A novel method for high-throughput printing and culturing of organoids

Abstract

The present invention discloses a novel method for high-throughput printing and culturing of organoids, comprising the following steps; S2: encapsulate cells in dispersed phase 1; S4: inject the dispersed phase 1 encapsulating cells and dispersed phase 2 into the organoid precursor preparation area of the microfluidic system respectively, and the dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized organoid precursor liquid droplets, and the sheared organoid precursor liquid droplets can sequentially enter the organoid droplet ripening area of the microfluidic system and can ripen into organoid spheres in the organoid droplet ripening area; S6: use the delivery pump of the microfluidic system to deliver the first organoid sphere in the organoid droplet ripening area to the printing nozzle of the 3D printing system through the delivery pipeline of the 3D printing system; S8: use the organoid recognition and detection unit of the 3D printing system to detect whether there is an organoid sphere at the printing nozzle. The present invention has good controllability, ensures the consistency of organoids, the organoids are of uniform size, and reduces the differences between individual organoids.

Description

Technical Field

[0001] The present invention relates to the field of medical devices, and specifically, to a novel method for high-throughput printing and culturing of organoids. Background Art

[0002] The culture of primary organoids contributes to the research and development of anti-cancer drugs and the screening of precise drug use for cancer patients.

[0003] Currently, the culture method of primary organoids, such as tumor organoids, is mainly divided into two steps: First, the preparation of tumor organoids, which usually depends on the skills of laboratory technicians. Second, the laboratory technician manually transfers the prepared tumor organoids into the wells of a high-throughput culture carrier, such as a well plate, for culture. This process is time-consuming and laborious, the operation process is uncontrollable, the sizes of the prepared organoids are not uniform, the consistency of the organoids cannot be guaranteed, and the differences between individual organoids are relatively large. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a novel method for high-throughput printing and culturing of organoids, which has good controllability, saves time and effort, has good stability, ensures the consistency of the organoids, and reduces the differences between individual organoids.

[0005] The technical solution adopted by the present invention to solve its technical problems is:

[0006] A novel method for high-throughput printing and culturing of organoids, comprising the following steps: S2, encapsulating cells in dispersed phase 1; S4, injecting the dispersed phase 1 encapsulating cells and dispersed phase 2 into the organoid precursor preparation area of the microfluidic system respectively. The dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized organoid precursor liquid droplets, and the sheared organoid precursor liquid droplets can sequentially enter the organoid droplet ripening area of the microfluidic system and can ripen into organoid spheres in this organoid droplet ripening area; S6, transporting the first organoid sphere in the organoid droplet ripening area to the printing nozzle of the 3D printing system through the transport pipeline of the 3D printing system by means of the transport pump of the microfluidic system; S8, detecting whether there is an organoid sphere at the printing nozzle through the organoid recognition and detection unit of the 3D printing system. When the organoid recognition and detection unit detects an organoid sphere at the printing nozzle, the organoid recognition and detection unit outputs an organoid signal to the control unit of the 3D printing system; S10, controlling the action of the driving unit of the 3D printing system through the control unit. By driving the printing nozzle to move down to the liquid surface, bottom, junction of the continuous phase and the bottom in the first hole of the well plate or inside the continuous phase, after staying for 1 - 2 seconds, the first organoid sphere will adsorb on the liquid surface, bottom, junction of the continuous phase and the bottom in the first hole of the well plate or inside the continuous phase. Thus, the first organoid sphere is printed into the first hole of the well plate. After completion, the driving unit drives the printing nozzle back to the initial position; S12, sequentially printing the remaining organoid spheres in the organoid droplet ripening area into the remaining holes of the well plate according to the method of steps S6 to S10; S14, culturing the organoid spheres in all the holes of the well plate.

[0007] As a preferred technical solution, the organoid precursor preparation area is a Y-shaped structure. The Y-shaped structure includes a first inlet pipeline, a second inlet pipeline and a connecting pipeline. The first ends of the first inlet pipeline and the second inlet pipeline are respectively connected to a first syringe and a second syringe. The first syringe is connected with a first injection pump, and the second syringe is connected with a second injection pump. The second ends of the first inlet pipeline and the second inlet pipeline are both connected to the first end of the connecting pipeline. The organoid droplet ripening area is an outlet pipeline, and the second end of the connecting pipeline is connected to the outlet pipeline; The step S4 includes the following steps; S42, placing the dispersed phase 1 and the dispersed phase 2 in the first syringe and the second syringe respectively; S44, injecting the dispersed phase 1 in the first syringe into the first inlet pipeline through the first injection pump, and injecting the dispersed phase 2 in the second syringe into the second inlet pipeline through the second injection pump. In the connecting pipeline, the dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized organoid precursor liquid droplets, and the sheared organoid precursor liquid droplets can sequentially enter the outlet pipeline and can ripen into organoid spheres in this outlet pipeline.

