Culture structure, culture method and culture chip

Abstract

The embodiment of the invention provides a culture structure, a culture method and a culture chip, and relates to but is not limited to the technical field of biology. The culture structure comprises a culture plate and a vibration signal generating part, and the vibration signal generating part is arranged on the culture plate; the culture plate comprises a plurality of containing structures, the containing structures are arranged to contain culture solutions, and each containing structure comprises a first containing cavity and at least one second containing cavity located at the bottom of the first containing cavity; the vibration signal generating part is arranged to generate a vibration signal, the orthographic projection of the vibration signal generating part on the culture plate is not overlapped with the plurality of accommodating structures, and the vibration signal is arranged to drive the culture solution in the plurality of accommodating structures to move. According to the culture structure, a culture solution can move, and oxygen and nutrients in the culture solution are balanced; in addition, the culture structure can avoid the transfer step of the to-be-cultured object in different growth stages, and avoid damage to the to-be-cultured object due to transfer.

Description

Technical Field

[0001] The present application relates to but is not limited to the field of biotechnology, and in particular to a culture structure and culture method, and a culture chip. Background Art

[0002] In recent years, with the development of cell biology and tissue engineering, three-dimensional cell models are gradually replacing traditional two-dimensional cell models. Organoids, as a new type of three-dimensional in vitro research model, are self-assembled by stem cells in vitro and grow and develop into three-dimensional aggregates similar to the structure and function of human tissues or organs, such as brain organoids, vascular organoids, liver organoids, kidney organoids and tumor organoids. Summary of the invention

[0003] To achieve the above objectives, the embodiments of the present application adopt the following technical solutions:

[0004] In a first aspect, an embodiment of the present application provides a culture structure, which includes: a culture plate and a vibration signal generating component, wherein the vibration signal generating component is disposed on the culture plate;

[0005] The culture plate comprises: a plurality of containing structures, the containing structures are configured to contain culture fluid, the containing structures comprise a first containing cavity and at least one second containing cavity located at the bottom of the first containing cavity; the volume of the first containing cavity is greater than the volume of the second containing cavity;

[0006] The vibration signal generating component is configured to generate a vibration signal, the orthographic projection of the vibration signal generating component on the culture plate does not overlap with the multiple containing structures, and the vibration signal is configured to drive the culture fluid in the multiple containing structures to move.

[0007] In at least one embodiment of the present application, the culture plate comprises a main structure and an extension portion located on at least one side of the main structure, the plurality of accommodating structures are arranged on the main structure, and the vibration signal generating component is arranged on the extension portion;

[0008] The extension part is connected to the main structure, and the vibration signal acts on the main structure through the extension part.

[0009] In at least one embodiment of the present application, the vibration signal generating component includes at least two piezoelectric transducers, the at least two piezoelectric transducers are arranged on the extension portion, and the propagation directions of the vibration signals generated by the at least two piezoelectric transducers intersect in the plane where the culture plate is located.

[0010] In at least one embodiment of the present application, the vibration signal generating component includes two piezoelectric transducers, and within the plane where the culture plate is located, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers is greater than or equal to 80° and less than or equal to 100°.

[0011] In at least one embodiment of the present application, the culture plate includes two extension parts, the two extension parts are located on two adjacent sides of the main structure, and the two extension parts are connected.

[0012] In at least one embodiment of the present application, the culture plate comprises a first surface, and the first accommodating cavity and the second accommodating cavity both comprise a bottom and an opening; the plane where the opening of the first accommodating cavity is located is coplanar with the first surface, and the plane where the opening of the second accommodating cavity is located is coplanar with the bottom of the first accommodating cavity;

[0013] Wherein, along a direction perpendicular to the first surface, a distance from an opening to a bottom of the first accommodating cavity is greater than a distance from an opening to a bottom of the second accommodating cavity.

[0014] In at least one embodiment of the present application, the second accommodating cavity includes a second axis, and an angle between the second axis of the second accommodating cavity and the first surface is greater than 0° and less than or equal to 90°.

[0015] In at least one embodiment of the present application, the angle between the second axis of the second accommodating cavity and the first surface is approximately 90°, and the area of ​​the orthographic projection of the bottom of the second accommodating cavity on the first surface is smaller than the area of ​​the region enclosed by the orthographic projection of the edge of the opening of the second accommodating cavity on the first surface.

[0016] In at least one embodiment of the present application, the angle between the second axis of the second accommodating cavity and the first surface is greater than 0° and less than 90°, and the area of ​​the orthographic projection of the bottom of the second accommodating cavity on the first surface is less than or equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening of the second accommodating cavity on the first surface.

[0017] In at least one embodiment of the present application, the first accommodating cavity includes a first axis, and the angle between the first axis of the first accommodating cavity and the first surface is approximately equal to the angle between the second axis of the second accommodating cavity and the first surface; the area of ​​the region enclosed by the orthographic projection of the bottom outer contour of the first accommodating cavity on the first surface is less than or equal to the area of ​​the region enclosed by the orthographic projection of the edge of the opening of the first accommodating cavity on the first surface.

[0018] In at least one embodiment of the present application, the edges of the openings of the first accommodating cavity and the second accommodating cavity are arcs, the outer contours of the bottoms of the first accommodating cavity and the second accommodating cavity are arcs, and the side walls of the first accommodating cavity and the second accommodating cavity are curved surfaces.

[0019] In at least one embodiment of the present application, the edges of the openings of the first accommodating cavity and the second accommodating cavity are both circular, and the outer contours of the bottoms of the first accommodating cavity and the second accommodating cavity are both circular;

[0020] The first accommodating chamber and the second accommodating chamber each include two first areas, the intensity of the vibration signal received by the culture fluid in the first area is greater than the intensity of the vibration signal received by the culture fluid in other areas, the circle includes a first diameter and a second diameter, and the first diameter and the second diameter are orthogonal; the extension direction of the first diameter is consistent with the propagation direction of the vibration signal generated by one of the piezoelectric transducers, and the extension direction of the second diameter is consistent with the propagation direction of the vibration signal generated by the other piezoelectric transducer;

[0021] The two first regions are both located on the same side of the first diameter, and the orthographic projections of the two first regions on the first surface are symmetrically distributed about the second diameter.

[0022] In at least one embodiment of the present application, along a direction perpendicular to the first surface, a distance from the opening of the first accommodating cavity to the bottom ranges from 250 μm to 1000 μm, and a distance from the opening of the second accommodating cavity to the bottom ranges from 200 μm to 300 μm;

[0023] The diameter of the figure enclosed by the outer contour of the bottom of the first accommodating cavity is in the range of 3 mm to 5 mm, and the diameter of the figure enclosed by the outer contour of the bottom of the second accommodating cavity is in the range of 600 μm to 1000 μm.

[0024] In at least one embodiment of the present application, the piezoelectric transducer input voltage signal is 0.3V to 0.8V, and the frequency of the input voltage signal is 150kHz to 400kHz or 700kHz to 1200kHz.

[0025] In at least one embodiment of the present application, the culture structure further includes an enclosure structure, a partial area of ​​the enclosure structure is located between the main structure and the extension portion, and the enclosure structure is arranged around the multiple containing structures.

