An organoid spheroid preparation device and method

Abstract

The application discloses an organoid sphere preparation device and method, and relates to the field of organoid sphere preparation.The organoid sphere preparation device comprises a platform, a microfluidic chip, a switching mechanism, a first liquid pushing / sucking mechanism and a second liquid pushing / sucking mechanism.The microfluidic chip comprises a chip body arranged at the top of the platform and a liquid inlet flow channel, a liquid storage flow channel, a liquid outlet flow channel, a sphere outlet flow channel and a continuous phase liquid inlet flow channel arranged in the chip body.The first end of the liquid inlet flow channel is connected with the first liquid pushing / sucking mechanism, the first end of the liquid storage flow channel is connected with the first liquid pushing / sucking mechanism, the first end of the sphere outlet flow channel is in communication with the first end of the liquid outlet flow channel and the first end of the continuous phase liquid inlet flow channel, the second end of the sphere outlet flow channel extends to the top of the chip body and is used for being connected with a sphere outlet connecting pipe, and the second end of the continuous phase liquid inlet flow channel is connected with the second liquid pushing / sucking mechanism.The application can realize high-throughput preparation of organoid spheres, has high automation degree and can realize large-scale production and commercial use.

Description

Technical Field

[0001] This invention relates to the field of medical devices, specifically to an apparatus and method for preparing organoid spheres. Background Technology

[0002] Cancer threatens the health of all humanity. The heterogeneity of cancer means that the same drug will elicit different response rates in different individuals. Traditional 2D culture methods suffer from low modeling success rates and the loss of the tumor microenvironment, which determines drug sensitivity, severely limiting their clinical application. While patient-specific xenograft models (PDX) can reconstruct the tumor microenvironment and be used to test the efficacy of different drugs, their high cost and long development cycle limit their widespread clinical application. Tumor organoids, which involve extracting cancer cells and the stromal cells constituting their microenvironment from a patient's tumor tissue and culturing them in vitro in 3D, have been shown to highly reconstruct the tumor microenvironment and maintain the phenotype of the patient's cancer cells, and can be used to predict drug sensitivity in patients.

[0003] The preparation of tumor organoids mainly involves the preparation of organoid spheroids, the maturation of organoid spheroids, and the culture of organoid spheroids. Current methods for preparing organoid spheroids typically involve manually adding a cell mixture to a pipette, then using the pipette tip to form a spherical droplet of the mixed cell matrix. The size of the droplet is controlled by adjusting factors such as injection speed, contact angle, and pipette tip diameter to create a uniform droplet that serves as the organoid spheroid. This method relies on manual operation, has low automation, and is difficult to achieve high-throughput preparation of organoid spheroids, making it unsuitable for large-scale production and commercial use. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides an organoid sphere preparation device and method, which can realize high-throughput preparation of organoid spheres, with a high degree of automation, and can realize large-scale production and commercial use.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] A first aspect of the present invention provides an organoid spheroid preparation apparatus, comprising a platform, a microfluidic chip, a switching mechanism, a first push / aspirate mechanism, and a second push / aspirate mechanism; the microfluidic chip includes a chip body disposed at the top of the platform and a liquid inlet channel, a liquid storage channel, a liquid outlet channel, a spheroid outlet channel, and a continuous phase liquid inlet channel disposed within the chip body; a first end of the liquid inlet channel is connected to the first push / aspirate mechanism, the first push / aspirate mechanism being used to connect to a sample injection tube; a first end of the liquid storage channel is connected to the first push / aspirate mechanism; a first end of the spheroid outlet channel is connected to the first end of the liquid outlet channel and the first end of the continuous phase liquid inlet channel, respectively; and a second end of the spheroid outlet channel extends to the chip body. The top of the chip body is used to connect to the ball outlet connecting pipe. The second end of the continuous phase liquid inlet channel is connected to the second push / suction mechanism, which is used to connect to the liquid storage container. The switching mechanism is disposed at the top of the chip body and has a connecting channel. The switching mechanism can rotate relative to the chip body between a first position and a second position. When the switching mechanism is in the first position, the first end of the connecting channel is connected to the second end of the liquid storage channel and the second end of the connecting channel is connected to the second end of the liquid inlet channel. When the switching mechanism is in the second position, the first end of the connecting channel is connected to the second end of the liquid storage channel and the second end of the connecting channel is connected to the second end of the liquid outlet channel.

[0007] As a preferred technical solution, the chip body is provided with a dispersed phase liquid inlet channel, a first liquid storage channel and a continuous phase liquid inlet channel. The first end of the dispersed phase liquid inlet channel is connected to the first end of the liquid inlet channel, and the second end of the dispersed phase liquid inlet channel extends to the top of the chip body. The first end of the first liquid storage channel is connected to the first end of the liquid storage channel, and the second end of the first liquid storage channel extends to the top of the chip body. The first end of the continuous phase liquid inlet channel is connected to the second end of the continuous phase liquid inlet channel, and the second end of the continuous phase liquid inlet channel extends to the top of the chip body.

