Cell culture system for multi-sensor data fusion and flow-controllable micro-fluidic chip

Abstract

The invention belongs to the technical field of cell culture, and relates to a multi-sensor data fusion and flow controllable micro-fluidic chip cell culture system, which comprises a micro-fluidic chip, a liquid exchange device, a gas injection device, a multi-sensor data fusion subsystem and a constant temperature subsystem, the cell culture fluid flow channel in the direction from the culture fluid injection port to the cell culture chamber is in a multi-branch fluid channel shape, the liquid exchange device is communicated to the culture fluid injection port through a pipeline, the gas injection device is communicated to the gas injection port through a pipeline, and the multi-sensor data fusion subsystem and the constant temperature subsystem are electrically connected with the circuit board respectively; the cell culture environment is monitored and controlled, the cell states of multiple different cell culture chambers can be monitored in real time, culture parameters are adjusted in time according to model feedback, the consistency of cell communities in the multiple cell culture chambers is ensured, and therefore the repeatability and reliability of experimental results are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and relates to a cell culture system of multi-sensor data fusion and flow-controllable microfluidic chips. Background Art

[0002] Three-dimensional cell culture is a cell culture technology that improves the understanding and research of cell behavior by simulating the in-vivo environment. This technology can maintain the inherent characteristics of cells, thus more accurately observing cell behavior and exploring its mechanism. Three-dimensional cell culture provides a three-dimensional growth space closer to the in-vivo environment for cells by constructing three-dimensional scaffolds or using materials such as hydrogels, which helps cells maintain their physiological functions and morphology. However, the culture process is relatively complex and the cost is relatively high. At the same time, although three-dimensional culture improves the cell growth environment, it is still difficult to accurately simulate the complex physiological microenvironment in the body, and the culture process is difficult to monitor in real time and precisely control.

[0003] In recent years, the emergence of microfluidic chip technology has brought new solutions for three-dimensional cell culture. This technology uses microfabrication processes to construct tiny channels and chambers on the chip, which can precisely control the microenvironment of cells and realize the culture, manipulation, and analysis of cells. Its advantages are significant. For example, it can simulate the physiological microenvironment in the body and provide growth conditions closer to the real situation for cells; it can realize real-time monitoring and precise control of the cell culture process; it can also greatly reduce the usage of reagents and cells and lower the experimental cost.

[0004] However, the existing cell culture systems based on microfluidic chips still have some deficiencies. First, in terms of multi-channel control, it is impossible to precisely control the flow rate of the input culture medium into multiple culture chambers simultaneously, increasing the error between experimental results. Second, in terms of cell parameter monitoring, it is difficult to simultaneously monitor multiple cell parameters, such as cell metabolic activity, gene expression, protein secretion, etc., which limits the comprehensive understanding of the physiological state of cells. In addition, due to the subtle differences between cell communities cultured in microfluidic chips, even under the same culture conditions, the observation results will still be different, which affects the repeatability and reliability of experimental results to a certain extent.

[0005] Therefore, a device or method that can both monitor and control the cell culture environment in real time and ensure the consistency of the experimental environment in multi-cell culture chambers is needed to solve the above technical problems. Summary of the Invention

[0006] The technical solution adopted by the present invention to solve the technical problems is: a cell culture system of multi-sensor data fusion and flow-controllable microfluidic chips, including:

[0007] A microfluidic chip for monitoring and / or controlling cell culture parameters in a cell culture chamber provided in the microfluidic chip; the cell culture parameters include: chamber flow rate, carbon dioxide concentration, oxygen concentration, temperature, metabolic activity, gene expression, protein secretion; the chamber flow rate, carbon dioxide concentration, oxygen concentration, and temperature are monitored and controlled in real time, and the metabolic activity, gene expression, and protein secretion are monitored in real time;

[0008] A liquid exchange device for injecting a cleaning solution into the microfluidic chip for cleaning during the cleaning stage and then sucking out the cleaning solution, and also for injecting a cell suspension and a culture medium into the microfluidic chip during the culture stage;

[0009] A gas injection device for quickly drying the microfluidic chip during the cleaning stage and also for delivering a culture gas to the cell culture chamber for cell culture during the culture stage; the culture gas includes: oxygen, carbon dioxide;

[0010] A multi-sensor data fusion subsystem for monitoring and analyzing cell parameters in multiple cell culture chambers, so as to comprehensively and accurately understand the physiological state of cells, and also for predicting the culture results of different cell communities, adjusting the culture conditions of each cell culture chamber in advance, reducing the result deviation caused by cell community differences, ensuring the consistency of cell culture, and thus improving the repeatability and reliability of experimental results;

[0011] A constant temperature subsystem for controlling the temperature of the cell culture chamber to ensure the stability of the cell culture environment;

[0012] The microfluidic chip is provided with a culture medium injection port and a cell culture chamber, and the cell culture medium flow channel in the direction from the culture medium injection port to the cell culture chamber is in the shape of multiple bifurcated fluid channels; the cell culture chamber is provided with a circuit board, a fluorescence sensor, a temperature sensor, a carbon dioxide sensor, an oxygen sensor, a serpentine resistance wire, and a gas injection port; the fluorescence sensor, the temperature sensor, the carbon dioxide sensor, the oxygen sensor, and the serpentine resistance wire are respectively electrically connected to the circuit board;

[0013] The cell culture system is provided with multiple microfluidic chips;

[0014] The liquid exchange device is connected to the culture medium injection port through a pipeline, the gas injection device is connected to the gas injection port through a pipeline, and the multi-sensor data fusion subsystem and the constant temperature subsystem are respectively electrically connected to the circuit board.

