Ultrasound based reproductive cell micro-manipulator

Abstract

Systems and methods are disclosed for manipulating cells using ultrasonic transducers with electrodes. A system includes a chamber configured to contain a cell and a processor in electronic communication with the ultrasonic transducer. The processor is configured to control the ultrasonic transducer to generate acoustic waves directed to the cell to manipulate the cell within the chamber. The acoustic waves generate an acoustic field that has a pattern defined by a shape of the electrodes of the ultrasonic transducer. The pattern may have a ring, polygonal, petal, crescent, or bottle shape.

Description

ULTRASOUND BASED REPRODUCTIVE CELL MICRO-MANIPULATORCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority to and the benefit of U. S. Provisional Patent Application Serial No. 63 / 714,963, filed November 1, 2024, the entire disclosure of which is hereby incorporated by reference.TECHNICAL FIELD

[0002] This disclosure relates to micro-manipulation of cells using acoustic waves, for example, micro-manipulation of reproductive cells using acoustic waves generated by ultrasonic transducers.BACKGROUND

[0003] Assisted reproductive technology (ART) includes in vitro fertilization (IVF) procedures which entail the insemination of cumulus oocyte complexes (COCs) with several thousand spermatozoa. A majority of IVF procedures now include intracytoplasmic sperm injection (ICSI). To perform ICSI, removal of cumulus corona cells from the oocyte(s) is needed. ICSI requires a skilled operator and a controlled setting such as predefined media and temperature as well as sophisticated equipment. Most importantly, ICSI requires direct manipulation of a gamete that starts with pre -treatment of the COCs. The pre-treatment includes cumulus cell removal (e.g., stripping) which allows visualization of the oocyte(s) while performing the controlled injection of spermatozoa.

[0004] In manual examples of ART, cumulus cell removal is executed by manually pipetting in and out using a properly sized capillary while immersed in hyaluronidasecontaining medium. If skills are inadequate, prolonged exposure to hyaluronidase may compromise the quality of the oocyte. On the other hand, excessive suction pressure through an improperly selected capillary size may exert mechanical stress on the COCs, resulting in damage to the zona pellucida in various degrees, such as cracking of the zona or yielding a completely zona-free oocyte.SUMMARY

[0005] This application describes acousto-hydrodynamic trapping (or tweezer) (AHT) modules designed for contactless manipulation of cells. For example, AHT may be used with reproductive cells to denude, trap, and move oocytes in a microfluidic system contained in a disposable dish. These functionalities have been evaluated through controlled experimental measurements and confirmed by biological proof. This complementary tool may increase the consistency of the oocyte denudation procedure, eventually leading to more reproducible and reliable outcomes among different clinics and operators. This is particularly relevant in an era of increased regulations and stringent needs to provide accessible reproductive care in an affordable manner, while granting standardized performance. The most relevant prerequisite of this work is the ability to prepare the oocyte for the ICSI procedure in a reliable, safe, and contactless manner in a disposable microsystem that paves the way to integration into a comprehensive ICSI-on-chip.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity.

[0007] FIG. 1 A shows an active piezoelectric substrate that generates an acoustic vortex that propagates into an open microfluidic dish through a glass layer and a thin layer of deionized (DI) water that supports the dish. The focalized acousto-fluidic flow is then used to trap, denude, and reposition trapped cells, such as oocytes.

[0008] FIG. IB shows numerical predictions of a normalized intensity field for (i), (ii), and (iii) ring-like (e.g., SAV, Fresnel, and R-14), (iv) polygonal-like (P-4), (v) circular-petal (T-3), and (vi) crescent-shaped petal fields (C-41).

[0009] FIG. 1C shows a perspective view of an iso-intensity level = 0.8 with phase information for R-15 and its propagation through layers from glass to oil inside the dish.

[0010] FIG. ID shows a three-dimensional schematic representation of the open microfluidic stmcture with embedded microchamber at the center for oocyte retention during active pumping. A representative photo of the open microfluidic dish and capillary holders for active pumping is also shown (bottom).

[0011] FIG. IE shows a scheme illustrating the working principle of acoustic (oracousto-) hydrodynamic traps (or tweezers) (AHT) and open microfluidic dish for oocyte preparation. First, (i) cells are loaded into the open microfluidic dish loaded with 20 lU / mL HA solution; second, (ii) the AHTs are used for the denudation procedure and denuded oocytes are translated to microchambers; and (iii) third, active pumping is used for quenching the enzymatic reaction.

[0012] FIGS. 2 A to 2F show strategies for plucking and shedding denudation strategies based on AHT. FIG. 2A and FIG. 2D show acousto-fluidic fields created in open and closed microfluidics following the displayed acoustic intensities shown at the bottom. FIG. 2B and FIG. 2E show that transverse repositioning of the AHT location is achieved by moving the dish either manually or by a scanning stage; vertical displacement of the AHT in the propagation axis is achieved by sweeping the input frequency around the designed frequency. FIG. 2C and FIG. 2F show a schematic and a photograph of plucking (FIG. 2C) denudation methods that target the cumulus cells and shedding (FIG. 2F) denudation method that target the oocytes.

[0013] FIG. 3A shows the effect of power on denudation time for various devices and denudation strategies.

[0014] FIG. 3B shows the effect of HA concentration on denudation time for P-4 (shedding strategy) at two power levels. There exist significant correlations between the power level, HA concentration, and denudation efficiency for both plucking (P value < 0.05) and shedding (P value < 0.05) methods. Given the operating conditions, there are no significant differences between the plucking and shedding strategies for denudation.

[0015] FIG. 3C shows representative COC clusters (the scale bar is 400 microns).

[0016] FIG. 3D shows completely denuded oocytes (the scale bar is 100 microns) using a Poly-4 AHT device (shedding strategy).

[0017] FIG. 4A and FIG. 4B show overall ICSI micromanipulation survival and embryo development data (mean and SD). The control is the manual protocol (MP). *P value > 0.1 indicates a comparable blastocyst rate between the control and all the acoustic devices operating at high or low power levels, respectively.

[0018] FIG. 4C shows overall IVF survival and embryo development data (mean and SD). Control is the manual protocol (MP). *P value > 0.1 indicates a comparable cleave and blastocyst rate between the control and all the acoustic devices operating at a power level of 20 dB, 15dB, and lOdB, respectively.

[0019] FIG. 4D shows time-lapse data for full pre-implantation embryo developmentfrom fertilized oocytes denuded by manual pipeting (control), P-4, and T-2 AHT. PB2=time to 2nd polar body extrusion; tPNa=time to pronuclear appearance; tPNf=time to pronuclear fading; t2-8=time to 2-cell... 8-cell; tM=time to morula compaction; and tB=time to blastocyst.

[0020] FIG. 5A(1) and FIG. 5B(1) show COC positioning strategies using intact cumulus cells as anchor points. FIG. 5C( 1) shows consecutive images of COC positioning, where the dashed white patch is the intended trajectory which turns to red as the cell moves along that trajectory. FIG. 5A(2) and FIG. 5B(2) show that PVx is used for out-of-the-plane rotation of embryonic cells. FIG. 5C(2) shows consecutive images of out-of-plane bovine zygote rotation. FIG. 5A(3) and FIG. 5B(3) show COC, oocyte, or embryo positioning using TVx swirling flows. FIG. 5C(3) shows consecutive images of COC translation along the intended trajectory (white dashed line). FIG. 5A(4) and FIG. 5B(4) show TVx of appropriate size for in-plane rotation of the mouse oocytes cells. FIG. 5C(4) shows consecutive images of in-plane oocyte rotation.

[0021] FIG. 6A shows a spherical Hankel beam at the top and Cylindrical Bessel beam at the botom used for driving the geometrical polar equations (Eq. 7-8).

[0022] FIG. 6B and FIG. 6C show phase wrapping by varying the height of the focal point as a function of frequency when FIG. 6B(i), s = 1 and FIG. 6B(ii), s =3. The isophases of cylindrical waves are shown in FIG. 6C(i) where (1, s) = (3,1) and FIG. 6C(ii), when (l, s) = (1,3) in Eq. 11-12.

[0023] FIGS. 7 A, 7B, 7C, and 7D show an example of electrode design, intensity profile, and phase of a generated acoustic field in a focal plane. For example, the patern of IDTs obtained using Eq, 7-8 for ring-like acoustic fields is shown in FIG. A(i), (l,s) = (1,1), in FIG.7B(i), (l,s) = (1.4), in FIG. 7C(i), (l,s) = (4.1), and in FIG. 7D(i). (l,s) = (2.3). In FIG. 7 A(ii) to FIG. 7D(ii), corresponding numerical predictions of normalized intensity are shown. In FIG 7 A(iii) to FIG. 7D(iii), phase of the acoustic wave at the designed focal length (z=l.l mm) following the IDT paterns in FIG. A(i) to FIG. 7D(i) is shown.

[0024] FIGS. 8 A, 8B, and 8C show a comparison of radius of intensity profiles. In FIG.8A. R In, in FIG. 8B P — n, when n = 1, 2,..., 8. and in FIG. 8C, the crescent shape C — In. for the acoustic filed when n = 2, 4, 6, 8.

[0025] FIG. 9 shows displacement of an axial position of a trapping point by frequency tuning. For frequencies above the designed frequency, the axial position of the trap is higher than the designed axial position and vice versa. The inset shows the linear dependency ofaxial displacement and frequency when the deviations are small.

[0026] FIGS. 10A, 10B, IOC, and 10D show an example of electrode design, intensity profile, and phase of a generated acoustic field in a focal plane. For example, the pattern of IDTs obtained using Eq, 11-12 for polygonal-shape acoustic fields is shown in FIG. 10A(i), s = 1, in FIG. 10B(i), s = 3, in FIG. 10C(i), s= 4, and in FIG. 10D(i), s = 8. FIGS. 10A(ii) to 10D(ii) show corresponding numerical predictions of normalized intensity. FIGS. lOA(iii) to lOD(iii) show phase of the acoustic wave at the designed focal length (z=l.1 mm) following the IDT patterns.

[0027] FIG. 11 shows intensity (I>0.9)-phase plots for a quadrilateral shape acoustic field obtained from Eq. 2-7 with n =4. Incorporating a negative helicity parameter ( / = -1 or -2 as in the upper plots) decreases the side’s length, while a positive helicity parameter ( / = or 2 as in the lower plots) increases the side’s length and near-zero pressure region.

[0028] FIGS. 12A, 12B, 12C, and 12D show an example of electrode design, intensity profile, and phase of a generated acoustic field in a focal plane. For example, the IDT pattern in FIG. 12A(i) is a T-2 (twin-tweezer), q = 2. The IDT patten in FIG. 12B(i) is a T-8, q — 8 acoustic field when the angular polarity is controlled by Eq. 13. The IDT pattern in FIG. 12C(i) is a C- 18 crescent-shaped acoustic field where the polarity and topological charge directivity' are controlled by Eq. 20. The IDT pattern in FIG. 12D(i) is a K-22 crescent-shaped field in which the phase wrapping direction in Eq. 11-13 is controlled by Eq. 13. FIGS. 12A(ii) to 12D(ii) are corresponding numerical predictions of normalized intensity. FIGS. 12 A(iii) to 12D(iii) are phase of the acoustic wave at the designed focal length (z=l.l mm) following the IDT patterns.

[0029] FIGS. 13A, 13B, 13C, and 13D show an example of electrode design, intensity' profile, and phase of a generated acoustic field in a focal plane. For example, FIG.13A(i) to 13D(i) show patterns of IDTs where a radial phase jump is introduced in the polarity of the connections where FIG. 13A(i) is a focused spherical beam, FIG. 13B(i) is R-ll, FIG. 13C(i) is T-2, and FIG. 13D(i) is R-22 following Eq. 21. FIGS. 13 A(ii) to 13D(ii) show the numeral predictions of the normalized intensity' in the xz plane with the curved (bottle shape) propagation path for each acoustic field in the beam axis direction. FIGS. 13 A(iii) to 13D(iii) show the perspective view of the iso-intensity’ level 0.8 of the normalized intensity following the designs in FIGS. 13A(i) to 13D(i).

[0030] FIGS. 14A, 14B, 14C, and 14D show force intensity and distribution. FIGS. 14A(i) to 14D(i), and FIGS 14A(ii) to 14D(ii) show the normalized force intensity anddistribution for R-14, P-4, T-6, and C-18 AHT when cumulus cells with an average diameter and oocytes with an average of 100 / 'mare considered as spherical scatterers. respectively.

[0031] FIGS. 15Aand 15B show normalized y-component (FIG. 15A) and z-component (FIG. 15B) of acoustic radiation force in the xz-plane acting on a 10-micron biological cell scatterer. The dotted curves show the x and y (FIG. 15 A) and z (FIG. 15B) components of the radiation force on the x = 0 line.

[0032] FIGS. 16A, 16B, 16C, 16D, 16E, 16F, 16G, and 16H show acoustic intensity, streamlines, and magnitude in a 200-mi cron-deep microchamber covered with oil. FIG. 16A shows R-01, FIG. 16B shows R-ll, FIG. 16C shows T-l, FIG. 16D shows Fresnel, FIG. 16E shows R-18, FIG. 16F shows P-18, FIG. 16G shows T-8, and FIG. 16A shows C-81.

[0033] FIG. 17 shows maximum temperature variation for the P-series AHTs used in denudation experiments at two power levels. The bigger temperature variation on the left is for continuous input power, while the smaller temperature variation is for pulsed input power at IKHz and 50% duty cycle.

[0034] FIGS. 18A, 18B, and 18C show working principles for open microfluidic channels. FIG. 18A shows a low-depth cylindrical microchamber where cells are directly loaded into the media plus HA solution and denuded with AHTs. FIG. 18B shows cell loading and HA exposure that occur in the bigger chamber on the right (FIGS. 18B(i) and 18B(ii)) and that denuded oocytes are positioned inside the microchamber embedded on the left side in the microchannels (FIGS. 18B(iii) and 18B(iv)). FIG. 18C shows HA exposure and denudation that occur after placing the COCs in the microchambers in the middle of the channel.

[0035] FIG. 19A shows cup-sleeve compartments used to create a stretched thin-film support at the bottom of a dish.

[0036] FIG. 19B shows that UV-curable polyacrylamide copolymers can be patterned at the top of the thin-film support to create open microfluidic structures.

