A method for preparing organoids based on single cell chips

Abstract

The application provides a preparation method of organoids based on a single cell chip, which comprises microfluidic chip preparation, a two-water-phase solution preparation, a single cell suspension preparation, microfluidic manipulation, single cell-loaded microgel generation and organoid construction and the like. The application takes a microfluidic chip integrated with a normally closed pneumatic pump valve as a technical platform, takes a single cell suspension pre-incubated with a crosslinking agent as a chemical reaction core, takes a water-in-water droplet as a forming template, and realizes efficient in-situ loading of single dispersed seed cells in a micro hydrogel carrier through a one-step method, and simply removes empty droplets without cells in a subsequent transfer step. The single cells in the microgel can be cultured, expanded and self-organized to form required organoids. The application improves the consistency of the starting point of the organoids, has the high-throughput characteristics of the droplet microfluidic technology, realizes efficient and highly reproducible in-vitro construction of the organoids, and can play a great role in the fields of basic research and transformation application related to the organoids.

Description

Technical Field

[0001] The present invention relates to the fields of microfluidics, material chemistry and bioengineering technology, and in particular to a method for preparing organoids based on a single-cell chip. Background Art

[0002] With the rapid development of the life sciences, traditional 2D cell and animal models are no longer able to meet the urgent needs of modern biomedical research and new drug development. Establishing new experimental medical models and research paradigms is crucial for a deeper understanding of human health and disease processes and the development of effective therapeutics. Organoids are a newly developed in vitro model. They primarily refer to functional cell-tissue complexes that differentiate through cellular self-organization under 3D culture conditions in vitro. Organoids typically possess some key structural and functional characteristics of human tissues or organs and have shown promising applications in areas such as tissue and organ development, disease modeling, drug screening, and regenerative medicine. In recent years, the organoid field has rapidly developed, with a series of organoid models for various human organs (such as the brain, liver, kidney, and intestine) reported and beginning to be used in research in biology and medicine. However, overall, existing organoid research systems still face numerous challenges in terms of cell-matrix homogeneity, controllable organoid morphology, and high-throughput analysis, which significantly restricts their wider application.

[0003] Droplet microfluidics, a downstream offshoot of traditional microfluidics, offers advantages such as high throughput, excellent reproducibility, low sample consumption, and rapid sample mixing. It is playing an increasingly important role in fields such as biochemical analysis and functional material preparation. Leveraging the size and throughput advantages of droplet microfluidics, it is possible to effectively fabricate 3D cell culture carriers ranging from micrometers to millimeters. In particular, aqueous two-phase droplet microfluidics systems, in addition to these advantages, offer a more biocompatible carrier preparation environment and simpler carrier post-processing. However, traditional droplet microfluidics techniques for cell loading and culture rely on probabilistic embedding of target cells within microcarriers, resulting in poor uniformity and efficiency. Previous studies have demonstrated that pretreatment of the target cells can act as a crosslinking agent, enabling the targeted generation of non-empty cell carriers. Furthermore, at a low enough cell concentration, the majority of non-empty carriers contain single cells. This approach enables the generation of highly consistent single-cell carriers, which can then be further expanded and developed in situ to create the final organoid model. If materials with known matrix composition are used, organoid construction with better matrix and organoid uniformity and higher throughput can be achieved in this system, thus providing a new technical platform for basic and translational application research related to organoids. Summary of the Invention

[0004] According to the above-mentioned traditional droplet microfluidics technology for cell loading and culture, all of them use probabilistic events to embed target cells in microcarriers, which has the technical problems of poor uniformity and effectiveness, and provide a method for preparing organoids based on single-cell chips. The method of the present invention uses a microfluidics chip integrated with a normally closed pneumatic pump valve as a technical platform, a single-cell suspension pre-incubated with a cross-linker as the chemical reaction core, and a water-in-water droplet as a forming template. The monodispersed seed cells are loaded into a micro-hydrogel carrier by a one-step method, and the empty droplets without cells are simply removed in the subsequent transfer step to achieve efficient single-cell in situ loading. The single cells in the microgel can be normally cultured, amplified and self-organized to form the desired organoid. This method improves the consistency of the starting point of the organoid, and also has the high-throughput characteristics of droplet microfluidics technology, realizing efficient and highly reproducible in vitro construction of organoids, which can play a huge role in the fields of basic research and translational applications related to organoids.

[0005] The technical means adopted in the present invention are as follows:

[0006] A method for preparing organoids based on a single-cell chip comprises the following steps:

[0007] S1. Preparation of microfluidic chips: Soft lithography was used to fabricate a polydimethylsiloxane (PDMS) chip with an integrated normally closed pneumatic pump valve, i.e., a microfluidic chip. This chip was used to generate aqueous two-phase droplet templates and prepare hydrogel microspheres (microgels) loaded with single cells.

[0008] S2. Preparation of the two-phase aqueous solution: preparing a reaction phase containing a crosslinker, a continuous phase, and a dispersed phase respectively;

[0009] S3. Preparation of single cell suspension: preparing a single cell suspension, and resuspending the single cell suspension in the dispersed phase prepared in step S2 at a certain concentration to obtain a dispersed phase solution containing cells;

[0010] S4. Microfluidics manipulation: delivering the crosslinker-containing reaction phase, the continuous phase, and the cell-containing dispersed phase into the microfluidic chip, and obtaining cell-containing dispersed phase droplets using a normally closed pneumatic pump valve;

[0011] S5. Production of single cell-loaded microgels: In a microfluidic chip, the cell-containing dispersed phase droplets formed in step S4 are allowed to meet and react with a reaction phase containing a crosslinker to form cell-loaded microgels;

[0012] S6. Construction of organoids: The single-cell loaded microgels obtained in step S5 are transferred to the corresponding cell culture medium and cultured and expanded long-term in a cell culture environment at 37°C and 5% CO2 or a low-oxygen environment, so that the single cells develop into monoclones and eventually form organoids.

