Disclosure of Invention
The invention provides a biological tissue culture system based on a micro-fluidic chip and an implementation and operation method thereof, and the system is combined with the micro-fluidic chip to form a living cell and biological tissue culture system which is small, portable, low in cost, simple to operate and capable of providing stable biological tissues and cell growth environmental conditions, so as to solve the problems.
A biological tissue culture system based on a microfluidic chip is characterized by comprising: the device comprises a temperature control device, a gas-liquid exchange device, a micro-fluidic chip, a transparent water tank and conductive glass; wherein,
the culture device comprises conductive glass and a transparent water tank, the transparent water tank is arranged on the upper surface of the conductive glass, and the microfluidic chip is arranged inside the transparent water tank;
the temperature control system is respectively electrically connected with the microfluidic chip and the conductive glass and is used for controlling the cell culture temperature;
the gas-liquid exchange device is communicated with the microfluidic chip and transmits a cell complete culture medium to the microfluidic chip;
the micro-fluidic chip is used for controlling the timing replacement of a complete cell culture medium and culturing cells and biological tissues.
Preferably, the upper surface of the transparent water tank is provided with symmetrically distributed windows; wherein,
the windows include a first window, a second window, a third window, and a fourth window.
Preferably, the temperature control device includes: the temperature probe, the digital temperature control plate and the switchable power supply; wherein,
the switchable power supply is an external adapter or a lithium battery;
the digital temperature control board is electrically connected with the switchable power supply through a first lead;
the temperature probe is inserted into the transparent water tank through a second window and is attached to the surface of the conductive glass, the surface of the conductive glass is provided with a PDMS film to obtain the real-time temperature in the microfluidic chip, and the temperature probe is electrically connected with the input end of the digital temperature control board and inputs the real-time temperature into the digital temperature control board;
the digital temperature control plate is used for displaying the temperature of the culture device and controlling the conductive glass to heat;
the digital temperature control plate is connected to two sides of the conductive glass through a first output lead and a second output lead and is used for temperature regulation through the conductive glass;
and the digital temperature control plate is also provided with a system switch.
Preferably, the gas-liquid exchange device comprises a first liquid storage bottle, a second liquid storage bottle 21, a first cell culture cavity and a second cell culture cavity; wherein,
the first cell culture cavity and the second cell culture cavity are arranged at the upper part of the transparent water tank;
the first liquid storage bottle is also provided with a first air inlet, and the second liquid storage bottle is also provided with a second air inlet; wherein,
the first air inlet and the second air inlet are externally connected with a gas compression bottle, compressed air is arranged in the gas compression bottle, and the compressed air is filtered compressed air or 5% CO2 compressed air;
when compressed air is introduced into the first liquid storage bottle, the first liquid inlet pipeline inputs a cell complete culture medium into the first cell culture cavity for replacement, one end of the first liquid inlet pipeline is connected with the first liquid outlet end of the first liquid storage bottle, and the other end of the first liquid inlet pipeline penetrates through the first window and is inserted into the microfluidic chip;
when compressed air is introduced into the second liquid storage bottle, a cell complete culture medium is input into the second cell culture cavity through the second liquid inlet pipeline for replacement, one end of the second liquid inlet pipeline is connected with the second liquid outlet end of the second liquid storage bottle, and the other end of the second liquid storage bottle penetrates through the first window and is inserted into the microfluidic chip.
Preferably, the system further comprises: a first control valve line, a second control valve line, a first liquid outlet line and a second liquid outlet line; wherein,
one end of the first control valve pipeline is inserted into the microfluidic chip, and the other end of the first control valve pipeline penetrates through the second window to be communicated with the outside;
one end of the second control valve pipeline is inserted into the microfluidic chip, and the other end of the second control valve pipeline penetrates through the third window to be communicated with the outside;
one end of the first liquid outlet pipeline is inserted into the microfluidic chip, and the other end of the first liquid outlet pipeline penetrates through the fourth window to be communicated with the outside;
one end of the second liquid outlet pipeline is inserted into the microfluidic chip, and the other end of the second liquid outlet pipeline penetrates through the fourth window to be communicated with the outside.
