Construction of three-dimensional cell sphere and application of three-dimensional cell sphere in drug screening

Abstract

The invention relates to construction of a three-dimensional cell sphere and application of the three-dimensional cell sphere in drug screening, and the method comprises the following steps: providing a pairing fusion chip which comprises a plurality of groups of capture structures arranged in a periodic array, each group of capturing structure comprises a cell sphere capturing micropore, a test liquid drop capturing micropore and a microchannel for communicating the cell sphere capturing micropore with the test liquid drop capturing micropore through a fluid; loading the liquid drops loaded with the 3D cell spheres into the cell sphere capturing micropores, loading and filling test liquid drops into the test liquid drop capturing micropores so as to pair the liquid drops loaded with the 3D cell spheres with the test liquid drops, and fusing the liquid drops loaded with the 3D cell spheres with the test liquid drops of the same group of capturing structures under an electrifying condition. According to the invention, the hydrogel microspheres with core-shell structures are generated with high throughput through a microfluidic technology, and rapid pairing and fusion of 3D tumor cell sphere-loaded liquid drops and test liquid drops are realized.

Description

Technical Field

[0001] The present invention relates to the field of biological monitoring technology, and in particular to a droplet fusion model based on three-dimensional cell spheres, a construction method and application thereof. Background Art

[0002] In recent years, cell-based analytical studies have been increasingly used in drug development to determine the phenotypes of potential drug candidates. Cell-based analytical studies can provide multi-parameter data for quantitative measurement of cellular responses. Currently, the most widely used cells in drug screening are monolayer cultured cells, that is, cells cultured in traditional well plates or culture dishes. However, although monolayer (2D) cell culture is widely used, this model lacks the interactions (signaling pathways) between cells and cells and cells and extracellular matrix, which are essential for the function of organisms. Compared with traditional two-dimensional culture models, cells cultured in three-dimensional (3D) mode can better simulate the actual complex environment in vivo in terms of extracellular matrix components, cell-cell, and cell-matrix interactions. At the same time, drug screening results in 3D mode are closer to clinical trials and can provide more realistic predictions for safety and risk assessment.

[0003] At present, a variety of 3D cell construction and culture methods have been developed to simulate the in vivo characteristics of physiological tissues and used in various drug studies. Zhao et al. used droplet microfluidics to co-encapsulate rabbit bone marrow-derived mesenchymal stem cells (BMSCs) and photo-crosslinked hydrogel gelatin methacryloyl (GelMA) into droplets, and then photopolymerized to quickly obtain cell-containing gel microspheres. After the cells proliferated into 3D cell spheres, they were injected into rabbit bone injury tissue for regeneration of damaged tissue. Wu et al. used droplet microfluidics and hydrogel scaffold materials to encapsulate tumor cells from breast cancer patients into droplets containing Matrigel. By controlling the cell concentration and droplet size, the number and composition of cells in each droplet can be precisely controlled; after removing the oil phase, a large number of tumor cell spheres can be formed within a few hours through the self-assembly of cells supported by the hydrogel matrix; using it for drug testing can obtain a rapid evaluation of patients' tumor treatment. Agarwa et al. constructed a vascularized 3D cell sphere and used it for drug testing. The researchers first encapsulated the tumor cells into the core of the core-shell gel microspheres. Once the 3D cell spheres were successfully constructed, endothelial cells and other matrix cells were introduced to construct large-scale 3D vascularized tumors. After using the 3D vascularized tumors for drug testing, they found that they had stronger drug resistance than non-vascularized 3D cell cultures and 2D cell cultures.

[0004] Although many advances have been made in drug screening based on 3D cell culture, the current 3D cell culture for drug screening still has some shortcomings, such as low throughput, low combination efficiency between 3D cells and drugs, and the inability to achieve high-throughput real-time, in situ observation in drug testing. Summary of the invention

[0005] In view of the shortcomings of the prior art mentioned above, the object of the present invention is to provide a droplet fusion model based on three-dimensional cell spheroids, a construction method and its use, so as to solve the problems of low efficiency of the combination of cell spheroids and drug droplets in existing 3D cell culture models and in situ, real-time, high-throughput observation during drug screening.

[0006] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0007] The present invention provides a droplet fusion model based on a three-dimensional cell sphere, comprising:

[0008] A paired fusion chip is provided, wherein the paired fusion chip comprises a plurality of capture structures arranged in a periodic array, each capture structure comprising a cell ball capture micropore, a test droplet capture micropore, and a microchannel for fluidly connecting the cell ball capture micropore and the test droplet capture micropore;

[0009] The droplets loaded with 3D cell spheroids are assembled in the cell spheroid capture microwells, and the test droplets are loaded and filled in the test droplet capture microwells to pair the droplets loaded with 3D cell spheroids with the test droplets. Under power-on conditions, the droplets loaded with 3D cell spheroids are fused with the test droplets of the same group of capture structures and absorb the contents of the test droplets.

[0010] Optionally, the 3D cell sphere is formed by in vitro culture of methacryloyl gelatin (GelMA) hydrogel microspheres that encapsulate tumor cells based on microfluidic technology.