[0026] In at least one embodiment of the present application, the thickness of the culture plate is 1.8 mm to 3.5 mm, the dimension of the enclosure structure along the thickness direction of the culture plate is 7 mm to 12 mm, and the dimension of the enclosure structure along the direction parallel to the plane of the culture plate is 1.5 mm to 3.5 mm.

[0027] In a second aspect, an embodiment of the present application provides a culture method, which is applied to the culture structure described in any one of the first aspects, wherein the culture structure comprises a culture plate and a vibration signal generating component, and the vibration signal generating component is arranged on the culture plate; the culture plate comprises a plurality of containing structures, and the containing structures are arranged to contain culture liquid, and the containing structures comprise a first containing cavity and at least one second containing cavity located at the bottom of the first containing cavity; the orthographic projection of the vibration signal generating component on the culture plate does not overlap with the plurality of containing structures; the method comprises:

[0028] generating a vibration signal;

[0029] The vibration signal drives the culture fluid in the multiple containing structures to move.

[0030] In at least one embodiment of the present application, the vibration signal drives the culture solution in the plurality of containing structures to move, including:

[0031] In the first stage, the second containing chamber is configured to also contain the object to be cultured, the frequency of the voltage signal input by the vibration signal generating component is 150kHz to 400kHz, and the vibration signal drives the object to be cultured and the culture liquid to move;

[0032] In the second stage, the first containing chamber is configured to contain the object to be cultured, the frequency of the voltage signal input to the vibration signal generating component is 700kHz to 1200kHz, and the vibration signal drives the object to be cultured and the culture fluid to move.

[0033] In a third aspect, an embodiment of the present application provides a culture chip, which includes a culture structure as described in any one of the first aspects.

[0034] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 and Figure 2 A schematic diagram of the three-dimensional structure of two culture structures provided in the embodiments of the present application;

[0037] Figure 3-Figure 9 for Figure 2 Schematic diagram of seven cross-sectional structures along the M1M2 direction;

[0038] Fig.10 for Figure 2 Schematic diagram of the cross-sectional structure along the M3M4 direction;

[0039] Fig.11 A schematic diagram of simulation results of an acoustic flow field of a culture structure provided in an embodiment of the present application;

[0040] Fig.12 A schematic diagram of simulation results of an acoustic flow field of another culture structure provided in an embodiment of the present application;

[0041] Fig.13 A schematic diagram of simulation results of particle tracking trajectories in an acoustic flow field of a culture structure provided in an embodiment of the present application;

[0042] Fig.14A , Fig. 14B and Fig. 14C Schematic diagram of top view of three containing structures provided in the embodiments of the present application. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0044] In the embodiments of the present application, words such as "first", "second", "third", and "fourth" are used to distinguish between identical or similar items with basically the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of the present application and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.

[0045] In the embodiments of the present application, the terms "upper", "lower", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0046] In the description of the specification, the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example" or "some examples" are intended to indicate that a specific feature, structure, material or characteristic associated with the embodiment or example is included in at least one embodiment or example of the present application. The schematic representation of the above terms does not necessarily refer to the same embodiment or example. In addition, the specific features, structures, materials or characteristics may be included in any one or more embodiments or examples in any appropriate manner.

[0047] In the embodiments of the present application, “plurality” means two or more, and “at least one” means one or more, unless otherwise clearly defined.

[0048] The features such as "parallel", "perpendicular", and "same" used in the embodiments of the present application include the features such as "parallel", "perpendicular", and "same" in a strict sense, as well as the cases where "approximately parallel", "approximately perpendicular", "approximately the same", etc. include certain tolerances, taking into account the tolerances related to the measurement of specific quantities (e.g., the limitations of the measurement system), and are expressed as being within the acceptable deviation range for a specific value determined by a person of ordinary skill in the art. For example, "approximately" can mean within one or more standard deviations, or within 10% or 5% of the value.

[0049] Unless the context requires otherwise, throughout the specification and claims, the term "including" is interpreted as an open, inclusive meaning, that is, "including, but not limited to". The polygons in this specification are not strictly defined, and may be approximate triangles, parallelograms, trapezoids, pentagons or hexagons, etc., and may have some small deformations caused by tolerances.

[0050] With the development of the understanding of diseases, the research on complex diseases such as major diseases of the nervous system has expanded from single cells to multi-cell and even tissue and organ levels. The in vitro simulation of the development of induced pluripotent stem cells (iPSC) into organoids and further disease and drug research has gradually become a hot topic in current research and industry.

[0051] However, in the process of culturing organoids, uneven growth and development of organoids often occur, and central hypoxia or lack of nutrients is prone to occur during the growth of organoids, leading to incomplete development and even organoid cell death. Therefore, it is necessary to introduce physical methods to change the distribution of nutrients in the environment to ensure its growth. Among them, organoids refer to organ-like tissue structures with relatively stable phenotypes and genetic characteristics cultured in vitro using 3D culture technology, which are of great significance in the study of growth and development, physiology and pathology, and drug effects.

[0052] It should be noted that the genetic characteristics resources involved in this specification are all genetic resources that are legitimately acquired or used in accordance with laws and administrative regulations.

[0053] To this end, the embodiments of the present application provide a culture structure, such as Figure 1 or Figure 2 As shown, the culture structure includes: a culture plate 1 and a vibration signal generating component (such as a piezoelectric transducer 2), and the vibration signal generating component is arranged on the culture plate 1;

[0054] The culture plate 1 comprises: a plurality of containing structures R, the containing structures R are configured to contain culture fluid, the containing structures R comprise a first containing cavity R1 and at least one second containing cavity R2 located at the bottom of the first containing cavity R1; the volume of the first containing cavity R1 is greater than the volume of the second containing cavity R2;

[0055] The vibration signal generating component 2 is configured to generate a vibration signal, the orthographic projection of the vibration signal generating component on the culture plate 1 does not overlap with the multiple containing structures R, and the vibration signal is configured to drive the culture fluid in the multiple containing structures R to move.

[0056] In the embodiments of the present application, the culture structure can be used to culture organoids or culture cells.

[0057] In the culture structure provided by the embodiment of the present application, the vibration signal generated by the vibration signal generating component drives the movement of the culture medium in the multiple containing structures R, which can provide sufficient oxygen to the organoids during their growth and balance the oxygen and nutrients in the culture medium, so that the organoids cultured in the culture medium can develop completely, solving the technical problem of organoid cell death due to lack of oxygen or nutrients in the center during the growth of the organoids.

[0058] In an exemplary embodiment, the vibration signal generated by the vibration signal generating component may include sound waves, such as ultrasonic waves. Specifically, the sound waves transmitted to the culture fluid may generate an acoustic flow field, and the acoustic flow field may drive the culture fluid in the containing structure R to move.

[0059] When the organoid is placed in the culture medium, the organoid rotates driven by the culture medium. The main force acting on the organoid is the drag force, which is calculated as follows:

[0060]

[0061] Where μ is the dynamic viscosity of the fluid, a is the cell radius (organoid radius), is the cell velocity (organoid velocity), is the liquid velocity. Drag is the force exerted by the fluid on the solid with relative velocity. This force is opposite to the direction of the solid relative to the fluid velocity and is actually the resistance to relative motion.

