A tumor organoid microarray for drug screening and a drug screening method

Abstract

This invention provides a microarray for drug screening of tumor organoids and a method for drug screening. The method includes microfluidic chip fabrication, preparation of a two-phase aqueous solution, preparation of a single-cell suspension, microfluidic manipulation, generation of single-cell loaded microgels, and high-throughput anticancer drug screening of pancreatic cancer organoids. This invention uses a microfluidic chip integrating a normally closed pneumatic pump valve as a technical platform, a pre-incubated single-cell suspension with a cross-linking agent as the core of the chemical reaction, and water-in-water droplets as molding templates. A one-step method loads monodisperse seed cells onto a microhydrogel carrier, achieving efficient in-situ loading of single cells, construction of pancreatic cancer organoids, and high-throughput drug screening. This method improves the consistency of the organoid culture environment and starting point, while also possessing the high-throughput characteristics of droplet microfluidic technology. It achieves efficient and highly reproducible in vitro organoid construction and anticancer drug screening, and can play a significant role in basic research and translational applications related to pancreatic cancer organoids.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics, bioengineering, and drug evaluation, and more particularly to a tumor organoid microarray for drug screening and a drug screening method. Background Technology

[0002] Pancreatic cancer is one of the most common malignant tumors of the digestive tract, with a five-year survival rate of approximately 10%, making it one of the malignant tumors with the worst prognosis. Furthermore, the early diagnosis rate of pancreatic cancer is low, the surgical mortality rate is high, and the cure rate is very low. Therefore, finding safe and effective treatments for pancreatic cancer is urgent. In pancreatic cancer drug screening, traditional 2D cell and animal models are far from meeting practical needs. Establishing new experimental medical models and research paradigms is of great significance for a deeper understanding of the pathogenesis of pancreatic cancer and the development of effective treatments. Organoids are a new type of in vitro model that has developed in recent years. They mainly refer to functional cell-tissue complexes differentiated under 3D in vitro culture conditions through cell self-organization. Organoids typically possess some key structural and functional characteristics of human tissues or organs and have shown significant application prospects in areas such as tissue and organ development, disease simulation, drug screening, and regenerative medicine. In recent years, pancreatic cancer organoids have also emerged and begun to be used in research in fields such as biology and medicine. However, overall, the existing pancreatic cancer organoid research system still faces many challenges in terms of cell matrix homogeneity, controllable organoid morphology, and high-throughput analysis.

[0003] As a downstream branch of traditional microfluidics, droplet microfluidic systems offer advantages such as high throughput, good reproducibility, low sample consumption, and rapid sample mixing, playing an increasingly important role in biochemical analysis and functional material preparation. Based on the size and throughput advantages of droplet microfluidics, 3D cell culture carriers at the micrometer to millimeter scale can be effectively prepared. In particular, aqueous two-phase droplet microfluidic systems, in addition to the aforementioned advantages, provide a more biocompatible carrier preparation environment and a simpler post-processing procedure. However, traditional droplet microfluidic techniques for cell loading and culture rely on probabilistic events to embed target cells within microcarriers, resulting in poor uniformity and effectiveness. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, this invention provides a tumor organoid microarray for drug screening and a drug screening method. Based on this microarray, through further amplification and development under conventional cell culture conditions, a more controllable in vitro pancreatic cancer organoid model can be obtained. Combining this model with well plates or high-throughput arrays enables rapid screening of multiple drugs and their anticancer effects at multiple concentrations. The technical means employed in this invention are as follows:

[0005] A drug screening tumor organoid microarray, the microarray comprising an upper and lower two-layer structure, wherein:

[0006] The upper structure is the liquid path section, including: an upper negative pressure interface, a reaction phase inlet containing a cross-linking agent, a reaction phase channel containing a cross-linking agent, a continuous phase inlet, a continuous phase channel, a dispersed phase inlet containing cells, a dispersed phase channel containing cells, a normally closed pneumatic valve, a droplet transport channel, a microgel formation channel, a microgel outlet, and intersections A and B.

[0007] The reaction phase inlet containing the crosslinking agent is connected to two reaction phase channels containing the crosslinking agent. The continuous phase inlet is connected to two continuous phase channels. The cell-containing dispersed phase inlet is connected to a cell-containing dispersed phase channel. The end of the cell-containing dispersed phase channel first intersects with the two continuous phase channels at intersection A, then intersects with the two reaction phase channels containing the crosslinking agent at intersection B, and subsequently connects to the microgel forming channel.

[0008] The microgel forming channel has a serpentine structure, with a microgel outlet at its end.

[0009] A normally closed pneumatic valve is installed on the cell-containing dispersion phase channel;

[0010] The lower structure is the gas path section, including the lower negative pressure interface, gas passage, and the working area of ​​the normally closed pneumatic pump valve.