[0013] Furthermore, in step S1, the polydimethylsiloxane chip is prepared by: using soft lithography technology, first preparing a SU-8 photoresist template on a single crystal silicon wafer or a dust-free glass sheet, then pouring a polydimethylsiloxane prepolymer on the SU-8 photoresist template, and heating it at 80°C for 1-3 hours for cross-linking polymerization to prepare a polydimethylsiloxane chip; the chip is based on a flow-focusing microfluidic droplet chip, integrated with a lower gas path portion; the chip includes an upper liquid path portion and a lower gas path portion;

[0014] The liquid circuit part includes an upper negative pressure interface, a first inlet, a reaction phase channel containing a cross-linking agent, a second inlet, a continuous phase channel, a third inlet, a dispersed phase channel containing cells, a normally closed pneumatic pump valve, a droplet transport channel, a microgel formation channel, a microgel outlet, an intersection A and an intersection B. The upper negative pressure interface is connected to an external vacuum device; the first inlet is used to transport the reaction phase containing a cross-linking agent into the chip, and the reaction phase channel containing a cross-linking agent has a conical structure, with two first channels in parallel, the inlet ends of the two first channels are connected to the first inlet, and the outlet ends of the two channels are located at the tip of the cone; the second inlet is used to transport the continuous phase into the chip, and the continuous phase channel is located at the cross-linking agent. The reaction phase channel of the crosslinker has two second channels connected in parallel, and the inlet end where the two second channels converge is connected to the second inlet; the third inlet is used to transport the dispersed phase containing cells into the chip, and the dispersed phase channel containing cells is located in the continuous phase channel, one end of which is connected to the third inlet, and the other end of which converges with the outlet ends where the two second channels converge to the inlet end of the droplet transport channel, forming a flow focusing intersection A; the droplet transport channel is located in the reaction phase channel containing the crosslinker, and its outlet end converges with the outlet ends where the two first channels converge to the inlet end of the microgel formation channel, forming an intersection B; the main body of the microgel formation channel is a serpentine curved channel, and its outlet end serves as a microgel outlet;

[0015] The air path portion includes a lower negative pressure interface, a gas channel and a normally closed pneumatic pump valve action area. The lower negative pressure interface is connected to the upper negative pressure interface. One end of the gas channel is connected to the lower negative pressure interface, and the other end is connected to the normally closed pneumatic pump valve action area. The normally closed pneumatic pump valve action area is located directly below the normally closed pneumatic pump valve. The dispersed phase channel is periodically opened through the two states of pump valve suction and rest, so that the dispersed phase intermittently enters the continuous phase, stably and efficiently forming two-phase aqueous droplets.

[0016] Furthermore, in step S2, polyethylene glycol (PEG) is dissolved in an acid solution as a reaction phase containing a cross-linking agent, the polyethylene glycol (PEG) isotonic solution is used as a continuous phase, and the polymer that can be cross-linked by high-valent metal cations is dissolved in a dextran isotonic solution as a dispersed phase.

[0017] Furthermore, the specific steps of step S3 are as follows:

[0018] Two-dimensional (2D) adherent or three-dimensional (3D) cultured cells are enzymatically digested to obtain a monodisperse cell suspension. An isotonic solution containing metal oxide or salt nanoparticles that has been previously ultrasonically dispersed is then thoroughly mixed with the cell suspension and incubated at room temperature or 37°C. Nanoparticles not adhered to the cell surface are then removed by centrifugation or filtration to obtain a single-cell suspension with surface-adhered nanoparticles. The single-cell suspension is resuspended in a dispersed phase at a certain concentration to obtain a dispersed phase solution containing cells.

[0019] Furthermore, the specific steps of step S4 are as follows:

[0020] The reaction phase containing the cross-linker enters the microfluidic chip through the first inlet, and then reaches intersection B along the reaction phase channel containing the cross-linker; the continuous phase enters the microfluidic chip through the second inlet, passes through the continuous phase channel, intersection A and the droplet transport channel to reach intersection B; the dispersed phase containing cells enters the microfluidic chip through the third inlet, passes through the dispersed phase channel containing cells, the normally closed pneumatic pump valve, intersection A and the droplet transport channel to reach intersection B; the external vacuum equipment is connected to the microfluidic chip through the upper negative pressure interface, passes through the lower negative pressure interface and the gas channel to reach the normally closed pneumatic pump valve action area, and periodically drives the normally closed pneumatic pump valve to open and close, thereby controlling the on and off of the dispersed phase to promote the formation of dispersed phase droplets and control their size.

[0021] Furthermore, the specific steps of step S5 are as follows:

[0022] The cell-containing dispersed phase droplets formed in step S4 meet the reaction phase containing the crosslinker (acid) at the intersection B of the chip. The hydrogen ions in the reaction phase enter the droplets due to free diffusion, pass through the intermediate continuous phase, and instantly interact with the nanoparticles adhered to the cell surface, releasing high-valent metal ions, thereby causing the polymers in the dispersed phase to crosslink and form cell-loaded microgels. The cell-free droplets, however, cannot crosslink because no metal ions are released and can naturally dissolve in the surrounding solution during the subsequent collection and transfer process.

[0023] Furthermore, the height of each channel in the chip is 50-900 μm, the width of each channel is 50-900 μm, the width of the normally closed pneumatic pump valve disconnection area is 20-500 μm, the thickness of the PDMS membrane in the normally closed pneumatic pump valve action area is 50-1000 μm, the length of the droplet transport channel is 0.3-1.5 cm, the number of serpentine bending channels in the microgel formation channel is 3-15, and their length is 3-15 cm.

[0024] Furthermore, the acid in step S2 is one of formic acid, acetic acid, sulfuric acid, hydrochloric acid, nitric acid or sulfurous acid, or a combination of more than one, and its total concentration is: 0.05-2% (v / v); the molecular weight of polyethylene glycol is: 8-20 kDa, and its concentration in the continuous phase and the reaction phase is: 10%-30% (w / v); the isotonic fluid is: PBS buffer or physiological saline; the molecular weight of dextran is: 70-500 kDa, and the concentration is: 10%-30% (w / v); the polymer that can be cross-linked by high-valent metal cations is: one of sodium alginate, pectin, carboxymethyl chitosan and unactivated FXIII-modified polymers, or a combination of more than one, with a molecular weight of 1-10000 kDa and a concentration of 0.1-5% (w / v).

[0025] Furthermore, the cells in step S3 are: various commercial cell lines, primary healthy or tumor tissue derived cells, various stem cells; the cell concentration during incubation is: 10 5 -10 8 / mL; the cell concentration in the dispersed phase is: 10 3 -10 6 / mL; the type of nanoparticles is one of calcium carbonate, barium carbonate, iron oxide, zinc oxide, and copper oxide, the particle diameter is 1-1000nm, and the concentration used for incubation is 0.1-10g / L; the incubation time of cells and nanoparticles is 5-60min; the centrifugal operation centrifugal force is 5-200g, and the time is 0.1-3min; the pore size of the filter membrane used in the filtration operation is 2-15μm.

[0026] Furthermore, in step S4, the external vacuum degree is: (-0.01) ~ (-0.1) MPa; the flow rate of the dispersed phase containing cells is: 0.01-5 μL / min; the flow rate of the continuous phase is: 1-20 μL / min; the flow rate of the reaction phase containing the crosslinker is: 1-20 μL / min.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The single-cell chip-based organoid preparation method provided by this invention can significantly improve the single-cell loading rate of microgels, thereby increasing the consistency of the starting point during organoid construction. This results in a more controllable extracellular matrix and organoid morphology, and a higher throughput for organoid construction. This method is expected to play an important role in basic and translational research, such as developmental studies, disease modeling, and efficient drug screening.