[0062] In an exemplary embodiment, since the second containing chamber R2 is disposed at the bottom of the first containing chamber R1D, the volume of the second containing chamber R2 is smaller than the volume of the first containing chamber R1, and the amount of culture fluid contained in the second containing chamber R2 is smaller than the amount of culture fluid contained in the first containing chamber R1.

[0063] In the culture structure provided in the embodiment of the present application, the volume of the second containing chamber R2 is smaller than that of the first containing chamber R1, and smaller organoids or tissues can be cultured in the second containing chamber R2, and larger organoids or tissues can be cultured in the first containing chamber R1; for example, in the initial stage of organoid development, it is placed in the second containing chamber R2, and in the later stage of organoid development, it is transferred from the second containing chamber R2 to the first containing chamber R1.

[0064] The specific material of the culture plate 1 is not limited here; illustratively, the material of the culture plate 1 can be a transparent or light-transmitting material to facilitate observation of the growth process of the organoid.

[0065] For example, the transparent or light-transmitting material may include glass, acrylic resin (polymethyl methacrylate, PMMA), polystyrene (PS), polycarbonate (PC), and polyethylene terephthalate (PET).

[0066] In an exemplary embodiment, the vibration signal generating component can be bonded to the culture plate 1.

[0067] The arrangement of the multiple containing structures R on the culture plate 1 is not limited here. For example, the multiple containing structures R can be arranged in an array.

[0068] In the related technology, the culture process of organoids involves the transfer of three culture plates. First, small balls are formed in a 96-well round-bottom culture plate, and then they develop into embryoid bodies. Then, they are transferred to a 24-well plate for differentiation and development. After they grow further, they are transferred to a 6-well plate for the final shaking development process. In other words, the traditional organoid culture process requires three transfers, and organoid loss is very likely to occur during the transfer process.

[0069] In the embodiment of the present application, since the containing structure R includes a first containing chamber R1 and at least one second containing chamber R2 located at the bottom of the first containing chamber R1, during the growth of the organoid, the organoids can be cultured at multiple stages in the culture structure, thereby avoiding the loss and damage of the organoids caused by the transfer of organoids at different developmental stages, thereby improving the culture efficiency.

[0070] The containing structure R (including the first containing cavity R1 and the second containing cavity R2) in the culture structure provided in the embodiment of the present application can adjust its corresponding size and shape according to actual needs, so as to match various types of application scenarios, such as matching the culture of different types of organoids. The culture structure of the embodiment of the present application has a simple overall structure and low preparation cost, and can be prepared in batches using processing technology, which is conducive to large-scale promotion and application.

[0071] In at least one embodiment of the present application, Figure 1 and Figure 2 As shown, the culture plate 1 includes a main structure 11 and an extension portion 12 located on at least one side of the main structure 11, a plurality of containing structures R are arranged on the main structure 11, and a vibration signal generating component is arranged on the extension portion 12; the extension portion 12 is connected to the main structure 11, and the vibration signal acts on the main structure 11 through the extension portion 12.

[0072] The planar shapes of the main structure 11 and the extension portion 12 in the above-mentioned culture plate 1 are not limited here. For example, the planar shapes of the main structure 11 and the extension portion 12 can be arc-shaped, polygonal, or a combination of arc-shaped and polygonal.

[0073] For example, the plane figures of the main structure 11 and the extension portion 12 may be polygons, including but not limited to quadrilaterals, pentagons, etc., wherein the polygons in this specification may include standard polygons and non-standard polygons, wherein the non-standard polygons may include rounded polygons;

[0074] For example, the planar shapes of the main structure 11 and the extension portion 12 may be arc-shaped, including but not limited to circular and elliptical shapes.

[0075] In an exemplary embodiment, the extension portion 12 and the main structure 11 can be made of the same material and in the same manufacturing process, and the two can be an integral whole.

[0076] In an embodiment of the present application, the extension portion 12 is connected to the main structure 11, and the vibration signal acts on the main structure 11 through the extension portion 12. Since multiple containing structures R are arranged on the main structure 11, the vibration signal can be transmitted to the culture medium through the main structure 11, thereby driving the movement of the culture medium in the multiple containing structures R, increasing the oxygen in the culture medium, and making the oxygen and nutrients in the culture medium evenly distributed.

[0077] When the extension portion 12 and the main structure 11 are integrated as a whole, it is more conducive to the propagation of the vibration signal and reduces the loss of the vibration signal during the propagation process.

[0078] In at least one embodiment of the present application, Figure 2 As shown, the vibration signal generating component includes at least two piezoelectric transducers 2, at least two piezoelectric transducers 2 are arranged on the extension part 12, and the propagation directions of the vibration signals generated by the at least two piezoelectric transducers 2 intersect in the plane where the culture plate 11 is located.

[0079] The core components of a piezoelectric transducer include a piezoelectric chip. The piezoelectric chip can be deformed under the action of pressure, which causes the chip itself to be polarized and positive and negative bound charges to appear on the surface of the chip. This effect is the piezoelectric effect. In addition, the piezoelectric effect is reversible, that is, when a voltage is applied to the chip, it will deform and generate a vibration signal. Among them, its vibration modes can include stretching vibration, bending vibration and torsional vibration.

[0080] The planar shape of the piezoelectric transducer includes, but is not limited to, rectangular, circular, annular, tubular, and the like.

[0081] In at least one embodiment of the present application, Figure 2 As shown, the vibration signal generating component includes two piezoelectric transducers 2. In the plane where the culture plate 1 is located, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 (for example, the angle between direction A and direction B) is greater than or equal to 80° and less than or equal to 100°.

[0082] Exemplarily, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 may be 80°, 85°, 88°, 90°, 93°, 95°, 98°, or 100°.

[0083] For example, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 is 90°, that is, the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 are orthogonal in the plane where the culture plate 1 is located.

[0084] In an embodiment of the present application, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 (for example, the angle between direction A and direction B) is set to be greater than or equal to 80° and less than or equal to 100°. In this way, for the culture fluid in the same containment structure R, the vibration signal received is asymmetric, causing the culture fluid in the containment structure R to move from an area with a strong received vibration signal to an area with a weak received vibration signal, thereby driving the movement of the culture fluid.

[0085] It should be noted that when the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 are set to be parallel and opposite, the vibration signals generated by the two cancel each other out in the culture medium. The culture medium may fluctuate but is difficult to move, making it difficult to achieve uniform distribution of nutrients and oxygen in the culture medium.

[0086] In the embodiment of the present application, the two piezoelectric transducers 2 have the same parameter settings when generating vibration signals. For example, the two piezoelectric transducers 2 have the same model and can use the same input voltage, the same frequency and the same phase. In the actual process of culturing organoids, when the frequency of the piezoelectric transducer 2 needs to be adjusted to change the size of the vibration signal, the two piezoelectric transducers 2 are adjusted at the same time.

[0087] In the related art, four interdigital transducers are arranged around the containing structure of the culture plate, and the frequency and phase of each interdigital transducer are adjusted to make the culture fluid in the containing structure undergo a double rotational motion in the plane formed by the Y direction and the Z direction (in the vertical plane). However, the vibration signals generated by the two interdigital transducers arranged relatively to each other are prone to cancel each other out, and the process of adjusting the frequency and phase of the four interdigital transducers is relatively complicated, which increases the difficulty of experimental operations during the culture of organoids.

