An organoid molding device and a method of operation thereof

Abstract

The application provides an organoid forming device. The organoid forming device comprises a pump, a spiral pipe connected to the pump and used for storing and transporting cell suspension, and a nozzle unit connected to the other end of the spiral pipe and used for extruding the cell suspension transported by the spiral pipe to form cell suspension microspheres through air jet action or high voltage electrostatic action. The application also provides a working method of the organoid forming device. The application can make the initial cell content of the obtained cell suspension sample droplets for organoid culture uniform and consistent, and further make the size of the finally constructed organoids uniform.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, more particularly, to an organoid forming device and a working method thereof. BACKGROUND

[0002] An organoid is a model that is highly similar to a tissue or organ in vivo, which is established based on a 3D in vitro cell culture system. These 3D in vitro culture systems can replicate the complex spatial morphology of differentiated tissues and can exhibit the interaction and spatial position morphology between cells and the surrounding matrix. The characteristics must include one or more cell types that are the same as the source organ; it should exhibit some functions unique to the source organ; and the organization of cells should be similar to the source organ. The organoid technology has a wide range of applications, including developmental biology, disease pathology, cell biology, regeneration mechanism, precision medicine, and drug toxicity and efficacy testing.

[0003] A typical method for preparing an organoid first separates the corresponding seed cells, such as tumor stem cells in a tumor or pluripotent stem cells from normal tissues, and then cultures them on a biocompatible material such as Matrigel or a manual extrusion spotting process. Growth factors and small molecules need to be added to the culture medium to activate or inhibit specific signaling pathways that depend on the formation of organoids, and different combinations of additives are used to prepare different organoids. After a period of culture, spherical cell clusters are formed. However, the traditional method is to culture directly in a culture dish, so the cells are spontaneously clustered, and the initial number of cells forming clusters is not controlled, so the size of the organoids formed is also inconsistent, and the position is random, as shown in Figure 1 More troublesome is that the cell forming clusters are suspended and grown, which makes it difficult to perform targeted analysis or uniform and batch drug screening. To solve the above problems, one method is to use a syringe to sample to obtain a relatively large array of sample droplets to ensure uniform size of each sample droplet, such as Chinese patent CN109613294A, which discloses a high-throughput droplet array microfluidic spotting liquid sample feeding device. However, using this syringe sampling method, cells will settle at the bottom of the syringe due to their own gravity, resulting in a higher concentration of cells in the initial sample droplets and a lower concentration of cells in the later sample droplets, which is a clear concentration difference, as shown in Figure 2 If the initial amount of cells is not uniform, it is also difficult to objectively compare the later drug evaluation. SUMMARY

[0004] In order to overcome the defects of the existing organoid culture cell suspension sample droplet obtained by using the syringe spotting method, the cells will be settled at the bottom of the syringe due to their own gravity, resulting in higher cell concentration in the initial sample droplet and lower cell concentration in the later sample droplet, and the obvious concentration difference, the present application provides an organoid forming device and a working method thereof. The present application can make the initial cell content of the obtained cell suspension sample droplet for organoid culture uniform, and further make the size of the finally constructed organoid uniform.

[0005] In order to solve the above technical problems, the technical scheme adopted by the present application is: an organoid forming device, comprising a pump, a spiral pipe connected to one end of the pump and used for storing and transporting cell suspension, and a nozzle unit connected to the other end of the spiral pipe and used for extruding the cell suspension transported by the spiral pipe to form cell suspension microspheres by air jet or high voltage electrostatic action. In the present application, the spiral pipe is a medical silicone rubber tube with an inner diameter of 0.7-3.6mm. The liquid in the tube maintains a certain stability due to adhesion and capillary adsorption with the tube wall and does not flow spontaneously. Therefore, the cell suspension can be stably stored in the spiral pipe, ensuring the uniformity of the cell concentration before and after the cell suspension microsphere forming process, and finally making the size of the constructed organoid uniform, which is suitable for high-throughput drug screening, immunotherapy, tumor occurrence and development research.