[0015] Preferably, the cell culture medium flow channel from the culture medium injection port to the cell culture chamber includes: the culture medium injection port is respectively connected to the inlets of two front-stage flow channels, the outlets of the two front-stage flow channels are respectively connected to the inlets of a differential balance flow channel, the outlet of the differential balance flow channel is connected to the inlet of a current-limiting flow channel, and the outlets of the current-limiting flow channels are respectively connected to the cell culture chamber and a flow storage pool.

[0016] Preferably, there are N culture medium inlets in the microfluidic chip, where 1 ≤ N ≤ 100 and N is an integer; the N culture medium inlets respectively correspond to 2N front-stage flow channels, 2N differential balance flow channels, 2N flow-limiting flow channels, 2N cell culture chambers, and 2N flow storage pools.

[0017] Preferably, the connection between the culture medium inlets and two front-stage flow channels is a "Y"-shaped bifurcation; this "Y"-shaped bifurcation has better flow balance and distribution, enabling the flow rate in the front-stage flow channels to be more evenly distributed.

[0018] The connection between the flow-limiting flow channels and the cell culture chambers and the flow storage pools is a "Y"-shaped bifurcation; this "Y"-shaped bifurcation has better flow distribution, enabling further distribution adjustment of the flow rates between the cell culture chambers and the flow storage pools.

[0019] Preferably, the two front-stage flow channels and the two differential balance flow channels are arranged side by side along the fluid flow direction. There is a differential balance diaphragm between the two differential balance flow channels. The differential balance diaphragm is made of an elastic nanomaterial and divides a fluid pipeline into two parallel differential balance flow channels along the fluid flow direction; the differential balance diaphragm is used to adjust the flow rate between adjacent differential balance flow channels.

[0020] Preferably, the connection mode of the flow-limiting flow channels to the cell culture chambers and the flow storage pools includes: the flow-limiting flow channels are sequentially provided with a front-stage flow-limiting flow channel, a cantilever beam, and a flow dividing pile along the fluid flow direction. The cantilever beam is made of an elastically deformable material and is arranged on the left and right inner walls of the front-stage flow-limiting flow channel. There are fluid passage openings reserved on the upper and lower sides of the cantilever beam. The cantilever beam is used to reduce the liquid flow rate by elastic deformation when the liquid flow rate is too fast. Therefore, the cantilever beam can be connected to the inner wall of the front-stage flow-limiting flow channel in various ways, such as by brackets or rib plates. Fluid passage openings can be preset on the upper, lower, left, and right sides of the cantilever beam, but at least the fluid passage openings on the upper and lower sides should be reserved; the flow dividing pile is arranged at the "Y"-shaped bifurcation where the flow-limiting flow channels are connected to the cell culture chambers and the flow storage pools, and the flow dividing pile is in the shape of a triangular frustum; the flow dividing pile is used to limit the flow rate into the flow channels of the cell culture chambers by means of flow division when the liquid flow rate is too fast.

[0021] More preferably, a cell culture chamber flow channel and a flow channel of a flow storage tank are provided at the flow dividing pile. The two ends of the cell culture chamber flow channel are respectively communicated with the front stage current limiting flow channel and the cell culture chamber, and the two ends of the flow channel of the flow storage tank are respectively communicated with the front stage current limiting flow channel and the flow storage tank; the inlet height of the cell culture chamber flow channel is lower than the inlet height of the flow channel of the flow storage tank; when the liquid passes through the flow dividing pile, when the flow rate is normal, since the flow channel of the flow storage tank is higher than the cell culture chamber flow channel, the liquid will flow into the cell culture chamber from the cell culture chamber flow channel; when the liquid flow rate is too large, the liquid level exceeds the bottom surface of the flow channel passing through the flow storage tank, and the liquid exceeding the height difference will flow into the flow storage tank from the flow channel of the flow storage tank, and the flow rate flowing into the cell culture chamber flow channel is controlled through the flow dividing effect.

[0022] Preferably, the liquid exchange device includes: a first micropump, a first hose, and a culture solution pool. The two ends of the first hose are respectively communicated with the culture solution injection port and the culture solution pool, and the first micropump is arranged on the first hose;

[0023] The gas injection device includes: a second micropump, a second hose, and a gas storage tank. The two ends of the second hose are respectively communicated with the gas injection port and the gas storage tank, and the second micropump is arranged on the second hose.