[0037] FIG. 19C shows that patterned medical-grade adhesive tapes of an appropriate size can be stuck to the top of the thin-film support to create microfluidic structures.

[0038] FIG. 19D shows that hot embossing a polystyrene film (0.2 mm) using a thermally aged PDMS can create the open microfluidic structures with a thin support film at the bottom in one step.

[0039] FIG. 19E shows a fully closed hydrogel-based microfluidic device made bycuring two layers on top of each other.

[0040] FIG. 20 A shows microbead attachment to a cumulus cell as an anchor point for an acoustic tweezer.

[0041] FIG. 20B shows that acoustic fields such as R-01 and T2 can be used to grab microbeads in closed microfluidics.

[0042] FIG. 20C shows a schematic representation of translation of a COC with attached microbeads and an R-01 acoustic vortex.

[0043] FIG. 20D shows a denuded oocyte and other stages of an embryo that can be manipulated by AHT.

[0044] FIG. 20E shows other AHT possibilities such as (top) large ring-like, polygonal-like, and (bottom) petal and crescent-shaped fields that can be used for ova translation.

[0045] FIG. 20F shows a schematic of a (top) large acoustic trap used for moving a 2-cell stage embryo and (bottom) a crescent-shape acousto-fluidic field used to push the blastocyst inside a microchannel.DETAILED DESCRIPTION

[0046] The claimed subject matter is described in terms of certain examples. But, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure. It is understood that other examples of the present disclosure may be made without departing from the scope of the present disclosure.

[0047] Unless stated otherwise, technical and scientific terms used in this specification carry the same meaning as generally understood by those having ordinary skill in the art. Methods and materials that are equivalent or comparable to those described herein may also be employed in practicing or testing the present disclosure.

[0048] Any patents or publications cited in this specification are referenced to describe the methods and / or materials related to the subject matter disclosed. Each cited reference is incorporated by reference as though set forth herein, but only to the extent that it pertains to the methods and / or materials described. Such incorporation by reference does not limit this disclosure. Any definition, description, or term appearing in the cited materials that is not explicitly repeated in this application should not be considered as limiting or defining the terms of the present disclosure or its claims. Additionally, the stated publication dates maydiffer from actual publication dates, which may require independent verification.General Definitions

[0049] As used herein, the singular forms “a”, “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0050] As used herein, the terms “about,” “approximately,” “substantially,” or the like, when used in connection with a measurable variable such as, for example, a parameter, an amount, a temporal duration, or the like, are meant to encompass variations of, for example, a specified value including, for example, those within experimental error (which can be determined by for example, a given data set, an art accepted standard, and / or with a given confidence interval (e.g., 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / -10% or less, + / -5% or less, + / -!% or less, and + / -0.1% or less of and from the specified value), insofar such variations are appropriate to perform in the context of the disclosure. As used herein, unless otherwise stated, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the sample claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, and the like, and other factors known to those of skill in the art such that, for example, equivalent results, effects, or the like are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter, or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not it is expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0051] As used herein, the terms “acoustic” or “acousto” refer to generation, propagation, and manipulation of mechanical pressure waves within a medium to exert controlled forces on materials, such as biological materials. In the context of assisted reproductive technologies (ART) and microfluidic systems, acoustic methods may employ surface acoustic waves (SAW s), bulk acoustic waves (B AWs), or focused acoustic beams to manipulate, trap, or translate cells, cumulus-oocyte complexes (COCs), or embryos in acontactless manner.

[0052] As used herein, the term “cell” refers to the basic structural and functional unit of living organisms, typically enclosed by a membrane and containing genetic material, organelles, and cytoplasm. Cells may exist as independent units of life or as part of multicellular organisms (e.g., in COCs). Cells may be live, fixed, lysed, or fragmented. Cells may include derivatives such as cell membranes, cytoplasmic fractions, nuclei, organelles, and other subcellular components. Cells may include, but are not limited to, reproductive cells.

[0053] As used herein, the term “contactless” refers to not requiring physical contact between two objects. For example, “contactless” may refer to achieving a result (agitating, mixing, stirring, moving, separating) without physical contact between a first component (e.g., cell, particle, molecule, or other material) and a second component (e.g., a wall, fin, paddle, or other solid or semi solid component) of a device or system.

[0054] As used herein, the term “holographic” refers to systems, devices, or methods that employ spatial phase modulation of acoustic or optical waves to form three-dimensional field distributions capable of contactless manipulation of particles or biological materials. Holographic configurations may enable dynamic control of acoustic fields within a planar, optically transparent device compatible with microscopy.

[0055] As used herein, the term “hydrodynamic” refers to controlled motion and manipulation of fluids and suspended materials through pressure-driven or flow-induced forces within a medium. For example, hydrodynamics may be utilized to guide, position, or stabilize biological entities such as gametes, oocytes, or embryos through fluid shear, laminar flow patterns, or localized vortices, either independently or in combination with acoustic fields.

[0056] As used herein, the term “interdigitated transducer” (IDT) refers to a device that is composed of arrays of electrodes. In an IDT, electrode fingers may be connected to opposing electrical potentials, such that when an alternating voltage is applied, the IDT generates localized acoustic waves such as surface acoustic waves (SAWs) or bulk acoustic waves (BAWs) within a substrate or an adjacent fluid medium. An IDT may be planar, curved, or have other suitable shapes or configurations sufficient to be shaped to achieve the described results. An IDT may be fabricated on a piezoelectric substrate such as lithium niobate, lithium tantalate, quartz, aluminum nitride, or gallium nitride. The electrodes may be composed of metals such as aluminum, gold, platinum, or copper. The configuration,pitch, and geometry of the IDT may determine the frequency, amplitude, and direction of generated acoustic waves.

[0057] As used herein, the term “glass” refers to any type of glass including, but not limited to silicate glasses (e.g., soda-lime glass, borosilicate glass, lead glass, aluminosilicate glass, glass- ceramics, and fiber glass), silica-free glasses (e.g., amorphous metals and polymers), and molecular liquids and molten salts. Glass may contain additives that can modify optical and thermal properties (e.g., transparency, insulation, color, refractivity etc.).

[0058] As used herein, the term “polymer” refers to a chemical compound composed of a plurality of repeating structural units, referred to as monomers, covalently bonded to form a macromolecular structure. The term encompasses homopolymers, consisting of identical monomeric units, and copolymers, comprising two or more chemically distinct monomeric units. In the context of microfluidic systems, polymers may be used as structural materials, substrates, coatings, or functional layers for fluidic channels, chambers, and integrated components. Polymers may include, without limitation, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin polymer (COP), polycarbonate (PC), polyethylene (PE), polypropylene (PP), polyimide (PI), and polyurethane (PU), as well as biocompatible or functionalized polymers designed to interact or support biological samples or reagents. Polymers may be formed by any polymerization method, including addition, condensation, or ring-opening reactions, to yield materials with predetermined mechanical, optical, or chemical properties for microfluidic device fabrication and operation.

[0059] As used herein, the term “range” may be used to set out a lower limit value and an upper limit value. Unless otherwise stated, a range includes the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also, unless otherwise stated, include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 0.5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0060] As used herein, the terms “trap,” “trapping,” or “trapped” refer to keeping objects, cells, particles or other components in a fixed position. For example, “trapping” may encompass any mechanism that allows precise spatial control or retention of biological materials in a vessel, dish, sample holder, microfluidic, or otherwise contactless system, facilitating manipulation while maintaining viability, integrity, and sterility of samples such as gametes and embryos. The trapping may be static, where a component remains in a substantially fixed position, or dynamic, wherein the position of the component can be selectively adjusted, translated, or released in response to controlled stimuli.

[0061] As used herein, the term “tunable” refers to being capable of being adjusted in one or more attributes to achieve a particular purpose. The term “tunable” may refer to the ability to control or vary physical, chemical, electrical, or mechanical parameters of a device or component. For example, tunable attributes may include channel geometry, flow rate, pressure, temperature, acoustic frequency or amplitude, electric or acoustic field strength, or polymer stiffness.OVERVIEW

[0062] Assisted reproductive technology (ART) has been widely adopted, and one of its procedures, intracytoplasmic sperm injection (ICSI), now represents at least 60% of all in vitro fertilization (IVF) procedures. To perform ICSI, removal of cumulus corona cells from the oocyte is required. This procedure needs to be performed in a proficient manner and in controlled settings to avoid any possible insult to the female gamete. This practice, because of the multifaceted components, is delicate and, at times, somewhat challenging. Indeed, it requires a proper amount of digestive enzyme, an adequate diameter of the pipette, and a skilled embryologist. The availability of a controlled and automated system, such asacoustic (or, acousto-) hydrodynamic trapping (or tweezing) (AHT) modules, would allow contactless removal of the cumulus cells from the COCs prior to ICSI in an expedited, safe, and reliable manner.

[0063] Global access and demand for assisted reproductive technologies (ART) have increased, reaching over 4 million cycles annually (1: Factsheets and infographics [cited 2024 Jul 28]: Available from: https: / / www.eshre.eu / Europe / Factsheets-and-infographics). IVF entails the insemination in a petri dish of cumulus oocyte complex (COC) with several thousands of spermatozoa and was initially intended to overcome tubal infertility (2:Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet [Internet] 1978 [cited 2024 Jul 28]; 2(8085): 366: Available from http: / / www.thelancet.com / article / S0140673678929574 / abstract). Nowadays, IVF addresses a variety of indications such as endocrine, immunological, and idiopathic infertility. For standard in vitro insemination, the retrieved oocyte requires a simple and partial trimming of the cumulus cell cluster carried out by small needles to enhance sperm-oocyte binding and facilitate fertilization.

[0064] In 1992, intracytoplasmic sperm injection (ICSI) was introduced to address male factor infertility, in all its aspects and variances (3: Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet [Internet] 1992; 340(8810): 17-8. Available from: http: / / dx.doi.org / 10.1016 / 0140-6736(92)92425-1). ICSI now has become very popular, ranging its application from 40 to 60% of all ART worldwide and it is not limited to male gamete dysfunction, but concurrently used with other ART aspects such as cryopreservation of oocytes, preimplantation genetic testing for single gene defect or advanced maternal age, poor responders women, and restrictive legislation (4: O’Neill CL, Chow S, Rosenwaks Z, Palermo GD. Development of ICSI. J Reprod Fertil [Internet] 2018 [cited 2024 Jul 28]; 156(1): F51-8. Available from: https: / / rep.bioscientifica.com / downloadpdf / view / journals / rep / 156 / 1 / REP-18-0011.pdf). ICSI requires direct manipulation of the gamete that after oocyte retrieval and rinsing off the follicular fluid, starts with pre-treatment of COCs. This entails the mechanical and concurrent chemical stripping of the surrounding cumulus and corona cells (5: Rienzi LF, Maggiulli R, Ubaldi FM. Oocyte Denuding [Internet]. In: Nagy ZP, Varghese AC, Agarwal A, editors. In Vitro Fertilization: A Textbook of Current and Emerging Methods and Devices. Cham: Springer International Publishing: 2019. p. 133-45: Available from:https: / / doi.org / 10.1007 / 978-3-319-43011-9_14) to allow proper visualization for a controlled injection.

[0065] The COC consists of a large oocyte of about 120 microns with a few layers of firmly attached cumulus cells embedded in the extracellular matrix (ECM) that spans 400-700 microns. This delicate procedure is heavily dependent on techniques and skills of the embryologist as well as proper hyaluronidase concentration and adequately sized microcapillary. Currently, cumulus cell removal is done by manually pipetting in and out COCs with properly sized capillaries in hyaluronidase-containing medium. Prolonged exposure to hyaluronidase may compromise the quality of the oocyte, as well as excessive suction pressure through improperly selected capillary size may exert mechanical stress on the COCs, resulting in damage to the zona pellucida in various degrees, ranging from cracking of the zona to yielding a completely zona-free oocyte. This multifaceted step of the ICSI procedure is somewhat challenging and often overlooked. The availability of a controlled and automated system that avoids any direct contact of a mechanical device while utilizing a very minute amount of enzyme would be ideal.

[0066] Attempts to automate ART are flawed by the attempt to mimic the hands of the embryologists with a robotic arm, which is cumbersome, large, and impractical.Microfluidic microsystems for denudation have been proposed. However, these technologies introduce additional preparation steps, increase the risk of oocyte loss and recovery and demands new skillsets. Among non-contact single cell manipulation techniques such as optical, optoelectronic, electrokinetic, magnetic, and acoustic (6-10: 6. Stewart MP, Langer R, Jensen KF %J CR. Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. Chem Rev [Internet] 2018; 118(16): 7409-531. Available from: http: / / dx.doi.org / 10.1021 / acs.chemrev.7b00678. 7. Kang P, Tian Z, Yang S, Yu W, Zhu H, Bachman H, et al. Acoustic tweezers based on circular, slanted-finger interdigital transducers for dynamic manipulation of micro-objects. Lab Chip [Internet] 2020; 20(5): 987-94. Available from: http: / / dx.doi.org / 10.1039 / c91c01124b. 8. Zhang Z, Wang X, Liu J, Dai C, Sun Y. Robotic Micromanipulation: Fundamentals and Applications. Annu Rev Control Robot Auton Syst [Internet] 2019;2(1): 181- 203. Available from: https: / / doi.org / 10.1146 / annurev-control-053018-023755. 9. Permana S, Grant E, Walker GM, Yoder JA. A Review of Automated Microinjection Systems for Single Cells in the Embryogenesis Stage. IEEE / ASME Trans Mechatron [Internet] 2016; 21(5): 2391-404. Available from: http: / / dx.doi.org / 10.1109 / TMECH.2016.2574871. 10. Wei Y, Xu Q. ASurvey of Force -Assisted Robotic Cell Microinjection Technologies. IEEE Trans Autom Sci Eng [Internet] 2019; 16(2): 931-45. Available from:https: / / ieeexplore. ieee.org / ielx7 / 8856 / 8681660 / 08540891. pdf?tp=&arnumber=8540891&isn umber=8681660&ref=). Acoustic trapping stands out as it is capable of non-invasive manipulation with minimal thermal insult compared with optical tweezers (11: Ashkin A. Acceleration and trapping of particles by radiation pressure. Phys Rev Lett [Internet] 1970 [cited 2024 Jul 28]; 24(4): 156-9. Available from:http: / / link.aps.org / pdf / 10.1103 / PhysRevLett.24.156).