[0011] The present invention also provides a method for constructing a droplet fusion model based on a three-dimensional cell sphere, comprising:

[0012] Hydrogel microspheres encapsulating tumor cells are constructed using a microfluidic chip, the microfluidic chip includes a flow channel layer, the surface of the flow channel layer is provided with an external phase fluid flow channel, an internal phase fluid flow channel and a dispersed phase flow channel, the external phase fluid flow channel is used to transport a solution containing a hydrogel prepolymer, the internal phase fluid flow channel is used to transport a suspension of tumor cells, the external phase fluid flow channel and the internal phase fluid flow channel merge into a laminar flow channel to form a laminar flow of a suspension loaded with tumor cells and a hydrogel prepolymer solution, the dispersed phase flow channel is configured to transport an oil phase solvent to generate oil phase droplets encapsulating the tumor cells at the intersection of the dispersed phase flow channel and the laminar flow channel, the oil phase droplets of the tumor cells are photo-crosslinked to form hydrogel microspheres with a core-shell structure, wherein the hydrogel includes one of methacrylated gelatin, sodium alginate hydrogel, and polyethylene glycol diacrylate hydrogel;

[0013] The hydrogel microspheres encapsulating tumor cells are placed in culture medium and cultured into 3D cell spheres;

[0014] A paired fusion chip is provided according to the above, wherein droplets carrying the 3D cell sphere and test droplets are sequentially loaded on multiple groups of capture structures of the paired fusion chip, and the droplets carrying the 3D cell sphere are fused with the test droplets of the same group of capture structures under power-on conditions, and the contents of the test droplets are absorbed.

[0015] Optionally, the flow channel layer of the microfluidic chip is prepared by soft photolithography, and the material of the flow channel layer includes: one of polydimethylsiloxane, polymethyl methacrylate, and polycarbonate.

[0016] Optionally, the step of preparing the paired fusion chip by a soft template method comprises:

[0017] Spin-coat photoresist on the surface of the silicon wafer, and perform photolithography and etching methods on the photoresist based on a photomask;

[0018] The polydimethylsiloxane monomer is mixed with a curing agent and poured onto the silicon wafer formed with a patterned photoresist, and the polydimethylsiloxane monomer is formed into a capture structure layer based on the patterned photoresist, and a plurality of capture structures arranged in a periodic array are provided on the surface of the capture structure layer;

[0019] The trapping structure layer is sealed onto the chip substrate using oxygen plasma treatment.

[0020] Optionally, the step of preparing the paired fusion chip also includes: determining the geometric shape and size of the cell ball capture micropores and the corresponding test droplet capture micropores of the paired fusion chip based on the size of the 3D cell ball, wherein the ratio of the diameter of the cell ball capture micropore to the test droplet capture micropore is not less than 1:1; and performing hydrophobic treatment on the surface of the paired fusion chip.

[0021] Optionally, the method further comprises: collecting oil phase droplets encapsulating the tumor cells at the outlet end of the flow channel layer of the microfluidic chip, initiating free radical cross-linking through ultraviolet light to form a hydrogel shell layer encapsulating the tumor cells; and demulsifying the oil phase droplets to obtain hydrogel microspheres with a core-shell structure.

[0022] Optionally, it also includes:

[0023] Before introducing the droplet loaded with the 3D cell spheroid and the test droplet, introducing an oil phase solvent to wet the paired fusion chip;

[0024] Passing the droplet loaded with the 3D cell spheroid into the paired fusion chip to fill the droplet into the cell spheroid capture micropores of the paired fusion chip; and,

[0025] The test droplets are generated by a microfluidics method, and the test droplets are assembled and filled in the test droplet capturing microwells.

[0026] Optionally, it also includes:

[0027] Loading one or more formulations of test droplets in each test droplet capture microwell of the paired fusion chip, wherein the formulation includes components, concentrations, and combinations thereof of the test droplets;

[0028] The paired fusion chip is contacted with a corona bar to fuse the droplets loaded with the 3D cells with the test droplets in each set of capture structures.

[0029] Optionally, the suspension of the 3D cell spheroid and the content of the test droplet contain fluorescein with different excitation wavelengths, and the fusion behavior of the droplet loaded with the 3D cell spheroid and the test droplet is traced by the fluorescein.

[0030] The present invention provides a method for using the aforementioned droplet pairing and fusion model based on three-dimensional cell spheres in drug screening.

[0031] Optionally, the use includes:

[0032] Using a microfluidic method to generate droplets of anti-tumor drugs, the droplets of the anti-tumor drugs are loaded into the test droplet capture micropores, and the fusion of the 3D cell sphere and the droplets of the anti-tumor drugs is performed under power-on conditions;

[0033] The suspension of the 3D cell sphere after fusion is aspirated, the supernatant is discarded by centrifugation, and staining analysis is performed;

[0034] The types, concentrations and combination formulations of anti-tumor drugs are screened by means of staining markers, wherein the staining markers include one of live / dead cell staining and protein expression staining.

[0035] Optionally, one or more formulations of anti-tumor drugs are tested simultaneously on the paired fusion chip, and the formulations of the anti-tumor drugs are tested for effectiveness and drug resistance in response to the cell survival rate or cell apoptosis rate marked by staining, wherein the formulations include types, concentrations and combinations of anti-tumor drugs.

[0036] In summary, the present invention provides a droplet fusion model based on three-dimensional cell spheroids, a construction method and its use, wherein tumor cells are encapsulated into methacrylated gelatin hydrogel (GelMA) by microfluidic technology to generate hydrogel microspheres with a core-shell structure in high throughput, which are used to construct 3D tumor cell spheres with uniform size and stable cell number; thereafter, after the droplets loaded with 3D tumor cell spheres are passed into a droplet pair-merge chip, the droplets loaded with 3D tumor cell spheres will be loaded into the cell sphere capture micropores due to the size effect; then, after the test droplets containing fluorescein are passed into the chip, they will be loaded into the test droplet capture micropores, thereby realizing the rapid pairing of the droplets loaded with 3D tumor cell spheres and the test droplets, and after the droplets loaded with 3D tumor cell spheres are fused with the test droplets under power-on conditions, in the application of selecting anti-tumor drugs to make drug droplets, the droplet fusion model based on three-dimensional cell spheres of the present invention can realize in situ, real-time, and high-throughput observation of tumor cell spheres and drug droplets. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shown is a schematic diagram of the process of constructing a droplet fusion model based on three-dimensional cell spheroids according to the present invention.

