Integrated in-situ observation stage cell culture apparatus and environmental control method

Abstract

The application discloses an integrated in-situ observation carrier table cell culture device, which comprises a surrounding containing mechanism, a cell carrier, a stress loading mechanism and a control circuit board. The application also discloses an environment control method based on the integrated in-situ observation carrier table cell culture device. The integrated in-situ observation carrier table cell culture device and the environment control method are used for cell in-vitro culture and can realize in-situ real-time microscopic observation of cells, and realize multi-stage regulation of gas and temperature in the integrated in-situ observation carrier table cell culture device. The heat distribution and the uniform distribution of gas concentration are realized through the internal mixing, internal circulation and external circulation integrated method. The application has the characteristics of high portability, high integration, convenient operation, high flexibility, low cost and multiple functions.

Description

Technical Field

[0001] This invention relates to the field of culture equipment technology, and in particular to an integrated in-situ observation stage cell culture equipment and its environmental control method. Background Technology

[0002] Cell biology is the study of cellular life processes. Early cell biology focused primarily on morphological description. Later, by integrating molecular biology, cell biology delved into the ultrastructural level, mainly studying biological processes such as cell growth, metabolism, and heredity. Currently, cell experiments are a crucial and frequently involved component in cell biology, life sciences, and related medical research. Living cells are the basic structural and functional units of living organisms; therefore, real-time observation and analysis of individual living cells are of great significance. To monitor cell activity, migration, proliferation, and changes in related protein expression over long periods in real time, a cell culture apparatus and corresponding control system that are compatible with the microscope stage structure are required.

[0003] However, current cell culture devices are difficult to make into convenient, miniaturized, fully integrated in-situ real-time cell observation instruments in terms of size and structure. Some foreign staged culture devices can realize in-situ observation of single-cell culture process, but they are expensive and have low integration with other operation modules. Furthermore, the control box is separated from the culture box, and the overall integration is still not high. In addition, the peripheral equipment connected by wires and pipes is large in size, and the local deviation of some control quantities is increased due to the connection distance.

[0004] Therefore, it is essential to invent an integrated small-scale culture stage and its environmental control method that is highly portable, easy to use, flexible, and includes temperature and gas control components. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides an integrated in-situ observation stage cell culture device, including a surrounding containment mechanism, a cell carrier, a stress loading mechanism, and a control circuit board;

[0007] The surrounding enclosure mechanism includes a transparent top cover, a top U-shaped cover, an inner enclosure structure, a middle enclosure structure, an outer enclosure structure, and a base plate. The inner enclosure structure, the middle enclosure structure, and the outer enclosure structure are sequentially nested from the inside to the outside to form an enclosure structure. The transparent top cover and the top U-shaped cover are sequentially arranged on the top of the enclosure structure from top to bottom. The enclosure structure is mounted on the base plate. The cell carrier is placed inside the enclosure structure and mounted on the base plate. The inner enclosure structure has gas exchange holes and a surrounding heating device and a fan are arranged on its outer side. The detection device and a serpentine gas inlet pipe are arranged between the inner enclosure structure and the middle enclosure structure. A bottom metal plate with through holes is installed on the lower side of the base plate. A flange is installed on the bottom metal plate. A flange connecting pipe and a variable diameter hose are sequentially connected to the lower end of the flange. An objective lens is arranged at the lower end of the variable diameter hose.

[0008] The stress loading mechanism includes a pressure sensing module, a vacuum pump, a detection connection pipe, and a connecting pipe. The vacuum pump is connected to the variable diameter hose through the connecting pipe, and the variable diameter hose is connected to the pressure sensing module through the detection connection pipe.

[0009] The control circuit board is disposed between the middle layer enclosure structure and the outer layer enclosure structure; the ambient heating device, the fan, the detection device, the pressure sensing module, and the vacuum pump are all electrically connected to the control circuit board.

[0010] Preferably, the inner layer surrounding structure is made of metal and is mounted on the base plate via a first recess in the base plate. The inner layer surrounding structure includes an inner front plate, an inner rear plate, an inner left plate, and an inner right plate. The fan is provided on the inner front plate, and an air filter membrane is provided on the side of the fan facing inward. The inner rear plate is provided with an adjustable gas inlet and an air exchange port. Both the inner left plate and the inner right plate include a gas exchange section and a sealing section. The sealing section is connected to the inner front plate, and the gas exchange section is connected to the inner rear plate. The gas exchange section is provided with a single or arrayed gas exchange port.

[0011] Preferably, the detection device includes at least one oxygen sensor, at least one carbon dioxide sensor, and at least one temperature sensor, wherein the temperature sensor is mounted on a base plate between the inner layer enclosure structure and the middle layer enclosure structure.

[0012] Preferably, the middle layer surrounding structure is made of heat-insulating material and is mounted on the base plate via a second recess on the base plate. The middle layer surrounding structure includes a middle layer front plate, a middle layer rear plate, a middle layer left plate, and a middle layer right plate. The middle layer front plate has mounting holes for installing oxygen and carbon dioxide sensors. The two mounting holes are respectively positioned opposite to the inner front plates on both sides of the fan. The inner side of the middle layer front plate opposite the fan outlet side has a trapezoidal groove with smooth horizontal stripes on the inner wall of the groove. The horizontal cross-sections between the horizontal stripes are arc-shaped, and there are multiple horizontal stripes. The middle layer surrounding structure has a return air inlet, and the middle layer rear plate has a first gas inlet, a second gas inlet, and an air exchange port. The air exchange port on the middle layer rear plate is sealed and connected to the air exchange port on the inner rear plate by a bent or meandering copper pipe or a variable diameter copper pipe.