[0008] As a preferred technical solution, the organoid precursor preparation area is a T-shaped structure. The T-shaped structure includes a first inlet pipe, a second inlet pipe, and a connecting pipe. The first end of the first inlet pipe and the first end of the second inlet pipe are respectively connected to a first syringe and a second syringe. The first syringe is connected to a first injection pump, and the second syringe is connected to a second injection pump. The second end of the first inlet pipe and the second end of the second inlet pipe are respectively connected to the first end of the connecting pipe. The organoid droplet ripening area is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe; the step S4 includes the following steps: S42, placing the dispersed phase 1 and the dispersed phase 2 in the first syringe and the second syringe respectively; S44, injecting the dispersed phase 1 in the first syringe into the first inlet pipe through the first injection pump, and injecting the dispersed phase 2 in the second syringe into the second inlet pipe through the second injection pump. In the connecting pipe, the dispersed phase 1 is sheared into uniformly sized organoid precursor fluid droplets by the dispersed phase 2. The sheared organoid precursor fluid droplets can sequentially enter the outlet pipe and ripen into organoid spheres in the outlet pipe.

[0009] As a preferred technical solution, the organoid precursor preparation area is a cross-shaped structure. The cross-shaped structure includes a first inlet pipe, two second inlet pipes arranged oppositely, and a connecting pipe. The first inlet pipe and the connecting pipe are arranged oppositely. The first end of the first inlet pipe is connected to a first syringe, and the second end of the first inlet pipe is connected to the first end of the connecting pipe. The first syringe is connected to a first injection pump. The first ends of the two second inlet pipes are respectively connected to two second syringes, and the second ends of the two second inlet pipes are respectively connected to the first end of the connecting pipe. The two second syringes are respectively connected to two second injection pumps. The organoid droplet ripening area is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe; the step S4 includes the following steps: S42, placing the dispersed phase 1 in the first syringe and placing the dispersed phase 2 in the two second syringes respectively; S44, injecting the dispersed phase 1 in the first syringe into the first inlet pipe through the first injection pump, and injecting the dispersed phase 2 in the two second syringes into the two second inlet pipes respectively through the two second injection pumps. In the connecting pipe, the dispersed phase 1 is sheared into uniformly sized organoid precursor fluid droplets by the dispersed phase 2 injected into the two second inlet pipes. The sheared organoid precursor fluid droplets can sequentially enter the outlet pipe and ripen into organoid spheres in the outlet pipe.

[0010] As a preferred technical solution, in step S10, the driving unit of the 3D printing system drives the printing nozzle to move downward to the bottom inside the first hole position of the orifice plate. After staying for 1 - 2 seconds, the first organoid sphere will adsorb to the bottom inside the first hole position. Before step S14, there is also step S13: adding a continuous phase into all the hole positions of the orifice plate.

[0011] As a preferred technical solution, in step S10, the driving unit of the 3D printing system drives the printing nozzle to move downward to the liquid level of the continuous phase inside the first hole position of the orifice plate. After staying for 1 - 2 seconds, the first organoid sphere will adsorb to the liquid level of the continuous phase inside the first hole position. The printing nozzle is wedge-shaped.

[0012] As a preferred technical solution, the wedge angle of the printing nozzle is 10 - 80 degrees.

[0013] As a preferred technical solution, in step S2, the cells are primary tumor tissue cells, stem cells or cell lines.

[0014] As a preferred technical solution, the material of the dispersed phase 1 includes Matrigel, collagen or gelatin, and the material of the dispersed phase 2 includes fluorinated oil or vegetable oil.

[0015] As a preferred technical solution, the continuous phase is a normal culture medium, a culture medium containing growth factors, a culture medium containing drugs or Dulbecco's phosphate buffered saline.

[0016] The beneficial effects of the present invention are as follows: The novel organoid high-throughput printing and culturing method provided by the present invention completes the preparation of organoids through a microfluidic system, and combines the microfluidic system with the 3D printing system, which can simply, quickly and accurately transfer the organoids into the hole positions of the orifice plate by printing for culturing. It has good controllability, saves time and effort, has good stability, the organoids are of uniform size, ensuring the consistency of the organoids and reducing the differences between individual organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below in conjunction with the drawings and embodiments.