Preferably, the microfluidic chip comprises: a cell culture layer and a chip control layer; wherein,
the number of the cell culture layers is M, and M is more than or equal to 1 and less than or equal to 10;
the number of the layers of the chip control layer is N, and N is more than or equal to 0 and less than or equal to 10;
the cell culture layer comprises liquid inlets and liquid outlets, the number of the liquid inlets and the liquid outlets is Q, and Q is more than or equal to 1 and less than or equal to 100; (1, the cell culture layer comprises cell culture chambers, the number of the cell culture chambers is S, and S is more than or equal to 1 and less than or equal to 10000;
the chip control layer comprises chip control valves and chip control valve liquid inlets, the number of the chip control valves and the chip control valve liquid inlets is W, and W is not less than 1 and not more than 100.
Preferably, the microfluidic chip and the conductive glass are bonded through plasma.
Preferably, the conductive glass is provided with a double-layer ITO (indium tin oxide) plated film, and the transparent water tank is an acrylic plate;
the culture device is also provided with a water tank outside.
Preferably, deionized water is filled between the microfluidic chip (12) and the transparent water tank (7) for providing humidity for the culture environment.
The invention discloses a method for implementing biological tissue culture based on a microfluidic chip, which is applicable to a system of the invention and comprises the following steps:
step 1: sterilizing the gas-liquid exchange device, the microfluidic chip, the first control valve pipeline, the second control valve pipeline, the first liquid output pipeline and the second liquid output pipeline (6);
step 2: injecting sterilized ionized water into the transparent water tank through the first window, the second window, the third window or the fourth window;
and step 3: opening the system switch, controlling the temperature through the temperature control device, and inoculating cells to be inoculated into a first cell culture cavity and a second cell culture cavity of the microfluidic chip from a liquid inlet of a cell culture layer of the microfluidic chip when the temperature is stabilized to a preset culture temperature;
and 4, step 4: compressed air is led into a first liquid storage bottle and a second liquid storage bottle through an external gas compression bottle, complete culture mediums in the first liquid storage bottle and the second liquid storage bottle are driven to enter a cell culture layer liquid inlet, the opening and closing of the cell culture layer liquid inlet and the cell culture layer liquid outlet are controlled through a chip control valve of a microfluidic chip, and the cell complete culture mediums are replaced at regular time;
and 5: controlling the air inlet pressure in the first liquid storage bottle and the second liquid storage bottle to be 1.5psi, and driving the complete cell culture medium in the first liquid storage bottle and the second liquid storage bottle to be input into the first cell culture cavity and the second cell culture cavity at the flow rate of less than or equal to 1.5 mm/s.
The invention has the beneficial effects that:
1. the invention constructs a micro and portable cell and biological tissue culture system based on the micro-fluidic chip, realizes the control and maintenance of the cell culture environment, can directly carry out independent cell culture in the micro-fluidic chip, and does not need any external auxiliary device;
2. the invention uses the self-designed water sealing structure with the acrylic transparent structure, so that the temperature is more stable after the water sealing structure is filled with the ionized water, and the inherent defects of the microfluidic chip material are effectively improved in the cell culture process, the aggregation of protein molecules caused by the evaporation phenomenon is inhibited, and the affinity to living cells is enough;
3. compared with the prior art, the invention directly controls the working state of the conductive glass, uniformly controls the temperature of the cell layer of the microfluidic chip at the target temperature in a surface heating mode, and ensures the temperature condition of cell culture;
4. according to the invention, the cell complete culture medium in the liquid storage bottle is directly driven by the compressed gas, so that on one hand, the flow rate of the liquid in the cell culture cavity can be controlled by controlling the pressure of the compressed gas, and on the other hand, the gas required in the cell survival process can be provided by the compressed gas.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and drawings.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Compared with the traditional living cell workstation and a living cell microscopic imaging system, the system has the characteristics of small volume, portability, simple preparation process and stable environmental conditions, and can support the culture and drug stimulation of living cells and biological tissues for a long time in the microscopic imaging environment; the invention is a small-sized and portable design, which can provide a stable and reliable growth environment for retaining and transporting biological samples, cells and biological tissues extracted from animals and human bodies, and avoid the influence on the cells caused by temperature and humidity fluctuation; the micro-fluidic chip is combined, so that necessary medicines, cell factors and nutritional factors can be uninterruptedly conveyed to cells and biological tissues, and environmental condition support is provided for long-distance and long-time conveying of biological samples.
