CN112501020A - Biological tissue culture system based on micro-fluidic chip and implementation operation method thereof - Google Patents

Abstract

The invention provides a biological tissue culture system based on a microfluidic chip and an implementation and operation method thereof. The invention comprises a temperature control device, a gas-liquid exchange device, a micro-fluidic chip, a transparent water tank and conductive glass, wherein the five parts are combined for cell culture. Compared with the traditional living cell workstation and a living cell microscopic imaging system, the invention 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 small and portable design can provide a stable and reliable growth environment for retaining and conveying 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.

Description

Biological tissue culture system based on microfluidic chip and its implementation method

technical field

The invention relates to the technical field of cell culture, in particular to a biological tissue culture system based on a microfluidic chip and a method for implementing the same.

Background technique

Currently, the application of microscopic imaging technology to observe the dynamic physical and biochemical reactions of cells and biological tissues in real time at the macro, micro and nano scales is crucial for our understanding of the pathogenic mechanisms of life processes and diseases. Traditional cell culture is carried out in a complete black box - a closed CO2 incubator, and we cannot effectively observe the biological cells in it. At present, the most widely used live cell workstations based on microscopic imaging systems can maintain a relatively transparent environment where cells can grow normally. The level of the incubator.

At present, the related research and technical patents for the in vitro culture of cells and biological tissues generally have the problems of complicated operation and high cost. For example, patent CN111808748A is a micro-cultivation system and method for microfluidic cell culture, and its operation process is complicated. Because the system temperature is controlled by heat exchange, the uniformity of temperature distribution is poor, and the method of cell culture has great limitations.

Animal and human biopsies in biomedical experiments are mainly carried out by means of blood, secretions and biopsies. Due to the limitations of technical conditions and experimental costs, proper growth conditions for the extracted biological samples, biological tissues and cells are mostly not maintained. This leads to the fact that the activity and functionality of cells and biological tissues are largely affected by environmental conditions, resulting in problems such as large variability and irreproducibility of experimental results.

SUMMARY OF THE INVENTION

The present invention provides a biological tissue culture system based on a microfluidic chip and its implementation and operation method. The present invention combines the microfluidic chip to form a small, portable, low-cost, simple to operate, and can provide stable Living cells and biological tissue culture systems for biological tissue and cell growth environmental conditions to address the above.

A biological tissue culture system based on a microfluidic chip, characterized by comprising: a temperature control device, a gas-liquid exchange device, a microfluidic chip, a transparent water tank and conductive glass; wherein,

The culture device includes 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 electrically connected to the microfluidic chip and the conductive glass respectively, and is used to control the cell culture temperature;

The gas-liquid exchange device is communicated with the microfluidic chip, and transmits the cell complete culture medium into the microfluidic chip;

The microfluidic chip is used to control the regular replacement of the cell complete medium for cell and biological tissue culture.

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: a temperature probe, a digital temperature control board and a switchable power supply; wherein,

The switchable power source is an external adapter or a lithium battery;

the digital temperature control board and the switchable power supply are electrically connected through a first wire;

The temperature probe is inserted into the transparent water tank through the second window, and is attached to the surface of the conductive glass. The surface of the conductive glass has a PDMS film to obtain the real-time temperature in the microfluidic chip. The probe is electrically connected to the input end of the digital temperature control board, and the real-time temperature is input into the digital temperature control board;

The digital temperature control panel is used to display the temperature of the culture device and control the heating of the conductive glass;

The digital temperature control board is connected to both sides of the conductive glass through a first output wire and a second output wire, and the temperature is adjusted through the conductive glass;

The digital temperature control board is also provided with a system switch.

Preferably, the gas-liquid exchange device includes a first liquid storage bottle, a second liquid storage bottle 21, a first cell culture chamber and a second cell culture chamber; wherein,

The first cell culture cavity and the second cell culture cavity are arranged on 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 connected to a gas compression bottle, and the gas compression bottle is provided with compressed air, and the compressed air is filtered compressed gas or 5% CO2 compressed gas;

When the inside of the first liquid storage bottle is filled with compressed air, the first liquid inlet pipeline inputs the cell complete medium into the first cell culture chamber for replacement, and one end of the first liquid inlet pipeline is connected to the The first liquid outlet end of the first liquid storage bottle is connected, and the other end is inserted into the microfluidic chip through the first window;

When the inside of the second liquid storage bottle is filled with compressed air, the complete cell culture medium is input into the second cell culture chamber through the second liquid inlet pipeline for replacement, and one end of the second liquid inlet pipeline is connected to the second liquid inlet pipeline. The second liquid outlet end of the second liquid storage bottle is connected, and the other end thereof is inserted into the microfluidic chip through the first window.

