Full-automatic cell dynamic real-time label-free online monitoring and analysis system

Abstract

The invention belongs to the technical field of biomedical detection and analysis, and particularly relates to a full-automatic cell dynamic real-time label-free online monitoring and analysis system. Comprising a cell culture cavity, an image acquisition module and a detection and analysis module, the cell culture cavity is of a double-cavity structure and comprises an inner cavity and an outer cavity, the top of the inner cavity is provided with a breathable film and is provided with a liquid supply pipeline and a waste liquid collection pipeline, the outer cavity is provided with a gas supply pipeline, the gas supply pipeline is provided with a gas detection module, and the cell culture cavity is used for culturing cells; the image acquisition module is used for acquiring cell images in the inner chamber; and the detection and analysis module stores the trained classification model and is used for extracting the texture, shadow and color information of the cell image, inputting the information into the classification model and outputting a cell activity classification result.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection and analysis, and particularly relates to a fully automatic cell dynamic real-time label-free online monitoring and analysis system. Background Art

[0002] In the fields of biotechnology and biomedical engineering, the online analysis system of cell morphology is an active and continuously developing research direction, which integrates multidisciplinary knowledge and technologies such as biology, materials science, electronic engineering, and computer science, aiming to develop equipment capable of real-time monitoring and morphological analysis of cells, and promoting the progress of biomedical research and clinical applications.

[0003] Currently, the acquisition of cell dynamic morphological information mainly relies on offline analysis, that is, sampling during or after the culture process, and then using instruments to sort out and analyze the process data. Among them, flow cytometry measures, stores, and displays characteristic parameters of suspended cells through its specific devices and technologies, and sorts cell subpopulations according to preset parameter ranges. In the field of biopharmaceuticals, process analytical technology (PAT) is used to online real-time monitor the cell culture process, optimize and control productivity and product quality, and can conduct online real-time observation and analysis of key product quality measurements and process parameters.

[0004] In addition, some efficient and low-cost methods such as using conventional counting plates and semi-automatic / automatic devices for cell counting, and using an inverted microscope to observe and record cell size and morphology. With the development of microscopic imaging technology, in-situ microscope imaging technology has been widely used because it can directly display local images. In terms of cell culture monitoring, some people use small bioreactors, such as installing an in-situ microscope with a special structure in the bioreactor to capture cell images, and outputting cell growth state information through image processing methods. In recent years, microfluidic technology, sensor technology, and wireless communication technology have developed rapidly, and a variety of cell culture monitoring systems have been developed. These systems integrate microfluidic chips, biochemical sensors, temperature control units, and image data acquisition and processing modules, and can real-time monitor the cell culture environment (such as pH value, oxygen concentration, nutrient concentration, etc.) and cell status (such as cell number, morphology, activity, etc.).

[0005] Traditional methods for cell morphology analysis mainly rely on manual observation and measurement. This approach requires a large amount of time and human resources and is vulnerable to subjective factors, leading to inaccurate results. Methods such as microscope observation, Enzyme Linked Immunosorbent Assay (ELISA), and flow cytometry have provided a great deal of important information for cell biology research in the past few decades. However, these methods often require removing cells from the culture environment, which may not only interfere with the cell state but also traditional methods cannot obtain real-time dynamic information of cells and cannot meet the requirements of biomedical research for real-time online monitoring and analysis. In recent years, with the rapid development of microfluidic technology, optical imaging technology, and biosensing technology, cell dynamic real-time monitoring methods based on these technologies have begun to be studied and applied. These methods can achieve real-time monitoring of various characteristics of cell growth status, morphological changes, biochemical properties, etc. without damaging cells. However, these systems are often costly and complex to operate, making it difficult to be widely applied in ordinary laboratories and clinical environments. Summary of the Invention

[0006] In view of this, the present invention provides a fully automatic cell dynamic real-time label-free online monitoring and analysis system, which can monitor the dynamic changes of cells in real time, timely obtain information about the cell state, and provide important support for the research of cell dynamic processes.

