High-throughput drug screening and organoid culture device

Abstract

The invention discloses a high-throughput drug screening and organoid culture device, and aims to overcome the defects that conventional drug testing in a laboratory mainly depends on a porous plate, operation is generally complex, cell culture and drug stimulation lack dynamic perfusion, and cross contamination is prone to occurring. The system comprises a drug concentration generation module and an organoid culture module, the drug concentration generation module comprises a plurality of drug concentration generation units, and the organoid culture module comprises a plurality of organoid culture units corresponding to the drug concentration generation units. A medicine inlet, a culture medium solution inlet and a plurality of liquid outlets are formed in the medicine concentration generation unit; the organoid culture unit comprises a plurality of groups of organoid culture chambers corresponding to the liquid outlets, and the organoid culture chambers in the same group are communicated with one another. The high-throughput drug screening and organoid culture device is convenient to operate, whole-course dynamic culture of organoid culture and drug stimulation can be achieved, and cross contamination is not prone to occurring.

Description

Technical Field

[0001] The present invention relates to the field of biomedical equipment, and more specifically, to a high-throughput drug screening and organoid culture device. Background Art

[0002] Tumors pose a serious threat to human life and health and have become the leading cause of death worldwide. The world has been working hard to find and develop effective drugs or methods to treat tumors. Typical drug research relies on traditional cell models and animal experiments, but they have limitations. Two-dimensional cell culture systems lack physiological conditions such as cell-cell interactions, extracellular matrix, dynamic nutrient supply, and tumor microenvironment. Animal models differ greatly from humans in genetics, epigenetics, and physiology, and are usually unable to accurately predict the true response of tumor patients to drugs. There is an urgent need for new in vitro bionic models that can effectively simulate human pathophysiological responses to improve the success rate of drug transformation from basic research to clinical practice.

[0003] Fully humanized models are becoming the preferred method for cancer drug development. Multicellular spheroid tumor organoids are widely used by scientists because of their highly similar organ-level structures. Among them, patient-derived tumor organoids (PDO) are considered to be an ideal model for anti-tumor drug screening. The self-assembled cell population formed by this 3D culture system can exhibit properties similar to the in vivo microenvironment. When used in drug testing, it can more truly reflect the sensitivity of tumor patients to drugs, greatly improving the accuracy and effectiveness of cancer drug screening.

[0004] Every year, biopharmaceutical companies and research institutions invest a lot of resources in new drug development and testing. High-throughput drug screening is an effective means to improve drug screening efficiency and shorten the new drug development cycle. Conventional high-throughput drug testing in laboratories mainly relies on multi-well plates (96-well or 384-well plates), but they generally have shortcomings such as complex operations, lack of dynamic perfusion for cell culture and drug stimulation, and easy cross-contamination. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings, the present invention provides a high-throughput drug screening and organoid culture device, which is easy to operate, can realize the full dynamic culture of organoid culture and drug stimulation, and can better simulate the human microenvironment. As an in vitro model, the results of drug screening experiments are more accurate and reliable, and it is not easy to cross-contaminate.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: a high-throughput drug screening and organoid culture device, comprising a drug concentration generation module and an organoid culture module, the drug concentration generation module comprises a plurality of drug concentration generation units, the organoid culture module comprises a plurality of organoid culture units corresponding to the drug concentration generation unit, a drug inlet, a culture medium solution inlet and a plurality of liquid outlets are arranged on the drug concentration generation unit; the organoid culture unit comprises a plurality of groups of organoid culture chambers corresponding to the liquid outlets, and the organoid culture chambers in the same group are interconnected.

[0007] During operation, the cells are mixed with the extracellular matrix and inoculated at the bottom of the organoid culture chamber of the organoid culture unit, the drug concentration generation module is covered, and then the entire device is cultured in a CO2 / temperature control box. After the extracellular matrix becomes a gel, the culture medium solution is injected through the culture medium solution inlet and flows into the organoid culture chamber from the outlet. The culture medium solution is driven by an integrated micropump. Since the organoid culture chambers of the same group are interconnected, when the culture medium solution continues to perfuse until the liquid level reaches the upper edge of the organoid culture chamber, the culture medium solution enters the next organoid culture chamber. Similarly, after the culture medium solution enters the last organoid culture chamber, all organoid culture chambers in the same group are perfused. When the external driving force is continuously given, the waste liquid after perfusion flows outward, completing the continuous perfusion of the organoid and cleaning up metabolic waste.

[0008] After the organoids have grown and developed well, drug stimulation is performed. Driven by the integrated micropump, the drug and culture solution are respectively introduced into the drug concentration generation unit from the drug inlet and the culture medium solution inlet, forming multiple drugs of different concentrations flowing out from different outlets and flowing into different groups of organoid culture chambers. In this way, a single drug stimulation unit can complete multiple concentration stimulations of a drug for organoids and multiple parallel experiments of drug stimulation at the same concentration. If drug type and concentration experiments are performed on multiple organoid culture units of the organoid culture module at the same time, high-throughput organoid drug testing can be achieved. After the drug stimulation is completed, the sample can be tested after adding a suitable detection reagent for incubation using an integrated micropump.