[0018] Figure 1 is a flowchart of a novel organoid high-throughput printing and culturing method provided by the first embodiment of the present invention;

[0019] Figure 2 is Figure 1 the flowchart diagram of a novel organoid high-throughput printing and culturing method shown;

[0020] Figure 3 is a schematic diagram of extracting primary tumor tissue cells from primary tumor tissue;

[0021] Figure 4 It is a schematic structural diagram of the first syringe and the second syringe;

[0022] Figure 5 It is a schematic plan view of the organoid precursor preparation area and the organoid droplet maturation area of the first solution of the microfluidic system;

[0023] Figure 6 It is a schematic plan view of the organoid precursor preparation area and the organoid droplet maturation area of the second solution of the microfluidic system;

[0024] Figure 7 It is a schematic plan view of the organoid precursor preparation area and the organoid droplet maturation area of the third solution of the microfluidic system;

[0025] Figure 8 It is a schematic plan view of the organoid precursor preparation area and the organoid droplet maturation area of the fourth solution of the microfluidic system;

[0026] Figure 9 It is a schematic structural diagram of the continuous phase liquid level where the organoid sphere is printed into the pore position of the well plate;

[0027] Figure 10 It is a schematic diagram of the effect from the first day to the sixth day after the organoid sphere is cultured;

[0028] Figure 11 It is a flowchart of a novel organoid high-throughput printing and culturing method provided by the second embodiment of the present invention;

[0029] Figure 12 It is Figure 11 The flowchart of the novel organoid high-throughput printing and culturing method shown;

[0030] Figure 13 It is a schematic structural diagram of the bottom of the pore position where the organoid sphere is printed into the well plate;

[0031] Figure 14 It is a schematic diagram of the effect from the first day to the sixth day after the organoid sphere is cultured;

[0032] Figure 15 It is a flowchart of a novel organoid high-throughput printing and culturing method provided by the third embodiment of the present invention;

[0033] Figure 16 It is Figure 15 The flowchart of the novel organoid high-throughput printing and culturing method shown;

[0034] Figure 17 It is a flowchart of a novel organoid high-throughput printing and culturing method provided by the fourth embodiment of the present invention;

[0035] Figure 18 is Figure 17 a schematic flow chart of a new type of organoid high-throughput printing and culturing method as shown Specific implementation manners

[0036] The concept, specific structure and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the drawings, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention. In addition, all the connection / connection relationships involved in the patent do not simply refer to the direct connection of components, but refer to the more optimal connection structure that can be formed by adding or reducing connection accessories according to the specific implementation situation. The various technical features in the present invention can be combined with each other without conflicting with each other.

[0037] The first embodiment

[0038] Please refer to Figure 1 and Figure 2 A new type of organoid high-throughput printing and culturing method provided by the first embodiment of the present invention includes the following steps:

[0039] S2. Wrap the cells in the dispersed phase 1.

[0040] In step S2, the cells are primary tumor tissue cells extracted from the primary tumor tissue, as Figure 3 shown. It can be understood that the cells can also be, for example, stem cells, cell lines, etc., and the type and quantity of the cells can be set according to the actual situation.

[0041] In this embodiment, the cell density in the dispersed phase 1 is 1×10 7 cells / ml. The cell densities compatible with the present invention include but are not limited to 1×10 5 cells / ml to 1×10 9 cells / ml, and can be flexibly adjusted according to the culture requirements and cell viability.

[0042] The material of the dispersed phase 1 includes Matrigel. It can be understood that the material of the dispersed phase 1 can also be natural materials including, for example, collagen, gelatin, etc., and the material of the dispersed phase 1 can also be synthetic materials, which can be set according to the actual situation.

[0043] S4. Inject the disperse phase 1 and disperse phase 2 wrapped with cells into the organoid precursor preparation area of the microfluidic system respectively. The disperse phase 1 is sheared by the disperse phase 2 into uniform-sized organoid precursor liquid droplets 100. The outside of the organoid precursor liquid droplets 100 is wrapped with a part of the disperse phase 2. The sheared organoid precursor liquid droplets 100 can successively enter the organoid droplet maturation area of the microfluidic system and can mature into organoid spheres 102 in this organoid droplet maturation area, and the organoids are thus prepared.

[0044] In step S4, the material of the disperse phase 2 includes fluorinated oil. Fluorinated oil can better produce droplet phenomena and has better shear force, enabling the disperse phase 1 wrapped with cells to form uniform-sized organoid precursor liquid droplets 100. Understandably, the disperse phase 2 can also be a material such as vegetable oil that has good compatibility with cells and is immiscible with water, and can be set according to actual situations.

[0045] The method of maturing the organoid precursor liquid droplets 100 into organoid spheres 102 includes methods such as temperature crosslinking method, enzymatic crosslinking method, ionic crosslinking method, photo-crosslinking method, and so on.

