example, coating the substrate with glucose can improve wetting and filling of the microcavity wells due to the affinity of water for polar molecules. However, both techniques are chemical processes and require additional handling and processing steps prior to cell culture.

[0021] In contrast, the device and method of the present invention use ultrasound to overcome surface tension. Thus, the present invention provides a mechanical method, rather than chemical treatment, that can be used to assist in wetting of microcavity wells. The device uses mechanical agitation, rather than chemical modification or surface modification, to remove trapped air from the bottom of the microcavity wells. In some embodiments of the present invention, a kit is provided that includes an ultrasonic device along with a cell culture vessel or bulk spheroid microcavity substrate. The device and method of the present invention simplify the manufacturing process of the microcavity substrate or vessel. By using mechanical agitation, the microcavity substrate no longer requires a chemical coating process, such as a polysaccharide coating process. Because a chemical coating is no longer required, special packaging of the microcavity substrate is also no longer necessary.

[0022] The devices and methods of the present invention utilize cavitation of gas bubbles in a liquid in response to ultrasound to remove trapped gas bubbles from the bottom of a microcavity well. In certain embodiments, a piezoelectric transducer vibrates at a determined frequency when an AC voltage waveform is applied, converting electrical energy into mechanical energy. In certain embodiments, the devices of the present invention include an ultrasonic piezoelectric transducer operating at frequencies ranging from approximately 220 kHz to frequencies of GHz. When air or gas bubbles are subjected to high-frequency vibrations, changes in wave pressure cause the bubbles contained within the fluid to rapidly expand and contract until they collapse, thereby releasing shock waves known as cavitation. By subjecting the microcavity substrate to ultrasonic frequencies, the devices of the present invention can remove gas bubbles from the bottom of a microcavity well by mechanical agitation, instead of the traditional chemical modification used.

[0023] The device and method of the present invention also simplify the processing of microcavity substrates for spheroid formation. The device of the present invention generates consistent and predictable mechanical agitation. Mechanical agitation applied to the surface of the microcavity substrate eliminates the need to manually "bang" or "bang" the cell culture vessel on a work surface, such as a laboratory bench, to remove trapped air bubbles. Furthermore, the size and small footprint of the device allow the ultrasonic device to be used inside a cell culture hood. Therefore, this device size eliminates the need to remove the substrate from a cell culture hood, such as a Class II cell culture hood, to use a tabletop centrifuge or other method to remove air bubbles during the cell culture process and seeding of cells into the cell culture vessel. The ability to remove air bubbles inside the cell culture hood reduces process and handling time while maintaining the microcavity substrate within the sterile environment of the cell culture hood.

[0024] The device and method of the present invention can also be found to be useful in the later stage of the spheroid or organoid generation process.The present invention can be used to remove the spheroid or organoid that adheres to the microcavity substrate after long-term culture.By using the device of the present invention to provide mechanical agitation, it is possible to remove the spheroid or organoid without destroying the spheroid or organoid or damaging the cells contained therein.

[0025] The ultrasonic device of the present invention includes a housing having an ultrasonic surface, an ultrasonic transducer disposed within the housing, and an ultrasonic horn that delivers and focuses energy from the ultrasonic transducer to the ultrasonic surface. The ultrasonic transducer may be a piezoelectric ultrasonic transducer. In some embodiments, the device also includes an activation switch, a power switch, a setting switch, an indicator light, or a combination thereof. In some embodiments, a kit is provided that includes an ultrasonic device of the present invention and a bulk microcavity substrate for generating spheroids or organoids.

[0026] The device of the present invention further includes a power source and an ultrasonic power driver board or control board for amplifying the power. The power source may be any suitable power source, such as a battery or a wall outlet. The device may include an AC input cable and plug or a battery input. Any suitable control device may be used. For example, the control device may include a control board, a power amplification module, or a combination thereof. An activation switch may be located on the periphery of the face of the ultrasonic horn. A power switch may be provided to power down the device to conserve energy and prevent undesired operation when the device is not in use.