[0007] The technical solution of the present invention is implemented as follows:

[0008] A fully automatic cell dynamic real-time label-free online monitoring and analysis system, comprising: a cell culture chamber, an image acquisition module, and a detection and analysis module;

[0009] The cell culture chamber has a double-chamber structure, including an inner chamber and an outer chamber. The top of the inner chamber is a breathable membrane, and a liquid supply pipeline and a waste liquid collection pipeline are provided. A gas supply pipeline is provided on the outer chamber, and a gas detection module is provided on the gas supply pipeline. The cell culture chamber is used for culturing cells;

[0010] The image acquisition module is used to acquire cell images in the inner chamber;

[0011] The detection and analysis module stores a trained classification model, which is used to extract cell image texture, shadow, and color information and input it into the classification model to output a cell activity classification result.

[0012] Optionally, the cell culture chamber of the present invention is a hollow columnar structure, the outer chamber wall is made of PMMA material, and the breathable membrane at the top of the inner chamber is made of PDMS material.

[0013] Optionally, when culturing cells in the present invention, physiological saline is stored in the outer chamber to maintain the osmotic pressure in the culture area.

[0014] Optionally, the liquid supply pipeline of the present invention is composed of an injection pump and a medical hose.

[0015] Optionally, the waste liquid collection pipeline of the present invention is composed of a peristaltic pump and a recovery container.

[0016] Optionally, the gas supply pipeline of the present invention includes a gas sampling bag, a peristaltic pump, a gas detection module, a gas preheating component, and a filter.

[0017] Optionally, the temperature sensors of the present invention are arranged at the top and bottom of the outer chamber, the heating films are arranged at the top and bottom inside the outer chamber, and a heating isolation plate is arranged between the heating film at the top and the wall of the outer chamber.

[0018] Optionally, the gas preheating component of the present invention consists of a microchannel plate and a heating film. The microchannel plate is made of aluminum alloy material through 3D printing technology. A layer of adhesive polyimide heating film is covered on one side of the microchannel plate, and the preheating temperature is controlled by controlling the voltage at both ends of the PI heating film.

[0019] Optionally, the gas detection module of the present invention consists of a gas sensor and a 3D printing kit. Among them, the inlet and outlet of the kit are directly connected to the gas conduit of the gas supply pipeline. The sensor is installed above the kit, and is bonded with sealant above the sensor. A base printed circuit board is provided at the pin of the sensor for reading sensor data. The gas inlet and outlet of the kit are designed in a butt-joint manner, and the kit is designed as a cylindrical cavity in the sensor detection area.

[0020] Optionally, a light source is provided on the image acquisition module of the present invention, and a design of cooperating an inverted microscope with a CCD camera and related connection control are adopted.

[0021] Advantages:

[0022] First, the device can have the ability to collect cell images in real time online, and use a deep learning model to identify and analyze the cell images. Through training with a large amount of cell image data, the system can continuously optimize the model and improve the recognition accuracy and speed of cell morphological features.

[0023] Second, through this system, the dynamic changes of cells can be monitored in real time, information about the cell state can be obtained in a timely manner, and important support is provided for the research on the dynamic process of cells.

[0024] Third, the device provides a good growth environment for cells, can culture sample cells, thereby reducing the consumption of expensive cell samples during the monitoring and analysis process, and reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 is a framework diagram of the online monitoring and analysis system;

[0027] Figure 2 is a schematic diagram of the cell culture cavity structure;

[0028] Figure 3 is a schematic diagram of the gas detection module;

[0029] Figure 4 is a working flow chart of the online monitoring and analysis system. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The embodiments of the present invention will be described in detail below with reference to the drawings.

[0031] It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other; and, based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present disclosure.

[0032] It should be noted that the following describes various aspects of the embodiments within the scope of the appended claims. It should be obvious that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is illustrative only. Based on the present disclosure, those skilled in the art should understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number of aspects described herein can be used to implement the device and / or practice the method. In addition, this device can be implemented and this method can be practiced using other structures and / or functions in addition to one or more of the aspects described herein.

[0033] As Figure 1-2 shown, an embodiment of the present application provides a fully automatic cell dynamic real-time label-free online monitoring and analysis system, including: a cell culture cavity, an image acquisition module, and a detection and analysis module; wherein,

[0034] The cell culture chamber has a double-chamber structure, including an inner chamber and an outer chamber. The top of the inner chamber is a breathable membrane, and a liquid supply pipeline and a waste liquid collection pipeline are provided. A gas supply pipeline is provided on the outer chamber, and a gas sensor is provided on the gas supply pipeline. The cell culture chamber is used for culturing cells;

[0035] The image acquisition module is used to acquire cell images in the inner chamber;

[0036] The detection and analysis module stores a trained classification model, which is used to extract cell image texture, shadow and color information and input it into the classification model to output the cell activity classification result.