[0029] 2. The single-cell chip-based organoid preparation method provided by this invention utilizes a microfluidic chip integrated with a normally closed pneumatic pump valve as the technical platform. A single-cell suspension pre-incubated with a crosslinker serves as the chemical reaction core, and water-in-water droplets serve as the forming template. Monodispersed seed cells are loaded into a microhydrogel carrier in a one-step process. The cell-free empty droplets are then simply removed during the subsequent transfer step, achieving efficient single-cell in situ loading. The single cells in the microgel can then be cultured, expanded, and self-organized to form the desired organoids.

[0030] In summary, the application of the technical solution of the present invention can solve the problem that traditional droplet microfluidics technology is used for cell loading and culture, which uses probabilistic events to embed target cells in microcarriers, but has poor uniformity and effectiveness.

[0031] Based on the above reasons, the present invention can be widely promoted in the fields of biology, medicine and pharmaceutical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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 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 labor.

[0033] Figure 1 This is a schematic diagram of the two-phase aqueous droplet microfluidic chip used to prepare single-cell loaded microgels of the present invention, which is an overall diagram of the combination of two layers of chips.

[0034] Figure 2 Schematic diagram of the upper liquid circuit chip of the present invention.

[0035] Figure 3 This is a schematic diagram of the lower gas path chip of the present invention.

[0036] Figure 4 Schematic diagram of the key units in the aqueous two-phase droplet microfluidic chip of the present invention. (a) is the working principle diagram of the normally closed pneumatic pump valve, and (b) is a schematic diagram of the droplet and microgel formation at intersections A and B.

[0037] Figure 5 This is a physical picture of the two-phase aqueous droplet microfluidic chip used to construct the organoid model in Example 1 of the present invention.

[0038] Figure 6 This is a fluorescence characterization image of the single cell-loaded microgel obtained in Example 2 of the present invention.

[0039] Figure 7 This is a bright field representation of the organoid-loaded microgel obtained in Example 3 of the present invention.

[0040] In the figure: 1. Upper negative pressure interface; 2. First inlet; 3. Reaction phase channel containing cross-linker; 4. Second inlet; 5. Continuous phase channel; 6. Third inlet; 7. Dispersed phase channel containing cells; 8. Normally closed pneumatic pump valve; 9. Droplet transport channel; 10. Microgel formation channel; 11. Microgel outlet; 12. Intersection A; 13. Intersection B; 14. Lower negative pressure interface; 15. Gas channel; 16. Normally closed pneumatic pump valve action area. DETAILED DESCRIPTION

[0041] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0042] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0044] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be clear that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The technology, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be considered as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0045] like Figures 1-4 As shown, the present invention provides a method for preparing organoids based on a single-cell chip, which is an efficient single-cell-derived organoid construction method based on two-phase aqueous droplet microfluidics.

[0046] The method for preparing organoids based on a single cell chip comprises the following steps:

[0047] (1) Preparation of microfluidic chip: A polydimethylsiloxane (PDMS) chip integrated with a normally closed pneumatic pump valve 8 was prepared using conventional soft lithography methods; the chip was used to generate a two-phase aqueous droplet template and prepare hydrogel microspheres (microgels) loaded with single cells. Its structure consists of two layers: the upper layer is the liquid circuit part, which consists of an upper negative pressure interface 1, a reaction phase inlet containing a crosslinker (first inlet 2), a reaction phase channel containing a crosslinker 3, a continuous phase inlet (second inlet 4), a continuous phase channel 5, a dispersed phase inlet containing cells (third inlet 6), a dispersed phase channel containing cells 7, a normally closed pneumatic pump valve 8, a droplet transport channel 9, a microgel formation channel 10, a microgel outlet 11, an intersection A12, and an intersection B13; the lower layer is the gas circuit part, which consists of a lower negative pressure interface 14, a gas channel 15, and a normally closed pneumatic pump valve action area 16. Among them, the upper negative pressure interface 1 is connected to the external vacuum equipment; the first inlet 2 is used to transport the reaction phase containing the crosslinker into the chip, and the reaction phase channel 3 containing the crosslinker is in a conical structure, with two first channels in parallel, the inlet end where the two first channels converge is connected to the first inlet 2, and the outlet end where the two channels converge is located at the tip of the cone; the second inlet 4 is used to transport the continuous phase into the chip, and the continuous phase channel 5 is located in the reaction phase channel 3 containing the crosslinker, and has two second channels in parallel, the inlet end where the two second channels converge is connected to the second inlet 4; the third inlet 6 is used to transport the dispersed phase containing cells into the chip, and the dispersed phase channel 7 containing cells is located in the continuous phase channel 5, one end of which is connected to the third inlet 6, and the other end is connected to the outlet end where the two second channels converge to converge to the droplet transport The inlet end of the channel 9 forms a flow focusing intersection A12; the droplet transport channel 9 is located in the reaction phase channel 3 containing the crosslinker, and its outlet end and the outlet ends of the two first channels converge to the inlet end of the microgel forming channel 10, forming an intersection B13; the main body of the microgel forming channel 10 is a serpentine curved channel, and its outlet end serves as a microgel outlet 11; the lower layer negative pressure interface 14 is connected to the upper layer negative pressure interface 1, one end of the gas channel 15 is connected to the lower layer negative pressure interface 14, and the other end is connected to the normally closed pneumatic pump valve action area 16, the normally closed pneumatic pump valve action area 16 is located directly below the normally closed pneumatic pump valve 8, and the dispersed phase channel is periodically opened through the two states of pump valve exhaust and rest, so that the dispersed phase intermittently enters the continuous phase, stably and efficiently forming two-phase aqueous droplets.

[0048] (2) Preparation of the double aqueous phase solution: Dissolve polyethylene glycol (PEG) in an acid solution as the reaction phase containing cross-linking agent, and use PEG isotonic solution as the continuous phase. Dissolve the polymer that can be cross-linked by high-valence metal cations in the dextran isotonic solution as the dispersed phase.

[0049] (3) Preparation of the single cell suspension: After enzymatic digestion of the two-dimensional (2D) adherent or three-dimensional (3D) cultured cells, a single dispersed cell suspension is obtained. Then, the pre-ultrasonically dispersed isotonic solution containing metal oxide or salt nanoparticles is thoroughly mixed with the cell suspension, and incubated at room temperature or 37°C. Subsequently, the nanoparticles not adhered to the cell surface are removed by centrifugation or filtration, and a single cell suspension with surface-attached nanoparticles is obtained. The single cell suspension is resuspended in the dispersed phase at a certain concentration to obtain a dispersed phase solution containing cells.