[0027] The surface of the housing may include an ultrasonic surface. The ultrasonic surface may be the top surface of the device. The ultrasonic surface may include an opening through which a face of an ultrasonic horn protrudes. The ultrasonic surface may receive a cell culture vessel. In some embodiments, the cell culture vessel includes a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. In certain embodiments, the ultrasonic device has a small footprint, allowing the device to be used inside a cell culture hood without occupying valuable space underneath the hood. In certain embodiments, the device is compact, modular, and has dimensions suitable for use inside a cell culture hood. In some embodiments, the device is shaped like a 6-inch (approximately 15 cm) cube. The device may be made with sealed internal components, allowing the device to be easily wiped down and sanitized for use inside a sterile cell culture hood. In some embodiments, the device is portable.

[0028] The housing may be formed from any suitable material that provides a rigid structure. For example, the housing may be formed from a rigid plastic material or other rigid non-conductive material. In some embodiments, at least a portion of the ultrasonic horn protrudes from the ultrasonic surface of the housing. The portion of the ultrasonic horn that protrudes from the ultrasonic surface may be the face of the ultrasonic horn.

[0029] The devices and methods of the present invention may use any suitable ultrasonic transducer operating at any suitable and desired ultrasonic frequency. In some embodiments, the ultrasonic frequency for cavitation ranges from about 25 kHz to about 100 kHz. In some embodiments, the ultrasonic frequency is about 40 kHz. The ultrasonic transducer may be a piezoelectric ultrasonic transducer. The ultrasonic transducer may be a 40 kHz 60 W ultrasonic transducer. In some embodiments, the ultrasonic frequency ranges from about 25 kHz to about 100 kHz. In some embodiments, the ultrasonic frequency is selected from 25 kHz, 40 kHz, and 80 kHz. In a preferred embodiment, the ultrasonic frequency is 40 kHz. It is preferred that ultrasonic frequencies be above the range of human hearing, which is up to about 20 kHz. In some embodiments, the ultrasonic transducer may be a 40 kHz piezoelectric ultrasonic transducer manufactured by KEMET Electronics Corporation (Fort Lauderdale, Florida) or a 40 kHz piezoelectric ultrasonic air transducer manufactured by Steiner & Martins, Inc. (Davenport, Florida). It should be understood that other ultrasonic transducers are contemplated and possible.

[0030] The horn may be any suitable ultrasonic horn capable of focusing the ultrasonic energy emitted from the ultrasonic transducer. The ultrasonic horn may be of any suitable size and shape and may be formed from any suitable material. In one embodiment, the ultrasonic horn is a metallic cylindrical ultrasonic horn. The ultrasonic horn may be fabricated from any suitable material, such as aluminum, titanium, or steel. By way of example, the ultrasonic horn may be shaped like a cylinder or a rectangular block. In one embodiment, the ultrasonic horn may be a cylindrical aluminum ultrasonic horn, such as those manufactured by Branson Ultrasonics Corp. (Danbury, Connecticut) or Sonitek Corporation (Milford, Connecticut). It should be understood that other ultrasonic horns are also contemplated and possible.

[0031] As described herein, the device of the present invention can be used to remove trapped air bubbles from the bottom of microcavity wells to aid in the even distribution of cell seeding for bulk spheroid formation. The device of the present invention can also be used to detach cells from microcavity wells.

[0032] In some embodiments, the devices and methods of the present invention are used with cell culture vessels or bulk spheroid microcavity substrates to "wet-out" or fully saturate the microcavity wells within the cell culture vessel with an aqueous solution. Using an ultrasonic transducer as a consistent and predictable means of mechanical agitation can remove trapped air bubbles from a series of microcavity wells, thereby "wetting out" the microcavity wells. "Wetting out" the microcavity wells allows for uniform cell seeding and cell spheroid formation across a series of microcavities for downstream processing.