[0024] Preferably, the multi-sensor data fusion subsystem further includes: a PC computer, a WiFi receiver, a data fusion subsystem, a data analysis subsystem, and a negative feedback closed-loop control subsystem. The WiFi receiver, the data fusion subsystem, the data analysis subsystem, and the negative feedback closed-loop control subsystem are arranged in the PC computer;

[0025] The constant temperature subsystem further includes: a regulated power supply and a power cord. The regulated power supply is electrically connected to the circuit board through the power cord.

[0026] Preferably, the operation method of the cell culture system includes the following steps:

[0027] Step 1: Inject a cleaning solution into the microfluidic chip through the liquid exchange device for cleaning, and then suck out the cleaning solution through the liquid exchange device after the cleaning is completed;

[0028] Step 2: Inject gas into the microfluidic chip through the gas injection device, and then suck out the remaining cleaning solution through the liquid exchange device;

[0029] Step 3: After the microfluidic chip is completely dried, inject a cell suspension into the cell culture chamber of the microfluidic chip through the liquid exchange device, and then inject a cell culture solution into the cell culture chamber of the microfluidic chip as needed. During the injection of the cell suspension and the cell culture solution, the microfluidic chip ensures that the flow rates entering multiple cell culture chambers are consistent through the differential balance flow channel, the current limiting flow channel, the flow storage tank and the structures included therein;

[0030] Step 4: Maintain the temperature of the cell culture chamber through the constant temperature subsystem, and monitor and analyze the cell parameters in multiple cell culture chambers through the multi-sensor data fusion subsystem.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The present invention establishes a miniaturized and intelligent cell culture system through multi-sensor data fusion and flow-controllable microfluidic chips, realizing the monitoring and control of the cell culture environment, and directly achieving cell culture and observation in the microfluidic chip.

[0033] 2. The differential balance flow channel, flow-limiting flow channel, flow storage pool and their included structures of the present invention can ensure the precise control of the flow rate in multiple cell culture chambers.

[0034] 3. The constant temperature subsystem of the present invention can precisely control the temperature of the cell culture chamber for a long time through the negative feedback closed-loop control subsystem, ensuring the stability of the cell culture environment.

[0035] 4. The multi-sensor data fusion subsystem adopted by the present invention can realize the simultaneous monitoring and in-depth analysis of multiple cell parameters. The machine learning algorithm in the system can quickly process and analyze these massive and complex data, mine the potential correlations and laws between the data, establish a mapping model between the cell physiological state and multiple parameters through the deep learning algorithm, so as to comprehensively and accurately understand the cell physiological state, find out the key factors affecting cell growth and experimental results, and further establish a prediction model based on the deep neural network according to these factors to predict the culture results of different cell communities before the experiment, adjust the culture conditions in advance, and reduce the result deviation caused by cell community differences. At the same time, the system of the present invention can monitor the cell state in real time and adjust the culture parameters in time according to the model feedback to ensure the consistency of the experimental conditions, thereby improving the repeatability and reliability of the experimental results. Description of the Drawings

[0036] Figure 1 is a schematic structural diagram of the cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip cell culture system of the present invention;

[0037] Figure 2 is a top view of the flow-controllable microfluidic chip when N = 5 in the embodiment of the present invention;

[0038] Figure 3 is a schematic partial structural diagram of the microfluidic chip in the embodiment of the present invention;

[0039] Figure 4 For the present invention Figure 3 is a schematic diagram of the A-A cross-section.

[0040] In the figure, 1 is a microfluidic chip; 100 is a culture medium injection port; 110 is a front flow channel; 111 is an injection port connecting flow channel; 112 is a horizontal flow channel; 120 is a differential balance flow channel; 121 is a differential balance diaphragm; 130 is a current limiting flow channel; 131 is a front current limiting flow channel; 132 is a cantilever beam; 133 is a flow dividing pile; 134 is a cell culture chamber flow channel; 135 is a flow storage pool flow channel; 140 is a cell culture chamber; 141 is a circuit board; 142 is a fluorescence sensor; 143 is a temperature sensor; 144 is a carbon dioxide sensor; 145 is an oxygen sensor; 146 is a serpentine resistance wire; 147 is a gas injection port; 148 is a wire through hole; 150 is a flow storage pool; 2 is a liquid exchange device; 210 is a first micropump; 220 is a first hose; 230 is a culture medium pool; 240 is a first sealing rubber ring; 3 is a gas injection device; 310 is a second micropump; 320 is a second hose; 330 is a gas storage tank; 340 is a second sealing rubber ring; 4 is a multi-sensor data fusion subsystem; 410 is a PC computer; 411 is a WiFi receiver; 412 is a data fusion subsystem; 413 is a data analysis subsystem; 414 is a negative feedback closed-loop control subsystem; 5 is a constant temperature subsystem; 510 is a regulated power supply; 520 is a power cord. Detailed implementation manners

