Organoid online monitoring culture device

Abstract

The application relates to the technical field of organoid culture, and discloses an online monitoring and culture device for organoids. The online monitoring and culture device for organoids comprises a base frame, a plurality of flow chambers, an optical coherence tomography imaging structure, a Raman spectrum imaging structure, a near-infrared spectrum imaging structure, an optical microscopic imaging structure, an enzyme-linked immunosorbent assay structure, a three-dimensional moving structure and a controller. The controller is electrically connected with the three-dimensional moving structure and is used for driving the three-dimensional moving structure to move to a specified position of the flow chamber so as to observe organoid cells and culture medium in the flow chamber. The controller is electrically connected with the enzyme-linked immunosorbent assay structure. The online monitoring and culture device for organoids can realize online in-situ monitoring of organoids and culture medium in the flow chamber, real-time feedback of data, adjustment of the environment in the flow chamber, dynamic monitoring of the growth state and the environment of the organoids, and long-term and homogenized culture.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and more specifically to an online monitoring and culture device for organoids. Background Technology

[0002] Organoids are three-dimensional (3D) cell cultures that contain some of the key characteristics of their representative organs. These in vitro culture systems consist of a self-renewing population of stem cells that can differentiate into multiple organ-specific cell types, possessing similar spatial organization to the corresponding organ and capable of reproducing some of its functions, thus providing a highly physiologically relevant system.

[0003] Because organoid development requires a long culture period, monitoring its environment and growth process is crucial. Current technologies for monitoring organoid culture typically employ static culture, where nutrients are artificially supplied. This requires manually extracting and analyzing large amounts of data to assess the organoid's growth environment and status. Direct human intervention can easily contaminate the organoid's growth environment, leading to biased analysis results. Another approach uses microtechnology for dynamic culture, which avoids manual operations such as changing the medium. However, this technology lacks integration of multiple functional modules, limiting the range of biochemical indicators that can be monitored online. It cannot achieve in-situ online detection of multiple indicators, which is detrimental to maintaining the organoid culture environment and its growth and development over long periods. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the shortcomings of the prior art, which is that direct human intervention can easily cause pollution of the organoid growth environment, leading to deviations in the analysis results, making it impossible to achieve in-situ online detection of multiple indicators, and making it unfavorable to maintain the organoid culture environment and its growth and development for a long time. The present invention provides an organoid online detection and culture device that can perform online in-situ monitoring of organoids and culture medium in the flow chamber, and provide real-time data feedback and adjust the environment in the flow chamber. Through dynamic monitoring, the growth status and environment of organoids are maintained, and long-term homogeneous culture is achieved.

[0005] Therefore, the present invention proposes an online monitoring and culture device for organoids, comprising:

[0006] A base frame, wherein a first placement layer and a second placement layer are arranged in parallel and spaced apart from top to bottom;

[0007] Multiple flow chambers are suitable for placing organoid cells and culture medium; the multiple flow chambers are arranged in a row along the length of the second placement stage on the second placement layer, and the flow chambers are respectively connected to an air tank and a culture medium storage device;

[0008] An optical coherence tomography (OCT) imaging structure is disposed on the first placement layer, with the imaging probe of the OCT imaging structure facing the organoid cells in the flow chamber;

[0009] A Raman spectroscopy imaging structure is disposed on the first placement layer, with the Raman fiber probe of the Raman spectroscopy imaging structure facing the culture medium in the flow chamber;

[0010] A near-infrared spectral imaging structure is disposed on the first placement layer, with the infrared fiber optic probe of the near-infrared spectral imaging structure facing the culture medium in the flow chamber;

[0011] An optical microscopy imaging structure is disposed on both sides of the flow chamber, with the optical microscopy imaging structure facing the organoid cells inside the flow chamber;

[0012] The enzyme-linked immunosorbent assay (ELISA) structure is connected to the flow chamber via a conduit;

[0013] A three-dimensional moving structure is mounted on the base frame, and the imaging probe, the Raman fiber probe, the infrared fiber probe, and the optical microscopic imaging structure are respectively fixedly mounted on the three-dimensional moving structure;

[0014] A controller, electrically connected to the three-dimensional moving structure, is used to drive the three-dimensional moving structure to a designated position in the flow chamber to observe organoid cells and culture medium within the flow chamber; the controller is also electrically connected to the enzyme-linked immunosorbent assay (ELISA) structure to control the ELISA structure to extract culture medium from the flow chamber for monitoring.

[0015] Furthermore, the enzyme-linked immunosorbent assay (ELISA) structure includes a fixture and a three-dimensional moving device;

[0016] The controller is electrically connected to the three-dimensional moving device and is used to control the movement of the three-dimensional moving device on the fixed frame;

[0017] The mounting frame is provided with a test area that communicates with the pipeline; the mounting frame is also provided with a consumable area suitable for storing the orifice plate; the mounting frame is also provided with a sample preparation area and a reagent area;

[0018] The three-dimensional moving device is equipped with a sampling needle and a clamping structure; the controller is electrically connected to the sampling needle and controls the sampling needle to extract culture medium and reagents; the controller is electrically connected to the clamping structure and controls the clamping structure to clamp the orifice plate.

[0019] Furthermore, the mounting frame is also provided with a cleaning area and a plate washing area;

[0020] The cleaning area is suitable for cleaning the sampling needle;

[0021] The plate washing area is suitable for cleaning the perforated plate.

[0022] Furthermore, the enzyme-linked immunosorbent assay (ELISA) structure also includes an optical analysis module; the controller is electrically connected to the optical analysis module and controls the optical analysis module to perform optical detection and analysis on the sample in the well plate.

