A device and method for establishing a multi-spheroid tissue model

Abstract

The embodiment of the present invention discloses a device and method for establishing a multi-spheroid tissue model. The device for establishing a multi-spheroid tissue model includes: a base, a stage, a photothermal microfluidic platform, a mobile platform, an optical component, and a spotting needle. The stage, the mobile platform, the optical component, and the spotting needle can move in different directions, thereby realizing multi-dimensional control of the spotting needle and the optical component for spotting and assembling the multi-spheroid tissue model on the photothermal microfluidic platform; the transparent glass of the photothermal microfluidic platform can be used to prepare gel droplets, and the combination of a graphene nanosheet layer with photothermal conversion capability, a chip layer with pyroelectric effect, and the optical component can generate a photothermal effect, thereby causing the gel droplets to be subjected to uneven force manipulation in an uneven electric field, thereby realizing controllable, non-contact construction of a multi-spheroid tissue model with a 3D structure, and the operation is simple. The obtained multi-spheroid tissue model has high biological activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a device and method for establishing a multi-spheroid tissue model. Background Art

[0002] Cell tissues in the human body and animals are all 3D structures. Using only 2D cell culture or animal models to understand the formation, function and pathology of cell tissues cannot achieve ideal results. Therefore, the establishment of in vitro 3D tissue models, as a technical means to well simulate the in vivo physiological environment, is of great significance to the development of tissue engineering and regenerative medicine. It not only provides a model system for experimental ideas and potential therapeutic interventions, but also may allow high-throughput drug screening of human tissues in vitro, promoting the development and screening of new therapies.

[0003] However, in the existing technology, although there are many methods for establishing in vitro 3D tissue models, these assembly methods still have many different problems. For example, using magnetic microrobots to encode hydrogel structures requires multiple flipping of the tissue model during operation, which is complicated and may damage the cell structure; using surface tension to assemble hydrogel structures in a direction is unstable and inconvenient for programming tissue models; using sound waves, magnetic fields and magnetic materials for non-contact manipulation and assembly usually has special requirements for the properties of the hydrogel or requires the addition of special substances to the hydrogel, and the introduction of these additional factors may interact with the encapsulated cells, affecting their viability and function. Summary of the Invention

[0004] The present invention provides a device and method for establishing a multi-spheroid tissue model, which is used to solve the problems that the construction operation process of in vitro 3D tissue models is complicated, the controllable construction of multi-spheroid tissue models is difficult to achieve, and the obtained multi-spheroid tissue models are easily destroyed.

[0005] On the one hand, the present application provides a device for establishing a multi-spheroid tissue model, comprising: a base, a stage, a photothermal microfluidic platform, a mobile platform, an optical component, and a spotting needle;

[0006] The base is provided with a slide rail, and the slide rail is movably connected to the loading platform;

[0007] The photothermal microfluidic platform is placed on the stage;

[0008] The base on both sides of the loading platform is provided with a first guide groove and a second guide groove respectively, and the movable platform is movably connected to the first guide groove and the second guide groove;

[0009] A third guide groove is provided on the movable platform, and the optical component and the spotting needle are movably connected to the third guide groove and are located above the photothermal and electromicrofluidic platform;

[0010] The photothermoelectric microfluidic platform comprises, from top to bottom, transparent glass, a wafer layer with pyroelectric effect, and a graphene nanosheet layer.

[0011] Preferably, the optical assembly comprises: a first light source and a second light source;

[0012] The wavelength of the first light source is 365nm or 405nm;

[0013] The wavelength of the second light source is 808 nm.

[0014] Preferably, the photothermoelectric microfluidic platform comprises, from top to bottom, transparent glass, a wafer layer with pyroelectric effect, and a graphene nanosheet layer.

[0015] Preferably, the graphene nanosheet layer comprises: graphene nanosheets and polydimethylsiloxane, and the mass fraction of the graphene nanosheets is 3% to 5%.

[0016] Another aspect of the present application provides a method for establishing a multispheroid tissue model, comprising the following steps:

[0017] Step 1: Performing super-amphiphobic treatment on transparent glass to obtain a super-amphiphobic functional layer;

[0018] Step 2: using a spotting needle to draw the cell-laden hydrogel solution and spot it on the super-amphiphobic functional layer to obtain cell-laden hydrogel droplets;

[0019] Step 3: using a first light source to irradiate the cell-laden hydrogel droplet for the first time; using a second light source to guide the cell-laden hydrogel droplet to move, thereby obtaining a multi-spheroid tissue model; and using the first light source to irradiate the multi-spheroid tissue model for the second time, thereby obtaining a solidified multi-spheroid tissue model.