[0041] Next, the relevant technologies in the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0042] Refer to Figures 1 to 4As shown, the cell culture system based on multi-sensor data fusion and flow-controllable microfluidic chip in this embodiment has the characteristics of miniaturization, portability and intelligence compared with the existing cell culture system based on microfluidic chip, realizes the monitoring and control of the cell culture environment, can directly realize cell culture and observation in the microfluidic chip, can ensure the precise control of the flow of multiple cell culture chambers, can accurately control the temperature of the cell culture chamber for a long time, and ensure the stability of the cell culture environment. The multi-sensor data fusion subsystem adopted can help to realize the simultaneous monitoring and in-depth analysis of multiple cell parameters. The machine learning algorithm in the system can quickly process and analyze these massive and complex data, explore the potential correlation and rules between the data, and establish a mapping model between the physiological state of cells and multiple parameters through the deep learning algorithm, so as to fully and accurately understand the physiological state of cells, find out the key factors affecting cell growth and experimental results, and further establish a prediction model based on deep neural network based on these factors. The culture results of different cell communities are predicted before the experiment, and the culture conditions are adjusted in advance to reduce the result deviation caused by differences in cell communities. At the same time, during the experiment, the system can monitor the cell status in real time and adjust the culture parameters in time according to the model feedback to ensure the consistency of experimental conditions, thereby improving the repeatability and reliability of the experimental results.

[0043] As attached Figure 1 As shown, the cell culture system based on multi-sensor data fusion and flow-controllable microfluidic chip of this specific embodiment can be used to simulate the human physiological environment to quickly screen drugs, evaluate the effects and toxicity of drugs on different cells, shorten the research and development cycle, and reduce costs; personalized treatment plans can be formulated according to the culture response of the patient's personal cells; it can be used to observe basic life activities such as cell growth, proliferation, differentiation, apoptosis, and real-time monitoring of cell behavior changes; it can be used to study the growth characteristics, metabolic laws and group behavior of microbial cells; and the study of direct contact and signal exchange between cells is helpful to reveal immune regulation and disease mechanisms.

[0044] The overall structure of this specific embodiment includes: a microfluidic chip 1, a liquid exchange device 2, a gas injection device 3, a multi-sensor data fusion subsystem 4 and a constant temperature subsystem 5;

[0045] This specific implementation establishes a miniaturized and intelligent cell culture system, realizes the monitoring and control of the cell culture environment, and can directly realize cell culture and observation in the microfluidic chip 1.

[0046] As attached Figure 2As shown, the microfluidic chip 1 includes N culture medium inlets 100, 2N front-stage flow channels 110, differential balance flow channels 120, current-limiting flow channels 130, cell culture chambers 140, and flow accumulation pools 150. One culture medium inlet 100 is connected to two front-stage flow channels 110, and N ranges from 5 to 20. The microfluidic chip 1 is formed by bonding multiple layers of PDMS materials. Specifically, as shown in the attached Figure 3 shown.

