A method and device for multi-flux in situ tracing of organoid metabolic flux based on molten DNP

Abstract

The present invention discloses a method and device for tracing organoid metabolic flux in situ by multi-flux based on melt DNP. The present invention develops organoid vitality maintenance technology, super-target molecular probe quality control and shunting technology, and CSI combined with selective excitation technology to establish a set of multi-flux in situ real-time metabolic flux tracing methods suitable for organoids, develops multi-flux multi-metabolism small molecule metabolic flux detection technology, and can provide more rapid and sensitive in situ tracing of organoid metabolic pathways than conventional solutions, which is expected to provide a new technical solution for the study of organoid models. The present invention can realize the detection and tracing of metabolic flux under in situ culture conditions of organoids, and can realize high-throughput detection and tracing with high sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear magnetic resonance technology, and in particular relates to a method and a device for tracing organoid metabolic flux in situ based on multi-flux molten DNP. Background Art

[0002] Tumor organoid models have irreplaceable advantages in anti-tumor drug screening. They can reduce the complexity of animal experiments and retain the relevant characteristics of tumors to the greatest extent. However, the current evaluation of their anti-tumor drug response is mainly focused on some phenotypic data, lacking in-depth real-time tracing at the molecular level. 13 C) The melt-DNP technology, which has increased the detection intensity by at least 4 orders of magnitude, is expected to achieve in situ non-destructive observation of drug-mediated changes in sub-second chemical reactions of molecular probes in organoids without background interference, and can evaluate the effects of drugs on tumor organoids in real time at the molecular level.

[0003] At present, the tracing objects of hyperpolarized molecular probes produced by melt DNP technology mainly include cells and living bodies (animals / humans). The detection objects of cell reactions are mainly cell suspensions, and hyperpolarized probe molecules can directly enter cells through interactions with cell membranes. In in vivo studies, probe molecules mainly enter the target detection location through the circulatory system. However, there are no reports on the application of this technology in organoid models internationally, and its key technologies need to be established and developed urgently.

[0004] Different from cells and living environments, organoids grow in 3D gels, and the gels are surrounded by rich culture fluids. To promote the application of melt-DNP technology to organoid models, there are several key technical issues that need to be solved: 1) Maintaining the vitality of organoids during metabolic flux detection: A major challenge of melt-DNP in situ metabolic flux in vivo tracing is to maintain the vitality of the living body during signal detection. Organoids that leave the cell culture incubator need to be provided with a constant temperature and sufficient oxygen supply environment. Therefore, we will develop key technologies for maintaining the vitality of organoids that are compatible with melt-DNP technology to meet this need; 2) Efficient and accurate (physiological) tracing: Generally, the melt-DNP experimental system can only observe one sample at a time, but in fact, the hyperpolarized molecular probe solution prepared at one time is enough to trace more than 10 samples, which leads to a huge waste of hyperpolarized probe molecular solution; on the other hand, for the organoid model itself, one sample at a time is not conducive to the consistency of parallel sample tracing results, nor is it conducive to the realization of multi-flux detection, thus affecting the detection efficiency; 3) Organoids grow in 3D The gel has its own unique physiological environment. The concentration and pH value of the hyperpolarized molecular solution entering the organoid tube need to be strictly controlled in a short period of time so as to detect the reaction of the molecular probe without interfering with the normal physiological state of the organoid and ensure the accuracy of molecular tracing. Summary of the invention

[0005] In response to the problems that need to be urgently solved in the above-mentioned prior art, the present invention provides a method and device for multi-flux in situ tracing of organoid metabolic flux based on molten DNP. The present invention can realize the detection and tracing of metabolic flux under in situ culture conditions of organoids, and can realize high-throughput detection and tracing with high sensitivity and accuracy.

[0006] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0007] A method for in situ tracing of organoid metabolic flux based on multi-flux molten DNP comprises the following steps:

[0008] S1. In situ high-throughput culture of organoids in the wells of a multi-well culture dish.