[0088] In the embodiment of the present application, by setting the parameter settings of the two piezoelectric transducers 2 to be the same when generating vibration signals, the steps of culturing organoids using the culture structure can be greatly simplified and the difficulty of the culture process can be reduced; in addition, the culture structure of the present application can generate a reflow field on the plane formed by the X direction and the Y direction (on the horizontal plane) and drive the culture fluid and organoids to move (see the introduction below for details), while using fewer transducers, which can reduce costs.

[0089] In at least one embodiment of the present application, Figure 2 As shown, the culture plate 1 includes two extension parts 12, and the two extension parts 12 are located on two adjacent sides of the main structure 11, and the two extension parts 12 are connected.

[0090] In an exemplary embodiment, the plane shape of the main structure 11 is a rectangle. In this case, the two extension portions 12 are located on two adjacent sides of the main structure 11, and a piezoelectric transducer 2 is arranged on one extension portion 12. In this way, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 (for example, the angle between direction A and direction B) can be greater than or equal to 80° and less than or equal to 100°.

[0091] In an embodiment of the present application, by providing two extension portions 12 located on both sides of the main structure 11 and connecting them together, the mechanical stability of the extension portions 12 and the main structure 11 can be improved, thereby avoiding damage to the culture plate 1 when the piezoelectric transducer 2 operates at a high frequency, and improving the service life of the culture structure.

[0092] In an embodiment of the present application, the extension portion 12 is used as a transmission medium for the vibration signal. The extension portion 12 transmits the vibration signal to the main structure 11, and then to the culture fluid in the containing structure R. In addition, the propagation directions of the vibration signals generated by the two piezoelectric transducers 2 are arranged to intersect in the plane where the culture plate 11 is located, thereby realizing the construction of a reflux field for the culture fluid in the containing structure R (including the first containing cavity R1 and the second containing cavity R2), so that the particles in the culture fluid reflux along the area with high vibration signals to the area with low vibration signals, ensuring uniform rotation of the culture fluid. The continuously rotating fluid ensures uniform distribution of nutrients and oxygen, provides sufficient supply to the organoids, and improves the survival rate of the organoids, thereby solving problems such as hypoxia of the organoids in the culture fluid on the culture plate.

[0093] In at least one embodiment of the present application, Figure 3-Figure 10 As shown, the culture plate 1 includes a first surface B1, and the first accommodating cavity R1 and the second accommodating cavity R2 both include a bottom and an opening; the plane where the opening K1 of the first accommodating cavity R1 is located is coplanar with the first surface B1, and the plane where the opening K2 of the second accommodating cavity R2 is located is coplanar with the bottom D1 of the first accommodating cavity R1;

[0094] Among them, Figure 3 As shown, along the direction perpendicular to the first surface B1, the distance h1 between the opening K1 of the first accommodating cavity R1 and the bottom D1 (the depth of the first accommodating cavity R1) is greater than the distance h2 between the opening K2 of the second accommodating cavity R2 and the bottom D2 (the depth of the second accommodating cavity R2).

[0095] In an embodiment of the present application, in the initial stage of organoid development, it is placed in the second accommodating chamber R2, and in the later stage of organoid development, it is transferred from the second accommodating chamber R2 to the first accommodating chamber R1. By setting the depth of the first accommodating chamber R1 to be greater than the depth of the second accommodating chamber R2, that is, the depth of the second accommodating chamber R2 is less than the depth of the first accommodating chamber R1, in the actual process of culturing organoids, under the action of the buoyancy and the backflow field (acoustic flow field) generated by the vibration signal, it is more conducive to the transfer of the organoid from the second accommodating chamber R2 to the first accommodating chamber R1, thereby avoiding the problem of death caused by hypoxia due to excessive development of the organoid due to difficulty in transfer.

[0096] In at least one embodiment of the present application, Figure 3-Figure 9As shown, the second accommodating cavity R2 includes a second axis O2, and an angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1 is greater than 0° and less than or equal to 90°.

[0097] It should be noted that, in this specification, the angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1 refers to a smaller angle between the second axis O2 of the second accommodating cavity R2 and the first surface B1.

[0098] In an exemplary embodiment, the first surface B1 and the surface of the culture solution are substantially parallel.

[0099] In the culture structure provided in the embodiment of the present application, the first accommodating chamber R1 and the second accommodating chamber R2 may both be axisymmetric structures, wherein the second axis O2 is the symmetry axis of the second accommodating chamber R2.

[0100] In some embodiments, Figure 3 , Figure 4 and Figure 5 As shown, the angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1 is substantially equal to 90°, that is, the extension direction of the second accommodating cavity R2 is substantially perpendicular to the surface of the culture solution.

[0101] In some embodiments, Figure 6 , Figure 7 , Figure 8 and Fig. 9 As shown, the angle β between the second axis O2 of the second accommodating chamber R2 and the first surface B1 is less than 90 degrees. That is, there is an acute angle between the extension direction of the second accommodating chamber R2 and the surface of the culture solution, and the second accommodating chamber R2 is inclined relative to the surface of the culture solution.

[0102] In the culture structure provided in the embodiment of the present application, the second containing chamber R2 is inclined relative to the surface of the culture medium. When the organoid is transferred from the second containing chamber R2 to the first containing chamber R1, the buoyancy and the backflow field (acoustic flow field) generated by the vibration signal are more conducive to the transfer of the organoid from the second containing chamber R2 to the first containing chamber R1, thereby avoiding the problem of death caused by hypoxia due to excessive development of the organoid due to difficulty in transfer.

[0103] In at least one embodiment of the present application, Figure 4 and Figure 5 As shown, the angle between the second axis O2 of the second accommodating cavity R2 and the first surface B1 is approximately 90°, and the area of ​​the orthographic projection of the bottom D2 of the second accommodating cavity R2 on the first surface B1 is smaller than the area enclosed by the orthographic projection of the edge of the opening K2 of the second accommodating cavity R2 on the first surface B1.

[0104] In an exemplary embodiment, the edge of the opening K2 may also be referred to as the edge of the opening K2 . Similar descriptions in the following text have the same meaning as here and are not repeated here.

[0105] In the culture structure provided in the embodiments of the present application, by setting the area of ​​the orthographic projection of the bottom D2 of the second accommodating chamber R2 on the first surface B1 to be smaller than the area of ​​the area enclosed by the orthographic projection of the edge of the opening K2 of the second accommodating chamber R2 on the first surface B1, the second accommodating chamber R2 is made to have a structure with a small bottom D2 and a large opening K2. When the organoid is transferred from the second accommodating chamber R2 to the first accommodating chamber R1, the buoyancy and the backflow field (acoustic flow field) generated by the vibration signal are more conducive to the transfer of the organoid from the second accommodating chamber R2 to the first accommodating chamber R1, thereby avoiding the problem of death caused by hypoxia due to excessive growth of the organoid due to difficulty in transfer.