[0020] The super amphiphobic treatment comprises: sequentially subjecting the transparent glass to a first vacuum deposition, a first heat treatment, a plasma treatment, a second vacuum deposition, and a second heat treatment.

[0021] Preferably, the solution used in the first vacuum deposition is tetraethoxysilane (TES) and an ammonia solution;

[0022] The solution used for the second vacuum deposition is selected from one or more of 1H,1H,2H,2H-perfluorodecyltriethoxysilane, 1H,1H,2H,2H-perfluorooctyltrichlorosilane, and 1H,1H,2H,2H-perfluorodecyltrichlorosilane;

[0023] The first vacuum deposition time is 18 to 24 hours;

[0024] The second vacuum deposition time is 2 to 4 hours.

[0025] Preferably, the cell-laden hydrogel solution is cells resuspended in a complete GelMA solution;

[0026] The complete GelMA solution was prepared by mixing 1% (w / v) LAP solution and 10% (w / v) GelMA solution in a volume ratio of 1:1.

[0027] Preferably, the temperature of the first heat treatment is 450-600°C;

[0028] The duration of the first heat treatment is 2 hours;

[0029] The temperature of the second heat treatment is 200-300°C;

[0030] The duration of the second heat treatment is 0.5 h.

[0031] Preferably, the first irradiation duration is 6 to 10 seconds, and the second irradiation duration is 10 to 20 seconds.

[0032] Preferably, after step 3, the method further comprises: culturing the multispheroid tissue model.

[0033] It can be seen from the above technical solutions that the present invention has the following advantages:

[0034] The present invention provides a device for establishing a multi-spheroid tissue model, comprising: a base, a stage, a photothermoelectric microfluidic platform, a mobile platform, an optical component and a spotting needle; the base is provided with a slide rail, which is movably connected to the stage; the photothermoelectric microfluidic platform is placed on the stage; the base on both sides of the stage is provided with a first guide groove and a second guide groove, respectively, and the mobile platform is movably connected to the first guide groove and the second guide groove; the mobile platform is provided with a third guide groove, and the optical component and the spotting needle are movably connected to the third guide groove and are located above the photothermoelectric conversion functional layer; the photothermoelectric microfluidic platform comprises, from top to bottom, transparent glass, a wafer layer with a pyroelectric effect, and a graphene nanosheet layer. In this scheme, the slide rail is connected to the stage so that the stage can move in the direction of the slide rail; the guide groove is connected to the mobile platform, and the optical component and the spotting needle are movably connected to the guide groove on the mobile platform, thereby realizing multi-dimensional movement of the optical component and the spotting needle, and facilitating batch preparation of multiple droplets; the optical component is set above the photothermal microfluidic platform. When the light source irradiates the photothermal microfluidic platform, the underlying graphene nanosheets quickly produce a local temperature rise, and the chip layer with pyroelectric effect produces free charge changes under the change of temperature field, forming a non-uniform electric field on the chip surface, inducing droplet polarization, so that the droplets are subjected to non-uniform force manipulation in the non-uniform electric field, realizing the controllable construction of a multi-spherical tissue model with a 3D structure, and the operation is simple, without contact with cell tissue, so that the obtained multi-spherical tissue model has high activity.

[0035] The present invention also provides a method for establishing a multi-spheroid tissue model, comprising the following steps: subjecting transparent glass to a super-amphiphobic treatment to obtain a super-amphiphobic functional layer; using a spotting needle to aspirate a cell-loaded hydrogel solution and spot sample on the super-amphiphobic functional layer to obtain a cell-loaded hydrogel droplet; using a first light source to irradiate the cell-loaded hydrogel droplet for the first time; using a second light source to guide the cell-loaded hydrogel droplet to move to obtain a multi-spheroid tissue model; using the first light source to irradiate the multi-spheroid tissue model for the second time to obtain a solidified multi-spheroid tissue model; the super-amphiphobic treatment comprises: subjecting the transparent glass to a first vacuum deposition, a first heat treatment, a plasma treatment, a second vacuum deposition, and a second heat treatment in sequence. In this scheme, the transparent glass is first subjected to a super-amphiphobic treatment to make the cell-loaded hydrogel droplet spherical, which is convenient for constructing a multi-spheroid tissue model, and then two light sources are used to respectively solidify the cell-loaded hydrogel droplet and construct a multi-spheroid tissue model with a 3D structure. The operation is simple, no contact with cell tissue is required, and the obtained multi-spheroid tissue model is intact and undamaged, and can be used to simulate different natural cell compartments, interactions, and signal transmission processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 Schematic diagram of a device for establishing a multi-spheroid tissue model provided in an embodiment of the present invention;

