Apparatus and method for manipulating aggregation of cells and particles in droplets based on acoustic waves

Abstract

The application provides a device and a method for manipulating cells and particles in a droplet based on acoustic waves. The device comprises a cavity for accommodating the droplet and a transducer for generating acoustic waves. The transducer is distributed around the cavity. The transducer can modulate the received acoustic wave signals to form a point-like node array in the middle of the cavity. The method uses the acoustic radiation force gradient generated by the acoustic potential well in the point-like node array to capture the droplet and manipulate the particles in the droplet, thereby realizing the manipulation of the cells or particles in the droplet and being suitable for efficiently constructing an in-vitro model.

Description

Technical Field

[0001] This application relates to the field of acoustic microfluidics, and in particular to a device and method for manipulating the aggregation of cells and particles within droplets based on acoustic waves. Background Technology

[0002] In fields such as tissue engineering and biomedicine, the construction of specific tissue structures and models is of great significance. For example, in the field of personalized medicine for tumor diseases with significant individual differences, constructing tumor models based on in vivo patient samples for subsequent culture and drug screening can greatly improve the therapeutic effectiveness and efficiency of drug combination strategies, maximizing prognosis and minimizing side effects. Therefore, in vitro model construction technologies based on various techniques have seen significant development in recent years. The construction of specific tissue structures and models requires efficient cell assembly and manipulation. Currently, the main technologies for efficient cell assembly and manipulation are traditional culture plates or microfluidic technology.

[0003] Compared to traditional culture plates or microfluidic technologies, droplet-based cell culture and testing technology has emerged as a high-throughput and high-efficiency platform. This is because droplet technology provides an independent three-dimensional environment, mimicking the extracellular matrix environment in vivo, and allows for high-throughput mass production, significantly improving efficiency and demonstrating immense potential for industrialization and clinical applications. Efficiently assembling or actively manipulating dispersed cells or substances within droplets to construct specific structures has always been a technical challenge in droplet-based in vitro model building. Traditional contact techniques cannot operate within independent droplets, while non-contact optical tweezers and magnetic methods suffer from low efficiency or require additional heterogeneous material labeling. Acoustic wave technology offers advantages such as non-contact, bio-friendly, and high efficiency. Although acoustic wave technology has been used to manipulate various cells, existing manipulation methods often manipulate droplets or particles simultaneously, making it difficult to achieve efficient in vitro model construction. Summary of the Invention

[0004] This application provides a device and method for manipulating the aggregation of cells and particles within droplets based on sound waves, in order to solve the aforementioned technical problems.

[0005] A device for manipulating the aggregation of cells and particles within a droplet based on sound waves includes a cavity for containing the droplet and transducers; the transducers are distributed around the cavity; the transducers can modulate the received sound wave signals, which are interfered with in the center of the cavity to form a point-like array of wave nodes sound field.

[0006] Furthermore, the transducer is an interdigital transducer or a piezoelectric ceramic transducer.

[0007] Furthermore, the device for controlling the aggregation of cells and particles within a droplet based on sound waves includes a lithium niobate substrate, on which four interdigital transducers are disposed orthogonally, and a cavity for accommodating the droplet is disposed between the four interdigital transducers.

[0008] Furthermore, the lithium niobate substrate is a 128° Y-cut lithium niobate substrate, and the interdigital transducer forms angles of 45° and 135° with the crystal axis Y-axis of the lithium niobate substrate, respectively.

[0009] Furthermore, the cavity is rectangular, and the four sides of the rectangular shape are piezoelectric ceramic transducers.

[0010] A method for manipulating the aggregation of cells and particles within droplets based on sound waves, using the aforementioned apparatus, the method comprising:

[0011] To prepare droplets containing cells or particles;

[0012] The emulsion is placed in the cavity, and the sound field generated by the transducer acts on the emulsion. The acoustic radiation force gradient generated by the acoustic potential trap in the point-shaped node array of the sound field on the droplets and the cells or particles is used to realize the aggregation and manipulation of the cells or particles in the droplets.