[0106] In at least one embodiment of the present application, Figure 6 , Figure 7 , Figure 8 and Fig. 9 As shown, the angle between the second axis of the second accommodating cavity and the first surface is greater than 0° and less than 90°, and the area of ​​the orthographic projection of the bottom D2 of the second accommodating cavity R2 on the first surface B1 is less than or equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening K2 of the second accommodating cavity R2 on the first surface B1.

[0107] Exemplarily, when the angle between the second axis and the first surface of the second containing cavity is greater than 0° and less than 90° (i.e., the second containing cavity R2 is inclined relative to the surface of the culture liquid), the area of ​​the orthographic projection of the bottom D2 of the second containing cavity R2 on the first surface B1 can be set to be smaller than the area of ​​the area enclosed by the orthographic projection of the edge of the opening K2 of the second containing cavity R2 on the first surface B1; or, the area of ​​the orthographic projection of the bottom D2 of the second containing cavity R2 on the first surface B1 can be set to be equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening K2 of the second containing cavity R2 on the first surface B1.

[0108] Under the condition of ensuring that the organoid can be smoothly transferred from the first containing chamber R1 to the second containing chamber R2, in order to simplify the design of the culture structure and reduce the difficulty of its preparation process, when the second containing chamber R2 is inclined relative to the surface of the culture fluid, the area of ​​the orthographic projection of the bottom D2 of the second containing chamber R2 on the first surface B1 can be set equal to the area of ​​the region enclosed by the orthographic projection of the edge of the opening K2 of the second containing chamber R2 on the first surface B1.

[0109] In the drawings provided in the embodiments of the present application, the area of ​​the orthographic projection of the bottom D2 of the second accommodating cavity R2 on the first surface B1 is drawn as an example in which the area enclosed by the orthographic projection of the edge of the opening K2 of the second accommodating cavity R2 on the first surface B1 is equal to the area enclosed by the orthographic projection of the edge of the opening K2 of the second accommodating cavity R2 on the first surface B1.

[0110] In at least one embodiment of the present application, Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, the first accommodating cavity R1 includes a first axis O1, and the angle α between the first axis O1 of the first accommodating cavity R1 and the first surface B1 is approximately equal to the angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1; the area of ​​the area enclosed by the orthographic projection of the outer contour of the bottom D1 of the first accommodating cavity R1 on the first surface B1 is less than or equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening K2 of the first accommodating cavity R2 on the first surface B1.

[0111] In an exemplary embodiment, if Figure 3 and Figure 5 As shown, the first accommodating cavity R1 includes a first axis O1, an angle α between the first axis O1 of the first accommodating cavity R1 and the first surface B1 and an angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1 are approximately 90°, and an area enclosed by the orthographic projection of the outer contour of the bottom D1 of the first accommodating cavity R1 on the first surface B1 is equal to an area enclosed by the orthographic projection of the edge of the opening K2 of the first accommodating cavity R2 on the first surface B1.

[0112] In an exemplary embodiment, if Figure 4 As shown, the first accommodating cavity R1 includes a first axis O1, an angle α between the first axis O1 of the first accommodating cavity R1 and the first surface B1 and an angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1 are approximately 90°, and an area enclosed by the orthographic projection of the outer contour of the bottom D1 of the first accommodating cavity R1 on the first surface B1 is smaller than an area enclosed by the orthographic projection of the edge of the opening K2 of the first accommodating cavity R2 on the first surface B1.

[0113] In an exemplary embodiment, if Figure 6 and Figure 7 As shown, the first accommodating cavity R1 includes a first axis O1, and the angle α between the first axis O1 of the first accommodating cavity R1 and the first surface B1 is approximately equal to the angle β between the second axis O2 of the second accommodating cavity R2 and the first surface B1, and both are less than 90°, and the area of ​​the area enclosed by the orthographic projection of the outer contour of the bottom D1 of the first accommodating cavity R1 on the first surface B1 is equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening K2 of the first accommodating cavity R2 on the first surface B1.

[0114] In the culture structure provided in the embodiment of the present application, by setting, the angle α between the first axis O1 and the first surface B1 of the first accommodating chamber R1 is approximately equal to the angle β between the second axis O2 and the first surface B1 of the second accommodating chamber R2, that is, the first axis O1 and the second axis O2 are collinear. In this way, when the organoid is transferred from the second accommodating chamber R2 to the first accommodating chamber R1, under the action of the buoyancy and the backflow field (acoustic flow field) generated by the vibration signal, it is more conducive to transfer the organoid from the second accommodating chamber R2 to the first accommodating chamber R1, avoiding the problem of death caused by hypoxia due to excessive development of the organoid due to difficulty in transfer.

[0115] In the following embodiment, when the organoid is small in size or the size of the containing structure R is large, the angle α between the first axis O1 and the first surface B1 of the first containing cavity R1 can be set to be different from the angle β between the second axis O2 and the first surface B1 of the second containing cavity R2, that is, there is an angle between the first axis O1 and the second axis O2, and the organoid can be transferred from the second containing cavity R2 to the first containing cavity R1 under the action of the acoustic flow field generated by the buoyancy and vibration signals, thereby avoiding the problem of death caused by hypoxia due to excessive development of the organoid due to difficulty in transfer.

[0116] In at least one embodiment of the present application, Fig.14A , Fig. 14B , Fig. 14C As shown, the edges of the openings K of the first accommodating chamber R1 and the second accommodating chamber R2 are both arcs;

[0117] In addition, the outer contours of the bottoms of the first accommodating chamber R1 and the second accommodating chamber R2 are both arcs, and the side walls of the first accommodating chamber R1 and the second accommodating chamber R2 are both arc surfaces.

[0118] It should be noted that Fig.14A , Fig. 14B , Fig. 14C What are shown are the outlines of the edges of the respective openings K of the culture structures when viewed from above.

[0119] In an exemplary embodiment, the edges of the openings K of the first accommodating cavity R1 and the second accommodating cavity R2 are both arc-shaped, wherein the arc shape may include a circle or an ellipse.

[0120] Likewise, the shapes formed by the outer contours of the bottoms of the first accommodating cavity R1 and the second accommodating cavity R2 are both arc-shaped, for example, circular or elliptical.

[0121] Here, there is no limitation on whether the pattern formed by the edge of the opening is the same as the pattern formed by the outer contour of the bottom, which can be determined according to needs.

[0122] In an exemplary embodiment, the first accommodation cavity R1 may be cylindrical, truncated cone, elliptical cylinder, elliptical cone, oblique cylindrical, oblique truncated cone, oblique elliptical cylinder or oblique elliptical cone.

[0123] In an exemplary embodiment, the second accommodation cavity R2 may be cylindrical, truncated cone, elliptical cylinder, elliptical cone, oblique cylindrical, oblique truncated cone, oblique elliptical cylinder or oblique elliptical cone.

[0124] In the culture structure provided in the embodiment of the present application, the edges of the openings K of the first accommodating chamber R1 and the second accommodating chamber R2 are both arcs; the outer contours of the bottoms of the first accommodating chamber R1 and the second accommodating chamber R2 are both arcs, and the side walls of the first accommodating chamber R1 and the second accommodating chamber R2 are both curved surfaces; when the moving culture fluid drives the organoid to rotate, the sharp structures in the first accommodating chamber R1 and the second accommodating chamber R2 are avoided from causing damage to the organoid, and while ensuring that the organoid absorbs oxygen and nutrients uniformly, the loss rate of the organoid is reduced and the survival rate of the organoid is improved.

