Preparation method and application of monodisperse thermosensitive hydrogel microspheres

Abstract

This invention discloses a method for preparing monodisperse temperature-sensitive hydrogel microspheres and their applications. The method utilizes the property that hydrophobic silica particles can effectively maintain the shape of water-in-oil droplets. Monodisperse water-in-oil droplets are prepared in a temperature-controlled microfluidic system within a solution of the temperature-sensitive hydrogel. Hydrophobic silica particles are pre-layered at the bottom of a container to receive the droplets. The microfluidically prepared droplets are then sequentially dropped into the container, which is then placed at a gelation temperature to gel, thus obtaining the monodisperse temperature-sensitive hydrogel microspheres. This method completely avoids the long-pipe heating step required in traditional preparation processes, solving problems such as droplet fusion, uneven spacing, long waiting times, and complex equipment. The resulting microspheres can be used for three-dimensional cell culture, organoid construction, and high-throughput drug screening, possessing significant biomedical application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for preparing and applying monodisperse temperature-sensitive hydrogel microspheres. Specifically, it involves utilizing the property that hydrophobic silica particles can effectively maintain the shape of water-in-oil droplets, and using a temperature-controlled microfluidic system to achieve in-situ gelation of shortened pipelines to prepare temperature-sensitive hydrogel microspheres, as well as their application in cell encapsulation, organoid culture, and drug screening. Background Technology

[0002] Hydrogel microspheres, as a three-dimensional cell culture carrier, can simulate the microenvironment of cells in vivo, showing great potential in tissue engineering, disease model construction, and drug screening. Thermosensitive hydrogels, such as Matrigel and collagen, are widely used to prepare cell-carrying microspheres due to their temperature-responsive gelation properties. Traditional methods for preparing thermosensitive hydrogel microspheres based on microfluidic technology typically require integrating a long heating zone to ensure that droplets gel before entering the collection device, thus maintaining their spherical shape and preventing fusion. For example, in the preparation of Matrigel microspheres, the equipment often needs to integrate heating pipes tens of meters long. This "long-pipe heating" mode has significant drawbacks: 1. Difficulty in droplet quality control: During long-distance transport and heating, droplets are prone to fusion, deformation, or breakage due to flow rate fluctuations or changes in pipe diameter, resulting in non-uniform particle size (high CV value); 2. 3. Poor system fault tolerance: Long pipelines are prone to blockage, and once blocked, the entire system needs to be shut down for cleaning, which is cumbersome; 4. Low process efficiency: The long waiting time from droplet generation to solidification is not conducive to rapid and high-throughput preparation; 5. Complex equipment: The integration of long pipelines makes the equipment bulky, making it difficult to achieve miniaturization and integration, which increases the cost of use and maintenance.

[0003] Currently, there is an urgent need in this field to research and develop a new method that can avoid heating through long pipelines, simplify the operation process, and at the same time prepare temperature-sensitive hydrogel microspheres with uniform particle size and good sphericity. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a method for preparing and applying temperature-sensitive hydrogel microspheres. This invention utilizes the property that hydrophobic silica particles can effectively maintain the shape of water-in-oil droplets. Monodisperse water-in-oil droplets are prepared in a temperature-controlled microfluidic system within a solution of the temperature-sensitive hydrogel (no emulsifier is required in the continuous phase). Hydrophobic silica particles are pre-layered at the bottom of a container to receive the water-in-oil droplets. The microfluidically prepared droplets are then sequentially dropped into the container, and the container is placed at a gelation temperature to allow gelation, thus obtaining monodisperse temperature-sensitive hydrogel microspheres. This method solves the problems of solidification of the temperature-sensitive hydrogel before droplet formation and addresses issues such as uneven droplet spacing, poor tolerance, long waiting time, and droplet fusion within the pipeline caused by gelation in complex long pipelines.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing monodisperse temperature-sensitive hydrogel microspheres, comprising the following steps: (1) Under temperature control, the cell suspension and colloidal solution are mixed evenly at a volume ratio of 1:2 to 2:1 to obtain a dispersed phase solution; (2) Coating the container for receiving water-in-oil droplets with polylysine aqueous solution, then adding an organic phase containing hydrophobic silica particles, and after standing, obtaining a receiving container with hydrophobic silica at the bottom. (3) The dispersed phase solution from step (1) is introduced into the dispersed phase inlet of the temperature-controlled microfluidic system, and the continuous phase is introduced into the continuous phase inlet of the temperature-controlled microfluidic system. Water-in-oil droplets are formed through the microfluidic system and dripped into the receiving container with hydrophobic silica at the bottom obtained in step (2) through the outlet of the microfluidic system to form monodisperse microdroplets. After solidification, hydrogel microspheres are obtained.