[0013] Furthermore, this includes altering the aggregation structure of cells or particles within the droplet by adjusting the frequency, intensity, and duration of the acoustic signal emitted by the transducer.

[0014] Furthermore, the preparation of droplets containing cells or particles is an emulsion comprising an oil phase and aqueous droplets dispersed in the oil phase, wherein the aqueous droplets contain cells or particles.

[0015] Furthermore, the aqueous droplets are photocurable hydrogel droplets.

[0016] Further, the oil phase is isopropyl palmitate; and / or the oil phase contains 3 wt% polypropylene glycol-10 (EM180).

[0017] The technical solutions provided in this application have the following advantages compared with the prior art:

[0018] The transducer described in this application can modulate the received acoustic wave signal, which is then interfered with in the middle of the cavity to form a point-like node array acoustic field. By utilizing the acoustic potential trap in the point-like node array of the acoustic field to generate an acoustic radiation force gradient on the droplets and the cells or particles, the droplets are captured while the particles within the droplets are manipulated, thereby achieving aggregation and manipulation of the cells or particles within the droplets. This is suitable for efficiently constructing in vitro models.

[0019] This application allows for flexible adjustment of the structure and size of cells or particles by controlling the frequency, intensity, and duration of the acoustic field, enabling active high-throughput aggregation and assembly of various cells or particles in droplets. It has broad application prospects in life sciences, droplet microfluidics, three-dimensional cell culture, and tumor micromodel construction.

[0020] This application uses acoustic wave technology, which is non-contact and has the advantages of being bio-friendly and highly efficient. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the device for controlling the aggregation of cells and particles in a droplet based on sound waves, provided in Embodiment 1 of this application. In the diagram, k1, k2, k3, and k4 are interdigitated transducers in four directions, 1 is a lithium niobate substrate, and 2 is a PDMS cavity.

[0024] Figure 2 This is a schematic diagram of a device for controlling the aggregation of cells and particles within a droplet based on sound waves, provided in Embodiment 2 of this application. In this diagram, k1, k2, k3, and k4 are piezoelectric ceramic transducers in four directions, and 1 is a cavity structure.

[0025] Figure 3 This is a schematic diagram of the device for manipulating the aggregation of cells and particles within a droplet based on sound waves, provided in Embodiment 3 of this application.

[0026] Figure 4 This is a schematic diagram of the acoustic potential trap array in the acoustic field generated by the acoustic wave-based device for controlling the aggregation of cells and particles within droplets, as provided in the embodiments of this application, controlling the aggregation of droplets and the substances therein.

[0027] Figure 5 This is a typical sound field distribution diagram generated by the acoustic wave-based device for manipulating the aggregation of cells and particles within droplets, based on an embodiment of this application (the sound field structure diagram generated by the acoustic bulk wave and surface acoustic wave device when the same frequency and amplitude sinusoidal wave signals are applied to the four transducers k1, k2, k3, and k4, with nodes in a grid pattern).

[0028] Figure 6This is a typical sound field distribution diagram generated by the acoustic wave-based device for manipulating the aggregation of cells and particles within droplets, based on an embodiment of this application (the sound field structure diagram generated by the acoustic bulk wave and surface acoustic wave device when the frequencies of the sinusoidal waves applied by k1 and k3 differ slightly from those applied by k2 and k4, with circular nodes).

[0029] Figure 7 Microscopic images of cell cluster structures in droplets constructed using the aggregation manipulation method provided in Embodiment 4 of this application (1 shows the structure of cells scattered in the droplet before the application of acoustic force; 2 shows the relatively loose cell cluster structure constructed in the droplet; 3 shows the tightly connected cell spheroid structure constructed in the droplet).