[0023] Furthermore, the three-dimensional moving device includes a first driving device, a second driving device, and a third driving device;

[0024] The first driving device is fixedly mounted on the fixed frame, and the fixed frame is provided with a first moving device connected to the first driving device. The controller is electrically connected to the first driving device and is used to control the first driving device to drive the first moving device to move along the length direction of the fixed frame.

[0025] The second driving device is fixedly mounted on the first moving device. The first moving device is provided with a second moving device connected to the second driving device. The controller is electrically connected to the second driving device and is used to control the second driving device to drive the second moving device to move along the width direction of the fixed frame.

[0026] The third driving device is fixedly mounted on the second moving device. The second moving device is provided with a third moving device connected to the third driving device. The controller is electrically connected to the third driving device and is used to control the third driving device to drive the third moving device to move along the height direction of the fixed frame. The third moving device is respectively provided with the sampling needle and the clamping structure.

[0027] Furthermore, the Raman spectroscopy imaging structure also includes a Raman spectroscopy analysis module, a Raman light source module, and a power supply box;

[0028] The Raman fiber optic probe is electrically connected to both the Raman spectroscopy analysis module and the Raman light source module; the power supply box is electrically connected to the Raman light source module.

[0029] Furthermore, the near-infrared spectral imaging structure also includes an infrared spectral analysis module and an infrared light source module; the infrared fiber optic probe is electrically connected to the infrared spectral analysis module and the infrared light source module, respectively.

[0030] Furthermore, the optical microscopic imaging structure includes: a substrate, a bright-field light source module, a fluorescence light source module, and a detection module;

[0031] The fluorescent light source module and the detection module are fixedly mounted on the substrate;

[0032] The bright-field light source module and the fluorescent light source module are respectively disposed on both sides of the flow chamber; the light signals emitted by the bright-field light source module and the fluorescent light source module pass through the flow chamber; the detection module and the fluorescent light source module are located on the same side of the flow chamber.

[0033] Furthermore, the base frame is also provided with a third placement layer, and the second placement layer is located between the first placement layer and the third placement layer;

[0034] The three-dimensional moving structure includes a first driving element, a second driving element, and a third driving element;

[0035] The first driving component is fixedly disposed on the third placement layer. A guide rail is installed on the third placement layer along the length direction of the third placement layer. A movable plate is correspondingly installed on the guide rail. The first driving component is connected to the movable plate. The substrate is movably disposed on the movable plate. The controller is electrically connected to the first driving component and is adapted to control the first driving component to drive the movable plate to move along the guide rail direction.

[0036] A second driving component is fixedly disposed on the movable plate. The second driving component is connected to the substrate. The controller is electrically connected to the second driving component and is adapted to control the second driving component to drive the substrate to move along the width direction of the third placement layer.

[0037] A connecting portion is fixedly disposed on the substrate, and a third driving member is disposed on the connecting portion. A moving portion is provided on the connecting portion, which moves along the height direction of the base frame. The imaging probe, the Raman fiber probe, the infrared fiber probe, and the bright field light source module are fixedly disposed on the moving portion. The third driving member is connected to the moving portion, and the controller is electrically connected to the third driving member, and is adapted to control the third driving member to drive the moving portion to move along the height direction of the base frame.

[0038] Furthermore, a biosensor is fixedly installed at the bottom of the flow chamber.

[0039] The technical solution of this invention has the following advantages:

[0040] 1. The organoid online monitoring and culture device provided by the present invention includes: a base frame, on which a first placement layer and a second placement layer are arranged in parallel at intervals, wherein the first placement layer and the second placement layer are arranged from top to bottom. Multiple flow chambers are adapted to be placed on the second placement layer, and the multiple flow chambers are arranged in a row along the length direction of the second placement layer. Organoid cells and culture medium are adapted to be placed in the flow chambers, and the flow chambers are respectively connected to an air tank and a culture medium storage tank. An optical coherence tomography (OCT) imaging structure is provided on the first placement layer, with the imaging probe of the OCT imaging structure facing the organoid cells inside the flow chamber. A Raman spectroscopy imaging structure is also provided on the first placement layer, with the Raman fiber optic probe of the Raman spectroscopy imaging structure facing the culture medium inside the flow chamber. A near-infrared spectroscopy imaging structure is also provided on the first placement layer, with an infrared fiber optic probe on the near-infrared spectroscopy imaging structure facing the culture medium inside the flow chamber. Optical microscopic imaging structures are located on both sides of the flow chamber, with the optical microscopic imaging structures facing the organoid cells inside the flow chamber. In addition, the flow chamber is connected to the enzyme-linked immunosorbent assay (ELISA) structure via tubing, and a controller is electrically connected to the ELISA structure to control the extraction of culture medium from the flow chamber for monitoring. A three-dimensional moving structure is also mounted on the frame, on which imaging probes, Raman fiber optic probes, infrared fiber optic probes, and optical microscopy imaging structures are fixed. The controller is electrically connected to the three-dimensional moving structure to control its movement to a designated position within the flow chamber for observation of organoid cells and culture medium.