[0006] As a preferred scheme, the nozzle unit comprises a nozzle hose arranged at the other end of the spiral pipe, a needle inserted into the inner cavity of the nozzle hose from the side wall of the nozzle hose and forming a coaxial structure with the nozzle hose, a first pinch valve and a second pinch valve arranged upstream and downstream of the position of the needle on the nozzle hose respectively, and a gas supply device connected to the tail end of the needle and capable of providing a gas pressure of 10-100kPa at the tail end of the needle. The nozzle hose is actually an integral structure with the spiral pipe and also adopts a medical silicone rubber tube, and the needle is a steel needle.

[0007] Further, the gas supply device comprises a gas pump connected to the needle through a gas pipe and an electromagnetic valve arranged on the gas pipe, the electromagnetic valve controls the on-off of the gas flow through the needle, and the pump is a peristaltic pump.

[0008] Further, the present scheme further comprises a controller and a data acquisition card connected to the controller, and the data acquisition card is connected to the pump, the first pinch valve, the second pinch valve and the electromagnetic valve respectively. The controller mainly sets, monitors and stores data of the whole device through a human-machine interface, the data acquisition card is connected to the controller through a special data cable, receives the control signal of the controller, and then transmits it to the peristaltic pump, the first pinch valve, the second pinch valve and the electromagnetic valve, so as to control the start and stop of these devices.

[0009] As another preferred solution, the nozzle unit comprises a nozzle hose arranged at the other end of the spiral tube, a needle connected to the nozzle hose away from the one end of the spiral tube, a third pinch valve arranged on the nozzle hose close to the needle, and a high-voltage direct current source connected to the needle.

[0010] Further, the needle is provided with a wire clamp, the high-voltage direct current source is connected to the wire clamp through a wire, and the pump is a constant flow pump.

[0011] Further, the solution further comprises a controller and a data acquisition card connected to the controller, and the data acquisition card is connected to the pump, the third pinch valve and the high-voltage direct current source respectively. The controller mainly sets parameters, monitors and stores data of the whole device through a man-machine interface, the data acquisition card is connected to the controller through a special data cable, receives a control signal of the controller, and then forwards the control signal to the constant flow pump and the third pinch valve, so as to control the start and stop of the devices.

[0012] Further, the solution further comprises a two-dimensional motion table arranged below the nozzle unit and used for containing cell suspension microspheres, and an optical detection unit arranged on one side of the two-dimensional motion table and used for detecting the diameter of the cell suspension microspheres, and the two-dimensional motion table and the optical detection unit are connected to the data acquisition card. When the cell suspension microspheres are contained, the culture device can be placed on the two-dimensional motion table, the two-dimensional motion table is controlled by the controller to move and drive the culture device to move together, so that the cell suspension microspheres can accurately fall on the set position of the culture surface. The optical detection unit can detect whether the diameter of the cell suspension microspheres is uniform in real time during the extrusion of the cell suspension microspheres. For example, the optical detection unit comprises an optical microscope, can collect an image after the microspheres are extruded, and transmit the image to the data acquisition card through a data line. The image is displayed on the screen of the controller and the diameter is measured. An operator can observe the aggregation, formation and maturation of the organoids in the microspheres on the screen of the controller. The diameter of the microspheres can also be detected, measured and recorded again after the organoids are mature and stable.

[0013] The application further provides a working method of the organoid forming device, which comprises the following steps.

[0014] S1. The passage of the nozzle unit is opened, and the pump is reversely rotated to suck the cell suspension containing cells or microtissues into the spiral tube for use.

[0015] S2. The outlet of the nozzle unit is closed, and the pump is forwardly rotated to extrude the cell suspension from the spiral tube to the nozzle unit.