[0037] In the line monitoring and analysis system of this embodiment, the inner chamber maintains the delivery of cell culture medium and the recovery of waste liquid in the inner cavity through the liquid supply pipeline and the waste liquid collection pipeline. The outer chamber maintains the gas update through the gas supply pipeline. The cell culture chamber provides the environmental conditions and nutritional conditions required for the normal growth activities of cells. The image acquisition module and the detection and analysis module can be used to monitor and analyze cells in real time.

[0038] Further, in another embodiment of this embodiment, the overall structure of the cell culture chamber is a hollow column, and the entire chamber structure is composed of an inner cavity and an outer cavity. The design principle and purpose are mainly to better simulate the natural growth environment of cells, effectively reduce the risk of external pollutants entering the culture area, improve the culture efficiency and quality, and at the same time facilitate the control and monitoring of the culture process. The outer chamber wall is made of PMMA material and is processed by an integrated computer numerical control (CNC) machine tool. The inner chamber is composed of a standard small-size culture dish and a PDMS breathable membrane, and the inner cavity can be used for directly culturing cells. The outer chamber is not only equipped with a temperature sensor and a heating film to ensure the internal and bottom temperatures, but also can maintain the osmotic pressure of the culture area through physiological saline.

[0039] Further, in another embodiment of the present application, the liquid supply pipeline is composed of an injection pump and a medical hose, and is used to transport the cell sample solution to be tested and nutrients into the culture chamber. The waste liquid collection pipeline is composed of a peristaltic pump and a recovery container, and is used for the recovery of waste liquid after a period of cell culture.

[0040] Furthermore, in another embodiment of the present application, the gas supply pipeline includes a gas sampling bag, a peristaltic pump, a gas detection module, a gas preheating assembly, and a filter, and is used to replace the gas in the cell culture cavity. In this embodiment, since continuous gas replacement is required during cell culture to ensure that the gas concentration in the environment meets the culture requirements, but the inflow and outflow of gas will cause certain fluctuations in the internal temperature, it is necessary to preheat the gas before it is introduced into the outer chamber of the cell culture device. The heating assembly is composed of a microchannel plate and a heating film. After the gas enters the microchannel plate with this structure, it will continuously move in the channel, so that there is enough time to be heated to a certain temperature. The microchannel plate is made of aluminum alloy material through a 3D printing process. A layer of adhesive polyimide (PI) heating film is covered on one side of the microchannel plate, and the preheating temperature is controlled by controlling the voltage at both ends of the PI heating film.

[0041] Furthermore, in another embodiment of the present application, temperature sensors are arranged at the top and bottom of the outer chamber, the heating films are arranged at the top and bottom inside the outer chamber, and a heating isolation plate is arranged between the heating film at the top and the outer chamber wall.

[0042] Furthermore, in another embodiment of the present application, the breathable membrane is made of PDMS material, and a nano-scale pore network is constructed on it. The gas permeation mechanism of this PDMS material breathable membrane: Traditional breathable materials often have difficulty in balancing gas permeability and barrier properties to other substances. However, the PDMS breathable membrane in this embodiment realizes high-efficiency gas permeation performance through unique microstructural design and material property optimization. It has a carefully constructed nano-scale pore network inside, and the sizes of these pores are precisely regulated, allowing gas molecules (such as oxygen, carbon dioxide, etc.) to diffuse and transmit at a fast and stable rate. Compared with conventional breathable membranes, the gas permeation rate of this PDMS breathable membrane can be increased significantly. While ensuring good gas permeability, it greatly shortens the time required for gas exchange, providing an ideal breathable solution for biological culture devices, etc. The barrier performance of this PDMS material breathable membrane: In terms of blocking substances such as aerosols and liquids that affect cell culture effects, this PDMS breathable membrane shows good effects. Through special surface treatment technology, a protective layer with low surface energy and high-density molecular chain arrangement is formed on the surface of the membrane. This protective layer can effectively prevent the penetration of aerosol particles (including microorganisms carriers such as viruses and bacteria) and various liquids (such as water, organic solvents, etc.). After strict testing, for common liquids such as normal saline and alcohol, there is no obvious penetration phenomenon under long-term contact. This excellent barrier performance makes this breathable membrane provide a good sterile culture environment for the cell culture area.