[0033] In some embodiments, the devices and methods of the present invention are used to remove spheroids or organoids from microcavity wells. Long-term cultured spheroids will remain firmly within microcavity wells, such as bulk spheroid microcavity substrates or microscale wells in cell culture vessels. Traditional methods for removing aggregated cells, such as spheroids or organoids, from microcavity wells include manual scraping or vigorous washing, which can damage the spheroids or organoids. The use of ultrasonic devices according to the present invention provides a consistent and predictable means of mechanical agitation, which allows for easier removal of long-term cultured spheroids compared to traditional manual or chemical processes.

[0034] In some embodiments, the devices of the present invention are suitable for use with cell culture vessels, such as microcavity substrate articles, non-limiting examples of which include T-25 flasks, T-75 flasks, open-well plates, and microcavity cell culture plates. Non-limiting examples of microplate products include EZSPHERE (Nacalai USA), AGGREWELL (STEMCELL Technologies), SPHER A (Nunclon), CELLSTAR (Greiner Bio-One), CELLCARRIER (PerkinElmer), and NANOCULTURE (MBL International), 96-well spheroid microplates (Corning Incorporated, Corning, NY), 384-well spheroid microplates (Corning Incorporated, Corning, NY), 1536-well spheroid microplates (Corning Incorporated, Corning, NY), and ELPLASIA plates (Corning Incorporated, Corning, NY). In some embodiments, the cell culture vessel comprises a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. In some embodiments, the cell culture vessel is a microcavity flask, including a T-25 flask, a T-75 flask, a T-175 flask, or a T-225 flask. In some embodiments, the cell culture vessel is a microcavity plate, including a 96-well spheroid microplate, a 384-well spheroid microplate, or a 1536-well spheroid microplate.In some embodiments, the cell culture vessel is a multi-layer microcavity cell culture vessel, including a "CellSTACK" vessel (manufactured by Corning Incorporated, Corning, NY), a "HYPERFlask" vessel (manufactured by Corning Incorporated, Corning, NY), or a "HYPERStack" vessel (manufactured by Corning Incorporated, Corning, NY). It should be understood that other cell culture vessels are also contemplated and possible.

[0035] When the device is turned on (for devices with a power switch, the power switch is turned on and power is supplied to the device by the power supply), the device enters standby mode until a cell culture vessel is placed on the face of the ultrasonic horn on the ultrasonic surface of the device. Once the cell culture vessel is placed on the face of the ultrasonic horn, the ultrasonic transducer is powered on. For devices with an activation switch, placing the cell culture vessel on the ultrasonic surface and face of the ultrasonic horn may also involve turning on the activation switch, which powers on the ultrasonic transducer. Once powered on, the ultrasonic transducer vibrates the surface of the bulk spheroid microcavity, thereby dislodging trapped air bubbles (or trapped spheroids) within the microcavity wells of the cell culture vessel. The mechanical action of the vibration is harsh enough to dislodge trapped air bubbles, but gentle enough to avoid catastrophic damage to the vessel or substrate.

[0036] Depending on the stage of the cell culture process, vibration may be used for cell seeding or cell harvesting. In cell seeding, vibration is used to dislodge trapped air bubbles by cavitation within the microcavity wells to enable the generation of spheroids or organoids. In cell harvesting, vibration followed by cavitation is used to detach cells from the microcavity wells to harvest the resulting spheroids or organoids.

[0037] Different settings of the device may be provided, such as continuous vibration or pulsed vibration. In some embodiments, the device provides continuous vibration. In some embodiments, the device provides pulsed vibration. Pulsed vibration may be pulsed intermittent vibration. The device may provide mechanical agitation or vibration in increments of about 15 seconds or less. In some embodiments, pulsed vibration occurs in increments of about 15 seconds or less. Pulsed vibration may be provided by alternating ON-OFF sequences or impulses of the ultrasonic transducer, or by programming timed intervals of ultrasonic vibration. Pulsed vibration may more effectively remove trapped air bubbles from the microcavities and limit the amount of heat and sound generated by operation of the device, thereby extending the life of the transducer.