[0038] Figure 2 Shown is a schematic structural diagram of a microfluidic chip for constructing three-dimensional cell spheres according to the present invention.

[0039] Figure 3 Shown are microscopic images of live / dead cell staining after fusion of a droplet loaded with a 3D cell spheroid and a test droplet in the droplet fusion model based on a three-dimensional cell spheroid of the present invention, wherein a is a bright field microscopic image, b is a fluorescence microscopic image of live cells, c is a fluorescence microscopic image of dead cells, and d is a fluorescence microscopic image of the overlapping fluorescence signals shown in b and c.

[0040] Figure 4 A schematic diagram showing the arrayed capture structure of the paired fusion chip of the present invention.

[0041] Figure 5A to Figure 5C It is a schematic diagram showing the pairing and fusion of a droplet loaded with 3D cell spheroids and a test droplet on a pairing and fusion chip according to an embodiment of the present invention.

[0042] Figure 6-7Shown are fluorescence microscopic images of the fusion behavior of blank cell droplets and blank test droplets in the control group 1 of the present invention using fluorescein tracing, wherein a is a bright field microscopic image, b is a fluorescence microscopic image of the blank test droplet, c is a fluorescence microscopic image of the blank cell droplet, and d is a fluorescence microscopic image of the overlapping fluorescence signals shown in b and c.

[0043] Fig. 8A Shown are fluorescence microscopic images of 3D cell spheroids after fusion with anti-tumor drugs of different concentrations in an embodiment of the present invention.

[0044] Figure 8B The figure is a graph illustrating the cell survival rate of 3D cell spheroids in the example of the present invention and the control group 3 after being acted upon by drugs of different concentrations. DETAILED DESCRIPTION

[0045] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0046] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0047] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0048] In order to construct a 3D tumor cell spheroid with stable size and solve the problem of low combination efficiency between 3D tumor cell spheroid and drug droplet, the present invention provides a droplet fusion model based on a three-dimensional cell spheroid, comprising:

[0049] A paired fusion chip is provided, wherein the paired fusion chip comprises a plurality of capture structures arranged in a periodic array, each capture structure comprising a cell ball capture micropore, a test droplet capture micropore, and a microchannel for fluidly connecting the cell ball capture micropore and the test droplet capture micropore;

[0050] The droplets loaded with 3D cell spheroids are assembled in the cell spheroid capture microwells, and the test droplets are loaded and filled in the test droplet capture microwells to pair the droplets loaded with 3D cell spheroids with the test droplets. Under power-on conditions, the droplets loaded with 3D cell spheroids are fused with the test droplets of the same group of capture structures and absorb the contents of the test droplets.

[0051] In an optional embodiment, the 3D cell sphere is formed by in vitro culture of methacrylated gelatin hydrogel microspheres that encapsulate tumor cells based on microfluidic technology.

[0052] The present invention provides a method for constructing a droplet fusion model based on a three-dimensional cell sphere, comprising:

[0053] A hydrogel microsphere encapsulating tumor cells is constructed by using a microfluidic chip, wherein the microfluidic chip comprises a flow channel layer, wherein the surface of the flow channel layer is provided with an external phase fluid flow channel, an internal phase fluid flow channel and a dispersed phase flow channel, wherein the external phase fluid flow channel is used to transport a solution containing a hydrogel prepolymer, and the internal phase fluid flow channel is used to transport a suspension of tumor cells, wherein the external phase fluid flow channel and the internal phase fluid flow channel merge into a laminar flow channel to form a laminar flow of a suspension of tumor cells and a hydrogel prepolymer solution, wherein the dispersed phase flow channel is configured to transport an oil phase solvent to generate oil phase droplets encapsulating the tumor cells at the intersection of the dispersed phase flow channel and the laminar flow channel, wherein the oil phase droplets of the tumor cells are photo-crosslinked to form hydrogel microspheres having a core-shell structure, wherein the hydrogel includes but is not limited to methacryloyl gelatin (GelMA), sodium alginate hydrogel, polyethylene glycol diacrylate hydrogel or similar hydrogel materials having good physical and chemical properties and biocompatibility;

[0054] The hydrogel microspheres encapsulating tumor cells are placed in culture medium and cultured into 3D cell spheres;

[0055] A paired fusion chip is provided according to the above, wherein droplets carrying the 3D cell sphere and test droplets are sequentially loaded on multiple groups of capture structures of the paired fusion chip, and the droplets carrying the 3D cell sphere are fused with the test droplets of the same group of capture structures under power-on conditions, and the contents of the test droplets are absorbed.

[0056] In an optional embodiment, the flow channel layer of the microfluidic chip is prepared by a soft template method, and the material of the flow channel layer includes: one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and polycarbonate (PC).

[0057] Preferably, the method further comprises: collecting oil phase droplets encapsulating the tumor cells at the outlet end of the flow channel layer of the microfluidic chip, initiating free radical cross-linking through ultraviolet light to form a hydrogel shell layer encapsulating the tumor cells; and demulsifying the oil phase droplets to obtain hydrogel microspheres with a core-shell structure.