[0009] The high-throughput drug screening and organoid culture device of this patent application is easy to operate, can realize the full dynamic culture of organoid culture and drug stimulation, and can better simulate the human microenvironment. As an in vitro model, the results of drug screening experiments are more accurate and reliable, and not prone to cross contamination.

[0010] Preferably, a waste liquid collection area is provided below the organoid culture module, and the waste liquid collection area receives liquid overflowing from the organoid culture chamber.

[0011] The waste liquid after perfusion in the organoid culture room overflows into the waste liquid collection area, completing the continuous perfusion of the organoids and cleaning up metabolic waste.

[0012] Preferably, a waste liquid collection dish is detachably mounted at the bottom of the organoid culture module, and a waste liquid collection area is formed inside the waste liquid collection dish.

[0013] The waste liquid collection dish and the lower part of the organoid culture module are detachable, which makes it easy to remove the waste liquid collection dish for cleaning the waste liquid.

[0014] Preferably, the bottom surface of the organoid culture chamber is concave to form a culture trough with a hemispherical structure.

[0015] The arc-shaped structure and volume of the culture trough are compatible with the shape and size of organoid spheroids, and can be used for artificial inoculation or 3D printing to construct organoid spheroids. It helps to generate spheroids of uniform size and good consistency in the organoid culture module, thereby increasing the repeatability of the experiment and reducing experimental errors caused by differences in spheroid morphology and volume.

[0016] Preferably, a buffer zone is arranged outside the culture tank on the bottom surface of the organoid culture chamber; the liquid outlet is arranged corresponding to the upper part of the organoid culture chamber, and the liquid outlet is staggered with the culture tank.

[0017] After the culture fluid reaches the buffer zone along the front wall of the organoid culture chamber, it slowly flows into the bottom of the organoid culture chamber to promote the growth and development of organoids. The buffer zone avoids direct contact between the culture fluid and the organoids, reducing the physical damage of the fluid flow to the organoids during the culture process.

[0018] Preferably, an overflow channel is provided between the organoid culture chambers in the same group, and the overflow channel is provided at the upper part of the organoid culture chamber.

[0019] During the perfusion process, the flow of liquid is achieved through the overflow channel set at the top of the organoid culture chamber, realizing the full dynamic culture of organoid culture and drug stimulation, which can better simulate the human microenvironment and is used as an in vitro model for drug screening experiments, making the results more accurate and reliable.

[0020] Preferably, the upper portion of the organoid culture chamber at one end of the same group is connected to the overflow hole, the overflow hole penetrates to the lower surface of the organoid culture unit, and the upper portion of the organoid culture chamber at the other end of the same group is aligned with the liquid outlet.

[0021] The culture medium and drugs are sent into the organoid culture chamber at the head end, and after being perfused through multiple organoid culture chambers, they flow out from the organoid culture chamber at the tail end through the overflow hole to ensure the perfusion effect.

[0022] Preferably, a positioning sleeve is provided at the lower edge of the drug concentration generating module, and the positioning sleeve is connected to the upper edge sleeve of the organoid culture module.

[0023] The positioning sleeve is used to realize the detachable connection between the drug concentration generation module and the organoid culture module, which is convenient for assembly and separation. When assembled together, the organoid culture and drug stimulation are carried out. When separated, the organoid culture module is placed in the microplate reader for detection.

[0024] Preferably, the drug concentration generating unit comprises a multi-stage branch microfluidic channel, the multi-stage branch microfluidic channel comprises a primary branch channel and a secondary branch channel, a plurality of primary microchannels are connected in parallel between the primary branch channel and the secondary branch channel, a plurality of secondary microchannels are connected in parallel to the secondary branch channel, the drug inlet and the culture medium solution inlet are both connected to the primary branch channel, and the plurality of secondary microchannel outlets are respectively connected to the plurality of liquid outlets.

[0025] The multi-stage branching microfluidic channel adopts the Christmas tree model concentration gradient formation principle. When the solution enters the main channel, the flow trend causes the solution to move to the upper and lower branches. Since the outer solution flows slower and the central solution flows faster, this speed difference causes the solution to diffuse. As the solution enters the branch channel, the shape and size changes of the branch channel will also cause changes in the fluid flow, resulting in changes in the speed of the liquid in the branch. The combined effect of these changes causes the solute in the solution to gradually form a concentration gradient in the channel.

[0026] Therefore, when the drug and culture medium solution are transported from the drug inlet and the culture medium solution inlet to the primary branch channel respectively, they pass through the primary microchannel, the secondary branch channel, and the secondary microchannel so that the liquid outlet outputs multiple drug solutions with different concentrations.

[0027] Preferably, the drug concentration generating module comprises a bottom plate and a top cover which are tightly connected together, the drug inlet and the culture medium solution inlet are arranged on the top cover, the liquid outlet is arranged on the bottom plate, and the multi-stage branch microfluidic channel is arranged on the lower surface of the top cover.

[0028] The drug concentration generation module, which is composed of a bottom plate and a top cover connected together, facilitates the processing of multi-stage branched microfluidic channels.