[0008] As a preferred technical solution, the top of the chip body is provided with a dispersed phase inlet connector, a storage connector, a continuous phase inlet connector, and a ball outlet connector. The dispersed phase inlet connector is connected to the second end of the dispersed phase inlet channel. The dispersed phase inlet connector is connected to the first push / suction mechanism through a first connecting tube. The first push / suction mechanism is used to connect to the sample injection tube through a second connecting tube. The storage connector is connected to the second end of the first storage channel. The storage connector is connected to the first push / suction mechanism through a third connecting tube. The continuous phase inlet connector is connected to the second end of the continuous phase inlet channel. The continuous phase inlet connector is connected to the second push / suction mechanism through a fourth connecting tube. The second push / suction mechanism is used to connect to the storage container through a fifth connecting tube. The ball outlet connector is connected to the second end of the ball outlet channel. The ball outlet connector is used to connect to the ball outlet connecting tube.

[0009] As a preferred technical solution, the chip body is provided with a dispersed phase outlet channel, a second liquid storage channel, and an outlet connection channel. The first end of the dispersed phase outlet channel is connected to the second end of the liquid inlet channel, and the second end of the dispersed phase outlet channel extends to the top of the chip body. The first end of the second liquid storage channel is connected to the second end of the liquid storage channel, and the second end of the second liquid storage channel extends to the top of the chip body. The first end of the outlet connection channel is connected to the second end of the outlet channel, and the second end of the outlet connection channel extends to the top of the chip body.

[0010] As a preferred technical solution, the switching mechanism includes a knob and a switching plate. A ring is formed at the top of the chip body. The second end of the dispersed phase outlet channel, the second end of the second storage channel, and the second end of the outlet connection channel are all located inside the ring. The bottom end of the knob is housed within the ring and can rotate relative to the chip body between a first position and a second position. The bottom end of the knob has a mounting cavity. The switching plate is disposed in the mounting cavity. The bottom end of the switching plate is in close contact with the top end of the chip body and has the connection channel. Rotation of the knob can drive the switching plate to rotate relative to the chip body between a first position and a second position. When the knob and the switching plate are in the first position, the first end of the connection channel is connected to the second end of the second storage channel, and the second end of the connection channel is connected to the second end of the dispersed phase outlet channel. When the knob and the switching plate are in the second position, the first end of the connection channel is connected to the second end of the second storage channel, and the second end of the connection channel is connected to the second end of the outlet connection channel.

[0011] As a preferred technical solution, it also includes a drive mechanism disposed at the top of the platform, the top of the knob being connected to the drive mechanism, and the drive mechanism being used to drive the knob to rotate between a first position and a second position.

[0012] As a preferred technical solution, the liquid storage channel is a serpentine channel.

[0013] As a preferred technical solution, the size of the ball outlet channel is larger than the size of the continuous phase inlet channel.

[0014] As a preferred technical solution, both the first push / suction mechanism and the second push / suction mechanism are injection pumps.

[0015] A second aspect of the present invention provides a method for preparing organoid spheroids, using the organoid spheroid preparation apparatus described above, comprising the following steps: S1, rotating a switching mechanism to a first position, such that the first end of the connecting channel is connected to the second end of the storage channel, and the second end of the connecting channel is connected to the second end of the inlet channel; S2, using a first push / suction mechanism to aspirate a dispersed phase containing cells from a sample inlet tube and pushing the aspirated dispersed phase through the inlet channel and the connecting channel into the storage channel, so as to store the dispersed phase through the storage channel; S3, rotating the switching mechanism to a second position, such that the first end of the connecting channel is connected to the second end of the storage channel, and The second end of the connecting channel is connected to the second end of the outlet channel; S4, the dispersed phase stored in the storage channel is pushed into the outlet channel through the connecting channel by the first push / suction mechanism. The dispersed phase in the outlet channel can enter the first end of the ball outlet channel. At the same time, the continuous phase in the storage container is sucked up by the second push / suction mechanism and pushed into the continuous phase inlet channel. When the continuous phase in the continuous phase inlet channel enters the first end of the ball outlet channel, the continuous phase can shear the dispersed phase in the first end of the ball outlet channel into organoid spheres of uniform size. The sheared organoid spheres can enter the ball outlet channel. Thus, the organoid spheres are prepared.