[0007] Based on the above technical solution, further, the cell types mentioned in step (1) include thyroid cells, breast cells, pancreatic cells, liver cells, thymocytes, lung cells, kidney cells, gastric cells, intestinal cells and their corresponding tumor cells.

[0008] Based on the above technical solution, further, the concentration of the cell suspension in step (1) is 10. 3 ~10 9 cell / mL.

[0009] Based on the above technical solution, the colloidal solution in step (1) further includes matrix gel, collagen solution, chitosan solution in low temperature solution state and gel state after heating, as well as agarose solution, agar solution, and carrageenan solution in high temperature solution state and gel state at room temperature.

[0010] Based on the above technical solution, further, the specific process of coating in step (2) is as follows: add a polylysine aqueous solution with a concentration of 0.01~1mg / mL into the well plate, incubate at 30~38℃ for 8~24h, discard the polylysine aqueous solution, continue incubation for 8~24h, and wash with PBS buffer 1~5 times before use.

[0011] Based on the above technical solution, further, the hydrophobic silica particles in step (2) include silanized hydrophobic modified silica particles and polydimethylsiloxaneized hydrophobic modified silica particles.

[0012] Based on the above technical solution, the silanized hydrophobically modified silica particles further include trimethylsilanized silica particles, perfluorosilanized silica particles, HMDS-modified silica particles, and dimethylsiloxy-modified silica particles, with a particle size of 7~200nm.

[0013] Based on the above technical solution, further, the solvent of the organic phase in step (2) includes n-hexane, low-boiling or high-boiling dimethyl silicone oil, undecane, dodecane, tridecane, electronic fluorinated liquid, soybean oil and corn oil, and the concentration of hydrophobic silica particles is 0.5~10 mg / mL.

[0014] Based on the above technical solution, further, the hydrogel microspheres described in step (3) are cultured in organoid culture medium to obtain organoids.

[0015] Based on the above technical solution, further, the inner diameter of the dispersed phase pipeline of the microfluidic system described in step (3) is 50~200 μm.

[0016] Based on the above technical solution, the continuous phase in step (3) further includes n-hexane, low-boiling-point or high-boiling-point dimethyl silicone oil, undecane, dodecane, tridecane, electronic fluorinated liquid, soybean oil and corn oil.

[0017] Based on the above technical solution, the temperature control in steps (1) and (3) further includes low temperature control and high temperature control. Low temperature control is achieved through an ice bath, cold table or refrigeration control box, and the low temperature is controlled at 0~9℃; high temperature control is achieved through an air bath or water bath, and the high temperature is controlled at 35~50℃.

[0018] Secondly, the present invention provides monodisperse thermosensitive hydrogel microspheres with particle size obtained by the above-described preparation method.

[0019] Thirdly, the present invention provides the application of the above-mentioned monodisperse thermosensitive hydrogel microspheres in three-dimensional cell culture, organoid construction, or drug screening.

[0020] Based on the above technical solution, the drug further includes an anti-tumor drug.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention utilizes the property that hydrophobic silica particles can effectively maintain the shape of water-in-oil droplets to solve the problems of easy blockage or bending of long pipelines, uneven droplet spacing, poor fault tolerance, long waiting time and droplet fusion in pipelines in the preparation of matrix glue hydrogel microspheres in the existing method. At the same time, the method of the present invention also has the advantage of not using surfactants in the droplet generation process.

[0022] (2) After the droplets prepared by the method of preparing matrix glue hydrogel microspheres of the present invention are solidified, the silica makes the hydrogel microspheres maintain a spherical shape and have good light transmittance during cell culture, avoiding the influence on cell growth during culture and making it easy to observe the internal cell growth behavior.

[0023] (3) The well plate coated with hydrophobic silica particles keeps the hydrogel microspheres suspended throughout the cell culture process, which has the advantage of fast mass transfer, can shorten the cell culture time, and save time for rapid clinical drug sensitivity tests. Attached Figure Description

[0024] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0025] Figure 1 This is a schematic diagram of the hydrogel microsphere preparation process.

[0026] Figure 2 The figure shows the effect of different concentrations of hydrophobic silica particles coating the perforated plate on the morphology of hydrogel droplets in Example 1.