[0050] (4) Microfluidic manipulation: The reaction phase containing cross-linking agent enters the microfluidic chip through the first inlet 2, and then reaches the intersection B13 along the cross-linking agent-containing reaction phase channel 3. The continuous phase enters the microfluidic chip through the second inlet 4, passes through the continuous phase channel 5, the intersection A12, and the droplet transport channel 9 in sequence, and then reaches the intersection B13. The dispersed phase containing cells enters the microfluidic chip through the third inlet 6, passes through the cell-containing dispersed phase channel 7, the normally closed pneumatic pump valve 8, the intersection A12, and the droplet transport channel 9 in sequence, and then reaches the intersection B13. The external vacuum device is connected to the microfluidic chip through the upper negative pressure interface 1, passes through the lower negative pressure interface 14 and the gas channel 15 in sequence, and then reaches the normally closed pneumatic pump valve action area 16. The normally closed pneumatic pump valve 8 is periodically driven to open and close, thereby controlling the on-off of the dispersed phase to promote the formation of dispersed phase droplets and control their size.

[0051] (5) Generation of single cell-loaded microgels: The cell-containing dispersed phase droplets formed in step (4) meet the reaction phase containing cross-linking agent (acid) at the intersection B13. Due to the free diffusion of hydrogen ions in the reaction phase, they pass through the intermediate continuous phase and enter the droplets, and instantly interact with the nanoparticles adhered to the cell surface, releasing high-valence metal ions, thereby cross-linking the polymers in the dispersed phase to form cell-loaded microgels. The droplets without cells cannot be cross-linked due to the absence of metal ion release, and can naturally dissolve in the surrounding solution during the subsequent collection and transfer process.

[0052] (6) Construction of organoids: The single cell-loaded microgels obtained in step (5) are transferred to the corresponding cell culture medium, and cultured and expanded in a conventional cell culture environment at 37°C and 5% CO2 or in a hypoxic environment, thereby developing from single cells to monoclonal cells and finally forming organoids.

[0053] Preferably, the chip channel height is 50-900 μm, the channel width is 50-900 μm, the normally closed pneumatic pump valve 8 off area width is 20-500 μm, the normally closed pneumatic pump valve action area 16 PDMS film thickness is 50-1000 μm, the droplet transport channel 9 length is 0.3-1.5 cm, the microgel formation channel 10 serpentine bending channel number is 3-15, and the length is 3-15 cm.

[0054] Preferably, in step (1), the method is: first, a SU-8 photoresist template is prepared on a single crystal silicon wafer or a dust-free glass sheet by using a conventional soft lithography method, then the PDMS prepolymer is poured on the SU-8 photoresist template, and after cross-linking polymerization at 80°C for 1-3 hours, a PDMS chip is prepared; the chip is based on a traditional flow focusing type microfluidic droplet chip, and is integrated with the lower air path part to form; the dispersed phase channel 7 containing cells and the continuous phase channel 5 converge at the droplet transport channel 9 to form a flow focusing intersection A12; the normally closed pneumatic pump valve 8 is located directly above the normally closed pneumatic pump valve action area 16 (the normally closed pneumatic pump valve action area 16 is opposite to the normally closed pneumatic pump valve 8 Figure 2 position), and the dispersed phase channel is periodically opened by pump valve suction and rest to make the dispersed phase intermittently enter the continuous phase, thereby stably and efficiently forming double aqueous phase droplets.

[0055] Preferably, in step (2), the acid is one or more of formic acid, acetic acid, sulfuric acid, hydrochloric acid, nitric acid, and sulfurous acid, and the total concentration is 0.05-2% (v / v); the PEG molecular weight is 8-20 kDa, and the concentration in the continuous phase and the reaction phase is 10%-30% (w / v); the isotonic solution is PBS buffer or normal saline; the dextran molecular weight is 70-500 kDa, and the concentration is 10%-30% (w / v); the high molecular weight that can be cross-linked by high valence metal cations is one or more of sodium alginate, pectin, carboxymethyl chitosan, and unactivated FXIII modified high molecular weight, the molecular weight is 1-10000 kDa, and the concentration is 0.1-5% (w / v).

[0056] Preferably, in step (3), the cells are one of various commercial cell lines, primary healthy or tumor tissue-derived cells, and various stem cells; the cell concentration during incubation is 10 5 -10 8 cells / mL; the cell concentration in the dispersed phase is 10 3 -10 6The concentration of the cells is 0.1-10 g / L, the incubation time is 5-60 min, the centrifugal force is 5-200 g, and the centrifugal time is 0.1-3 min, and the pore size of the filter membrane is 2-15 μm.

[0057] Preferably, the external vacuum degree in step (4) is (-0.01) to (-0.1) MPa, the flow rate of the cell-containing dispersed phase is 0.01-5 μL / min, the flow rate of the continuous phase is 1-20 μL / min, and the flow rate of the crosslinking agent-containing reaction phase is 1-20 μL / min.

[0058] First, a microfluidic chip with appropriate size is designed and processed according to actual needs; then, nanoparticles are pre-processed for cells of a desired type, and an appropriate type of matrix material is selected; finally, the generation and size of single-cell microgels are regulated by combining a pump-valve control system, and cells are further expanded and developed to obtain a desired type of organoids. The present application is further described below in combination with the accompanying drawings and Examples 1-2.

[0059] Example 1

[0060] A new technology for preparing organoids based on a single-cell chip, comprising the following steps:

[0061] (1) Preparation of a microfluidic chip: a PDMS chip integrated with a normally closed pneumatic pump valve is prepared by a conventional soft lithography method. Specifically, a SU-8 photoresist template is first prepared on a single-crystal silicon wafer, and then PDMS pre-polymer is poured onto the SU-8 photoresist template. After cross-linking polymerization at 80°C for 1 hour, a PDMS chip is prepared. The chip is used to generate a double-aqueous phase droplet template and prepare single-cell-loaded microgels. The structure comprises two layers: an upper layer and a lower layer. The upper layer is a liquid path part, which comprises an upper negative pressure interface 1, a crosslinking agent-containing reaction phase inlet (first inlet 2), a crosslinking agent-containing reaction phase channel 3, a continuous phase inlet (second inlet 4), a continuous phase channel 5, a cell-containing dispersed phase inlet (third inlet 6), a cell-containing dispersed phase channel 7, a normally closed pneumatic pump valve 8, a droplet transportation channel 9, a microgel formation channel 10, a microgel outlet 11, a cross 12, and a cross 13. The lower layer is a gas path part, which comprises a lower negative pressure interface 14, a gas channel 15, and a normally closed pneumatic pump valve action area 16. Figures 1-3). Wherein the dispersed phase channel 7 and the continuous phase channel 5 converge at the droplet transport channel 9 to form a flow-focusing intersection A12; the position of the normally closed pneumatic pump valve 8 is directly above the normally closed pneumatic pump valve action zone 16, periodically opening the dispersed phase channel by pump valve suction and static state, so that the dispersed phase intermittently enters the continuous phase, stably and efficiently forming double aqueous phase droplets Figure 4 ).