[0011] The lower negative pressure interface is connected to the operating area of ​​the normally closed pneumatic pump valve through a gas channel.

[0012] The upper and lower structures are used together. When in use, the lower negative pressure interface is connected to the upper negative pressure interface, and the position of the normally closed pneumatic valve's operating area is adapted to the position of the normally closed pneumatic valve.

[0013] Furthermore, the height of each connection channel on the chip is 50-900μm, and the channel width is 50-900μm;

[0014] The normally closed pneumatic valve has a disconnection area width of 20-500μm and a PDMS film thickness of 50-1000μm in the working area of ​​the pneumatic valve.

[0015] The droplet transport channel is 0.3-1.5 cm long, and the number of serpentine channels forming the microgel is 3-15, with a length of 3-15 cm.

[0016] This invention also discloses a drug screening method, comprising the following steps:

[0017] S1. Fabrication of microfluidic chip: A polydimethylsiloxane chip integrating a normally closed pneumatic pump valve was fabricated using soft photolithography. The polydimethylsiloxane chip is the drug screening tumor organoid chip described in claim 1.

[0018] S2. Preparation of aqueous two-phase solution: Dissolve polyethylene glycol in acetic acid solution as the reaction phase containing crosslinking agent, use polyethylene glycol isotonic solution as continuous phase, and dissolve sodium alginate and natural protein components in dextran isotonic solution as dispersion phase.

[0019] S3. Preparation of single-cell suspension: Two-dimensional adherent or three-dimensional cultured pancreatic cancer cells are digested by enzyme to obtain a monodisperse cell suspension. Then, an isotonic solution of calcium carbonate nanoparticles that have been pre-dispersed by ultrasound is thoroughly mixed with the cell suspension and incubated at room temperature or 37°C. Subsequently, the nanoparticles that are not attached to the cell surface are removed by centrifugation to obtain a single-cell suspension with nanoparticles attached to the surface. The single-cell suspension is resuspended in a dispersion phase at a certain concentration to obtain a dispersion phase solution containing cells.

[0020] S4. Microfluidic Manipulation: The reaction phase containing the crosslinking agent enters the microfluidic chip through the reaction phase inlet containing the crosslinking agent, and then reaches intersection B along the reaction phase channel containing the crosslinking agent. The continuous phase enters the microfluidic chip through the continuous phase inlet, and passes through the continuous phase channel, intersection A, and droplet transport channel to reach intersection B. The dispersed phase containing cells enters the microfluidic chip through the dispersed phase inlet containing cells, and passes through the dispersed phase channel containing cells, normally closed pneumatic pump valve, intersection A, and droplet transport channel to reach intersection B. The external vacuum device is connected to the microfluidic chip through the upper negative pressure interface, and passes through the lower negative pressure interface and gas channel to reach the working area of ​​the normally closed pneumatic valve, periodically driving the pneumatic pump valve 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.

[0021] S5. Generation of single-cell loaded microgels for pancreatic cancer: The cell-containing dispersed phase droplet meets the reaction phase containing the cross-linking agent at intersection B. Hydrogen ions in the reaction phase diffuse freely, passing through the intermediate continuous phase and entering the droplet. They then instantaneously interact with the nanoparticles adhering to the cell surface, releasing Ca2+. 2+ Ions crosslink the sodium alginate in the dispersed phase, forming a microgel loaded with pancreatic cancer cells along with the remaining protein components. Cell-free droplets, lacking calcium,... 2+ The released components cannot cross-link and can naturally dissolve in the surrounding solution during subsequent collection and transfer.

[0022] S6. High-throughput anticancer drug screening for pancreatic cancer organoids: Single cells loaded with microgels were transferred to the corresponding cell culture medium and cultured and expanded under conventional cell culture conditions of 37°C and 5% CO2 for a long period of time, thereby developing from single cells into monoclonal cells and finally forming pancreatic cancer organoids. The organoids were then screened for high-throughput efficacy by adding different concentrations of anticancer drugs in commercial well plates or microfluidic arrays using automated or manual pipetting methods. The tumor-killing effect was then evaluated based on CCK-8 assays, cell viability assays, etc.

[0023] Furthermore, the fabrication of microfluidic chips specifically includes:

[0024] A template of SU-8 photoresist is prepared on a single-crystal silicon wafer or a dust-free glass plate. Then, PDMS prepolymer is poured onto the SU-8 photoresist template and crosslinked and polymerized by heating at 80°C for 1-3 hours to prepare a PDMS chip.

[0025] Furthermore, in the preparation steps of the aqueous two-phase solution:

[0026] Acetic acid concentration: 0.05-2% (v / v);

[0027] The molecular weight of PEG is 8-20 kDa, and its concentration in the continuous phase and the reaction phase is 10%-30% (w / v).