[0027] Figure 3 The image shows the cell survival results of the cells in the hydrogel microspheres prepared in Examples 5-6 as the number of culture days increased.

[0028] Figure 4 The graph shows the sensitivity results of gemcitabine to the A549 lung cancer organoids in Example 8. Detailed Implementation

[0029] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0030] Example 1 This embodiment provides a method for preparing matrix adhesive hydrogel microspheres, such as... Figure 1As shown, it includes the following steps: (1) Coat a 96-well plate with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, incubate at 37°C for 12 h, remove the plate, aspirate the polylysine, and incubate for 12 h. Wash the plate 3 times with PBS before use. (2) Trimethylsilanediol dioxide particles (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) were dispersed in dimethyl silicone oil to prepare concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.5 mg / mL, 5 mg / mL and 10 mg / mL respectively. The particles were then ultrasonically treated to ensure uniform dispersion. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare the culture medium and resuspend the cell pellet to obtain a cell suspension (10). 6 (cell / mL) (5) The cell suspension and the commercially available matrix gel solution thawed on ice were mixed at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the matrix gel solution accounted for 50% of the total volume of the precursor solution. The mixing process was carried out on ice. (6) Draw the dispersed phase solution from step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 180 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate coated with hydrophobic silica obtained in step (3). The water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate, forming microdroplets, such as... Figure 2 As shown.

[0031] (10) Microdroplets are placed in an incubator at 37°C for 15 min to solidify, and matrix adhesive microspheres are obtained.

[0032] This embodiment also provides another method for preparing matrix adhesive hydrogel microspheres based on a perforated plate coated with polydimethylsiloxane-modified silica particles. The specific process is the same as the above method for preparing matrix adhesive hydrogel microspheres, except that in step (2), polydimethylsiloxane-modified silica particles (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) are dispersed in dimethyl silicone oil and prepared at concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.5 mg / mL, 5 mg / mL and 10 mg / mL, respectively. The particles are then ultrasonically treated to ensure uniform dispersion.

[0033] Example 2 This embodiment provides a method for preparing type I collagen hydrogel microspheres, such as... Figure 1 As shown, it includes the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare the culture medium and resuspend the cell pellet to obtain a cell suspension (10). 6 (cell / mL) (5) Mix the cell suspension with a type I collagen solution (solvent is water, concentration is 5 mg / mL) adjusted to pH 7 on ice and mix them evenly at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the type I collagen solution should be 50% of the total volume of the precursor solution. The mixing process should be carried out on ice. (6) Draw the dispersed phase solution from step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 180 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3). The water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate to form microdroplets. (10) After the microdroplets are placed in an incubator at 37°C for 15 min to solidify, type I collagen microspheres can be obtained.

[0034] Example 3 This embodiment provides a method for preparing agarose hydrogel microspheres, such as... Figure 1 As shown, it includes the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare the culture medium and resuspend the cell pellet to obtain a cell suspension (10). 6 (cell / mL) (5) The cell suspension and the liquid agarose solution (concentration of 1%) that has been heated in a microwave oven until there is no flocculent matter and then cooled to 37°C are mixed evenly at a volume ratio of 1:1. The volume of the agarose solution accounts for 50% of the total volume of the precursor solution. The mixing process is carried out in a metal bath at 37°C. (6) Draw the precursor solution of the hydrogel microspheres in step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a heating module; (7) Connect a stainless steel capillary tube with an inner diameter of 180 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3). The water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate to form microdroplets. (10) After the microdroplets are solidified at room temperature for 15 min, agarose microspheres can be obtained.

[0035] Example 4 This embodiment provides a method for preparing matrix adhesive hydrogel microspheres for breast cancer organoids, including the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare breast cancer organoid culture medium and resuspend the breast cancer MDA-MB-231 cell pellet to obtain a cell suspension (10 6 (cell / mL) (5) The cell suspension and the thawed matrix gel solution on ice were mixed at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the matrix gel solution should be 50% of the total volume of the precursor solution. The mixing process should be carried out on ice. (6) Draw the dispersed phase solution from step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 180 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3). The water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate to form microdroplets. (10) After the microdroplets are placed in an incubator at 37°C for 15 min to solidify, breast cancer matrix microspheres can be obtained; (11) Add the microspheres from step (10) to the breast cancer organoid culture medium and incubate them in an incubator for 5 days. You can see that breast cancer organoids are initially formed in the microspheres.