[0009] S2, preparing a hyperpolarized target molecule probe;

[0010] S3. After the organoids are cultured, but before the hyperpolarized target molecule probes are prepared, the multi-well culture dish is placed in a detection magnet of a nuclear magnetic resonance spectrometer;

[0011] S4. Continuously or intermittently injecting a culture medium for culturing the organoid into each culture well of the multi-well culture dish, and continuously or intermittently discharging waste liquid generated during the culture process, thereby maintaining the vitality of the organoid;

[0012] S5, injecting a hyperpolarized target molecule probe into each culture well of the multi-well culture dish;

[0013] S6. Use CSI combined with selective excitation technology to detect the NMR signals of the target molecular probe and its metabolites, thereby tracing the metabolic flux of the target molecular probe.

[0014] Furthermore, before the multi-well culture dish is placed in the detection magnet of the nuclear magnetic resonance spectrometer, the organoids in different culture wells can be treated with the same drug or method, or with different drugs and methods.

[0015] The target molecule probe is a small molecule involved in endogenous metabolism of an organism, and the target molecule probe is selected from at least one of pyruvate, lactate, and glucose. A multi-flux in situ tracing organoid metabolic flux device based on melt DNP, comprising an organoid vitality maintenance unit, a hyperpolarized target molecule probe injection unit, a multi-porous culture dish, and a control module;

[0016] The multi-well culture dish comprises a culture plate, on which a plurality of culture holes are evenly arranged, each culture hole is provided with a hole cover adapted thereto, and the hole cover is respectively provided with a first through hole, a second through hole and a third through hole;

[0017] The organoid vitality maintenance unit includes a CO2 supply unit, a culture fluid supply unit, a first shunt unit and a waste liquid discharge unit, the first shunt unit includes a plurality of first shunt pipelines, the waste liquid discharge unit includes a plurality of waste liquid discharge pipelines, the CO2 supply unit and the culture fluid supply unit are respectively connected to the upstream end of each first shunt pipeline, the downstream end of each first shunt pipeline is respectively connected to the first perforation on the corresponding hole cover, and the upstream end of each waste liquid discharge pipeline is respectively connected to the third perforation on the corresponding hole cover;

[0018] The hyperpolarized target molecule probe injection unit includes a mass control unit and a second shunt pipeline, the second shunt unit includes a plurality of second shunt pipelines, the upstream end of each second shunt pipeline is respectively connected to the mass control unit, and the downstream end of each second shunt pipeline is respectively connected to the second perforation on the corresponding hole cover;

[0019] The control module is respectively connected to and controls the operation of the CO2 supply unit, the culture solution supply unit, each first shunt pipeline, each waste liquid discharge pipeline, the quality control unit and the second shunt pipeline.

[0020] The CO2 supply unit includes a CO2 supply pipeline, an air supply pipeline, a gas mixer and a mixed gas supply pipeline. The CO2 supply pipeline includes a CO2 cylinder, a CO2 supply pipeline and a first solenoid valve. The upstream end of the CO2 supply pipeline is connected to the CO2 cylinder, and the first solenoid valve is installed on the CO2 supply pipeline. The air supply pipeline includes an air cylinder, an air supply pipeline and a second solenoid valve. The upstream end of the air supply pipeline is connected to the air cylinder, and the second solenoid valve is installed on the air supply pipeline. The downstream ends of the CO2 supply pipeline and the air supply pipeline are respectively connected to the gas mixer. The mixed gas supply pipeline includes a mixed gas supply pipeline, a third solenoid valve and a CO2 concentration detector. The upstream end of the mixed gas supply pipeline is connected to the mixed gas supply pipeline, and the third solenoid valve and the CO2 concentration detector are respectively installed on the mixed gas supply pipeline. The downstream end of the mixed gas supply pipeline is connected to the upstream end of each first diversion pipeline. The control module connects and controls the operation of the first solenoid valve, the second solenoid valve, the third solenoid valve, the gas mixer and the CO2 concentration detector.

[0021] The culture fluid supply unit includes a culture fluid supply pipeline, a culture fluid container, a constant temperature magnetic stirrer and a first liquid pump. The culture fluid container is located on the constant temperature magnetic stirrer. The upstream end of the culture fluid supply pipeline is connected to the culture fluid container. The first liquid pump is installed on the culture fluid supply pipeline. The control module is connected to and controls the operation of the first liquid pump.

[0022] The first diversion pipeline includes a first diversion pipe, and the first diversion pipe is provided with a one-way valve, a quantitative tank and a second liquid pump in sequence along the direction from the upstream end to the downstream end. The upstream end of the first diversion pipe is connected to the downstream end of the culture fluid supply pipe or the culture fluid supply unit, and the downstream end of the first diversion pipe is connected to the first perforation on the corresponding hole cover. The control module is connected to and controls the operation of the second liquid pump and the one-way valve.