[0058] In another optional embodiment, the step of preparing the paired fusion chip by a soft template method comprises:

[0059] Spin-coat photoresist on the surface of the silicon wafer, and perform photolithography and etching methods on the photoresist based on a photomask;

[0060] The polydimethylsiloxane monomer is mixed with a curing agent and poured onto the silicon wafer formed with a patterned photoresist, and the polydimethylsiloxane monomer is formed into a capture structure layer based on the patterned photoresist, and a plurality of capture structures arranged in a periodic array are provided on the capture structure layer;

[0061] The trapping structure layer is sealed onto the chip substrate using oxygen plasma treatment.

[0062] Preferably, the step of preparing the paired fusion chip also includes: determining the geometric shape and size of the cell ball capture micropores and the corresponding test droplet capture micropores of the paired fusion chip based on the size of the 3D cell ball, wherein the ratio of the diameter of the cell ball capture micropore to the test droplet capture micropore is not less than 1:1; and performing hydrophobic treatment on the surface of the paired fusion chip.

[0063] In an optional embodiment, it also includes:

[0064] Before introducing the droplet loaded with the 3D cell spheroid and the test droplet, introducing an oil phase solvent to wet the paired fusion chip;

[0065] Passing the droplet loaded with the 3D cell spheroid into the paired fusion chip to fill the droplet into the cell spheroid capture micropores of the paired fusion chip; and,

[0066] The test droplets are generated by a microfluidics method, and the test droplets are assembled and filled in the test droplet capturing microwells.

[0067] Preferably, the method further comprises: loading one or more formulations of test droplets in each test droplet capture microwell of the paired fusion chip; wherein the formulations include components, concentrations and combinations thereof of the test droplets;

[0068] The paired fusion chip is contacted with a corona bar to fuse the droplets loaded with the 3D cells with the test droplets in each set of capture structures.

[0069] More preferably, the method further comprises: the suspension of the 3D cell spheroid and the content of the test droplet contain fluorescein with different excitation wavelengths, and the fusion behavior of the droplet loaded with the 3D cell spheroid and the test droplet is traced by the fluorescein.

[0070] The present invention also provides a use of a droplet pairing fusion model based on a three-dimensional cell sphere in drug screening.

[0071] In an optional embodiment, the use includes:

[0072] Using a microfluidic method to generate droplets of anti-tumor drugs, the droplets of the anti-tumor drugs are loaded into the test droplet capture micropores, and the fusion of the 3D cell sphere and the droplets of the anti-tumor drugs is performed under power-on conditions;

[0073] The suspension of the 3D cell sphere after fusion is aspirated, the supernatant is discarded by centrifugation, and staining analysis is performed;

[0074] The types, concentrations and combination formulations of anti-tumor drugs are screened by means of staining markers, wherein the staining markers include one of live / dead cell staining and protein expression staining.

[0075] Preferably, one or more formulations of anti-tumor drugs are tested simultaneously on the paired fusion chip, and the formulations of the anti-tumor drugs are tested for effectiveness and drug resistance in response to the results of the stained cell survival rate or cell apoptosis rate, wherein the formulations include types, concentrations and combinations of anti-tumor drugs.

[0076] The above technical solutions and technical effects are further explained and verified through specific examples and implementation effect data below.

[0077] Example

[0078] (1) Three-dimensional cell spheroid model based on microfluidics technology

[0079] See also Figure 1 to Figure 2 , a method for constructing a three-dimensional cell spheroid model using microfluidic technology, comprising:

[0080] First, step S11 is performed to draw the droplet generation channel, fluid channel and outflow channel of the microfluidic chip using graphic software, and a photomask is made based on the graphic.

[0081] Then, step S12 is performed, in which photoresist is spin-coated on the surface of the silicon wafer, and photolithography and etching methods are performed on the photoresist based on the photomask. After coating, spinning, pre-baking, exposure, post-baking, developing, and hard baking, a SU-8 template defining a channel pattern is prepared. The specific process flow is: SU-8 3025 photoresist is spin-coated on a 3-inch silicon wafer at a speed of 1500 rpm, with a thickness of ≤60 μm; then, the photoresist is pre-baked at a temperature of 95° C. for 20 minutes; a photomask containing a channel pattern is covered on the silicon wafer, and exposed to a 120 mW ultraviolet lamp (M365L2, Thorlabs) for about 450 seconds to transfer the photomask pattern to the silicon wafer; then, the photoresist is baked at 95° C. for 10 minutes to further solidify the photoresist; and the exposed photoresist is developed in SU-8 developer (MicroChem) for 15 minutes.

[0082] Then, step S13 is performed. Based on the SU-8 template, polydimethylsiloxane (PDMS) monomer is mixed with a curing agent and poured to form a flow channel layer. The specific process flow is: the photolithographic silicon wafer is cleaned with isopropanol and ethanol, and blown dry with a nitrogen gun; PDMS monomer (SYLGARD 184, Dow Corning) and a curing agent are mixed with a curing agent at a weight ratio of 10:1 (w:w) PDMS, and poured into a culture dish with a silicon wafer; then, the curing reaction is maintained at 60°C in a constant temperature box for ~8 hours; the cured PDMS is peeled off from the silicon wafer, and a shell fluid flow channel inlet, a cell fluid flow channel inlet, an oil phase flow channel inlet and an oil phase droplet outlet are created in the flow channel layer using a 0.7 mm punch.