[0046] In this embodiment, the organoid precursor preparation area is a T-shaped structure, as shown in Figure 5 . The T-shaped structure includes a first inlet pipe 42, a second inlet pipe 43, and a connecting pipe 44. The first end of the first inlet pipe 42 and the first end of the second inlet pipe 43 are respectively connected to the first syringe 32 (see Figure 4 ), the second syringe 34 (see Figure 4 ). The first syringe 32 is connected with a first injection pump, the second syringe 34 is connected with a second injection pump, and the second end of the first inlet pipe 42 and the second end of the second inlet pipe 43 are respectively connected to the first end of the connecting pipe 44. The axis of the first inlet pipe 42 and the axis of the connecting pipe 44 are on the same horizontal line, and the second inlet pipe 43 is perpendicularly arranged with respect to the first inlet pipe 42 and the connecting pipe 44. The second inlet pipe 43 is located above the first inlet pipe 42 and the connecting pipe 44. The organoid droplet maturation area is an outlet pipe 50, and the second end of the connecting pipe 44 is connected to the outlet pipe 50. The connecting pipe 44 and the outlet pipe 50 are connected as a whole.

[0047] Step S4 specifically includes the following steps:

[0048] S42. Place the disperse phase 1 and the disperse phase 2 into the first syringe 32 and the second syringe 34 respectively.

[0049] S44. Inject the dispersed phase 1 in the first syringe 32 into the first inlet pipe 42 through the first syringe pump, and inject the dispersed phase 2 in the second syringe 34 into the second inlet pipe 43 through the second syringe pump. In the connecting pipe 44, the dispersed phase 1 is sheared into uniformly sized precursor organoid fluid droplets 100 by the dispersed phase 2. The sheared precursor organoid fluid droplets 100 can sequentially enter the outlet pipe 50 and can mature into organoid spheres 102 in the outlet pipe 50, as Figure 2 and Figure 5 shown.

[0050] In this embodiment, the flow rate of the dispersed phase 1 is usually set to 20 μL / min, and the flow rate of the dispersed phase 2 is usually set to 120 μL / min. The present invention can adjust the flow rates and the flow rate ratio of the two dispersed phases according to requirements, thereby adjusting the shape of the generated precursor organoid fluid droplets 100 (for example, circular or oval with unequal lengths), the spacing between the precursor organoid fluid droplets 100, and the overall speed of preparing the precursor organoid fluid droplets 100.

[0051] In the first alternative, as Figure 6 shown, the precursor organoid preparation area is also a T-shaped structure. Different from the Figure 5 precursor organoid preparation area, the axes of the first inlet pipe 42 and the second inlet pipe 43 are on the same horizontal line, the connecting pipe 44 is perpendicular to the first inlet pipe 42 and the second inlet pipe 43, and the connecting pipe 44 is located below the first inlet pipe 42 and the second inlet pipe 43.

[0052] In the second alternative, as Figure 7 shown, the precursor organoid preparation area is a Y-shaped structure. The Y-shaped structure includes the first inlet pipe 42, the second inlet pipe 43, and the connecting pipe 44. The first end of the first inlet pipe 42 and the first end of the second inlet pipe 43 are respectively connected to the first syringe 32 and the second syringe 34. The second ends of the first inlet pipe 42 and the second inlet pipe 43 are both connected to the first end of the connecting pipe 44. The first inlet pipe 42 and the second inlet pipe 43 form a V-shaped structure, and the connecting pipe 44 is located below the first inlet pipe 42 and the second inlet pipe 43. The organoid droplet maturation area is an outlet pipe 50, and the second end of the connecting pipe 44 is connected to the outlet pipe 50. The connecting pipe 44 and the outlet pipe 50 are connected as a whole.

[0053] Step S4 includes the following steps:

[0054] S42. Place the dispersed phase 1 and the dispersed phase 2 in the first syringe 32 and the second syringe 34 respectively;

[0055] S44. Inject the dispersed phase 1 in the first syringe 32 into the first inlet pipe 42 through the first syringe pump, and inject the dispersed phase 2 in the second syringe 34 into the second inlet pipe 43 through the second syringe pump. In the connecting pipe 44, the dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized precursor organoid fluid droplets 100. The sheared precursor organoid fluid droplets 100 can sequentially enter the outlet pipe 50 and can mature into organoid spheres 102 in the outlet pipe 50.