[0023] The waste liquid discharge pipeline includes a waste liquid discharge pipe, a third liquid pump and a waste liquid container. The upstream end of the waste liquid discharge pipe is connected to the third perforation on the corresponding hole cover, and the downstream end of the waste liquid discharge pipe is connected to the waste liquid container. The third liquid pump is installed on the waste liquid discharge pipe, and the control module is connected to and controls the operation of the third liquid pump.

[0024] The quality control unit includes a receiving container, a buffer injection unit and a quality detection unit. The buffer injection unit includes a buffer injection pipeline, a buffer container and a fourth liquid pump. The upstream end of the buffer injection pipeline is connected to the buffer container, and the downstream end of the buffer injection pipeline is connected to the receiving container. The fourth liquid pump is installed on the buffer injection pipeline. The quality detection unit includes a mass detection pool, a mass detection pipeline and a fifth liquid pump. The upstream end of the mass detection pipeline is connected to the receiving container, and the downstream end of the mass detection pipeline is connected to the mass detection pool. The fifth liquid pump is installed on the mass detection pipeline. The mass detection pool is equipped with a pH meter for detecting the pH of the hyperpolarized target molecule probe in the mass detection pool, a concentration photometer and a temperature thermometer. The control module is connected to and controls the fourth liquid pump, the fifth liquid pump, the pH meter, the photometer and the thermometer to work.

[0025] The second shunt pipeline comprises a second shunt pipe, the upstream end of which is connected to the receiving container or the quality control unit, and the downstream end of which is connected to the second perforation on the corresponding hole cover.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0027] 1. The present invention develops organoid vitality maintenance technology, super-target molecular probe quality control and diversion technology, and uses CSI combined with selective excitation technology to establish a set of multi-flux in situ real-time metabolic flux tracing methods suitable for organoids, develops multi-flux multi-metabolism small molecule metabolic flux detection technology, and can provide more rapid and sensitive in situ tracing of organoid metabolic pathways than conventional solutions, which is expected to provide a new technical solution for the study of organoid models.

[0028] 2. The organoids of the present invention can maintain the same environment as during culture during the metabolic flux tracing detection of small molecules, thereby maintaining the vitality of the organoids during culture. Not only can metabolic flux tracing be performed in the real culture environment of the organoids, making the tracing detection results highly accurate, but the organoids can continue to be cultured and utilized after the tracing detection, thus avoiding the waste of organoids, saving the cost of organoid model construction, and thus saving the cost of organoid model detection.

[0029] 3. The present invention can achieve sub-second tracing of metabolic flux of small molecules in organoids, greatly improving sensitivity.

[0030] 4. The quality control unit of the present invention can strictly control the concentration, temperature and pH value of the hyperpolarized target molecule solution entering the organoid tube and automatically adjust it to a set range, so as to detect the reaction of the target molecule probe without interfering with the normal physiological state of the organoid, thereby ensuring the accuracy of the target molecule probe tracing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the structure of the device for multi-flux in situ tracing of organoid metabolic fluxes based on molten DNP.

[0032] Figure 2 Schematic diagram of the structure of a multi-well culture dish.

[0033] Figure 3 These are the changes in HE staining during the growth of colon organoids, as well as DAPI staining of organoid cell nuclei, growth factor Ki67 staining, and differentiation factor CK20 staining.

[0034] Among them, 1-multi-porous culture dish: 101-culture plate, 102-culture hole, 103-hole cover, 104-first perforation, 105-second perforation, 106-third perforation; 2-CO2 supply unit: 201-CO2 gas cylinder 、 202-air bottle, 203-first output solenoid valve, 204-second solenoid valve, 205-gas mixer, 206-third solenoid valve, 207-CO2 concentration detector, 208-CO2 discharge check valve; 3-culture fluid supply unit: 301-culture fluid supply pipeline, 302-culture fluid container, 303-constant temperature magnetic stirrer, 304-first liquid pump, 305-buffer tank; 4-first shunt unit: 401-first shunt pipeline: 4011-first shunt pipeline, 4012-shunt check valve, 4013-quantitative tank, 4014-second liquid pump; 5-waste liquid discharge unit: 501-waste liquid discharge pipeline: 5011-waste liquid discharge pipeline, 5012-third liquid pump, 501 3-waste liquid container; 6-quality control unit: 601-receiving container; 602-buffer injection unit: 6021-buffer injection pipeline, 6022-buffer container, 6023-fourth liquid pump; 603-quality detection unit: 6031-quality detection pool, 6032-quality detection pipeline, 6033-fifth liquid pump, 6034-pH meter, 6035-photometer, 6036-thermometer; 7-second shunt pipeline: 701-second shunt pipeline, 702-sixth liquid pump; 8-control module. DETAILED DESCRIPTION