[0038] 1 shows an ultrasonic device 100 according to an embodiment of the present invention. The ultrasonic device 100 includes a housing 15 having an ultrasonic surface 25, an ultrasonic transducer disposed within the housing 15, and an ultrasonic horn 35 that delivers and focuses energy from the ultrasonic transducer to the ultrasonic surface 25. The ultrasonic horn 35 has a face 37 that protrudes from the ultrasonic surface 25. A power switch 55 having an OFF-ON mode is provided on the front of the ultrasonic device 100.

[0039] 2 shows an ultrasonic device 200 according to an embodiment of the present invention, further including an activation switch 50 around the face 37 of the ultrasonic horn 35 on the ultrasonic surface 25. Any suitable activation switch or button may be used. For example, a button may be used that is depressed when the cell culture vessel is pressed, thereby activating the ultrasonic transducer. An indicator light 53, such as a light that indicates the power level of the device's batteries, is shown on the front of the ultrasonic device.

[0040] FIG. 3 illustrates an ultrasonic device 300 according to an embodiment of the present invention. The device 300 includes a rigid housing 15 having an ultrasonic surface 25. A lid 23 is also provided that covers the ultrasonic surface 25. The lid may be any suitable lid, such as a hinged lid (as shown) or a clasp or interlocking lid. Other types of lids are contemplated. The ultrasonic surface 25 may be a flat surface, such as a tray, suitable for receiving cell culture vessels of various sizes and dimensions. A face 37 of an ultrasonic horn protrudes from the ultrasonic surface 25 of the housing 15. A rectangular ultrasonic surface face 37 is shown.

[0041] A power switch 55 is provided on the front of the ultrasonic device 300. In some embodiments, the power switch has an ON position and an OFF position. When in the ON position, the ultrasonic device is activated and in standby mode, and the device provides mechanical agitation when an item contacts the ultrasonic surface. When the power switch is in the OFF position, the ultrasonic device is not operational. A setting switch 49 is provided on the front of the ultrasonic device 300. The setting switch allows different settings of the device to be activated. Exemplary settings include pulse agitation, continuous agitation, and a time selection setting. Indicator lights 51, 52, and 54 are provided on the front of the ultrasonic device 300. These indicator lights may be associated with the power switch, the setting switch, and the ultrasonic surface. For example, indicator light 51 indicates the mode of setting switch 49, indicator light 52 indicates the mode of power switch 55, and indicator light 54 indicates whether the ultrasonic transducer is activated and / or providing mechanical agitation to the ultrasonic surface 25.

[0042] FIG. 4 shows an exploded view of an ultrasonic device 100. The device 100 includes a housing 15. In some embodiments, the housing 15 may be shaped like a cube, as shown. The housing may be made of a non-conductive material, such as a rigid plastic housing. Disposed within the housing 15 are a controller or control board 40, an ultrasonic transducer 30, an ultrasonic horn 35 having a face 37, and insulating material (not shown). The controller may be any suitable controller, such as an ultrasonic power driver board. The top surface of the housing 15 serves as an ultrasonic surface 25. The ultrasonic surface 25 has an opening 21 through which the face 37 of the ultrasonic horn 35 protrudes. The illustrated power source 45 is a receptacle. An electrical cord and power plug 47 provides power from the power source 45 to the device 100 when a power switch 55 is placed in the ON position. The control board 40 and ultrasonic transducer 30 convert electrical power into ultrasonic output in the form of mechanical agitation, which is focused by the ultrasonic horn 35 and provided to a cell culture vessel positioned at ultrasonic surface 25 flush with face 37 of the ultrasonic horn 35.

[0043] According to one aspect of the present invention, a method for removing trapped air bubbles from a cell culture vessel includes positioning the cell culture vessel on an ultrasonic surface of an ultrasonic device, the cell culture vessel containing trapped air bubbles in a liquid within a microcavity well of the cell culture vessel, and performing mechanical agitation on the ultrasonic surface to remove the trapped air bubbles from the microcavity well.