[0038] Figure 2 A scanning electron microscope image of a super amphiphobic functional layer provided in an embodiment of the present invention;

[0039] Figure 3 This is a diagram showing the effect of a hydrophobic needle vacuum-sucking a hydrogel droplet provided in an embodiment of the present invention;

[0040] Figure 4 This is a diagram showing the effect of using a spotting needle to absorb a solution onto a super-amphiphobic functional layer provided in an embodiment of the present invention;

[0041] Figure 5 Schematic diagram of the principle of an 808 nm light source guiding the movement of a hydrogel droplet provided in an embodiment of the present invention;

[0042] Figure 6 This is a diagram showing the process of hydrogel droplets moving and fusing under the guidance of an 808nm light source provided in an embodiment of the present invention;

[0043] Figure 7 This is a fluorescence staining effect diagram of a multi-spheroid tissue model prepared based on the method for establishing a multi-spheroid tissue model provided in an embodiment of the present invention;

[0044] Figure 8 Analysis of cell viability in a multispheroid tissue model prepared based on the method for establishing a multispheroid tissue model provided in an embodiment of the present invention;

[0045] Figure 9 An analysis of the controllable angles in the multi-spheroid tissue model prepared based on the method for establishing a multi-spheroid tissue model provided in the embodiment of the present invention;

[0046] Figure 1In the figure, 1 is the base; 2 is the first driven pulley seat; 3 is the first guide groove; 4 is the first belt; 5 is the fifth driven pulley seat; 6 is the first fixed clamp; 7 is the first drive motor; 8 is the first motor seat; 9 is the second driven pulley seat; 10 is the driving shaft; 11 is the third guide groove; 12 is the second fixed clamp; 13 is the 365nm ultraviolet light source; 14 is the 808nm fiber laser; 15 is the spotting needle; 16 is the third belt; 17 is the third driven pulley seat 18 is a second drive motor; 19 is a second motor seat; 20 is a sixth driven pulley seat; 21 is a second guide groove; 22 is a second belt; 23 is a fourth driven pulley seat; 24 is a third drive motor; 25 is a fixed plate; 26 is a third motor seat; 27 is a slide rail; 28 is a slider; 29 is a stage; 30 is a wafer layer spin-coated with a mixture of graphene nanosheets; 31 is transparent glass; 32 is a cell-carrying hydrogel droplet; 33 is a lead screw; 34 is a fourth guide groove;

[0047] Figure 9 In the figure, the left side shows four multi-spherical tissue models, and the right side shows the angle test results in the four multi-spherical tissue models. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solution of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0049] In the description of this application, it should be noted that the terms "upper," "lower," "front," "back," "inner," "outer," "both sides," and the like, indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate the description of this application and simplify the description. They do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on this application. Relational terms such as "first," "second," "third," and "fourth" are used solely to distinguish one entity from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities.

[0050] Unless otherwise expressly specified or limited, the terms "installed," "connected," "fixed," and "disposed" should be interpreted broadly. For example, they may refer to fixed, detachable, or integral connections; they may refer to mechanical connections, direct connections, indirect connections through an intermediary, or internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in this application based on the specific circumstances.

[0051] For easier understanding, see Figure 1 In this embodiment, a device for establishing a multi-spheroid tissue model is provided. A device for establishing a multi-spheroid tissue model includes: a base 1, a stage 29, a photothermoelectric microfluidic platform, a mobile platform, an optical component, and a spotting needle 15. A slide rail 27 is provided on the base 1, and the slide rail 27 is movably connected to the stage 29, allowing the stage to move back and forth. The photothermoelectric microfluidic platform is placed on the stage 29. A first guide groove and a second guide groove are provided on the base 1 on both sides of the stage 29, respectively. The mobile platform is movably connected to the first and second guide grooves, and the guide grooves enable the mobile platform to move up and down. A third guide groove is provided on the mobile platform. The optical component and the spotting needle are movably connected to the third guide groove and are located above the photothermoelectric conversion functional layer to facilitate spotting and irradiation of cell-carrying hydrogel droplets and control the movement of the cell-carrying hydrogel droplets. The photothermoelectric microfluidic platform includes, from top to bottom, transparent glass 31, a wafer layer with a pyroelectric effect, and a graphene nanosheet layer. In this scheme, the optical components and the spotting needle can move in multiple dimensions. Combined with the photothermal microfluidic platform, the light source can be used to irradiate the photothermal microfluidic platform, so that the graphene nanosheets at the bottom of the photothermal microfluidic platform can quickly produce a local temperature rise. The chip layer with pyroelectric effect produces free charge changes under the change of temperature field, forming a non-uniform electric field on the chip surface, inducing droplet polarization, so that the droplets are subjected to non-uniform force manipulation in the non-uniform electric field, realizing the controllable construction of multi-spherical tissue models. The operation is simple and does not require contact with cell tissue, so that the obtained multi-spherical tissue model has high activity.