As shown in the attached figure 1, the invention is a biological tissue culture system based on a microfluidic chip, and the invention is suitable for the preservation, transportation, micromanipulation experiment and the like of a patient biopsy sample in clinical medical treatment; the precise control of the microscopic imaging environment of living cells and biological tissues in biomedical experiments keeps the activity and the biological function of the cells.
The structure of the invention comprises: the device comprises a temperature control device, a gas-liquid exchange device, a micro-fluidic chip 12, a transparent water tank 7 and conductive glass 13; wherein,
the transparent water tank 7 is arranged on the upper surface of the conductive glass 13, and the microfluidic chip 12 is arranged inside the transparent water tank 7;
the temperature control system is respectively electrically connected with the microfluidic chip 12 and the conductive glass 13 and is used for controlling the cell culture temperature;
the gas-liquid exchange device is communicated with the microfluidic chip 12 and transmits a complete cell culture medium to the microfluidic chip 12;
the microfluidic chip 12 is used for controlling the timing replacement of a complete cell culture medium and performing cell and biological tissue culture.
The principle of the technical scheme is as follows: the invention relates to a temperature control device, a gas-liquid exchange device, a transparent water tank, conductive glass and a microfluidic chip, wherein the temperature control device is used for providing a controllable constant temperature condition for the whole culture environment, is a constant temperature adjusting device with detection, display and automatic adjustment, and can adjust the temperature of the culture environment by controlling the heating state of the conductive glass. The gas-liquid exchange device is used for storing a complete cell culture medium, and is also used for introducing compressed gas into the first liquid storage bottle 20 and the second liquid storage bottle 21 through an external gas compression bottle, inputting the complete cell culture medium into the microfluidic chip 12 by driving the complete cell culture medium in the first liquid storage bottle 20 and the second liquid storage bottle 21, and periodically replacing the complete cell culture medium in the first cell culture cavity 18 and the second cell culture cavity 19 by controlling the opening and closing of the cell culture layer liquid inlet and the cell culture layer liquid outlet through a chip control valve of the microfluidic chip 12. The micro-fluidic chip 12 is arranged inside the transparent water tank 7 and bonded with the conductive glass 13 through plasma. The transparent water tank 7 is arranged above the conductive glass 13 of the bonded microfluidic chip 12 to provide an environment for storing water.
The beneficial effect of above-mentioned technique lies in: 1. the invention constructs a micro and portable cell and biological tissue culture system based on the micro-fluidic chip, realizes the control and maintenance of the cell culture environment, can directly carry out independent cell culture in the micro-fluidic chip, and does not need any external auxiliary device;
2. the invention uses the self-designed water sealing structure with the acrylic transparent structure, so that the temperature is more stable after the water sealing structure is filled with the ionized water, and the inherent defects of the microfluidic chip material are effectively improved in the cell culture process, the aggregation of protein molecules caused by the evaporation phenomenon is inhibited, and the affinity to living cells is enough;
3. compared with the prior art, the invention directly controls the working state of the conductive glass, uniformly controls the temperature of the cell layer of the microfluidic chip at the target temperature in a surface heating mode, and ensures the temperature condition of cell culture;
4. according to the invention, the cell complete culture medium in the liquid storage bottle is directly driven by the compressed gas, so that on one hand, the flow rate of the liquid in the cell culture cavity can be controlled by controlling the pressure of the compressed gas, and on the other hand, the gas required in the cell survival process can be provided by the compressed gas.
As an embodiment of the present invention: the upper surface of the transparent water tank 7 is provided with symmetrically distributed windows; wherein,
the windows include a first window 8, a second window 9, a third window 10 and a fourth window 11.