[0044] The mechanical agitation may be pulsed vibration. The mechanical agitation may be continuous vibration. The generated mechanical agitation may include using an ultrasonic horn to provide and focus ultrasonic energy from an ultrasonic transducer. The mechanical agitation may be applied to the cell culture vessel by holding the cell culture vessel against the ultrasonic horn on an ultrasonic surface. The mechanical agitation may be applied to the cell culture vessel in increments of about 15 seconds or less.

[0045] In some embodiments, the face of the ultrasonic horn protrudes from the ultrasonic surface. In some embodiments, the cell culture vessel is positioned by holding the cell culture vessel flush against the face of the ultrasonic horn to apply ultrasonic energy in the form of mechanical agitation to the cell culture vessel, thereby removing trapped air bubbles. In some embodiments, the bottom surface of the cell culture vessel is held flush against the ultrasonic horn. In some embodiments, the cell culture vessel is manually manipulated by a user so that different portions of the bottom surface of the cell culture vessel are flush with the face of the ultrasonic horn.

[0046] In some embodiments, the cell culture vessel is a microcavity substrate or bulk microcavity substrate for generating spheroids or organoids. In some embodiments, the cell culture vessel comprises a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. In some embodiments, the cell culture vessel is a microcavity flask, including a T-25 flask, a T-75 flask, a T-175 flask, or a T-225 flask. In some embodiments, the cell culture vessel is a microcavity plate, including a 96-well spheroid microplate, a 384-well spheroid microplate, or a 1536-well spheroid microplate. In some embodiments, the cell culture vessel is a multi-layer microcavity cell culture vessel, including a "CellSTACK" vessel (manufactured by Corning Incorporated, Corning, NY), a "HYPERFlask" vessel (manufactured by Corning Incorporated, Corning, NY), or a "HYPERStack" vessel (manufactured by Corning Incorporated, Corning, NY). It should be understood that other cell culture vessels are also contemplated and possible.

[0047] 5 illustrates a method 400 of using an apparatus according to an embodiment of the present invention. The ultrasonic apparatus 100 includes a housing 15 disposed around an ultrasonic transducer and an ultrasonic horn 35. A face 37 of the ultrasonic horn protrudes from the ultrasonic surface 25 of the housing 15. Prior to use, the ultrasonic apparatus is in the "OFF" position to wet the wells of a microcavity substrate. The "OFF" and "ON" modes may be indicated by a power switch 35. When the apparatus's power switch 35 is turned to the "ON" position, it remains in standby mode until activated by a cell culture vessel 60.

[0048] The cell culture vessel may be any suitable cell culture vessel, such as a microcavity flask, a microcavity plate, a multi-layer microcavity cell culture vessel, or other cell culture vessel capable of culturing cells within microcavity wells. The cell culture vessel may include a microcavity substrate with microcavity wells. The cell culture vessel may include a bulk spheroid microcavity substrate.

[0049] When cell culture medium 70 or other liquid is added to the cell culture vessel, the size of the microcavity wells prevents the wells from filling with cell culture medium 70, resulting in trapped air bubbles 65 within the microcavity wells. In method 400, when the cell culture vessel 60 containing the trapped air bubbles 65 is placed flush against the face 37 of the ultrasonic horn, the ultrasonic device 100 is activated, generating an ultrasonic waveform 80. The ultrasonic waveform 80 provides mechanical agitation, vibrating the cell culture vessel 60 and dislodging any trapped air bubbles 65 within the microcavity wells. The ultrasonic energy applied may be continuous or pulsed. The cell culture vessel 60 may be held against the ultrasonic surface 25 until the trapped air bubbles 65 are removed. Once the trapped air bubbles 65 are removed, the cell culture vessel 60 is removed from the ultrasonic surface 25 of the ultrasonic device 100, at which point the ultrasonic waveform 80 is stopped and the ultrasonic device 100 enters standby mode.