[0030] Figure 8 Microscopic images of cell cluster structures in droplets constructed using the aggregation manipulation method provided in Embodiment 5 of this application (1 shows the structure of cells randomly distributed in the droplet before the application of acoustic force; 2 shows the relatively loose cell cluster structure constructed in the droplet; 3 shows the tightly connected cell spheroid structure constructed in the droplet).

[0031] Figure 9 Micrograph of cell cluster structure in droplets constructed using the aggregation manipulation method provided in Embodiment 6 of this application.

[0032] Figure 10 Micrograph of cell cluster structure in droplets constructed using the aggregation manipulation method provided in Embodiment 7 of this application.

[0033] Figure 11 Micrograph of cell cluster structure in droplets constructed using the aggregation manipulation method provided in Embodiment 8 of this application.

[0034] Figure 12 This is a typical sound field distribution diagram of the acoustic wave-based device for manipulating the aggregation of cells and particles within droplets provided in Embodiment 3 of this application (the sound field structure diagram generated by the acoustic bulk wave and surface acoustic wave device when six transducers apply sinusoidal wave signals of the same frequency and amplitude, with nodes in a grid pattern). Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0036] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0037] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0038] This application provides a device for manipulating the aggregation of cells and particles within a droplet based on sound waves, including a cavity for containing the droplet and transducers; the transducers are distributed around the cavity; the transducers can modulate the received sound wave signals, which are interfered with in the center of the cavity to form a point-like array of wave nodes sound field.

[0039] The cavity and the arrangement of transducers distributed around it are selected from cubes, cuboids, triangular prisms, specific hexagons, etc., but are not limited to these shapes, as long as they can form a point-like nodal array sound field in the center of the cavity. The acoustic radiation force in the nodes of the point-like nodal array sound field all points towards the center. The acoustic potential traps in the point-like nodal array generate an acoustic radiation force gradient on the droplets and cells or particles, simultaneously capturing the droplets and manipulating the particles within them. This can aggregate cells or organelles into clusters, achieving aggregation and manipulation of cells or particles within the droplets, suitable for efficiently constructing in vitro models. Existing devices typically cannot form this point-like nodal array sound field; they can only form sound fields of other shapes, and can only arrange cells into certain shapes, making it difficult to achieve high-throughput, rapid, non-destructive manipulation of cells or particles. Existing acoustic tweezers are difficult to manipulate particles in droplets, and often only manipulate droplets or particles. However, the device of this application generates a point-like array of acoustic fields, producing acoustic junctions that can capture droplets, allowing manipulation of droplets and particles within droplets at the same time.

[0040] In one embodiment, the transducer is an interdigital transducer or a piezoelectric ceramic transducer, but is not limited thereto.

[0041] In one embodiment, the acoustic wave-based device for manipulating the aggregation of cells and particles within a droplet includes a lithium niobate substrate. Four orthogonal interdigital transducers are disposed on the lithium niobate substrate, with a cavity for accommodating the droplet located between the four interdigital transducers. A sinusoidal wave signal of a corresponding frequency is applied through the four orthogonal interdigital transducers, thereby generating an interference sound field in the central region of the cavity. Due to the use of a two-dimensional sound field, by changing the cavity size, a large-area sound field and an array of thousands of point nodes can be generated simultaneously, enabling high-throughput manipulation of cells or particles within the droplet. Furthermore, by adjusting the signal frequency and the transducer combination, nodes with different structures can be generated, thereby achieving the assembly of cells or particles with different structures within the droplet, such as spherical and linear shapes, thus meeting different downstream model construction requirements.

[0042] In one embodiment, the lithium niobate substrate is a 128° Y-cut lithium niobate substrate, and the interdigital transducer forms angles of 45° and 135° with the crystal axis Y-axis of the lithium niobate substrate, respectively.