[0047] The culture medium injection port 100 is cylindrical with a diameter of 1 - 1.5 mm. The front - stage flow channel 110 includes an injection port connecting flow channel 111 and a horizontal flow channel 112, which are used to transmit the culture medium from the culture medium injection port 100 to the differential balance flow channel 120. The injection port connecting flow channel 111 has a width of 300 um and an angle of 22.5 degrees with the horizontal direction, and is used to connect the culture medium injection port 100 and the horizontal flow channel 112. The horizontal flow channel 112 has a width of 300 um and connects the injection port connecting flow channel 111 and the differential balance flow channel 120. The differential balance flow channel 120 has a width of 500 um and is used to connect the front - stage flow channel 110 and the flow - limiting flow channel 130. The flow - channel cross - sectional area of the differential balance flow channel 120 is larger than that of the front - stage flow channel 110 and the flow - limiting flow channel 130. A differential balance diaphragm 121 is provided between adjacent differential balance flow channels 120, which is used to adjust the flow rate between adjacent differential balance flow channels 120, ensure the same flow rate in adjacent differential balance flow channels 120, and at the same time transmit the culture medium to the flow - limiting flow channel 130. The thickness of the differential balance diaphragm 121 is about 200 um, and nanoparticles are injected to increase its elasticity. The flow - limiting flow channel 130 connects the differential balance flow channel 120, the cell culture chamber 140 and the flow - accumulating pool 150, and is used to limit the flow rate of the culture medium entering the cell culture chamber 140 when the flow rate is too high. The flow - limiting flow channel 130 is in a "Y" shape and includes a front - stage flow - limiting flow channel 131, a cantilever beam 132, a flow - dividing pile 133, a cell culture chamber flow channel 134, and a flow - accumulating pool flow channel 135. The front - stage flow - limiting flow channel 131 has a width of 300 um, connects the differential balance flow channel 120, the cell culture chamber flow channel 134 and the flow - accumulating pool flow channel 135, and the flow - channel cross - sectional area is the same as that of the front - stage flow channel. The cantilever beam 132 is arranged in the front - stage flow - limiting flow channel 131, with a length of 200 um and a width of 50 um, fixed on the side wall of the flow channel and not connected to the upper and lower planes of the flow channel. It is used to reduce the liquid flow rate through elastic deformation when the liquid flow rate is too fast. The flow - dividing pile 133 is located between the cell culture chamber flow channel 134 and the flow - accumulating pool flow channel 135, and is used to limit the flow rate flowing into the cell culture chamber flow channel 134 through flow - dividing when the liquid flow rate is too fast. The flow - accumulating pool flow channel 135 has a width of 300 um, is located at one of the bifurcations of the "Y" - shaped flow - limiting flow channel, connects the front - stage flow - limiting flow channel 131 and the flow - accumulating pool 150, the flow - channel cross - sectional area is smaller than that of the front - stage flow - limiting flow channel 131, and the bottom surface of the flow channel is raised by 1 / 3 compared with the front - stage flow - limiting flow channel. The cell culture chamber flow channel 134 has a width of 300 um, is located at the other bifurcation of the "Y" - shaped flow - limiting flow channel, connects the front - stage flow - limiting flow channel 131 and the cell culture chamber flow channel 134, and the flow - channel cross - sectional area is the same as that of the front - stage flow - limiting flow channel 131.The cell culture chamber 140 is cuboid-shaped. The bottom and top surfaces of the cell culture chamber 140 are rounded rectangles, with a length of 5 mm, a width of 2 mm, and a rounded corner radius of 0.1 mm. A circuit board 141, a fluorescence sensor 142, a temperature sensor 143, a carbon dioxide sensor 144, an oxygen sensor 145, a serpentine resistance wire 146, a gas injection port 147, and a wire through-hole 148 are provided on the top. The circuit board 141 is used for fixing and powering the fluorescence sensor 142, the temperature sensor 143, the carbon dioxide sensor 144, the oxygen sensor 145, and the serpentine resistance wire 146. The fluorescence sensor 142 is used for collecting multi-dimensional data such as the metabolic activity, gene expression, and protein secretion of cells. The carbon dioxide sensor 144 is used for real-time monitoring of the carbon dioxide concentration in the cell culture chamber 140. The oxygen sensor 145 is used for real-time monitoring of the oxygen concentration in the cell culture chamber 140. The temperature sensor 143 is used for real-time monitoring of the temperature in the cell culture chamber 140. The serpentine resistance wire 146 is used for heating and temperature control of the temperature in the cell culture chamber 140. The gas injection port 147 is cylindrical, with a diameter of 1 - 1.5 mm, and is used for connecting a gas injection device and introducing it into the cell culture chamber 140. The wire through-hole 148 is cylindrical, with a diameter of 0.25 mm, and is used for leading out the power line. The current storage pool 150 is a rounded rectangle, with a length of 5 mm, a width of 1 mm, and a rounded corner radius of 0.1 mm.

[0048] In the actual use of the microfluidic chip 1, when the flow rates in two adjacent differential balance channels 120 are different, the differential balance diaphragm 121 is subjected to pressure, and the pressure direction is from the channel with a larger flow rate to the channel with a smaller flow rate. Since nanoparticles are injected into the differential balance diaphragm 121 to increase its elasticity, under the action of the pressure, the differential balance diaphragm 121 will deform, resulting in an increase in the width of the channel with a larger flow rate. According to the continuity equation Q = vA (where Q is the flow rate, v is the flow velocity, and A is the cross-sectional area of the channel), the instantaneous cross-sectional area A of the channel with an increased width increases, and the flow velocity decreases, while the width of the channel with a smaller flow rate decreases, the cross-sectional area decreases, and the flow velocity increases; the flow rates of two adjacent differential balance channels 120 can be kept consistent when reaching the flow-limiting channel 130; the cantilever beam 132 in the subsequent flow-limiting channel 130 will not deform and will not function when the flow rate is normal, but can play a role in reducing the flow rate and thus reducing the flow rate through elastic deformation when the flow rate is too large; at the same time, the liquid passes through the shunt pile 133 between the cell culture chamber channel 134 and the current storage pool channel 135. When the flow rate is normal, since the bottom surface of the current storage pool channel 135 is 1 / 3 higher than the previous flow-limiting channel 131, as shown in the appendix Figure 4As shown, all the liquid will flow from the flow channel 134 of the cell culture chamber into the cell culture chamber 140. When the liquid flow rate is too high, the liquid level exceeds the bottom surface of the flow channel passing through the flow channel 135 of the flow accumulation pool, and part of the liquid will flow from the flow channel 135 of the flow accumulation pool into the flow accumulation pool 150, and the flow rate flowing into the flow channel 134 of the cell culture chamber is controlled through the shunt effect. The differential balance flow channel 120, the flow limiting flow channel 130, the flow accumulation pool 150 and the structures included in the present invention can ensure the precise control of the flow rates of multiple cell culture chambers.