The principle of the technical scheme is as follows: in the present invention, since the cell complete culture medium needs to be introduced into the microfluidic chip 12 during cell culture, the present invention guides the cell complete culture medium through the window by setting the window.
The beneficial effects of the above technical scheme are: according to the invention, through the arrangement of the window on the transparent water tank 7, under the condition of water storage, the flow guide of the cell culture layer and the chip control layer of the microfluidic chip 12 can be realized.
As an embodiment of the present invention: the temperature control device includes: a temperature probe 22, a digital temperature control board 25 and a switchable power supply 23; wherein,
the switchable power supply 23 is an external adapter or a lithium battery;
the digital temperature control board 25 is electrically connected with the switchable power supply 23 through a first lead 24;
the temperature probe 22 is inserted into the transparent water tank 7 through the second window 9 and is attached to the surface of the conductive glass 13, the surface of the conductive glass 13 is provided with a PDMS film to obtain the real-time temperature in the microfluidic chip 12, and the temperature probe 22 is electrically connected with the input end of the digital temperature control board 25 and inputs the real-time temperature into the digital temperature control board 25;
the digital temperature control plate 25 is used for displaying the temperature of the culture device and controlling the conductive glass 13 to generate heat;
the digital temperature control board 25 is connected to two sides of the conductive glass 13 through a first output lead 26 and a second output lead 27, and temperature adjustment is performed through the conductive glass 13;
the digital temperature control board is also provided with a system switch 28.
The principle of the technical scheme is as follows: in the invention, because the temperature in the culture system needs to be regulated, the temperature control device provided by the invention comprises a first lead 24, a switchable power supply 23, a digital temperature control board 25, a system main switch 28, a first output lead 26, a second output lead 27, a temperature probe 22 and a conductive glass 13; the temperature probe 22 detects the temperature in the system and feeds the detected temperature information back to the digital temperature control board 25, the digital temperature control board 25 preferably has a display screen, and can be used for temperature setting, so that the temperature can be displayed, and automatic temperature control can be realized through a preset editing program. The digital temperature control plate 25 is also connected with the conductive glass 13, and controls the working state of the conductive glass by controlling whether current is input to the conductive glass 13 or not, so as to realize temperature regulation. The switchable power supply 23 is an external adapter or a lithium battery; under long-term culture conditions that do not require movement, an external power source is used to supply power. In the process of moving the culture device, a built-in lithium battery is used for supplying power. In the case of considering portability and miniaturization of devices, the external adapter selects 5-50V or the lithium battery selects 3.7V, 2200 mAh.
The specific process is as follows: the switchable power supply 28 is connected to the digital temperature control board 25, and the digital temperature control board 25 controls the conductive glass 13. Meanwhile, the digital temperature control board 25 obtains real-time temperature data through the temperature probe 22 disposed on the conductive glass 13 in the transparent water tank, stops heating the conductive glass 13 when the temperature is higher than a preset temperature, and starts heating the conductive glass 13 when the temperature is lower than the preset temperature, thereby stabilizing the temperature in the chip at the preset temperature. The predetermined temperature is the temperature most suitable for cell or biological tissue culture.
The beneficial effects of the above technical scheme are that: in order to enable cells to have a proper temperature environment in the culture process, the temperature control device is arranged, the self-feedback type temperature regulation and control can be realized, the temperature is further increased to the most proper culture temperature, the temperature is controlled to rise while detection is carried out, the temperature is ensured to be accurate based on real-time contrast control, and the normalized temperature can be kept.