[0062] As shown in Figure 5 , the chip channel height is: 100 μm, the channel width is: 100 μm, the normally closed pneumatic pump valve 8 break area width is: 100 μm, the normally closed pneumatic pump valve action zone 16 PDMS film thickness is: 100 μm, the droplet transport channel 9 length is: 0.3 cm, the microgel formation channel 10 serpentine bending channel number is: 5, and the length is: 5 cm.

[0063] (2) Preparation of double aqueous phase solution: dissolve PEG as a crosslinking agent-containing reaction phase in an acid solution, use PEG isotonic solution as the continuous phase, and dissolve the high polymer crosslinkable by high valence metal cations in dextran isotonic solution as the dispersed phase.

[0064] The acid used here is formic acid, with a concentration of: 1% (v / v); the molecular weight of PEG is: 8 kDa, and the concentration in the continuous phase and the reaction phase is: 30% (w / v); the isotonic solution is: PBS buffer; the molecular weight of dextran is: 70 kDa, and the concentration is: 30% (w / v); the high polymer crosslinkable by high valence metal cations is: pectin, with a molecular weight of: 50 kDa and a concentration of: 4% (w / v).

[0065] (3) Preparation of single cell suspension: after enzymatic digestion of 2D adherent cultured cells, a single dispersed cell suspension is obtained, then the pre-ultrasonically dispersed isotonic solution containing metal oxide nanoparticles is thoroughly mixed with the cell suspension, incubated at room temperature, and then filtered to remove nanoparticles not adhered to the cell surface, obtaining a single cell suspension with surface-adhered nanoparticles. Resuspend the single cell suspension in the dispersed phase at a certain concentration to obtain a cell-containing dispersed phase solution.

[0066] The cells used here are commercial HepG2 cell lines; the cell concentration during incubation is: 10 8 × 10 5 / mL; the cell concentration in the dispersed phase is: 5 × 10 The type of nanoparticles is: iron oxide, with a particle diameter of: 50 nm, and the concentration used for incubation is: 1 g / L; the incubation time of cells and nanoparticles is: 10 min; the pore size of the filter used for filtration is: 8 μm.

[0067] (4) Manipulation of microfluidics: the reaction phase containing cross-linking agent enters the microfluidic chip through the first inlet 2, and then reaches the intersection B13 along the reaction phase containing cross-linking agent channel 3; the continuous phase enters the microfluidic chip through the second inlet 4, and then reaches the intersection B13 through the continuous phase channel 5, the intersection A12 and the droplet transport channel 9 in sequence; the dispersed phase containing cells enters the microfluidic chip through the third inlet 6, and then reaches the intersection B13 through the dispersed phase containing cells channel 7, the normally closed pneumatic pump valve 8, the intersection A12 and the droplet transport channel 9 in sequence; the external vacuum device is connected to the microfluidic chip through the upper negative pressure interface 1, and then reaches the normally closed pneumatic pump valve action area 16 through the lower negative pressure interface 14 and the gas channel 15 in sequence, and periodically drives the normally closed pneumatic pump valve 8 to open and close, so as to control the on-off of the dispersed phase, so as to promote the formation of the dispersed phase droplets and control the size thereof;

[0068] The external vacuum degree is -0.03 MPa, the flow rate of the dispersed phase containing cells is 0.1 μL / min, the flow rate of the continuous phase is 2 μL / min, and the flow rate of the reaction phase containing cross-linking agent is 2 μL / min.

[0069] (5) Generation of single-cell loaded microgels: the dispersed phase droplets containing cells formed in step (4) meet the reaction phase containing formic acid at the intersection B, and the hydrogen ions in the reaction phase diffuse freely and enter the droplets through the intermediate continuous phase, and instantaneously interact with the nanoparticles adhered to the surface of the cells to release high-valence metal ions Fe 3+ , so that the polymers in the dispersed phase are cross-linked to form cell-loaded microgels. The droplets without cells cannot be cross-linked because there is no release of metal ions therein, and can be naturally dissolved in the surrounding solution in the subsequent collection and transfer process;

[0070] (6) Construction of organoids: the single-cell loaded microgels obtained in step (5) are transferred to the corresponding cell culture medium, and long-term culture and amplification are carried out in a conventional cell culture environment at 37°C and 5% CO2, so that the single cells develop into single clones and finally form liver organoids.

[0071] Example 2

[0072] A new technology for preparing organoids based on a single-cell chip, comprising the following steps:

[0073] (1) Fabrication of microfluidic chip: The PDMS chip integrated with normally closed pneumatic pump valve was fabricated by conventional soft lithography. Firstly, the SU-8 photoresist template was prepared on a single crystal silicon wafer. Then, the PDMS pre-polymer was poured on the SU-8 photoresist template, and the PDMS chip was prepared after cross-linking polymerization at 80℃ for 2 hours. The chip was used to generate the double aqueous phase droplet template and prepare the single cell-loaded microgel. The structure of the chip includes two layers: the upper layer is the liquid path part, which is composed of the upper negative pressure interface 1, the reaction phase inlet containing cross-linking agent (the first inlet 2), the reaction phase channel containing cross-linking agent 3, the continuous phase inlet (the second inlet 4), the continuous phase channel 5, the dispersed phase inlet containing cells (the third inlet 6), the dispersed phase channel containing cells 7, the normally closed pneumatic pump valve 8, the droplet transport channel 9, the microgel formation channel 10, the microgel outlet 11, the intersection A 12 and the intersection B 13; the lower layer is the gas path part, which is composed of the lower negative pressure interface 14, the gas channel 15 and the normally closed pneumatic pump valve action area 16. Figures 1-3 ). The dispersed phase channel 7 and the continuous phase channel 5 converge at the droplet transport channel 9 to form the flow focusing intersection A 12; the normally closed pneumatic pump valve 8 is located directly above the normally closed pneumatic pump valve action area 16, and the dispersed phase channel is periodically opened by pump valve suction and static state, so that the dispersed phase intermittently enters the continuous phase, and double aqueous phase droplets are stably and efficiently formed. Figure 4

[0074] The chip channel height is 400 μm, the channel width is 400 μm, the normally closed pneumatic pump valve 8 break area width is 300 μm, the normally closed pneumatic pump valve action area 16 PDMS film thickness is 400 μm, the droplet transport channel 9 length is 0.8 cm, and the microgel formation channel 10 serpentine bending channel number is 10, and the length is 10 cm.