[0083] like Figure 2 As shown, the microfluidic chip includes a flow channel layer, and the surface of the flow channel layer is provided with an external phase fluid flow channel, an internal phase fluid flow channel and a dispersed phase flow channel, the external phase fluid flow channel is used to transport a solution 120 containing a hydrogel prepolymer, the internal phase fluid flow channel is used to transport a suspension 130 of tumor cells, the external phase fluid flow channel and the internal phase fluid flow channel merge into a laminar flow channel to form a laminar flow of a suspension loaded with tumor cells and a hydrogel prepolymer solution, the dispersed phase flow channel is configured to transport an oily solvent 110 as a dispersed phase to generate oil phase droplets encapsulating the tumor cells at the intersection of the dispersed phase flow channel and the laminar flow channel, and the oil phase droplets 140 encapsulating the tumor cells are collected at the outlet end of the flow channel layer.

[0084] Then, step S14 is performed to seal the flow channel layer on the chip substrate using oxygen plasma treatment; the microfluidic chip is placed at 100° C. for 30 minutes to enhance the bonding effect between the upper flow channel layer and the chip substrate.

[0085] Specifically, step S14 includes: before using the microfluidic chip, the channel surface of the channel layer is hydrophobically treated, for example, using a commercial water repellent Aquapel (PPG Industries) for hydrophobic treatment, and after hydrogel treatment, the microfluidic chip is set to 60° C. 10 minutes before use.

[0086] Then, step S15 is performed to construct a three-dimensional cell sphere based on the microfluidic chip. The specific process flow is: the tumor cells are mixed with 1% methyl cellulose (MC) solution as the inner phase, 1% methacrylated gelatin (gelma) solution as the outer phase, and HFE-7500 fluorinated oil containing 2% PEG-PFPE surfactant as the dispersed phase. Figure 2 As shown, the three are introduced into a microfluidic chip, and the flow rate (μl / h) of the fluid is set to oil phase: gelma phase: MC phase = 1000:200:100 to prepare oil phase droplets encapsulating tumor cells, wherein the tumor cells form a loaded core layer, and the hydrogel prepolymer encapsulating the tumor cells is subsequently solidified to form a shell layer.

[0087] Step S15 also includes: collecting oil phase droplets encapsulating the tumor cells at the outlet end 140 of the flow channel layer; irradiating the collected oil phase droplets under 365nm ultraviolet light for 30s, and forming a solidified hydrogel shell layer by ultraviolet light-induced free radical cross-linking, thereby forming a 3D cell sphere with core-loaded tumor cells, wherein the core size, shell thickness and overall size of the synthesized oil phase droplets are controlled by adjusting the fluid flow rate of the external phase fluid flow channel, the internal phase fluid flow channel and the dispersed phase flow channel; then, the oil phase droplets are demulsified to obtain hydrogel microspheres with a core-shell structure.

[0088] For example, HFE-7500 reagent containing 20% ​​perfluorooctanol (PFO) is used for demulsification, and PBS solution is used for washing three times to obtain core-shell gel microspheres containing cells.

[0089] Then, step S16 is performed to obtain hydrogel microspheres encapsulating tumor cells, and then the hydrogel microspheres are placed in a culture medium containing DMEM culture medium for culture. After 3-7 days of culture, 3D cell spheres loaded with tumor cells can be obtained. (2) Construction method of droplet fusion model based on three-dimensional cell spheres

[0090] The three-dimensional cell spheroid-based droplet fusion model mentioned above in this article can be implemented according to the following method for constructing a three-dimensional cell spheroid-based droplet fusion model.

[0091] First, step S21 is performed to provide a paired fusion chip, and the droplets encapsulating the 3D cell spheroids obtained in step S16 and the test droplets are sequentially loaded on the multiple capture structures of the paired fusion chip.

[0092] See also Figure 4 The paired fusion chip includes a plurality of capture structures arranged in a periodic array, each capture structure includes a cell ball capture micropore T, a test droplet capture micropore D, and a microchannel for fluidly connecting the cell ball capture micropore and the test droplet capture micropore. For example, the paired fusion chip may be provided with from dozens to tens of thousands of capture structures.

[0093] Specifically, similar to the preparation method of the microfluidic chip for preparing hydrogel microspheres, the paired fusion chip is prepared by a soft template method, and the paired fusion chip includes a capture structure layer and a chip substrate. The steps of preparing the paired fusion chip are as follows: using graphic software to draw a plurality of capture structures arranged in a periodic array, and making a photomask based on the graphic; spin coating photoresist on the surface of a silicon wafer, performing photolithography and etching methods on the photoresist based on the photomask, and preparing a SU-8 template defining a micropore array pattern through coating, spinning, pre-baking, exposure, post-baking, developing, and hard baking; mixing polydimethylsiloxane monomer with a curing agent and pouring them on the silicon wafer formed with a patterned photoresist, and forming the polydimethylsiloxane monomer into a capture structure layer based on the patterned photoresist, and the surface of the capture structure layer is provided with a plurality of capture structures arranged in a periodic array; and using oxygen plasma treatment to seal the capture structure layer to the chip substrate.

[0094] In this embodiment, the step of preparing the paired fusion chip by the soft template method also includes: spin coating SU-8 3025 photoresist on a 3-inch silicon wafer at a rotation speed of 800 rpm, and the depth of the prepared capture structure layer is close to or about 90 μm.

[0095] Then, step S22 is performed, in which the droplets of the 3D cell ball are sequentially loaded into the cell ball capture microwells using the paired fusion chip, and the test droplets are loaded and filled into the droplet loading microwells.

[0096] Step S22 also includes: determining the geometry and size of the cell ball capture micropore T and the corresponding test droplet capture micropore D of the paired fusion chip based on the size of the 3D cell ball, wherein the diameter ratio of the cell ball capture micropore T to the test droplet capture micropore D is not less than 1.5:1; preferably, the diameter ratio of the two is set to 2:1; and performing hydrophobic treatment on the capture structure surface of the paired fusion chip. For example, the diameter of the cell ball capture micropore T is close to or about 120 μm, and the diameter of the test droplet capture micropore D is close to or about 60 μm.