Preferably, the system further comprises: a first control valve pipeline, a second control valve pipeline, a first liquid outlet pipeline and a second liquid outlet pipeline; wherein,

One end of the first control valve pipeline is inserted into the microfluidic chip, and the other end passes through the second window to communicate with the outside world;

One end of the second control valve pipeline is inserted into the microfluidic chip, and the other end is connected to the outside world through the third window;

One end of the first liquid outlet pipeline is inserted into the microfluidic chip, and the other end is connected to the outside world through the fourth window;

One end of the second liquid outlet pipeline is inserted into the microfluidic chip, and the other end thereof passes through the fourth window to communicate with the outside world.

Preferably, the microfluidic chip includes: a cell culture layer and a chip control layer; wherein,

The number of layers of the cell culture layer is M layers, and 1≤M≤10;

The number of layers of the chip control layer is N, and 0≤N≤10;

The cell culture layer includes a liquid inlet and a liquid outlet, and the number of the liquid inlet and the liquid outlet is Q, and 1≤Q≤100; (1, the interior of the cell culture layer includes a cell culture cavity, The number of the cell culture chambers is S, and 1≤S≤10000;

The chip control layer includes a chip control valve and a chip control valve liquid inlet, and the number of the chip control valve and the chip control valve liquid inlet is W, and 1≤W≤100.

Preferably, the microfluidic chip and the conductive glass are bonded by plasma.

Preferably, the conductive glass has a double-layer ITO-plated film, and the transparent water tank is an acrylic plate;

A water tank is also provided outside the culture device.

Preferably, deionized water is filled between the microfluidic chip (12) and the transparent water tank (7) for providing humidity in the culture environment.

A method for implementing biological tissue culture based on a microfluidic chip, applicable to the system of the present invention, the method includes:

Step 1: Sterilize 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: inject sterilized ionized water into the transparent water tank through the first window, the second window, the third window or the fourth window;

Step 3: Turn on the system switch, control the temperature through the temperature control device, and when the temperature stabilizes to the preset culture temperature, inoculate the cells to be seeded into the microfluidic chip from the cell culture layer inlet of the microfluidic chip. in the first cell culture chamber and the second cell culture chamber of the fluidic chip;

Step 4: Introduce compressed air into the first liquid reserve bottle and the second liquid reserve bottle through an external gas compression bottle, drive the complete medium in the first liquid reserve bottle and the second liquid reserve bottle into the cell culture layer liquid inlet, and Control the opening and closing of the liquid inlet of the cell culture layer and the liquid outlet of the cell culture layer through the chip control valve of the microfluidic chip, and replace the cell complete medium regularly;

Step 5: Control the inlet pressure in the first liquid reserve bottle and the second liquid reserve bottle to 1.5psi, and drive the cell complete medium in the first liquid reserve bottle and the second liquid reserve bottle to be ≤1.5mm/ The flow rate of s is input into the first cell culture chamber and the second cell culture chamber.

The beneficial effects of the present invention are:

1. The present invention constructs a miniature, portable cell and biological tissue culture system based on the microfluidic chip, realizes the control and maintenance of the cell culture environment, and can directly carry out independent cell culture in the microfluidic chip without any external auxiliary devices;

2. The present invention uses a self-designed acrylic transparent structure to seal the water. After filling with ionized water, on the one hand, the temperature is more stable, and on the other hand, the inherent defects of the microfluidic chip material are effectively improved during the cell culture process, and evaporation is suppressed. The aggregation of protein molecules caused by the phenomenon shows sufficient affinity for living cells;

3. Compared with the prior art, the present invention directly controls the working state of the conductive glass, and uniformly controls the temperature of the cell layer of the microfluidic chip at the target temperature by means of surface heating to ensure the temperature conditions for cell culture;

4. The present invention drives the cell complete culture medium in the liquid storage bottle directly by the compressed gas, on the one hand, the flow rate of the liquid in the cell culture chamber can be controlled by controlling the pressure of the compressed gas, and on the other hand, the cell survival process can be provided by the compressed gas. required gas.

Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.