[0016] S3. The cell suspension breaks through the surface tension under the action of air jet or high voltage electrostatic force to form cell suspension microspheres, which finally drop and adhere to the designated position on the culture surface;

[0017] S4. Repeat steps S1, S2 and S3 until the target number of cell suspension microspheres are formed on the culture surface;

[0018] S5. Add a hydrogel containing organoid inducers to the culture surface, and culture the cell suspension microspheres on the culture surface to form organoids.

[0019] The concentration of cells in the cell suspension is 0.1-10*106 / ml, and the types of cells include primary tumor cells, stem cells, adult cells, etc. The hydrogel is methacrylated gelatin (3%-20%), or gelatin (2%-20%), collagen (0.1%-20%), fibrinogen (0.2%-5%), sodium alginate (0.5%-5%), etc. The addition of these hydrogels not only maintains the shape of the cell clusters initially, but also provides a good three-dimensional microenvironment for cell adhesion and growth. The hydrogel also contains organoid inducers, which include various growth factors or small molecule substances. Different types of organoids use different inducers. For example, in the culture of small intestine organoids, EGF (epidermal growth factor), R-spondin-1, Wnt-3A and Noggin are required.

[0020] After one week of culture and induction, uniform and stable organoid structures can be formed, with uniform overall size and regular arrangement.

[0021] Further, in steps S3, S4 and S5, the optical detection unit detects the diameter of the cell suspension microspheres in real time, and then feeds back to the data acquisition card. In actual application, the optical detection unit first detects whether the size of the extruded cell suspension microspheres is uniform in real time. After passing the detection, the organoid aggregation, formation and maturation also need to be detected during the culture process. After it is mature and stable, the diameter of the sphere is detected and measured again. Then anti-tumor drugs are added, and the change of the diameter of the organoid is detected in real time. If the drug is effective, the volume remains stable or shrinks. Conversely, if the diameter of the tumor organoid continues to develop and become larger, it means that the drug is ineffective. The occurrence and development of tumors can also be studied by cell live and dead staining, specific fluorescent staining and other methods.

[0022] Further, the working method of the above organoid forming device, such as forming cell suspension microspheres by air jet, includes the following specific steps:

[0023] S1. The controller controls the first pinch valve and the second pinch valve to open, and controls the pump to rotate reversely to suck the cell suspension containing cells or microtissues into the spiral pipe through the nozzle hose for standby;

[0024] S2. The controller controls the first pinch valve to keep open and the second pinch valve to close, and controls the pump to rotate forwardly for one beat to extrude the cell suspension from the spiral pipe to fill the space of the nozzle hose;

[0025] S3. The controller controls the first pinch valve to close and the second pinch valve to open, and controls the electromagnetic valve to open, and the filtered high-pressure gas is released through the needle by the air pump, so that a certain volume of cell suspension in the nozzle hose is pushed out to form a cell suspension microsphere, and finally drops and adheres to the set position of the culture surface.

[0026] Further, the working method of the organoid forming device as described above to form a cell suspension microsphere by high-voltage electrostatic action comprises the following specific steps:

[0027] S1. The controller controls the third pinch valve to open, and controls the pump to start reversely to suck the cell suspension containing cells or microtissues into the spiral pipe through the needle and the nozzle hose for standby;

[0028] S2. The controller controls the pump to start forwardly to push the cell suspension into the nozzle hose at low speed and stably;

[0029] S3. The high-voltage direct current source is opened, the needle is electrified, and then the third pinch valve is stably opened and closed at a certain frequency, when the third pinch valve is closed, the cell suspension at the end of the nozzle hose is extruded to the end of the needle, and then the cell suspension breaks through the surface tension to fly out to form a cell suspension microsphere under the action of high-voltage electrostatic force, and finally drops and adheres to the set position of the culture surface.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The present application adopts a spiral pipe to store and transport cell suspension, so that the cell suspension can be stably stored in the spiral pipe to ensure the uniformity of cell concentration before and after the cell suspension microsphere forming process, and the pump and the pinch valve can be started and stopped to extrude microspheres of uniform size, so that the size of the constructed organoids is uniform, which is suitable for high-throughput drug screening, immunotherapy, tumor development and other researches.