[0067] However, mainstream acoustical trap synthesis systems require rather complex transducer arrays and electronics (12-14: 12. Marzo A, Caleap M, Drinkwater BW. Acoustic Virtual Vortices with Tunable Orbital Angular Momentum for Trapping of Mie Particles. Phys Rev Lett [Internet] 2018; 120(4): 044301. Available from: http: / / dx.doi.org / 10.1103 / PhysRevLett.120.044301; 13. Baresch D, Thomas J-L, Marchiano R. Observation of a Single-Beam Gradient Force Acoustical Trap for Elastic Particles: Acoustical Tweezers. Phys Rev Lett [Internet] 2016; 116(2): 024301. Available from: http: / / dx.doi.org / 10.1103 / PhysRevLett.116.024301. 14. Jiang X, Li Y, Liang B, Cheng J-C, Zhang L. Convert Acoustic Resonances to Orbital Angular Momentum. Phys Rev Lett [Internet] 2016; 117(3): 034301. Available from: http: / / dx.doi.org / 10.1103 / PhysRevLett.117.034301).

[0068] Holographic methods using a single interdigitated transducer (IDT) have also been introduced, significantly reducing wave modulation complexity. They are flat, transparent, and compatible with microscopes and disposable dishes (15-16: 15. Riaud A, Baudoin M, Bou Matar O, Becerra L, Thomas J-L. Selective Manipulation of Microscopic Particles with Precursor Swirling Rayleigh Waves. Phys Rev Applied [Internet] 2017; 7(2): 024007. Available from: https: / / link.aps. Org / doi / 10.1103 / PhysRevApplied.7.024007. 16. Baudoin M, Thomas J-L, Sahely RA, Gerbedoen J-C, Gong Z, Sivery A, et al. Spatially selective manipulation of cells with single-beam acoustical tweezers. Nat Commun [Internet] 2020; 11(1): 4244. Available from: http: / / dx.doi.org / 10.1038 / s41467-020-18000-y), but must operate at lower frequencies with increased helicity (topological order) to manipulate larger cells such as oocytes, resulting in devices that are both hard to fabricate and incompatible with optical setups (17: Baudoin M, Gerbedoen J-C, Riaud A, Matar OB, Smagin N, Thomas J-L. Folding a focalized acoustical vortex on a flat holographic transducer: Miniaturized selective acoustical tweezers. Sci Adv [Internet] 2019; 5(4): eaav1967. Available from: http: / / dx.doi.org / 10.1126 / sciadv.aavl967).

[0069] Previously, a device was introduced that uses acoustic forces to remove the cumulus cells, however, it does not offer the use of a disposable dish and was designed for denudation of COCs in bulk (18: Mokhtare A, Davaji B, Xie P, Yaghoobi M, Rosenwaks Z, Lal A, et al. Non-contact ultrasound oocyte denudation. Lab Chip [Internet] 2022; 22(4): 777-92. Available from: http: / / dx.doi.org / 10.1039 / dllc00715g).ACOUSTIC-HYDRODYNAMIC TRAPPING (AHT) MODULES

[0070] The present disclosure provides examples of systems and methods for cell manipulation using acoustic waves. The systems may include assisted reproductive technology (ART) devices for manipulation of reproductive cells. The systems and methods may be configured for use in in vitro fertilization (IVF) procedures, such as, for example, intracytoplasmic sperm injection (ICSI). The systems and methods may be acoustichydrodynamic trapping (AHT) modules capable of manipulating single cells, performing removal of cumulus corona cells, translating the cleaned oocyte into specific microchambers, and rotating the oocytes or the conceptuses in the equatorial or axial plane in a non-contact fashion, all carried out in disposable dishes or other removable or reusable vessels. To support evaluation of the techniques described herein, denuded oocytes were ICSI inseminated and allowed to progress through full pre-implantation development under time-lapse microscopy. The resulting blastocysts were transferred to recipient mice, resulting in live births. All the pups weaned normally and are fertile. This work opens the possibility to utilize this device with all forms of female gamete manipulation in mammals and eventually in humans.

[0071] In various examples, a system comprises an ultrasonic transducer comprising electrodes. The system further comprises a chamber configured to contain a cell. The system further comprises a processor in electronic communication with the ultrasonic transducer. The processor is configured to control the ultrasonic transducer to generate acoustic waves directed to the cell to manipulate the cell within the chamber. The acoustic waves may propagate through the chamber. The acoustic waves may generate an acoustic field with a pattern defined by a shape of the electrodes of the ultrasonic transducer.

[0072] In various examples, cell manipulation may include one or more of cell translation (e.g., pick and place) in any arbitrary direction in the chamber, cell rotation in one or more in-plane (toroidal) and / or out-of-plane (poloidal) directions, and / or cellstimulation by pressure waves (e.g., applying force on the cell to deform or excite the cell based on sensitivity to a mechanical stimuli), or the like.

[0073] In various examples, the system may be an assisted reproductive technology (ART) device. The system may be configured for use in in vitro fertilization (IVF) procedures, such as, for example, intracytoplasmic sperm injection (ICSI).

[0074] In various examples, the electrodes may be intertwined. The electrodes may be micromachined. The electrodes may be piezoelectric. The electrodes may be capacitive. The ultrasonic transducer may comprise a capacitive micromachined ultrasonic transducer (CMUT), a piezoelectric micromachined ultrasonic transducer (PMUT), an interdigitated transducer (IDT), or the like. The ultrasonic transducer may comprise a single transducer or an array of transducers. The ultrasonic transducer may not comprise an array of transducers.

[0075] In various examples, the acoustic waves may be modulated, focused waves that generates an acoustic field, such as, for example, an acoustic vortex. In various examples, the pattern of the acoustic field may be predefined by the shape of the electrodes disposed on the substrate. The processor may be configured to control an intensity of the shape or pattern and / or control its vertical position according to an input signal transmitted to the substrate. The input signal may have a frequency of about 40 MHz to about 45 MHz, including all 0.1 MHz values and ranges therebetween (e.g., about 44 MHz), to manipulate cells from a few microns (e.g., about 5 microns) to a few hundred microns (e.g., about 300 microns), including all 0.1 micron values and ranges therebetween. Other frequencies (e.g., about 10 MHz to about 200 MHz, including all 0.1 MHz values and ranges therebetween) may be used to manipulate cells of other sizes.

[0076] In various examples, the cell may comprise one or more cells or a cell culture. The cell culture may comprise human or non-human cells contained in an extracellular matrix (ECM). The ECM may comprise hyaluronic acid, proteoglycans, various glycoproteins, and / or other components. The cells may comprise gametes or somatic cells. The cell culture may comprise cumulus-oocytes-complex (COCs) including an oocyte having a layer of cumulus cells attached thereto. The COCs may be about 400 microns to about 700 microns in size, including all 0.1 micron values and ranges therebetween. The oocyte may be about 100 microns in size to 200 microns in size, such as 120 microns in size.

[0077] In various examples, the chamber may be disposed on the ultrasonic transducer. The chamber may be movable with respect to the ultrasonic transducer, such as translatableor rotatable with respect to the ultrasonic transducer. The chamber may be or define a void within another vessel, such as a void within petri dish.

[0078] In various examples, the system may comprise a substrate disposed between the ultrasonic transducer and the chamber. The substrate may support motion between the chamber and the ultrasonic transducer. The substrate may comprise a piezoelectric substrate. The substrate may be formed from glass. The substrate may be disposed on the electrodes of the ultrasonic transducer. The acoustic waves generated by the ultrasonic transducer may be configured to propagate an acoustic field that extends through the substrate to manipulate the cell within the chamber.

[0079] In various examples, the pattern of the acoustic field has a ring shape. The ring shape may be generated by the intersection of a spherical beam and a cylindrical beam with a helicity of / or a superposition of.y cylindrical beams at the same frequency and helicity to increase or decrease the size of the acoustic trap. The ring shape may be present near a surface of the substrate that supports the chamber.

[0080] In various examples, the pattern of the acoustic field has a polygonal shape. The polygonal shape may be generated by combining single or multiple complementary phase wrappings (s) by including an azimuthal angular-dependent wave number. Phase wrapping by controlling the helicity of the cylindrical waves and the wave number can be used to control the size of the polygonal shape and number of the sides. The polygonal shape may include three or more (such as, for example, three, four, five, six, seven, eight, or more) substantially straight sections connected through annular junctions. The polygonal shape may be present near a surface of the substrate that supports the chamber.

[0081] In various examples, the intensity of the acoustic field may be higher at the vertices of the polygonal-shape. Increasing a number of vertices and a size of the polygonal shape may increase the intensity of the overall acoustic field. Alternatively, increasing the length of the sides while keeping the number of vertices fixed may increase the size of the polygonal shape to target larger cells.

[0082] In various examples, the pattern of the acoustic field has a petal shape (or pattern). The petal shape or pattern may be generated by including angular 7i-phase jumps ( / 2 steps) at discrete angles in the electrodes of a focused beam to create evenly split high intensity regions. The petal shape or pattern may include two or more (such as, for example, two, three, four, five, six, seven, eight, or more) petal sections. Use of a different number of petals may control the size of the petal pattern to target different size cells (e.g., smallcumulus cells or the oocyte cells of the COCs). The petal shape or pattern size may be smaller than an oocyte. The petal shape may be present near a surface of the substrate that supports the chamber.

[0083] In various examples, the chamber may be or include an open microfluidic system. The petal pattern may create a circulating fluidic stream in the vertical direction, which may trap and roll the entire COC in its periphery, and the rolling and fluid shear may disperse the cumulus cells from the oocyte. By increasing the number of petals of the petal shape or pattern, the oocyte may be locked inside a few circulating vortices, which may disperse the cumulus cells from the oocyte.

[0084] In various examples, the chamber may be a closed microfluidic system, and the petal pattern may be configured to only trap the cells without rotation, which may be used to grab the cumulus cells for oocyte denudation.

[0085] In various examples, the pattern of the acoustic field has a crescent shape.Including a single angular phase jump in the intertwined electrodes and switching in the directivity of the helicity at the same discrete angle can generate a crescent-shaped petal pattern with tunable arc length. The acoustic field may push or pull the cell with an edge of the crescent shape. The crescent shape may be present near a surface of the substrate that supports the chamber.

[0086] In various examples, the pattern of the acoustic field has a bottle shape. The bottle shape can be generated by including 7i-phase jumps ( / 2 steps) in the radial direction at a discrete radius that splits the area of the intertwined electrodes into two equals areas, which results in destructive interference in the beam propagation direction at the focal point. The bottle shape may be present at varied z-heights (up-down) within the chamber.

[0087] In various examples, the acoustic fields having a ring shape, a polygonal shape, a petal shape or pattern, or a bottle shape may produce an enclosed region defined by the acoustic fields. Acoustic forces of the acoustic fields may be directed toward the center inside the enclosed region (i.e., attract) and switch direction outside the enclosed region (i.e., repel). The size of the enclosed region compared to the cell (e.g., the COC) may define the manipulation of the cell (e.g., the mechanism of denudation and how cumulus cells may be detached from the oocyte).

[0088] In various examples, the size of the enclosed region may be similar to the cumulus cell (e.g., about 10 microns to about 15 microns, including all 0.1 microns and ranges therebetween), and the acoustic field may attract a few cumulus cells to the center ofthe enclosed region while repelling the oocyte to denude the oocyte.

[0089] In various examples, the size of the enclosed region may be similar to the oocyte (e.g., about 200 to 300 microns). The oocyte and the cumulus cells may be trapped simultaneously within the enclosed region, such that shear and agitation from rotation of the acoustic field may disperse the cumulus cells to denude the oocyte.

[0090] In various examples, the size of the enclosed region may be small, the acoustic field may lock the oocyte on its periphery, and shear forces may disperse the cumulus cells.

[0091] In various examples, the size of the enclosed region may be large, and the COC may be trapped inside the enclosed region such that rotating shear forces disperse the cumulus cells.

[0092] In various examples, the chamber may be open, such as in an open microfluidic system. Fluidic shear forces may be used to denude the oocyte in the open chamber. The chamber may be accessible to instruments, such as a pipette. The chamber may include a cavity that allows access to samples inside the chamber.

[0093] In various examples, the chamber may contain two immiscible fluids. The two immiscible fluids may be used when the chamber is open. The two immiscible fluids may comprise a water solution and an oil. The cell may be disposed in the water solution. The oil may be, for example, paraffin oil, fluorinated oils, or the like. The acoustic field may press the cell against the interface between the two immiscible fluids to manipulate the cell.

[0094] In various examples, the chamber may be closed, such as in a closed microfluidic system. Acoustic radiation forces may be used to denude the oocyte in the closed chamber. The chamber may be inaccessible to outside instruments. The chamber may be non-permanently closed, such that the chamber can be opened to introduce or retrieve the cell from the chamber.

[0095] In various examples, the chamber may contain sound-absorbing material. In the case of a closed chamber, the chamber may be made of or filled with sound-absorbing material. The sound-absorbing material may be one or more polymers. The sound-absorbing materials may be formed from polyester or polyacrylamide. The chamber may contain materials that do not absorb sound.

[0096] In various examples, the system may comprise a stage configured to move the chamber relative to the ultrasonic transducer or move the ultrasonic transducer relative to the chamber. The processor may be in electronic communication with the stage. The processor may be configured to control the stage to move the chamber relative to theultrasonic transducer or move the ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to manipulate the cell within the chamber. The chamber may be held in a fixed position while the stage moves the ultrasonic transducer relative to the chamber.

[0097] In various examples, the ultrasonic transducer may include a piezo element. The piezo element may be embedded in a glass plate to form the ultrasonic transducer. The piezo element may be configured to touch the bottom of the chamber, such as to contact a bottom of a dish. The piezo element may be coupled to the bottom of the dish through various media. The piezo element may be coupled to the bottom of the dish via a layer of DI water, ultrasound gel, or silicone oil.

[0098] In various examples, the ultrasonic transducer may be a piezo probe configured to enter the chamber to manipulate the cell within the chamber. The piezo probe may be movable to different locations within the chamber to adjust a position within the chamber at which the acoustic waves are targeted. The piezo probe may be manually controlled. The piezo probe may be used with an open chamber.

[0099] In various examples, moving the chamber relative to the ultrasonic transducer or moving the ultrasonic transducer relative to the chamber may mix the liquid in the chamber and denude COCs. Moving the chamber relative to the ultrasonic transducer may move denuded oocytes away from dispersed cumulus cells.