The technical solutions of the present invention will be further described in detail below through the accompanying drawings and embodiments.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention. In the attached image:

1 is a schematic structural diagram of a biological tissue culture system based on a microfluidic chip in an embodiment of the present invention;

FIG. 2 is a flow chart of a method for implementing an operating method for biological tissue culture based on a microfluidic chip according to an embodiment of the present invention.

Among them, 1 is the first liquid inlet pipeline, 2 is the second liquid inlet pipeline, 3 is the first control valve pipeline, 4 is the second control valve pipeline, 5 is the first liquid outlet pipeline, and 6 is the second outlet pipeline Liquid port pipeline, 7 is the transparent water tank, 8 is the first window, 9 is the second window, 10 is the third window, 11 is the fourth window, 12 is the microfluidic chip, 13 is the conductive glass, 14 is the first outlet Liquid end, 15 is the second liquid outlet, 16 is the first air inlet, 17 is the second air inlet, 18 is the first cell culture chamber, 19 is the second cell culture chamber, and 20 is the first liquid storage bottle , 21 is the second liquid storage bottle, 22 is the temperature probe, 23 is the switchable power supply, 24 is the first wire, 25 is the digital temperature control board; 26 is the first output wire, 27 is the second output wire, 28 is the system switch.

Detailed ways

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

The present invention is a system for culturing cells and biological tissues. Compared with the traditional live cell workstation and live cell microscopic imaging system, the present invention has the characteristics of small size, portability, simple preparation process and stable environmental conditions, and can Supports long-term culture and drug stimulation of living cells and biological tissues in a microscopic imaging environment; the present invention is a small, portable design that can be used to retain and transport biological samples, cells and biological tissues extracted from animals and humans. A stable and reliable growth environment avoids the impact on cells caused by temperature and humidity fluctuations; combined with microfluidic chips, necessary drugs, cytokines and nutritional factors can be continuously delivered to cells and biological tissues for long-distance and long-term delivery. Biological samples provide support for environmental conditions.

As shown in FIG. 1, the present invention is a biological tissue culture system based on a microfluidic chip, and the function of the present invention is to be suitable for the preservation, transportation and micromanipulation experiments of patient biopsy samples in clinical medicine; biomedical Precise control of the microscopic imaging environment of living cells and biological tissues in experiments to maintain cell viability and biological function.

The structure of the present invention includes: a temperature control device, a gas-liquid exchange device, a microfluidic chip 12, a transparent water tank 7 and a 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 for controlling the cell culture temperature;

The gas-liquid exchange device communicates with the microfluidic chip 12, and transmits the cell complete culture medium to the microfluidic chip 12;

The microfluidic chip 12 is used to control the timed replacement of the cell complete medium for culturing cells and biological tissues.

The principle of the above technical solution is: the temperature control device, the gas-liquid exchange device, the transparent water tank, the conductive glass and the microfluidic chip of the present invention, the temperature control device is used to provide a controllable constant temperature condition for the entire culture environment, and the temperature control device It is a thermostat with detection, display and automatic adjustment, which can adjust the temperature of the culture environment by controlling the heating state of the conductive glass. The gas-liquid exchange device is used to store the cell complete culture medium, and is also used to introduce the compressed gas into the first liquid storage bottle 20 and the second liquid storage bottle 21 through an external gas compression bottle, and by driving the first liquid storage bottle 20 and the second liquid storage bottle 20 and the second liquid The complete medium in the storage bottle 21 is input into the microfluidic chip 12, and the opening and closing of the liquid inlet of the cell culture layer and the liquid outlet of the cell culture layer are controlled by the chip control valve of the microfluidic chip 12, and the first cell is replaced regularly. Complete medium of cells in the culture chamber 18 and the second cell culture chamber 19 . The microfluidic chip 12 is disposed inside the transparent water tank 7 and is bonded to the conductive glass 13 by plasma. The transparent water tank 7 is arranged above the conductive glass 13 bonded to the microfluidic chip 12 to provide a water storage environment.