[0050] In one embodiment, the ultrasonic device of the present invention includes an activation button or switch. An ultrasonic device for wetting wells of a microcavity substrate in a cell culture vessel is set to the "OFF" position before use. When the power switch of the device is set to the "ON" position, the device enters standby mode until the activation switch is depressed by the cell culture vessel. When the activation switch is depressed, the ultrasonic device is activated, and mechanical energy from the ultrasonic transducer is focused by the ultrasonic horn. When the cell culture vessel is positioned or held flush against the surface of the ultrasonic horn, ultrasonic energy is applied to the cell culture vessel. Thus, when a cell culture vessel containing a bulk spheroid microcavity substrate is placed against the ultrasonic surface of the ultrasonic device, the activation switch is depressed, and the cell culture vessel is held flush against the surface of the ultrasonic horn, an ultrasonic waveform is generated, vibrating the cell culture vessel and dislodging air bubbles trapped in the microcavity wells. The cell culture vessel may be manually manipulated during application of ultrasonic energy so that different areas of the surface of the cell culture vessel are positioned flush against the surface of the ultrasonic horn. Once the trapped air bubbles are removed, the cell culture vessel is removed from the ultrasonic surface of the ultrasonic device, at which point the ultrasonic waveform is stopped and the ultrasonic device goes into standby mode.

[0051] FIG. 6 shows schematic diagrams of a cell culture vessel, such as a culture flask, (A) before undergoing the method of the present invention and (B) after applying the method of the present invention. As can be seen in (A), the culture flask 60, which may have a cap or lid 63, contains a bulk spheroid microcavity substrate 75. Upon addition of cell culture medium 70 or other fluid used to wet out the surface of the substrate, trapped air bubbles 65 form at the bottom of the microcavity wells of the microcavity substrate 75. In (B), the culture flask 60 is shown after activation of an ultrasonic device. After activation of the device, cavitation of the air bubbles occurs. As can be seen in (B), trapped air bubbles cavitate, and the microcavity substrate 75 now contains fluid-filled microcavities 85. Thus, the microcavities are wetted out and filled with fluid prior to adding cells to form spheroids.

[0052] A method for releasing cell aggregates from a cell culture vessel is provided. For example, the cell aggregates may be spheroids or organoids. When culturing spheroids, the aggregated spheroid culture may grow too large and become trapped in the microcavity. Using the ultrasonic device of the present invention, the aggregated spheroid culture can be removed without damaging the spheroids, thus allowing the spheroids to be harvested. According to one aspect of the present invention, the method for releasing cell aggregates from a cell culture vessel includes positioning a cell culture vessel containing cell aggregates in a microcavity well on an ultrasonic surface of an ultrasonic device, and releasing the cell aggregates from the microcavity well by applying mechanical agitation from the ultrasonic device to the surface of the cell culture vessel.

[0053] The cell aggregates may be spheroids or organoids. The surface of the cell culture vessel may be a bottom surface. In some embodiments, the cell culture vessel is a bulk substrate for generating spheroids or organoids. In some embodiments, the cell culture vessel comprises a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel. In some embodiments, the cell culture vessel is a microcavity flask, including a T-25 flask, a T-75 flask, a T-175 flask, or a T-225 flask. In some embodiments, the cell culture vessel is a microcavity plate, including a 96-well spheroid microplate, a 384-well spheroid microplate, or a 1536-well spheroid microplate. In some embodiments, the cell culture vessel is a multi-layer microcavity cell culture vessel, including a "CellSTACK" vessel (manufactured by Corning Incorporated, Corning, NY), a "HYPERFlask" vessel (manufactured by Corning Incorporated, Corning, NY), or a "HYPERStack" vessel (manufactured by Corning Incorporated, Corning, NY). It should be understood that other cell culture vessels are also contemplated and possible.