[0029] Compared with the prior art, the beneficial effects of the present invention are: (1) The high-throughput drug screening and organoid culture device of the present patent application is easy to operate, can realize the whole process of dynamic culture of organoid culture and drug stimulation, can better simulate the human microenvironment, and is used as an in vitro model for drug screening experiments. The results are more accurate and reliable, and not prone to cross-contamination; (2) A hemispherical culture tank is arranged at the bottom of the organoid culture chamber. The arc structure and volume of the culture tank are compatible with the shape and size of the organoid sphere, which can be suitable for artificial inoculation or 3D printing to construct organoid spheres, which helps to generate spheres with uniform size and good consistency in the organoid culture module, thereby increasing the repeatability of the experiment and reducing the experimental errors caused by the differences in the morphology and volume of the spheres; (3) After the culture fluid reaches the buffer zone along the front wall of the organoid culture chamber, it slowly flows into the bottom of the organoid culture chamber to promote the growth and development of the organoid. The buffer avoids direct contact between the culture fluid and the organoids, reducing the physical damage of the fluid flow to the organoids during the culture process; (4) The drug concentration generation module includes several drug concentration generation units, and the organoid culture module includes several organoid culture units corresponding to the drug concentration generation units. Drugs of different concentrations can be freely combined to meet various test needs, effectively improving the utilization rate of the well plate; (5) The multi-level branch microfluidic channel adopts the concentration gradient formation principle of the Christmas tree model, allowing multiple drugs and concentration gradient drug tests to be carried out simultaneously, providing a high-throughput experimental platform for drug screening, solving the problem of low throughput of current drug screening devices, and can quickly identify candidate drugs and evaluate the therapeutic effect of drugs on individual patients; (6) The integrated micropump system drives fluid flow and drug concentration gradient formation, so that a stable flow rate is formed in each microchannel and micropore, realizing the full dynamic culture of organoid culture and drug stimulation, better simulating the human microenvironment, and being used as an in vitro model for drug screening experiments. The results are more accurate and reliable; (7) After the drug stimulation is completed, the organoid culture module is disassembled and placed in an ELISA instrument for detection, or the ELISA instrument detection system is integrated into the device to achieve an integrated device from drug addition to result output. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a structural schematic diagram of the present invention.

[0031] Figure 2 It is a cross-sectional view of the present invention.

[0032] Figure 3 It is an exploded view of the present invention.

[0033] Figure 4 1 is a top view of the organoid culture unit of the present invention.

[0034] Figure 5 is a cross-sectional view of the organoid culture unit of the present invention.

[0035] Figure 6It is a structural diagram of the multi-stage branched microfluidic channel of the present invention.

[0036] Figure 7 It is a connection diagram of the simulation board of Example 2 of the present invention.

[0037] In the figure: 1. drug concentration generating module, 11. drug concentration generating unit, 110. drug inlet, 111. culture medium solution inlet, 112. liquid outlet, 113. multi-stage branch microfluidic channel, 1131. primary branch channel, 1132. secondary branch channel, 1133. primary microchannel, 1134. secondary microchannel, 12. top cover, 13. bottom plate, 14. positioning sleeve, 2. organoid culture module, 20. organoid culture unit, 200. organoid culture chamber, 201. culture tank, 202. buffer zone, 203. overflow channel, 204. overflow hole, 205. overflow tank, 3. waste liquid collection area, 30. waste liquid collection dish, 31. fixing buckle, 4. simulation board, 40. rotating disk, 41. connecting rod. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described in detail below through specific embodiments and in conjunction with the accompanying drawings: Example 1: A high-throughput drug screening and organoid culture device (see Figures 1 to 6 ), including a drug concentration generating module 1 and an organoid culture module 2, the drug concentration generating module 1 is placed above the organoid culture module 2, the drug concentration generating module 1 includes a plurality of drug concentration generating units 11, and the plurality of drug concentration generating units 11 are arranged in a rectangular array, the organoid culture module 2 includes a plurality of organoid culture units 20 corresponding one to one to the drug concentration generating units 11, and the plurality of organoid culture units 20 are also arranged in a rectangular array.

[0039] The drug concentration generating unit 11 is provided with a drug inlet 110 , a culture medium solution inlet 111 and a plurality of liquid outlets 112 ; the organoid culture unit 20 includes a plurality of groups of organoid culture chambers 200 corresponding to the liquid outlets 112 , and the organoid culture chambers 200 of the same group are interconnected.

[0040] A waste liquid collection area 3 is provided below the organoid culture module 2, and the waste liquid collection area 3 receives the liquid overflowing from the organoid culture chamber 200. The waste liquid overflow after the perfusion of the organoid culture chamber 200 enters the waste liquid collection area 3, completing the continuous perfusion of the organoid and cleaning of metabolic waste. A waste liquid collection dish 30 is detachably mounted on the lower part of the organoid culture module 2, and a waste liquid collection area 3 is formed inside the waste liquid collection dish 30. A fixing buckle 31 is provided on the inner wall of the waste liquid collection dish 30, and the lower part of the organoid culture module 2 is supported on the fixing buckle 31.

[0041] The bottom surface of the organoid culture chamber 200 is concave to form a hemispherical culture tank 201. The arc-shaped structure and volume of the culture tank 201 are compatible with the shape and size of the organoid sphere, and can be used for artificial inoculation or 3D printing to construct organoid spheres, which helps to generate spheres with uniform size and good consistency in the organoid culture module 2, thereby increasing the repeatability of the experiment and reducing the experimental errors caused by the differences in the morphology and volume of the spheres.