[0043] In one embodiment, the cavity is rectangular, with piezoelectric ceramic transducers on all four sides. A sinusoidal signal is applied through the four piezoelectric ceramic transducers, resulting in an interference sound field structure in the center of the cavity. By controlling the frequencies of the signals perpendicular to two directions, a grid-like or square lattice-like node array can be obtained. Within the nodes, water droplets dispersed in the oil phase, due to their higher density than the oil phase, experience a radiating force pointing towards the node center. Simultaneously, cells or particles within the droplets also experience a radiating force pointing towards the node center due to their higher density than the surrounding water phase. Therefore, under the influence of the node array in the sound field, the droplets can be arranged within the array, and the material within the droplets also aggregates according to the shape of the nodes, forming three-dimensional clusters and spheres. Furthermore, by controlling the sound field intensity and the application time, the density and size of the aggregated clusters can be controlled.

[0044] In one embodiment, the cavity material is polydimethylsiloxane (PDMS).

[0045] This application provides a method for manipulating the aggregation of cells and particles within droplets based on sound waves. Using the aforementioned apparatus, the method includes:

[0046] To prepare droplets containing cells or particles;

[0047] The emulsion is placed in the cavity, and the sound field generated by the transducer acts on the emulsion. The acoustic radiation force gradient generated by the acoustic potential trap in the point-shaped node array of the sound field on the droplets and the cells or particles is used to realize the aggregation and manipulation of the cells or particles in the droplets.

[0048] In one embodiment, the method includes altering the aggregation structure of cells or particles within the droplet by adjusting the frequency, intensity, and duration of an acoustic signal emitted by the transducer. Frequency and intensity affect the droplet's trapping effect and the degree of particle aggregation within the droplet. Different intensities also affect the speed of particle assembly within the droplet, and the duration of signal application also affects the degree of particle aggregation. Conversely, the degree of particle dispersion within the droplet can be controlled by adjusting the application time and intensity.

[0049] In one embodiment, the preparation of droplets containing cells or particles is an emulsion comprising an oil phase and aqueous droplets dispersed in the oil phase, wherein the aqueous droplets contain cells or particles.

[0050] In one embodiment, the aqueous droplet is a photocurable hydrogel droplet, which allows the material to be manipulated and assembled using sound waves before cross-linking and curing. Once assembled, the structure can be fixed by light curing, facilitating downstream applications.

[0051] In one embodiment, the oil phase is isopropyl palmitate; and / or 3 wt% polypropylene glycol-10 (EM180) is added to the oil phase to stabilize the droplet morphology and prevent them from merging.

[0052] Example 1

[0053] like Figure 1 As shown, a device for manipulating the aggregation of cells and particles within a droplet based on sound waves is a surface acoustic wave (SAW) device. The SAW device includes a lithium niobate substrate 1 with a 128° Y-section. Four interdigital transducers (IDTs) (k1, k2, k3, k4) are disposed on the lithium niobate substrate 1. The interdigital transducers form angles of 45° and 135° with the crystal axis Y-axis of the lithium niobate substrate 1, respectively. A cavity for accommodating the droplet is disposed between the four interdigital transducers.

[0054] Example 2

[0055] like Figure 2 As shown, a device for manipulating the aggregation of cells and particles within a droplet based on sound waves is a bulk acoustic wave device. The bulk acoustic wave device includes a cuboid cavity 1 and four piezoelectric ceramic transducers (k1, k2, k3, k4) respectively disposed around the cuboid cavity 1.

[0056] Example 3

[0057] like Figure 3 As shown, a device for manipulating the aggregation of cells and particles within a droplet based on sound waves includes a lithium niobate substrate 1, on which six piezoelectric ceramic transducers are disposed, the six piezoelectric ceramic transducers forming a regular hexagonal cavity.

[0058] Example 4

[0059] A method for manipulating the aggregation of cells and particles within droplets based on sound waves, comprising:

[0060] (1) Aqueous droplets with a size of 120 μm were prepared from 6 wt% GelMA 60 gel solution using a T-type microfluidic chip. 15 μm polystyrene microspheres were dispersed within the droplets. The oil phase consisted of isopropyl palmitate with 3 wt% Degussa polypropylene glycol-10 (EM180). The aqueous droplets were dispersed in the oil phase to obtain an emulsion. Figure 7-1 The image shows a micrograph of the particles agglomerated into relatively large, loosely structured droplets at this point.