[0052] Furthermore, the optical assembly includes a first light source and a second light source. The first light source has a wavelength of 365nm or 405nm, and the second light source has a wavelength of 808nm. The first light source is an ultraviolet light source, primarily used for solidifying the cell hydrogel droplets. The longer the wavelength, the lower the frequency and the lower the energy. The second light source is an infrared light source, used to control the movement of the cell hydrogel droplets, facilitating the preparation of multispherical cell models.

[0053] Furthermore, the wafer layer having the pyroelectric effect is a lithium niobate wafer.

[0054] Furthermore, the graphene nanosheet layer includes: graphene nanosheets and polydimethylsiloxane, and the mass fraction of the graphene nanosheets is 3% to 5%.

[0055] Furthermore, when establishing a multi-spherical tissue model, in order to obtain spherical droplets, the transparent glass 31 is generally subjected to a super-amphiphobic treatment.

[0056] Furthermore, in the photothermoelectric microfluidic platform, the transparent glass 31 is directly placed on the upper surface of the chip layer with the pyroelectric effect, and the graphene nanosheet layer is prepared by first preparing a graphene nanosheet mixture and then spin-coating the graphene nanosheet mixture on the lower surface of the chip layer with the pyroelectric effect.

[0057] Specifically, the method for preparing the graphene nanosheet mixture is as follows:

[0058] First, a polydimethylsiloxane (PDMS) prepolymer mixture was prepared with a prepolymer: curing agent ratio of 10:1; then, graphene nanosheets were evenly dispersed in the polydimethylsiloxane (PDMS) prepolymer mixture by stirring and oscillating. The mass fraction of graphene nanosheets in the mixture was controlled at 3% to 5% to ensure a good photothermal response.

[0059] Specifically, the graphene nanosheet mixture is spin-coated on the lower surface of the wafer layer having the pyroelectric effect in the following manner:

[0060] The above-mentioned graphene nanosheet mixture is evenly spin-coated on a wafer with a pyroelectric effect at a rotation speed of 1000r for 20s to ensure uniform spin coating; the spin-coated wafer is then placed in a plastic vacuum dryer and connected to a vacuum pump for 20 minutes to remove bubbles; the wafer after degassing is then placed horizontally in an oven at 50°C for curing for 1 hour to obtain a wafer layer 30 coated with the graphene nanosheet mixture.

[0061] Furthermore, the slide rail 27 is movably connected to the loading platform 29. Specifically, the base 1 is provided with a fourth guide slot 34, in which a lead screw 33 is installed. The slide rail 27 is disposed on both sides of the lead screw 33, and sliders 28 are disposed within the slide rail 27. The sliders 28 are connected to the lead screw 33. The third drive motor 24 controls the rotation of the lead screw 33, thereby controlling the forward and backward movement of the sliders, thereby achieving the forward and backward movement of the loading platform 29. To ensure that the loading platform 29 is stably connected to the base and moves in the operating direction, a fixing plate 25 and a third motor base 26 are also provided to fix the positions of the fourth guide slot 34 and the third drive motor 24, respectively, and control the stable forward and backward movement of the forward and backward movable platform according to the control instructions.

[0062] Furthermore, the movable platform is movably connected to the first guide groove and the second guide groove, specifically comprising: a first driven pulley seat 2 and a fourth driven pulley seat 23 are fixedly provided on both sides of the base 1, a first guide groove 3 is provided on the first driven pulley seat 2 on one side, a first belt 4 is provided in the first guide groove 3, a first fixed clamping block 6 is provided on the first belt 4, the first fixed clamping block 6 is fixedly connected to the movable platform, the top end of the first guide groove 3 is fixedly connected to the second driven pulley seat 9, and the first drive motor 7 is fixed to the right side of the second driven pulley seat 9 through the first motor seat 8; Similarly, a second guide groove 21 is provided on the fourth driven pulley seat 23 on the other side, and a second belt 22 is provided in the second guide groove 21. The top of the second guide groove 21 is fixedly connected to the first driven pulley seat 17, and the second drive motor 18 is fixed to the first driven pulley seat 17 through the second motor seat 19; and a driving shaft 10 is provided between the third driven pulley seat 17 and the second driven pulley seat 9, and the driving shaft is controlled to rotate by the motor, thereby driving the first fixed clamp 6 on the belt to move up and down, and then driving the movable platform connected to the first fixed clamp 6 to move up and down.