[0125] In at least one embodiment of the present application, the edges of the openings of the first accommodating cavity R1 and the second accommodating cavity R2 are both circular, and the outer contours of the bottoms of the first accommodating cavity R1 and the second accommodating cavity R2 are both circular;

[0126] Fig.11 A simulation effect diagram of the acoustic flow field in the second accommodating cavity R2 in two-dimensional directions (X direction and Y direction) is provided. Fig.12 A simulation effect diagram of the acoustic flow field in the first accommodating chamber R1 in two-dimensional directions (X direction and Y direction) is provided, wherein the unit of the simulation effect diagram of the acoustic flow field is mm / s, the larger the area marked by the arrow in the figure, the stronger the acoustic flow, and the direction of the arrow represents the direction of the acoustic flow (culture fluid flow).

[0127] like Fig.11 and Fig.12 As shown, the first accommodating cavity R1 and the second accommodating cavity R2 each include two first areas Area1, the intensity of the vibration signal received by the culture fluid in the first area Area1 is greater than the intensity of the vibration signal received by the culture fluid in other areas (that is, the intensity of the acoustic flow and the flow rate of the culture fluid in the first area Area1 are greater than those in other areas), the circle includes a first diameter L1 and a second diameter L2, and the first diameter L1 and the second diameter L2 are orthogonal; the two first areas Area1 are both located on the same side of the first diameter L1, and the orthographic projections of the two first areas Area1 on the first surface B1 are symmetrically distributed about the second diameter L2.

[0128] In an exemplary embodiment, if Fig.11As shown, the intensity of the acoustic flow field in the two first areas Area1 is the largest, and the culture fluid in the second containing chamber R2 performs a swirling motion from outside to inside, and drives the particles or cells in the culture fluid to perform a swirling motion from outside to inside. Fig.11 The two first areas Area are both located on the upper side of the first diameter L1, that is, for the culture fluid in the second accommodating chamber R2, the left half area is driven by the acoustic flow field in the first area Area to rotate clockwise; the right half area is driven by the acoustic flow field in the first area Area to rotate counterclockwise. It is precisely because the intensity of the acoustic flow field is asymmetrically distributed in the left half area that the culture fluid in the left half area is driven to rotate clockwise, and the intensity of the acoustic flow field is asymmetrically distributed in the right half area that the culture fluid in the right half area is driven to rotate counterclockwise, that is, both of them make a cyclotron motion from outside to inside.

[0129] In an exemplary embodiment, if Fig.12 As shown, since the first accommodating chamber R1 is larger than the second accommodating chamber R2, it can accommodate more culture fluid. At this time, the input frequency in the first accommodating chamber R1 increases, and the tendency of the culture fluid in the first accommodating chamber R1 to perform a cyclotron motion from outside to inside is more significant. Similarly, Fig.12 The two first areas Area are both located on the upper side of the first diameter L1, that is, for the culture fluid in the first containing chamber R1, the left half area rotates clockwise under the drive of the acoustic flow field in the first area Area; the right half area rotates counterclockwise under the drive of the acoustic flow field in the first area Area.

[0130] Fig.13 A schematic diagram of the simulation results of the particle tracking trajectory of a 500 μm particle in a containment structure is provided, the unit is μm / s, and the input frequency of the piezoelectric transducer 2 is 800 kHz at this time; wherein, Fig.13 The dotted arrow in the figure is the direction of particle movement. Fig.13 From the simulation results, it can be seen that particles with a diameter of 500 μm can complete two rotations within 3 seconds without colliding with the side wall of the containing structure. It can be seen that through the culture structure provided by the embodiment of the present application, the organoids can rotate well therein, so that during the growth of the organoids, the culture medium can provide oxygen and nutrients to the organoids in a balanced manner, solving the technical problem of organoid cell death due to lack of oxygen or nutrients in the center during the growth of the organoids.

[0131] In at least one embodiment of the present application, along a direction perpendicular to the first surface B1, a distance h1 between the opening K1 of the first accommodating cavity R1 and the bottom D1 ranges from 250 μm to 1000 μm, and a distance h2 between the opening K2 of the second accommodating cavity R2 and the bottom D2 ranges from 200 μm to 300 μm;

[0132] The diameter of the figure enclosed by the outer contour of the bottom D1 of the first accommodating cavity R1 is in the range of 3 mm to 5 mm, and the diameter of the figure enclosed by the outer contour of the bottom of the second accommodating cavity R2 is in the range of 600 μm to 1000 μm.

[0133] Exemplarily, the diameter of the figure enclosed by the outer contour of the bottom D1 of the first accommodating cavity R1 may be the same as the diameter of the figure enclosed by the edge of the opening K1 of the first accommodating cavity R1; or, the diameter of the figure enclosed by the outer contour of the bottom D1 of the first accommodating cavity R1 may be smaller than the diameter of the figure enclosed by the edge of the opening K1 of the first accommodating cavity R1.

[0134] Exemplarily, the diameter of the figure enclosed by the outer contour of the bottom D2 of the second accommodating cavity R2 may be the same as the diameter of the figure enclosed by the edge of the opening K2 of the second accommodating cavity R2; or, the diameter of the figure enclosed by the outer contour of the bottom D2 of the second accommodating cavity R2 may be smaller than the diameter of the figure enclosed by the edge of the opening K2 of the second accommodating cavity R2.

[0135] Exemplarily, the distance h1 (i.e., depth) between the opening K1 of the first accommodating cavity R1 and the bottom D1 can be 300μm, 350μm, 380μm, 400μm, 420μm, 450μm, 480μm, 500μm, 530μm, 550μm, 580μm, 600μm, 630μm, 660μm, 680μm, 700μm, 730μm, 750μm, 780μm, 800μm, 850μm, 880μm, 900μm, 930μm, 950μm, or 980μm.

[0136] Exemplarily, the distance h2 (ie, the depth) between the opening K2 and the bottom D2 of the second accommodating cavity R2 may be 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, or 290 μm.

[0137] Exemplarily, the diameter of the figure enclosed by the outer contour of the bottom D1 of the first accommodating cavity R1 may be 3.1 mm, 3.2 mm, 3.3 mm, 3.5 mm, 3.8 mm, 4.0 mm, 4.2 mm, 4.5 mm, 4.8 mm, or 4.9 mm.

[0138] Exemplarily, the diameter of the figure enclosed by the outer contour of the bottom of the second accommodating cavity R2 may be 600μm, 620μm, 650μm, 680μm, 700μm, 720μm, 750μm, 780μm, 800μm, 830μm, 850μm, 880μm, 900μm, 930μm, 950μm, or 980μm.

[0139] It should be noted that, when the figure formed by the outer contour of the bottom D1 of the first accommodating cavity R1 is an arc, the maximum diameter of the arc is 3mm to 5mm, for example, 3.1mm, 3.2mm, 3.3mm, 3.5mm, 3.8mm, 4.0mm, 4.2mm, 4.5mm, 4.8mm, 4.9mm; when the figure formed by the outer contour of the bottom of the second accommodating cavity R2 is an arc, the maximum diameter of the arc is 600μm to 1000μm, for example, 620μm, 650μm, 680μm, 700μm, 720μm, 750μm, 780μm, 800μm, 830μm, 850μm, 880μm, 900μm, 930μm, 950μm, 980μm.