[0100] In various examples, the chamber includes one or more microchambers in fluid communication with the chamber. The processor may be configured to control the stage to move the chamber relative to the ultrasonic transducer or move ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to move the cell (e.g., a denuded oocyte) from the chamber to one of the one or more microchambers.

[0101] In various examples, the chamber includes a microfluidic channel in fluid communication with the chamber and the one or more microchambers. The processor may be further configured to control the stage to move the chamber relative to the ultrasonic transducer or move the ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to move the cell (e.g., a denuded oocyte) from the chamber to one of the microchambers via the microfluidic channel.

[0102] In various examples, the chamber may include one or more microchambers and / or one or more microchannels in fluid communication with each other. The microchamber(s) may be defined within the microfluidic channel(s). The microchamber(s)may be radially connected to the microfluidic channel(s).

[0103] In various examples, the size and arrangement of the one or more microchambers and the one or more microchannels may be dependent on the use of the system and the and the cells. The system may be used for single-cell manipulation (e.g., IVF / ICSI procedures), and the chamber may include a dedicated number of microchambers configured to contain and separate each cell (e.g., denuded oocyte). The microchamber(s) may have a large diameter (such as, for example, about 50 times the cell diameter) and a small ratio of height to width (such as, for example, less than about 2 times the cell diameter). The microfluidic channel(s) may have a height and width similar to the size of one cell (e.g., a height less than about 2 times the cell diameter and a width is greater than the cell diameter).

[0104] In various examples, a method comprises disposing electrodes on a substrate to form an ultrasonic transducer. The method further comprises disposing a chamber on the ultrasonic transducer. The method further comprises generating, with the ultrasonic transducer, an acoustic field directed to the chamber to manipulate the cell within the chamber. The acoustic field may propagate through the chamber. The acoustic field has a pattern defined by a shape of the electrodes disposed on the substrate.

[0105] In various examples, the method may include partially filling the chamber with a liquid solution. The liquid solution may comprise a hyaluronic acid (HA) solution. The concentration of the HA solution may be about 1 lU / mL to about 40 lU / mL, including all 0.1 lU / mL values and ranges therebetween. The concentration of the HA solution may be about 15 lU / mLto about 20 lU / mL, including all 0.1 lU / mL values and ranges therebetween.

[0106] In various examples, the chamber may be filled with the liquid solution to about 75% of its capacity (e.g., about 50% to about 80%, including all 0.1% values and ranges therebetween). Filling the chamber with the liquid solution to less than capacity (i.e., less than about 100%) may prevent the liquid solution from forming a bulged upper surface, which can create a lens during inspection.

[0107] In various examples, the method may comprise loading the cell into the chamber. The chamber may be filled (to less than capacity) with the liquid solution before the cell is loaded into the chamber. The cell may be loaded into the liquid solution within the chamber.

[0108] In various examples, the method may comprise covering the liquid solution with an immiscible liquid. The immiscible liquid may be an oil. The oil may be, for example,paraffin oil, fluorinated oil, or the like. The liquid solution may be covered with the immiscible liquid after the cell is loaded into the liquid solution in the chamber. The cell may be contained in the liquid solution that is covered with the immiscible liquid. The acoustic field may press the cell against the interface between the liquid solution and the immiscible liquid to manipulate the cell within the chamber. A pipette may be inserted into the chamber, through the immiscible liquid, to extract the cell or other materials in the cell from the chamber.

[0109] In various examples, the method may comprise pumping the chamber to quench the cell. Quenching the cell may be an enzymatic reaction. Quenching may refer to diluting the digestive enzyme concentration by adding culture medium to the chamber. The cell may be quenched while the cell is being manipulated by the acoustic field (e.g., during a denudation procedure). The quenching may be stopped when the manipulation of the cell is stopped (e.g., the denudation procedure is finished).

[0110] In various examples, pumping the chamber to quench the cell may comprise pumping media into the chamber via an inlet port of the chamber and extracting media from the chamber via an outlet port of the chamber. The cell may comprise a COC, and the extracted media may comprise cumulus debris removed from the oocyte of the COC.

[0111] In various examples, the method may comprise injecting a denuded oocyte of the cell with spermatozoa.

[0112] In various examples, the chamber may include one or more microchambers in fluid communication with the chamber. The acoustic waves may generate an acoustic field configured to assist in moving the cell from the chamber to one of the microchambers. The acoustic field may have a shape or pattern that assists in moving the cell to one of the microchambers.

[0113] In various examples, the chamber may be freely movable on the substrate. The chamber may be a void within a vessel, such as within a dish, and the dish may be freely movable with respect to the substrate.

[0114] In various examples, the substrate may be disposed between the electrodes and the chamber. The substrate may be formed from glass. The electrodes may be embedded within the glass of the substrate. The electrodes may be secured to the substrate, such as with an adhesive. The acoustic waves generated by the ultrasonic transducer may be configured to propagate an acoustic field that extends through the substrate to manipulate the cell within the chamber.

[0115] In various examples, the acoustic field may be configured to translate or rotate the cell within the chamber. The acoustic field may be configured to translate and rotate the cell within the chamber.

[0116] In various examples, the method may be performed under observation of a microscope.

[0117] The steps of the methods described in the various examples disclosed herein are sufficient to carry out a method of the present disclosure. Thus, in various examples, a method consists essentially of a combination of the steps of the methods disclosed herein. In various other examples, a method consists of such steps.AHT MODULE EXAMPLEExperimental Implementation

[0118] An experimental set-up consisted of two parts, the AHT and a disposable dish such as a glass petri dish. As in conventional manual denudation, where the stripper (precision pipette) serves as the primary reusable tool and microcapillaries and dishes are disposable, the AHT serves as the primary reusable component that can generate the desired acoustic fields inside the disposable dishes that are placed on top of it. See FIG. 1 A.Generation ofAHTs

[0119] Holographic principles of Fresnel lenses and wave synthesis with IDTs were used fabricate flat, transparent and compact AHTs. In short, the phase of an AHT is encoded into the shape of a pair of intertwined electrodes (IDT) with opposite polarities and deposited on top a piezoelectric substrate (see 15-17). Other methods of shaping the electrodes are also possible, such as with capacitive (CMUT) or piezoelectric (PMUT) micromachining. The frequency range of 40-45 MHz was chosen as the working frequency considering the size of cumulus cells (10-15 microns) and the designing guideline of d / X ~ 3 for three-dimensional trapping (see 13 and 16), where d is the diameter of the particle and X is the wavelength.

[0120] To achieve larger intensity wells that can trap oocytes (100 to 120 microns) and COCs (400 to 700 microns), operated at the same frequency (about 45 MHz) and focal length (z = 1.1 mm), we introduced additional phase singularities to increase the size of the intensity wells generated by the spherical acoustical vortices. The first method uses the intersection of a spherical beam and a cylindrical beam with helicity of I or a superposition of.v cylindrical beams at the same frequency and helicity to increase the size of theacoustical trap. We refer to these AHTs as ring-like vortices and denote them with R-ls. FIG.lB(iii) shows the normalized intensity for R-14 where a 2.8 times larger low intensity (zero-pressure) central area is achieved in comparison to a spherical acoustic vortex (SAV, FIG. IB(i)) at the same frequency (see Supplementary Notes, Section 1). The second method combines single or multiple complementary phase wrappings.v by introducing an azimuthal angular-dependent wave number. In this method the azimuthal height of the trapping point along the propagation axis varies linearly with the circumferential angle. By superposition of several phase wrappings s and the fact the azimuthal height of the trapping point can be tuned by frequency (19: Gong Z, Baudoin M. Three-Dimensional Trapping and Dynamic Axial Manipulation with Frequency-Tuned Spiraling Acoustical Tweezers: A Theoretical Study. Phys Rev Appl [Internet] 2021; 16(2): 024034. Available from:https: / / link.aps.org / doi / lO.1103 / I}hysRev Applied.16.024034). the generated acoustical fields take a polygonal-like shape and are denoted by P-s where s is the number of superimposed fields. FIG. IB(iv) shows the normalized intensity for P-4 where a similar 2.8 increase in the size of the low intensity (zero-pressure) central area is observed (see Supplementary Notes, Section 2).

[0121] Another method introduces angular n-phase jumps (X 12 steps) at discrete angles in the IDT design of a focused beam (FIG. IB(ii)) to create evenly split high intensity region to achieve petal-like fields. These petal -like fields are denoted by T-q where 2q is the number of petals. FIG. IB(v) shows the normalized intensity for T-3 where 3 symmetrical phase singularity plans result in 6 high intensity spots and doubles the area of the low intensity (zero-pressure) region in comparison to SAV (see Supplementary Notes, Section 3). Incorporation of single angular phase jump into design of R-ls AHTs and switching in the directivity of the helicity at the same discrete angle result in crescent shaped petal fields with tunable arc length (see Supplementary Notes, Section 4). These crescent-shaped fields are denoted by C-ls where I and are the helicity and number of superimposed cylindrical beams respectively. FIG. IB(vi) shows the normalized intensity profile for C-81. The asymmetrical constructive interference from changing the helicity direction with destructive interference from angular n-phase jump result in a crescent petal profile that can be used to only push bioparticles with limited directivity. Lastly, another method introduces n -phase jumps ( I 2 steps) in the radial direction at a discrete radius that splits the area of the IDT into two equals areas. This method results in destructive interference in the beam propagation direction at the focal point creating bottle shape fields (see SupplementaryNotes, Section 4).Open Microchamber-Microfluidic System

[0122] An under-oil open microfluidic chamber and microfluidic system were developed to provide the same benefits as the droplet-oil system while leveraging the benefits of confining fluids in the ten-hundred-micron compartments and channels to mimic the natural dimensions of the reproductive tract. These under-oil open systems, devoid of a ceiling, are free to external tools at any arbitrary location (see FIGS. 1C and ID). This system also minimizes the evaporation and osmolarity changes in the culture medium and depending on the volume of the oil and culture medium, minimizes the temperature variation. Lastly, unlike traditional closed microfluidic systems, this system has minimal bubbling, clogging, evaporation, and airborne contamination issues. The system also has a low adoption barrier and low manufacturing cost.

[0123] The suggested open microfluidic devices have at least one loading section with a large diameter (50 times the COC diameter) for effortless loading of the COCs. Generally, the small ratio of the height to width of the microchannels (h / w) makes it possible to achieve cellular confinements in relatively large channel widths suitable for COC and oocyte handling. In the suggested design in FIG. ID, the difference between the radius of the inlet and the outlet is used to create a passive pumping system based on Laplace pressure difference between the inlet and outlet. The instant volumetric flow can be described bs the channel’s hydrodynamic resistance as a function of channel dimensions and Toii / media isthe oil-media interfacial tension (20: Li C, Hite Z, Warrick JW, Li J, Geller SH, Trantow VG, et al. Under oil open channel microfluidics empowered by exclusive liquid repellency. Sci Adv [Internet] 2020:6(16): eaay9919. Available from: http: / / dx.doi.org / 10.1126 / sciadv.aay9919).

[0124] For acoustic assisted denudation, first, the cylindrical well is filled 75% of its capacity with media+HA (20 lU / mL) and covered with oil to prevent evaporation. Then, the COCs are collected with a pipette at the remaining 25% volume of the cylindrical well and are added to the cylindrical well under oil (see FIG. 1 E(i)) After COC loading, the dish is placed on the AHT, and the denudation procedure starts. The acoustic vortices and movements of its location mix the liquid and denude the COCs based on one of the methods described in the denudation strategies section. Then denuded oocytes are moved away from dispersed cumulus cells to the embedded microchambers and are washed by fresh mediausing active pumping (see FIGS. lE(ii) to lE(iii)). For active pumping, an inlet capillary is placed atop the inlet port to passively pump fresh media (no HA) into the channel (see FIG. lE(iii)). Similarly, an outlet capillary is placed inside the COC loading section to remove the excess media and cumulus debris (Media+HA+Cumulus).

[0125] In other scenarios it is possible to manually collect the denuded COCs and move them to another droplet (see FIGS. 18A, 18B, 18C). It is also possible to first move the COCs to microchambers and then expose them to HA by active pumping. These scenarios are discussed in Supplementary Notes, Section 7.In-vitro Acoustic MicromanipulationAcoustic Assisted Denudation Strategies

[0126] Two strategies were investigated as shown in FIGS. 2A to 2E to develop an efficient yet safe method for performing non-contact denudation. In the first strategy (FIGS.2A, 2B, 2C), the AHTs target the cumulus cells at the individual level while pushing the oocyte away. This method, referred to as a plucking method hereafter, detaches the cumulus cells that fall inside the inner radius of the first ring of the acoustic trap. At the same time, the oocyte, due to its size, is always repelled by the outward-directed forces (see FIG. 2C). As shown in FIG. 2B, repositioning the acoustic traps around the periphery of the COC by moving the dish and applying a swept frequency input signal to vertically alternate the focal point is necessary to increase the efficiency of the denudation procedure. This denudation strategy is most effective when the microfluidic device is closed and made of a soundabsorbing material.

[0127] Acoustic streaming and accompanying fluid shear forces are the main underlying mechanisms of action in the case of open microfluidics. The vortex-based traps that carry 0AM create vortices with a rotating axis parallel to the beam axis, referred to as toroidal vortices (TVx) hereafter, (see FIGS. 16A, 16B, 16E, 16F, and 16H). In contrast, focused, twin-trap, and petal acoustical fields create fluidic vortices with a rotating axis perpendicular to the beam axis, referred to as poloidal vortices (PVx) hereafter, (see FIGS.16C, 16D, and 16G). Like closed microfluidics, small TVx tend to trap the cumulus into the vortex center while repelling the oocyte. Cumulus trapping is significantly hampered by acoustic streaming and COCs need to be chased constantly. As such, a denudation procedure with small TVx in open microfluidics requires more movements than other methods. In contrast, small PVx vortices lock the COCs in their periphery while rolling them in the outof plane direction. This rolling motion loosens the cumulus cells while keeping the COCs in place. It is still necessary to reposition the PVx location for a complete removal of cumulus cells from oocyte.

[0128] In the second strategy, shown in FIGS. 2D, 2E, and 2F, the AHT traps aim to directly capture the oocytes. In this method, referred to as a shedding method hereafter, once a COC is trapped, the accompanying swirling fluid shear, originating from the 0AM transfer, starts to shed the cumulus cells. Then, the trapped oocytes can be moved to a new location while most cumulus cells are left behind. A distinct feature of the shedding method is that the inward rotational forces prevent the perivitelline space expansion that may complicate the ICSI procedure later.