[0041] This online monitoring and culture device for organoids places organoid cells and culture medium in a flow chamber for online culture. The flow chamber is connected to a gas tank, allowing the injection of the necessary gases for organoid culture. The flow chamber is also connected to a culture medium storage device, allowing the injection of the required culture medium. Furthermore, a controller moves a three-dimensional moving structure to a designated position within the flow chamber to observe the organoid cells. Optical coherence tomography (OCT) imaging structure analyzes the images to detect cell morphology and size online, thus monitoring the growth status of the organoid cells. Additionally, the controller moves a Raman fiber optic probe to a designated position within the flow chamber to observe the culture medium, acquiring its Raman spectrum. The Raman spectroscopy imaging structure processes and analyzes this spectrum to detect specific indicators such as glucose, lactic acid, and glutamine in the culture medium online. Furthermore, the controller can move the infrared fiber optic probe to a designated position in the flow chamber via a three-dimensional moving structure to observe the culture medium within the flow chamber, collect its infrared spectrum, and process and analyze the collected infrared spectrum using a near-infrared spectroscopy imaging structure to achieve online detection of amino acids, acetic acid, and calcium, potassium, and magnesium ion concentrations in the culture medium. Additionally, the controller can move the optical microscopy imaging structure to a designated position in the flow chamber via the three-dimensional moving structure to perform optical imaging of the organoid cells within the flow chamber, enabling online detection of indicators such as organoid cell development tracking and the number of live / dead cells. The controller can also control the enzyme-linked immunosorbent assay (ELISA) structure to extract culture medium from the flow chamber for monitoring, enabling online detection of indicators such as alkaline phosphatase (ALP), transforming growth factor β1 (TGF-β1), and tumor necrosis factor α (TNF-α). In short, during organoid culture monitoring, the online organoid monitoring and culture device can perform online in-situ monitoring of organoids and culture medium within the flow chamber, providing real-time data feedback and adjusting the environment within the flow chamber. Through dynamic monitoring, it maintains the organoid growth status and environment, achieving long-term, homogeneous culture. During organoid culture, the elimination of the need for manual data extraction, processing, analysis, and evaluation prevents contamination of the organoid production environment due to direct human intervention, thus ensuring accurate analytical results and enabling in-situ online detection of multiple indicators. This facilitates the long-term maintenance of the organoid culture environment and its growth and development. Furthermore, by controlling the three-dimensional moving structure, online in-situ monitoring of organoids and culture media in multiple flow chambers can be achieved, improving organoid culture efficiency.

[0042] 2. The organoid online monitoring and culture device provided by this invention includes an enzyme-linked immunosorbent assay (ELISA) structure comprising a fixed frame and a three-dimensional moving device. A controller is electrically connected to the three-dimensional moving device to control its movement on the fixed frame. The fixed frame has a test area connected to a consumable area for storing well plates, a sample preparation area, and a reagent area. The three-dimensional moving device has a sampling needle and a clamping structure. The controller is electrically connected to the sampling needle to control its extraction of culture medium and reagents; the controller is also electrically connected to the clamping structure to control its clamping of the well plates, thus enabling automated sample addition and preparation for the ELISA assay.

[0043] 3. The organoid online monitoring and culture device provided by the present invention is further provided with a cleaning area and a plate washing area on the fixed frame. The sampling needle is cleaned in the cleaning area and the plate washing area is cleaned in the plate washing area, so that the enzyme-linked immunosorbent assay structure can be automatically cleaned.

[0044] 4. The organoid online monitoring and culture device provided by the present invention further includes an optical analysis module in the enzyme-linked immunosorbent assay (ELISA) structure. The controller is electrically connected to the optical analysis module to control the optical analysis module to perform optical detection and analysis on the samples in the well plate, thereby enabling automated data analysis of the ELISA structure.

[0045] 5. The organoid online monitoring and culture device provided by the present invention includes a three-dimensional moving device comprising a first driving device, a second driving device, and a third driving device. The first driving device is fixedly mounted on a fixed frame, and a first moving device is movably mounted on the fixed frame and connected to the first driving device. A controller is electrically connected to the first driving device, and the controller controls the first driving device to move the first moving device along the length direction of the fixed frame. The second driving device is fixedly mounted on the first moving device, and a second moving device connected to the second driving device is mounted on the first moving device. A controller is electrically connected to the second driving device, and the controller controls the second driving device to move the second moving device along the width direction of the fixed frame. The third driving device is fixedly mounted on the second moving device, and a third moving device is mounted on the second moving device. The third moving device is connected to the third driving device, and the controller is electrically connected to the third driving device, and the controller controls the third moving device to move along the height direction of the fixed frame. A sampling needle and a clamping structure are mounted on the third moving device. That is, the controller controls the first driving device, the second driving device, and the third driving device respectively, enabling the sampling needle and the clamping structure to move in three dimensions.

[0046] 6. The organoid online monitoring and culture device provided by the present invention further includes a Raman spectroscopy imaging structure comprising a Raman spectroscopy analysis module, a Raman light source module, and a power supply box. A Raman fiber optic probe is electrically connected to both the Raman spectroscopy analysis module and the Raman light source module. The power supply box is electrically connected to the Raman light source module. The power supply box controls the opening and closing of the Raman light source module. The light signal emitted by the Raman light source module is transmitted to the Raman fiber optic probe, enabling the probe to perform a two-dimensional scan of the culture medium above the flow chamber to acquire the Raman spectrum of the culture medium. The acquired Raman spectrum is transmitted to the Raman spectroscopy analysis module for spectral information processing and analysis, enabling online monitoring of specific indicators such as glucose, lactic acid, and glutamine in the culture medium.

[0047] 7. The organoid online monitoring and culture device provided by the present invention includes an infrared spectral analysis module and an infrared light source module in its near-infrared spectral imaging structure. The infrared fiber optic probe is electrically connected to the infrared spectral analysis module and the infrared light source module, respectively. By controlling the three-dimensional moving structure, the infrared fiber optic probe performs a two-dimensional scan of the culture medium above the flow chamber to collect the infrared spectrum of the culture medium. The collected infrared spectrum is transmitted to the infrared spectral analysis module for spectral information processing and analysis, thereby realizing online monitoring of indicators such as amino acid, acetic acid, and calcium, potassium and magnesium ion concentrations in the culture medium.