[0140] In at least one embodiment of the present application, the input voltage signal of the piezoelectric transducer 2 is 0.3V to 0.8V, and the frequency of the input voltage signal is 150kHz to 400kHz or 700kHz to 1200kHz.

[0141] It should be noted that the two piezoelectric transducers 2 have the same input voltage signal, the same frequency and the same phase. The input voltage signal can be 0.4V, 0.5V, 0.6V, 0.7V; the input frequency can be 160kHz, 180kHz, 200kHz, 220kHz, 240kHz, 260kHz, 290kHz, 300kHz, 330kHz, 350kHz, 380kHz, 720kHz, 750kHz, 780kHz, 800kHz, 820kHz, 850kHz, 880kHz, 900kHz, 960kHz, 980kHz, 1000kHz, 1050kHz, 1080kHz, 1100kHz, 1150kHz.

[0142] The input voltage signals of the two piezoelectric transducers 2 and the frequencies of the input voltage signals can be set according to the types of cultured organoids and the different stages of organoid development, which are not specifically limited here.

[0143] In at least one embodiment of the present application, in combination Figure 2 and Fig.10 As shown, the culture structure further includes an enclosure structure 3, a portion of which is located between the main structure 11 and the extension portion 12, and the enclosure structure 3 is arranged around a plurality of containing structures R. Fig.10 for Figure 2 Schematic diagram of the cross-sectional structure along the M3M4 direction.

[0144] In an exemplary embodiment, the enclosing structure 3 and the first surface B1 form a third accommodating cavity, which is located on the side of the first accommodating cavity R1 away from the second accommodating cavity R2. The size of the third accommodating cavity is larger than the first accommodating cavity R1. When the organoid further develops and becomes larger in size, the first accommodating cavity R1 in the organoid can be further transferred to the third accommodating cavity, which is beneficial to the further development and absorption of nutrients of the organoid and also beneficial to the removal of the organoid at the end of the culture.

[0145] The shape of the edge of the opening of the third accommodating cavity and the outline of the bottom are not limited here. For example, it can be any one of a circle, an ellipse, a quadrilateral (including a rectangle or a square), and a pentagon.

[0146] In an exemplary embodiment, the enclosure structure 3 can be made of the same material as the culture plate 1 .

[0147] In at least one embodiment of the present application, Fig.10 As shown, the thickness h3 of the culture plate 1 is 1.8 mm to 3.5 mm, the dimension h4 of the enclosure structure 3 along the thickness direction of the culture plate 1 is 7 mm to 12 mm, and the dimension h5 of the enclosure structure 3 along the direction parallel to the plane of the culture plate 1 is 1.5 mm to 3.5 mm.

[0148] Exemplarily, the thickness h3 of the culture plate 1 can be 2 mm, 2.5 mm, or 3 mm. The dimension h4 of the enclosure structure 3 along the thickness direction of the culture plate 1 (i.e., the height of the enclosure structure 3) can be 8 mm, 9 mm, 10 mm, or 11 mm. The dimension h5 of the enclosure structure 3 along the direction parallel to the plane where the culture plate 1 is located (i.e., the thickness of the enclosure structure 3) can be 2 mm, 2.5 mm, 2.8 mm, 3.0 mm, or 3.3 mm.

[0149] In an exemplary embodiment, a size of a planar pattern of the piezoelectric transducer 2 is smaller than a size of a planar pattern of the extending portion 2 .

[0150] Exemplarily, the plane shape of the main structure 11 may be a square, and the side length thereof may be 50 mm±0.5 mm, and the plane shape of the extension portion 2 may be a rectangle, and the long side of the rectangle may be 50 mm±0.5 mm.

[0151] The culture structure provided in the embodiment of the present application can adjust its corresponding size and shape according to actual needs to match various types of application scenarios, such as matching the culture of different types of organoids. The overall structure of the culture structure in the embodiment of the present application is simple, the preparation cost is low, and it can be prepared in batches using processing technology, which is conducive to large-scale promotion and application.

[0152] The embodiment of the present application provides a culture method, which is applied to the culture structure described above, wherein the culture structure includes a culture plate 1 and a vibration signal generating component, and the vibration signal generating component is arranged on the culture plate 1; the culture plate 1 includes a plurality of containing structures R, and the containing structures R are arranged to contain culture liquid, and the containing structures R include a first containing cavity R1 and at least one second containing cavity R2 located at the bottom of the first containing cavity R1; the orthographic projection of the vibration signal generating component on the culture plate 1 does not overlap with the plurality of containing structures R; the method includes:

[0153] S801, generating a vibration signal;

[0154] Wherein, by applying a voltage signal to the vibration signal generating component, the vibration signal generating structure generates a vibration signal according to the voltage signal;

[0155] S802. The vibration signal drives the culture fluid in the multiple containing structures to move.

[0156] In the culture structure provided by the embodiment of the present application, the vibration signal generated by the vibration signal generating component drives the movement of the culture medium in the multiple containing structures R, which can provide sufficient oxygen to the organoids during their growth and balance the oxygen and nutrients in the culture medium, so that the organoids cultured in the culture medium can develop completely, solving the technical problem of organoid cell death due to lack of oxygen or nutrients in the center during the growth of the organoids.

[0157] In at least one embodiment of the present application, step S802: the vibration signal drives the culture solution in the plurality of containing structures R to move, comprises:

[0158] S8021. In the first stage, the second accommodating chamber R2 is configured to further accommodate the object to be cultured, the frequency of the voltage signal input by the vibration signal generating component is 150kHz to 400kHz, and the vibration signal drives the object to be cultured and the culture solution to move;

[0159] S8022. In the second stage, the first containing chamber R1 is configured to contain the object to be cultured, the frequency of the voltage signal input to the vibration signal generating component is 700kHz to 1200kHz, and the vibration signal drives the object to be cultured and the culture fluid to move.

[0160] The development degree and size of the cultured object in the second stage are greater than those in the first stage. The cultured object may include cells or organoids, for example, brain organoids.

[0161] In the related technology, the culture process of organoids involves the transfer of three culture plates. First, small balls are formed in a 96-well round-bottom culture plate, and then they develop into embryoid bodies. Then, they are transferred to a 24-well plate for differentiation and development. After they grow further, they are transferred to a 6-well plate for the final shaking development process. In other words, the traditional organoid culture process requires three transfers, and organoid loss is very likely to occur during the transfer process.

[0162] In the embodiments of the present application, since the containing structure R includes a first containing chamber R1 and at least one second containing chamber R2 located at the bottom of the first containing chamber R1, in the first stage of the organoid growth process, the organoid is cultivated and grown in the second containing chamber R2; in the second stage, the organoid is cultivated and grown in the first containing chamber R1, thereby avoiding the loss and damage of the organoids caused by the transfer of organoids at different developmental stages and improving the culture efficiency.

[0163] An embodiment of the present application provides a culture chip, which includes the culture structure as described above.