[0028] Isotonic solutions include PBS buffer or physiological saline.

[0029] The molecular weight of dextran is 70-500 kDa, and the concentration is 10%-30% (w / v).

[0030] Sodium alginate has a molecular weight of 1-10000 kDa and a concentration of 0.1-4% (w / v).

[0031] Natural protein molecules are one or more of collagen, laminin, and fibroin, with a concentration of 0.5-10 mg / mL for a single protein.

[0032] Furthermore, in the preparation steps of the single-cell suspension:

[0033] The pancreatic cancer cells are one of the following: various commercial cell lines, cells derived from primary tumor tissue, and various stem cells;

[0034] The cell concentration during incubation was: 10 5 -10 8 cells / mL;

[0035] The cell concentration in the dispersed phase is: 10 3 -10 6 cells / mL;

[0036] The diameter of the nanoparticles is 1-1000 nm, and the concentration used for incubation is 0.1-10 g / L;

[0037] The incubation time between cells and nanoparticles is 5-60 min;

[0038] Centrifugation operation: centrifugal force: 5-200g, time: 0.1-3min.

[0039] Furthermore, in the microfluidic manipulation steps:

[0040] The external vacuum level is (-0.01) - (-0.1) MPa;

[0041] The flow rate of the cell-containing dispersion was 0.01-5 μL / min;

[0042] The continuous phase flow rate is 1-20 μL / min;

[0043] The flow rate of the reaction phase containing the crosslinking agent is 1-20 μL / min.

[0044] Furthermore, in the high-throughput anticancer drug screening steps for pancreatic cancer organoids:

[0045] The anticancer drugs are one or more of gemcitabine, erlotinib hydrochloride, olaparib, everolimus, and sunitinib, with a concentration of 1-10000 nM.

[0046] Compared with the prior art, the present invention has the following advantages:

[0047] This invention can significantly improve the consistency of the starting point in the construction of pancreatic cancer organoids, including the certainty of matrix materials and the uniformity of single cells, thereby increasing the reproducibility and reliability of organoids in the drug screening process. Furthermore, the use of droplet microfluidics technology ensures the high-throughput characteristics of organoid production and application. Therefore, in addition to its advantages in efficient drug screening, this approach is expected to play an important role in basic and translational research, such as developmental studies and disease simulation.

[0048] Based on the above reasons, this invention can be widely promoted in the fields of biological, medical and pharmaceutical research and application related to pancreatic cancer organoids. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is a schematic diagram of a tumor organoid microarray structure for drug screening according to the present invention.

[0051] Figure 2 This is a schematic diagram of the upper layer structure of the tumor organoid chip for drug screening in this invention.

[0052] Figure 3 This is a schematic diagram of the lower layer structure of the tumor organoid chip for drug screening in this invention.

[0053] Figure 4 This is a schematic diagram illustrating the working principle of the normally closed pneumatic pump valve of the present invention.

[0054] Figure 5 This is a schematic diagram of the formation of droplets and microgels at intersections A and B of the present invention.

[0055] Figure 6 This is a physical image of the two-phase aqueous droplet microfluidic chip used to construct the organoid model in Example 1.

[0056] Figure 7 This is a bright-field characterization image of the organoid loaded microgel obtained in Example 2.

[0057] Figure 8 This is a diagram showing the changes in cell activity after treatment with gemcitabine in Example 3.

[0058] In the diagram: 1 Upper negative pressure interface; 2 Reaction phase inlet containing crosslinking agent; 3 Reaction phase channel containing crosslinking agent; 4 Continuous phase inlet; 5 Continuous phase channel; 6 Dispersed phase inlet containing cells; 7 Dispersed phase channel containing cells; 8 Normally closed pneumatic valve; 9 Droplet transport channel; 10 Microgel formation channel; 11 Microgel outlet; 12 Crossroads A; 13 Crossroads B; 14 Lower negative pressure interface; 15 Gas channel; 16 Normally closed pneumatic pump valve operating area. Detailed Implementation

[0059] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The invention will now be described in detail with reference to the accompanying drawings and embodiments. To make the objectives, technical solutions, and advantages of the embodiments of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0060] The application of this invention first requires the design and fabrication of a microfluidic chip of suitable size according to actual needs; then, the desired cell types undergo nanoparticle pretreatment, and suitable matrix materials are selected; finally, a pump-valve control system is used to regulate the generation and size of single-cell microgels, and pancreatic cancer organoids are obtained through further cell expansion and development, which are then used for high-throughput screening of anticancer drugs in conjunction with commercial well plates or microfluidic arrays. The invention will be further described below with reference to the accompanying drawings and embodiments.