[0063] Furthermore, the mobile platform includes: a fifth driven pulley seat 5, a third guide groove 11, a third belt 16, and a sixth driven pulley seat 20. The left side of the fifth driven pulley seat 5 is fixedly connected to the third guide groove 11, and the other end of the third guide groove 11 is fixedly connected to the sixth driven pulley seat 20. A third belt 16 is provided inside the third guide groove 11, and the fifth driven pulley seat 5 is fixedly connected to the first fixed clamp 6.

[0064] Furthermore, the optical component and the spotting needle are movably connected to the mobile platform, specifically including: a second fixed clamp 12 is provided on the third belt 16, and the optical component and the spotting needle 15 are provided on the second fixed clamp 12. The second fixed clamp 12 can be controlled to move left and right by the third belt 16, thereby realizing the left and right movement of the optical component and the spotting needle, facilitating the spotting operation and controlling the movement of the droplets, making it easier to establish the tissue model.

[0065] The following is a method for establishing a multi-spheroid tissue model provided in another embodiment of the present application.

[0066] All reagents and raw materials used in the following examples were commercially available or homemade. Tetraethoxysilane (TES) (98%) and 1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFDTS) (96%) were purchased from Shanghai MacLean Biochemical Co., Ltd.; PDMS (components A and B) was purchased from Dow Corning; the superhydrophobic coating NeverWet was an AUSUM brand, model Rust-Oleum; and GelMA (gelatin methyl acrylate) and the photoinitiator LAP were purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0067] A method for establishing a multi-spheroid tissue model provided by one embodiment of the present invention is as follows:

[0068] Step 1: Perform super-amphiphobic treatment on transparent glass to obtain a super-amphiphobic functional layer. The specific operations of this step are as follows:

[0069] ① Pretreatment: Use a candle to burn the transparent glass to evenly adhere the soot coating. It should be noted that the transparent glass is thinner to facilitate the light source to pass through, better photothermal conversion, and better force effect of the uneven electric field under the photothermoelectric effect. The candle is an ordinary candle used for daily lighting. Only one side of the transparent glass needs to be evenly adhered to the soot coating. The soot coating is formed by the deposition of incompletely burned carbon particles into a loose porous sediment network. This porous sediment network can serve as a skeleton template for the preparation of the superamphiphobic functional layer.

[0070] ② Perform the first vapor deposition: Place the transparent glass after the candle is burned and two open containers containing 1 ml of tetraethoxysilane (TES) and 1 ml of ammonia solution respectively in a vacuum desiccator for vapor deposition, and keep the deposition in a vacuum environment for 24 hours. In the vacuum desiccator, ammonia will catalyze the hydrolysis and condensation of TES on the surface of the transparent glass, thereby forming a layer of silica shell on the porous sediment network of the transparent glass. Among them, the shell thickness of the silica shell can be adjusted by the duration of vapor deposition. The general reaction deposition time is 18 to 24 hours. In this scheme, the vacuum desiccator item number is PC-250.

[0071] ③ Perform the first heat treatment: Transfer the transparent glass after vacuum deposition to a muffle furnace and calcine at 550°C for 2 hours. Then, close the muffle furnace to allow the soot to thermally degrade and anneal and slowly cool to room temperature. At this point, the silica shell on the transparent glass is hollow. It should be noted that the combustion temperature of carbon black is required to be between 310°C and 400°C, so the calcination temperature must be maintained above 400°C. At the same time, calcination temperatures that are too high may cause problems such as deformation of thin glass. Therefore, the preferred calcination temperature is 450°C to 600°C. The calcination time mainly depends on whether the carbon black formed by the soot deposition completely disappears in the form of CO2. In this solution, the preferred calcination time is about 2 hours.

[0072] ④ Plasma treatment: The cooled transparent glass is treated with air plasma for 100s-300s to generate more hydrophilic groups on the sample surface.