[0097] Specifically, step S22 also includes: before introducing the droplets carrying the 3D cell sphere and the test droplets, introducing an oil phase solvent to wet the paired fusion chip; using a microfluidic method to generate droplets carrying the 3D cell sphere from the suspension of the 3D cell sphere, and assembling the droplets of the 3D cell sphere into the cell sphere capture micropores; and, using a microfluidic method to generate the test droplets, and the droplets of the 3D cell sphere are assembled and filled in the test droplet capture micropores, wherein the microfluidic method includes using a microfluidic chip to provide an oily solvent as a dispersed phase to generate oil phase droplets encapsulating the 3D cell sphere, and the microfluidic chip here can use a conventional microfluidic chip including a dispersed phase flow channel and a continuous phase flow channel. Preferably, the droplets loaded with 3D cell spheroids have a particle size close to that of the cell spheroid capture micropores, and the test droplets have a particle size close to that of the test droplet capture micropores. The 3D cell spheroids are loaded into droplets through microfluidic technology, and test droplets are generated. By optimizing the device and flow rate, a high proportion of loading rate and highly uniform droplets can be achieved.

[0098] Then, step S23 is performed to fuse the 3D cell sphere with the test droplet of the same capture structure under power-on conditions, and absorb the contents of the test droplet. The specific process flow is: after the droplet carrying the 3D cell sphere is paired with the test droplet, a corona rod is used to contact the paired fusion chip, and the droplet carrying the 3D cell sphere is fused with the test droplet in each capture structure. Preferably, the test droplet contains fluorescein, and fluorescein is used to trace the transfer and fusion of the test droplet. The fluorescent microscopic image of live / dead cell staining after the fusion of the droplet carrying the 3D cell sphere and the test droplet based on the droplet fusion model of the three-dimensional cell sphere is shown in Figure 3 , the droplets loaded with 3D cell spheroids and the test droplets in the embodiment are paired and fused on the paired fusion chip. Figure 5A to Figure 5C .

[0099] In order to facilitate the real-time observation of the fusion behavior of the droplet loaded with 3D cell spheroids and the test droplet, the suspension of the 3D cell spheroids and the test droplet contents contain fluorescein with different excitation wavelengths, and the fusion behavior of the droplet loaded with 3D cell spheroids and the test droplet is traced by fluorescein. The specific process flow is as follows: ① Alexa546 dye is incorporated into the suspension of the 3D cell spheroids; a microfluidic method is used to generate droplets of the 3D cell spheroids wrapped in an oil phase; the generated droplets of the 3D cell spheroids wrapped in an oil phase are passed into a paired fusion chip, and the droplets of the 3D cell spheroids wrapped in an oil phase are assembled into the cell spheroid capture micropores; ② Alexa488 dye is added to the solution used to generate test droplets; a microfluidic method is used to generate oil-phase-encapsulated test droplets; before introducing the test droplets, a clean oil phase is introduced to flush the excess droplets encapsulating the 3D cell sphere, the oil-phase-encapsulated test droplets are loaded and filled in the test droplet capture micropores, and a clean oil phase is introduced to flush the excess test droplets, thereby achieving the pairing of the droplets loaded with the 3D cell sphere on the fusion chip and the test droplets; ③ A fluorescence microscope is used to observe in situ the fusion behavior of the oil-phase-encapsulated 3D cell sphere droplets and the test droplets under power-on conditions.

[0100] Depend on Figure 3 and Figure 5A to Figure 5C It can be seen that based on the size effect of the 3D cell spheroid, the cell spheroid capture micropores on the paired fusion chip can effectively capture the 3D cell spheroid, and the test droplet capture micropores can achieve a controllable loading amount for the test droplet.

[0101] (3) Validation of the droplet fusion model based on three-dimensional cell spheroids

[0102] In order to verify the effectiveness of the droplet fusion model on the paired fusion chip, a control group 1 was set up, and two fluorescent substances were used to track the fusion behavior between blank droplets on the paired fusion chip. The rest of the operation process was the same as the construction method of the droplet fusion model based on the three-dimensional cell spheroid. The obtained fluorescence microscopy image is shown in the figure, wherein: ① The specific steps for making blank cell droplets are: adding Alexa546 dye to ordinary ultrapure water to obtain a first blank solution with a final concentration of 1 μM; then using a microfluidic method to encapsulate it into an oil-in-water droplet with a particle size of about 120 μm; ② The specific steps for making blank test droplets are: adding Alexa488 dye to ordinary ultrapure water to obtain a second blank solution with a final concentration of 1 μM, and then using a microfluidic method to encapsulate it into an oil-in-water droplet with a particle size of about 60 μm; ③ The generated blank cell droplets and blank test droplets are successively passed into the paired fusion chip, the blank cell droplets are assembled into the cell spheroid capture micropores, and the blank test droplets are assembled and filled in the cell spheroid capture micropores. After the blank cell droplet and the blank test droplet are paired, the corona rod is used to contact the chip, and the fluorescence microscopic image of the fusion behavior of the blank cell droplet and the blank test droplet is shown in FIG. Figure 6-7 middle.

[0103] The results show that the pairing fusion chip can effectively capture droplets loaded with 3D cell spheroids, and based on the droplet pairing model of three-dimensional cell spheroids, the droplets loaded with 3D cell spheroids and the test droplets can be quickly paired and directional fused; in addition, the microfluidic method is used to generate high-throughput generation of 3D cell spheroids with uniform size. By making the droplets loaded with 3D cell spheroids and the test droplets contain fluorescent substances with different excitation wavelengths, the fusion behavior of multiple groups of droplets loaded with 3D cell spheroids and test droplets can be observed in situ and in real time.