[0164] In the culture chip provided in the embodiment of the present application, since the containing structure R includes a first containing cavity R1 and at least one second containing cavity R2 located at the bottom of the first containing cavity R1, during the growth of organoids, organoids can be cultured at multiple stages in the culture structure, thereby avoiding the loss and damage of organoids caused by the transfer of organoids at different developmental stages and improving the culture efficiency.

[0165] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A culture structure, wherein: include: A culture plate and a vibration signal generating component, wherein the vibration signal generating component is arranged on the culture plate; The culture plate comprises: a plurality of containing structures, the containing structures are configured to contain culture fluid, the containing structures comprise a first containing cavity and at least one second containing cavity located at the bottom of the first containing cavity; the volume of the first containing cavity is greater than the volume of the second containing cavity; The vibration signal generating component is configured to generate a vibration signal, the orthographic projection of the vibration signal generating component on the culture plate does not overlap with the multiple containing structures, and the vibration signal is configured to drive the culture fluid in the multiple containing structures to move.

2. The culture structure according to claim 1, wherein: The culture plate comprises a main body structure and an extension portion located on at least one side of the main body structure, the plurality of accommodating structures are arranged on the main body structure, and the vibration signal generating component is arranged on the extension portion; The extension part is connected to the main structure, and the vibration signal acts on the main structure through the extension part.

3. The culture structure according to claim 2, wherein: The vibration signal generating component includes at least two piezoelectric transducers, which are arranged on the extension part, and the propagation directions of the vibration signals generated by the at least two piezoelectric transducers intersect in the plane where the culture plate is located.

4. The culture structure according to claim 3, wherein: The vibration signal generating component includes two piezoelectric transducers. In the plane where the culture plate is located, the angle formed by the propagation directions of the vibration signals generated by the two piezoelectric transducers is greater than or equal to 80° and less than or equal to 100°.

5. The culture structure according to claim 4, wherein: The culture plate comprises two extension parts, the two extension parts are located on two adjacent sides of the main structure, and the two extension parts are connected.

6. The culture structure according to claim 4, wherein: The culture plate comprises a first surface, and the first accommodating cavity and the second accommodating cavity both comprise a bottom and an opening; the plane where the opening of the first accommodating cavity is located is coplanar with the first surface, and the plane where the opening of the second accommodating cavity is located is coplanar with the bottom of the first accommodating cavity; Wherein, along a direction perpendicular to the first surface, a distance from an opening to a bottom of the first accommodating cavity is greater than a distance from an opening to a bottom of the second accommodating cavity.

7. The culture structure according to claim 6, wherein: The second accommodating cavity comprises a second axis, and an angle between the second axis of the second accommodating cavity and the first surface is greater than 0° and less than or equal to 90°.

8. The culture structure according to claim 7, wherein: The angle between the second axis of the second accommodating cavity and the first surface is approximately 90°, and the area of ​​the orthographic projection of the bottom of the second accommodating cavity on the first surface is smaller than the area of ​​the region enclosed by the orthographic projection of the edge of the opening of the second accommodating cavity on the first surface.

9. The culture structure according to claim 7, wherein: The angle between the second axis of the second accommodating cavity and the first surface is greater than 0° and less than 90°, and the area of ​​the orthographic projection of the bottom of the second accommodating cavity on the first surface is less than or equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening of the second accommodating cavity on the first surface.

10. The culture structure according to claim 8 or 9, wherein: The first accommodating cavity includes a first axis, and the angle between the first axis of the first accommodating cavity and the first surface is approximately equal to the angle between the second axis of the second accommodating cavity and the first surface; the area of ​​the area enclosed by the orthographic projection of the bottom outer contour of the first accommodating cavity on the first surface is less than or equal to the area of ​​the area enclosed by the orthographic projection of the edge of the opening of the first accommodating cavity on the first surface.

11. The culture structure according to any one of claims 6 to 9, wherein The edges of the openings of the first accommodating cavity and the second accommodating cavity are arcs, the outer contours of the bottoms of the first accommodating cavity and the second accommodating cavity are arcs, and the side walls of the first accommodating cavity and the second accommodating cavity are arc surfaces.

12. The culture structure according to claim 11, wherein: The edges of the openings of the first accommodating cavity and the second accommodating cavity are both circular, and the outer contours of the bottoms of the first accommodating cavity and the second accommodating cavity are both circular; The first accommodating chamber and the second accommodating chamber each include two first areas, the intensity of the vibration signal received by the culture fluid in the first area is greater than the intensity of the vibration signal received by the culture fluid in other areas, the circle includes a first diameter and a second diameter, and the first diameter and the second diameter are orthogonal; the extension direction of the first diameter is consistent with the propagation direction of the vibration signal generated by one of the piezoelectric transducers, and the extension direction of the second diameter is consistent with the propagation direction of the vibration signal generated by the other piezoelectric transducer; The two first regions are both located on the same side of the first diameter, and the orthographic projections of the two first regions on the first surface are symmetrically distributed about the second diameter.

13. The culture structure according to claim 12, wherein: Along the direction perpendicular to the first surface, the distance between the opening of the first accommodating cavity and the bottom ranges from 250 μm to 1000 μm, and the distance between the opening of the second accommodating cavity and the bottom ranges from 200 μm to 300 μm; The diameter of the figure enclosed by the outer contour of the bottom of the first accommodating cavity is in the range of 3 mm to 5 mm, and the diameter of the figure enclosed by the outer contour of the bottom of the second accommodating cavity is in the range of 600 μm to 1000 μm.

14. The culture structure according to claim 2, wherein: The culture structure further includes an enclosure structure, a partial area of ​​which is located between the main structure and the extension portion, and the enclosure structure is arranged around the plurality of containing structures.

15. The culture structure according to claim 14, wherein: The thickness of the culture plate is 1.8 mm to 3.5 mm, the dimension of the enclosure structure along the thickness direction of the culture plate is 7 mm to 12 mm, and the dimension of the enclosure structure along the direction parallel to the plane where the culture plate is located is 1.5 mm to 3.5 mm.

16. A culture method, applied to the culture structure according to any one of claims 1 to 15, wherein: The culture structure comprises a culture plate and a vibration signal generating component, wherein the vibration signal generating component is arranged on the culture plate; the culture plate comprises a plurality of containing structures, wherein the containing structures are arranged to contain culture liquid, and the containing structures comprise a first containing cavity and at least one second containing cavity located at the bottom of the first containing cavity; the orthographic projection of the vibration signal generating component on the culture plate does not overlap with the plurality of containing structures; the method comprises: generating a vibration signal; The vibration signal drives the culture fluid in the multiple containing structures to move.

17. The culture method according to claim 16, wherein: The vibration signal drives the culture fluid in the plurality of containing structures to move, including: In the first stage, the second containing chamber is configured to also contain the object to be cultured, the frequency of the voltage signal input by the vibration signal generating component is 150kHz to 400kHz, and the vibration signal drives the object to be cultured and the culture liquid to move; In the second stage, the first containing chamber is configured to contain the object to be cultured, the frequency of the voltage signal input to the vibration signal generating component is 700kHz to 1200kHz, and the vibration signal drives the object to be cultured and the culture fluid to move.

18. A culture chip, wherein: A culture structure comprising any one of claims 1 to 15.

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