[0073] ⑤ Perform a second vapor deposition: Place the transparent glass after the plasma cleaning mentioned above in a vacuum desiccator together with an open container containing 100-200 μl of 1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFDTS), keep it in a vacuum environment, and deposit it for 2-4 hours. It should be noted that PFDTS can react with the hydrophilic groups on the surface of the transparent glass to introduce strong hydrophobic groups containing fluorine, so that a hydrophobic film layer is formed on the surface of the transparent glass. After the reaction, the unreacted silane residue in the vacuum desiccator is removed by removing the container containing the residual 1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFDTS) from the vacuum desiccator and vacuuming it again; the unreacted silane residue on the transparent glass is removed by heating the transparent glass after the above vacuum deposition at 100-150°C in a microwave oven for about 30 minutes.

[0074] ⑥ Perform a second heat treatment: Heat the transparent glass in a muffle furnace at 200-300°C for approximately 30 minutes to promote condensation and lateral crosslinking of the silanol groups. After the reaction is complete, close the muffle furnace and slowly cool the transparent glass to room temperature to obtain a super-amphiphobic functional layer. The super-amphiphobic functional layer is then placed back onto the wafer layer. It is understood that the super-amphiphobic functional layer is the transparent glass after the super-amphiphobic treatment.

[0075] It should be noted that when applying super-amphiphobic treatment to transparent glass, the treatment can be applied to only one surface of the transparent glass. When the super-amphiphobic functional layer is placed on the wafer layer, the hydrophobic side faces upward, and the non-hydrophobic side is in close contact with the wafer layer. After the super-amphiphobic treatment, the transparent glass can be used repeatedly until the super-amphiphobic functional layer wears out or has other problems, at which point the transparent glass can be treated again.

[0076] See also Figure 2 , a scanning electron microscope image of a super-amphiphobic functional layer provided in one embodiment of the present invention. From the image, it can be seen that a hydrophobic film layer is formed on the surface of the transparent glass.

[0077] Step 2: Use a spotting needle to absorb the cell-loaded hydrogel solution and spot it on the super-amphiphobic functional layer to obtain cell-loaded hydrogel droplets. The specific operations of this step are as follows:

[0078] 1) Preparation of a hydrophobic spotting needle: spray a spotting needle with super hydrophobic coating NeverWet, let it stand for 1 hour to obtain a hydrophobic spotting needle, and then install the hydrophobic spotting needle on a spotting needle tube to obtain a hydrophobic spotting needle.

[0079] See also Figure 3 , the effect diagram of the hydrophobic needle vacuum aspirating hydrogel droplets provided in an embodiment of the present invention, from the figure it can be seen that the hydrogel droplets prepared by the hydrophobic spotting needle are spherical and have a good gel state.

[0080] 2) Preparation of cell-laden hydrogel solution:

[0081] ① Prepare 1% (w / v) initiator standard solution: Mix 1 ml of phosphate buffered saline (PBS) and 0.01 g of photoinitiator LAP, and heat in a 40-50°C water bath in the dark for 15 minutes, shaking several times during the process;

[0082] ② Prepare 10% (w / v) gelatin methyl acrylate (GelMA) standard solution: Mix 0.1g of GelMA with 1ml of PBS and heat in a 60-70℃ water bath for 20 minutes, shaking several times during the process.

[0083] ③ Prepare a complete GelMA solution: Mix the 1% (w / v) initiator standard solution and the 10% (w / v) GelMA standard solution prepared above at a volume ratio of 1:1 to obtain a complete GelMA solution containing 0.5% (w / v) LAP and 5% (w / v) GelMA. Immediately filter the prepared complete GelMA solution through a 0.22 μm sterile syringe filter to sterilize.

[0084] ④ Digest the adherent cells in the culture flask with 0.25% trypsin to obtain the required number of cells, and resuspend them in the prepared GelMA complete solution.

[0085] 3) Sample:

[0086] The cell-laden hydrogel solution was aspirated using the hydrophobic spotting needle and spotted on the super-hydrophobic-coated glass on the constructed photothermal microfluidic platform to prepare volume-controllable cell-laden hydrogel droplets with high throughput.

[0087] See also Figure 4 , provided in an embodiment of the present invention, a diagram of the effect of using a spotting needle to draw a solution and spotting it on the super-amphiphobic functional layer. Different droplets were prepared on the super-amphiphobic surface of the super-amphiphobic functional layer, including pure water droplets, phosphate buffer droplets, bovine serum albumin solution droplets, high-glucose culture medium droplets, GelMA hydrogel droplets, dimethyl sulfoxide droplets, liquid paraffin droplets, and polydimethylsilicone oil droplets. It can be seen that the condensation state of various droplets formed on the surface of the super-amphiphobic functional layer is complete spherical, indicating that the super-amphiphobic functional layer prepared by the present invention has a good super-amphiphobic effect.