[0075] (2) Preparation of double aqueous phase solution: PEG was dissolved in acid solution as the reaction phase containing cross-linking agent, PEG isotonic solution was used as the continuous phase, and the high molecular polymer cross-linked by high valence metal cation was dissolved in dextran isotonic solution as the dispersed phase.

[0076] The acid used here is acetic acid, and the concentration is 1.5% (v / v); the molecular weight of PEG is 10 kDa, and the concentration in the continuous phase and the reaction phase is 15% (w / v); the isotonic solution is PBS buffer; the molecular weight of dextran is 200 kDa, and the concentration is 15% (w / v); the high molecular polymer cross-linked by high valence metal cation is carboxymethyl chitosan, the molecular weight is 1000 kDa, and the concentration is 2% (w / v).

[0077] ​(3) Preparation of single-cell suspension: 3D spheroid cultured cells are enzymatically digested to obtain a monodispersed cell suspension. The cell suspension is then thoroughly mixed with an isotonic solution containing metal salt nanoparticles that has been previously ultrasonically dispersed. The mixture is incubated at 37°C and then centrifuged to remove nanoparticles that are not adhered to the cell surface. This results in a single-cell suspension with surface-adhered nanoparticles. The single-cell suspension is resuspended in a dispersed phase at a specific concentration to obtain a dispersed phase solution containing cells.

[0078] The cells used here are human induced pluripotent stem cells (hiPSCs); the cell concentration during incubation is: 10 7 / mL; the cell concentration in the dispersed phase is: 10 5 / mL; the type of nanoparticles is barium carbonate, the particle diameter is 600nm, and the incubation concentration is 9g / L; the incubation time of cells and nanoparticles is 50min; the centrifugal force of the centrifugal operation is 150g, and the time is 0.3min.

[0079] (4) Control of microfluidics: The reaction phase containing the crosslinker enters the microfluidic chip through the first inlet 2, and then reaches the intersection B13 along the reaction phase channel 3 containing the crosslinker; the continuous phase enters the microfluidic chip through the second inlet 4, and passes through the continuous phase channel 5, the intersection A12 and the droplet transport channel 9 to reach the intersection B13; the dispersed phase containing cells enters the microfluidic chip through the third inlet 6, and passes through the dispersed phase channel 7 containing cells, the normally closed pneumatic pump valve 8, the intersection A12 and the droplet transport channel 9 to reach the intersection B13; the external vacuum equipment is connected to the microfluidic chip through the upper negative pressure interface 1, and passes through the lower negative pressure interface 14 and the gas channel 15 to reach the normally closed pneumatic pump valve action area 16, and periodically drives the normally closed pneumatic pump valve 8 to open and close, thereby controlling the on-off of the dispersed phase to promote the formation of dispersed phase droplets and control their size;

[0080] Here, the external vacuum degree is: -0.05 MPa; the flow rate of the dispersed phase containing cells is: 1 μL / min; the flow rate of the continuous phase is: 10 μL / min; the flow rate of the reaction phase containing the cross-linking agent is: 10 μL / min.

[0081] (5) Production of single cell loaded microgels: The dispersed phase droplets containing cells formed in step (4) meet the reaction phase containing acetic acid at the intersection B. The hydrogen ions in the reaction phase enter the droplets due to free diffusion movement through the intermediate continuous phase and instantly interact with the nanoparticles attached to the cell surface, releasing high-valent metal ions Ba 2+ , so that the polymer in the dispersed phase is cross-linked to form a cell-loaded microgel ( Figure 6). Droplets without cells cannot be cross-linked because no metal ions are released, and can naturally dissolve in the surrounding solution during the subsequent collection and transfer process;

[0082] (6) Organoid construction: The single cell-loaded microgel obtained in step (5) is transferred to the corresponding cell culture medium and cultured for a long time, expanded, and induced to differentiate in the pancreatic direction in a conventional cell culture environment at 37°C and 5% CO2, so that the single cell develops into a monoclone and finally forms a pancreatic organoid.

[0083] Example 3

[0084] A new single-cell chip-based organoid preparation technology includes the following steps:

[0085] (1) Preparation of microfluidic chip: A PDMS chip with an integrated normally closed pneumatic pump valve was prepared using conventional soft lithography. Specifically, a SU-8 photoresist template was first prepared on a single-crystal silicon wafer. Then, a PDMS prepolymer was poured onto the SU-8 photoresist template. After cross-linking polymerization by heating at 80°C for 3 hours, a PDMS chip was prepared. The chip is used to generate aqueous two-phase droplet templates and prepare microgels loaded with single cells. Its structure consists of two layers: the upper layer is the liquid circuit part, which consists of an upper negative pressure interface 1, a reaction phase inlet containing a crosslinker (first inlet 2), a reaction phase channel containing a crosslinker 3, a continuous phase inlet (second inlet 4), a continuous phase channel 5, a dispersed phase inlet containing cells (third inlet 6), a dispersed phase channel containing cells 7, a normally closed pneumatic pump valve 8, a droplet transport channel 9, a microgel formation channel 10, a microgel outlet 11, an intersection A12 and an intersection B13; the lower layer is the gas circuit part, which consists of a lower negative pressure interface 14, a gas channel 15 and a normally closed pneumatic pump valve action area 16 ( Figures 1-3 The dispersed phase channel 7 and the continuous phase channel 5 converge at the droplet transport channel 9 to form a flow focusing intersection A12; the normally closed pneumatic pump valve 8 is located directly above the normally closed pneumatic pump valve action area 16. The dispersed phase channel is periodically opened through the pump valve in the two states of pumping and resting, so that the dispersed phase intermittently enters the continuous phase, stably and efficiently forming two-phase aqueous droplets ( Figure 4 ).

[0086] The chip channel height is 800 μm, the channel width is 800 μm, the width of the disconnection area of ​​the normally closed pneumatic pump valve 8 is 400 μm, the thickness of the PDMS film in the normally closed pneumatic pump valve action area 16 is 800 μm, the length of the droplet transport channel 9 is 1.2 cm, and the number of serpentine bending channels in the microgel formation channel 10 is 12, and its length is 12 cm.

[0087] (2) Preparation of aqueous two-phase solution: PEG is dissolved in an acid solution as the reaction phase containing the crosslinker, the PEG isotonic solution is used as the continuous phase, and the polymer that can be cross-linked by high-valent metal cations is dissolved in a dextran isotonic solution as the dispersed phase.