[0016] The beneficial effects of this invention are: by providing liquid inlet channels, liquid storage channels, liquid outlet channels, ball outlet channels, and continuous phase liquid inlet channels within the chip body, and by providing a first push / suction mechanism, a second push / suction mechanism, and a switching mechanism, this invention can achieve high-throughput preparation of organoid spheres, with a high degree of automation, improved preparation efficiency, and the ability to achieve large-scale production and commercial use. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Figure 1 This is a schematic diagram of the structure of an organoid spheroid preparation device provided in an embodiment of the present invention;

[0019] Figure 2 yes Figure 1 A schematic diagram of the organoid spheroid preparation device after removing the refrigeration mechanism;

[0020] Figure 3 yes Figure 1 A top view schematic diagram of the microfluidic chip and switching mechanism of the organoid spheroid preparation device shown;

[0021] Figure 4 yes Figure 3 A cross-sectional schematic diagram of the microfluidic chip in the organoid spheroid preparation device shown;

[0022] Figure 5 yes Figure 3 An exploded view of the microfluidic chip and switching mechanism of the organoid spheroid preparation device from a first angle;

[0023] Figure 6 yes Figure 3 An exploded view of the microfluidic chip and switching mechanism of the organoid spheroid preparation device from a second angle;

[0024] Figure 7 Based on Figure 1 The illustrated organoid spheroid preparation apparatus provides a flowchart of a method for preparing organoid spheroids. Detailed Implementation

[0025] The following will clearly and completely describe the concept, specific structure, and technical effects of the present invention in conjunction with embodiments and accompanying 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, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Furthermore, all connections / linkages involved in the patent do not simply refer to direct contact between components, but rather to the ability to form a better connection structure by adding or reducing connecting accessories according to specific implementation conditions. The various technical features in this invention can be combined interactively without contradicting each other.

[0026] Please refer to Figure 1 and Figure 2 An embodiment of the present invention provides an organoid spheroid preparation device, comprising a platform 10, a microfluidic chip 20, a switching mechanism 30, a first push / suction mechanism 60, a second push / suction mechanism 50, a driving mechanism 70, and a cooling mechanism 80.

[0027] Platform 10 is positioned at the top of the cooling mechanism 80. The cooling mechanism 80 is an existing structure. The cooling mechanism 80 mainly consists of a custom-designed thermoelectric cooling (Peltier semiconductor cooling) module, a heat sink, and a temperature sensor. The microfluidic chip 20 and the drive mechanism 70 are both positioned at the top of platform 10. The switching mechanism 30 is positioned at the top of the microfluidic chip 20. The first push / suction mechanism 60 and the second push / suction mechanism 50 are positioned on one side of platform 10.

[0028] Combination Figures 3 to 6 As shown, the microfluidic chip 20 includes a chip body 21 disposed at the top of the platform 10, and liquid inlet channel 22, liquid storage channel 23, liquid outlet channel 24, ball outlet channel 262, dispersed phase liquid inlet channel 263, dispersed phase liquid outlet channel 266, first liquid storage channel 264, second liquid storage channel 267, liquid outlet connection channel 268, continuous phase liquid inlet channel 25, and continuous phase liquid inlet channel 265 disposed within the chip body 21.

[0029] The chip body 21 is a plate-like structure, made of materials such as PC (polycarbonate). In this embodiment, the top of the platform 10 is provided with a mounting slot, and the chip body 21 is disposed in the mounting slot.

[0030] In this embodiment, there are two microfluidic chips 20, which are spaced apart along the length of the platform 10. The number of mounting slots on the switching mechanism 30 and the platform 10 also corresponds to the number of microfluidic chips 20, which are also two each. It can be understood that the number of microfluidic chips 20 can be set according to actual conditions.

[0031] The inlet channel 22, the storage channel 23, the outlet channel 24, the ball outlet channel 262, and the continuous phase inlet channel 25 each have a first end and a second end. The dispersed phase inlet channel 263, the dispersed phase outlet channel 266, the first storage channel 264, the second storage channel 267, the outlet connection channel 268, and the continuous phase inlet channel 265 each have a first end and a second end.

[0032] Among them, the first end and the second end of the dispersed phase inlet channel 263, the dispersed phase outlet channel 266, the first liquid storage channel 264, the second liquid storage channel 267, the outlet connection channel 268, the continuous phase inlet channel 265, and the ball outlet channel 262 are respectively a closed end and an open end.

[0033] The first end of the dispersed phase inlet channel 263 is connected to the first end of the inlet channel 22, and the second end of the dispersed phase inlet channel 263 extends to the top of the chip body 21. The first end of the dispersed phase outlet channel 266 is connected to the second end of the inlet channel 22, and the second end of the dispersed phase outlet channel 266 extends to the top of the chip body 21. The first end of the first storage channel 264 is connected to the first end of the storage channel 23, and the second end of the first storage channel 264 extends to the top of the chip body 21. The first end of the second storage channel 267 is connected to the second end of the storage channel 23, and the second end of the second storage channel 267 extends to the top of the chip body 21. The first end of the outlet connection channel 268 is connected to the second end of the outlet channel 24, and the second end of the outlet connection channel 268 extends to the top of the chip body 21. The first end of the ball outlet channel 262 is connected to the first end of the continuous phase inlet channel 25 and the first end of the outlet channel 24, respectively, and the second end of the ball outlet channel 262 extends to the top of the chip body 21. The first end of the continuous phase inlet channel 265 is connected to the second end of the continuous phase inlet channel 25, and the second end of the continuous phase inlet channel 265 extends to the top of the chip body 21.