[0048] 8. The organoid online monitoring and culture device provided by this invention includes an optical microscopy imaging structure comprising a substrate, a bright-field light source module, a fluorescence light source module, and a detection module. The fluorescence light source module and the detection module are fixedly mounted on the substrate, while the bright-field light source module and the fluorescence light source module are respectively positioned on opposite sides of the flow chamber. The light signals emitted by the bright-field light source module and the fluorescence light source module pass through the flow chamber. The detection module and the fluorescence light source module are located on the same side of the flow chamber. The light signals emitted by the bright-field light source module and the fluorescence light source module pass through the flow chamber, and the detection module performs optical imaging on various regions of the flow chamber, enabling high-resolution bright-field and multi-channel fluorescence imaging. This allows for online monitoring of indicators such as organoid development tracking (size, etc.), live / dead cell count, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and specific markers.

[0049] 9. The organoid online monitoring and culture device provided by the present invention further includes a third placement layer on the base frame, the third placement layer being arranged parallel to the second placement layer, and the second placement layer being disposed between the third placement layer and the first placement layer. The three-dimensional moving structure includes a first driving component, a second driving component, and a third driving component. The first driving component is fixedly mounted on a third placement layer. A guide rail is installed on the third placement layer, extending along the length of the third placement layer. A movable plate is correspondingly mounted on the guide rail. The first driving component is connected to the movable plate. A substrate is movably mounted on the movable plate. A controller is electrically connected to the first driving component and controls the first driving component to drive the movable plate to move along the guide rail. A second driving component is fixedly mounted on the movable plate and connected to the substrate. The controller is electrically connected to the second driving component and controls the second driving component to drive the substrate to move along the width of the third placement layer. A connecting part is fixedly mounted on the substrate. The third driving component is mounted on the connecting part, which has a movable part that moves along the height of the base frame. An imaging probe, a Raman fiber probe, an infrared fiber probe, and a bright field light source module are fixedly mounted on the movable plate. The third driving component is connected to the movable part, and the controller is electrically connected to the third driving component and controls the third driving component to drive the movable part to move along the height of the base frame. That is, the first driving component, the second driving component, and the third driving component are controlled by the controller to enable the imaging probe, the Raman fiber probe, the infrared fiber probe, and the optical microscopic imaging structure to move in three dimensions.

[0050] 10. The organoid online monitoring and culture device provided by the present invention has a biosensor fixedly installed at the bottom of the flow chamber, which enables in-situ online collection of indicators such as pH, voltage, current, ATP, albumin, glucose, and endotoxin within the flow chamber. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This is a schematic diagram of the structure of the organoid online monitoring and culture device in an embodiment of the present invention;

[0053] Figure 2 This is a schematic diagram of the hidden portion of the base frame of the organoid online monitoring and culture device in an embodiment of the present invention;

[0054] Figure 3 This is a schematic diagram of the optical coherence tomography imaging structure in the organoid online monitoring and culture device according to an embodiment of the present invention;

[0055] Figure 4 This is a schematic diagram of the Raman spectroscopy imaging structure in the organoid online monitoring and culture device according to an embodiment of the present invention;

[0056] Figure 5 This is a schematic diagram of the near-infrared spectral imaging structure in the organoid online monitoring and culture device according to an embodiment of the present invention;

[0057] Figure 6 This is a schematic diagram of the optical microscopic imaging structure in the organoid online monitoring and culture device in this embodiment of the invention;

[0058] Figure 7 This is a schematic diagram of the optical microscopic imaging structure in the organoid online monitoring and culture device in this embodiment of the invention;

[0059] Figure 8 This is a side view of the optical microscopic imaging structure in the organoid online monitoring and culture device in an embodiment of the present invention;

[0060] Figure 9 This is a schematic diagram of the structure of the enzyme-linked immunosorbent assay (ELISA) in the organoid online monitoring and culture device of this invention.

[0061] Figure 10 This is a schematic diagram of the structure of the fixation frame for the hidden part of the enzyme-linked immunosorbent assay (ELISA) in the organoid online monitoring and culture device of this invention.

[0062] Figure 11 This is a schematic diagram of the flow chamber in the organoid online monitoring and culture device according to an embodiment of the present invention.

[0063] Explanation of reference numerals in the attached figures:

[0064] 1. Base frame; 2. Gas tank;

[0065] 3. Optical coherence tomography imaging structure; 301. Imaging analysis module; 302. Imaging probe; 303. First connecting line;

[0066] 4. Raman spectroscopy imaging structure; 401. Raman spectroscopy analysis module; 402. Raman light source module; 403. Power supply box; 404. Raman fiber optic probe; 405. Second connecting cable; 406. Third connecting cable; 407. Fourth connecting cable;

[0067] 5. Near-infrared spectral imaging structure; 501. Infrared spectral analysis module; 502. Infrared light source module; 503. Infrared fiber optic probe; 504. Fifth connecting line; 505. Sixth connecting line;

[0068] 6. Optical microscopic imaging structure; 601. Substrate; 602. Bright field light source module; 603. Fluorescent light source module; 604. Detection module;

[0069] 7. Flow chamber; 701. Biosensor;

[0070] 8. Guide rail; 9. Culture medium storage container;

[0071] 10. Enzyme-linked immunosorbent assay (ELISA) structure; 101. Fixture; 102. Consumables area; 103. Plate washing area; 104. Three-dimensional moving device; 105. Sampling needle; 106. Test area; 107. Cleaning area; 108. Reagent area; 109. Optical analysis module; 1010. Sample preparation area; 1011. Clamping structure. Detailed Implementation