[0061] Example 1

[0062] This embodiment discloses a method for screening tumor organoid microarrays based on high-throughput drug screening, comprising the following steps:

[0063] (1) Fabrication of the microfluidic chip: A PDMS chip integrating a normally closed pneumatic pump valve was fabricated using conventional soft lithography. Specifically, a template of SU-8 photoresist was first fabricated on a single-crystal silicon wafer. Then, the PDMS prepolymer was poured onto the SU-8 photoresist template and crosslinked and polymerized at 80°C for 1 hour to produce the PDMS chip. This chip is used to generate a two-phase droplet template and to prepare microgels loaded with single cells. Its structure consists of two layers: the upper layer is the liquid path, which consists of an upper negative pressure interface 1, a reaction phase inlet containing crosslinking agent 2, a reaction phase channel containing crosslinking agent 3, a continuous phase inlet 4, a continuous phase channel 5, a dispersed phase inlet containing cells 6, a dispersed phase channel containing cells 7, a normally closed pneumatic 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 path, which consists of a lower negative pressure interface 14, a gas channel 15, and a normally closed pneumatic pump valve action area 16. Figure 1 The dispersed phase channel 7 and the continuous phase channel 5 converge at the droplet transport channel 9 to form a flow focusing intersection. The normally closed pneumatic pump valve 8 is located directly above the pneumatic valve's operating area 16. It periodically opens the dispersed phase channel through both pumping and resting states, allowing the dispersed phase to intermittently enter the continuous phase, stably and efficiently forming two-phase aqueous droplets, such as... Figure 5 As shown.

[0064] like Figure 6 As shown, the chip channel height is 100μm, the channel width is 100μm, the width of the normally closed pneumatic valve disconnection area is 100μm, the PDMS film thickness in the pneumatic valve action area is 100μm, the length of droplet transport channel 9 is 0.3cm, and the number of serpentine channels in microgel formation channel 10 is 5, with a length of 5cm.

[0065] (2) Preparation of aqueous two-phase solution: PEG is dissolved in acetic acid solution as the reaction phase containing cross-linking agent, and sodium alginate and natural protein components are dissolved in dextran isotonic solution as the dispersion phase.

[0066] The acid concentration used here is 0.5% (v / v); the molecular weight of PEG is 8 kDa, and its concentration in the continuous phase and reaction phase is 30% (w / v); the isotonic solution is PBS buffer; the molecular weight of dextran is 70 kDa, and its concentration is 30% (w / v); the molecular weight of sodium alginate is 10 kDa, and its concentration is 4% (w / v); the natural protein component is type IV collagen, and its concentration is 2 mg / mL.

[0067] (3) Preparation of single-cell suspension: 2D adherent cells were enzymatically digested to obtain a monodisperse cell suspension. Then, an isotonic solution of pre-dispersed calcium carbonate nanoparticles was thoroughly mixed with the cell suspension and incubated at room temperature. Subsequently, centrifugation was used to remove nanoparticles not adhering to the cell surface, resulting in a single-cell suspension with adhered nanoparticles. The single-cell suspension was resuspended in a dispersion phase at a specific concentration to obtain a dispersed phase solution containing cells.

[0068] The cells used here are the commercial PANC-1 cell line; the cell concentration during incubation was 10-1. 8 Cells / mL; Cell concentration in the dispersed phase: 5 × 10⁻⁶ 5 Cells / mL; Nanoparticle diameter: 100 nm; Incubation concentration: 1 g / L; Incubation time between cells and nanoparticles: 10 min; Centrifugation force: 180 g; Centrifugation time: 0.5 min.

[0069] (4) Microfluidic manipulation: The reaction phase containing the crosslinking agent enters the microfluidic chip through inlet 2, and then reaches the intersection B13 along channel 3; the continuous phase enters the microfluidic chip through inlet 4, and passes through channel 5, intersection A12 and channel 9 to reach intersection B13; the dispersed phase containing cells enters the microfluidic chip through inlet 6, and passes through channel 7, normally closed pneumatic pump valve 8, intersection A12 and channel 9 to reach intersection B13; the external vacuum device is connected to the microfluidic chip through interface 1, and passes through interface 14 and channel 15 to reach the working area of ​​the normally closed pneumatic pump valve, periodically driving the 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.

[0070] The external vacuum level here 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 the crosslinking agent is 2 μL / min.

[0071] (5) Generation of single-cell loaded microgels: The cell-containing dispersed phase droplets formed in (4) meet the reaction phase containing acetic acid at intersection B. Hydrogen ions in the reaction phase diffuse freely, passing through the intermediate continuous phase and entering the droplets. They then instantly interact with the calcium carbonate nanoparticles adhering to the cell surface, releasing Ca2+. 2+ This causes the sodium alginate and protein components in the dispersed phase to cross-link, forming a cell-loaded microgel. Cell-free droplets, lacking Ca2+, do not... 2+ The released components cannot cross-link and can naturally dissolve in the surrounding solution during subsequent collection and transfer.