[0061] (2) Cell assembly was performed using the acoustic wave-based device for manipulating the aggregation of cells and particles within droplets as described in Application Example 2. The cavity of the acoustic wave-based device for manipulating the aggregation of cells and particles within droplets is a 1cm*1cm*1cm cuboid, the piezoelectric ceramic transducer is 1cm*0.6cm in size, and the resonant frequency is 3.75MHz. The piezoelectric ceramic transducer is attached to the periphery of the cavity. The emulsion is injected into the cavity through a syringe to make the liquid surface level.

[0062] (3) Then, a 3.75MHz sine wave is generated by the function signal generator, and after passing through the power amplifier, a signal with an amplitude of 15Vpp is output and applied to the piezoelectric ceramic transducers k1 and k3; at the same time, a 3.7MHz sine wave is generated by the signal generator, and after passing through the power amplifier, a signal with an amplitude of 15Vpp is output and applied to the piezoelectric ceramic transducers k2 and k4, which will generate a circular node array in the central region of the cavity;

[0063] (4) When the sound wave is applied for 10 seconds, the water droplets are pushed down the region of the wave node by the sound field, and the particles in the droplets are gradually gathered under the action of sound force.

[0064] (5) If the sound wave continues to be applied for 20 seconds, the particles further aggregate at the nodes, forming a more compact spherical structure (e.g., Figure 7-2 (as shown);

[0065] (6) Continuing to increase the sound wave application time to 40 seconds, the particles further aggregate, forming a very dense spherical structure (e.g., Figure 7-3 (As shown).

[0066] After cell assembly within the droplets is complete, the droplets are irradiated with 405 nm light for 20 seconds to cross-link the gel solution, completing gelation and preserving the cell sphere structure. The gel microspheres are then transferred from the oil phase to the aqueous phase by centrifugation, allowing for further long-term culture and drug testing.

[0067] Example 5

[0068] A method for manipulating the aggregation of cells and particles within droplets based on sound waves, comprising:

[0069] (1) Aqueous droplets with a size of 120 μm were prepared from 6 wt% GelMA 60 gel solution using a T-type microfluidic chip. 15 μm polystyrene microspheres were dispersed within the droplets. The oil phase consisted of isopropyl palmitate with 3 wt% Degussa polypropylene glycol-10 (EM180). The aqueous droplets were dispersed in the oil phase to obtain an emulsion (droplet micrograph shown in Figure 1). Figure 8-1 (as shown);

[0070] (2) Cell assembly was performed using the acoustic wave-based aggregation manipulation device for cells and particles within droplets as described in Application Example 1. The surface acoustic wave device includes a lithium niobate substrate with a 128° Y-cut section. Four interdigital transducers orthogonally arranged on the lithium niobate substrate are provided. The interdigital transducers form angles of 45° and 135° with the crystal axis Y-axis of the lithium niobate substrate, respectively. A cavity for dissolving the emulsion is provided in the middle of the four interdigital transducers. The cavity is a 1cm*1cm*1cm PDMS cuboid cavity. The emulsion is injected into the cavity through a syringe to make the liquid surface flush.

[0071] (3) Then, a 3.75MHz sine wave is generated by the function signal generator, and after passing through the power amplifier, a signal with an amplitude of 15Vpp is output and applied to the interdigital transducers k1 and k3; at the same time, a 3.7MHz sine wave is generated by the signal generator, and after passing through the power amplifier, a signal with an amplitude of 15Vpp is output and applied to the interdigital transducers k2 and k4, which will generate a circular node array in the central region of the cavity;

[0072] (4) When the sound wave is applied continuously for 10 seconds, the water droplets are pushed down the region of the wave node by the sound field, and the particles in the droplets gradually gather under the action of sound force (e.g. Figure 8-2 (as shown);

[0073] (5) If the sound wave continues to be applied for 40 seconds, the particles aggregate to form a very dense spherical structure (e.g., Figure 8-3 (As shown).