[0056] In the third alternative, as Figure 8 shown, the precursor organoid preparation area is a cross-shaped structure. The cross-shaped structure includes a first inlet pipe 42, two second inlet pipes 43 arranged oppositely, and a connecting pipe 44. The first inlet pipe 42 and the connecting pipe 44 are arranged oppositely. The first end of the first inlet pipe 42 is connected to the first syringe 32, and the second end of the first inlet pipe 42 is connected to the first end of the connecting pipe 44. The first syringe 32 is connected to a first syringe pump. The first ends of the two second inlet pipes 43 are respectively connected to the two second syringes 34, and the second ends of the two second inlet pipes 43 are respectively connected to the first end of the connecting pipe 44. The two second syringes 34 are respectively connected to two second syringe pumps. The organoid droplet maturation area is an outlet pipe 50, and the second end of the connecting pipe 44 is connected to the outlet pipe 50. The connecting pipe 44 and the outlet pipe 50 are integrally connected.

[0057] Step S4 specifically includes the following steps:

[0058] S42. Place the dispersed phase 1 in the first syringe 32, and place the dispersed phase 2 in the two second syringes 34 respectively;

[0059] S44. Inject the dispersed phase 1 in the first syringe 32 into the first inlet pipe 42 through the first syringe pump, and inject the dispersed phase 2 in the two second syringes 34 into the two second inlet pipes 43 through the two second syringe pumps respectively. In the connecting pipe 44, the dispersed phase 1 is sheared by the dispersed phase 2 injected into the two second inlet pipes 43 into uniformly sized precursor organoid fluid droplets 100. The sheared precursor organoid fluid droplets 100 can sequentially enter the outlet pipe 50 and can mature into organoid spheres 102 in the outlet pipe.

[0060] S6. Through the delivery pump of the microfluidic system, deliver the first organoid sphere 102 in the organoid droplet maturation area to the printing nozzle 62 of the 3D printing system through the delivery pipe 52 of the 3D printing system.

[0061] In step S6, the printing nozzle 62 is wedge-shaped, and the wedge angle of the printing nozzle 62 is 10 - 80 degrees, preferably 45 degrees.

[0062] The 3D printing system is a 3D bioprinter.

[0063] S8. The organoid recognition and detection unit 64 of the 3D printing system detects whether there is an organoid sphere 102 at the printing nozzle 62. When the organoid recognition and detection unit 64 detects an organoid sphere 102 at the printing nozzle 62, the organoid recognition and detection unit 64 outputs an organoid signal to the control unit of the 3D printing system.

[0064] In step S8, the organoid recognition and detection unit 64 is opposite to the printing nozzle 62. As Figure 2 shown, the organoid recognition and detection unit 64 detects, for example, the color, geometric contour, fluorescence signal, etc. of the organoid sphere 102, so as to detect that there is an organoid sphere 102 at the printing nozzle 62. The organoid recognition and detection unit 64 is, for example, a CCD camera.

[0065] S10. The control unit controls the driving unit of the 3D printing system to act, and drives the printing nozzle 62 to move downward to the liquid surface of the continuous phase in the first hole position 72 of the well plate 70. Since the printing nozzle 62 is wedge-shaped, the wedge-shaped printing nozzle 62 can break the surface tension of the continuous phase liquid surface. After staying for 1 - 2 seconds, preferably 1 second, under the adhesion of the first organoid sphere 102 and the continuous phase, the first organoid sphere 102 will adsorb on the liquid surface of the continuous phase in the first hole position 72, forming an air-liquid culture interface of the organoid. Thus, the printing of the air-liquid interface of the first organoid sphere 102 is realized, and the first organoid sphere 102 is printed into the first hole position 72 of the well plate 70. As Figure 9 shown, after completion, the driving unit drives the printing nozzle 62 to move upward to return to the initial position, and the initial position is the position where the printing nozzle 62 is opposite to the organoid recognition and detection unit 64.

[0066] In this embodiment, the moving speed range of the printing nozzle 62 is generally 0 - 100 mm / s (millimeters per second) for the XY axis, and the Z-axis speed is 0 - 80 mm / s. The moving speed and acceleration of the printing nozzle 62 can be adjusted flexibly in real time according to the printing speed requirements and accuracy requirements.

[0067] In step S10, the continuous phase is preferably a normal culture medium. It can be understood that the continuous phase can also be, for example, a culture medium containing growth factors, a culture medium containing drugs, Dulbecco's phosphate buffer solution, other special culture media, etc., which can be set according to the actual situation.

[0068] S12. Sequentially print the remaining organoid spheres 102 in the organoid droplet maturation area into the remaining well positions 72 of the well plate 70 according to the method of steps S6 to S10, thus completing the high-throughput printing of the organoid air-liquid interface. In this embodiment, the number of well positions 72 of the well plate 70 is 96. Understandably, the number of well positions 72 of the well plate 70 can be set according to the actual situation.