[0036] Example 5 This embodiment provides a method for preparing matrix adhesive hydrogel microspheres for breast cancer organoids, including the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare breast cancer organoid culture medium and resuspend the breast cancer MDA-MB-231 cell pellet to obtain a cell suspension (10 6 (cell / mL) (5) The cell suspension and the thawed matrix gel solution on ice were mixed at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the matrix gel solution should be 50% of the total volume of the precursor solution. The mixing process should be carried out on ice. (6) Draw the dispersed phase solution from step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 60 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3), and the water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate. (10) Microdroplets are placed in an incubator at 37°C for 15 minutes and then solidified to obtain breast cancer matrix microspheres; (11) Add the microspheres from step (10) to the breast cancer organoid culture medium and incubate them in an incubator for 5 days. Preliminary formation of breast cancer organoids can be observed in the microspheres. After staining with live and dead cells, the results are shown in the figure. Figure 3 The results showed that the cells in the breast cancer organoids prepared by this method had a very high survival rate.

[0037] Example 6 This embodiment provides a method for preparing lung cancer organoid matrix glue hydrogel microspheres, including the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare lung cancer organoid culture medium and resuspend lung cancer A549 cell pellet to obtain cell suspension (10 6 (cell / mL) (5) The cell suspension and the thawed matrix gel solution on ice were mixed at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the matrix gel solution should be 50% of the total volume of the precursor solution. The mixing process should be carried out on ice. (6) Draw the precursor solution of the hydrogel microspheres in step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 60 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3), and the water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate. (10) After the microdroplets are placed in an incubator at 37°C for 15 minutes to solidify, lung cancer matrix microspheres can be obtained.

[0038] (11) Add the microspheres from step (10) to the lung cancer organoid culture medium and incubate them in an incubator for 5 days. Preliminary formation of lung cancer organoids can be observed in the microspheres. After staining with live and dead cells, the results are shown in the figure. Figure 3 The results showed that the lung cancer organoids prepared by this method had a very high cell survival rate.

[0039] Example 7 This embodiment provides a method for preparing colorectal cancer hydrogel microspheres, including the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare colon cancer organoid culture medium and resuspend the cell pellet to obtain a cell suspension (10). 6 (cell / mL) (5) The cell suspension and the thawed matrix gel solution on ice were mixed at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the matrix gel solution should be 50% of the total volume of the precursor solution. The mixing process should be carried out on ice. (6) Draw the dispersed phase solution from step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 60 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous phase, connect the tubing, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3), and the water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate. (10) After the microdroplets are placed in an incubator at 37°C for 15 min to solidify, colorectal cancer matrix microspheres can be obtained; (11) Add the microspheres from step (10) to the colorectal cancer organoid culture medium and incubate them in an incubator for 5 days. You can see that colorectal cancer organoids are initially formed in the microspheres.

[0040] Example 8 This embodiment provides a method for preparing lung cancer matrix glue hydrogel microspheres and applying them to tumor drug screening, including the following steps: (1) Take a 96-well plate, coat it with 0.1 mg / mL polylysine aqueous solution, add 100 μL to each well, place it in an incubator at 37°C for 12 h, take it out, aspirate the polylysine and place it in an incubator for 12 h, wash it with PBS 3 times before use. (2) Disperse trimethylsilanediol dioxide particles in dimethyl silicone oil at a concentration of 5 mg / mL, and sonicate to make the particles uniformly dispersed. (3) Take the suspension from step (2) and add 50 μL to each well of the polylysine-coated plate obtained in step (1), and let it stand for 10 minutes. (4) Prepare lung cancer organoid culture medium and resuspend lung cancer A549 cell pellet to obtain cell suspension (10 6 (cell / mL) (5) The cell suspension and the thawed matrix gel solution on ice were mixed at a volume ratio of 1:1 to prepare a dispersed phase. The volume of the matrix gel solution should be 50% of the total volume of the precursor solution. The mixing process should be carried out on ice. (6) Draw the dispersed phase solution from step (5) into a 1 mL syringe and install the syringe into a micro-injection pump with a cooling module; (7) Connect a stainless steel capillary tube with an inner diameter of 60 μm to the head of a 1 mL syringe, set the flow rate to 1 μL / min, and connect it to the dispersed phase inlet of the microfluidic system. (8) Draw dimethyl silicone oil into a 20ml syringe as a continuous connection pipeline, set the flow rate to 100μL / min, and connect it to the continuous phase inlet of the microfluidic system; (9) Move the outlet of the capillary of the microfluidic system above the well plate covered with hydrophobic silica obtained in step (3). The water-in-oil hydrogel microdroplets prepared by the microfluidic system fall and suspend in the well plate to form microdroplets. After repeating the operation, 3 hydrogel microspheres are distributed in each well. (10) After the microdroplets were placed in an incubator at 37°C for 15 min to solidify, lung cancer matrix microspheres were obtained; (11) Add the microspheres from step (10) to the lung cancer organoid culture medium and incubate them in an incubator for 5 days. You can see that lung cancer organoids are initially formed in the microspheres. (12) Prepare a series of gemcitabine drug culture media of different concentrations, replace the old culture media with drug-containing culture media, incubate in an incubator for 72 hours, add CellTiter-Glo 3D for cell viability assay, and the results are shown in the figure. Figure 4 .