The beneficial effects of the above technology are: 1. The present invention builds a miniature, portable cell and biological tissue culture system based on a microfluidic chip, realizes the control and maintenance of the cell culture environment, and can be directly carried out independently in the microfluidic chip. Cell culture without any external aids;

2. The present invention uses a self-designed acrylic transparent structure to seal the water. After filling with ionized water, on the one hand, the temperature is more stable, and on the other hand, the inherent defects of the microfluidic chip material are effectively improved during the cell culture process, and evaporation is suppressed. The aggregation of protein molecules caused by the phenomenon shows sufficient affinity for living cells;

3. Compared with the prior art, the present invention directly controls the working state of the conductive glass, and uniformly controls the temperature of the cell layer of the microfluidic chip at the target temperature by means of surface heating to ensure the temperature conditions for cell culture;

4. The present invention drives the cell complete culture medium in the liquid storage bottle directly by the compressed gas, on the one hand, the flow rate of the liquid in the cell culture chamber can be controlled by controlling the pressure of the compressed gas, and on the other hand, the cell survival process can be provided by the compressed gas. required 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 above technical solution is: in the present invention, because the cell culture medium needs to be introduced into the microfluidic chip 12 during cell culture, the present invention provides a window to conduct flow of the cell complete medium through the window.

The beneficial effects of the above technical solutions are as follows: the present invention can also conduct flow to the cell culture layer and the chip control layer of the microfluidic chip 12 under the condition that water can be stored through the setting of the window on the transparent water tank 7 .

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 source 23 is an external adapter or a lithium battery;

The digital temperature control board 25 and the switchable power supply 23 are electrically connected through the first wire 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 has a PDMS film to obtain the real-time temperature in the microfluidic chip 12. temperature, the temperature probe 22 is electrically connected to the input end of the digital temperature control board 25, and the real-time temperature is input to the digital temperature control board 25;

The digital temperature control panel 25 is used to display the temperature of the culturing device and control the heat generation of the conductive glass 13;

The digital temperature control board 25 is connected to both sides of the conductive glass 13 through the first output wire 26 and the second output wire 27, and the temperature is adjusted through the conductive glass 13;

The digital temperature control board is also provided with a system switch 28 .

The principle of the above technical solution is: in the present invention, because the temperature inside the culture system needs to be adjusted, the temperature control device provided by the present invention includes a first wire 24, a switchable power supply 23, a digital temperature control board 25, and a system master switch 28. The first output wire 26, the second output wire 27, the temperature probe 22, the conductive glass 13; the temperature probe 22 detects the temperature in the system, and feeds back the detected temperature information to the digital temperature control board 25, the digital temperature control board 25. A temperature control panel with a display screen and temperature setting is preferred, which can realize the display of the temperature, and can also realize automatic temperature control through the pre-editing program. The digital temperature control board 25 is also connected to the conductive glass 13 , and the working state of the conductive glass is controlled by controlling whether to input current to the conductive glass 13 , thereby realizing temperature adjustment. The switchable power source 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 for power supply. In the process of moving the culture equipment, the built-in lithium battery is used for power supply. Considering the portability and miniaturization of the device, choose 5-50V for the external adapter or 3.7V for the lithium battery, 2200mAh.

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 . At the same time, the digital temperature control board 25 obtains real-time temperature data through the temperature probe 22 placed on the conductive glass 13 in the transparent water tank, stops the heating of the conductive glass 13 when the temperature is greater than the preset temperature, and starts heating when the temperature is lower than the preset temperature Conductive glass 13, thereby stabilizing the temperature in the chip at a preset temperature. The preset temperature is the most suitable temperature for cell or biological tissue culture.

The beneficial effects of the above technical solutions are: in order to make the cells have a suitable temperature environment during the culturing process, the present invention is provided with a temperature control device, which can realize self-feedback adjustment and control of the temperature, and then let the temperature rise to the most suitable temperature for culturing. Detection, while controlling the temperature rise, based on real-time comparison control, ensures the temperature accuracy and can maintain a normalized temperature.

Preferably, the gas-liquid exchange device includes a first liquid storage bottle 20, a second liquid storage bottle 21, a first cell culture chamber 18 and a second cell culture chamber 19; wherein,

The first cell culture cavity 18 and the second cell culture cavity 19 are arranged on 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 connected to a gas compression bottle, and the gas compression bottle is provided with compressed air, and the compressed air is filtered compressed gas or 5% CO2 compressed gas;

When compressed air is introduced into the first liquid storage bottle 20, the first liquid inlet line 1 inputs the cell complete medium into the first cell culture chamber 18 for replacement, and the first liquid inlet line 1 One end is connected to the first liquid outlet end 14 of the first liquid storage bottle 20, and the other end is inserted into the microfluidic chip 12 through the first window 8;

When the compressed air is introduced into the second liquid storage bottle 21, the cell complete medium is input into the second cell culture chamber 19 through the second liquid inlet pipeline 2 for replacement, and the second liquid inlet pipeline 2 One end is connected to the second liquid outlet end 15 of the second liquid storage bottle 20 , and the other end is inserted into the microfluidic chip 12 through the first window 8 .