[0069] S14. Cultivate the organoid spheres 102 in all well positions 72 of the well plate 70. The organoid spheres 102 are stably cultured at the liquid surface of the continuous phase.

[0070] In step S14, when cultivating the organoid spheres 102, since the material of the dispersed phase 2 includes fluorinated oil, the dispersed phase 2 will volatilize by itself.

[0071] Figure 10 It is a schematic diagram of the effect from the first day to the sixth day after cultivating the organoid spheres 102. As can be seen from Figure 10 it, the organoids have good consistency and small differences among individuals.

[0072] The novel organoid high-throughput printing and cultivation method provided by the present invention completes the preparation of organoids through a microfluidic system, and combines the microfluidic system with a 3D printing system, which can simply, quickly, and accurately transfer the organoids to the liquid surface of the continuous phase in the well positions 72 of the well plate 70 by printing for cultivation. It has good controllability, saves time and effort, has good stability, the organoids are of uniform size, ensures the consistency of the organoids, and reduces the differences among organoid individuals.

[0073] Second Embodiment

[0074] Please refer to Figure 11 and Figure 12 . Steps S2 to S8 and step S14 of this embodiment are the same as those of the first embodiment and will not be elaborated here. The difference between this embodiment and the first embodiment is that in step S10, the control unit controls the driving unit of the 3D printing system to act, and the driving unit drives the printing nozzle 62 to move downward to the bottom of the first well position 72 of the well plate 70. After staying for 1 - 2 seconds, preferably 1 second, under the adhesion of the first organoid sphere 102 to the bottom of the first well position 72, the first organoid sphere 102 will adsorb to the bottom of the first well position 72, thus realizing the printing of the air-solid interface of the first organoid sphere 102. The first organoid sphere 102 is printed into the first well position 72 of the well plate 70. As shown in Figure 13 , after completion, the driving unit drives the printing nozzle 62 back to the initial position.

[0075] Step S12: Sequentially print the remaining organoid spheres 102 in the organoid droplet maturation area into the remaining well positions 72 of the well plate 70 according to the method of steps S6 to S10. In this way, the high-throughput printing of the organoid gas-solid interface is completed.

[0076] Before step S14, there is also step S13: Add a continuous phase to all well positions 72 of the well plate 70. The organoid spheres 102 are then stably cultured at the bottom within the well positions 72 of the well plate 70.

[0077] Figure 14 It is a schematic diagram of the effect from the first day to the sixth day after culturing the organoid spheres 102. As can be seen Figure 10 from it, the organoids have good consistency and small differences among individuals.

[0078] In addition, the printing nozzle 62 of this embodiment may not be set as a wedge shape.

[0079] The novel organoid high-throughput printing and culturing method provided by the present invention completes the preparation of organoids through a microfluidic system, and combines the microfluidic system with a 3D printing system, which can simply, quickly, and precisely transfer the organoids to the bottom within the well positions 72 of the well plate 70 by printing for culturing. It has good controllability, saves time and effort, has good stability, the organoids are of uniform size, ensures the consistency of the organoids, and reduces the differences among organoid individuals.

[0080] Third Embodiment

[0081] Please refer to Figure 15 and Figure 16 In this embodiment, steps S2 to step 8 and step S14 are the same as those in the first embodiment and will not be elaborated here. The difference between this embodiment and the first embodiment is that in step S10, the control unit controls the driving unit of the 3D printing system to act, and the driving unit drives the printing nozzle 62 to move downward to the junction of the continuous phase and the bottom within the first well position 72 of the well plate 70. After staying for 1 - 2 seconds, preferably 1 second, under the adhesion of the first organoid sphere 102 to the bottom within the first well position 72, the first organoid sphere 102 will adsorb at the junction of the continuous phase and the bottom within the first well position 72. In this way, the printing of the liquid-solid interface of the first organoid sphere 102 is achieved, and the first organoid sphere 102 is printed into the first well position 72 of the well plate 70. After completion, the driving unit drives the printing nozzle 62 back to the initial position.

[0082] Step S12: Print the remaining organoid spheres 102 in the organoid droplet maturation area into the remaining well positions 72 of the well plate 70 in sequence according to the method of steps S6 to S10. In this way, the high-throughput printing of the organoid liquid-solid interface is completed. The organoid spheres 102 are then cultured stably at the junction between the continuous phase in the well positions 72 and the bottom of the well positions 72.

[0083] In addition, the printing nozzle 62 of this embodiment may not be set to a wedge shape.

[0084] The novel organoid high-throughput printing and culturing method provided by the present invention completes the preparation of organoids through a microfluidic system, and combines the microfluidic system with a 3D printing system, which can simply, quickly, and precisely transfer the organoids to the junction between the continuous phase in the well positions 72 of the well plate 70 and the bottom of the well positions 72 for culturing by printing. It has good controllability, saves time and effort, has good stability, the organoids are of uniform size, ensuring the consistency of the organoids and reducing the differences between individual organoids.