[0042] A buffer zone 202 is arranged outside the culture tank 201 at the bottom of the organoid culture chamber 200; the liquid outlet 112 is arranged corresponding to the upper part of the organoid culture chamber 200, and the liquid outlet 112 is offset from the culture tank 201. After the liquid flowing out of the liquid outlet 112 reaches the buffer zone 202 along the front wall of the organoid culture chamber 200, it slowly flows into the bottom of the organoid culture chamber 200 to promote the growth and development of the organoid, instead of falling directly to the culture tank 201, thereby avoiding direct contact between the liquid and the organoid and reducing the physical damage of the liquid flow to the organoid during the culture process.

[0043] An overflow channel 203 is provided between the organoid culture chambers 200 of the same group, and the overflow channel 203 is provided at the upper part of the organoid culture chamber 200. The upper part of the organoid culture chamber 200 at one end of the same group is connected to the overflow hole 204, and the overflow hole 204 passes through the lower surface of the organoid culture unit 20, and the upper part of the organoid culture chamber 200 at the other end of the same group is aligned with the liquid outlet 112, and the liquid outlet 112 is close to the inner wall of the organoid culture chamber 200. A plurality of organoid culture chambers 200 are provided on the organoid culture module 2 to form a perforated plate-like structure. The upper end of the organoid culture chamber 200 is open and the lower end is closed.

[0044] A positioning sleeve 14 is provided at the lower edge of the drug concentration generating module 1, and the positioning sleeve 14 is connected to the upper edge of the organoid culture module 2. The positioning sleeve 14 realizes the detachable connection between the drug concentration generating module 1 and the organoid culture module 2, which is convenient for assembly and separation. When assembled together, the organoids are cultured and stimulated by drugs. When separated, the organoid culture module 2 is placed in an ELISA instrument for detection. The drug concentration generating unit 11 includes a multi-stage branch microfluidic channel 113, and the multi-stage branch microfluidic channel 113 includes a primary branch channel 1131 and a secondary branch channel 1132. A plurality of primary microchannels 1133 are connected in parallel between the primary branch channel 1131 and the secondary branch channel 1132, and a plurality of secondary microchannels 1134 are connected in parallel to the secondary branch channel 1132. The drug inlet 110 and the culture medium solution inlet 111 are both connected to the primary branch channel 1131, and the outlets of the plurality of secondary microchannels 1134 are respectively connected to the plurality of liquid outlets 112. The multi-stage branched microfluidic channel 113 adopts the Christmas tree model concentration gradient formation principle. When the solution enters the main channel, the flow trend causes the solution to move to the upper and lower branches. Since the outer solution flows slower and the central solution flows faster, this speed difference causes the solution to diffuse. As the solution enters the branch channel, changes in the shape and size of the branch channel will also cause changes in the fluid flow, resulting in changes in the speed of the liquid in the branch. The combined effect of these changes causes the solute in the solution to gradually form a concentration gradient in the channel.

[0045] Therefore, when the drug and culture medium solution are transported from the drug inlet 110 and the culture medium solution inlet 111 to the primary branch channel 1131 respectively, they pass through the primary microchannel 1133, the secondary branch channel 1132, and the secondary microchannel 1134 so that the liquid outlet 112 outputs multiple drug solutions of different concentrations.

[0046] The drug concentration generating module 1 includes a bottom plate 13 and a top cover 12 which are tightly connected together, a drug inlet 110 and a culture medium solution inlet 111 are arranged on the top cover 12, a liquid outlet 112 is arranged on the bottom plate 13, a multi-stage branched microfluidic channel 113 is arranged on the lower surface of the top cover 12, the drug inlet 110 and the culture medium solution inlet 111 are connected to the upper surface of the top cover 12, and the liquid outlet 112 is connected to the lower surface of the bottom plate 13.

[0047] Specifically, Figure 1 , Figure 2As shown, the drug concentration generation module 1, the organoid culture module 2, and the waste liquid collection dish 30 are arranged in sequence from top to bottom, the top cover 12 is sealed and connected with the bottom plate 13 to form the drug concentration generation module 1, the positioning sleeve 14 extends downward from the edge of the bottom plate 13 and is connected with the outer wall of the organoid culture module 2, and the liquid outlet 112 on the bottom plate 13 is arranged corresponding to the upper opening of the organoid culture chamber 200, and the liquid outlet 112 is arranged close to the inner wall of the organoid culture chamber 200. Three organoid culture chambers 200 form a group, the liquid outlet 112 corresponds to an organoid culture chamber 200 at the head end, the overflow hole 204 corresponds to an organoid culture chamber 200 at the tail end, and the overflow groove 205 is arranged on the upper part of the organoid culture chamber 200 at the tail end, and the upper end of the overflow hole 204 is connected to the bottom of the overflow groove 205. The bottom of the overflow channel 203 is flush with the bottom of the overflow groove 205. The waste liquid collection dish 30 is connected to the lower outer wall of the organoid culture module 2, and the fixing buckle 31 supports and positions the organoid culture module 2. A distance is set between the lower surface of the organoid culture module 2 and the bottom surface of the waste liquid collection dish 30 for storing waste liquid.