[0035] In order to facilitate those skilled in the art to understand and implement the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the implementation examples described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0036] The device for multi-flux in situ tracing of organoid metabolic flux based on molten DNP of the present invention is described in detail below with reference to the accompanying drawings.

[0037] Example 1

[0038] The present embodiment provides a multi-flux in situ tracing device for organoid metabolic flux based on molten DNP. Figure 1 As shown, it includes an organoid vitality maintenance unit, a hyperpolarized target molecule probe injection unit, a multi-well culture dish and a control module. The organoid vitality maintenance unit continuously or intermittently provides 5% CO2 and culture fluid to the organoid during the target metabolic molecule metabolic flux tracing to maintain the vitality of the organoid and simulate the organoid culture environment. The hyperpolarized target molecule probe injection unit is used to provide the organoid with a hyperpolarized target small molecule probe with controllable quality.

[0039] The multi-porous culture dish 1 includes a culture plate 101, which is made of quartz and is cylindrical. The size of the culture plate 101 can be designed according to different detection instruments. In this embodiment, the diameter of the culture plate 101 is 30 mm. The culture plate 101 is provided with five culture holes 102 distributed in a cross, and the culture holes 102 are circular holes. One of the culture holes 102 is located in the center of the culture plate 101, and the culture hole 102 is coaxial with the culture plate 101. The other four culture holes 102 are distributed in a square, and the center point of the square is located on the axis of the culture plate 101. According to actual needs, the five culture holes 102 can culture the same type of organoids, or different organoids, and the organoids of different culture holes 102 can be treated with the same drug or method, or with different drugs and methods. Each culture hole 102 is provided with a hole cover 103 adapted thereto, and the hole cover 103 is respectively provided with a first perforation 104, a second perforation 105 and a third perforation 106. The number of culture wells 102 can be increased or decreased according to actual conditions, and their distribution can also be optimized according to actual conditions.

[0040] The organoid vitality maintenance unit includes a CO2 supply unit, a culture fluid supply unit, a first diversion unit and a waste fluid discharge unit.

[0041] The CO2 supply unit 2 includes a CO2 cylinder 2001, a CO2 supply pipeline, a first solenoid valve 203, an air cylinder 202, an air supply pipeline, a second solenoid valve 204, a gas mixer 205, a mixed gas supply pipeline, a third solenoid valve 206, a CO2 concentration detector 207 and a CO2 discharge check valve 208. The upstream end of the CO2 supply pipeline is connected to the CO2 cylinder 201, and the first solenoid valve 203 is installed on the CO2 supply pipeline. The upstream end of the air supply pipeline is connected to the air cylinder 202, and the second solenoid valve 204 is installed on the air supply pipeline. The downstream ends of the CO2 supply pipeline and the air supply pipeline are respectively connected to the gas mixer 205. The upstream end of the mixed gas supply pipeline is connected to the gas mixer 205, and the third solenoid valve 206 and the CO2 concentration detector 207 are respectively installed on the mixed gas supply pipeline.

[0042] The CO2 cylinder 201 and the air cylinder 202 provide the gas mixer 205 with the air and CO2 required for mixing into a volume fraction of 5% CO2, wherein the output of the two gases is controlled by opening and closing the first solenoid valve 203 and the second solenoid valve 204. The CO2 concentration detector 207 is responsible for monitoring the concentration of CO2 introduced into the culture medium container 302 and feeding back the relevant concentration value to the control module 8, and the control module 8 controls the opening and closing of the first solenoid valve 203, the second solenoid valve 204, the gas mixing proportioner 205 and the third solenoid valve 206 according to the detected concentration value. When the third solenoid valve 206 is opened, the CO2 gas with a volume percentage concentration of 5% can be input into the culture medium container 302, and the CO2 discharge check valve 208 is opened at this time.