[0088] The acid used here is hydrochloric acid, and its concentration is: 0.2% (v / v); PEG molecular weight is: 20kDa, and its concentration in the continuous phase and the reaction phase is: 10% (w / v); the isotonic fluid is: normal saline; the molecular weight of dextran is: 500kDa, and its concentration is: 10% (w / v); the polymer that can be cross-linked by high-valent metal cations is: sodium alginate, molecular weight: 5000kDa, and its concentration is: 1% (w / v).

[0089] (3) Preparation of single-cell suspension: 3D spheroid cultured cells are enzymatically digested to obtain a monodispersed cell suspension. The cell suspension is then thoroughly mixed with an isotonic solution containing metal salt nanoparticles that has been previously ultrasonically dispersed. The mixture is incubated at 37°C and then centrifuged to remove nanoparticles that are not adhered to the cell surface. This results in a single-cell suspension with surface-adhered nanoparticles. The single-cell suspension is resuspended in a dispersed phase at a specific concentration to obtain a dispersed phase solution containing cells.

[0090] The cells used here are cells derived from primary pancreatic cancer tissue; the cell concentration during incubation is 106 cells / mL; the cell concentration in the dispersed phase is 104 cells / mL; the type of nanoparticles is calcium carbonate, the particle diameter is 800nm, and the concentration used for incubation is 6g / L; the incubation time of cells and nanoparticles is 25min; the centrifugal force of the centrifugal operation is 50g, and the time is 2min.

[0091] (4) Control of microfluidics: The reaction phase containing the crosslinker enters the microfluidic chip through the first inlet 2, and then reaches the intersection B13 along the reaction phase channel 3 containing the crosslinker; the continuous phase enters the microfluidic chip through the second inlet 4, and passes through the continuous phase channel 5, the intersection A12 and the droplet transport channel 9 to reach the intersection B13; the dispersed phase containing cells enters the microfluidic chip through the third inlet 6, and passes through the dispersed phase channel 7 containing cells, the normally closed pneumatic pump valve 8, the intersection A12 and the droplet transport channel 9 to reach the intersection B13; the external vacuum equipment is connected to the microfluidic chip through the upper negative pressure interface 1, and passes through the lower negative pressure interface 14 and the gas channel 15 to reach the normally closed pneumatic pump valve action area 16, and periodically drives the normally closed pneumatic pump valve 8 to open and close, thereby controlling the on-off of the dispersed phase to promote the formation of dispersed phase droplets and control their size;

[0092] Here, the external vacuum degree is: -0.08 MPa; the flow rate of the dispersed phase containing cells is: 3 μL / min; the flow rate of the continuous phase is: 20 μL / min; the flow rate of the reaction phase containing the cross-linking agent is: 20 μL / min.

[0093] (5) Production of single cell loaded microgels: The dispersed phase droplets containing cells formed in step (4) meet the reaction phase containing hydrochloric acid at the intersection B. The hydrogen ions in the reaction phase enter the droplets due to free diffusion movement through the intermediate continuous phase and instantly interact with the nanoparticles attached to the cell surface, releasing high-valent metal ions Ca 2+ , causing the polymers in the dispersed phase to cross-link and form cell-loaded microgels. Droplets without cells, however, cannot cross-link because no metal ions are released, and can naturally dissolve in the surrounding solution during subsequent collection and transfer.

[0094] (6) Organoid construction: The single cell-loaded microgel obtained in step (5) was transferred to the corresponding cell culture medium and cultured and expanded for a long time in a conventional cell culture environment at 37°C and 5% CO2, thereby developing from a single cell into a monoclonal cell and finally forming a pancreatic cancer organoid ( Figure 7 ).

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing organoids based on a single cell chip, characterized in that: Using a microfluidic chip integrated with a normally closed pneumatic pump valve as the technical platform, a single-cell suspension pre-incubated with a crosslinker as the chemical reaction core, and water-in-water droplets as the molding template, the monodispersed seed cells are loaded into the micro-hydrogel carrier in a one-step method. The cell-free empty droplets are simply removed in the subsequent transfer step to achieve efficient single-cell in situ loading. The process includes the following steps: S1. Preparation of microfluidic chip: A polydimethylsiloxane chip integrated with a normally closed pneumatic pump valve (8), i.e., a microfluidic chip, is prepared using soft lithography technology. The chip is used to generate a two-phase aqueous droplet template and prepare hydrogel microspheres loaded with single cells. The chip comprises an upper layer of a liquid path portion and a lower layer of an air path portion, wherein the air path portion periodically opens a dispersed phase channel in two states: pumping and resting through a normally closed pneumatic pump valve (8), thereby allowing the dispersed phase to intermittently enter the continuous phase, thereby stably and efficiently forming two-phase aqueous droplets. S2. Preparation of a two-phase aqueous solution: Separately preparing a reaction phase containing a crosslinker, a continuous phase, and a dispersed phase; dissolving a polymer crosslinkable by high-valent metal cations in a dextran isotonic solution as the dispersed phase; dissolving polyethylene glycol in an acid solution as the reaction phase containing a crosslinker, and using the polyethylene glycol isotonic solution as the continuous phase; S3. Preparation of single cell suspension: preparing a single cell suspension, and resuspending the single cell suspension in the dispersed phase prepared in step S2 at a certain concentration to obtain a dispersed phase solution containing cells; S4. Microfluidics manipulation: delivering the reaction phase containing the crosslinker, the continuous phase, and the dispersed phase containing cells into the microfluidic chip, and obtaining the dispersed phase droplets containing cells using a normally closed pneumatic pump valve (8); S5. Production of single cell-loaded microgels: In a microfluidic chip, the cell-containing dispersed phase droplets formed in step S4 are allowed to meet and react with a reaction phase containing a crosslinker to form cell-loaded microgels; S6. Organoid construction: The single-cell loaded microgel obtained in step S5 is transferred to the corresponding cell culture medium and cultured and expanded long-term in a cell culture environment at 37°C and 5% CO2 or a hypoxic environment, so that the single cell develops into a monoclonal clone and finally forms an organoid; The specific steps of step S3 are as follows: The two-dimensional adherent or three-dimensional cultured cells are enzymatically digested to obtain a monodisperse cell suspension. An isotonic solution containing metal oxide or salt nanoparticles, which has been previously dispersed by ultrasound, is then thoroughly mixed with the cell suspension and incubated at room temperature or 37°C. Nanoparticles not adhered to the cell surface are then removed by centrifugation or filtration to obtain a single-cell suspension with surface-adhered nanoparticles. The single-cell suspension is resuspended in a dispersed phase at a certain concentration to obtain a dispersed phase solution containing cells. The specific steps of step S5 are as follows: The cell-containing dispersed phase droplets formed in step S4 meet the reaction phase containing the crosslinker at the intersection B (13) of the chip. The hydrogen ions in the reaction phase enter the droplets due to free diffusion movement through the intermediate continuous phase and instantly interact with the nanoparticles adhered to the cell surface, releasing high-valent metal ions, thereby causing the polymers in the dispersed phase to crosslink and form cell-loaded microgels. The cell-free droplets, however, cannot crosslink because no metal ions are released and can naturally dissolve in the surrounding solution during the subsequent collection and transfer process. The type of the nanoparticles is one of calcium carbonate, barium carbonate, iron oxide, zinc oxide, and copper oxide.