[0129] The denudation efficiency is defined as—■^■treated / A-untreated where A is the area of the cumulus residues after performing denudation determined visually or through image processing reported in our previous work (18). Unless due to human error, the device's yield (oocyte recovery) from the open microfl ui die dish is 100%.

[0130] Four different devices were used, namely, R-01 and T-2 for plucking denudation and P-4 and P-8 for shedding denudation performance evaluation. These AHTs were chosen based on the initial performance tests that are not presented here. The denudation tests were done on different days using different frozen and fresh COC cohorts with AHTs from different devices to test for device-to-device variability. Denudation was performed at two different intensity levels controlled by the input signal modulation. At the higher intensity level, the input electrical signal is continuous, while the lower intensity level is generated by pulsing the input signal at 1 KHz and 50% duty cycle (^rms, pulsed — Vrms, continuousxAs shown in FIG. 3A, a significant correlation exists between the power levels and complete denudation time. At the same time, the population means for various devices and denudation strategies are not significantly different. FIG. 3B shows that complete denudation time is a function of HA concentration and exposure time. As expected, the higher power level and HA concentration result in shorter denudation procedure times.COC Denudation and Viability

[0131] A total of 235 mouse oocytes from 6 hyperovulated B6D2F1 mice were retrieved and allocated to the conventional denudation method by mechanical pipetting with 40 IU cumulase (n=65) or denuded using a contact-free denudation system with 15 IU cumulase (n=170). Based on screening experiments, the 15 IU cumulase concentration was chosen toachieve the same denudation performance of conventional stripping at a 62.5% reduction in cumulase concentration. It is important to note that denudation efficiency, or the total time required to complete denudation, is a function of both cumulase concentration and acoustic power, (see FIG. 3B). 15 IU was chosen to perform the acoustic-assisted denudation procedures, yielding a complete denudation time like manual pipetting. All oocytes were denuded completely confirmed visually by a trained embryologist, see, for example, FIGS.3C and 3D. No oocyte was lost during the experiment. Denuded oocytes were then injected with spermatozoa from B6D2F1 male mice by Piezo-ICSI, and post-injection oocytes were loaded in a time-lapse incubator to monitor embryo development.

[0132] As shown in FIGS. 4A and 4B, the control cohort yielded a survival rate of 86.2% (56 / 65) and a fertilization rate of 96.3 (54 / 56). These embryos cleaved to the 2-cell stage at 92.9% (52 / 56) and an eventual blastocyst rate of 85.7% (48 / 56). The experimental cohort generally obtained a comparable piezo-ICSI survival rate of 85.9% (146 / 170) and a fertilization rate of 90.7% (117 / 129, P>0.05). The subsequent 2-cell cleavage and blastocyst rates were like the control at 83.6% (122 / 146) and 76.0% (98 / 129).

[0133] When time-lapse data are analyzed, the prokinetics between the experimental and control cohorts are almost identical, demonstrating that the introduction of soundwave does not impact embryo development (see FIG. 4D). For experimental cohort, a total of 65 embryos were transferred to eight recipient mice that resulted in 45 live births (69%) comparable to 21 embryo transfers to two recipient mice for the control group that resulted in 15 (71%) live births. All pups weaned without any development complications.Acoustic-Assisted Positioning and Rotation Strategies

[0134] Selective micromanipulation, including arbitrary positioning of either COCs or denuded oocytes, is achieved using two methods. For applications such as conventional IVF, it is necessary to position the COCs in the desired positions while the cumulus mass is still intact and attached to the oocyte. Thus, we can leverage the firm attachments of cumulus cells as an anchor point to pull or push the COCs in arbitrary positions using acoustical traps that target cumulus cell size (see FIGS. 5A, 5B, and 5C). Similarly, attaching glass or polystyrene microbeads to COCs and using those as an anchor point is possible (see FIG. 20A, green microbeads). In this method, the microbeads will naturally stick to the cumulus mass mainly due to a rich extracellular matrix composed of hyaluronic acid, proteoglycans, and various glycoproteins. These firm attachments further provide abetter anchoring handle with a better acoustical contrast factor that can be trapped by acoustical traps. The main advantage of this technique is that cumulus cells will act as another protective layer against acoustic radiation pressure, acoustic streaming shear, and temperature fluctuations. Lastly, this method only performs well in the closed microfluidic chamber where absorptive or reflective material is used at the top (ceiling) of the microfluidic chamber. FIG. 5C(1) shows the sequence of moving a COC along an arbitrary path without disturbing the neighboring COCs.

[0135] In the case of the ICSI procedure, the COC denudation is performed before insemination. So, most of the positioning and translation happens on the denuded oocytes lacking cumulus mass (see FIGS. 5A(3) and 5B(3) and FIGS. 16D, 16E, and 16F). In this case, acoustical traps of appropriate size can trap and move the oocytes. Similarly, it is possible to use the repelling forces of acoustic streaming of the crescent-shaped acoustical fields to push the oocyte without trapping them inside a microchannel (see FIGS. 16H).

[0136] In and out of plane rotation is crucial for visual assessment and correct oocyte injection position and could be used for further assessment during time-lapse measurements. TVx of appropriate size originating from either R or P AHTs (ring-shaped or polygonal acoustical traps) can be used for rotating oocytes and embryos normal to the plane of observation (see FIG. 5A(4), 5B(4) and 5C(4)). The same holds true for PVx fluidic fields in the open microfluidic devices. Similarly, PVx originating from T or P AHTs (twin-trap and petal-like fields) can roll the oocytes and embryos in the out-of-plane direction (see Error! Reference source not found. A(2), 5B(2) and 5C(2)).Oocyte Positioning and Rotation Viability

[0137] Similarly, cumulus-free (denuded by conventional vortexing) fertilized bovine zygotes, which are much larger than mouse zygotes and have a similar size as human zygotes, are used to conduct translation studies to investigate the AHT’s effects on the fertilization potential. A total of 293 zygotes from three replicate experiments are cultured for two different translation strategies and three acoustic power levels (see FIG. 4C). No zygotes were missed during the micromanipulation and dish transfers. All the micromanipulated zygotes in all groups generally obtained comparable cleavage and blastocyst rates. Double -tailed t-tests confirmed no significant differences among the groups compared to the control group.Discussion and Outlook

[0138] It is recognized that one of the most relevant ART procedures is ICSI with its ability to identify and inject an individual spermatozoon directly into an oocyte. This procedure to be reliable and successful requires sophisticated equipment and attention to details in relation to all its aspects. One of the most relevant is the denudation of the oocyte that permits visualization of oocyte maturity and of the cytoplasm during the execution of the procedure. This is accomplished by the removal of the cumulus corona cells by the action of an enzyme and concurrent mechanical stripping.

[0139] Most of the components of IVF have been simplified by the development and commercialization of reagents such as handling and denudation media, as well as specialty solutions for time-lapse microscopy, cumulus-cell stripping capillaries, embryo transfer pipettes, and micromanipulation tools, for the oocytes. In addition, equipment for the assessment of the male gamete have been marketed as well as media and devices for selection of the most motile spermatozoa. All of these additions have the main purpose of simplifying the workload while promoting standardization. One of the most popular contributions is the ability to monitor around the clock all preimplantation development of the conceptus by time-lapse.

[0140] The removal of the cumulus cells remains an artisanal procedure and requires manual skills that are highly dependent on the individual hand dexterity of the embryologist as well as on proper training. This characteristic is responsible for a sensible inter-technician variability resulting in unwanted outcome ranging from large perivitelline space to a fractured zona or all the way to a zona free egg. Most concerning is, however, an incomplete removal of the cumulus cells with consequent increased difficulty in ICSI execution due to poor visualization, leading to a suboptimal outcome.

[0141] The difficulty in performing a proper and complete stripping of cumulus corona cells has even induced some laboratories to promote the partial removal of the cumulus cells for oocysts undergoing ICSI and / or cryopreservation (21-22: 21. Ebner T, Yaman C, Moser M, Sommergruber M, Jesacher K, Tews G. A prospective study on oocyte survival rate after ICSI: influence of injection technique and morphological features. J Assist Reprod Genet [Internet] 2001 [cited 2024 Aug 9]; 18(12): 623-8. Available from: https: / / link.springer.eom / artide / 10.1023 / A:1013171505702. 22. Ebner T, Moser M, Sommergruber M, Shebl O, Tews G. Incomplete denudation of oocytes prior to ICSI enhances embryo quality and blastocyst development. Hum Reprod [Internet] 2006 [cited2024 Aug 9]; 21(11): 2972-7. Available from: https: / / academic.oup.com / humrep / article-pdf / 21 / 1 l / 2972 / 9683753 / del272.pdf). In these circumstances, the incomplete removal induces difficulties in performing ICSI on fresh or cryopreserved eggs. This embryologists’ convenience has possibly contributed to a recent fashion to promote a reduction in the number of ICSI procedures performed, relegating the procedure only to the most severe and undisputable form of male factor infertility (23: Jin H-X, Song W-Y, Xin Z-M, Dai S-J, Chen Z-J, Sun Y-P. Effects of cumulus cells on vitreous cryopreservation of human mature oocytes and clinical pregnancy outcomes. Reprod Sci [Internet] 2012 [cited 2024 Aug 9]; 19(2): 216-20. Available from:https: / / link.springer.com / article / 10.1177 / 1933719111424450).

[0142] The availability of a simple automated device that would void acquired individual skills would be highly desirable especially in the cunent age of automation and Al that is surfacing not only in ART laboratories but in all human activities (24: Lattin MT, Djandji AS, Kronfeld MT, Samsel T, Ling R, Ciskanik M, et al. Development and validation of an automated robotic system for preparation of embryo culture dishes. Fertil Steril [Internet] 2024 [cited 2024 Aug 9]; 122(2): 297-303. Available from: http: / / www.fertstert.org / article / S0015028224002449 / abstract).

[0143] The principle of acoustic manipulation of small objects has been known (15-17) and (25-26: Thomas J-L, Marchiano R, Baresch D. Acoustical and optical radiation pressure and the development of single beam acoustical tweezers. J Quant Spectrosc Radiat Transf [Internet] 2017; 195: 55-65. Available from: https: / / linkinghub.elsevier.com / retrieve / pii / S0022407316306409. 26. Ghanem MA, Maxwell AD, Wang Y-N, Cunitz BW, Khokhlova VA, Sapozhnikov OA, et al. Noninvasive acoustic manipulation of objects in a living body. Proc Natl Acad Sci U S A [Internet] 2020; 117(29): 16848-55. Available from: http: / / dx.doi.org / 10.1073 / pnas.2001779117), and here, various specific beams are synthesized based on only a single electrical input signal on transparent transducers. Manipulating cumulus cells up to individual COCs for performing denudation, rotation and transportation within a microfluidic channel is demonstrated. In this device, the disposable dish with embedded open micro-chamber / channels at its base can be easily moved on a heating stage under a stereo microscope.

[0144] A few microfluidic-based technologies have been proposed, specifically designed for oocyte denudation. One method jammed the COCs through microchannels with a similar width to the oocyte diameter, hoping to detach surrounding cells by suctionfrom side ports. Another method traps the COCs in microchannels flooded with hyaluronidase, and the cumulus cells are removed by fluid shear force.(27-28: 27. Zeringue HC, Beebe DJ. Microfluidic removal of cumulus cells from Mammalian zygotes. In: Germ Cell Protocols. Springer; 2004. p. 365-73. 28. Zeringue HC, Rutledge JJ, Beebe DJ. Early mammalian embryo development depends on cumulus removal technique. Lab Chip [Internet] 2005; 5(1); 86-90. Available from: http: / / dx.doi.org / 10.1039 / b316494m) More recently, microfluidic channels with jagged side walls have also been proposed (29: Weng L, Lee GY, Liu J, Kapur R, Toth TL, Toner M. On-chip oocyte denudation from cumulusoocyte complexes for assisted reproductive therapy. Lab Chip [Internet] 2018; 18(24): 3892-902. Available from: http: / / dx.doi.org / 10.1039 / c81c01075g), as well as oscillatory, pressure-controlled fluid flow. (30-31: 30. Mokhtare A, Xie P, Abbaspourrad A, Rosenwaks Z, Palermo G. Toward an ICSI chip: automated microfluidic oocyte denudation module. In: HUMAN REPRODUCTION. OXFORD UNIV PRESS GREAT CLARENDON ST, OXFORD OX26DP, ENGLAND; 2020. p. 71-2. 31. Mokhtare A, Xie P, Abbaspourrad A, Rosenwaks Z, Palermo GD. EMBRYOLOGY LAB-ON-A-CHIP: AUTOMATED OOCYTE DENUDATION MICROFLUIDIC DEVICE. Fertil Steril [Internet] 2020; 114(3): e76. Available from: https: / / doi. Org / 10.1016 / j.fertnstert.2020.08.234) All the above-mentioned techniques require preparation and loading step and allowed the COCs to exit the field of view. (32: Swain JE, Lai D, Takayama S, Smith GD. Thinking big by thinking small: application of microfluidic technology to improve ART. Lab Chip [Internet] 2013; 13(7): 1213-24. Available from: https: / / pubs.rsc.org / en / content articlepdf / 2013 / lc / c31c41290c)

[0145] A method proposed conventional pipetting through a robotic system that still requires a dedicated microscope and an automated manipulator, therefore rendering it expensive and cumbersome. (33: Zhai R, Shan G, Dai C, Hao M, Zhu J, Ru C, et al.Automated Denudation of Oocytes. Micromachines [Internet] 2022 [cited 2022 Aug 17]; 13(8): 1301. Available from: https: / / www.mdpi.eom / 2072-666X / 13 / 8 / 1301 / htm) To address these issues, we recently reported a batch denudation of COCs inside microwells using acoustic streaming-induced shear to process up to 20 COCs at once in a few minutes (18) and (34: Xie P, Mokhtare A, Davaji B, Rosenwaks Z, Abbaspourrad A, Palermo GD. An expedited and safe oocyte denudation system based on soundwaves in a microfluidic chip. Fertil Steril [Internet] 2021; 116(3): el52-3. Available from: https: / / linkinghub.elsevier.com / retrieve / pii / S0015028221010128). However, it lacked theselectivity and precision required for individual ova denudation. In this current study, these limitations have been addressed by using a device that can focus on a single oocyte and utilize a disposable dish.