[0088] Step 3: Use a first light source to irradiate the cell-laden hydrogel droplet for the first time; use a second light source to guide the movement of the cell-laden hydrogel droplet to obtain a multi-spheroid tissue model; use the first light source to irradiate the multi-spheroid tissue model for the second time to obtain a solidified multi-spheroid tissue model.

[0089] Specifically, in step 3, the wavelength of the first light source is 365nm or 405nm, and the wavelength of the second light source is 808nm. The first irradiation duration is 6 to 10 seconds, so that the cell-loaded hydrogel droplets are not completely solidified. The second light source is used to guide the movement of the cell-loaded hydrogel droplets in order to assemble multiple cell-loaded hydrogel droplets into a multi-spherical tissue model. The second irradiation duration is 10 to 20 seconds, so that the multi-spherical tissue model is completely solidified to form a stable multi-spherical tissue model.

[0090] See also Figure 5 Schematic diagram of the principle of guiding the movement of hydrogel droplets using an 808nm light source, as provided in an embodiment of the present invention. The principle of 808nm infrared laser guidance of hydrogel droplet movement is as follows: 808nm light irradiates the photothermal microfluidic platform, causing a rapid local temperature rise in the underlying graphene nanosheets. The lithium niobate crystals generate free charge changes under the changing temperature field, forming a non-uniform electric field on the wafer surface, inducing droplet polarization. Consequently, the droplets are subjected to non-uniform forces in the non-uniform electric field, guiding the movement of the hydrogel droplets in a controllable manner.

[0091] See also Figure 6 A diagram illustrating the process of hydrogel droplet migration and fusion guided by an 808nm light source, as provided in an embodiment of the present invention. By guiding the migration and fusion of hydrogel droplets using an 808nm infrared laser, programmable construction of in vitro 3D tissue models is achieved. The resulting tissue models are intact, highly active, and simple to operate, enabling batch preparation and high-throughput construction of multispheroid tissue cells.

[0092] Furthermore, to facilitate long-term culture and observation of the multispheroid tissue model, after step 3, the method further includes: culturing the multispheroid tissue model. Specifically, the assembled multispheroid tissue model is transferred to a low-adhesion plate for long-term culture by vacuum aspiration to facilitate long-term observation.

[0093] Furthermore, the multi-spheroid tissue model prepared in the above embodiment was tested, and the specific test results are as follows:

[0094] See also Figure 7 , an image showing the fluorescence staining effect of a multispheroid tissue model prepared based on the method for establishing a multispheroid tissue model provided in an embodiment of the present invention. Fluorescence imaging of the multispheroid tissue model revealed that cells within the hydrogel can express fluorescent protein under normal physiological conditions, demonstrating that the method of the present invention can guide the movement of cell-laden hydrogel microspheres into different multispheroid tissue models using infrared lasers while maintaining a high cell survival rate.

[0095] See also Figure 8, analysis of cell viability in a multi-spheroid tissue model prepared based on the method for establishing a multi-spheroid tissue model of the present application provided in an embodiment of the present invention. By performing CCK-8 cell testing on the constructed multi-spheroid tissue model, it is shown that while the method of the present invention successfully constructs a multi-spheroid tissue model, the cells in the hydrogel can significantly enhance the survival activity of the cells as the culture time increases. It should be noted that the Cell Counting Kit-8 (abbreviated as CCK-8) cell test is a rapid and highly sensitive detection reagent based on WST-8 that is widely used in cell proliferation and cytotoxicity, wherein 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt (abbreviated as WST-8) is a compound similar to MTT, which is reduced to a highly water-soluble orange-yellow formazan product (formazan) by dehydrogenases in mitochondria in the presence of electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-MethoxyPMS). The more and faster the cells proliferate, the darker the culture medium color. The specific detection steps of CCK-8 cell test are as follows:

[0096] 1) Add 10 μl of CCK-8 solution to each well of a 96-well plate. Gently shake the plate to mix thoroughly. Avoid adding a small amount of CCK-8, which could cause errors due to the reagent sticking to the well walls. It should be noted that in another embodiment, culture medium containing 10% CCK-8 can also be directly added as a liquid replacement. Also, avoid creating bubbles during the addition process.

[0097] 2) Place the culture plate in an incubator and incubate for 1-4 hours. Because different cell types produce varying amounts of formazan, if color development is insufficient, extend the incubation time.

[0098] 3) Using a microplate reader, measure the absorbance (OD) of the culture medium at 450 nm after incubation of the culture plate.