[0048] like Figure 3 , Figure 4 , Figure 5 As shown, the drug concentration generating module 1 includes eight drug concentration generating units 11, and the organoid culture module 2 includes eight organoid culture units 20. In the multi-stage branch microfluidic channel 113 of the drug concentration generating unit 11, three primary microchannels 1133 are connected in parallel between the primary branch channel 1131 and the secondary branch channel 1132, and four secondary microchannels 1134 are connected in parallel between the secondary branch channel 1132. The four secondary microchannels 1134 correspond to four liquid outlets 112. The primary microchannels 1133 and the secondary microchannels 1134 are both serpentine tubular structures. Each organoid culture unit 20 is provided with four groups of organoid culture chambers 200. The drug inlet 110 and the culture medium solution inlet 111 are both connected to the primary branch channel 1131, and the outlets of the four secondary microchannels 1134 are respectively connected to the four liquid outlets 112. The drug inlet 110 and the culture medium solution inlet 111 are respectively connected to two integrated micropumps through pipelines.

[0049] During operation, the first step is to assemble, test and sterilize. After the drug concentration generation module 1 and the organoid culture module 2 are assembled, a 75% alcohol solution is passed into the drug concentration generation module 1 and the organoid culture module 2 under the drive of the integrated micropump to observe whether the liquid flow runs normally according to the channel to check the sealing of the device and the smoothness of the channel. After the test, air is introduced to discharge the residual alcohol solution. After disassembling the device, dry it at 50°C and then sterilize it with ultraviolet light for more than 30 minutes.

[0050] The second step is to carry out organoid culture and dynamic perfusion, taking a drug screening experiment as an example. The four sides of the disinfected organoid culture module 2 are clamped in the fixed buckle 31 position on the waste liquid collection, so that it falls flat on the waste liquid collection area 3. The cells are mixed with the extracellular matrix and inoculated at the bottom of the organoid culture chamber 200. Cover the drug concentration generation module 1 and place it in a CO2 / temperature control box for culture. After the extracellular matrix becomes a gel, the culture medium is injected through the culture medium solution inlet 111, and the driving force is provided by an integrated micropump. After the culture fluid passes through the liquid outlet 112 and reaches the buffer zone 202 along the front wall of the organoid culture chamber 200, it slowly flows into the bottom of the organoid culture chamber 200 to promote the growth and development of the organoid. The buffer zone 202 avoids direct contact between the culture fluid and the organoid, reducing the physical damage of the liquid flow to the organoid during the culture process. When the culture solution continues to perfuse until the liquid level reaches the upper edge of the rear wall of the organoid culture chamber 200, the culture solution flows into the next organoid culture chamber 200 through the overflow channel 203, so that the entire organoid culture chamber 200 is perfused. When the external driving force is continuously applied, the waste liquid after perfusion enters the waste liquid collection dish 30 through the overflow groove 205 and the overflow hole 204, completing the continuous perfusion of the organoid and cleaning of metabolic waste.

[0051] The third step is to perform drug stimulation of the organoids. After the organoids grow and develop well, drug stimulation is performed. Driven by the integrated micropump, the drug and the culture solution are respectively introduced into the multi-stage branch microfluidic channel 113 of the drug concentration generation unit 11 from the drug inlet 110 and the culture medium solution inlet 111. After the drug and the culture medium solution are mixed through the primary branch channel 1131, the drug mixture with three concentrations is fully mixed through the primary microchannel 1133, and then enters the secondary branch channel 1132 to form a drug mixture with four concentrations. After passing through four secondary microchannels 1134, the drug mixture of four concentrations flows into drug stimulation group one, drug stimulation group two, drug stimulation group three and drug stimulation group four from four liquid outlets 112, and each drug stimulation group has three organoid culture chambers 200. In this way, a single drug concentration generation unit 11 can complete four concentration stimulations of a drug of the organoid and three parallel experiments of drug stimulation with the same concentration. If the eight organoid culture units 20 of the organoid culture module 2 are simultaneously subjected to drug type and concentration experiments, high-throughput organoid drug testing can be achieved.

[0052] The fourth step is to test the samples after drug stimulation. After adding appropriate detection reagents and incubating them using an integrated micropump, the samples can be tested. The organoid culture module 2 is compatible with existing microplate readers. When conducting experimental tests based on microplate readers, the organoid culture module 2 can be removed and directly placed in the microplate reader for testing.

[0053] Example 2: A high-throughput drug screening and organoid culture device (see Figures 1 to 6), including a drug concentration generating module 1 and an organoid culture module 2, the drug concentration generating module 1 is placed above the organoid culture module 2, the drug concentration generating module 1 includes a plurality of drug concentration generating units 11, and the plurality of drug concentration generating units 11 are arranged in a rectangular array, the organoid culture module 2 includes a plurality of organoid culture units 20 corresponding one to one to the drug concentration generating units 11, and the plurality of organoid culture units 20 are also arranged in a rectangular array.

[0054] The drug concentration generating unit 11 is provided with a drug inlet 110 , a culture medium solution inlet 111 and a plurality of liquid outlets 112 ; the organoid culture unit 20 includes a plurality of groups of organoid culture chambers 200 corresponding to the liquid outlets 112 , and the organoid culture chambers 200 of the same group are interconnected.