[0085] Fourth Embodiment

[0086] Please refer to Figure 17 and Figure 18 In this embodiment, steps S2 to S8 and step S14 are the same as those in the first embodiment and will not be elaborated here. The difference between this embodiment and the first embodiment is that in step S10, the driving unit of the 3D printing system is controlled by the control unit to act, and the printing nozzle 62 is driven by the driving unit to move downward to the inside of the continuous phase in the first well position 72 of the well plate 70, preferably to the middle position inside the continuous phase. After staying for 1 - 2 seconds, preferably 1 second, since the continuous phase has a certain viscosity, the first organoid sphere 102 will be adsorbed inside the continuous phase in the first well position 72 and be in a suspended state. In this way, the suspended printing of the first organoid sphere 102 is achieved. The first organoid sphere 102 is then printed into the first well position 72 of the well plate 70. After completion, the driving unit drives the printing nozzle 62 back to the initial position.

[0087] Step S12: Print the remaining organoid spheres 102 in the organoid droplet maturation area into the remaining well positions 72 of the well plate 70 in sequence according to the method of steps S6 to S10. In this way, the high-throughput printing of the organoid suspension is completed. The organoid spheres 102 are then cultured stably and suspended inside the continuous phase in the well positions 72.

[0088] In addition, the printing nozzle 62 of this embodiment may not be set to a wedge shape.

[0089] The novel organoid high-throughput printing and culturing method provided by the present invention prepares organoids through a microfluidic system, and combines the microfluidic system with a 3D printing system, enabling the organoids to be simply, quickly, and precisely transferred into the interior of the continuous phase at the pore position 72 of the well plate 70 by printing for culturing. It has good controllability, saves time and effort, has good stability, uniform organoid size, ensures the consistency of organoids, and reduces the differences between individual organoids.

[0090] The above is a specific description of the preferred embodiment of the present invention, but the present invention is not limited to the described embodiment. Those skilled in the art can make various equivalent deformations or substitutions without departing from the spirit of the present invention, and these equivalent deformations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A novel method for high-throughput printing and culturing of organoids, characterized in that, it includes the following steps: S2. Wrap cells in dispersed phase 1; S4. Inject the dispersed phase 1 wrapped with cells and dispersed phase 2 into the organoid precursor preparation area of the microfluidic system respectively. The dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized organoid precursor liquid droplets. The sheared organoid precursor liquid droplets can sequentially enter the organoid droplet ripening area of the microfluidic system and can ripen into organoid spheres in this organoid droplet ripening area; S6. Through the delivery pump of the microfluidic system, the first organoid sphere in the organoid droplet ripening area is delivered to the printing nozzle of the 3D printing system through the delivery pipeline of the 3D printing system; S8. Use the organoid recognition and detection unit of the 3D printing system to detect whether there is an organoid sphere at the printing nozzle. When the organoid recognition and detection unit detects an organoid sphere at the printing nozzle, the organoid recognition and detection unit outputs an organoid signal to the control unit of the 3D printing system; S10. Control the action of the driving unit of the 3D printing system through the control unit. Drive the printing nozzle to move downward to the liquid surface, bottom, interface between the continuous phase and the bottom of the first hole position in the well plate, or the inside of the continuous phase through the driving unit. After staying for 1-2 seconds, the first organoid sphere will adsorb on the liquid surface, bottom, interface between the continuous phase and the bottom of the first hole position in the well plate, or the inside of the continuous phase. In this way, the first organoid sphere is printed into the first hole position of the well plate. After completion, drive the printing nozzle back to the initial position through the driving unit; S12. Sequentially print the remaining organoid spheres in the organoid droplet ripening area into the remaining hole positions of the well plate according to the method of steps S6 to S10; S14. Culture the organoid spheres in all hole positions of the well plate; In the step S4, the method of ripening the organoid precursor liquid droplets into organoid spheres includes enzymatic crosslinking method, ionic crosslinking method or photocrosslinking method; The material of the dispersed phase 1 includes matrix gel, collagen or gelatin, and the material of the dispersed phase 2 includes fluorinated oil or vegetable oil.