Preferably, the gas-liquid exchange device comprises a first liquid storage bottle 20, a second liquid storage bottle 21, a first cell culture cavity 18 and a second cell culture cavity 19; wherein,
the first cell culture cavity 18 and the second cell culture cavity 19 are arranged at the upper part of the transparent water tank 7;
the first liquid storage bottle 20 is also provided with a first air inlet 16, and the second liquid storage bottle 21 is also provided with a second air inlet 17; wherein,
the first air inlet 16 and the second air inlet 17 are externally connected with a gas compression bottle, compressed air is arranged in the gas compression bottle, and the compressed air is filtered compressed air or 5% CO2 compressed air;
when compressed air is introduced into the first liquid storage bottle 20, the first liquid inlet pipeline 1 inputs a cell complete culture medium into the first cell culture chamber 18 for replacement, one end of the first liquid inlet pipeline 1 is connected with the first liquid outlet end 14 of the first liquid storage bottle 20, and the other end of the first liquid inlet pipeline passes through the first window 8 and is inserted into the microfluidic chip 12;
when compressed air is introduced into the second liquid storage bottle 21, the cell complete culture medium is input into the second cell culture cavity 19 through the second liquid inlet pipeline 2 for replacement, one end of the second liquid inlet pipeline 2 is connected with the second liquid outlet end 15 of the second liquid storage bottle 20, and the other end of the second liquid inlet pipeline passes through the first window 8 and is inserted into the microfluidic chip 12.
The principle of the technical scheme is as follows: because toxic substances can be generated in the cell metabolism and death processes, and the cells also need nutrient substances, the gas-liquid exchange device of the invention externally connects compressed air through the first air inlet 16 and the second air inlet 17 to input gas into the microfluidic chip 12, and the cell complete culture medium in the liquid storage bottle is conveyed into the microfluidic chip 12 through a pipeline connected with a liquid inlet of a cell culture layer of the chip; the liquid inlet of the cell culture layer sequentially enters the micro-fluidic chip 12 to replace the complete culture medium in the cell culture cavity, and finally the cell culture layer flows out of the liquid outlet of the cell culture layer, wherein the liquid inlet and the liquid outlet of the control layer can be independently controlled by the chip control valve through the liquid inlet of the chip control valve, so that the replacement of the complete culture medium of the cells is realized.
In this process, the pressure is embodied in the form of a gas, and the gas is also intended to keep the cell culture fluid stable in the culture system, for example: the gas to maintain the stable pH and oxygen content of the cell culture broth may be filtered compressed air (0.1-40MPa) mixed with 5% CO2 (for the purpose of filtration to prevent contamination of the complete medium in the storage bottle). Or 5% CO2 compressed gas (0.5 MPa ═ 0.5MPa CO2 (purity 99.9%) +7.5MPa N2 (purity 99.995%) +2MPa O2 (purity 99.9%)). The driving gas pressure for conveying fresh culture solution in the chip is adjusted according to different microfluidic chip structures, so that the flow speed of the cell complete culture medium in the chip is less than or equal to 1.5 mm/s.
The beneficial effects of the above technical scheme are that: the invention conveys the cell complete culture medium to the cell culture layer of the microfluidic chip 12 through the gas-liquid exchange device, toxic substances are brought out through liquid exchange, substances beneficial to cell growth can be added, and if the introduced gas is stable gas for keeping the cell culture liquid in the culture system, the culture system has a good gas environment.
Preferably, the system further comprises: a first control valve line 3, a second control valve line 4, a first outlet line 5 and a second outlet line 6; wherein,
one end of the first control valve pipeline 3 is inserted into the microfluidic chip 12, and the other end of the first control valve pipeline penetrates through the second window 9 to be communicated with the outside;
one end of the second control valve pipeline 4 is inserted into the microfluidic chip 12, and the other end of the second control valve pipeline passes through the third window 10 and is communicated with the outside;
one end of the first liquid outlet pipeline 5 is inserted into the microfluidic chip 12, and the other end of the first liquid outlet pipeline penetrates through the fourth window 10 to be communicated with the outside;
one end of the second liquid outlet pipeline 6 is inserted into the microfluidic chip 12, and the other end of the second liquid outlet pipeline passes through the fourth window 10 and is communicated with the outside.
The principle of the technical scheme is as follows: when the cell complete culture medium in the culture system is replaced, the original cell complete culture medium needs to be discharged and flowed to the outside. Therefore, the invention is provided with: the first control valve pipeline 3, the second control valve pipeline 4, the first liquid outlet pipeline 5 and the second liquid outlet pipeline 6 are controlled by the micro-fluidic chip 12, and the original cell complete culture medium is replaced.