[0146] It is understood that acoustic waves induce heat. In our device, however, the average denudation time for each COC is less than 8 sec, implying that the temperature variation is less than 1.2°C (see, Supplementary Notes, Section 6), and this is within the regulatory limits defined for the application of sonographic waves on human tissue (35: Duck FA. Medical and non-medical protection standards for ultrasound and infrasound. Prog Biophys Mol Biol [Internet] 2007 [cited 2024 Aug 9]; 93(1-3): 176-91. Available from: http: / / dx.doi. Org / 10.1016 / j.pbiomolbio.2006.07.008) In this device, it can be estimated that only <3% of the acoustic energy is converted into thermal energy (16, 18). Moreover, we carried out a biological assessment to confirm that no signs of mechanical or thermal damage to the oocytes were incurred. This system employed an open microfluidic dish devoid of a ceiling to match the benefits of the droplet-under-oil that allowed external probes, while minimizing evaporation and fluctuation in osmolarity and temperature. This setup limits contamination and permits easy extraction of the cells from the device, if desired.

[0147] A limitation to be considered is the need for a thin layer of medium under oil in order for the device to be effective. In addition, the size of the COC may induce it to spin and slip away from the trap.

[0148] Our approach presents a different paradigm, characterized by an acoustic device and protocol that obviated the need for a physical pipette and reduces the level of operator training, skillset, and reliance on an embryologist all together. Indeed, the precision and resolution of the presented AHT surpasses the human capabilities while the applied shear force can be controlled electronically to minimize human errors.MethodsOva Micromanipulation Module Fabrication and Operation

[0149] Each micromanipulation module consists of two parts: the AHT and the microfluidic system. AHTs are fabricated based on electrode deposition on transparent lithium niobate transducers, with details of the fabrication process explained in our previous work (18). We followed the steps explained in Baudoin et al. (16) to glue the glass substrate on top of the transducer. As emphasized in their work, having a uniform and thin layer ofepoxy is critical to minimize acoustic energy loss and heat dissipation in the viscoelastic layer. This is achieved by dispensing a known volume of epoxy (to achieve a thickness of ~1 micron) between the transducer and glass substrate and letting it spread by capillary action. The holographic shape of electrodes on the lithium niobate substrate is changed to achieve different acoustical fields inside the dish for different procedures.

[0150] In all the experiments, a customized petri dish with embedded microchannels on its bottom is placed on top of the AHT part that holds the samples. The details of microfluidic dish fabrication are discussed in Supplementary Notes, Section 8. Deionized water is used as an ultrasound coupling to facilitate the acoustic wave transmission from glass to dishes. After each experiment, the cells in petri dishes are removed for culturing or further analysis. While Petri dishes are disposable, the AHTs can be used many times for subsequent assays.Animal Use, Acquisition of Mouse Gametes, and ICSI Procedure

[0151] Murine oocytes were collected from superovulated B6D2F1 mice. To induce superovulation, ~4-week-old female mice received an intraperitoneal (IP) injection of pregnant mare serum gonadotropin (PMSG)Zinhibin mix. Forty -eight hours later, they were administered human chorionic gonadotropin (hCG) via IP injection to trigger final oocyte maturation and ovulation. The mice were euthanized 18 hours later, and their oviducts were excised, placed in KSOM media, and micro-dissected to extract cumulus-oocyte complexes (COCs) for experimentation.

[0152] Murine spermatozoa were retrieved from 6-12 week-old male mice of the same strain. The mice were euthanized, their cauda epididymis excised and dissected in HTF media to release spermatozoa. The sperm cells were diluted to 3 million / ml and incubated until use.

[0153] Piezo-actuated ICSI was performed to fertilize denuded oocytes. A custom piezo-ICSI micropipette was prepared, inserted into a micropipette holder, and aligned for the procedure. Prior to injection, the pipette was primed with PVP During ICSI, sperm heads were isolated and aspirated into the pipette. The oocyte's metaphase II spindle was positioned at 9 o’clock, a laser pulse created a breach at 3 o’clock, and the injection pipette was advanced to 80% of the oocyte. A piezo pulse penetrated the membrane, and the sperm head was deposited into the ooplasm. The pipette was retracted while gently aspirating to seal the oocyte membrane. Post-ICSI embryos were loaded into a time-lapse microscope tomonitor full pre-implantation development.

[0154] All experimentations on murine models were approved by the IACUC of Weill Cornell Medicine, protocol number 2023-0008.Acquisition of Bovine Gametes and Embryo Production

[0155] Bovine zygotes were produced from slaughterhouse ovaries (Wyalusing, PA, USA). Ovaries were soaked in saline during transport to the laboratory. Follicles measuring 2-8 mm were aspirated using a custom manifold system, and the follicular contents were collected in a conical tube. The follicular contents were filtered through a cell strainer with a mesh pore size of 75 um and washed with commercial embryo flush media. Grade 1 cumulus-oocyte complexes (COCs) defined as those surrounded with three or more layers of compact cumulus cells and dark homogeneous ooplasm were selected for the experiment.

[0156] In vitro bovine embryo production was accomplished using a commercial (IVF Bioscience, Falmouth, UK) media and protocols as previously described (36: Abdelhady AWA, Aguiar LH, Lee YL, Guo Z, Bovell RT, Crane PL, et al. Rho-associated coiled-coil containing kinase inhibitor improves outcomes of direct-transfer slow-cooled bovine blastocysts. Theriogenology [Internet] 2023 [cited 2024 Aug 19]; 211: 19-27. Available from: http: / / dx.doi. Org / 10.1016 / j.theriogenology.2023.07.030). Briefly, COCs were matured for 18-22 hours at 38.5°C in humidified atmosphere supplemented with 6.2% CO2 then fertilized with cryopreserved bull semen of known fertility. Semen straws were warmed in a 37°C water bath for 30 seconds, then the sperm was washed to remove cryoprotectants by two rounds of dilution in SemenPrep media, centrifugation (350 g, 5 minutes), and removing the supernatant. Sperm motility was confirmed by subjective evaluation under phase contrast microscopy and used for fertilization of the COCs at a concentration of 2 million sperm / mLfor 18 hours in humidified atmosphere with 6.2% CO2. Cumulus cells were removed from the zygotes by vortexing in BO-wash media. Zygotes were randomly assigned to different groups and exposed to acoustic treatment or loaded onto the chambers without turning on the acoustic tweezer system as controls. Embryos were then cultured in BO-IVC media in 6.2% O2, 6.2% CO2, and 87.6% N2at 38.5°C.SUPPLEMENTARY NOTES - AHT MODULE EXAMPLE

[0157] The following supplementary notes are divided into sections. Reference materials within the supplementary notes are enumerated after the letter “S” in front of thereference number to distinguish from the references cited with respect to the previously described examples where reference numbers alone are used.

[0158] Spherical acoustic beams can be described by equation (1) where Ptypically a zero, first, or higher order axisymmetric Bessel function of the first kind that satisfies the wave equation and results in spherically focused and acoustical vortices, respectively. ) are polar coordinates, w is the angular frequency, and e2”^ describes the helicoidal propagation along the z-axis where < *, and m are the azimuthal angle and chirality of the wavefront propagation, respectively. (S 1: Gong, Z. & Baudoin, M. Three-Dimensional Trapping and Assembly of Small Particles with Sy nchronized Spherical Acoustical Vortices. Physical Review Applied 14, (2020).) Increasing the helicity (m > 1) has been used to trap Mie particles (« > where a, and are the particle radius and the wavelength, respectively) (S2-S3: S2. Maxwell, A. D. et al. Vortex beams and radiation torque for kidney stone management. J. Acoust. Soc. Am. 139, 2040-2040 (2016). S3. Marzo, A., Caleap, M. & Drinkwater, B. W. Acoustic Virtual Vortices with Tunable Orbital Angular Momentum for Trapping of Mie Particles. Phys. Rev. Lett. 120. 044301 (2018).)wt) (!)

[0159] Here, instead of directly increasing the helicity of the spherical beam, we used three other methods for creating large ring-like, polygonal-like, crescent-like, petal-like, and bottle-shaped acoustical fields. We chose the frequency range of 40-45 MHz as the working frequency considering the size of cumulus cells (10-15 microns) and the designing guideline~ ^-3 forthree-dimensional trapping (S4-S5: S4. Baresch. D., Thomas, J.-L. & Marchiano, R. Observation of a Single-Beam Gradient Force Acoustical Trap for Elastic Particles: Acoustical Tweezers. Phys. Rev. Lett. 116, 024301 (2016). S5. Baudoin, M. et al. Spatially selective manipulation of cells with single-beam acoustical tweezers. Nat.Commun. 11, 4244 (2020).). The terms d and A are the diameter and wavelength of the particle, respectively.Section 1: Ring Torus-like Acoustical Vortices

[0160] The spiraling electrode equations for ring-torus-like acoustical fields are derived from the intersection of the 3-dimensional approximation of a converging Hankel vortex beam with a cylindrical Bessel beam that is a focal distance away from the desired trapping point. (See FIGS. 6A, 6B, 6C).

[0161] The mathematical expression of an outgoing (converging) Hankel spherical vortex in spherical coordinate (r> ‘ and a Bessel beam in cylindrical coordinates (p, <t>c, z) jsgiven jnequations (2) and (3):where, A > 0 is the wave amplitude, — ' / cis the wave number, c is the sound speed, ^ is the Legendre polynomial of order (n>mw,is the frequency, t is the time, hnis the spherical Hankel function of the first kind, Ji is the Bessel function of order I, krand C, are the radial and axial wave numbers, respective!}' related to dispersion relationship k'r + k2z— k2S6- Baudoin, M. et al. Folding a focalized acoustical vortex on a flat holographic transducer: Miniaturized selective acoustical tweezers. Sei Adv 5, eaavl967 (2019).)

[0162] In the far field, the Hankel function of the first kind can be approximated by,6rwhere r — kr is the dimensionless parameter introduced for simplicity. Thus, the iso-phase for the spherical vortex can be derived in equation (4) where Ci is a constant for an arbitrary iso-phase.

[0163] Similarly, the asymptotic approximation of the Bessel function whenThus, the iso-phase for the cylindrical Bessel beam can be derived in equation (5) where C2 is a constant for an arbitrary' iso-phase.

[0164] In an arbitrary instance of time, the isophase of the spherical beam reduces to asimple expression ofLikewise, in an arbitrary instant of time and fixed focal length the isophase for cylindrical beam reduces to ‘h. = Ipc. Thus, by equating the two iso-phases, the expression for the intersection of two beams at a fixed focal length becomes as shown in equation (6).r — m<l> T — lpc— Cs (5)_ __

[0165] Replacing r in —(0Hod the intersection of this function with a plane in cylindrical coordinates results in the polar equations of the isophase spirals that can be used for electrode patterning. Since electrical potentials are converted to mechanical vibrations due to the piezoelectric effect, following the principles of metallic electrode fabrications, these spiral polar tracks can be deposited on a piezoelectric substrate as isopotential tracks that can generate undulation on its surface and launch bulk acoustic w aves in the axis perpendicular to the surface. Therefore, the geometrical shapes of the holographic electrodes becomes as shown in equations (6) and (7) where (m> s) and 0c) are the helicity and azimuthal angles in spherical and cylindrical coordinates, respectively. k — w / caisthe wavenumber,fasound speed, C a constant, and z is the axis of wave propagation normal to the transducer plane.

[0166] To create IDT designs with angular sy mmctry and fields with more uniform angular intensity distribution, we linearly superposed cylindrical Bessel beams to create periodic helicoidal surfaces (FIG. 5C(ii)). For s cylindrical beams, the cylindrical azimuthal (Pt) is periodic for the interval [0, '- represented by equation (8) where I ■ J is the floor function to handle the periodicity. In this w ay, when m — I — 1, the resulting IDT designs have s arms at discrete angles Pc (n) are determined by equation (9).

[0167] The generated ring torus-like acoustical fields are denoted by R — Is, where I, and s are the topological charge of the cylindrical Bessel beam, and s is the number of superimposed beams, respectively. FIG. 7A(i) shows the IDT design for R-ll (m=l and 1=1). The complementary increase in the chirality of the cylindrical Bessel beam resulted in central phase singularity7evolution that goes from —?r to TT twice when goes from 0-2~ (FIG. 7 A(iii)) and increases the diameter of the first ring of the intensity7well by %170. FIG. 7B(i) shows the IDT design, normalized intensity7, and phase of the acoustic field of R-14. The phase profile (FIG. 7B(iii)) shows a central phase singularity surrounded by five (I • s + m) other side phase singularities. An almost identical intensity7profile (FIG. 7C(iii)) with slightly less angular intensity uniformity can be built by increasing the cylindrical beam chirality (topological charge) (1=4) without superimposing several beams (s=l). For example, FIG. 8A shows the radius of the peak intensity for different R-ln (n=l-8). By increasing the number of segments, the secondary ring distance and intensity that occurs immediately after the first ring also decreases.