[0072] (6) High-throughput anticancer drug screening of pancreatic cancer organoids: The single-cell loaded microgels obtained in (5) were transferred to the corresponding cell culture medium and cultured and expanded under conventional cell culture conditions of 37°C and 5% CO2 for a long period of time, so that single cells developed into monoclonal cells and eventually formed pancreatic cancer organoids. The obtained organoids can be screened for high-throughput efficacy by adding 10 groups of olaparib drugs at 1-1000 nM in commercial well plates using automated pipetting, and the tumor killing effect was evaluated based on CCK-8, cell death and survival kits, etc.

[0073] Example 2

[0074] This embodiment discloses a drug screening method based on high-throughput drug screening tumor organoid microarrays, comprising the following steps:

[0075] (1) Fabrication of the microfluidic chip: A PDMS chip integrating a normally closed pneumatic pump valve was fabricated using conventional soft lithography. Specifically, a template of SU-8 photoresist was first fabricated on a single-crystal silicon wafer. Then, the PDMS prepolymer was poured onto the SU-8 photoresist template and crosslinked and polymerized at 80°C for 2 hours to produce the PDMS chip. This chip is used to generate a two-phase droplet template and to prepare microgels loaded with single cells. Its structure consists of two layers: the upper layer is the liquid path, which consists of an upper negative pressure interface 1, a reaction phase inlet 2 containing crosslinking agent, a reaction phase channel 3 containing crosslinking agent, a continuous phase inlet 4, a continuous phase channel 5, a dispersed phase inlet 6 containing cells, a dispersed phase channel 7 containing cells, a normally closed pneumatic 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 path, which consists of a lower negative pressure interface 14, a gas channel 15, and a normally closed pneumatic pump valve action area 16. Figure 1 The dispersed phase channel 7 and the continuous phase channel 5 converge at the droplet transport channel 9 to form a flow focusing intersection. The normally closed pneumatic pump valve 8 is located directly above the pneumatic valve's operating area 16. It periodically opens the dispersed phase channel through both pumping and resting states, allowing the dispersed phase to intermittently enter the continuous phase, stably and efficiently forming two-phase aqueous droplets, such as... Figure 5 As shown.

[0076] The chip channel height is 400μm, the channel width is 400μm, the width of the normally closed pneumatic valve disconnection area is 300μm, the PDMS film thickness in the pneumatic valve action area is 400μm, the length of droplet transport channel 9 is 0.8cm, and the number of serpentine channels 10 for microgel formation is 10, with a length of 10cm.

[0077] (2) Preparation of aqueous two-phase solution: PEG is dissolved in acetic acid solution as the reaction phase containing cross-linking agent, and sodium alginate and natural protein components are dissolved in dextran isotonic solution as the dispersion phase.

[0078] The acetic acid concentration here is 1.5% (v / v); the molecular weight of PEG is 10 kDa, and its concentration in the continuous phase and reaction phase is 15% (w / v); the isotonic solution is PBS buffer; the molecular weight of dextran is 200 kDa, and its concentration is 15% (w / v); the molecular weight of sodium alginate is 5000 kDa, and its concentration is 1% (w / v); the natural protein component is fibrin, and its concentration is 5 mg / mL.

[0079] (3) Preparation of single-cell suspension: Cells cultured in 3D spheroids were digested by enzymes to obtain a monodisperse cell suspension. Then, an isotonic solution containing calcium carbonate nanoparticles, which had been pre-dispersed by ultrasound, was thoroughly mixed with the cell suspension and incubated at 37°C. Subsequently, the nanoparticles not adhering to the cell surface were removed by centrifugation to obtain a single-cell suspension with nanoparticles adhering to the cell surface. The single-cell suspension was resuspended in a dispersion phase at a certain concentration to obtain a dispersion phase solution containing cells.

[0080] The cells used here are primary human pancreatic ductal carcinoma cells; the cell concentration during incubation was 10-1. 7 Cells / mL; Cell concentration in the dispersed phase: 10 5 Cells / mL; Nanoparticle diameter: 600 nm; Incubation concentration: 9 g / L; Incubation time between cells and nanoparticles: 50 min; Centrifugation force: 100 g; Centrifugation time: 1 min.

[0081] (4) Microfluidic manipulation: The reaction phase containing the crosslinking agent enters the microfluidic chip through inlet 2, and then reaches the intersection B13 along channel 3; the continuous phase enters the microfluidic chip through inlet 4, and passes through channel 5, intersection A12 and channel 9 to reach intersection B13; the dispersed phase containing cells enters the microfluidic chip through inlet 6, and passes through channel 7, normally closed pneumatic pump valve 8, intersection A12 and channel 9 to reach intersection B13; the external vacuum device is connected to the microfluidic chip through interface 1, and passes through interface 14 and channel 15 to reach the working area of ​​the normally closed pneumatic pump valve, periodically driving the 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;

[0082] The external vacuum level here 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; and the flow rate of the reaction phase containing the crosslinking agent is 10 μL / min.