[0043] Furthermore, in another embodiment of the present application, the gas detection module consists of a gas sensor and a 3D printing kit. Since the carbon dioxide concentration in the mixed gas required by cells is much higher than that of the commonly used carbon dioxide mixed gas, a sensor with a high concentration range is needed for measurement. Since this type of sensor in the prior art is relatively large and cannot be embedded in the gas collection bag and the cell culture cavity, a 3D printing kit is designed for the gas sensor in this embodiment. By connecting the outlet of the gas collection bag to the inlet of the gas sensor kit, the volume of the gas in the gas collection bag can be accurately measured.

[0044] As Figure 3 shown, the inlet and outlet of the kit are directly connected to the gas conduit of the gas supply pipeline. A sensor is installed above the kit, and is bonded with sealant above the sensor. A base printed circuit board is provided at the pin of the sensor for reading sensor data. Among them, the gas inlet and outlet of the kit are designed with opposite ports (that is, the outlet and the inlet are on the same straight line in space, and the gas can directly pass through, with better fluidity). The kit is designed as a cylindrical cavity in the sensor detection area, which can make the gas entering the module more evenly distributed, and is more conducive to the measurement of the internal detection elements of the sensor. Connect the gas inlets and outlets of the culture device to the inlet of the CO 2 sensor kit respectively, and the gas concentration inside the culture device can be accurately measured.

[0045] Furthermore, in another embodiment of the present application, a light source is provided on the image acquisition module, and an inverted microscope and a CCD camera are used in combination with relevant connection control.

[0046] Furthermore, in another embodiment of the present application, a trained classification model is stored in the detection and analysis module, and cell label-free viability detection is realized by using the classification model.

[0047] During the cell culture process, the viability of cells will change due to environmental changes. If the viability of cells can be monitored in real time during this process and adjusted according to the current situation, the effect of cell culture will be greatly improved. At present, the conventional methods for detecting cell viability include colorimetry, cell counting kit method, etc. Most of these methods require processes such as digestion, resuspension, and staining and labeling of cells at specific time points. There are many steps and limited detection points, often missing the time points of cell changes and increasing the detection blind area. However, in addition to being toxic, the staining method of fluorescence imaging will also damage the cell structure, resulting in cell abnormalities or death. This makes precious cell samples unable to be reused, causing cost losses. In addition, many instruments and operation processes require professional personnel to complete and are expensive.

[0048] This embodiment proposes a method for automatically classifying the activity of label-free cells based on deep learning, which is specifically applied to the analysis of cell bright-field images. This method focuses on key features such as image texture, shadow, and color in cell bright-field images. During the training phase, only the operation of classifying and labeling cells is required, and the model constructs a classification model based on these features. When the model is trained and enters the prediction phase, the task of classifying cell activity can be achieved only by relying on the image texture, shadow, and color information contained in the bright-field images of cells. This method greatly simplifies the cell activity detection process and effectively avoids the risk of damage to cell samples and inability to reuse them due to the use of toxic staining agents to process cell samples, thus providing a more efficient, non-destructive, and accurate technical means for cell activity research.

[0049] As Figure 4 shown, the working process of this system is as follows:

[0050] Gas delivery process: After preparing the mixed gas, it is necessary to deliver the gas to the cell culture chamber. After the gas is prepared, it is filled into the gas collection bag. When starting the air exchange, open the pinch valve at the outlet of the culture system, and then start the peristaltic pump to deliver the mixed gas after passing through the gas sensor kit to the microchannel plate with a heating film for preheating, and finally enter the culture area through the gas filter membrane. After the air exchange is completed, close the pinch valve.