[0049] The liquid exchange device 2 includes a first micropump 210, a first hose 220, a culture medium pool 230, and a first sealing rubber ring 240, and is used for injecting the cell suspension and the culture medium into the culture medium injection port 100 of the microfluidic chip 1 and injecting the cleaning solution into the culture medium injection port 100 of the microfluidic chip 1 for cleaning and sucking out the cleaning solution. Among them, the first micropump 210 is used for transporting the cell suspension and the culture medium in the culture medium pool 230 to the microfluidic chip 1 through the first hose 220 and injecting and sucking out the cleaning solution. The first hose 220 is used for transporting the cell suspension, the culture medium and the cleaning solution, and the inner diameter is 0.25 - 0.5 mm. The culture medium pool 230 is connected to the microfluidic chip 1 through the first hose 220 and the first micropump 210, and is used for storing the initial cell suspension, the subsequent cell culture medium and the cleaning solution. The first sealing rubber ring 240 is used for sealing the connection between the first hose 220 and the microfluidic chip 1 to prevent external impurities from entering, and the diameter is 1.5 mm.

[0050] The gas injection device 3 includes a second micropump 310, a second hose 320, a gas storage tank 330, and a second sealing rubber ring 340, and is used for injecting gas into the gas injection port 147 in the cell culture chamber 140 of the microfluidic chip 1. The gas adopts a ratio of 3% oxygen, 5% carbon dioxide and 92% nitrogen. This gas can be used for quickly drying the microfluidic chip 1 during the cleaning stage and providing a necessary culture environment during the cell culture stage. The second micropump 310 is used for transporting the gas to the cell culture chamber 140. The second hose 320 is used for transporting the gas, and the inner diameter is 0.25 - 0.5 mm. The gas storage tank 330 is connected to the cell culture chamber 140 through the second micropump 310 and the second hose 320, and is used for storing the gas. The second sealing rubber ring 340 is used for sealing the connection between the second hose 320 and the cell culture chamber 140 to prevent external impurities from entering, and the diameter is 1.5 mm.

[0051] The multi-sensor data fusion subsystem 4 includes fluorescence sensors 142, temperature sensors 143, carbon dioxide sensors 144, oxygen sensors 145 in the cell culture chambers 140 of multiple microfluidic chips 1, and a PC computer 410. The PC computer 410 communicates and transmits data with the multiple fluorescence sensors 142, temperature sensors 143, carbon dioxide sensors 144, and oxygen sensors 145 through the WiFi communication protocol. The PC computer contains a WiFi receiver 411, a data fusion subsystem 412, and a data analysis subsystem 413. The data fusion subsystem 412 is located in the PC computer 410 and is used to collect and label the monitoring data of the multiple fluorescence sensors 142, temperature sensors 143, carbon dioxide sensors 144, and oxygen sensors 145, and filter and classify the noise. The data analysis subsystem 413 is located in the PC computer 410 and uses the monitoring data processed by the data fusion subsystem 412 to train a deep neural network, mine the potential associations and rules between the data, and establish a prediction model based on the deep neural network.

[0052] The multi-sensor data fusion subsystem 4 of this specific embodiment can achieve simultaneous monitoring and in-depth analysis of multiple cell parameters. The machine learning algorithm in the system can quickly process and analyze these massive and complex data, mine the potential associations and rules between the data, establish a mapping model between the cell physiological state and multiple parameters through the deep learning algorithm, so as to comprehensively and accurately understand the cell physiological state, find out the key factors affecting cell growth and experimental results, and further establish a prediction model based on the deep neural network according to these factors to predict the culture results of different cell communities before the experiment, adjust the culture conditions in advance, and reduce the result deviation caused by cell community differences. At the same time, the multi-sensor data fusion subsystem 4 of this embodiment can monitor the cell state in real time and adjust the culture parameters in a timely manner according to the model feedback to ensure the consistency of cell culture, thereby improving the repeatability and reliability of experimental results.

[0053] The constant temperature subsystem 5 includes a negative feedback closed-loop control subsystem 414, a temperature sensor 143, a regulated power supply 510, and a power cord 520. Among them, the regulated power supply 510 is used to supply power to the fluorescence sensor 142, temperature sensor 143, carbon dioxide sensor 144, oxygen sensor 145, and serpentine resistance wire 146 on the circuit board 141 of multiple microfluidic chips 1. The power cord 520 is introduced into the cell culture chamber 140 in the microfluidic chip 1 through a wire through-hole 148. The power cord 520 enters the microfluidic chip 1 through the wire through-hole 148 on the microfluidic chip 1 and is used to connect the regulated power supply 510 to the fluorescence sensor 142, temperature sensor 143, carbon dioxide sensor 144, oxygen sensor 145, and serpentine resistance wire 146 on the circuit board 141. The negative feedback closed-loop control subsystem 414 is located in the PC computer 410. It uses a PI structure as the controller of the system, the temperature sensor 143 in the cell culture chamber 140 as the signal input of the system, and the serpentine resistance wire 146 as the actuator of the system to form a complete negative feedback closed-loop control subsystem 414. After setting the temperature in the PC computer 410, the negative feedback closed-loop control subsystem 414 is used to keep the temperature of the cell culture chamber 140 constant.