[0034] In this embodiment, the inlet channel 22, the storage channel 23, the outlet channel 24, and the continuous phase inlet channel 25 are arranged horizontally. The dispersed phase inlet channel 263 and the dispersed phase outlet channel 266 are arranged perpendicularly to the inlet channel 22. The first storage channel 264 and the second storage channel 267 are arranged perpendicularly to the storage channel 23. The outlet connection channel 268 is arranged perpendicularly to the outlet channel 24. The ball outlet channel 262 is arranged perpendicularly to the outlet channel 24 and the continuous phase inlet channel 25, forming a T-shaped structure between the ball outlet channel 262, the outlet channel 24, and the continuous phase inlet channel 25. The continuous phase inlet channel 265 is arranged perpendicularly to the continuous phase inlet channel 25.

[0035] The chip body 21 has a dispersed phase inlet connector 212, a reservoir connector 213, a continuous phase inlet connector 215, and a ball outlet connector 214 at its top. The dispersed phase inlet connector 212 is connected to the second end of the dispersed phase inlet channel 263. The dispersed phase inlet connector 212 is connected to the first push / suction mechanism 60 through the first connecting tube 92. The first push / suction mechanism 60 is used to connect to the sample injection tube through the second connecting tube. The sample injection tube is used to hold the dispersed phase containing cells, i.e., the cell mixture. The cells are primary tumor tissue cells extracted from primary tumor tissue. Understandably, the cells can also be, for example, stem cells, cell lines, etc., and the type and number of cells can be set according to the actual situation. The density of cells encapsulated in the dispersed phase is 1×107 cells / mL. Understandably, the cell density can be set according to the actual situation. The dispersed phase is preferably a matrix gel. Understandably, the dispersed phase can also be, for example, hydrogel, collagen, hyaluronic acid, alginate, etc., and can be set according to the actual situation.

[0036] The liquid storage connector 213 is connected to the second end of the first liquid storage channel 264. The liquid storage connector 213 is connected to the first push / suction mechanism 60 through the third connecting pipe. The first push / suction mechanism 60 is used to draw the dispersed phase in the sample inlet tube through the second connecting pipe and push the drawn dispersed phase through the first connecting pipe 92, the dispersed phase inlet connector 212, the dispersed phase inlet channel 263, the inlet flow channel 22, the dispersed phase outlet channel 266, the connecting channel 331 of the switching mechanism 30, and the second liquid storage channel 267 into the liquid storage flow channel 23, so as to store the dispersed phase through the liquid storage flow channel 23. The continuous phase inlet connector 215 is connected to the second end of the continuous phase inlet channel 265. The continuous phase inlet connector 215 is connected to the second push / suction mechanism 50 through the fourth connecting pipe 93. The second push / suction mechanism 50 is used to connect to the first interface 41 of the liquid storage container 40 through the fifth connecting pipe 94. The second push / suction mechanism 50 is used to draw the continuous phase from the storage container 40 via the fifth connecting pipe 94 and push the drawn continuous phase into the continuous phase inlet channel 25 via the second end of the fourth connecting pipe 93, the continuous phase inlet connector 215, and the continuous phase inlet channel 265. The storage container 40 can be located between the first push / suction mechanism 60 and the second push / suction mechanism 50. The storage container 40 is used to hold the continuous phase, which is preferably an electronically fluorinated liquid. The electronically fluorinated liquid has good shear force and can shear the dispersed phase containing cells into uniformly sized organoid spheres. Understandably, the continuous phase can also be a material with good cell compatibility and immiscibility with water, such as vegetable oil, and can be set according to the actual situation.

[0037] The ball outlet connector 214 is connected to the second end of the ball outlet channel 262. The ball outlet connector 214 is used to connect to the ball outlet connecting tube 95. The end of the ball outlet connecting tube 95 away from the ball outlet connector 214 is wound into several turns and then placed in the receiving cavity of the heat curing device.

[0038] The first push / suction mechanism 60 and the second push / suction mechanism 50 are both injection pumps, and the injection pumps are existing structures.

[0039] In this embodiment, the angle between the line connecting the second end of the dispersed phase outlet channel 266 and the second end of the second liquid storage channel 267 and the line connecting the second end of the outlet connection channel 268 and the second end of the second liquid storage channel 267 is 90 degrees. Understandably, the angle can also be other.