The principle of the above technical solution is: because in the process of cell metabolism and death, there will be toxic substances produced, and cells also need nutrients, the gas-liquid exchange device of the present invention, through the first air inlet 16 and the second air inlet 17 The external compressed air is input into the microfluidic chip 12, and the cell complete culture medium in the liquid storage bottle is transported into the microfluidic chip 12 through the pipeline connected to the liquid inlet of the cell culture layer of the chip; The control chip 12 enters the liquid inlet of the cell culture layer in turn, replaces the complete medium in the cell culture chamber, and finally flows out of the cell culture layer from the liquid outlet of the cell culture layer. It is independently controlled by the liquid in the control layer entering the liquid inlet of the chip control valve, thereby realizing the replacement of the complete cell culture medium.

In this process, the pressure is embodied in the form of gas, and the gas is also intended to maintain the stability of the cell culture medium in the culture system. For example, in order to maintain the stable pH and oxygen content of the cell culture medium, the gas can be filtered compressed air ( 0.1-40MPa) mixed with 5% CO2 (the purpose of filtration is to prevent contamination of the complete medium in the stock bottle). It can also be 5% CO2 compressed gas (10MPa=0.5MPa CO2 (purity 99.9%)+7.5MPa N2 (purity 99.995%)+2MPaO2 (purity 99.9%)). The driving gas pressure for delivering fresh culture medium in the chip is adjusted according to different microfluidic chip structures, so that the flow rate of the complete cell culture medium in the chip is ≤1.5mm/s.

The beneficial effects of the above technical solutions are: the present invention transports the cell complete culture medium to the cell culture layer of the microfluidic chip 12 through the gas-liquid exchange device, brings out the toxic substances through the liquid exchange, and at the same time can add substances that are beneficial to cell growth, If the gas introduced can also be a stable gas for maintaining the cell culture liquid in the culture system, the culture system has a good gas environment.

Preferably, the system further includes: a first control valve pipeline 3, a second control valve pipeline 4, a first liquid outlet pipeline 5 and a second liquid outlet pipeline 6; wherein,

One end of the first control valve pipeline 3 is inserted into the microfluidic chip 12, and the other end thereof passes through the second window 9 to communicate with the outside world;

One end of the second control valve pipeline 4 is inserted into the microfluidic chip 12, and the other end thereof passes through the third window 10 to communicate with the outside world;

One end of the first liquid outlet pipeline 5 is inserted into the microfluidic chip 12, and the other end thereof passes through the fourth window 10 to communicate with the outside world;

One end of the second liquid outlet pipeline 6 is inserted into the microfluidic chip 12 , and the other end thereof passes through the fourth window 10 to communicate with the outside world.

The principle of the above technical solution is that when the complete cell culture medium in the culture system is replaced, the original complete cell culture medium needs to be discharged to the outside. Therefore, the present invention provides: 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 , and the first control valve pipeline 3 is connected to the first control valve pipeline 3 through the microfluidic chip 12 . . The pipeline control of the second control valve pipeline 4 realizes the replacement of the original complete cell culture medium.

The beneficial effect of the above technical scheme is that: by discharging the cell complete medium and toxic substances in the cell culture chamber, the nutrient substance in the cell culture chamber can be maintained sufficient, and the content of toxic substances in the cell culture chamber 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 layers of the cell culture layer is M layers, and 1≤M≤10;

The number of layers of the chip control layer is N, and 0≤N≤10;

The cell culture layer includes a liquid inlet and a liquid outlet, and the number of the liquid inlet and the liquid outlet is Q, and 1≤Q≤100; (1, the interior of the cell culture layer includes a cell culture cavity, The number of the cell culture chambers is S, and 1≤S≤10000;

The chip control layer includes a chip control valve and a chip control valve liquid inlet, and the number of the chip control valve and the chip control valve liquid inlet is W, and 1≤W≤100.

The principle of the above technical solution is: in the present invention, efficient cell culture is realized inside the microfluidic chip 12, and the cell culture layer and the chip control layer in the present invention can be arranged in a combination of human and arrangement, thereby realizing different microfluidics. Functional expansion of the fluid control chip 12 .