[0055] A waste liquid collection area 3 is provided below the organoid culture module 2, and the waste liquid collection area 3 receives the liquid overflowing from the organoid culture chamber 200. The waste liquid overflow after the perfusion of the organoid culture chamber 200 enters the waste liquid collection area 3, completing the continuous perfusion of the organoid and cleaning of metabolic waste. A waste liquid collection dish 30 is detachably mounted on the lower part of the organoid culture module 2, and a waste liquid collection area 3 is formed inside the waste liquid collection dish 30. A fixing buckle 31 is provided on the inner wall of the waste liquid collection dish 30, and the lower part of the organoid culture module 2 is supported on the fixing buckle 31.

[0056] The bottom surface of the organoid culture chamber 200 is concave to form a hemispherical culture tank 201. The arc-shaped structure and volume of the culture tank 201 are compatible with the shape and size of the organoid sphere, and can be used for artificial inoculation or 3D printing to construct organoid spheres, which helps to generate spheres with uniform size and good consistency in the organoid culture module 2, thereby increasing the repeatability of the experiment and reducing the experimental errors caused by the differences in the morphology and volume of the spheres.

[0057] A buffer zone 202 is arranged outside the culture tank 201 at the bottom of the organoid culture chamber 200; the liquid outlet 112 is arranged corresponding to the upper part of the organoid culture chamber 200, and the liquid outlet 112 is offset from the culture tank 201. After the liquid flowing out of the liquid outlet 112 reaches the buffer zone 202 along the front wall of the organoid culture chamber 200, it slowly flows into the bottom of the organoid culture chamber 200 to promote the growth and development of the organoid, instead of falling directly to the culture tank 201, thereby avoiding direct contact between the liquid and the organoid and reducing the physical damage of the liquid flow to the organoid during the culture process.

[0058] An overflow channel 203 is provided between the organoid culture chambers 200 of the same group, and the overflow channel 203 is provided at the upper part of the organoid culture chamber 200. The upper part of the organoid culture chamber 200 at one end of the same group is connected to the overflow hole 204, and the overflow hole 204 passes through the lower surface of the organoid culture unit 20, and the upper part of the organoid culture chamber 200 at the other end of the same group is aligned with the liquid outlet 112, and the liquid outlet 112 is close to the inner wall of the organoid culture chamber 200. A plurality of organoid culture chambers 200 are provided on the organoid culture module 2 to form a perforated plate-like structure. The upper end of the organoid culture chamber 200 is open and the lower end is closed.

[0059] A positioning sleeve 14 is provided at the lower edge of the drug concentration generating module 1, and the positioning sleeve 14 is connected to the upper edge of the organoid culture module 2. The positioning sleeve 14 realizes the detachable connection between the drug concentration generating module 1 and the organoid culture module 2, which is convenient for assembly and separation. When assembled together, the organoids are cultured and stimulated by drugs. When separated, the organoid culture module 2 is placed in an ELISA instrument for detection. The drug concentration generating unit 11 includes a multi-stage branch microfluidic channel 113, and the multi-stage branch microfluidic channel 113 includes a primary branch channel 1131 and a secondary branch channel 1132. A plurality of primary microchannels 1133 are connected in parallel between the primary branch channel 1131 and the secondary branch channel 1132, and a plurality of secondary microchannels 1134 are connected in parallel to the secondary branch channel 1132. The drug inlet 110 and the culture medium solution inlet 111 are both connected to the primary branch channel 1131, and the outlets of the plurality of secondary microchannels 1134 are respectively connected to the plurality of liquid outlets 112. The multi-stage branched microfluidic channel 113 adopts the Christmas tree model concentration gradient formation principle. When the solution enters the main channel, the flow trend causes the solution to move to the upper and lower branches. Since the outer solution flows slower and the central solution flows faster, this speed difference causes the solution to diffuse. As the solution enters the branch channel, changes in the shape and size of the branch channel will also cause changes in the fluid flow, resulting in changes in the speed of the liquid in the branch. The combined effect of these changes causes the solute in the solution to gradually form a concentration gradient in the channel.

[0060] Therefore, when the drug and culture medium solution are transported from the drug inlet 110 and the culture medium solution inlet 111 to the primary branch channel 1131 respectively, they pass through the primary microchannel 1133, the secondary branch channel 1132, and the secondary microchannel 1134 so that the liquid outlet 112 outputs multiple drug solutions of different concentrations.

[0061] The drug concentration generating module 1 includes a bottom plate 13 and a top cover 12 which are tightly connected together, a drug inlet 110 and a culture medium solution inlet 111 are arranged on the top cover 12, a liquid outlet 112 is arranged on the bottom plate 13, a multi-stage branched microfluidic channel 113 is arranged on the lower surface of the top cover 12, the drug inlet 110 and the culture medium solution inlet 111 are connected to the upper surface of the top cover 12, and the liquid outlet 112 is connected to the lower surface of the bottom plate 13.