[0040] The switching mechanism 30 is located at the top of the chip body 21.

[0041] Specifically, the switching mechanism 30 includes a knob 32 and a switching plate 33. An upwardly extending ring 2111 is formed at the top of the chip body 21. The second end of the dispersed phase outlet channel 266, the second end of the second liquid storage channel 267, and the second end of the outlet connection channel 268 are all located inside the ring 2111. The bottom end of the knob 32 is adapted to the ring 2111. The bottom end of the knob 32 is housed within the ring 2111 and is in close contact with the top of the chip body 21. The knob 32 can rotate relative to the chip body 21 between a first position and a second position. The bottom end of the knob 32 has a mounting cavity 322, and the switching plate 33 is disposed within the mounting cavity 322, with the bottom end of the switching plate 33 flush with the bottom end of the knob 32. The bottom end of the switching plate 33 is in close contact with the top of the chip body 21 and has a connection channel 331. The connecting channel 331 has a first end 331a and a second end 331b. The first end 331a of the connecting channel 331 is located at the center of the bottom end of the switching plate 33, and the second end 331b of the connecting channel 331 is located to one side of the center of the bottom end of the switching plate 33. The first end 331a of the connecting channel 331 is connected to the second end of the second liquid storage channel 267. Rotation of the knob 32 can drive the switching plate 33 to rotate relative to the chip body 21 between a first position and a second position. Since the first end 331a of the connecting channel 331 is located at the center of the bottom end of the switching plate 33, the first end 331a of the connecting channel 331 can always remain connected to the second end of the second liquid storage channel 267 during the rotation of the switching plate 33. That is, when the knob 32 and the switching plate 33 are in the first position, the first end 331a of the connecting channel 331 is connected to the second end of the second liquid storage channel 267. Thus, the first end 331a of the connecting channel 331 can be connected to the second end of the liquid storage channel 23 through the second liquid storage channel 267, and the second end 331b of the connecting channel 331 is connected to the second end of the dispersed phase outlet channel 266. The second end of the connecting channel 331 is connected to the second end of the inlet channel 22 through the dispersed phase outlet channel 266. When the knob 32 and the switching plate 33 are in the second position, the first end 331a of the connecting channel 331 is connected to the second end of the second storage channel 267, so the first end 331a of the connecting channel 331 is connected to the second end of the storage channel 23 through the second storage channel 267, and the second end 331b of the connecting channel 331 is connected to the second end of the outlet connecting channel 268, so the second end of the connecting channel 331 is connected to the second end of the outlet channel 24 through the outlet connecting channel 268.

[0042] A knob cover 2112 is fixedly sleeved on the outer periphery of the ring portion 2111. The knob 32 is housed within the knob cover 2112. An elastic element 34, preferably a spring, is sleeved on the outer periphery of the knob 32. One end of the elastic element 34 is connected to the knob 32, and the other end is connected to the top inside the knob cover 2112. The elastic element 34 is in a compressed state, and it applies pressure to the knob 32, ensuring tight contact between the knob 32, the switching plate 33, and the top of the chip body 21 to achieve a seal. Furthermore, the length of the elastic element 34 can be dynamically adjusted during subsequent rotation operations to prevent interference, friction, and damage from compression between the knob 32, the switching plate 33, and the chip body 21.

[0043] A drive mechanism 70 is located at the top of the platform 10. Specifically, two mounting posts 12 are provided on one side of the microfluidic chip 20 at the top of the platform 10. A mounting plate 13 is fitted around the outer periphery of the two mounting posts 12, and the mounting plate 13 is located above the microfluidic chip 20 and the switching mechanism 30. The drive mechanism 70 can be, for example, a rotary cylinder or a motor. The top of the knob 32 has a groove 323, and the top of the knob cover 2112 has an opening corresponding to the groove 323. The output end of the drive mechanism 70 passes through the through hole of the mounting plate 13 and the opening of the knob cover 2112 and engages with the groove 323 of the knob 32. The drive mechanism 70 is used to drive the knob 32 to rotate relative to the chip body 21 between a first position and a second position, thereby driving the switching plate 33 to rotate relative to the chip body 21 between the first position and the second position. The number of mounting posts 12 can be set according to actual conditions. Through the drive mechanism, the rotation of the knob 32 and the switching plate 33 between the first position and the second position can be automated and is easy to use.