The beneficial effects of the above technical scheme are that: by discharging the complete culture medium and the toxic substances of the cells in the cell culture cavity, the nutrient substances in the cell culture cavity can be maintained sufficient, and the content of the toxic substances in the cell culture cavity can be reduced.
As an embodiment of the present invention: the microfluidic chip 12 includes: a cell culture layer and a chip control layer; wherein,
the number of the cell culture layers is M, and M is more than or equal to 1 and less than or equal to 10;
the number of the layers of the chip control layer is N, and N is more than or equal to 0 and less than or equal to 10;
the cell culture layer comprises liquid inlets and liquid outlets, the number of the liquid inlets and the liquid outlets is Q, and Q is more than or equal to 1 and less than or equal to 100; (1, the cell culture layer comprises cell culture chambers, the number of the cell culture chambers is S, and S is more than or equal to 1 and less than or equal to 10000;
the chip control layer comprises chip control valves and chip control valve liquid inlets, the number of the chip control valves and the chip control valve liquid inlets is W, and W is not less than 1 and not more than 100.
The principle of the technical scheme is as follows: in the invention, efficient cell culture is realized inside the microfluidic chip 12, and the cell culture layer and the chip control layer in the invention can be arranged by human and array combination, thereby realizing the functional expansion of different microfluidic chips 12.
The beneficial effects of the above technical scheme are that: : it constructs the living space in the process of culturing cells and biological tissues and is a key part of the micro-culture system. The main functions are to separate the cell culture environment from the liquid in the water tank, provide the space necessary for the growth of cells and biological tissues, and replace the culture solution at regular time and quantity. The basic structure of the invention is a single-channel or multi-channel structure of one or more cell culture chambers, and cell culture chips with different structures are constructed.
Preferably, the microfluidic chip 12 and the conductive glass 13 are bonded by plasma.
The principle and the beneficial effects of the technical scheme are as follows: according to the invention, the micro-fluidic chip 12 and the conductive glass 13 are bonded through plasma, so that the direct temperature supply of the conductive glass 13 to the micro-fluidic chip 12 is realized.
Preferably, the conductive glass 13 is provided with a double-layer ITO-plated film, and the transparent water tank 7 is an acrylic plate;
the culture device is also provided with a water tank outside.
The principle and the beneficial effects of the technical scheme are as follows: the ITO film of the conductive glass 13 has high conductivity, high visible light transmittance, high mechanical hardness and good chemical stability, the acrylic plate seals water through the transparent structure, observation is convenient, the temperature is more stable after deionized water is filled, and the inherent defects of the microfluidic chip material are effectively overcome in the cell culture process.
As an embodiment of the present invention, a method of making,
deionized water is filled between the microfluidic chip 12 and the transparent water tank 7 and is used for providing humidity for the culture environment.
The principle and the beneficial effects of the technical scheme are as follows: 100% humidity is required in the culture environment, and therefore, by constructing a transparent water tank around the chip, the evaporation phenomenon in the chip and the excessive environmental protein concentration caused by evaporation are suppressed.
As shown in fig. 2, a method for implementing biological tissue culture based on a microfluidic chip is applicable to the system of the present invention, and the method includes:
step 1: sterilizing the gas-liquid exchange device, the microfluidic chip 12, the first control valve pipeline 3, the second control valve pipeline 4, the first liquid output pipeline 5 and the second liquid output pipeline 6;
step 2: injecting sterilized ionized water into the transparent water tank 7 through the first window 8, the second window 9, the third window 10 or the fourth window 11;
and step 3: the system switch 28 is turned on, the temperature control device is used for controlling the temperature, and when the temperature is stabilized to the preset culture temperature, the cells to be inoculated are inoculated into the first cell culture cavity 18 and the second cell culture cavity 19 of the microfluidic chip 12 from the liquid inlet of the cell culture layer of the microfluidic chip 12;
and 4, step 4: compressed air is led into the first liquid storage bottle 20 and the second liquid storage bottle 21 through an external gas compression bottle, complete culture mediums in the first liquid storage bottle 20 and the second liquid storage bottle 21 are driven to enter the liquid inlet of the cell culture layer, the on and off of the liquid inlet of the cell culture layer and the liquid outlet of the cell culture layer are controlled through a chip control valve of the microfluidic chip 1, and the cell complete culture mediums are replaced at regular time;
and 5: controlling the air inlet pressure in the first liquid storage bottle 20 and the second liquid storage bottle 21 to be 1.5psi, and driving the cell complete culture medium to be input into the first cell culture cavity 18 and the second cell culture cavity 19 at the flow rate of less than or equal to 1.5 mm/s.