[0083] (5) Generation of single-cell loaded microgels: The cell-containing dispersed phase droplets formed in (4) meet the reaction phase containing acetic acid at intersection B. Hydrogen ions in the reaction phase diffuse freely, passing through the intermediate continuous phase and entering the droplets. They then interact instantaneously with the nanoparticles adhering to the cell surface, releasing high-valence metal ions Ca. 2+ This causes the polymers in the dispersed phase to cross-link, forming cell-loaded microgels. Cell-free droplets, on the other hand, lack Ca2+. 2+ The released components cannot cross-link and can naturally dissolve in the surrounding solution during subsequent collection and transfer.

[0084] (6) High-throughput anticancer drug screening for pancreatic cancer organoids: The single-cell loaded microgels obtained in (5) were transferred to the corresponding cell culture medium and cultured and expanded under conventional cell culture conditions of 37°C and 5% CO2 for a long period of time, thereby developing from single cells into monoclonal cells and eventually forming pancreatic cancer organoids, such as Figure 7 As shown. The obtained organoids can be used in a microfluidic array chip to perform high-throughput pharmacodynamic screening by adding 10 groups of sunitinib drugs ranging from 1-1000 nM using manual pipetting. The tumor-killing effect is evaluated based on CCK-8 and cell liveness assay kits.

[0085] Example 3

[0086] This embodiment discloses a drug screening method based on high-throughput drug screening tumor organoid microarrays, comprising the following steps:

[0087] (1) Fabrication of the microfluidic chip: A PDMS chip integrating a normally closed pneumatic pump valve was fabricated using conventional soft lithography. Specifically, a template of SU-8 photoresist was first fabricated on a single-crystal silicon wafer. Then, the PDMS prepolymer was poured onto the SU-8 photoresist template and crosslinked and polymerized at 80°C for 3 hours to produce the PDMS chip. This chip is used to generate a two-phase droplet template and to prepare microgels loaded with single cells. Its structure consists of two layers: the upper layer is the liquid path, which consists of an upper negative pressure interface 1, a reaction phase inlet containing crosslinking agent 2, a reaction phase channel containing crosslinking agent 3, a continuous phase inlet 4, a continuous phase channel 5, a dispersed phase inlet containing cells 6, a dispersed phase channel containing cells 7, a normally closed pneumatic 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 path, which consists of a lower negative pressure interface 14, a gas channel 15, and a normally closed pneumatic pump valve action area 16. Figure 1 The dispersed phase channel 7 and the continuous phase channel 5 converge at the droplet transport channel 9 to form a flow focusing intersection. The normally closed pneumatic pump valve 8 is located directly above the pneumatic valve's operating area 16. It periodically opens the dispersed phase channel through both pumping and resting states, allowing the dispersed phase to intermittently enter the continuous phase, stably and efficiently forming two-phase aqueous droplets, such as... Figure 5 As shown.

[0088] The chip channel height is 800μm, the channel width is 800μm, the width of the normally closed pneumatic valve disconnection area is 400μm, the PDMS film thickness in the pneumatic valve action area is 800μm, the droplet transport channel 9 has a length of 1.2cm, and the number of serpentine channels 10 for microgel formation is 12, with a length of 12cm.

[0089] (2) Preparation of aqueous two-phase solution: PEG is dissolved in acetic acid solution as the reaction phase containing cross-linking agent, and sodium alginate and natural protein components are dissolved in dextran isotonic solution as the dispersion phase.

[0090] The acetic acid concentration here is 1% (v / v); the molecular weight of PEG is 20 kDa, and its concentration in the continuous phase and the reaction phase is 10% (w / v); the isotonic solution is physiological saline; the molecular weight of dextran is 500 kDa, and its concentration is 10% (w / v); the molecular weight of sodium alginate is 500 kDa, and its concentration is 2% (w / v); the natural protein component is laminin, and its concentration is 8 mg / mL.

[0091] (3) Preparation of single-cell suspension: Cells cultured in 3D spheroids were digested by enzymes to obtain a monodisperse cell suspension. Then, an isotonic solution containing calcium carbonate nanoparticles, which had been pre-dispersed by ultrasound, was thoroughly mixed with the cell suspension and incubated at 37°C. Subsequently, the nanoparticles not adhering to the cell surface were removed by centrifugation to obtain a single-cell suspension with nanoparticles adhering to the cell surface. The single-cell suspension was resuspended in a dispersion phase at a certain concentration to obtain a dispersion phase solution containing cells.