[0044] With the above structure, in practical application, the knob 32 and the switching plate 33 are first driven to rotate to the first position by the drive mechanism 70. At this time, the first end 331a of the connecting channel 331 is connected to the second end of the liquid storage channel 23 through the second liquid storage channel 267, and the second end 331b of the connecting channel 331 is connected to the second end of the liquid inlet channel 22 through the dispersed phase liquid outlet channel 266. Then, the first push / suction mechanism 60 draws the dispersed phase from the sample inlet tube through the second connecting tube and pushes the drawn dispersed phase into the storage channel 23 through the first connecting tube 92, the dispersed phase inlet connector 212, the dispersed phase inlet channel 263, the inlet flow channel 22, the dispersed phase outlet channel 266, the connecting channel 331, and the second storage channel 267, so as to store the dispersed phase through the storage channel 23. At this time, the cooling mechanism 80 cools the dispersed phase stored in the platform 10 and the storage channel 23 to ensure that the liquid temperature of the dispersed phase is between 0-10℃, thereby preventing the dispersed phase from solidifying and ensuring its low viscosity physical state for microdroplet formation. Then, the drive mechanism 70 drives the knob 32 and the switching plate 33 to rotate to the second position. At this time, the first end 331a of the connecting channel 331 is connected to the second end of the liquid storage channel 23 through the second liquid storage channel 267, and the second end 331b of the connecting channel 331 is connected to the second end of the liquid outlet channel 24 through the liquid outlet connecting channel 268. Then, the first push / suction mechanism 60 pushes the dispersed phase stored in the liquid storage channel 23 into the liquid outlet channel 24 through the second liquid storage channel 267, the connecting channel 331, and the liquid outlet connecting channel 268 via the third connecting pipe. The dispersed phase in the liquid outlet channel 24 can enter the first end of the ball outlet channel 262. Simultaneously, the second push / suction mechanism 50 draws the continuous phase from the storage container 40 through the fifth connecting pipe 94 and pushes the drawn continuous phase into the continuous phase inlet channel 25 via the fourth connecting pipe 93, the continuous phase inlet connector 215, and the second end of the continuous phase inlet channel 265. When the continuous phase in the continuous phase inlet channel 25 enters the first end of the outlet channel 262, under the pressure of the first push / suction mechanism 60 and the second push / suction mechanism 50, the continuous phase can shear the dispersed phase entering the first end of the outlet channel 262 into uniformly sized organoid spheres. The sheared organoid spheres can then enter the outlet channel 262, thus completing the preparation of the organoid spheres. Afterward, the organoid spheres can enter the outlet connecting pipe 95 via the second end of the outlet channel 262 and the outlet connector 214. Then, the organoid spheres can be heated and matured by a heating curing device, and then the matured organoid spheres can be cultured.

[0045] The present invention enables high-throughput preparation of organoid spheres with a high degree of automation, and can achieve large-scale production and commercial use by using a liquid inlet channel 22, a liquid storage channel 23, a liquid outlet channel 24, a sphere outlet channel 262, and a continuous phase liquid inlet channel 25 provided in the chip body 21, as well as a first push / suction mechanism 60, a second push / suction mechanism 50, and a switching mechanism 30. Furthermore, the inlet channel 22, storage channel 23, outlet channel 24, ball outlet channel 262, and continuous phase inlet channel 25 are all located within the chip body 21, resulting in a small size and low cost. The rotation of the switching mechanism 30 connects the inlet channel 22 to the storage channel 23 or vice versa. This allows the dispersed phase to be transferred to the storage channel 23 for storage under the action of the first push / suction mechanism 60, and then transferred to the outlet channel 24 under the same action of the first push / suction mechanism 60. This transfer of the dispersed phase occurs on the same carrier, minimizing phase loss and improving preparation efficiency. Simultaneously, the storage channel 23, with its small volume, prevents waste of the dispersed phase. This invention is particularly suitable for applications with small dispersed phase volumes, enabling the micro-preparation of organoid spheres.

[0046] In this embodiment, the liquid storage channel 23 is preferably a serpentine channel, which can reduce the size of the chip body 21. The inlet channel 22, the liquid storage channel 23, the outlet channel 24, and the continuous phase inlet channel 25 all have square cross-sectional shapes, with a side length of, for example, 1 mm. The dispersed phase inlet channel 263, the dispersed phase outlet channel 266, the first liquid storage channel 264, the second liquid storage channel 267, the outlet connection channel 268, the ball outlet channel 262, and the continuous phase inlet channel 265 all have circular cross-sectional shapes. Among them, the inner diameter of the ball outlet channel 262 is, for example, 0.45 mm. The size of the ball outlet channel 262 is larger than that of the continuous phase inlet channel 25, which allows the cut organoid spheres to smoothly enter the ball outlet channel 262 and avoids the phenomenon of sphere jamming. The size of the continuous phase inlet channel 25 is the same as that of the outlet channel 24.

[0047] Please refer to Figure 7 Based on the above-mentioned organoid spheroid preparation apparatus, this invention also provides a method for preparing organoid spheroids, comprising the following steps:

[0048] S1. Rotate the switching mechanism 30 to the first position so that the first end 331a of the connecting channel 331 is connected to the second end of the liquid storage channel 23, and the second end of the connecting channel 331 is connected to the second end of the liquid inlet channel 22.