[0032] Compared with the device used in the traditional method, the device of the present application has high automation degree, and the size of the formed cell suspension microsphere and the number of cells are controllable, which can obviously shorten the induction time, and the appropriate microsphere size and appropriate cell number can be beneficial to high-throughput production and ensure the organoid forming rate.

[0033] Compared with the traditional method, the present application can significantly shorten the induction time and improve the success rate. The traditional method needs one to two weeks to induce and culture cells into organoid spheroids, while the present application can observe the phenomenon of cell aggregation into groups in about two days.

[0034] In the present application, the amount of organoid inducer used is small and the effect on cells is mild. In the traditional method, the inducer is directly added to the culture medium, and the initial concentration is high in order to maintain the corresponding concentration in subsequent events. Moreover, the liquid exchange operation every two days will cause multiple high-concentration shocks to the cells, which is very unfavorable. In the present application, the growth factor is slowly released within the hydrogel, which has a process from low to high, is uniform and gentle, and will not cause damage to the cells due to high concentration. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is an effect diagram of an organoid constructed by using a traditional culture dish culture method.

[0036] Figure 2 is a schematic diagram of the sedimentation of cells in a cell suspension due to their own gravity when obtaining a cell suspension microsphere by using a syringe spotting method.

[0037] Figure 3 is a schematic diagram of the overall structure of Example 1 of the present application.

[0038] Figure 4 is a schematic diagram of the structure of the spiral tube and the nozzle hose in the present application.

[0039] Figure 5 is a schematic diagram of the structure of the nozzle hose, the needle, the first pinch valve, and the second pinch valve in Example 1 of the present application.

[0040] Figure 6 is a schematic diagram of the overall structure of Example 2 of the present application.

[0041] Figure 7 is a schematic diagram of the structure of the nozzle hose, the needle, and the third pinch valve in Example 2 of the present application.

[0042] Figure 8 is an effect diagram of an organoid constructed by using Example 3 and Example 4 of the present application. DETAILED DESCRIPTION

[0043] The drawings are only used for illustrative description and cannot be understood as a limitation of the present patent; in order to better illustrate the present embodiment, some components in the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted. The positional relationship described in the drawings is only used for illustrative description and cannot be understood as a limitation of the present patent.

[0044] Example 1

[0045] As shown in Figure 3 and Figure 4 , an organoid forming device, comprising a pump 1, a spiral tube 2 connected to one end of the pump 1 and used for storing and transporting cell suspension, and a nozzle unit 3 connected to the other end of the spiral tube 2 and used for extruding the cell suspension transported by the spiral tube 2 to form cell suspension microspheres by air jet or high voltage electrostatic action. In the present application, the spiral tube 2 is a medical silicone rubber tube with an inner diameter of 0.7-3.6 mm. The liquid in the tube is stable and will not flow spontaneously due to adhesion and capillary adsorption to the tube wall. Thus, the cell suspension can be stably stored in the spiral tube 2 to ensure the uniformity of cell concentration before and after the cell suspension microsphere forming process, and ultimately make the size of the constructed organoids uniform, which is suitable for high-throughput drug screening, immunotherapy, tumor development and other researches.

[0046] As shown in Figure 4 and Figure 5 , the nozzle unit 3 comprises a nozzle hose 31 arranged at the other end of the spiral tube 2, a needle 32 inserted into the inner cavity of the nozzle hose 31 from the side wall of the nozzle hose 31 and forming a coaxial structure with the nozzle hose 31, a first pinch valve 33 and a second pinch valve 34 arranged on the nozzle hose 31 respectively upstream and downstream of the position of the needle 32, and a gas delivery device connected to the tail end of the needle 32, which can provide a gas pressure of 10-100 kPa to the tail end of the needle. The nozzle hose 31 is actually an integral structure with the spiral tube 2 and is also a medical silicone rubber tube. The needle 32 is a steel needle.