[0043] The culture solution supply unit 3 includes a culture solution supply pipeline 301, a culture solution container 302, a constant temperature magnetic stirrer 303, a first liquid pump 304 and a buffer tank 305. The culture solution container 302 is located on the constant temperature magnetic stirrer 303, and the constant temperature magnetic stirrer 303 can mix and heat the culture solution in the culture solution container 302. The culture solution container 302, the first liquid pump 304 and the buffer tank 305 are distributed in sequence from the upstream end to the downstream end of the culture solution supply pipeline 301, and the upstream end of the culture solution supply pipeline 301 is connected to the culture solution container 302.

[0044] The first flow diversion unit 4 includes five first flow diversion pipelines 401, and the first flow diversion unit 4 is used to divert the culture solution. The first flow diversion pipeline 401 includes a first flow diversion pipeline 4011, and the first flow diversion pipeline 4011 is provided with a diversion check valve 4012, a quantitative buffer tank 4013 and a second liquid pump 4014 in sequence along the direction from the upstream end to the downstream end thereof. The upstream end of the first flow diversion pipeline 4011 is connected to the downstream end of the culture solution supply pipeline 301, and the downstream end of the first flow diversion pipeline 4011 is respectively connected to the first through-holes 104 on the corresponding hole cover 103.

[0045] The waste liquid discharge unit 5 includes five waste liquid discharge pipelines 501, and the waste liquid discharge pipeline 501 includes a waste liquid discharge pipeline 5011, a third liquid pump 5012 and a waste liquid container 5013. The upstream end of the waste liquid discharge pipeline 5011 is connected to the third perforation 106 on the corresponding hole cover 103, and the downstream end of the waste liquid discharge pipeline 5011 is connected to the waste liquid container 5013. The third liquid pump 5012 is installed on the waste liquid discharge pipeline 5011.

[0046] The hyperpolarized target molecule probe injection unit comprises a quality control unit 6 and a second shunt pipeline 7. The quality control unit is used to monitor various indicators of the hyperpolarized target molecule probe and make adjustments according to the monitoring results.

[0047] The quality control unit 6 includes a receiving container 601, a buffer injection unit 602, a quality detection unit 603 and a melt injection pipeline 604. The downstream end of the melt injection pipeline 604 is connected to the receiving container 601, and the receiving container 601 is used to receive the melt hyperpolarized target molecule probe melt. The buffer injection unit 602 includes a buffer injection pipeline 6021, a buffer container 6022 and a fourth liquid pump 6023, the upstream end of the buffer injection pipeline 6021 is connected to the buffer container 6022, the downstream end of the buffer injection pipeline 6021 is connected to the receiving container 601, and the fourth liquid pump 6023 is installed on the buffer injection pipeline 6021. The mass detection unit 603 includes a mass detection pool 6031, a mass detection pipeline 6032 and a fifth liquid pump 6033. The upstream end of the mass detection pipeline 6032 is connected to the receiving container 601, the downstream end of the mass detection pipeline 6032 is connected to the mass detection pool 6031, and the fifth liquid pump 6033 is installed on the mass detection pipeline 6032. The mass detection pool 6031 is equipped with a pH meter 6034 for detecting the pH value of the hyperpolarized target molecule probe in the mass detection pool, a concentration photometer 6035, and a temperature thermometer 6036.

[0048] The second diversion pipeline 7 includes a second diversion pipeline 701 and a sixth liquid pump 702. The upstream end of the second diversion pipeline 701 is connected to the receiving container 601, and the downstream end of the second diversion pipeline 701 is connected to the second perforation 105 on the corresponding hole cover 103. The sixth liquid pump 702 is installed on the second diversion pipeline 701.

[0049] The control module 8 connects and controls the operation of the first solenoid valve 203, the second solenoid valve 204, the gas mixing proportioner 205, the 5% CO2 gas output solenoid valve 206, the CO2 concentration detector 207, the first liquid pump 304, each second liquid pump 4014, each third liquid pump 5012, the fourth liquid pump 6023, the fifth liquid pump 6033, each sixth liquid pump 702 and each one-way valve 4012.