[0051] Liquid delivery process: The nutritional conditions for cell culture mainly include culture medium and other nutritional components. Before cell culture, the culture medium and other required nutritional components are mixed, and the update of the mixed nutrients is carried out by perfusion. In order to achieve uniform internal liquid nutrients and complete the harvest of samples, after the cells are resuscitated, the culture medium is first added and delivered to the culture area of the culture device by controlling the syringe pump. After culturing for a period of time, the waste liquid at the top of the cell culture solution is pumped out by the peristaltic pump and transferred to the recovery container. Then, new culture substances are delivered to the culture area by the syringe pump to form a complete cycle system for updating the culture substances.

[0052] Cell culture environment monitoring and real-time image acquisition process: Cell image acquisition is mainly achieved by using a common inverted microscope in the laboratory in cooperation with a CCD camera. Such a design scheme can greatly reduce costs while ensuring the imaging quality of cell images. When in use, directly place the cell culture cavity device on the stage and align the microscope lens directly with the imaging area of the cells.

[0053] The computer controls the microcontroller through a remote connection. The microcontroller is connected to the CCD camera with an image sensor on the inverted microscope in a wired manner, and connects to various sensors through serial communication to collect cell environment state parameters (such as temperature and gas concentration, etc.). The microcontroller controls the CCD camera through the software development kit of the camera, takes real-time pictures of the cells in the cell culture chamber device, and transmits the image files back to the microcontroller. The microcontroller processes the images to obtain the morphological feature information of the cells (cell image texture, shadow and color information) and transmits it to the host computer. The detection and analysis module on the host computer stores the trained classification model, inputs the morphological feature information into the classification model, outputs the cell viability classification result, and views it in real time on the computer. If there is a situation that cannot be processed due to the performance limitation of the Raspberry Pi, a computer with stronger performance can also be directly remotely called for processing.

[0054] In summary, the above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fully automatic real-time label-free online monitoring and analysis system for cell dynamics, characterized in that: include: Cell culture chamber, image acquisition module and detection and analysis module; The cell culture chamber is a double-chamber structure, including an inner chamber and an outer chamber. The top of the inner chamber is a gas-permeable membrane and is provided with a liquid supply pipeline and a waste liquid collection pipeline. The outer chamber is provided with a gas supply pipeline, and the gas supply pipeline is provided with a gas detection module. The cell culture chamber is used to culture cells; The image acquisition module is used to acquire images of cells in the inner chamber; The detection and analysis module stores a trained classification model, which is used to extract cell image texture, shadow and color information and input them into the classification model to output cell activity classification results.

2. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 1, characterized in that: The cell culture chamber is a hollow columnar structure, the outer chamber wall is made of PMMA material, and the air permeable membrane on the top of the inner chamber is made of PDMS material.

3. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 1, characterized in that: When cell culture is performed, physiological saline is stored in the outer chamber to maintain the osmotic pressure of the culture area.

4. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 1, characterized in that: The liquid supply pipeline is composed of a syringe pump and a medical hose.

5. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 1, characterized in that: The waste liquid collection pipeline is composed of a peristaltic pump and a recovery container.

6. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 1, characterized in that: The gas supply pipeline comprises a gas sampling bag, a peristaltic pump, a gas detection module, a gas preheating component and a filter.

7. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 6, characterized in that: The gas preheating component consists of two parts: a microchannel plate and a heating film. The microchannel plate is made of aluminum alloy material through a 3D printing process. One side of the microchannel plate is covered with a layer of polyimide heating film with glue. The preheating temperature is controlled by controlling the voltage at both ends of the PI heating film.

8. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 7, characterized in that: The temperature sensors are arranged at the top and bottom of the outer chamber, the heating film is arranged at the top and bottom of the outer chamber, and a heating isolation plate is arranged between the heating film at the top and the wall of the outer chamber.

9. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 6, characterized in that: The gas detection module consists of a gas sensor and a 3D printed kit; wherein, the inlet and outlet of the kit are directly connected to the gas duct of the gas supply pipeline, the sensor is installed above the kit, and the sensor is bonded with a sealant, and a base printed circuit board is provided at the pin of the sensor for sensor data reading; the gas inlet and outlet of the kit are designed to be ported, and the kit is designed as a cylindrical cavity in the sensor detection area.

10. The fully automatic cell dynamics real-time label-free online monitoring and analysis system according to claim 1, characterized in that: The image acquisition module is provided with a light source, and adopts a design of matching an inverted microscope with a CCD camera and related connection control.

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