[0047] As shown in Figure 3 , the gas delivery device comprises a gas pump 35 connected to the needle 32 through a gas pipe and an electromagnetic valve 36 arranged on the gas pipe. The electromagnetic valve 36 controls the on-off of the gas flow through the needle 32. The pump 1 is a peristaltic pump 1.

[0048] As shown in Figure 3 , the present embodiment further comprises a controller 4 and a data acquisition card 5 connected to the controller 4. The data acquisition card 5 is connected to the pump 1, the first pinch valve 33, the second pinch valve 34 and the electromagnetic valve 36 respectively. The controller 4 mainly sets parameters, monitors and stores data of the whole device through a human-machine interface. The data acquisition card 5 is connected to the controller 4 through a special data cable, receives the control signal of the controller 4 and then transmits it to the peristaltic pump 1, the first pinch valve 33, the second pinch valve 34 and the electromagnetic valve 36, so as to control the start and stop of these devices.

[0049] As shown in Figure 3As shown, the embodiment also includes a two-dimensional motion table 6 arranged below the spray head unit 3 and used for containing the cell suspension microspheres, an optical detection unit 7 arranged on one side of the two-dimensional motion table 6 and used for detecting the diameter of the cell suspension microspheres, and the two-dimensional motion table 6 and the optical detection unit 7 are connected with the data acquisition card 5. When containing the cell suspension microspheres, the culture device can be placed on the two-dimensional motion table 6, and the two-dimensional motion table 6 is controlled to move by the controller 4, thereby driving the culture device to move together, so that the cell suspension microspheres can accurately fall on the set position of the culture surface. The optical detection unit 7 can detect in real time whether the diameter of the cell suspension microspheres is uniform during the extrusion process of the cell suspension microspheres, and the optical detection unit 7 can detect the aggregation, formation and maturation of the organoids during the culture process. After the organoids are mature and stable, the diameter of the spheres is detected and measured again.

[0050] Embodiment 2

[0051] The embodiment is similar to embodiment 1, and the difference lies in that, as shown in Figure 6 and Figure 7 , the spray head unit 3 includes a spray head hose 31 arranged at the other end of the spiral pipe 2, a needle 32 connected to the end of the spray head hose 31 away from the spiral pipe 2, a third pinch valve 37 arranged on the spray head hose 31 close to the needle 32, and a high-voltage direct current source 38 connected to the needle 32. The spray head hose 31 is actually an integral structure with the spiral pipe 2, and is also a medical silicone rubber tube. The needle 32 is a steel needle 32, and the high-voltage direct current source 38 can connect 1kv-15kv high-voltage direct current to the needle 32, thereby forming a high-voltage electrostatic field below the needle 32.

[0052] As shown in Figure 7 , a wiring clamp 39 is arranged on the needle 32, the high-voltage direct current source 38 is connected to the wiring clamp 39 through a wire, and the pump 1 is a constant-flow pump 1.

[0053] As shown in Figure 6 , the embodiment also includes a controller 4 and a data acquisition card 5 connected with the controller 4, and the data acquisition card 5 is connected with the pump 1, the third pinch valve 37 and the high-voltage direct current source 38 respectively. The controller 4 mainly sets, monitors and stores data of the whole device through a human-computer interface. The data acquisition card 5 is connected with the controller 4 through a special data cable, receives a control signal of the controller 4, and then transmits the control signal to the constant-flow pump 1 and the third pinch valve 37, so as to control the start and stop of these devices.