[0054] The constant temperature subsystem 5 of this specific embodiment can accurately control the temperature of the cell culture chamber 140 for a long time through the negative feedback closed-loop control subsystem 414. When the temperature is greater than the preset temperature, the heating of the serpentine resistance wire 146 is stopped; when the temperature is less than the preset temperature, the serpentine resistance wire 146 starts to heat; thereby keeping the temperature of the cell culture chamber 140 constant and ensuring the stability of the cell culture environment.

[0055] This specific embodiment also relates to an implementation method for cell culture based on multi-sensor data fusion and a flow-controllable microfluidic chip, which is applicable to the cell culture system of this specific embodiment. The method includes:

[0056] Step 1: Inject the cleaning solution into the microfluidic chip 1 through the liquid exchange device 2 for cleaning, and then suck out the cleaning solution through the liquid exchange device 2 after the cleaning is completed;

[0057] Step 2: Inject gas into the microfluidic chip 1 through the gas injection device 3, and then suck out the remaining cleaning solution through the liquid exchange device 2.

[0058] Step 3: After the microfluidic chip 1 is completely dried, inject the cell suspension into the cell culture chamber 140 of the microfluidic chip 1 through the liquid exchange device 2. Subsequently, inject the cell culture medium into the cell culture chamber 140 of the microfluidic chip 1 through the liquid exchange device 2 as needed. During the injection process of the cell suspension and the cell culture medium, the microfluidic chip 1 ensures that the flow rates entering multiple cell culture chambers 140 are consistent through the differential balance flow channel 120, the flow limiting channel 130, the flow storage pool 150, and the structures they contain.

[0059] Step 4: Keep the temperature of the cell culture chamber 140 constant at the set value through the thermostatic subsystem 5. At the same time, simultaneously monitor and deeply analyze the cell parameters in multiple cell culture chambers 140 through the multi-sensor data fusion subsystem 4, and establish a prediction model.

[0060] In summary, the present invention establishes a miniaturized and intelligent cell culture system through multi-sensor data fusion and a microfluidic chip with controllable flow rate, realizes the monitoring and control of the cell culture environment, and can directly achieve cell culture and observation in the microfluidic chip. At the same time, the present invention can monitor the cell states of multiple different cell culture chambers in real time and adjust the culture parameters in a timely manner according to the model feedback to ensure the consistency of the cell communities in multiple cell culture chambers, thereby improving the repeatability and reliability of experimental results. Therefore, the present invention has broad application prospects in the field of cell culture.

[0061] It should be emphasized that the above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A cell culture system for multi-sensor data fusion and flow-controllable microfluidic chips, characterized in that, Comprising: A microfluidic chip (1) for monitoring and / or controlling cell culture parameters of a cell culture chamber (140) provided within the microfluidic chip (1); the cell culture parameters include: chamber flow rate, carbon dioxide concentration, oxygen concentration, temperature, metabolic activity, gene expression, protein secretion; A liquid exchange device (2) for injecting a cleaning liquid into the microfluidic chip (1) for cleaning and sucking out the cleaning liquid during a cleaning stage, and also for injecting a cell suspension and a culture medium into the microfluidic chip (1) during a culture stage; A gas injection device (3) for drying the microfluidic chip (1) during a cleaning stage, and also for delivering a culture gas to the cell culture chamber (140) for cell culture during a culture stage; the culture gas includes: oxygen, carbon dioxide; A multi-sensor data fusion subsystem (4) for monitoring and analyzing cell parameters of multiple cell culture chambers (140), and also for predicting the culture results of different cell communities and adjusting the culture conditions of each cell culture chamber (140); A constant temperature subsystem (5) for controlling the temperature of the cell culture chamber (140); The microfluidic chip (1) is provided with a culture medium injection port (100) and a cell culture chamber (140), and the cell culture medium flow channel in the direction from the culture medium injection port (100) to the cell culture chamber (140) is in the shape of multiple bifurcated fluid channels; a circuit board (141), a fluorescence sensor (142), a temperature sensor (143), a carbon dioxide sensor (144), an oxygen sensor (145), a serpentine resistance wire (146), and a gas injection port (147) are provided within the cell culture chamber (140); the fluorescence sensor (142), the temperature sensor (143), the carbon dioxide sensor (144), the oxygen sensor (145), and the serpentine resistance wire (146) are respectively electrically connected to the circuit board (141); The liquid exchange device (2) is connected to the culture medium injection port (100) through a pipeline, the gas injection device (3) is connected to the gas injection port (147) through a pipeline, and the multi-sensor data fusion subsystem (4) and the constant temperature subsystem (5) are respectively electrically connected to the circuit board (141).