[0050] The method of the present invention for multi-flux in situ tracing of organoid metabolic fluxes based on molten DNP is described in detail below in conjunction with the above-mentioned device.

[0051] Example 2

[0052] 1. Anesthetize the mouse and dissect the colon tissue to obtain the cell inoculum after digestion, resuspending, separation and filtration. Mix the matrix gel and cell inoculum to obtain a mixed suspension. Use a pipette to pipette the mixed suspension into five culture wells of a multi-well culture dish and place it in a cell culture incubator for culture. Figure 3As shown, including the growth change graph of colon organoids under HE staining (the day of inoculation, the second day of inoculation, the fifth day of inoculation, and the seventh day of inoculation), DAPI staining of organoid nuclei (fifth day of inoculation), organoid growth factor Ki67 staining (fifth day of inoculation), and organoid differentiation factor CK20 staining (fifth day of inoculation). It can be seen from the growth change graph of colon organoids under HE staining, DAPI staining of organoid nuclei, organoid growth factor Ki67 staining, and organoid differentiation factor CK20 staining that the cultured organoids are in good growth condition.

[0053] 2. After UV irradiation, 300ul of [1- 13 The [C]-pyruvic acid sample was placed in a melt-type DNP spectrometer for polarization and melting.

[0054] 3. Add the culture medium for culturing organoids into the culture medium container 302, and add the phosphate buffer (pH=7.0) into the buffer container 6022. After the organoids are cultured, 13 Before the preparation of the [C]-pyruvic acid probe is completed, according to the connection relationship of the above device (such as Figure 1 (As shown in the figure) The device for multi-flux in situ tracing of organoid metabolic flux based on molten DNP is assembled and connected, except that the multi-well culture dish is not connected and the multi-well culture dish remains in the cell culture incubator.

[0055] 4. Turn on the gas mixer 205 and set the gas mixer 205 so that after the CO2 and air are mixed in the gas mixer, the volume percentage concentration of CO2 is 5%. Turn on the CO2 cylinder, the first solenoid valve 203, the air cylinder 202 and the second solenoid valve 204 to mix the CO2 and air evenly.

[0056] 5. Open the third solenoid valve 206, the CO2 concentration detector 207 and the CO2 discharge check valve 208, and introduce a mixed gas with a CO2 volume fraction of 5% into the culture liquid container 302. In this process, if the CO2 concentration detector 207 detects that the volume percentage concentration of CO2 in the mixed gas is not equal to 5%, the control module 8 controls the third solenoid valve 206 to close after receiving the feedback signal from the CO2 concentration detector 207, and the control module 8 controls the gas mixer 205 to adjust the volume percentage concentration of CO2 to 5% again, and then opens the third solenoid valve 206 again, introduces the mixed gas into the culture liquid container 302, and detects the volume percentage concentration of CO2 again through the CO2 concentration detector 207, and repeats the cycle until the volume percentage concentration of CO2 is adjusted to 5%.

[0057] 6. Connect the multi-porous culture dish to the device according to the connection relationship described in Example 1, and then place it in the detection magnet of the nuclear magnetic resonance spectrometer, turn on the first liquid pump 304, each diversion one-way valve 4012, each second liquid pump 4014 and each third liquid pump 5012, and input culture fluid into each culture well 102 to maintain the culture environment of the organoid, thereby maintaining the vitality of the organoid in each culture well 102, and at the same time discharge the waste liquid generated during the culturing of the organoid in each culture well 102 through each waste liquid discharge pipeline.

[0058] 7. The molten hyperpolarized pyruvate probe is introduced into the receiving container 601 through the molten liquid injection pipe 604, and then the fifth liquid pump 6033, the pH meter 6034, the photometer 6035 and the thermometer 6036 are turned on. The pH meter 6034, the photometer 6035 and the thermometer 6036 feed back the detection results to the control module 8. The control module controls the fourth liquid pump 6023 to be turned on and the fifth liquid pump 6033 to be turned off according to the measured acidity and concentration results. If the measured acidity and concentration are too high, the control module 8 controls the fourth liquid pump 6023 to be turned on and a certain amount of phosphate buffer is pumped into the receiving container 601. Then the control module 8 controls the fourth liquid pump 6023 to be turned off and the fifth liquid pump 6033 to be turned on, and the acidity, concentration and temperature of the hyperpolarized pyruvate probe diluted by the phosphate buffer are detected again. This cycle is repeated until the acidity and concentration of the hyperpolarized pyruvate probe meet the requirements.