[0041] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; modifications may be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some or all of the technical features therein; however, such 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 method for preparing monodisperse thermosensitive hydrogel microspheres, characterized in that, Includes the following steps: (1) Under temperature control, the cell suspension and colloidal solution are mixed evenly at a volume ratio of 1:2 to 2:1 to obtain a dispersed phase solution; (2) Coating the container for receiving water-in-oil droplets with polylysine aqueous solution, then adding an organic phase containing hydrophobic silica particles, and after standing, obtaining a receiving container with hydrophobic silica at the bottom. (3) The dispersed phase solution from step (1) is introduced into the dispersed phase inlet of the temperature-controlled microfluidic system, and the continuous phase is introduced into the continuous phase inlet of the temperature-controlled microfluidic system. Water-in-oil droplets are formed through the microfluidic system and dripped into the receiving container with hydrophobic silica at the bottom obtained in step (2) through the outlet of the microfluidic system to form monodisperse microdroplets. After solidification, hydrogel microspheres are obtained.

2. The preparation method according to claim 1, characterized in that, The cell types mentioned in step (1) include thyroid cells, breast cells, pancreatic cells, liver cells, thymocytes, lung cells, kidney cells, gastric cells, intestinal cells, and their corresponding tumor cells; the concentration of the cell suspension is 10. 3 ~10 9 cell / mL.

3. The preparation method according to claim 1, characterized in that, The colloidal solution mentioned in step (1) includes matrix gel, collagen solution, chitosan solution in low temperature solution state and gel state after heating, as well as agarose solution, agar solution, and carrageenan solution in high temperature solution state and gel state at room temperature.

4. The preparation method according to claim 1, characterized in that, The specific process of coating in step (2) is as follows: add a polylysine aqueous solution with a concentration of 0.01~1 mg / mL into the well plate, incubate at 30~38℃ for 8~24 h, discard the polylysine aqueous solution, continue incubation for 8~24 h, and wash with PBS buffer 1~5 times before use.

5. The preparation method according to claim 1, characterized in that, The hydrophobic silica particles mentioned in step (2) include silanized hydrophobic modified silica particles and polydimethylsiloxane-modified hydrophobic modified silica particles; the solvent of the organic phase includes n-hexane, dimethyl silicone oil, undecane, dodecane, tridecane, electronic fluorinated liquid, soybean oil and corn oil, and the concentration of the hydrophobic silica particles is 0.5~10 mg / mL.

6. The preparation method according to claim 1, characterized in that, The hydrogel microspheres described in step (3) are cultured in organoid culture medium to obtain organoids.

7. The preparation method according to claim 1, characterized in that, The inner diameter of the dispersed phase pipeline of the microfluidic system in step (3) is 50~200 μm; the continuous phase includes n-hexane, dimethyl silicone oil, undecane, dodecane, tridecane, electronic fluorinated liquid, soybean oil and corn oil.

8. The preparation method according to claim 1, characterized in that, The temperature control described in steps (1) and (3) includes low temperature control and high temperature control. Low temperature control is achieved through an ice bath, cold table or refrigeration control box, with the low temperature controlled at 0~9℃; high temperature control is achieved through an air bath or water bath, with the high temperature controlled at 35~50℃.

9. Monodisperse thermosensitive hydrogel microspheres with particle size prepared by the preparation method according to any one of claims 1-8.

10. The application of the monodisperse thermosensitive hydrogel microspheres of claim 9 in three-dimensional cell culture, organoid construction, or drug screening.

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