2. The cell culture system of the multi-sensor data fusion and flow controllable microfluidic chip according to claim 1, wherein The cell culture medium flow channel from the culture medium injection port (100) to the cell culture chamber (140) includes: the culture medium injection port (100) is respectively connected to the inlets of two front-stage flow channels (110), the outlets of the two front-stage flow channels (110) are respectively connected to the inlet of a differential balance flow channel (120), the outlet of the differential balance flow channel (120) is connected to the inlet of a current-limiting flow channel (130), and the outlets of the current-limiting flow channel (130) are respectively connected to the cell culture chamber (140) and a flow storage pool (150).

3. The cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip according to claim 2, characterized in that, The microfluidic chip (1) is provided with N culture medium injection ports (100), where 1 ≤ N ≤ 100 and N is an integer.

4. The cell culture system of the multi-sensor data fusion and flow controllable microfluidic chip according to claim 2, characterized in that, The connection between the culture medium injection port (100) and the two front-stage flow channels (110) is in a "Y"-shaped bifurcation; The connecting points where the flow-through and flow-limiting flow channels (130) are connected to the cell culture chamber (140) and the flow storage tank (150) are bifurcated in a "Y" shape.

5. The cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip according to claim 4, characterized in that, The two front section flow channels (110) and the two differential balancing flow channels (120) are arranged side by side along the fluid flow direction; a differential balancing diaphragm (121) is arranged between the two differential balancing flow channels (120); the differential balancing diaphragm (121) is made of elastic nanomaterial; and the differential balancing diaphragm (121) divides a fluid pipeline into two parallel differential balancing flow channels (120) along the fluid flow direction.

6. The cell culture system of the multi-sensor data fusion and flow controllable microfluidic chip according to claim 4, characterized in that, The communication method of the flow-through and flow-limiting channel (130) connecting the cell culture chamber (140) and the flow reservoir (150) comprises: the flow-through and flow-limiting channel (130) is provided with: a front-end flow-limiting channel (131), a cantilever beam (132), and a flow-dividing pile (133) in sequence along the fluid flow direction; the cantilever beam (132) is made of elastic deformation material; the cantilever beam (132) is arranged on the left and right sides of the inner wall of the front-end flow-limiting channel (131); and fluid passage openings are reserved on the upper and lower sides of the cantilever beam (132); The flow-dividing pile (133) is arranged at a "Y"-shaped bifurcation where the flow-limiting flow channel (130), the cell culture chamber (140), and the flow storage tank (150) are connected, and the flow-dividing pile (133) is in the shape of a triangular truncated cone.

7. The cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip according to claim 6, characterized in that, The flow diversion pile (133) is provided with a cell culture chamber flow channel (134) and a flow reservoir flow channel (135); the two ends of the cell culture chamber flow channel (134) are respectively connected to the front-end flow limiting flow channel (131) and the cell culture chamber (140); the two ends of the flow reservoir flow channel (135) are respectively connected to the front-end flow limiting flow channel (131) and the flow reservoir (150); the inlet height of the cell culture chamber flow channel (134) is lower than the inlet height of the flow reservoir flow channel (135).

8. The cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip according to claim 1, wherein The liquid exchange device (2) comprises: a first micro pump (210), a first hose (220), and a culture solution pool (230), wherein two ends of the first hose (220) are respectively connected to a culture solution injection port (100) and the culture solution pool (230), and the first micro pump (210) is arranged on the first hose (220); The gas injection device (3) comprises: a second micro pump (310), a second hose (320), and a gas storage tank (330); the two ends of the second hose (320) are respectively connected to a gas injection port (147) and the gas storage tank (330); and the second micro pump (310) is arranged on the second hose (320).

9. The cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip according to claim 1, characterized in that, The multi-sensor data fusion subsystem (4) comprises: a PC (410), a WiFi receiver (411), a data fusion subsystem (412), a data analysis subsystem (413), and a negative feedback closed-loop control subsystem (414); the WiFi receiver (411), the data fusion subsystem (412), the data analysis subsystem (413), and the negative feedback closed-loop control subsystem (414) are arranged in the PC (410); The constant temperature subsystem (5) further includes: a voltage stabilizer power supply (510) and a power cord (520). The voltage stabilizer power supply (510) is electrically connected to the circuit board (141) through the power cord (520).

10. The cell culture system of the multi-sensor data fusion and flow-controllable microfluidic chip according to claim 1, characterized in that, The operation method of the cell culture system includes the following steps: Step 1: Inject a cleaning solution into the microfluidic chip (1) through the liquid exchange device (2) for cleaning, and then suck out the cleaning solution through the liquid exchange device (2) after the cleaning is completed; Step 2: Inject gas into the microfluidic chip (1) through the gas injection device (3), and then suck out the remaining cleaning solution through the liquid exchange device (2); Step 3: After the microfluidic chip (1) is completely dry, inject the cell suspension into the cell culture chamber (140) of the microfluidic chip (1) through the liquid exchange device (2); Step 4: Maintain the temperature of the cell culture chamber (140) through the constant temperature subsystem (5), and monitor and analyze the cell parameters in multiple cell culture chambers (140) through the multi-sensor data fusion subsystem (4).

Images