[0054] Embodiment 3

[0055] A working method of an organoid forming device based on embodiment 1, wherein the method includes the following steps:

[0056] S1. The controller 4 controls the first pinch valve 33 and the second pinch valve 34 to open, and the controller 4 controls the pump 1 to rotate reversely to suck the cell suspension containing cells or microtissues into the spiral tube 2 through the nozzle hose 31 for standby;

[0057] S2. The controller 4 controls the first pinch valve 33 to keep open, and the second pinch valve 34 to close, and the pump 1 to rotate forwardly for one beat to extrude the cell suspension from the spiral tube 2 to fill the space of the nozzle hose 31;

[0058] S3. The controller 4 controls the first pinch valve 33 to close, and the second pinch valve 34 to open, and the controller 4 controls the electromagnetic valve 36 to open, and the filtered high-pressure gas is released through the needle 32 by the air pump 35, so as to push a certain volume of the cell suspension in the nozzle hose 31 out, and the cell suspension in the process of flying in the air will shrink into a sphere to form a cell suspension microsphere due to the surface tension, and finally drop and adhere to the set position of the culture surface;

[0059] S4. Repeat steps S1, S2 and S3 until the target number of cell suspension microspheres on the culture surface is reached;

[0060] S5. Add the hydrogel containing organoid inducer to the culture surface, and culture the cell suspension microspheres on the culture surface to form organoids.

[0061] The concentration of the cells in the cell suspension is 0.1-10*10 6 / ml, and the cell type is primary tumor cells, or stem cells, adult cells, etc. The hydrogel is methacrylated gelatin (3%-20%), or gelatin (2%-20%), collagen (0.1%-20%), fibrinogen (0.2%-5%), sodium alginate (0.5%-5%), etc. The addition of these hydrogels is not only to maintain the shape of the cell cluster initially, but also to provide a good three-dimensional microenvironment for the adhesion and growth of cells. The hydrogel also contains organoid inducers, which include various growth factors or small molecule substances, and different organoid types use different inducers. For example, for the culture of small intestine organoids, EGF (epidermal growth factor), R-spondin-1, Wnt-3A and Noggin growth factors need to be added.

[0062] After one week of culture and induction, uniform and stable organoid structures can be formed, and the overall size is uniform and arranged regularly, as shown in Figure 8 .

[0063] In the steps S3, S4 and S5, the optical detection unit 7 detects the diameter of the cell suspension microspheres in real time, and then feeds back to the data acquisition card 5. In actual application, the optical detection unit 7 first detects whether the size of the extruded cell suspension microspheres is uniform in real time. After the detection passes, the organoid aggregation, formation and maturation need to be detected during the culture process. After the maturation and stability, the diameter of the microspheres is detected and measured again. Then the anti-tumor drug is added, and the change of the diameter of the organoids is detected in real time. If the drug is effective, the volume remains stable or shrinks. On the contrary, if the diameter of the tumor organoids continues to develop and become larger, it means that the drug is ineffective. The occurrence and development of tumors can also be studied by cell live and dead staining, specific fluorescent staining and other methods.

[0064] Embodiment 4

[0065] A working method based on the organoid forming device of embodiment 2, comprising the following steps:

[0066] S1. The controller 4 controls the third pinch valve 37 to open, and the controller 4 controls the pump 1 to start in reverse to suck the cell suspension containing cells or microtissues into the spiral pipe 2 through the needle 32 and the nozzle hose 31 for standby;

[0067] S2. The controller 4 controls the pump 1 to start in forward direction at low speed to stably push the cell suspension, and the high-voltage direct current source 38 is turned on at the same time, and then the third pinch valve 37 is stably opened and closed at a certain frequency;

[0068] S3. When the third pinch valve 37 closes the liquid path, the cell suspension at the end of the nozzle hose 31 is extruded to the end of the needle 32, and then the cell suspension breaks through the surface tension under the action of high-voltage electrostatic field to form a cell suspension microsphere, and finally drops and adheres to the set position on the culture surface;

[0069] S4. Repeat steps S1, S2 and S3 until the target number of cell suspension microspheres on the culture surface is reached;

[0070] S5. Add the hydrogel containing organoid inducer on the culture surface to culture the cell suspension microspheres on the culture surface to form organoids.