[0059] 8. Run the CSI-based MRS sampling sequence, specifically: perform 3*3 phase encoding on the area to be detected with a field of view (FOV) of 30mm*30mm, where the layer thickness is 10mm, TE is 35ms, and TR is 110ms. For pyruvate, the pulse angle of selective excitation is 10 degrees; for lactate, alanine or carbonate molecules, the angle of the selective excitation pulse is 20 degrees. Turn on each sixth liquid pump 702, and introduce a hyperpolarized pyruvate probe into each culture well 102. First, sample pyruvate twice at a small angle, then selectively excite lactate, carbonate and alanine signals respectively, and then excite pyruvate-lactate-carbonate-alanine in turn, and repeat this cycle to observe the hyperpolarized molecular probe [1- 13 Real-time metabolic flux of [C]-pyruvate in colon organoids.

Claims

1. A method for in situ tracing of organoid metabolic flux based on multi-flux melting DNP, characterized in that The steps include: S1. In situ high-throughput culture of organoids in the wells of a multi-well culture dish. S2, preparing a hyperpolarized target molecule probe; S3. After the organoids are cultured, but before the hyperpolarized target molecule probes are prepared, the multi-well culture dish is placed in a detection magnet of a nuclear magnetic resonance spectrometer; S4. Continuously or intermittently injecting a culture medium for culturing the organoid into each culture well of the multi-well culture dish, and continuously or intermittently discharging waste liquid generated during the culture process, thereby maintaining the vitality of the organoid; S5, injecting a hyperpolarized target molecule probe into each culture well of the multi-well culture dish; S6. Use CSI combined with selective excitation technology to detect the NMR signals of the target molecular probe and its metabolites, thereby tracing the metabolic flux of the target molecular probe.

2. The method of multi-flux in situ tracing of organoid metabolic flux based on melt DNP according to claim 1, characterized in that: Before the multi-well culture dish is placed in the detection magnet of the nuclear magnetic resonance spectrometer, the organoids in different culture wells can be the same type of organoid or different types of organoids; wherein the cultured organoids can be treated with the same drug or method or with different drugs and methods.

3. The method of multi-flux in situ tracing of organoid metabolic flux based on melt DNP according to claim 1, characterized in that: The target molecule probe is a small molecule involved in endogenous metabolism of an organism, and the target molecule probe is selected from at least one of pyruvate, lactate, and glucose.

4. A multi-flux in situ tracing device for organoid metabolic flux based on melt DNP, characterized in that: It includes an organoid vitality maintenance unit, a hyperpolarized target molecule probe injection unit, a multi-well culture dish and a control module; The multi-well culture dish comprises a culture plate, on which a plurality of culture holes are evenly arranged, each culture hole is provided with a hole cover adapted thereto, and the hole cover is respectively provided with a first through hole, a second through hole and a third through hole; The organoid vitality maintenance unit includes a CO2 supply unit, a culture fluid supply unit, a first shunt unit and a waste liquid discharge unit, the first shunt unit includes a plurality of first shunt pipelines, the waste liquid discharge unit includes a plurality of waste liquid discharge pipelines, the CO2 supply unit and the culture fluid supply unit are respectively connected to the upstream end of each first shunt pipeline, the downstream end of each first shunt pipeline is respectively connected to the first perforation on the corresponding hole cover, and the upstream end of each waste liquid discharge pipeline is respectively connected to the third perforation on the corresponding hole cover; The hyperpolarized target molecule probe injection unit includes a mass control unit and a second shunt pipeline, the second shunt unit includes a plurality of second shunt pipelines, the upstream end of each second shunt pipeline is respectively connected to the mass control unit, and the downstream end of each second shunt pipeline is respectively connected to the second perforation on the corresponding hole cover; The control module is respectively connected to and controls the operation of the CO2 supply unit, the culture solution supply unit, each first shunt pipeline, each waste liquid discharge pipeline, the quality control unit and the second shunt pipeline.