[0054] Mechanical agitation may be applied to the cell culture vessel in increments of 15 seconds or less. Mechanical agitation may be applied to the cell culture vessel continuously. Mechanical agitation may be applied to the cell culture vessel in pulsed increments. In some embodiments, the ultrasonic device is pulsed in increments of several hundred milliseconds to about 15 seconds. By operating the ultrasonic transducer intermittently rather than continuously, the methods of the present invention allow for more efficient cavitation action in removing microcavity wells.

[0055] In one example, the device was run continuously for approximately 15 seconds and then shut off. From this point onward, the power switch was alternately toggled between the ON and OFF positions. When the power switch was turned OFF, the trapped gas bubbles were observed floating to the surface of the liquid in the cell culture vessel. The frequency at which the ultrasonic transducer operated resulted in a pause in the animation for the gas bubbles, which were released from the microcavities but remained in place within the fluid. Once the ultrasonic vibrations were turned off, the gas bubbles escaped to the gas-liquid interface.

[0056] While numerous embodiments of the present disclosure are illustrated in the accompanying drawings and described in the foregoing detailed description, it should be understood that the present disclosure is not limited to the disclosed embodiments, but is capable of numerous rearrangements, modifications, and substitutions without departing from the disclosure as set forth and defined in the following claims.

[0057] Preferred embodiments of the present invention will be described below in detail.

[0058] Embodiment 1 1. An ultrasound device comprising: a housing having an ultrasonic surface; an ultrasonic transducer disposed within the housing; and an ultrasonic horn for delivering and focusing energy from the ultrasonic transducer to the ultrasonic surface.

[0059] Embodiment 2 2. The device of embodiment 1, wherein the ultrasonic transducer is an ultrasonic piezoelectric transducer.

[0060] Embodiment 3 2. The apparatus of embodiment 1, wherein the ultrasonic surface is capable of receiving a cell culture vessel.

[0061] Embodiment 4 4. The apparatus of embodiment 3, wherein the cell culture vessel comprises a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel.

[0062] Embodiment 5 2. The device of embodiment 1, wherein the ultrasonic surface includes an opening through which a face of the ultrasonic horn protrudes.

[0063] Embodiment 6 2. The device of embodiment 1, wherein the ultrasound frequency is in the range of 25 kHz to 100 kHz.

[0064] Embodiment 7 7. The device of embodiment 6, wherein the ultrasound frequency is 40 kHz.

[0065] Embodiment 8 10. The apparatus of embodiment 1, wherein the ultrasound device further comprises an ultrasound power driver board.

[0066] Embodiment 9 10. The device of embodiment 1, wherein a power switch is located on the surface of the housing.

[0067] Embodiment 10 10. The device of embodiment 1, wherein an indicator light is disposed on a surface of the housing.

[0068] Embodiment 11 10. The device of embodiment 1, further comprising an activation switch on the ultrasound surface.

[0069] Embodiment 12 2. The device of embodiment 1, wherein the ultrasound device provides continuous vibrations.

[0070] Embodiment 13 2. The device of embodiment 1, wherein the ultrasound device applies pulsed vibrations.

[0071] Embodiment 14 2. The device of embodiment 1, wherein the ultrasound device is portable.

[0072] Embodiment 15 1. A method for removing trapped air bubbles from a cell culture vessel, comprising: positioning a cell culture vessel on an ultrasonic surface of an ultrasonic device, the cell culture vessel containing air bubbles trapped in a liquid within a microcavity well of the cell culture vessel; and performing mechanical agitation with the ultrasonic surface to remove trapped air bubbles from the microcavity wells; The method comprising:

[0073] Embodiment 16 16. The method of embodiment 15, wherein the mechanical agitation is pulsating.

[0074] Embodiment 17 16. The method of embodiment 15, wherein said mechanical agitation is continuous vibration.

[0075] Embodiment 18 16. The method of embodiment 15, wherein said mechanical agitation comprises using an ultrasonic horn to provide and focus ultrasonic energy from an ultrasonic transducer.