The beneficial effects of the above-mentioned technical solution are: it constructs a living space in the process of culturing cells and biological tissues, and is a key component of the micro-culture system. The main function is to separate the cell culture environment from the liquid in the water tank, to provide the space necessary for the growth of cells and biological tissues, and to replace the culture medium regularly and quantitatively. Its basic structure is the single-channel or multi-channel structure of one or more cell culture chambers in the present invention, 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 beneficial effects of the above technical solutions are as follows: the microfluidic chip 12 and the conductive glass 13 of the present invention are bonded by plasma to realize the direct temperature supply of the conductive glass 13 to the microfluidic chip 12 .

Preferably, the conductive glass 13 has a double-layer ITO-plated film, and the transparent water tank 7 is an acrylic plate;

A water tank is also provided outside the culture device.

The principle and beneficial effects of the above technical solutions are: the ITO film of the conductive glass 13 has high electrical conductivity, high visible light transmittance, high mechanical hardness and good chemical stability, and the acrylic plate seals water through a transparent structure, which is convenient for observation. , and after filling with deionized water, on the one hand, the temperature is more stable, and on the other hand, the inherent defects of the microfluidic chip material are effectively improved during the cell culture process.

As an embodiment of the present invention,

Deionized water is filled between the microfluidic chip 12 and the transparent water tank 7 to provide humidity for the culture environment.

The principle and beneficial effects of the above technical solution are: 100% humidity is required in the culture environment, so by constructing a transparent water tank around the chip, the evaporation phenomenon in the chip and the high concentration of environmental proteins caused by evaporation are suppressed.

As shown in FIG. 2, an implementation method of biological tissue culture based on a microfluidic chip is applicable to the system of the present invention, and the method includes:

Step 1: Disinfect the gas-liquid exchange device, the microfluidic chip 12, the first control valve line 3, the second control valve line 4, the first liquid output line 5 and the second liquid output line 6;

Step 2: inject 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;

Step 3: Turn on the system switch 28, perform temperature control through the temperature control device, and when the temperature stabilizes to the preset culture temperature, inoculate the cells to be seeded through the liquid inlet of the cell culture layer of the microfluidic chip 12 into the first cell culture chamber 18 and the second cell culture chamber 19 of the microfluidic chip 12;

Step 4: Introduce the compressed air into the first liquid storage bottle 20 and the second liquid storage bottle 21 through an external gas compression bottle, and drive the complete medium in the first liquid storage bottle 20 and the second liquid storage bottle 21 into the cell culture layer. and control the opening and closing of the liquid inlet of the cell culture layer and the liquid outlet of the cell culture layer through the chip control valve of the microfluidic chip 1, and replace the complete cell culture medium regularly;

Step 5: Control the inlet pressure in the first liquid storage bottle 20 and the second liquid storage bottle 21 to be 1.5 psi, and drive the cell complete medium to enter the first cell culture chamber 18 at a flow rate of ≤1.5 mm/s and inside the second cell culture chamber 19 .

The principles and beneficial effects of the above technical solutions are:

In the process of cell culture, bacteria are the factors that can most affect the effect of cell and biological tissue culture, so any component in the system of the present invention will be sterilized by high temperature and high pressure when it is used. The implementation method of the present invention is a method for constructing an environment suitable for the growth of cells and biological tissues based on the system, including the removal of temperature, humidity, gas and toxic substances. In step 1, the gas-liquid exchange device, the first liquid inlet line 1, the second liquid inlet line 2, the first liquid output line 5 and the second liquid output line 6 of the gas-liquid exchange device and the microfluidic chip 12 are subjected to high temperature and high pressure sterilization to construct sterile environment. Step 2: Inject 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 from being generated. The sterility of the cell culture environment is maintained. Step 3: Through the temperature control device, the temperature of the cell culture environment is regulated and adjusted to a target environment temperature suitable for cell culture in a self-feedback manner. In step 4, the gas conditions in the culture environment are maintained by passing in a specific compressed gas. Gas conditions can be introduced into the cell safety medium or drugs by connecting compressed air or 5% CO2 compressed gas, so as to maintain sufficient O2 and stable acidity and alkalinity in the liquid environment. In step 5, the pressure is adjusted to a specific value, which reduces the influence of the shear force generated when the cell complete medium is replaced in the culture system on the cell state, and realizes the timed quantitative replacement of the cell complete medium through automatic control. Metabolites are excreted with the replaced complete medium.