[0092] The cells used here are derived from primary pancreatic ductal carcinoma tissue; the cell concentration during incubation was 10-1. 6 Cells / mL; Cell concentration in the dispersed phase: 10 4 Cells / mL; Nanoparticle diameter: 800 nm; Incubation concentration: 6 g / L; Incubation time between cells and nanoparticles: 25 min; Centrifugation force: 50 g; Centrifugation time: 2 min.

[0093] (4) Microfluidic manipulation: The reaction phase containing the crosslinking agent enters the microfluidic chip through inlet 2, and then reaches the intersection B13 along channel 3; the continuous phase enters the microfluidic chip through inlet 4, and passes through channel 5, intersection A12 and channel 9 to reach intersection B13; the dispersed phase containing cells enters the microfluidic chip through inlet 6, and passes through channel 7, normally closed pneumatic pump valve 8, intersection A12 and channel 9 to reach intersection B13; the external vacuum device is connected to the microfluidic chip through interface 1, and passes through interface 14 and channel 15 to reach the working area of ​​the normally closed pneumatic pump valve, periodically driving the 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.

[0094] The external vacuum level here 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; and the flow rate of the reaction phase containing the crosslinking agent is 20 μL / min.

[0095] (5) Generation of single-cell loaded microgels: The cell-containing dispersed phase droplets formed in (4) meet the reaction phase containing acetic acid at intersection B. Hydrogen ions in the reaction phase diffuse freely, passing through the intermediate continuous phase and entering the droplets. They then interact instantaneously with the nanoparticles adhering to the cell surface, releasing metal ions Ca. 2+ This causes the polymers in the dispersed phase to cross-link, forming cell-loaded microgels. Cell-free droplets, on the other hand, lack Ca2+. 2+ The released components cannot cross-link and can naturally dissolve in the surrounding solution during subsequent collection and transfer.

[0096] (6) High-throughput anticancer drug screening of pancreatic cancer organoids: The single-cell loaded microgels obtained in (5) were transferred to the corresponding cell culture medium and cultured and expanded under conventional cell culture conditions of 37°C and 5% CO2 for a long period of time, so that single cells developed into monoclonal cells and eventually formed pancreatic cancer organoids. The obtained organoids can be screened for high-throughput drug efficacy by adding 10 groups of gemcitabine drugs between 1-1000 nM in commercial well plates using an automated pipetting method. The tumor killing effect was evaluated based on CCK-8, cell viability test kits, etc., such as... Figure 8 As shown.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 drug screening method, characterized in that, Includes the following steps: S1. Fabrication of the microfluidic chip: A polydimethylsiloxane chip integrating a normally closed pneumatic pump valve was fabricated using soft photolithography. The polydimethylsiloxane chip comprises an upper and lower two-layer structure, wherein: The upper structure is the liquid path section, including: upper negative pressure interface (1), reaction phase inlet containing crosslinking agent (2), reaction phase channel containing crosslinking agent (3), continuous phase inlet (4), continuous phase channel (5), dispersed phase inlet containing cells (6), dispersed phase channel containing cells (7), normally closed pneumatic valve (8), droplet transport channel (9), microgel formation channel (10), microgel outlet (11), cross A (12), and cross B (13). The reaction phase inlet (2) containing the crosslinking agent is connected to two reaction phase channels (3) containing the crosslinking agent. The continuous phase inlet (4) is connected to two continuous phase channels (5). The cell-containing dispersed phase inlet (6) is connected to the cell-containing dispersed phase channel (7). The end of the cell-containing dispersed phase channel (7) first intersects with the two continuous phase channels (5) at intersection A (12), and then intersects with the two reaction phase channels (3) containing the crosslinking agent at intersection B (13), and then connects to the microgel forming channel (10). The microgel forming channel (10) has a serpentine structure, with a microgel outlet (11) at its end. A normally closed pneumatic valve (8) is provided on the cell-containing dispersed phase channel (7). The lower structure is the gas path section, including the lower negative pressure interface (14), gas passage (15) and the normally closed pneumatic pump valve working area (16). The lower negative pressure interface (14) is connected to the normally closed pneumatic pump valve operating area (16) through the gas channel (15). The upper and lower structures are used together. When in use, the lower negative pressure interface (14) is connected to the upper negative pressure interface (1). The position of the normally closed pneumatic valve action area (16) is adapted to the position of the normally closed pneumatic valve (8). S2. Preparation of aqueous two-phase solution: Dissolve polyethylene glycol in acetic acid solution as the reaction phase containing crosslinking agent, use polyethylene glycol isotonic solution as continuous phase, and dissolve sodium alginate and natural protein components in dextran isotonic solution as dispersion phase; the natural protein is one or more of collagen, laminin and fibroin, and the concentration of a single protein is 0.5-10 mg / mL; S3. Preparation of single-cell suspension: Two-dimensional adherent or three-dimensional cultured pancreatic cancer cells are digested by enzyme to obtain a monodisperse cell suspension. Then, an isotonic solution of calcium carbonate nanoparticles that have been pre-dispersed by ultrasound is thoroughly mixed with the cell suspension and incubated at room temperature or 37°C. Subsequently, the nanoparticles that are not attached to the cell surface are removed by centrifugation to obtain a single-cell suspension with nanoparticles attached to the surface. The single-cell suspension is resuspended in a dispersion phase at a certain concentration to obtain a dispersion phase solution containing cells. S4. Microfluidic manipulation: The reaction phase containing the crosslinking agent enters the microfluidic chip through the reaction phase inlet (2) containing the crosslinking agent, and then reaches the intersection B (13) along the reaction phase channel (3) containing the crosslinking agent. The continuous phase enters the microfluidic chip through the continuous phase inlet (4), and passes through the continuous phase channel (5), intersection A (12) and droplet transport channel (9) to reach the intersection B (13). The dispersed phase containing cells enters the microfluidic chip through the dispersed phase inlet (6) containing cells, and passes through the dispersed phase channel (7), normally closed pneumatic pump valve (8), intersection A (12) and droplet transport channel (9) to reach the intersection B (13). The external vacuum device is connected to the microfluidic chip through the upper negative pressure interface (1), and passes through the lower negative pressure interface (14) and gas channel (15) to reach the normally closed pneumatic valve action area (16), periodically driving the 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. S5. Generation of single-cell loaded microgels for pancreatic cancer: Cell-containing dispersed phase droplets meet the reaction phase containing a cross-linking agent at intersection B. Hydrogen ions in the reaction phase, due to free diffusion, pass through the intermediate continuous phase and enter the droplets, instantly interacting with nanoparticles adhering to the cell surface, releasing... Ions crosslink the sodium alginate in the dispersed phase, forming a microgel loaded with pancreatic cancer cells along with the remaining protein components, while containing cell-free droplets. The released components cannot cross-link and can naturally dissolve in the surrounding solution during subsequent collection and transfer. S6. High-throughput anticancer drug screening of pancreatic cancer organoids: Single cells loaded with microgels were transferred to the corresponding cell culture medium and cultured and expanded under conventional cell culture conditions of 37°C and 5% CO2 for a long period of time, thereby developing from single cells into monoclonal cells and finally forming pancreatic cancer organoids. The organoids were then screened for high-throughput efficacy by adding different concentrations of anticancer drugs in commercial well plates or microfluidic arrays using automated or manual pipetting methods. The tumor killing effect was then evaluated based on CCK-8 and cell liveness assay kits.