[0072] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0074] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0075] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0076] This embodiment provides an online monitoring and culture device for organoids, such as... Figure 1 and Figure 2As shown, the system includes: a base frame 1, with a first placement layer and a second placement layer arranged parallel to each other on the base frame 1, wherein the first and second placement layers are arranged from top to bottom. This layered arrangement allows for full utilization of the space in the base frame 1 and facilitates the orderly placement of various components. The second placement layer is suitable for placing multiple flow chambers 7, which are arranged in rows along the length of the second placement layer. The flow chambers 7 are suitable for placing organoid cells and culture medium. The flow chambers 7 are respectively connected to a gas tank 2 and a culture medium storage device 9, wherein the gas tank 2 and the culture medium storage device 9 can be located on the base frame 1 or around the base frame 1. The first placement layer is provided with an optical coherence tomography (OCT) imaging structure 3, with its imaging probe 302 facing the organoid cells inside the flow chamber 7. The first placement layer is also provided with a Raman spectroscopy imaging structure 4, with its Raman fiber optic probe 404 facing the culture medium inside the flow chamber 7. A near-infrared spectral imaging structure 5 is also provided on the first placement layer. An infrared fiber optic probe 503 is mounted on the near-infrared spectral imaging structure 5, facing the culture medium inside the flow chamber 7. Optical microscopic imaging structures 6 are located on both sides of the flow chamber 7, facing the organoid cells inside the flow chamber 7. Furthermore, the flow chamber 7 is connected to an enzyme-linked immunosorbent assay (ELISA) structure 10 via a pipe. A controller is electrically connected to the ELISA structure 10 to control the ELISA structure 10 to extract the culture medium inside the flow chamber 7 for monitoring. A three-dimensional moving structure is also provided on the base frame 1. An imaging probe 302, a Raman fiber optic probe 404, an infrared fiber optic probe 503, and the optical microscopic imaging structure 6 are fixedly mounted on the three-dimensional moving structure. The controller is electrically connected to the three-dimensional moving structure to control its movement to a designated position within the flow chamber 7 to observe the organoid cells and culture medium inside the flow chamber 7.

[0077] This type of online monitoring and culture device for organoids places organoid cells and culture medium in a flow chamber 7 for online culture. The flow chamber 7 is connected to a gas tank 2, which can be used to inject the gases required for organoid culture into the flow chamber 7. The gas tank 2 can contain gases such as carbon dioxide and nitrogen. The flow chamber 7 is also connected to a culture medium storage device 9, which can be used to inject the culture medium required for organoid culture into the flow chamber 7. Furthermore, a controller moves a three-dimensional moving structure to a designated position in the flow chamber 7 to observe the organoid cells. An optical coherence tomography (OCT) imaging structure 3 performs image analysis to detect cell morphology and size online, thus observing the growth status of the organoid cells. Additionally, a controller moves a three-dimensional moving structure to a designated position in the flow chamber 7 to observe the culture medium and collect its Raman spectrum. A Raman spectroscopy imaging structure 4 processes and analyzes the Raman spectrum to detect specific indicators such as glucose, lactic acid, and glutamine in the culture medium online. Furthermore, the controller can move the infrared fiber optic probe 503 to a designated position in the flow chamber 7 via a three-dimensional moving structure to observe the culture medium within the flow chamber 7 and collect its infrared spectrum. The collected infrared spectrum is then processed and analyzed by the near-infrared spectroscopy imaging structure 5 to achieve online detection of amino acids, acetic acid, and calcium, potassium, and magnesium ion concentrations in the culture medium. Additionally, the controller can move the optical microscopy imaging structure 6 to a designated position in the flow chamber 7 via the three-dimensional moving structure to perform optical imaging of the organoid cells within the flow chamber 7, enabling online detection of indicators such as organoid cell development tracking and the number of live / dead cells. The controller can also control the enzyme-linked immunosorbent assay (ELISA) assay structure 10 to extract culture medium from the flow chamber 7 for monitoring, enabling online detection of indicators such as alkaline phosphatase (ALP), transforming growth factor β1 (TGF-β1), and tumor necrosis factor α (TNF-α). In the process of organoid culture monitoring, the online organoid monitoring and culture device enables online in-situ monitoring of organoids and culture medium within flow chamber 7, providing real-time data feedback and adjusting the environment within flow chamber 7. Dynamic monitoring maintains the growth status and environment of organoids, achieving long-term, homogeneous culture. During organoid culture, there is no need for manual extraction, processing, analysis, and evaluation of large amounts of data, preventing direct human intervention that could contaminate the organoid production environment. This ensures accurate analytical results and allows for in-situ online detection of multiple indicators, facilitating the long-term maintenance of the organoid culture environment and its growth and development. Furthermore, by controlling the three-dimensional moving structure, online in-situ monitoring of organoids and culture medium within multiple flow chambers 7 can be performed, improving organoid culture efficiency.

[0078] Specifically, such as Figure 9 and Figure 10As shown, the enzyme-linked immunosorbent assay (ELISA) structure 10 includes a fixture 101 and a three-dimensional moving device 104. A controller is electrically connected to the three-dimensional moving device 104 to control its movement on the fixture 101. A test area 106 is provided on the fixture 101, which is connected to a consumable area 102 suitable for storing well plates. The fixture 101 also includes a sample preparation area 1010 and a reagent area 108. A sampling needle 105 and a clamping structure 1011 are provided on the three-dimensional moving device 104. The controller is electrically connected to the sampling needle 105 to control its extraction of culture medium and reagents; the controller is also electrically connected to the clamping structure 1011 to control its clamping of the well plates, thus enabling automated sample addition and preparation in the ELISA assay structure 10.

[0079] Specifically, such as Figure 9 and Figure 10 As shown, the fixture 101 is also provided with a cleaning area 107 and a plate washing area 103. The sampling needle 105 is cleaned by the cleaning area 107 and the plate washing area 103 is cleaned by the plate washing area, so that the enzyme-linked immunosorbent assay structure 10 can be automatically cleaned.