[0062] Specifically, Figure 1 , Figure 2 As shown, the drug concentration generation module 1, the organoid culture module 2, and the waste liquid collection dish 30 are arranged in sequence from top to bottom, the top cover 12 is sealed and connected with the bottom plate 13 to form the drug concentration generation module 1, the positioning sleeve 14 extends downward from the edge of the bottom plate 13 and is connected with the outer wall of the organoid culture module 2, and the liquid outlet 112 on the bottom plate 13 is arranged corresponding to the upper opening of the organoid culture chamber 200, and the liquid outlet 112 is arranged close to the inner wall of the organoid culture chamber 200. Three organoid culture chambers 200 form a group, the liquid outlet 112 corresponds to an organoid culture chamber 200 at the head end, the overflow hole 204 corresponds to an organoid culture chamber 200 at the tail end, and the overflow groove 205 is arranged on the upper part of the organoid culture chamber 200 at the tail end, and the upper end of the overflow hole 204 is connected to the bottom of the overflow groove 205. The bottom of the overflow channel 203 is flush with the bottom of the overflow groove 205. The waste liquid collection dish 30 is connected to the lower outer wall of the organoid culture module 2, and the fixing buckle 31 supports and positions the organoid culture module 2. A distance is set between the lower surface of the organoid culture module 2 and the bottom surface of the waste liquid collection dish 30 for storing waste liquid.

[0063] like Figure 3 , Figure 4 , Figure 5 As shown, the drug concentration generating module 1 includes eight drug concentration generating units 11, and the organoid culture module 2 includes eight organoid culture units 20. In the multi-stage branch microfluidic channel 113 of the drug concentration generating unit 11, three primary microchannels 1133 are connected in parallel between the primary branch channel 1131 and the secondary branch channel 1132, and four secondary microchannels 1134 are connected in parallel between the secondary branch channel 1132. The four secondary microchannels 1134 correspond to four liquid outlets 112. The primary microchannels 1133 and the secondary microchannels 1134 are both serpentine tubular structures. Each organoid culture unit 20 is provided with four groups of organoid culture chambers 200. The drug inlet 110 and the culture medium solution inlet 111 are both connected to the primary branch channel 1131, and the outlets of the four secondary microchannels 1134 are respectively connected to the four liquid outlets 112. The drug inlet 110 and the culture medium solution inlet 111 are respectively connected to two integrated micropumps through pipelines.

[0064] like Figure 7As shown, a simulation plate 4 is arranged below the waste liquid collection dish 30, and the drug concentration generation module 1, the organoid culture module 2 and the waste liquid collection dish 30 are supported on the simulation plate 4 after being assembled, and the simulation plate 4 is connected to a driving mechanism, and the driving mechanism includes two rotating disks 40, and a connecting rod 41 is connected to the rotating disk 40, and the connecting rod 41 is rotatably connected to the simulation plate 4, and the connecting rod 41 is arranged in parallel, and the two rotating disks 40 are connected by transmission. There is a belt transmission between the two rotating disks 40. The rotating disk 40 and the connecting rod 41 are arranged on the front side of the simulation plate 4, and an auxiliary frame is installed on the rear side of the simulation plate 4. The auxiliary frame includes two auxiliary disks, and an auxiliary rod is arranged on the auxiliary disk. The auxiliary rod is rotatably connected to the simulation plate 4, and the auxiliary rod is parallel to the connecting rod 41. The auxiliary disk and the rotating disk 40 are arranged one by one coaxially, and the rotating disk 40 and the auxiliary disk are rotatably installed in the CO2 / temperature control box. The driving motor drives the two rotating disks 40 to rotate. During drug stimulation, the rotating disk 40 rotates, thereby realizing the rotation and lifting of the simulation plate 4, simulating the walking and living state of the human body when the drug acts on the human body, making the environment of drug-stimulated organoids closer to the actual drug use environment, which is beneficial to the accuracy of the experimental data.

[0065] During operation, the first step is to assemble, test and sterilize. After the drug concentration generation module 1 and the organoid culture module 2 are assembled, a 75% alcohol solution is passed into the drug concentration generation module 1 and the organoid culture module 2 under the drive of the integrated micropump to observe whether the liquid flow runs normally according to the channel to check the sealing of the device and the smoothness of the channel. After the test, air is introduced to discharge the residual alcohol solution. After disassembling the device, dry it at 50°C and then sterilize it with ultraviolet light for more than 30 minutes.

[0066] The second step is to carry out organoid culture and dynamic perfusion, taking a drug screening experiment as an example. The four sides of the disinfected organoid culture module 2 are clamped in the fixed buckle 31 position on the waste liquid collection, so that it falls flat on the waste liquid collection area 3. The cells are mixed with the extracellular matrix and inoculated at the bottom of the organoid culture chamber 200. Cover the drug concentration generation module 1 and place it in a CO2 / temperature control box for culture. After the extracellular matrix becomes a gel, the culture medium is injected through the culture medium solution inlet 111, and the driving force is provided by an integrated micropump. After the culture fluid passes through the liquid outlet 112 and reaches the buffer zone 202 along the front wall of the organoid culture chamber 200, it slowly flows into the bottom of the organoid culture chamber 200 to promote the growth and development of the organoid. The buffer zone 202 avoids direct contact between the culture fluid and the organoid, reducing the physical damage of the liquid flow to the organoid during the culture process. When the culture solution continues to perfuse until the liquid level reaches the upper edge of the rear wall of the organoid culture chamber 200, the culture solution flows into the next organoid culture chamber 200 through the overflow channel 203, so that the entire organoid culture chamber 200 is perfused. When the external driving force is continuously applied, the waste liquid after perfusion enters the waste liquid collection dish 30 through the overflow groove 205 and the overflow hole 204, completing the continuous perfusion of the organoid and cleaning of metabolic waste.