[0104] (4) Application of droplet fusion model based on three-dimensional cell spheroids in drug screening

[0105] Anti-tumor drugs are selected as test droplets, and a droplet fusion model based on three-dimensional cell spheroids is used to pair and fuse droplets loaded with 3D cell spheroids with droplets of anti-tumor drugs on the pairing fusion chip in turn, thereby constructing a droplet fusion model based on three-dimensional cell spheroids for use in drug screening.

[0106] Specifically, a microfluidic method is used to generate anti-tumor drug droplets, the anti-tumor drug droplets are loaded into the test droplet capture micropores, and the 3D cell spheres and the anti-tumor drug droplets are fused under power-on conditions; the suspension of the 3D cell spheres after the fusion is absorbed, the supernatant is discarded by centrifugation and staining analysis is performed; the types, concentrations and combination formulas of anti-tumor drugs are screened with the help of staining markers, wherein the staining markers include one of live / dead cell staining and protein expression staining.

[0107] The use also includes: performing effectiveness and drug resistance tests on the formulation of the anti-tumor drug in response to the results of the cell survival rate or cell apoptosis rate marked by staining, wherein the formulation includes the types, concentrations and combinations of anti-tumor drugs.

[0108] In this embodiment, different concentrations of doxorubicin (Doxorubicin) drugs (0nM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM) were set, and pairing and fusion of 3D cell spheroids with different concentrations of doxorubicin drugs were performed on the pairing fusion chip. After the 3D cell spheroids were fused with the drugs, they were placed in an incubator and incubated for 48 hours. The 3D cell spheroids after the drug action were aspirated from the cell spheroid capture micropores, the supernatant was discarded by centrifugation, and then live / dead cell staining analysis was performed.

[0109] In order to verify the advantages of the droplet pairing model based on three-dimensional cell spheroids in drug efficacy and drug resistance testing, control groups 2 and 3 were set up. After the 3D cell spheroids were mixed evenly, they were inoculated into a 24-well microplate, in which 3×7=21 microwells were used; then, 30μl of a predetermined concentration of doxorubicin drug solution was added to each well, and the culture medium was added to make the total volume 300μl; after the 3D cell spheroids and the drugs were mixed evenly, they were placed in an incubator and incubated for 48h. The following operations were performed on the tumor cell spheroids after the drug action:

[0110] ① Control group 2, live / dead cell staining was performed directly on the tumor cell spheres: the tumor cell spheres in the microwells were aspirated, the supernatant was discarded after centrifugation, and then live / dead cell dye was added for staining analysis.

[0111] ② Control group 3, enzymatic hydrolysis of tumor cell spheres for live / dead cell staining and counting: aspirate the tumor cell spheres in the microwells, centrifuge and discard the supernatant, then add trypsin for enzymatic hydrolysis, centrifuge and discard the supernatant after enzymatic hydrolysis, and add live / dead cell dye for staining and counting.

[0112] The 3D cell spheroids of the droplet pairing model of this embodiment and the control group 2 were co-incubated with different concentrations of doxorubicin for 48 hours, and then live / dead cell staining analysis was performed and the results were statistically analyzed. The obtained fluorescence microscopy images are shown in Fig. 8A In this example and control group 3, the 3D cell spheres were co-incubated with different concentrations of doxorubicin for 48 hours, and then live / dead cell staining analysis was performed and the results were statistically analyzed. The obtained cell survival rate results are shown in Figure 8B middle.

[0113] Result analysis:

[0114] The live / dead cell staining results of the embodiment show that after the 3D cell spheroids in the droplet pairing model based on the three-dimensional cell spheroids of the embodiment acted with different concentrations of drugs, the cell activity decreased with the increase of the concentration of doxorubicin drug. Therefore, the cell activity of the 3D cell spheroids incubated for 48 hours after the drug action can be used to evaluate the sensitivity and effectiveness of the drug.

[0115] The cell survival rate results of Example 3 and Control Group 3 showed that the cell survival rate after drug action had a tendency to decrease with the increase of the concentration of doxorubicin, as shown in the following: Figure 8B As shown, when the concentration of doxorubicin used for testing increased from 100 nM to 1 μM, the cell survival rate of the tumor cell spheres after fusion based on the paired fusion chip in this example decreased from 90% to 60%, and the cell survival rate of the control group decreased from 78% to 43%.

[0116] Through the technical solution of the present invention, drug screening can be performed based on three-dimensional tumor cell spheroids. Compared with traditional two-dimensional cell models, the drug screening model based on three-dimensional tumor cell spheroids can more realistically and better simulate the drug effects in the actual complex environment of the body. At the same time, high-throughput drug testing allows batch execution of drug evaluation and test analysis of multiple formulations.

[0117] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A droplet fusion model based on three-dimensional cell spheroids, characterized in that: include: A paired fusion chip is provided, wherein the paired fusion chip comprises a plurality of capture structures arranged in a periodic array, each capture structure comprising a cell ball capture micropore, a test droplet capture micropore, and a microchannel for fluidly connecting the cell ball capture micropore and the test droplet capture micropore; The droplets loaded with 3D cell spheroids are assembled in the cell spheroid capture microwells, and the test droplets are loaded and filled in the test droplet capture microwells to pair the droplets loaded with 3D cell spheroids with the test droplets. Under power-on conditions, the droplets loaded with 3D cell spheroids are fused with the test droplets of the same group of capture structures and absorb the contents of the test droplets.