[0071] The concentration of cells in the cell suspension is 0.1-10*10 6The cell concentration is 1*10<6> / ml, and the cell type is primary tumor cells, stem cells, adult cells, etc. The hydrogel is methacrylated gelatin (3%-20%), or gelatin (2%-20%), collagen (0.1%-20%), fibrinogen (0.2%-5%), sodium alginate (0.5%-5%), etc. The addition of these hydrogels is to maintain the shape of the cell cluster in the initial stage, and to provide a good three-dimensional microenvironment for the adhesion and growth of cells. The hydrogel also contains organoid inducers, which include various growth factors or small molecules, and different organoid types use different inducers. For example, for the culture of small intestine organoids, EGF (epidermal growth factor), R-spondin-1, Wnt-3A and Noggin growth factors need to be added.

[0072] After one week of culture and induction, uniform and stable organoid structures can be formed, and the overall size is uniform and arranged regularly, as shown in Figure 8

[0073] In the step S3, S4 and S5, the optical detection unit 7 detects the diameter of the cell suspension microspheres in real time, and then feeds back to the data acquisition card 5. In actual application, the optical detection unit 7 first detects whether the size of the extruded cell suspension microspheres is uniform in real time. After the detection passes, the organoid aggregation, formation and maturation also need to be detected during the culture process. After it is mature and stable, the diameter of the microsphere is detected and measured again. Then the anti-tumor drug is added, and the change of the diameter of the organoid is detected in real time. If the drug is effective, the volume remains stable or shrinks. On the contrary, if the diameter of the tumor organoid continues to develop and become larger, it means that the drug is ineffective. The occurrence and development of tumors can also be studied by cell live and dead staining, specific fluorescence staining and other methods.

[0074] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application should be included in the protection scope of the claims of the present application.​

Claims

1. An organoid molding device, characterized by, The device comprises a pump (1), a spiral tube (2) connected to the pump (1) and used for storing and transporting cell suspension, and a nozzle unit (3) connected to the other end of the spiral tube (2) and used for forming cell suspension microspheres by air jet or high voltage electrostatic action. When the cell suspension microspheres are formed by air jet, the nozzle unit (3) comprises a nozzle hose (31) arranged at the other end of the spiral tube (2), a needle (32) inserted into the inner cavity of the nozzle hose (31) from the side wall of the nozzle hose (31) and coaxially arranged with the nozzle hose (31), and a first pinch valve (33) and a second pinch valve (34) arranged on the nozzle hose (31) respectively at the upstream and downstream of the position of the needle (32). When the cell suspension microspheres are formed by high voltage electrostatic action, the nozzle unit (3) comprises a nozzle hose (31) arranged at the other end of the spiral tube (2), a needle (32) connected to the nozzle hose (31) at the end away from the spiral tube (2), and a third pinch valve (37) arranged on the nozzle hose (31) close to the needle (32), and the needle (32) is connected with a high voltage direct current source (38).

2. The organoid forming device of claim 1, wherein The gas conveying device comprises a gas pump (35) connected with the needle (32) through a gas pipe and an electromagnetic valve (36) arranged on the gas pipe, and the pump (1) is a peristaltic pump.

3. The organoid forming device of claim 2, wherein The device further comprises a controller (4) and a data acquisition card (5) connected with the controller (4), and the data acquisition card (5) is connected with the pump (1), the first pinch valve (33), the second pinch valve (34) and the electromagnetic valve (36) respectively.

4. The organoid-forming device of claim 1, wherein When the cell suspension microspheres are formed by high voltage electrostatic action, a wire clamp (39) is arranged on the needle (32), the high voltage direct current source (38) is connected with the wire clamp (39) through a wire, and the pump (1) is a constant flow pump.