[0099] Note: The measured value here is the cell viability OD value in our data.

[0100] See also Figure 9 , analysis of the controllable angles in the multi-spheroid tissue models prepared based on the method for establishing a multi-spheroid tissue model of the present application provided in an embodiment of the present invention. In the figure, four multi-spheroid tissue models are shown on the left, and the angle test results in the four multi-spheroid tissue models are shown on the right. Analysis of the test results shows that the platform constructed based on the present invention is capable of controllably assembling multi-spheroid tissue models with different angles, further illustrating that the establishment method of the present application can achieve controllable construction of multi-spheroid tissue models.

[0101] The above is a detailed introduction to the device and method for establishing a multi-spheroid tissue model provided by the present invention. For those skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A device for establishing a multi-spheroid tissue model, characterized in that: include: Base, stage, photothermal and electro-microfluidic platform, mobile platform, optical components and spotting needle; The base is provided with a slide rail, and the slide rail is movably connected to the loading platform; The photothermal microfluidic platform is placed on the stage; The base on both sides of the loading platform is provided with a first guide groove and a second guide groove respectively, and the movable platform is movably connected to the first guide groove and the second guide groove; A third guide groove is provided on the movable platform, and the optical component and the spotting needle are movably connected to the third guide groove to achieve multi-dimensional movement of the optical component and the spotting needle; The optical component and the spotting needle are located above the photothermal and electro-microfluidic platform; The photothermoelectric microfluidic platform includes, from top to bottom, transparent glass, a wafer layer with pyroelectric effect, and a graphene nanosheet layer; The optical component includes: a first light source and a second light source; the wavelength of the first light source is 365 nm or 405 nm; the wavelength of the second light source is 808 nm.

2. The device according to claim 1, characterized in that The wafer layer having the pyroelectric effect is a lithium niobate wafer.

3. The device according to claim 1, characterized in that The graphene nanosheet layer comprises graphene nanosheets and polydimethylsiloxane, and the mass fraction of the graphene nanosheets is 3% to 5%.

4. A method for establishing a multi-spheroid tissue model based on the device according to any one of claims 1 to 3, characterized in that: The following steps are included: Step 1: The transparent glass is subjected to a super-amphiphobic treatment to obtain a super-amphiphobic functional layer, and the transparent glass with the super-amphiphobic functional layer is placed on a photothermal microfluidic platform; Step 2: using a spotting needle located above the photothermal microfluidic platform to draw a cell-loaded hydrogel solution and spot it on the super-amphiphobic functional layer to obtain cell-loaded hydrogel droplets; Step 3: Using a first light source in an optical assembly located above the photothermal microfluidic platform to irradiate the cell-laden hydrogel droplet for the first time; after the first light source irradiation is completed, moving the optical assembly located above the photothermal microfluidic platform to allow a second light source to guide the cell-laden hydrogel droplet to move, thereby obtaining a multi-spheroid tissue model; using the first light source to irradiate the multi-spheroid tissue model for a second time, thereby obtaining a solidified multi-spheroid tissue model; The super amphiphobic treatment comprises: sequentially subjecting the transparent glass to a first vacuum deposition, a first heat treatment, a plasma treatment, a second vacuum deposition, and a second heat treatment.

5. The method according to claim 4, characterized in that The solution used in the first vacuum deposition is tetraethoxysilane (TES) and ammonia solution; The solution used for the second vacuum deposition is selected from one or more of 1H,1H,2H,2H-perfluorodecyltriethoxysilane, 1H,1H,2H,2H-perfluorooctyltrichlorosilane, and 1H,1H,2H,2H-perfluorodecyltrichlorosilane; The first vapor deposition time is 18 to 24 hours; The second vapor deposition time is 2 to 4 hours.

6. The method according to claim 4, characterized in that The cell-laden hydrogel solution is cells resuspended in a complete GelMA solution; The complete GelMA solution was prepared by mixing 1% (w / v) LAP solution and 10% (w / v) GelMA solution in a volume ratio of 1:

1.

7. The method according to claim 4, characterized in that The temperature of the first heat treatment is 450-600°C; The duration of the first heat treatment is 2 hours; The temperature of the second heat treatment is 200-300°C; The duration of the second heat treatment is 0.5 h.

8. The method according to claim 4, characterized in that The first irradiation duration is 6 to 10 seconds, and the second irradiation duration is 10 to 20 seconds.

9. The method according to claim 4, characterized in that After step 3, the method further includes: culturing the multispheroid tissue model.

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