[0168] It is important to note that for higher chirality7(I > 1) and segments (s > 1), not all the IDT segments can be connected to the two mam signal ports, making some of the IDTs redundant. As such, we used angular gap spaces between each segment to connect the similar IDT lines together. As shown in FIGS. 7D(i) to 7D(iii), the IDT design takes a ninja star shape that expands anticlockwise, originating from spherical topological order (m=l).Section 2: Polygonal -like Acoustical Vortices

[0169] Recently, both theoretically (S7: Gong, Z. & Baudoin, M. Three-Dimensional Trapping and Dynamic Axial Manipulation with Frequency -Tuned Spiraling Acoustical Tweezers: A Theoretical Study. Phys. Rev. Appl. 16, 024034 (2021)) and in airborne experiments (S8: Muelas-Hurtado, R. D., Ealo, J. L. & Volke-Sepiilveda, K. Active-spiral Fresnel zone plate with tunable focal length for airborne generation of focused acoustic vortices. Appl. Phys. Lett. 116, 114101 (2020), it has been show n that the focal point of avortex beam can be axially displaced when the transducer is operated at an arbitrary' frequency other than the frequency it is designed for. Furthermore, recent explorations of polygonal vortices in optics and airborne acoustics reported that polygonal optical vortices with variable intensity at their vertices may make micromanipulation easier. (S9-S11: S9. Wang, G. et al. Generation of a polygonal perfect optical vortex array by optical pen. J. Opt. Soc. Am. B 39, 2056 (2022). S10. Wang, C. et al. Generating a new type of polygonal perfect optical vortex. Opt. Express 29, 14126-14134 (2021). Sil. Sun, Y. et al. Acoustic polygonal Bessel vortices based on metasurfaces. Phys. Rev. Appl. 21, 044029 (2024))

[0170] Gong et al. (S7) demonstrated that there exists a region where the displacement position remains a linear function of frequency with sufficient axial trapping force, (see also FIG. 9 in this application) Using this knowledge, we chose a narrower frequency range of 4 MHz for creating a complementary’ phase wrapping in the focal point by an angular dependent wave number (Error! Reference source not found. FIG. 9). First, the maximum frequency deviation from the design frequency can’t exceed 4 MHz to minimize distortion (see FIG. 9). Second, the 4 MHz range is then linearly mapped to 0 - 2TT azimuthal angular range to create the phase wrapping such as in equation (13). Then the angular dependent frequency is used to calculate the deviation ratio from the designed frequency7and corresponding wave number in equation (13). This phase wrapping based on wave number translates to introducing a helical axial trapping (screw shape) focus length as shown in FIGS. 6B and 6C. Therefore, the geometrical shapes of the holographic electrodes become equations (10) and (11).

[0171] Here, (< *)>are the asymmetric helicoidal wave number deviated from thedesigned wave number in equation (12) and the wave number of the designed frequency, respectively. N is the number of revolutions of the spiral iso-phase lines, which also determines the maximum radius of the IDT. The frequency range is determined from the region where the axial variation of the focal point has a linear relationship with deviation from the designed frequency, a is the linear coefficient where the 2TT phase wrapping occurs to introduce a helicity based on the wave number.

[0172] We construct the polygonal -like acoustic vortices by linear superposition of several beams with asymmetric wavenumbers based on equation (14) where (n) defines the discrete angles at which this variation occurs as in equation (14).

[0173] The generated polygonal-like acoustical fields are denoted by P — s, where s is the number of superimposed beams. FIG. 10A(i) shows the IDT design for P-1. Like R-ll, the additional phase wrapping through wave number resulted in the evolution of central phase singularity from -TT to TT twice when goes from < Pso - (<f>so + 2TT), where <t>so is the initial azimuthal angle (FIG. lOA(iii)). FIG. 10B(i) shows the IDT design of P-3, where all four sections are connected through small angular gap junctions. The normalized intensity' profile resembles a trilateral-like shape (Reuleaux Triangle). The phase profile has a central phase singularity surrounded by five (•? + m) side phase singulanties. FIG. 8B shows the radius of the biggest circle that can be inscribed in each polygonal-like intensity' field for different P-ln(n=l-8). The normalized intensity7is show n at the y=0 line, which differs for various shapes while it reaches the maximum at the vertices for each polygonal field (FIG.8B). FIG. 8C shows the crescent shape C - In for the acoustic filed when n = 2, 4, 6, 8. See also FIGS. 10C and 10D that show7the IDT designs, normalized simulated intensity7, and phase profile for P-3 and P8, respectively.

[0174] The combination of both methods, i.e., the phase w rapping by controlling the helicity of the cylindrical w aves and the wave number, can be used to control the size of the polygonal-like acoustic fields. For example, FIG. 11 shows that for a quadrilateral -type acoustic field, decreasing or increasing the length of the sides is possible by introducingpositive or negative helicity parameters in cy lindrical coordinates (1).Section 3: Circular and Crescent -shaped Petal Fields

[0175] Focused petal fields of different radii can be achieved for stable (rotation-free) 2D manipulation in both Rayleigh and Mie regimes by incorporating severalsteps (TT-phase jumps) in the hologram of the focused spherical beams. The following iso-phase lines for a focused acoustic field with the polarity coefficient are used to determine the polarity of each sector when the lens is divided into <7 sections as shown in equations (15), (16), and (17).

[0176] The generated petal-like acoustic fields are denoted bywhere <7 > 1 is the number of petals. The case of q — 2 results in an acoustic field known as twin-trap tweezer with a pair of high intensity regions (FIG. 12A(ii)) and a plane of singularity (FIG.12A(iii)). Error! Reference source not found. IG. 12B(i) shows the IDT design for T-8 (q — 4) based on equation (17), which results in eight (2<7) evenly split high-intensity cylindrical regions (petals, FIG. 12B(ii)). The phase profile is also split into eight sectors with 7r-phase shift between adjacent sectors (FIG. 12B(iii)).

[0177] The pressure (amplitude) gradient is the leading force for exerting trapping force in one axis and the restraining forces in the perpendicular and propagation axis come from velocity gradients. Analogously, the phase of petal-shaped fields exhibits planes of singularity based on the number of petals that extend into the propagation direction.

[0178] To create crescent-shaped petal beams, we superimpose an even number of beams (see equations (6) and (7)) such that the topological helical charge ( / ) and polarity of the electrodes change from positive to negative according to equation (19).

[0179] FIGS. 12C and 12D show crescent-shaped fields denoted by C - Is where I is the topological charge of cylindrical Bessel beams and s is the number of superimposed beams. FIG 12C(i) shows the IDT pattern for the C — 18 crescent-shaped acoustical field where the polarity and topological charge directivity are controlled by equation (20).

[0180] The symmetrical angular opposition in the chirality and polarity result in asymmetrical constructive interference that result in the creation of crescent field shape. The polarity switching ensures that the region inside of the crescent has a close to zero intensity' (FIGS. 12C(ii)-12C(iii)).

[0181] Another method to create crescent shape petal beams is to change the direction of phase wrapping and the polarity of the electrodes at a discrete angle (ty)to create an axisymmetric IDT design based on equation (20). This method also results in almost identical crescent shape petal fields like C acoustic family. The crescent-shaped fields resulting from wavenumber folding are denoted by K — Is where I is the topological charge of cylindrical Bessel beams and q is the number of superimposed beams. FIGS. 12C(i) to 12D(iii) show IDT pattern, normalized intensity, and phase profile for the K-22 crescentshaped field in which the phase wrapping direction in equations (11)-(13) is controlled by equation (20).Section 4: Bottle-shape Fields

[0182] We further used a 7r-phase jump at properly chosen values of the holographic iso-phase radius to introduce destructive interference in the focal spot of either focused or vortical Bessel beams to create bottle-shaped acoustic fields. It is worth noting that creating such fields in acoustics has been mainly achieved with either complex phased arrays or metalenses. However, we simply achieved the necessary phase shift by swapping the polarity of IDTs when connecting to the main positive and negative electrodes.

[0183] Here, we only considered a single possibility for dividing the circular shape of the holographic lenses into symmetrical parts by introducing the following polaritycoefficients in equation (21).

[0184] In equation (21), (r) determines the phase (polarity of electrodes) of the holographic IDT.ri, andrmaj; are the radii where the area of the acoustic lens is divided into two equal parts and the maximum radius of the acoustic lens, respectively. FIG. 13A(i) shows a focused spherical beam, FIG. 13B(i) R-ll, FIG. 13C(i), T-2, and FIG. 13C(i), R-22 following equation (21). FIGS. 13C and 13D show the effect of radial 7r-phase jump on the iso-intensity profiles of Fresnel, R-ll, T-2, and R22, respectively. In the case of acoustic bottle beams, the minimum pressure region is enclosed by a bottle shape intensity shell expanding in three dimensions. Although not confirmed experimentally, such a curved propagation path may relax the limitation of having a thin film liquid for 2D manipulation of particles.Section 5: Consideration of Acoustic Intensity and Dissipated Energy

[0185] Despite decades of practical use and data on its innocuity to biological tissues, the safety of ultrasound on biological cells is not absolute. It depends on many factors, such as frequency, intensity', pulse duration, etc. The FDA suggested safety indices, including mechanical Index (MI), thermal Index (TI), and spatial-peak temporal-average to indicate the probability of ultrasound on biological tissue are determined for specific conditions and instruments and cannot be directly applied here. However, most clinical ultrasound devices' intensity falls between 0.03 and 1.0 W / cm². Further, it has been shown that intensities of about 3000 W / cm² can potentially induce cell apoptosis and damage the cells (S 12— S 14: S12. Xin, Z., Lin, G., Lei, H., Lue, T. F. & Guo, Y. Clinical applications of low-intensity pulsed ultrasound and its potential role in urology. Transl. Androl. Urol. 5, 255-266 (2016). SI 3. Church, C. C.. Labuda, C. & Nightingale, K. A theoretical study of inertial cavitation from acoustic radiation force impulse imaging and implications for the mechanical index. Ultrasound Med. Biol. 41, 472-485 (2015). S14. Ando, H. et al. An echo-contrast agent, Levovist, lowers the ultrasound intensity required to induce apoptosis of human leukemia cells. Cancer Lett. 242, 37-45 (2006)). As such, it is better to develop devices that operateat levels at least ten times lower than the toxicity threshold.

[0186] For both cases of vortex and petal fields, the surface displacements were measured using a Polytech vibrometer with values ranging between 0.05 - 1.5 nm for different power levels. Theoretical formulae for calculation of the acoustic radiation force of an arbitrary acoustic field exerting on an elastic sphere are provided by Sapozhnikov et al. (27) and the software provided by Krokhmal et al. (28) is used here for visualization of the force field on the target plane. The normalized force intensity and distribution for R-14, P-4, T-6, and R-18 AHT, when cumulus cells with an average diameter of 10 / im and oocytes with an average of 100 / im are considered spherical scatterers, are given in FIGS. 14A, 14B, 14C, and 14D. The exact force largely depends on the properties of the studied oocytes and cumulus cells, which are unknown. In all cases, as shown in FIGS. 14 A(iii) to 14D(iii), the cells are located inside the circle where the lateral force direction changes from negative to positive, and the cells are pushed to the center. Otherwise, cells are repelled outside of this region. This distance defines the spatial selectivity of the tweezer, where tw o parti cles / cells can be individually separated and manipulated.

[0187] The normalized transverse force intensity- distribution in the xz plane for R-14 AHT indicates an acoustical trap zone that pushes the scatterers toward the beam axis (see FIG. 15 A). At the same time, the longitudinal z-component is always positive and pushes the scatterer in the beam axis direction (see FIG. 15B). In the case of invitro fertilization, an oil layer positioned at an appropriate height on top of the media can create an effective acoustic-hydrodynamic trap for biological scatterers of large size. The reasons for choosing an oil-based system are twofold. First, an oil layer de-risks the accessibility and mass transport requirements, and second, due to the small difference betw een the density of the media and oocytes, the trapped oocytes can be easily pushed away in the beam axis direction. However, an immiscible oil layer will create an efficient barrier where oocytes are restrained by the acousto-hydrodynamic trap.

[0188] FIGS. 16Ato 16H show' the exploded views of acoustic intensity (bottom) and acousto-fluidic streaming (top) for R-01, R-ll, T-l, Fresnel, R-18, P-8, T-8 and C-18. respectively. For the family of R and P devices (FIGS. 16A. 16B, 16E, and 16F), acoustical vortices create hydrodynamic fields that combine steady torque and pushing forces, mainly depending on the topological order of the vortex. Further, the flow' streamline indicates the flow' is essentially axisymmetric in the beam axis direction. For the family of T, C, and Fresnel devices, the helicity is zero, and most of the acoustic power is concentrated at asingle or a few locations. As such, the acoustic energy is converted to push the fluid upward. When the fluid reaches the oil / water interface, it recirculates back and creates a poloidal vortex perpendicular to the beam axis. Due to the significant computational load of the whole Multiphysics problem, we only considered the wave propagation inside the dish (open microchamber). We used the angular spectrum method for wave propagation and COMSOL Multiphysics for acousto-fluidic simulation. The complex pressure values at the interface of the dish are exported as a boundary condition to COMSOL. Then, the thermo-viscous and pressure physics, combined with the coupled acoustic streaming equation, are solved to calculate the velocity field inside the microchamber.Section 6: Ultrasound-induced Thermal Effects

[0189] The most sensitive cellular structure to temperature variation is the miotic spindle (MS). Chemical and physical stresses can induce microtubule disassembly and injure MS. Therefore, it is necessary to prevent temperature fluctuation during denudation, observation, and manipulation (SI 5: In Vitro Fertilization: A Textbook of Current and Emerging Methods and Devices. (Springer, Cham, 2019). doi: 10.1007 / 978-3-319-43011-9.).

[0190] When sound propagates from one medium to another, the radiated acoustic energy results in a steady fluid flow known as acoustic streaming and viscous dissipation that leads to heating effects. The heating effect in ultrasound-based technologies for biomedical applications has always been a point of concern. The much-discussed issue is whether the increase in temperature will negatively affect the biological samples or even result in evaporation in open microfluidic settings. The heating effect stems from two main sources of acoustic energy dissipation, which depend on the acoustic power and fluid viscosity, and Joule heating that occurs in the IDTs responsible for the reverse piezo-electric effect (S16-S17: SI 6. Mokhtare, A. et al. Non-contact ultrasound oocyte denudation. Lab Chip 22, 777-792 (2022). S17. Collignon, S., Manor, O. & Friend, J. Improving and predicting fluid atomization via hysteresis-free thickness vibration of lithium niobate. Adv. Fund. Mater. 28, 1704359 (2018)).

[0191] For temperature measurements, we confined a droplet of the culture medium between two thin film membranes with a gap the same as the microchamber dishes. The same setup of function generator and amplifier used for manipulation tests is used for temperature variation tests. The signal generator was on for a period of 120 seconds, three times the complete denudation procedure time. We measured the temperature variationsover time using a thermal camera (Fotric 600 R& D). For each measurement, we measured the maximum temperature in an area three times the radius of a COC (1.8 mm) and subtracted the average temperature from it. The results were exported as a function of time and power. During the stimulation period, the temperature for different AHTs reached a stable temperature condition that can be predicted by W —(1 ~exP (^ / r)), where Tss is the steady state temperature and T is the characteristic time constant that temperature reached 67% of the steady -state temperature.

[0192] FIG. 17 shows the maximum temperature variation of media from its initial temperature (room temperature) for different powers and AHT. For both power levels, the temperature reaches a relatively steady state after -12 seconds (-220% of the denudation time for an individual COC). Temperature also returns to the initial temperature (before stimulation) with a similar characteristic time constant of -16 seconds. In these experiments, the spurious Joule heating also contributes to the heating through conduction over the glass substrate, and that's the main reason for the slower rise of the temperature and delay in reaching a steady state, especially at the high-power level. These results indicate a need for a better experimental setup to isolate the IDT-induced heat effects.