The present invention also includes a specific embodiment:

In this example, first, prepare a microfluidic chip (the chip is divided into 4 areas, each area has 320 cell culture chambers, a total of 1280, 8 liquid inlets, 8 liquid outlets, 50 control valves) and 2 sterilized 1.5mL liquid storage bottles, multiple 10cm long sterilization tubes, and 1 1mL syringe;

Insert all the sterilization tubes into the microfluidic chip through the window of the transparent structure (all sizes are 2*25mm2), and then contact the temperature probe on the conductive glass in the lower left corner of the microfluidic chip through the window, and fix it with transparent tape ;

Assemble the transparent structure and the microfluidic chip, and inject sterilized deionized water (60mL) into the transparent structure through any window; connect the power supply to the digital temperature control board, and then connect the output wire of the digital temperature control board to the conductive On the glass, finally turn on the switch of the culture system to control the temperature;

After the temperature is stabilized to 37°C, the prepared HepG2 cells to be seeded (take a 50mL culture flask filled with cells, and take 1mL of cells diluted by centrifugation) are injected into a 1mL needle tube and connected to the liquid inlet pipeline. The liquid inlet of the cell culture layer of the microfluidic chip is inoculated into the cell culture chamber of the microfluidic chip;

Connect the liquid storage bottle filled with complete cell culture medium to the inlet and outlet of the cell culture layer of the microfluidic chip through a sterilization tube, and use the microfluidic chip control valve to control the inlet and outlet of the cell culture layer to close. Periodic replacement of cell stabilization medium;

Adjust the inlet CO2 pressure of the liquid reserve bottle to 1.5psi, and start to automatically control the inlet and outlet control valves to drive the timed replacement of the cell complete medium in the cell culture chamber.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these 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 these modifications and variations.

Claims (10)

1. 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 (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 micro-fluidic chip (12) is arranged inside the transparent water tank (7);

the temperature control system is respectively and 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 into the microfluidic chip (12);

the microfluidic chip (12) is used for controlling the timing replacement of a complete cell culture medium and culturing cells and biological tissues.

2. The microfluidic chip and biological tissue culture based system according to claim 1, wherein 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).

3. The microfluidic chip based biological tissue culture system according to claim 2, wherein the temperature control device comprises: the temperature probe (22), the digital temperature control board (25) and the 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 a second window (9) and is attached to the surface of the conductive glass (13), a PDMS (polydimethylsiloxane) film is arranged on the surface of the conductive glass (13) 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 plate (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 regulation is carried out through the conductive glass (13);

the digital temperature control plate is also provided with a system switch (28).

4. The microfluidic chip-based and biological tissue culture system according to claim 2, wherein 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);

a first air inlet (16) is further formed in the first liquid storage bottle (20), and a second air inlet (17) is further formed in the second liquid storage bottle (21); 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 cavity (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), a 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 storage bottle penetrates through the first window (8) and is inserted into the microfluidic chip (12).

5. The microfluidic chip and biological tissue culture based system according to claim 2, further comprising: 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 penetrates through the third window (10) to be 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 penetrates through the fourth window (10) to be communicated with the outside.

6. A microfluidic chip and biological tissue culture based system according to claim 1, wherein the microfluidic chip (12) 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; (1-100), 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.

7. A microfluidic chip and biological tissue culture based system according to claim 1, wherein the microfluidic chip (12) is plasma bonded to the conductive glass (13).

8. The biological tissue culture system based on the microfluidic chip as claimed in claim 1, wherein the conductive glass has a double-layer ITO-coated thin film, and the transparent water tank (7) is an acrylic plate.

9. The biological tissue culture system based on the microfluidic chip as claimed in claim 1, wherein deionized water is filled between the microfluidic chip (12) and the transparent water tank (7) for providing humidity for the culture environment.

10. An operation method for implementing biological tissue culture based on a microfluidic chip, which is suitable for the system of any one of claims 1 to 9, and comprises the following steps:

step 1: sterilizing the gas-liquid exchange device, the micro-fluidic 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 opened, the temperature control is carried out through the temperature control device, and when the temperature is stabilized to the preset culture temperature, 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 a first liquid storage bottle (20) and a 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 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 (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 in the first liquid storage bottle (20) and the second liquid storage bottle (21) to be input into the first cell culture cavity (18) and the second cell culture cavity (19) at a flow rate of less than or equal to 1.5 mm/s.

Images