5. The device for in situ tracing of organoid metabolic flux based on molten DNP according to claim 4, characterized in that: The CO2 supply unit includes a CO2 supply pipeline, an air supply pipeline, a gas mixer and a mixed gas supply pipeline. The CO2 supply pipeline includes a CO2 cylinder, a CO2 supply pipeline and a first solenoid valve. The upstream end of the CO2 supply pipeline is connected to the CO2 cylinder, and the first solenoid valve is installed on the CO2 supply pipeline. The air supply pipeline includes an air cylinder, an air supply pipeline and a second solenoid valve. The upstream end of the air supply pipeline is connected to the air cylinder, and the second solenoid valve is installed on the air supply pipeline. The downstream ends of the CO2 supply pipeline and the air supply pipeline are respectively connected to the gas mixer. The mixed gas supply pipeline includes a mixed gas supply pipeline, a third solenoid valve and a CO2 concentration detector. The upstream end of the mixed gas supply pipeline is connected to the mixed gas supply pipeline, and the third solenoid valve and the CO2 concentration detector are respectively installed on the mixed gas supply pipeline. The downstream end of the mixed gas supply pipeline is connected to the upstream end of each first diversion pipeline. The control module connects and controls the operation of the first solenoid valve, the second solenoid valve, the third solenoid valve, the gas mixer and the CO2 concentration detector.

6. The device for multi-flux in situ tracing of organoid metabolic fluxes based on molten DNP according to claim 4, characterized in that: The culture fluid supply unit includes a culture fluid supply pipeline, a culture fluid container, a constant temperature magnetic stirrer and a first liquid pump. The culture fluid container is located on the constant temperature magnetic stirrer. The upstream end of the culture fluid supply pipeline is connected to the culture fluid container. The first liquid pump is installed on the culture fluid supply pipeline. The control module is connected to and controls the operation of the first liquid pump.

7. The device for in situ tracing of organoid metabolic flux based on molten DNP according to any one of claims 4 or 5, characterized in that: The first diversion pipeline includes a first diversion pipe, and the first diversion pipe is provided with a one-way valve, a quantitative tank and a second liquid pump in sequence along the direction from the upstream end to the downstream end. The upstream end of the first diversion pipe is connected to the downstream end of the culture fluid supply pipe or the culture fluid supply unit, and the downstream end of the first diversion pipe is connected to the first perforation on the corresponding hole cover. The control module is connected to and controls the operation of the second liquid pump and the one-way valve.

8. The device for in situ tracing of organoid metabolic flux based on molten DNP according to claim 4, characterized in that: The waste liquid discharge pipeline includes a waste liquid discharge pipe, a third liquid pump and a waste liquid container. The upstream end of the waste liquid discharge pipe is connected to the third perforation on the corresponding hole cover, and the downstream end of the waste liquid discharge pipe is connected to the waste liquid container. The third liquid pump is installed on the waste liquid discharge pipe, and the control module is connected to and controls the operation of the third liquid pump.

9. The device for multi-flux in situ tracing of organoid metabolic fluxes based on molten DNP according to claim 4, characterized in that: The quality control unit includes a receiving container, a buffer injection unit and a quality detection unit. The buffer injection unit includes a buffer injection pipeline, a buffer container and a fourth liquid pump. The upstream end of the buffer injection pipeline is connected to the buffer container, and the downstream end of the buffer injection pipeline is connected to the receiving container. The fourth liquid pump is installed on the buffer injection pipeline. The quality detection unit includes a mass detection pool, a mass detection pipeline and a fifth liquid pump. The upstream end of the mass detection pipeline is connected to the receiving container, and the downstream end of the mass detection pipeline is connected to the mass detection pool. The fifth liquid pump is installed on the mass detection pipeline. The mass detection pool is equipped with a pH meter for detecting the pH of the hyperpolarized target molecule probe in the mass detection pool, a concentration photometer and a temperature thermometer. The control module is connected to and controls the fourth liquid pump, the fifth liquid pump, the pH meter, the photometer and the thermometer to work.

10. The device for in situ tracing of organoid metabolic flux based on molten DNP according to any one of claims 4 or 8, characterized in that: The second diversion pipeline includes a second diversion pipeline and a sixth liquid pump. The upstream end of the second diversion pipeline is connected to the receiving container or the quality control unit, and the downstream end of the second diversion pipeline is connected to the second perforation on the corresponding hole cover. The sixth liquid pump is installed on the second diversion pipeline.

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