[0080] Specifically, such as Figure 9 and Figure 10 As shown, the enzyme-linked immunosorbent assay (ELISA) structure 10 also includes an optical analysis module 109, which is electrically connected to the optical analysis module 109 via a controller. The controller controls the optical analysis module 109 to perform optical detection and analysis on the samples in the well plate, thereby enabling the ELISA structure 10 to perform automated data analysis.

[0081] Specifically, such as Figure 10As shown, the three-dimensional moving device 104 includes a first driving device, a second driving device, and a third driving device. The first driving device is fixedly mounted on a fixed frame 101, and the first moving device is movably mounted on the fixed frame 101. The first moving device is connected to the first driving device, and a controller is electrically connected to the first driving device. The controller controls the first driving device to drive the first moving device to move along the length direction of the fixed frame 101. The second driving device is fixedly mounted on the first moving device, and the first moving device is provided with a second moving device connected to the second driving device. The controller is electrically connected to the second driving device, and the controller controls the second driving device to drive the second moving device to move along the width direction of the fixed frame 101. The third driving device is fixedly mounted on the second moving device, and the second moving device is provided with a third moving device. The third moving device is connected to the third driving device, and a controller is electrically connected to the third driving device. The controller controls the third driving device to drive the third moving device to move along the height direction of the fixed frame 101. The sampling needle 105 and the clamping structure 1011 are provided on the third moving device. Specifically, the first, second, and third driving devices are controlled by a controller to enable three-dimensional movement of the sampling needle 105 and the gripping structure 1011. The driving devices can be motors, which drive the moving devices. For example, the motor is connected to a conveyor belt via pulleys, and the conveyor belt is connected to the moving device. The rotation of the motor drives the pulleys, causing the conveyor belt to rotate, which in turn moves the moving device. Alternatively, a lead screw transmission method can be used, where a motor drives the lead screw to rotate, thus moving the moving device.

[0082] Specifically, such as Figure 3 As shown, the optical coherence tomography imaging structure 3 includes an imaging analysis module 301 and an imaging probe 302. The imaging analysis module 301 and the imaging probe 302 are connected by a first connecting line 303. The imaging probe 302 is driven by a three-dimensional moving structure to scan and image organoid cells in the flow chamber 7 above the flow chamber 7. The imaging signal collected by the imaging probe 302 is transmitted to the imaging analysis module 301 through the first connecting line 303. The imaging analysis module 301 converts the collected signal into two-dimensional or three-dimensional images. Through image analysis, online monitoring of organoid cell or tissue morphology, size and other indicators can be achieved.

[0083] Specifically, such as Figure 4As shown, the Raman spectroscopy imaging structure 4 also includes a Raman spectroscopy analysis module 401, a Raman light source module 402, and a power supply box 403. The Raman fiber optic probe 404 is connected to the Raman spectroscopy analysis module 401 via a second connecting line 405, the Raman fiber optic probe 404 is connected to the Raman light source module 402 via a third connecting line 406, and the power supply box 403 is connected to the Raman light source module 402 via a fourth connecting line 407. The Raman light source module 402 is turned on and off by controlling the power supply box 403. The light signal emitted by the Raman light source module 402 is transmitted to the Raman fiber optic probe 404, which performs a two-dimensional scan of the culture medium above the flow chamber 7 to collect the Raman spectrum of the culture medium. The collected Raman spectrum is transmitted to the Raman spectroscopy analysis module 401 for spectral information processing and analysis, which can realize online monitoring of specific indicators such as glucose, lactic acid, and glutamine in the culture medium.

[0084] Specifically, such as Figure 5 As shown, the near-infrared spectral imaging structure 5 also includes an infrared spectral analysis module 501 and an infrared light source module 502. The infrared fiber optic probe 503 is connected to the infrared spectral analysis module 501 via a fifth connecting line 504, and the infrared fiber optic probe 503 is connected to the infrared light source module 502 via a sixth connecting line 505. By controlling the three-dimensional moving structure, the infrared fiber optic probe 503 performs a two-dimensional scan of the culture medium above the flow chamber 7 to collect the infrared spectrum of the culture medium. The collected infrared spectrum is transmitted to the infrared spectral analysis module 501 for spectral information processing and analysis, thereby realizing online monitoring of indicators such as amino acid, acetic acid, and calcium, potassium, and magnesium ion concentrations in the culture medium.

[0085] Specifically, such as Figures 6-8 As shown, the optical microscopy imaging structure 6 includes a substrate 601, a bright-field light source module 602, a fluorescence light source module 603, and a detector module 604. The fluorescence light source module 603 and the detector module 604 are fixedly mounted on the substrate 601. The bright-field light source module 602 and the fluorescence light source module 603 are respectively positioned on opposite sides of the flow chamber 7. The light signals emitted by the bright-field light source module 602 and the fluorescence light source module 603 pass through the flow chamber 7. The detector module 604 and the fluorescence light source module 603 are located on the same side of the flow chamber 7. The light signals emitted by the bright-field light source module 602 and the fluorescence light source module 603 pass through the flow chamber 7, and the detector module 604 performs optical imaging on each region of the flow chamber 7, enabling high-resolution bright-field and multi-channel fluorescence imaging. This allows for online monitoring of indicators such as organoid development tracking (size, etc.), live / dead cell count, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and specific markers.