2. The droplet fusion model according to claim 1, characterized in that; The 3D cell sphere is formed by in vitro culture of methacrylated gelatin hydrogel microspheres that encapsulate tumor cells based on microfluidic technology.

3. A method for constructing a droplet fusion model based on a three-dimensional cell spheroid, characterized in that: include: A hydrogel microsphere encapsulating tumor cells is constructed by using a microfluidic chip, wherein the microfluidic chip comprises a flow channel layer, wherein the surface of the flow channel layer is provided with an external phase fluid flow channel, an internal phase fluid flow channel and a dispersed phase flow channel, wherein the external phase fluid flow channel is used to transport a solution containing a hydrogel prepolymer, wherein the internal phase fluid flow channel is used to transport a suspension of tumor cells, wherein the external phase fluid flow channel and the internal phase fluid flow channel merge into a laminar flow channel to form a laminar flow of a suspension of tumor cells and a hydrogel prepolymer solution, wherein the dispersed phase flow channel is configured to transport an oil phase solvent to generate oil phase droplets encapsulating the tumor cells at the intersection of the dispersed phase flow channel and the laminar flow channel, wherein the oil phase droplets of the tumor cells are photo-crosslinked to form hydrogel microspheres having a core-shell structure, wherein the hydrogel comprises one of methacrylated gelatin, sodium alginate hydrogel and polyethylene glycol diacrylate hydrogel; The hydrogel microspheres encapsulating tumor cells are placed in culture medium and cultured into 3D cell spheres; A paired fusion chip according to any one of claims 1 and 2 is provided, wherein droplets carrying the 3D cell spheroids and test droplets are sequentially loaded on multiple groups of capture structures of the paired fusion chip, and the droplets carrying the 3D cell spheroids are fused with the test droplets of the same group of capture structures under power-on conditions, and the contents of the test droplets are absorbed.

4. The construction method according to claim 3, characterized in that: The flow channel layer of the microfluidic chip is prepared by a soft template method, and the material of the flow channel layer includes: one of polydimethylsiloxane, polymethyl methacrylate, and polycarbonate.

5. The construction method according to claim 4, characterized in that: The steps of preparing the paired fusion chip by using a soft template method include: Spin-coat photoresist on the surface of the silicon wafer, and perform photolithography and etching methods on the photoresist based on a photomask; The polydimethylsiloxane monomer is mixed with a curing agent and poured onto the silicon wafer formed with a patterned photoresist, and the polydimethylsiloxane monomer is formed into a capture structure layer based on the patterned photoresist, and a plurality of capture structures arranged in a periodic array are provided on the surface of the capture structure layer; The trapping structure layer is sealed onto the chip substrate using oxygen plasma treatment.

6. The construction method according to claim 5, characterized in that: The step of preparing the paired fusion chip also includes: The geometric shape and size of the cell ball capture micropores and the corresponding test droplet capture micropores of the paired fusion chip are determined based on the size of the 3D cell ball, wherein the ratio of the diameter of the cell ball capture micropores to the test droplet capture micropores is not less than 1:1; and the surface of the paired fusion chip is hydrophobically treated.

7. The construction method according to claim 3, characterized in that: The step of preparing the paired fusion chip also includes: The oil phase droplets encapsulating the tumor cells are collected at the outlet end of the flow channel layer of the microfluidic chip, and free radical cross-linking is initiated by ultraviolet light to form a hydrogel shell layer encapsulating the tumor cells; the oil phase droplets are demulsified to obtain hydrogel microspheres with a core-shell structure.

8. The construction method according to claim 3 or 7, characterized in that: Also includes: Before introducing the droplet loaded with the 3D cell spheroid and the test droplet, introducing an oil phase solvent to wet the paired fusion chip; Passing the droplet loaded with the 3D cell spheroid into the paired fusion chip to fill the droplet into the cell spheroid capture micropores of the paired fusion chip; The test droplets are generated by a microfluidics method, and the test droplets are assembled and filled in the test droplet capturing microwells.

9. The construction method according to claim 3, characterized in that: Also includes: Loading one or more formulations of test droplets in each test droplet capture microwell of the paired fusion chip; wherein the formulation includes the components, concentrations and combinations thereof of the test droplets; The paired fusion chip is contacted with a corona bar to fuse the droplets loaded with the 3D cells with the test droplets in each set of capture structures.

10. The construction method according to claim 8, characterized in that: Also includes: The suspension of the 3D cell spheroid and the content of the test droplet contain fluorescein with different excitation wavelengths, and the fusion behavior of the droplet loaded with the 3D cell spheroid and the test droplet is traced by the fluorescein.

11. Use of the droplet pairing fusion model based on three-dimensional cell spheroids according to any one of claims 3 to 10 in drug screening.

12. The use according to claim 11, characterized in that The uses include: Using a microfluidic method to generate droplets of anti-tumor drugs, the droplets of the anti-tumor drugs are loaded into the test droplet capture micropores, and the fusion of the 3D cell sphere and the droplets of the anti-tumor drugs is performed under power-on conditions; The suspension of the 3D cell sphere after fusion is aspirated, the supernatant is discarded by centrifugation, and staining analysis is performed; The types, concentrations and combination formulations of anti-tumor drugs are screened by means of staining markers, wherein the staining markers include one of live / dead cell staining and protein expression staining.

13. The use according to claim 12, characterized in that: One or more formulations of anti-tumor drugs are tested simultaneously on the paired fusion chip, and the formulations of the anti-tumor drugs are tested for effectiveness and drug resistance in response to the cell survival rate or cell apoptosis rate marked by staining, wherein the formulations include types, concentrations and combinations of anti-tumor drugs.

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