[0049] Specifically, the drive mechanism 70 drives the knob 32 and the switching plate 33 to rotate to the first position, so that the first end 331a of the connecting channel 331 is connected to the second end of the liquid storage channel 23 through the second liquid storage channel 267, and the second end 331b of the connecting channel 331 is connected to the second end of the liquid inlet channel 22 through the dispersed phase outlet channel 266.

[0050] S2. The first push / suction mechanism 60 draws the dispersed phase from the sample inlet tube and pushes it through the first connecting tube 92, dispersed phase inlet connector 212, dispersed phase inlet channel 263, inlet flow channel 22, dispersed phase outlet channel 266, connecting channel 331, and second storage channel 267 into the storage flow channel 23, so as to store the dispersed phase through the storage flow channel 23. The cooling mechanism 80 can cool the dispersed phase stored in the platform 10 and the storage flow channel 23.

[0051] S3. Rotate the switching mechanism 30 to the second position so that the first end of the connecting channel 331 is connected to the second end of the liquid storage channel 23, and the second end of the connecting channel 331 is connected to the second end of the liquid outlet channel 24.

[0052] Specifically, the drive mechanism 70 drives the knob 32 and the switching plate 33 to rotate to the second position, so that the first end 331a of the connecting channel 331 is connected to the second end of the liquid storage channel 23 through the second liquid storage channel 267, and the second end 331b of the connecting channel 331 is connected to the second end of the liquid outlet channel 24 through the liquid outlet connecting channel 268.

[0053] S4. The dispersed phase stored in the liquid storage channel 23 is pushed into the liquid outlet channel 24 through the second liquid storage channel 267, the connecting channel 331, and the liquid outlet connecting channel 268 by the first push / suction mechanism 60. The dispersed phase in the liquid outlet channel 24 can enter the first end of the ball outlet channel 262. At the same time, the continuous phase in the liquid storage container 40 is drawn by the second push / suction mechanism 50 and pushed into the continuous phase inlet channel 25 through the fourth connecting pipe 93, the continuous phase inlet connector 215, and the second end of the continuous phase inlet channel 265. When the continuous phase in the continuous phase inlet channel 25 enters the first end of the ball outlet channel 262, under the pressure of the first push / suction mechanism 60 and the second push / suction mechanism 50, the continuous phase can shear the dispersed phase entering the first end of the ball outlet channel 262 into organoid spheres of uniform size. The sheared organoid spheres can enter the ball outlet channel 262, thus completing the preparation of the organoid spheres.

[0054] Afterwards, the organoid spheres can enter the outlet connecting tube 95 through the second end of the outlet channel 262 and the outlet connector 214. Then, the organoid spheres can be heated and matured by the heating curing device, and then the matured organoid spheres can be cultured.

[0055] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. An apparatus for preparing organoid spheres, characterized in that, Includes a platform, a microfluidic chip, a switching mechanism, a first push / suction mechanism, and a second push / suction mechanism; The microfluidic chip includes a chip body disposed at the top of the platform and a liquid inlet channel, a liquid storage channel, a liquid outlet channel, a ball outlet channel, and a continuous phase liquid inlet channel disposed within the chip body. The first end of the liquid inlet channel is connected to the first push / suction mechanism, which is used to connect to the sample injection tube. The first end of the liquid storage channel is connected to the first push / suction mechanism. The first end of the ball outlet channel is connected to the first end of the liquid outlet channel and the first end of the continuous phase liquid inlet channel, respectively. The second end of the ball outlet channel extends to the top of the chip body and is used to connect to the ball outlet connecting tube. The second end of the continuous phase liquid inlet channel is connected to the second push / suction mechanism, which is used to connect to the liquid storage container. The switching mechanism is disposed at the top of the chip body and has a connection channel. The switching mechanism can rotate relative to the chip body between a first position and a second position. When the switching mechanism is in the first position, the first end of the connection channel is connected to the second end of the liquid storage channel and the second end of the connection channel is connected to the second end of the liquid inlet channel. When the switching mechanism is in the second position, the first end of the connection channel is connected to the second end of the liquid storage channel and the second end of the connection channel is connected to the second end of the liquid outlet channel.

2. The organoid spheroid preparation apparatus according to claim 1, characterized in that, The chip body is provided with a dispersed phase liquid inlet channel, a first liquid storage channel and a continuous phase liquid inlet channel. The first end of the dispersed phase liquid inlet channel is connected to the first end of the liquid inlet channel, and the second end of the dispersed phase liquid inlet channel extends to the top of the chip body. The first end of the first liquid storage channel is connected to the first end of the liquid storage channel, and the second end of the first liquid storage channel extends to the top of the chip body. The first end of the continuous phase liquid inlet channel is connected to the second end of the continuous phase liquid inlet channel, and the second end of the continuous phase liquid inlet channel extends to the top of the chip body.