[0074] After cell assembly within the droplets is complete, the droplets are irradiated with 405 nm light for 20 seconds to cross-link the gel solution, completing gelation and preserving the cell sphere structure. The gel microspheres are then transferred from the oil phase to the aqueous phase by centrifugation, allowing for further long-term culture and drug testing.

[0075] Example 6

[0076] The intensity of the sinusoidal signal applied to transducers k1, k2, k3, and k4 in Example 4 was changed to 3.0 Vpp, and the application time was 40 s. The particle aggregation situation is as follows. Figure 9 As shown.

[0077] Example 7

[0078] The intensity of the sinusoidal signal applied to transducers k1, k2, k3, and k4 in Example 4 was changed to 3.5 Vpp, and the application time was 40 s. The particle aggregation situation is as follows. Figure 10 As shown.

[0079] Example 8

[0080] The intensity of the sinusoidal signal applied to transducers k1, k2, k3, and k4 in Example 4 was changed to 4.0 Vpp, and the application time was 40 s. The particle aggregation situation is as follows. Figure 11 As shown.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A device for manipulating the aggregation of cells and particles within droplets based on sound waves, characterized in that, The device includes a cavity for containing droplets and transducers. The transducers are distributed around the cavity. The transducers can modulate the received acoustic wave signals, which are then interfered with in the center of the cavity to form a point-like node array acoustic field. The acoustic radiation force in the nodes of the point-like node array acoustic field all points towards the center. The sound field of the point-like node array is used to form an acoustic potential trap. The acoustic potential trap generates an acoustic radiation force gradient on both the droplets and the cells or particles. While capturing the droplets, it also manipulates the particles inside the droplets, agglomerating cells or organelles into clusters, thereby achieving aggregation and manipulation of cells or particles within the droplets. The invention includes a lithium niobate substrate, on which four orthogonal interdigital transducers are disposed, and a cavity for accommodating the droplet is disposed between the four interdigital transducers. The lithium niobate substrate is a 128° Y-cut lithium niobate substrate, and the interdigital transducer forms angles of 45° and 135° with the crystal axis Y-axis of the lithium niobate substrate, respectively. The cavity material is polydimethylsiloxane; By applying sinusoidal signals of corresponding frequencies through four mutually orthogonal interdigital transducers, an interference sound field is obtained in the central region of the cavity, generating a large-area sound field and an array of thousands of point nodes, enabling high-throughput manipulation of cells or particles in droplets; by adjusting the frequency of the signal and the combination of transducers, nodes with different structures are generated, enabling the assembly of cells or particles with different structures in droplets.

2. A method for manipulating the aggregation of cells and particles within droplets based on sound waves, characterized in that, The method using the apparatus of claim 1 comprises: To prepare droplets containing cells or particles; The droplet is placed in the cavity, and the sound field generated by the transducer acts on the droplet. The acoustic radiation force gradient generated by the acoustic potential trap in the point-shaped node array of the sound field on the droplet and the cell or particle is used to capture the droplet and manipulate the particles in the droplet at the same time, and gather the cells or organelles into clusters to achieve the aggregation and manipulation of the cells or particles in the droplet. This includes altering the aggregation structure of cells or particles within the droplet by adjusting the frequency, intensity, and duration of the acoustic signal emitted by the transducer.

3. The method according to claim 2, characterized in that, The preparation of droplets containing cells or particles involves configuring an emulsion comprising an oil phase and aqueous droplets dispersed in the oil phase, wherein the aqueous droplets contain cells or particles.

4. The method according to claim 3, characterized in that, The aqueous droplets are photocurable hydrogel droplets.

5. The method according to claim 3, characterized in that, The oil phase is isopropyl palmitate; and / or the oil phase contains 3 wt% polypropylene glycol-10.

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