2. The drug screening method according to claim 1, characterized in that, The fabrication of microfluidic chips specifically includes: A template of SU-8 photoresist is prepared on a single-crystal silicon wafer or a dust-free glass plate. Then, PDMS prepolymer is poured onto the SU-8 photoresist template and crosslinked and polymerized by heating at 80°C for 1-3 hours to prepare a PDMS chip.

3. The drug screening method according to claim 1, characterized in that, In the preparation steps of an aqueous two-phase solution: Acetic acid concentration: 0.05-2% (v / v); The molecular weight of PEG is 8-20 kDa, and its concentration in the continuous phase and the reaction phase is 10%-30% (w / v). Isotonic solutions include PBS buffer or physiological saline. The molecular weight of dextran is 70-500 kDa, and the concentration is 10%-30% (w / v). Sodium alginate has a molecular weight of 1-10000 kDa and a concentration of 0.1-4% (w / v).

4. The drug screening method according to claim 1, characterized in that, In the preparation steps of single-cell suspension: The pancreatic cancer cells are one of the following: various commercial cell lines, cells derived from primary tumor tissue, and various stem cells; The cell concentration during incubation was: 10 5 -10 8 cells / mL; The cell concentration in the dispersed phase is: 10 3 -10 6 cells / mL; The diameter of the nanoparticles is 1-1000 nm, and the concentration used for incubation is 0.1-10 g / L; The incubation time between cells and nanoparticles is 5-60 min; Centrifugation operation: centrifugal force: 5-200g, time: 0.1-3min.

5. The drug screening method according to claim 1, characterized in that, In the microfluidic manipulation steps: The external vacuum level is: (-0.01) - (-0.1) MPa; The flow rate of the cell-containing dispersion was 0.01-5 μL / min; The continuous phase flow rate is 1-20 μL / min; The flow rate of the reaction phase containing the crosslinking agent is 1-20 μL / min.

6. The drug screening method according to claim 1, characterized in that, In the high-throughput anticancer drug screening process for pancreatic cancer organoids: The anticancer drugs are one or more of gemcitabine, erlotinib hydrochloride, olaparib, everolimus, and sunitinib, with a concentration of 1-10000 nM.

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