The principle and the beneficial effects of the technical scheme are as follows:
during the cell culture process, bacteria are the most influential factors in the cell and biological tissue culture, and therefore any part of the system of the present invention is autoclaved at the time of use. The operation method of the invention is a method for constructing an environment suitable for the growth of cells and biological tissues based on the system, and comprises the removal of temperature, humidity, gas and toxic substances. Step 1, the gas-liquid exchange device, a first liquid inlet pipeline 1, a second liquid inlet pipeline 2, a first liquid output pipeline 5 and a second liquid output pipeline 6 of the microfluidic chip 12 are subjected to high-temperature high-pressure sterilization, and a sterile environment is constructed. Step 2: and injecting sterilized deionized water into the transparent water tank 7 through the first window 8, the second window 9, the third window 10 or the fourth window 11 to prevent bacteria. The sterility of the cell culture environment is maintained. And step 3: the temperature of the cell culture environment is regulated and controlled by the temperature control device, and the temperature is regulated to a target environment temperature suitable for cell culture in a self-feedback mode. In step 4, the gas conditions in the culture environment are maintained by introducing specific compressed gas. The gas condition can be introduced into the cell safety culture medium or the medicine by connecting compressed air or 5% CO2 compressed gas, thereby maintaining enough O2 and stable acidity and alkalinity in the liquid environment. In the step 5, the pressure is adjusted to a specific value, the influence of the shearing force generated when the cell complete culture medium in the culture system is replaced on the cell state is reduced, the cell complete culture medium is replaced at regular time and quantity through automatic control, and cell metabolites are discharged along with the replaced complete culture medium.
The invention also includes a specific embodiment:
in this example, first, a microfluidic chip (the chip is divided into 4 regions, each region has 320 cell culture chambers, 1280, 8 liquid inlets, 8 liquid outlets, and 50 control valves) and 2 sterilized 1.5mL liquid storage bottles, a plurality of 10cm long sterilization tubes, and 1mL needle tube are prepared;
all the sterilizing tubes are inserted into the microfluidic chip through a window (the size is 2 x 25mm2) of a transparent structure, and then the temperature probe is contacted on the conductive glass at the lower left corner of the microfluidic chip through the window and is fixed by using a transparent adhesive tape;
assembling the transparent structure and the microfluidic chip, and injecting sterilized deionized water (60mL) into the transparent structure through any window; connecting a power supply to the digital temperature control plate, connecting an output lead of the digital temperature control plate to the conductive glass, and finally turning on a switch of the culture system to control the temperature;
after the temperature is stabilized to 37 ℃, injecting the prepared HepG2 cells to be inoculated (a 50mL culture bottle full of cells is taken, 1mL of cells after centrifugal dilution is taken) into a 1ML needle tube and connecting to a liquid inlet pipeline, and inoculating the cells into a microfluidic chip cell culture cavity through a liquid inlet of a microfluidic chip cell culture layer;
connecting a liquid storage bottle filled with a cell complete culture medium to a liquid inlet of a cell culture layer of the microfluidic chip through a sterilizing tube, controlling the liquid inlet and the liquid outlet of the cell culture layer to be closed by using a microfluidic chip control valve, and setting the cell complete culture medium to be replaced at regular time for 2 min/time;
adjusting the pressure of the inlet CO2 of the liquid storage bottle to 1.5psi, and starting to automatically control the liquid inlet and outlet control valve to drive the timed replacement of the cell complete culture medium in the cell culture cavity.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.