2. The novel method for high-throughput printing and culturing of organoids according to claim 1, characterized in that, The organoid precursor preparation area is a Y-shaped structure. The Y-shaped structure includes a first inlet pipeline, a second inlet pipeline and a connecting pipeline. The first ends of the first inlet pipeline and the second inlet pipeline are respectively connected to a first syringe and a second syringe. The first syringe is connected with a first injection pump, and the second syringe is connected with a second injection pump. The second ends of the first inlet pipeline and the second inlet pipeline are both connected to the first end of the connecting pipeline. The organoid droplet ripening area is an outlet pipeline, and the second end of the connecting pipeline is connected to the outlet pipeline; The step S4 includes the following steps; S42. Place the dispersed phase 1 and the dispersed phase 2 in the first syringe and the second syringe respectively; S44. Inject the dispersed phase 1 in the first syringe into the first inlet pipe through the first injection pump, and inject the dispersed phase 2 in the second syringe into the second inlet pipe through the second injection pump. In the connecting pipe, the dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized precursor organoid fluid droplets, and the sheared precursor organoid fluid droplets can sequentially enter the outlet pipe and mature into organoid spheres in the outlet pipe.

3. The novel organoid high-throughput printing and culturing method according to claim 1, characterized in that, the precursor organoid preparation area is a T-shaped structure, and the T-shaped structure includes a first inlet pipe, a second inlet pipe, and a connecting pipe. The first end of the first inlet pipe and the first end of the second inlet pipe are respectively connected to the first syringe and the second syringe. The first syringe is connected to a first injection pump, and the second syringe is connected to a second injection pump. The second end of the first inlet pipe and the second end of the second inlet pipe are respectively connected to the first end of the connecting pipe. The organoid droplet maturation area is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe; The step S4 includes the following steps: S42. Place the dispersed phase 1 and the dispersed phase 2 in the first syringe and the second syringe respectively; S44. Inject the dispersed phase 1 in the first syringe into the first inlet pipe through the first injection pump, and inject the dispersed phase 2 in the second syringe into the second inlet pipe through the second injection pump. In the connecting pipe, the dispersed phase 1 is sheared by the dispersed phase 2 into uniformly sized precursor organoid fluid droplets, and the sheared precursor organoid fluid droplets can sequentially enter the outlet pipe and mature into organoid spheres in the outlet pipe.

4. The novel organoid high-throughput printing and culturing method according to claim 1, characterized in that, the precursor organoid preparation area is a cross-shaped structure, and the cross-shaped structure includes a first inlet pipe, two second inlet pipes arranged oppositely, and a connecting pipe. The first inlet pipe and the connecting pipe are arranged oppositely. The first end of the first inlet pipe is connected to the first syringe, and the second end of the first inlet pipe is connected to the first end of the connecting pipe. The first syringe is connected to a first injection pump. The first ends of the two second inlet pipes are respectively connected to two second syringes, and the second ends of the two second inlet pipes are respectively connected to the first end of the connecting pipe. The two second syringes are respectively connected to two second injection pumps. The organoid droplet maturation area is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe; The step S4 includes the following steps: S42. Place the dispersed phase 1 in the first syringe and place the dispersed phase 2 in the two second syringes respectively; S44. Inject the dispersed phase 1 in the first syringe into the first inlet pipe through the first injection pump, and inject the dispersed phase 2 in the two second syringes into the two second inlet pipes through the two second injection pumps respectively. In the connecting pipe, the dispersed phase 1 is sheared into uniformly sized precursor organoid liquid droplets by the dispersed phase 2 injected into the two second inlet pipes. The sheared precursor organoid liquid droplets can sequentially enter the outlet pipe and can mature into organoid spheres in the outlet pipe.

5. The novel organoid high-throughput printing and culturing method according to claim 1, characterized in that, in step S10, the driving unit of the 3D printing system drives the printing head to move downward to the bottom in the first hole position of the well plate, and after staying for 1-2 seconds, the first organoid sphere will adsorb to the bottom in the first hole position; Before step S14, there is also step S13: adding a continuous phase into all the hole positions of the well plate.

6. The novel organoid high-throughput printing and culturing method according to claim 1, characterized in that, in step S10, the driving unit of the 3D printing system drives the printing head to move downward to the liquid level of the continuous phase in the first hole position of the well plate, and after staying for 1-2 seconds, the first organoid sphere will adsorb to the liquid level of the continuous phase in the first hole position; The printing head is wedge-shaped.

7. The novel organoid high-throughput printing and culturing method according to claim 6, characterized in that, the wedge angle of the printing head is 10-80 degrees.

8. The novel organoid high-throughput printing and culturing method according to claim 1, characterized in that, in step S2, the cells are primary tumor tissue cells, stem cells or cell lines.

9. The novel organoid high-throughput printing and culturing method according to claim 1, characterized in that, the continuous phase is ordinary culture medium, growth factor-containing culture medium, drug-containing culture medium or Dulbecco's phosphate buffer solution.

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