[0086] Specifically, such as Figure 1 and Figure 2As shown, a third placement layer is also provided on the base frame 1. The third placement layer is arranged parallel to the second placement layer, and the second placement layer is located between the third placement layer and the first placement layer. The three-dimensional moving structure includes a first driving component, a second driving component, and a third driving component. The first driving component is fixedly mounted on the third placement layer. A guide rail 8 is installed on the third placement layer and extends along the length direction of the third placement layer. A moving plate is correspondingly mounted on the guide rail 8. The first driving component is connected to the moving plate. The base plate 601 is movably mounted on the moving plate. The controller is electrically connected to the first driving component and controls the first driving component to drive the moving plate to move along the direction of the guide rail 8. A second driving component is fixedly mounted on the moving plate and is connected to the base plate 601. The controller is electrically connected to the second driving component. The driving components are electrically connected, and the controller controls the second driving component to move the substrate 601 along the width direction of the third placement layer. A connecting part is fixedly provided on the substrate 601, and the third driving component is disposed on the connecting part. The connecting part is provided with a moving part that moves along the height direction of the base frame 1. An imaging probe 302, a Raman fiber probe 404, an infrared fiber probe 503, and a bright field light source module 602 are respectively fixedly disposed on the moving part. The third driving component is connected to the moving part, and the controller is electrically connected to the third driving component. The controller controls the third driving component to drive the moving part to move along the height direction of the base frame 1. That is, the controller controls the first, second, and third driving components to enable the imaging probe 302, the Raman fiber probe 404, the infrared fiber probe 503, and the optical microscopic imaging structure 6 to achieve three-dimensional movement. Among them, the first, second, and third driving components can all be driven by motors.

[0087] Specifically, such as Figure 11 As shown, a biosensor 701 is fixedly installed at the bottom of the flow chamber 7. The biosensor 701 can be a 3D electrode, an array electrode, a flexible electrode, or an electrochemical electrode, etc. The biosensor 701 enables in-situ online collection of indicators such as pH, voltage, current, ATP, albumin, glucose, and endotoxin in the flow chamber 7.

[0088] The principle of enzyme-linked immunosorbent assay (ELISA) for structure 10 in this embodiment is as follows:

[0089] The enzyme-linked immunosorbent assay (ELISA) structure 10 is connected in series with the flow chamber 7 via a pipe. The culture medium inside the flow chamber 7 is extracted and transferred to the test area 106 via the pipe. The three-dimensional moving device 104 controls the gripping structure 1011 to grab the well plate in the consumable area 102 and place it in the sample preparation area 1010. Culture medium and reagents can be placed in the well plate. The controller controls the three-dimensional moving device 104 to move the sampling needle 105, extracting the culture medium from the test area 106 to the test sample well in the reagent area 108. ELISA test reagents are placed in other wells in the reagent area 108. The sampling needle 105 continuously takes samples from each well in the reagent area 108 and repeatedly cleans the sampling needle 105 in the cleaning chamber before adding the sample to the well plate in the sample preparation area 1010. The clamping structure 1011 places the well plate into the optical analysis module 109 for incubation. After incubation, the clamping structure 1011 clamps the well plate into the washing area 103 for washing. After washing, the well plate is placed back into the sample preparation area 1010, and the sampling needle 105 performs sampling and sample addition operations again. Finally, a well plate sample is prepared in the sample preparation area 1010. Then, the clamping structure 1011 places the well plate from the sample preparation area 1010 into the optical analysis module 109 for optical detection and analysis. Online ELISA systems can automate operations from sample addition, preparation, and cleaning to data analysis, enabling online ELISA-related detection and monitoring of indicators such as alkaline phosphatase (ALP), transforming growth factor β1 (TGF-β1), and tumor necrosis factor α (TNF-α).

[0090] The principle of the organoid online monitoring and culture device in this embodiment is:

[0091] The controller moves the three-dimensional moving structure imaging probe 302 to a designated position in the flow chamber 7 to observe organoid cells within the flow chamber 7. The optical coherence tomography imaging structure 3 performs image analysis to detect cell morphology and size online, thereby observing the growth status of organoid cells.

[0092] The Raman fiber probe 404 is moved to a designated position in the flow chamber 7 by a three-dimensional moving structure controlled by the controller to observe the culture medium in the flow chamber 7 and collect the Raman spectrum of the culture medium. The Raman spectrum of the culture medium is then processed and analyzed by the Raman spectroscopy imaging structure 4 to realize the online detection of specific indicators such as glucose, lactic acid, and glutamine in the culture medium.

[0093] The infrared fiber optic probe 503 is moved to a designated position in the flow chamber 7 by a three-dimensional moving structure controlled by the controller to observe the culture medium in the flow chamber 7, collect the infrared spectrum of the culture medium, and process and analyze the collected infrared spectrum through the near-infrared spectral imaging structure 5 to realize online detection of amino acids, acetic acid, calcium, potassium and magnesium ion concentrations in the culture medium.

[0094] The controller moves the optical microscopy imaging structure 6 to a designated position in the flow chamber 7 using a three-dimensional moving structure, enabling optical imaging of organoid cells within the flow chamber 7 and online detection of indicators such as organoid cell development tracking and the number of live / dead cells. The controller also controls the enzyme-linked immunosorbent assay (ELISA) structure 10 to extract culture medium from the flow chamber 7 for monitoring, enabling online detection of indicators such as alkaline phosphatase (ALP), transforming growth factor β1 (TGF-β1), and tumor necrosis factor α (TNF-α).

[0095] Based on the test results of various indicators, control the gas tank 2 and the culture medium storage 9 to add gas and culture medium into the flow chamber 7 to maintain the culture environment in the flow chamber 7.

[0096] During the monitoring of organoid culture, the online monitoring and culture device can monitor the organoids and culture medium in the flow chamber 7 online in situ, provide real-time data feedback, and adjust the environment in the flow chamber 7. Through dynamic monitoring, the growth status and environment of the organoids are maintained, achieving long-term, homogeneous culture.