5. The organoid-forming device of claim 1, wherein When the cell suspension microspheres are formed by high voltage electrostatic action, the device further comprises a controller (4) and a data acquisition card (5) connected with the controller (4), and the data acquisition card (5) is connected with the pump (1), the third pinch valve (37) and the high voltage direct current source (38) respectively.

6. The organoid forming device according to claim 3 or 5, wherein The device further comprises a two-dimensional motion table (6) arranged below the nozzle unit (3) and used for containing the cell suspension microspheres, and an optical detection unit (7) arranged on one side of the two-dimensional motion table (6) and used for detecting the diameter of the cell suspension microspheres, and the two-dimensional motion table (6) and the optical detection unit (7) are connected with the data acquisition card (5).

7. A method of operating an organoid molding apparatus according to any one of claims 1 to 6, characterized by, The device comprises the following steps: S1. The passage of the nozzle unit (3) is opened, and the pump (1) is reversely rotated to suck the cell suspension containing cells or microtissues into the spiral tube (2) through the nozzle unit (3) for standby; S2. The outlet of the nozzle unit (3) is closed, and the pump (1) is forwardly rotated to extrude the cell suspension from the spiral tube (2) to the nozzle unit (3). S3. The cell suspension breaks through the surface tension and flies out to form cell suspension microspheres under the action of air jet or high voltage electrostatic force, and finally drops and adheres to the designated position on the culture surface; S4. Repeat steps S1, S2 and S3 until the target number of cell suspension microspheres on the culture surface is reached; S5. Add a hydrogel containing organoid inducers to the culture surface, and culture the cell suspension microspheres on the culture surface to form organoids.

8. The method of operating an organoid molding device of claim 7, wherein, Further comprising the following steps: in steps S3, S4 and S5, the optical detection unit (7) detects the diameter of the cell suspension microspheres in real time, and then feeds back to the data acquisition card (5).

9. The method of operating an organoid molding device according to claim 7 or 8, wherein, The specific steps include: S1. The controller (4) controls the first pinch valve (33) and the second pinch valve (34) to open, and the controller (4) controls the pump (1) to rotate reversely to suck the cell suspension containing cells or microtissues into the spiral pipe (2) through the nozzle hose (31) for standby; S2. The controller (4) controls the first pinch valve (33) to remain open and the second pinch valve (34) to close, and the pump (1) rotates forwardly for one beat to extrude the cell suspension from the spiral pipe (2) to fill the space of the nozzle hose (31); S3. The controller (4) controls the first pinch valve (33) to close and the second pinch valve (34) to open, and the controller (4) controls the electromagnetic valve (36) to open, so that the filtered high-pressure gas is released through the needle (32) by the air pump (35), thereby pushing a certain volume of cell suspension in the nozzle hose (31) out to form cell suspension microspheres, and finally dropping and adhering to the designated position on the culture surface.

10. The method of operating an organoid forming device according to claim 7 or 8, wherein, The specific steps include: S1. The controller (4) controls the third pinch valve (37) to open, and the controller (4) controls the pump (1) to start reversely to suck the cell suspension containing cells or microtissues into the spiral pipe (2) through the needle (32) and the nozzle hose (31) for standby; S2. The controller (4) controls the pump (1) to start forwardly at low speed to stably push the cell suspension into the nozzle hose (31); S3. The high-voltage direct current source (38) is turned on to electrify the needle (32), and then the third pinch valve (37) is stably opened and closed at a certain frequency. When the third pinch valve (37) is closed, the cell suspension at the end of the nozzle hose (31) is extruded to the end of the needle (32), and then the cell suspension breaks through the surface tension and flies out to form cell suspension microspheres under the action of high-voltage electrostatic force, and finally drops and adheres to the designated position on the culture surface.

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