[0067] The third step is to perform drug stimulation of the organoids. After the organoids grow and develop well, drug stimulation is performed. Driven by the integrated micropump, the drug and the culture solution are respectively introduced into the multi-stage branch microfluidic channel 113 of the drug concentration generation unit 11 from the drug inlet 110 and the culture medium solution inlet 111. After the drug and the culture medium solution are mixed through the primary branch channel 1131, the drug mixture with three concentrations is fully mixed through the primary microchannel 1133, and then enters the secondary branch channel 1132 to form a drug mixture with four concentrations. After passing through four secondary microchannels 1134, the drug mixture of four concentrations flows into drug stimulation group one, drug stimulation group two, drug stimulation group three and drug stimulation group four from four liquid outlets 112, and each drug stimulation group has three organoid culture chambers 200. In this way, a single drug concentration generation unit 11 can complete four concentration stimulations of a drug of the organoid and three parallel experiments of drug stimulation with the same concentration. If the eight organoid culture units 20 of the organoid culture module 2 are simultaneously subjected to drug type and concentration experiments, high-throughput organoid drug testing can be achieved. During this process, the simulation board 4 rotates and rises and falls periodically to simulate the walking and living state of the human body when the drug acts on the human body, making the environment of the drug-stimulated organoids closer to the actual medication environment, which is beneficial to the accuracy of the experimental data.

[0068] The fourth step is to test the samples after drug stimulation. After adding appropriate detection reagents and incubating them using an integrated micropump, the samples can be tested. The organoid culture module 2 is compatible with existing microplate readers. When conducting experimental tests based on microplate readers, the organoid culture module 2 can be removed and directly placed in the microplate reader for testing.

[0069] The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims

1. A high-throughput drug screening and organoid culture device, characterized in that: It includes a drug concentration generation module and an organoid culture module. The drug concentration generation module includes a plurality of drug concentration generation units. The organoid culture module includes a plurality of organoid culture units corresponding to the drug concentration generation units. The drug concentration generation units are provided with a drug inlet, a culture medium solution inlet and a plurality of liquid outlets. The organoid culture units include a plurality of groups of organoid culture chambers corresponding to the liquid outlets, and the organoid culture chambers in the same group are interconnected.

2. A high-throughput drug screening and organoid culture device according to claim 1, characterized in that: A waste liquid collection area is provided below the organoid culture module, and the waste liquid collection area receives liquid overflowing from the organoid culture chamber.

3. A high-throughput drug screening and organoid culture device according to claim 2, characterized in that: A waste liquid collection dish is detachably mounted at the bottom of the organoid culture module, and a waste liquid collection area is formed inside the waste liquid collection dish.

4. A high-throughput drug screening and organoid culture device according to claim 1, characterized in that: The bottom surface of the organoid culture chamber is concave to form a hemispherical culture trough.

5. A high-throughput drug screening and organoid culture device according to claim 4, characterized in that: A buffer zone is arranged outside the culture tank on the bottom surface of the organoid culture chamber; the liquid outlet is arranged corresponding to the upper part of the organoid culture chamber, and the liquid outlet is staggered with the culture tank.

6. A high-throughput drug screening and organoid culture device according to claim 1, characterized in that: An overflow channel is arranged between the organoid culture chambers of the same group, and the overflow channel is arranged at the upper part of the organoid culture chamber.

7. A high-throughput drug screening and organoid culture device according to claim 1, characterized in that: The upper part of the organoid culture chamber at one end of the same group is connected to the overflow hole, and the overflow hole penetrates to the lower surface of the organoid culture unit. The upper part of the organoid culture chamber at the other end of the same group is aligned with the liquid outlet.

8. The high-throughput drug screening and organoid culture device according to claim 1, characterized in that: A positioning sleeve is arranged at the lower edge of the drug concentration generating module, and the positioning sleeve is connected to the upper edge sleeve of the organoid culture module.

9. A high-throughput drug screening and organoid culture device according to any one of claims 1 to 8, characterized in that: The drug concentration generating unit includes a multi-stage branch microfluidic channel, which includes a primary branch channel and a secondary branch channel. A plurality of primary microchannels are connected in parallel between the primary branch channel and the secondary branch channel, and a plurality of secondary microchannels are connected in parallel to the secondary branch channel. Both the drug inlet and the culture medium solution inlet are connected to the primary branch channel, and the plurality of secondary microchannel outlets are connected to the plurality of liquid outlets respectively.

10. A high-throughput drug screening and organoid culture device according to claim 9, characterized in that: The drug concentration generating module comprises a bottom plate and a top cover which are tightly connected together, a drug inlet and a culture medium solution inlet are arranged on the top cover, a liquid outlet is arranged on the bottom plate, and a multi-stage branch microfluidic channel is arranged on the lower surface of the top cover.

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