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An online monitoring and culture device for organoids, characterized in that, include: The base frame (1) is provided with a first placement layer, a second placement layer and a third placement layer arranged in parallel from top to bottom; Multiple flow chambers (7) are suitable for placing organoid cells and culture medium; multiple flow chambers (7) are arranged in a row along the length of the second placement platform on the second placement layer, and the flow chambers (7) are respectively connected to the gas tank (2) and the culture medium storage (9), and a biosensor (701) is fixedly installed at the bottom of the flow chambers (7). An optical coherence tomography (OCT) imaging structure (3) is disposed on the first placement layer, and the imaging probe (302) of the OCT imaging structure (3) faces the organoid cells in the flow chamber (7); A Raman spectroscopy imaging structure (4) is disposed on the first placement layer, and the Raman fiber probe (404) of the Raman spectroscopy imaging structure (4) faces the culture medium inside the flow chamber (7); Near-infrared spectral imaging structure (5) is disposed on the first placement layer, and the infrared fiber optic probe (503) of the near-infrared spectral imaging structure (5) faces the culture medium in the flow chamber (7); An optical microscopy imaging structure (6) is disposed on both sides of the flow chamber (7), the optical microscopy imaging structure (6) facing the organoid cells in the flow chamber (7), the optical microscopy imaging structure (6) includes: a substrate (601), a bright field light source module (602), a fluorescence light source module (603) and a detection module (604), the fluorescence light source module (603) and the detection module (604) are fixedly disposed on the substrate (601); The bright field light source module (602) and the fluorescent light source module (603) are respectively disposed on both sides of the flow chamber (7); the light signals emitted by the bright field light source module (602) and the fluorescent light source module (603) pass through the flow chamber (7); the detection module (604) and the fluorescent light source module (603) are located on the same side of the flow chamber (7); The enzyme-linked immunosorbent assay (ELISA) structure (10) is connected to the flow chamber (7) via a pipe. The ELISA structure (10) includes a fixture (101) and a three-dimensional moving device (104). The fixture (101) is provided with a test area (106) connected to the pipe. The fixture (101) is also provided with a consumable area (102) suitable for storing well plates, a sample preparation area (1010), a reagent area (108), a cleaning area (107), a plate washing area (103), and an optical analysis module (109). The three-dimensional moving device (104) is provided with a sampling needle (105) and a clamping structure (1011). A three-dimensional moving structure is provided on the base frame (1). The three-dimensional moving structure includes a first driving member, a second driving member and a third driving member. The first driving member is fixedly disposed on the third placement layer. A guide rail (8) is installed on the third placement layer along the length direction of the third placement layer. A moving plate is correspondingly installed on the guide rail (8). The first driving member is connected to the moving plate. The base plate (601) is movably disposed on the moving plate. A second driving member is fixedly disposed on the moving plate. The second driving member is connected to the base plate (601). A connecting part is fixedly provided on the substrate (601), the third driving member is provided on the connecting part, the connecting part is provided with a moving part that moves along the height direction of the base frame (1), and the imaging probe (302), the Raman fiber probe (404), the infrared fiber probe (503) and the bright field light source module (602) are respectively fixedly provided on the moving part. The controller is electrically connected to the first drive unit, the second drive unit and the third drive unit, and is adapted to control the first drive unit to drive the moving plate to move along the direction of the guide rail (8), control the second drive unit to drive the substrate (601) to move along the width direction of the third placement layer, and control the third drive unit to drive the moving part to move along the height direction of the base frame (1) so as to drive the imaging probe (302), the Raman fiber probe (404), the infrared fiber probe (503) and the optical microscopic imaging structure (6) to move to the designated position of the flow chamber (7) so as to observe the organoid cells and culture medium in the flow chamber (7); The controller is electrically connected to the enzyme-linked immunosorbent assay (ELISA) structure (10) and is used to control the ELISA structure (10) to extract the culture medium in the flow chamber (7) for monitoring, control the sampling needle (105) to extract the culture medium and reagents, control the clamping structure (1011) to clamp the well plate, and control the optical analysis module (109) to perform optical detection and analysis on the sample in the well plate.

2. The organoid online monitoring and culture device according to claim 1, characterized in that, The three-dimensional moving device (104) includes a first driving device, a second driving device and a third driving device; The first driving device is fixedly mounted on the fixed frame (101). The fixed frame (101) is provided with a first moving device connected to the first driving device. The controller is electrically connected to the first driving device and is used to control the first driving device to drive the first moving device to move along the length direction of the fixed frame (101). The second driving device is fixedly mounted on the first moving device. The first moving device is provided with a second moving device connected to the second driving device. The controller is electrically connected to the second driving device and is used to control the second driving device to drive the second moving device to move along the width direction of the fixed frame (101). The third driving device is fixedly mounted on the second moving device. The second moving device is provided with a third moving device connected to the third driving device. The controller is electrically connected to the third driving device and is used to control the third driving device to drive the third moving device to move along the height direction of the fixed frame (101). The third moving device is respectively provided with the sampling needle (105) and the clamping structure (1011).

3. The organoid online monitoring and culture device according to claim 2, characterized in that, The Raman spectroscopy imaging structure (4) also includes a Raman spectroscopy analysis module (401), a Raman light source module (402), and a power supply box (403). The Raman fiber optic probe (404) is electrically connected to the Raman spectroscopy analysis module (401) and the Raman light source module (402) respectively; the power supply box (403) is electrically connected to the Raman light source module (402).

4. The organoid online monitoring and culture device according to claim 3, characterized in that, The near-infrared spectral imaging structure (5) further includes an infrared spectral analysis module (501) and an infrared light source module (502); the infrared fiber optic probe (503) is electrically connected to the infrared spectral analysis module (501) and the infrared light source module (502) respectively.

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