[0193] It is worth mentioning that the total electrical power transmission requires perfect impedance matching between the amplifier and acoustic tweezer. In our case, none of the tweezers are ideally 50 ohms, so only a part of the electrical power is transmitted to the transducer. In these situations, electrical energy does not reflect true acoustic power.

[0194] It is important to note that this measured temperature variation can be considered as the worst-case scenario since the culture medium is confined in a cavity between two membranes where almost all the acoustic energy is reflected from the top membrane-air interface. Due to constant reflections in this cavity, a larger amount of energy is lost in comparison to when an oil layer is placed on top of the thin culture medium. Further, the oil layer acts like a huge heat sink contributing to removal of the generated heat from the culture layer.Section 7: Dish Loading and other Open microfluidic Configurations

[0195] To minimize the effect of osmolarity, we loaded the dishes with a culture medium that was taken out of the refrigerator. The dish preparation is done on the nonheated surface of the laminar flow hood and an oil layer is placed on the culture medium immediately after.

[0196] Three other open microfluidic-based possibilities are also tested. In the first configuration (FIG. 18A), the dish only consists of a microchamber of the same diameter as the microfluidic loading port. The microchamber is first loaded up to 75% of its capacity with a 20 lU / mL HA solution. The COCs are then picked up with a precision micropipette and transferred to the microchamber. AHTs are then used to mix the fresh media and denudation simultaneously, as shown in FIGS. 18 A(ii) to 18A(iv). In the case of a microchamber placed on the side of the microchannel (FIG. 18B), the COCs are first loaded to the inlet, then a droplet of HA is added. Due to the difference in the curvature of the outlet and loading, the added HA media remains in the inlet. After performing denudation, oocytes are translated to a microchamber embedded in the side of the microchannel. Then, the medium can be switched with the active pumping method. This configuration is mainly suitable for oocyte freezing, where the media is changed with a cryoprotectant. After exposure, the oocyte can be loaded onto the straws for freezing.

[0197] Lastly, the microfluidic channel with embedded microchambers in the centerline (FIG. 18C) can be used in another scenario where the HA exposure can be performed by active pumping. In this case, the microfluidic channel is loaded with culture medium without HA, and COCs are transferred to microchambers embedded in the microchannel center. After the COCs are secured, HA can be pumped into the channel for a desired time and washed with a medium without HA. Due to COC confinement inside the microchambers and low fluid shear during active pumping, fluid shear doesn’t contribute significantly to denudation. Finally, the HA-exposed COCs can be denuded by AHTs as before. It should be noted that without HA presence, the COCs may stick to the bottom or side walls of the microfluidic, which prevents their manipulation.Section 8: Microfluidic Chamber Fabrication

[0198] Three methods have been used to create microfluidic dishes that perform best with the AHTs. All methods use a thin plastic film support to minimize sound reflection and heat generation.

[0199] In the first method, a polyester thin film with a thickness ranging from 3 microns to 30 microns is stretched between a cup-sleeve that fits together. This thin film creates the bottom layer of the open microfluidic structures (FIG. 19A). The side walls of the open microfluidic structure are created either by curing a photosensitive polyacrylamide copolymer (FIG. 19B) or by sticking a patterned medical-grade tape to the thin-film supportlayer (FIG. 19C).

[0200] UV-curable hydrogels (polyacrylamide) make it possible to create closed microfluidics entirely from hydrogels. This system has the advantage of providing sufficient confinement material for cells while acting as a good sound absorber. In an emergency, either glass or stainless-steel capillaries can easily rupture the hydrogels to access the cells (FIG. 19E) (SI 8: Chan, D. et al. Combinatorial Polyacrylamide Hydrogels for Preventing Biofouling on Implantable Biosensors. Adv. Mater. 34, e2109764 (2022)). To compare the open and enclosed microfluidic devices, conventional PDMS-based microfluidic devices are used.

[0201] In the second method, the thin-film support and microfluidic structures are fabricated simultaneously by hot embossing an aged PMDS mold to a polystyrene film, as shown in FIG. 19D.Section 9: Process Overview

[0202] FIG. 20A shows microbead attachment to cumulus cells as an anchor point for an acoustic tweezer. FIG. 20B shows acoustic fields such as R-01 and T2 can be used to grab microbeads in closed microfluidics. FIG. 20C shows a schematic representation of translation of a COC with attached microbeads and an R-01 acoustic vortex. FIG. 20D shows a denuded oocyte and other stages of the embryo that can be manipulated by AHT. FIG. 20E shows other AHT possibilities such as (top) large ring-like, polygonal-like, and (bottom) petal and crescent-shaped fields that can be used for ova translation. FIG. 20F shows (top) a schematic of large acoustic trap used for moving 2-cell stage embryo and (bottom) a crescent-shape acousto-fluidic field used to push the blastocyst inside a microchannel.NUMBERED ASPECTS

[0203] Although specific embodiments have been presented for purposes of illustration, the invention is not confined to these examples. Rather, the claimed subject matter encompasses additional embodiments and modifications that will be apparent to a person having ordinary skill in the art. Numerous changes, substitutions, and alternative implementations of the devices, systems, and methods described herein may be made without departing from the fundamental concepts. The present disclosure is intended to extend to all practical variations and adaptations that adhere to the underlying principles andthat reflect established and accepted practices within the relevant technical field, provided such variations remain consistent with the concepts disclosed herein.

[0204] Further details, embodiments, and variations of the invention can be appreciated with reference to the following enumerated aspects. Any element or feature disclosed in connection with one aspect is likewise applicable to other aspects or to combinations thereof, as supported by the corresponding antecedent description and logical combinations recognized by those having skill in the art. The enumerated aspects are as follows:1. A system comprising an ultrasonic transducer comprising electrodes; a chamber configured to contain a cell; and a processor in electronic communication with the ultrasonic transducer, wherein the processor is configured to control the ultrasonic transducer to generate acoustic waves directed to the cell to manipulate the cell within the chamber, and wherein the acoustic waves generate an acoustic field that has a pattern defined by a shape of the electrodes of the ultrasonic transducer.2. The system of aspect 1, wherein the electrodes are intertwined.3. The system of any of aspects 1-2, wherein the electrodes are micromachined.4. The system of any of aspects 1-3, wherein the electrodes are piezoelectric. 5. The system of any of aspects 1-3, wherein the electrodes are capacitive.6. The system of any of aspects 1-5, wherein the chamber is disposed on the ultrasonic transducer.7. The system of any one of aspects 1-6, wherein the chamber is movable with respect to the ultrasonic transducer.8. The system of any one of aspects 1-7, further comprising a glass substrate disposed between the ultrasonic transducer and the chamber and that supports motion between the chamber and the ultrasonic transducer.9. The system of any one of aspects 1-8, wherein the chamber is open.10. The system of any one of aspects 1-9, wherein the chamber contains two immiscible fluids.11. The system of any one of aspects 1-8 or 10, wherein the chamber is closed. 12. The system of any one of aspects 1-11, wherein the chamber contains soundabsorbing material.13. The system of any one of aspects 1-12, wherein the pattern of the acoustic field has a ring shape.14. The system of any one of aspects 1-12, wherein the pattern of the acoustic fieldhas a polygonal shape.15. The system of any one of aspects 1-12, wherein the pattern of the acoustic field has a petal shape.16. The system of any one of aspects 1-12, wherein the pattern of the acoustic field has a crescent shape.17. The system of any one of aspects 1-12, wherein the pattern of the acoustic field has a bottle shape.18. The system of any one of aspects 1-17, further comprising: a stage configured to move the chamber relative to the ultrasonic transducer or move the ultrasonic transducer relative to the chamber, wherein the processor is in electronic communication with the stage, and wherein the processor is further configured to control the stage to move the chamber relative to the ultrasonic transducer or move the ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to manipulate the cell within the chamber.19. The system of aspect 18, wherein the chamber includes one or more microchambers in fluid communication with the chamber, and wherein the processor is further configured to control the stage to move the chamber relative to the ultrasonic transducer or move ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to move the cell from the chamber to one of the one or more microchambers. 20. A method comprising disposing electrodes on a substrate to form an ultrasonic transducer; disposing a chamber on the ultrasonic transducer; and generating, with the ultrasonic transducer, acoustic waves directed to the chamber to manipulate a cell within the chamber, wherein the acoustic waves generate an acoustic field that has a pattern defined by a shape of the electrodes disposed on the substrate.21. The method of aspect 20, further comprising partially filling the chamber with a liquid solution.22. The method of aspect 21, further comprising covering the liquid solution with an immiscible liquid.23. The method of any of aspects 20-22, wherein the electrodes are intertwined. 24. The method of any of aspects 20-23, wherein the electrodes are micromachined.25. The method of any of aspects 20-24, wherein the electrodes are piezoelectric. 26. The method of any of aspects 20-24, wherein the electrodes are capacitive. 27. The method of any one of aspects 20-26, wherein the chamber includes one or more microchambers in fluid communication with the chamber, and wherein the acousticfield is configured to assist in moving the cell from the chamber to one of the microchambers.28. The method of any one of aspects 20-27, wherein the chamber is freely movable on the substrate.29. The method of any one of aspects 20-28, wherein the substrate is formed from glass.30. The method of aspect 29, wherein the electrodes are embedded within the glass.31. The method of any one of aspects 20-30, wherein the substrate is disposed between the electrodes and the chamber.32. The method of any one of aspects 20-31, wherein the acoustic field is configured to translate or rotate the cell within the chamber.33. The method of any one of aspects 20-31, wherein the acoustic field is configured to translate and rotate the cell within the chamber.34. The method of any one of aspects 20-33, wherein the pattern of the acoustic field has a ring shape.35. The method of any one of aspects 20-33, wherein the pattern of the acoustic field has a polygonal shape.36. The method of any one of aspects 20-33, wherein the pattern of the acoustic field has a petal shape.37. The method of any one of aspects 20-33, wherein the pattern of the acoustic field has a crescent shape.38. The method of any one of aspects 20-33, wherein the pattern of the acoustic field has a bottle shape.

Claims

What is claimed is:

1. A system, comprising:an ultrasonic transducer comprising electrodes;a chamber configured to contain a cell; anda processor in electronic communication with the ultrasonic transducer, wherein the processor is configured to control the ultrasonic transducer to generate acoustic waves directed to the cell to manipulate the cell within the chamber, and wherein the acoustic waves generate an acoustic field that has a pattern defined by a shape of the electrodes of the ultrasonic transducer.

2. The system of claim 1, wherein the electrodes are intertwined.

3. The system of claim 1, wherein the electrodes are micromachined.

4. The system of claim 3, wherein the electrodes are piezoelectric.

5. The system of claim 3, wherein the electrodes are capacitive.

6. The system of claim 1, wherein the chamber is disposed on the ultrasonic transducer.

7. The system of claim 6, wherein the chamber is movable with respect to the ultrasonic transducer.

8. The system of claim 7, further comprising:a glass substrate disposed between the ultrasonic transducer and the chamber and that supports motion between the chamber and the ultrasonic transducer.

9. The system of claim 1, wherein the chamber is open.

10. The system of claim 9, wherein the chamber contains two immiscible fluids.

11. The system of claim 1, wherein the chamber is closed.

12. The system of claim 11, wherein the chamber contains sound-absorbing material.

13. The system of claim 1, wherein the pattern of the acoustic field has a ring shape.

14. The system of claim 1, wherein the pattern of the acoustic field has a polygonal shape.

15. The system of claim 1, wherein the pattern of the acoustic field has a petal shape.

16. The system of claim 1, wherein the pattern of the acoustic field has a crescent shape.

17. The system of claim 1, wherein the pattern of the acoustic field has a bottle shape.

18. The system of claim 1, further comprising:a stage configured to move the chamber relative to the ultrasonic transducer or move the ultrasonic transducer relative to the chamber,wherein the processor is in electronic communication with the stage, and wherein the processor is further configured to control the stage to move the chamber relative to the ultrasonic transducer or move the ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to manipulate the cell within the chamber.

19. The system of claim 18, wherein the chamber includes one or more microchambers in fluid communication with the chamber, and wherein the processor is further configured to control the stage to move the chamber relative to the ultrasonic transducer or move ultrasonic transducer relative to the chamber to direct the acoustic field to the cell to move the cell from the chamber to one of the one or more microchambers.

20. A method, comprising:disposing electrodes on a substrate to form an ultrasonic transducer;disposing a chamber on the ultrasonic transducer; andgenerating, with the ultrasonic transducer, acoustic waves directed to the chamber to manipulate a cell within the chamber,wherein the acoustic waves generate an acoustic field that has a pattern defined by ashape of the electrodes disposed on the substrate.

21. The method of claim 20, further comprising:partially filling the chamber with a liquid solution.

22. The method of claim 21, further comprising:covering the liquid solution with an immiscible liquid.

23. The method of claim 20, wherein the electrodes are intertwined.

24. The method of claim 20, wherein the electrodes are micromachined.

25. The method of claim 24, wherein the electrodes are piezoelectric.

26. The method of claim 24, wherein the electrodes are capacitive.

27. The method of claim 20, wherein the chamber includes one or more microchambers in fluid communication with the chamber, and wherein the acoustic field is configured to assist in moving the cell from the chamber to one of the microchambers.

28. The method of claim 20, wherein the chamber is freely movable on the substrate.

29. The method of claim 28, wherein the substrate is formed from glass.

30. The method of claim 29, wherein the electrodes are embedded within the glass.

31. The method of claim 20, wherein the substrate is disposed between the electrodes and the chamber.

32. The method of claim 20, wherein the acoustic field is configured to translate or rotate the cell within the chamber.

33. The method of claim 20, wherein the acoustic field is configured to translate androtate the cell within the chamber.

34. The method of claim 20, wherein the pattern of the acoustic field has a ring shape.

35. The method of claim 20, wherein the pattern of the acoustic field has a polygonal shape.

36. The method of claim 20, wherein the pattern of the acoustic field has a petal shape.

37. The method of claim 20, wherein the pattern of the acoustic field has a crescent shape.

38. The method of claim 20, wherein the pattern of the acoustic field has a bottle shape.

Images