[0076] Embodiment 19 20. The method of embodiment 18, wherein the mechanical agitation is imparted to the cell culture vessel by holding the cell culture vessel against the ultrasonic horn at the ultrasonic surface.

[0077] Embodiment 20 16. The method of embodiment 15, wherein said cell culture vessel is a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel.

[0078] Embodiment 21 21. The method of embodiment 20, wherein the cell culture vessel comprises a microcavity substrate for generating spheroids or organoids.

[0079] Embodiment 22 16. The method of embodiment 15, wherein said mechanical agitation is imparted to said cell culture vessel in increments of 15 seconds or less.

[0080] Embodiment 23 1. A method for releasing cell aggregates from a cell culture vessel, comprising: positioning a cell culture vessel containing cell aggregates within microcavity wells on an ultrasonic surface of an ultrasonic device; and releasing the cell aggregates from the microcavity wells by applying mechanical agitation from the ultrasonic device to the surface of the cell culture vessel; The method comprising:

[0081] Embodiment 24 24. The method of embodiment 23, wherein the surface of the cell culture vessel is the bottom surface.

[0082] Embodiment 25 24. The method of embodiment 23, wherein said mechanical agitation is applied in increments of 15 seconds or less.

[0083] Embodiment 26 26. The method of embodiment 25, wherein said mechanical agitation is applied continuously.

[0084] Embodiment 27 26. The method of embodiment 25, wherein the mechanical agitation is applied in pulses.

[0085] Embodiment 28 24. The method of embodiment 23, wherein the cell aggregate is a spheroid or an organoid.

[0086] Embodiment 29 24. The method of embodiment 23, wherein said cell culture vessel comprises a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel.

[0087] Embodiment 30 A kit comprising: an ultrasound device according to embodiment 1; and A cell culture vessel comprising a microcavity substrate for generating spheroids or organoids, Kit including: [Explanation of symbols]

[0088] 15 Case 23 Lid 25 Ultrasonic Surface 30 Ultrasonic transducer 35 Ultrasonic Horn 37 sides 40 Control Board 45 Power supply, outlet 47 Electrical cords and power plugs 49 Setting Switch 50 Start switch 51, 52, 54 indicator light 55 Power switch 60 cell culture vessels 63 Cap or lid 65 Trapped bubbles 70 Cell Culture Media 80 Ultrasound Waveforms 85 Microcavity 100, 200, 300 ultrasound equipment

Claims

1. 1. A method for removing trapped air bubbles from a cell culture vessel, comprising: Positioning a cell culture vessel containing air bubbles trapped in a liquid within a microcavity well of the cell culture vessel on an ultrasonic surface of an ultrasonic device, the ultrasonic device comprising: a housing having an ultrasonic surface and an opening; an ultrasonic transducer disposed within the housing; an ultrasonic horn having a surface projecting from the opening, the ultrasonic horn delivering focused energy from the ultrasonic transducer to the ultrasonic surface; and automatically activating the ultrasonic transducer when the cell culture vessel is positioned flush against the face of the ultrasonic horn to provide mechanical agitation at the ultrasonic surface to remove trapped air bubbles from the microcavity wells; The method comprising:

2. The method of claim 1 , wherein the mechanical agitation is pulsating.

3. The method of claim 1 , wherein the mechanical agitation is continuous vibration.

4. The method of claim 1 , wherein the step of mechanically agitating comprises using an ultrasonic horn to provide and focus ultrasonic energy from an ultrasonic transducer.

5. 5. The method of claim 4, wherein the mechanical agitation is imparted to the cell culture vessel by holding the cell culture vessel against the ultrasonic horn at the ultrasonic surface.

6. The method of claim 1 , wherein the cell culture vessel is a microcavity plate, a microcavity flask, or a multi-layer microcavity cell culture vessel.

7. The method of claim 6, wherein the cell culture vessel comprises a microcavity substrate for generating spheroids or organoids.

8. 10. The method of claim 1, wherein the mechanical agitation is applied to the cell culture vessel in increments of 15 seconds or less.

Images