3. The organoid spheroid preparation apparatus according to claim 2, characterized in that, The top of the chip body is provided with a dispersed phase inlet connector, a storage connector, a continuous phase inlet connector, and a ball outlet connector. The dispersed phase inlet connector is connected to the second end of the dispersed phase inlet channel. The dispersed phase inlet connector is connected to the first push / suction mechanism through a first connecting tube. The first push / suction mechanism is used to connect to the sample injection tube through a second connecting tube. The storage connector is connected to the second end of the first storage channel. The storage connector is connected to the first push / suction mechanism through a third connecting tube. The continuous phase inlet connector is connected to the second end of the continuous phase inlet channel. The continuous phase inlet connector is connected to the second push / suction mechanism through a fourth connecting tube. The second push / suction mechanism is used to connect to the storage container through a fifth connecting tube. The ball outlet connector is connected to the second end of the ball outlet channel. The ball outlet connector is used to connect to the ball outlet connecting tube.

4. The organoid spheroid preparation apparatus according to claim 1, characterized in that, The chip body is provided with a dispersed phase outlet channel, a second liquid storage channel and an outlet connection channel. The first end of the dispersed phase outlet channel is connected to the second end of the liquid inlet channel, and the second end of the dispersed phase outlet channel extends to the top of the chip body. The first end of the second liquid storage channel is connected to the second end of the liquid storage channel, and the second end of the second liquid storage channel extends to the top of the chip body. The first end of the outlet connection channel is connected to the second end of the outlet channel, and the second end of the outlet connection channel extends to the top of the chip body.

5. The organoid spheroid preparation apparatus according to claim 4, characterized in that, The switching mechanism includes a knob and a switching plate. A ring is formed at the top of the chip body. The second end of the dispersed phase outlet channel, the second end of the second storage channel, and the second end of the outlet connection channel are all located inside the ring. The bottom end of the knob is housed within the ring and can rotate relative to the chip body between a first position and a second position. The bottom end of the knob has a mounting cavity. The switching plate is disposed in the mounting cavity. The bottom end of the switching plate is in close contact with the top of the chip body and has the connection channel. Rotation of the knob can drive the switching plate to rotate relative to the chip body between a first position and a second position. When the knob and the switching plate are in the first position, the first end of the connection channel is connected to the second end of the second storage channel, and the second end of the connection channel is connected to the second end of the dispersed phase outlet channel. When the knob and the switching plate are in the second position, the first end of the connection channel is connected to the second end of the second storage channel, and the second end of the connection channel is connected to the second end of the outlet connection channel.

6. The organoid spheroid preparation apparatus according to claim 5, characterized in that, It also includes a drive mechanism disposed at the top of the platform, the top of the knob being connected to the drive mechanism, the drive mechanism being used to drive the knob to rotate between a first position and a second position.

7. The organoid spheroid preparation apparatus according to claim 1, characterized in that, The liquid storage channel is a serpentine channel.

8. The organoid spheroid preparation apparatus according to claim 1, characterized in that, The size of the ball outlet channel is larger than the size of the continuous phase inlet channel.

9. The organoid spheroid preparation apparatus according to claim 1, characterized in that, Both the first push / suction mechanism and the second push / suction mechanism are syringe pumps.

10. A method for preparing organoid spheroids, using the organoid spheroid preparation apparatus as described in any one of claims 1-9, characterized in that, Includes the following steps: S1. Rotate the switching mechanism to the first position so that the first end of the connecting channel is connected to the second end of the liquid storage channel, and the second end of the connecting channel is connected to the second end of the liquid inlet channel. S2. The dispersed phase containing cells is drawn from the sample tube by the first push / suction mechanism and pushed into the storage channel through the liquid inlet channel and connecting channel to store the dispersed phase. S3. Rotate the switching mechanism to the second position so that the first end of the connecting channel is connected to the second end of the liquid storage channel, and the second end of the connecting channel is connected to the second end of the liquid outlet channel. S4. The dispersed phase stored in the liquid storage channel is pushed into the liquid outlet channel through the connecting channel by the first push / suction mechanism. The dispersed phase in the liquid outlet channel can enter the first end of the ball outlet channel. At the same time, the continuous phase in the liquid storage container is sucked up by the second push / suction mechanism and pushed into the continuous phase inlet channel. When the continuous phase in the continuous phase inlet channel enters the first end of the ball outlet channel, the continuous phase can shear the dispersed phase in the first end of the ball outlet channel into organoid spheres of uniform size. The sheared organoid spheres can enter the ball outlet channel. In this way, the organoid spheres are prepared.

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