2. The method for preparing organoids based on a single cell chip according to claim 1, characterized in that: In step S1, the polydimethylsiloxane chip is prepared by first preparing a SU-8 photoresist template on a single crystal silicon wafer or a dust-free glass sheet using soft lithography technology, then pouring a polydimethylsiloxane prepolymer onto the SU-8 photoresist template, and heating it at 80° C. for 1-3 hours for cross-linking polymerization to prepare a polydimethylsiloxane chip; the chip is based on a flow-focusing microfluidic droplet chip and is integrated with a lower gas path portion; The liquid circuit portion comprises an upper negative pressure interface (1), a first inlet (2), a reaction phase channel containing a crosslinker (3), a second inlet (4), a continuous phase channel (5), a third inlet (6), a dispersed phase channel containing cells (7), a normally closed pneumatic pump valve (8), a droplet transport channel (9), a microgel formation channel (10), a microgel outlet (11), an intersection A (12) and an intersection B (13), wherein the upper negative pressure interface (1) is connected to an external vacuum device; the first inlet (2) is used to transport the reaction phase containing a crosslinker into the chip; the reaction phase channel containing a crosslinker (3) is in a conical structure and has two first channels connected in parallel; the inlet ends of the two first channels are connected to the first inlet (2), and the outlet ends of the two channels are located at the tip of the cone; the second inlet (4) is used to transport the continuous phase into the chip; the continuous phase channel (5) is located in the reaction phase channel (3) containing a crosslinker, and has two second channels connected in parallel, and the inlet ends of the two second channels are connected to the second inlet (4); the third inlet (6) is used to transport the dispersed phase containing cells into the chip, and the dispersed phase channel (7) containing cells is located in the continuous phase channel (5), one end of which is connected to the third inlet (6), and the other end of which is connected to the outlet ends of the two second channels and converges to the inlet end of the droplet transport channel (9), forming a flow focusing intersection A (12); the droplet transport channel (9) is located in the reaction phase channel (3) containing a crosslinker, and its outlet end and the outlet ends of the two first channels converge to the inlet end of the microgel forming channel (10), forming an intersection B (13); the body of the microgel forming channel (10) is a serpentine curved channel, and its outlet end serves as a microgel outlet (11); The gas circuit portion comprises a lower negative pressure interface (14), a gas channel (15) and a normally closed pneumatic pump valve action area (16), wherein the lower negative pressure interface (14) is connected to the upper negative pressure interface (1), one end of the gas channel (15) is connected to the lower negative pressure interface (14), and the other end is connected to the normally closed pneumatic pump valve action area (16), and the normally closed pneumatic pump valve action area (16) is located directly below the normally closed pneumatic pump valve (8).

3. The method for preparing organoids based on a single cell chip according to claim 2, characterized in that: The specific steps of step S4 are as follows: The reaction phase containing the crosslinker enters the microfluidic chip through the first inlet (2), and then reaches the intersection B (13) along the reaction phase channel (3) containing the crosslinker; the continuous phase enters the microfluidic chip through the second inlet (4), passes through the continuous phase channel (5), the intersection A (12) and the droplet transport channel (9) to reach the intersection B (13); the dispersed phase containing cells enters the microfluidic chip through the third inlet (6), passes through the dispersed phase channel containing cells (7), the normally closed pneumatic pump valve (8), the intersection A (12) and the droplet transport channel (9) to reach the intersection B (13); the external vacuum equipment is connected to the microfluidic chip through the upper negative pressure interface (1), passes through the lower negative pressure interface (14) and the gas channel (15) to reach the normally closed pneumatic pump valve action area (16), and periodically drives the normally closed pneumatic pump valve (8) to open and close, thereby controlling the on-off of the dispersed phase to promote the formation of dispersed phase droplets and control their size.

4. The method for preparing organoids based on a single cell chip according to claim 2 or 3, characterized in that: The height of each channel in the chip is 50-900 μm, the width of each channel is 50-900 μm, the width of the disconnection area of ​​the normally closed pneumatic pump valve (8) is 20-500 μm, the thickness of the PDMS film in the normally closed pneumatic pump valve action area (16) is 50-1000 μm, the length of the droplet transport channel (9) is 0.3-1.5 cm, the number of serpentine curved channels in the microgel formation channel (10) is 3-15, and the length thereof is 3-15 cm.

5. The method for preparing organoids based on a single cell chip according to claim 1, characterized in that: The acid in step S2 is one of formic acid, acetic acid, sulfuric acid, hydrochloric acid, nitric acid or sulfurous acid, or a combination of more than one, and the total concentration thereof is 0.05-2% v / v; the molecular weight of polyethylene glycol is 8-20 kDa, and its concentration in the continuous phase and the reaction phase is 10%-30% w / v; the isotonic fluid is PBS buffer or normal saline; the molecular weight of dextran is 70-500 kDa, and the concentration is 10%-30% w / v; the polymer capable of cross-linking with high-valent metal cations is one of sodium alginate, pectin, carboxymethyl chitosan and a polymer modified with inactivated FXIII, or a combination of more than one, and the molecular weight is 1-10,000 kDa, and the concentration is 0.1-5% w / v.

6. The method for preparing organoids based on a single cell chip according to claim 1, characterized in that: The cells in step S3 are: various commercial cell lines, primary healthy or tumor tissue derived cells, various stem cells; the cell concentration during incubation is: 10 5 -10 8 / mL; the cell concentration in the dispersed phase is: 10 3 -10 6 The nanoparticles have a particle diameter of 1-1000 nm and a concentration of 0.1-10 g / L for incubation. The incubation time between cells and nanoparticles is 5-60 min. The centrifugal force for the centrifugation operation is 5-200 g and the time is 0.1-3 min. The pore size of the filter membrane used for the filtration operation is 2-15 μm.

7. The method for preparing organoids based on a single cell chip according to claim 3, characterized in that: In step S4, the external vacuum degree is: -0.01 to -0.1 MPa; the flow rate of the dispersed phase containing cells is: 0.01-5 μL / min; the flow rate of the continuous phase is: 1-20 μL / min; the flow rate of the reaction phase containing the cross-linking agent is: 1-20 μL / min.

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