[0013] Preferably, the outer enclosure structure is made of metal and includes an outer front plate, an outer rear plate, and outer side plates. The outer side plates include an outer left plate and an outer right plate. The outer left or right plate has inlet / outlet holes for wire harnesses and conduits, or it may simultaneously have air exchange pores on the outer left or right plate, with an air filter membrane or filter cotton installed on these pores. These air exchange pores on the outer plate can be gaps between wires at the inlet / outlet of the wire conduit, or they can be tiny pores on the outer plate. The air exchange pores can be single or arranged in an array on the outer plate. A display screen and buttons are installed on the outer side or transparent top cover of the outer enclosure structure. Both the display screen and buttons are electrically connected to the control circuit board. A thermally conductive and electrically insulating layer is provided on the inner side of the outer enclosure structure.

[0014] Preferably, the corners of the wall panels of the outer, middle, or inner surrounding structures are rounded or have a certain curvature.

[0015] Preferably, the cell carrier is made of non-metallic material and has a glass slide as its bottom surface. A glass slide gasket is provided between the glass slide and the flange, and an elastic film is installed in the lower part of the hole of the cell carrier.

[0016] Preferably, the surrounding heating device is a heating element or a heating film, and a bottom heating device is also provided. The bottom heating device includes a first heating film and a second heating film. The first heating film is installed on the inner or outer side of the variable diameter hose wrapping layer or wrapped around the objective lens surface. The second heating film is attached to the lower surface of the bottom metal sheet, and the lower edge of the second heating film is coated with a heat-insulating coating. A transparent heating film is provided on the lower surface of the glass slide. The transparent heating film, the first heating film, and the second heating film are all electrically connected to the control circuit board.

[0017] Preferably, the gas inlet pipe is provided with a first gas inlet pipe and a second gas inlet pipe, and a first solenoid valve and a second solenoid valve are respectively provided on the first gas inlet pipe and the second gas inlet pipe. The gas inlet pipe is provided with a curved angle parallel flow channel module, which includes a straight flow channel and several parallel curved flow channels. The angles of the several curved flow channels increase sequentially and each of them is provided with a curved flow channel valve. A straight flow channel valve is provided on the straight flow channel. The first solenoid valve, the second solenoid valve, the curved flow channel valve, and the straight flow channel valve are all electrically connected to the control circuit board.

[0018] An environmental control method based on the above-mentioned integrated in-situ observation stage cell culture device.

[0019] The homogenization process is as follows:

[0020] The fan on the inner front plate draws carbon dioxide, oxygen, and air from the cell culture chamber through an air filter membrane into the area between the inner and middle front plates.

[0021] A portion of the airflow flows along the narrow slit between the inner and middle enclosing structures to the gas exchange section of the inner left plate, and the airflow re-enters the cell culture chamber through single or arrayed gas exchange pores.

[0022] Another airflow flows to the gas exchange section of the inner right plate, and the airflow re-enters the cell culture chamber through single or arrayed gas exchange pores.

[0023] Meanwhile, a small portion of the airflow passes through the return air inlet of the middle layer enclosure structure and then through the gap between the middle and outer layers enclosure structures back to the gas exchange holes on the outer side plate of the outer layer enclosure structure, thus achieving gas circulation and stabilizing the pressure inside the cell culture chamber.

[0024] Temperature control process:

[0025] Set the desired temperature value, coarse temperature difference value, fine temperature difference value, and interval time;

[0026] The actual temperature value is detected at intervals. When the difference between the actual temperature value and the set temperature value is greater than or equal to the coarse adjustment temperature difference value, the coarse adjustment heating mode is activated, that is, the heating power is controlled by switching the heating elements or heating film in the surrounding heating device on and off.

[0027] When the difference between the actual temperature value and the set temperature value is less than the coarse adjustment temperature difference value but greater than or equal to the fine adjustment temperature difference value, the medium frequency PWM mode in the fine adjustment heating working mode is activated.

[0028] When the difference between the actual temperature value and the set temperature value is less than the fine-tuning temperature difference value, the high-frequency PWM mode in the fine-tuning heating working mode is activated;

[0029] The gas concentration control methods and processes are as follows:

[0030] 1) Set the connection method of the parallel flow channel module with bending angle, which includes two methods:

[0031] ① Each gas path passes through a parallel flow channel module with a bending angle before entering the oxygen solenoid valve and the carbon dioxide solenoid valve. This means that each gas path can be individually speed-regulated in terms of gas flow rate. At this time, two parallel flow channel modules with bending angles are required.

[0032] ② Only one of the oxygen solenoid valve or carbon dioxide solenoid valve is retained. Then, a curved angle parallel flow channel module is connected before the gas enters this valve. At this time, the curved angle parallel flow channel module completes the selection of multiple gas channels and the stepped speed regulation of gas flow. Only one curved angle parallel flow channel module is needed.

[0033] 2) Based on the current concentration values ​​of the two gases, output their respective continuous control quantities. When the second connection method is used, carbon dioxide and oxygen are selected through solenoid valves; after selecting a specific gas, the gas flow rate is steppedly regulated by the parallel flow channel module with bending angle and the on / off status of the corresponding solenoid valves.

[0034] 3) Every 2-5 seconds, based on the continuous control quantity output by the corresponding PID algorithm, the closest tiered discretized digital control quantity value is selected for output. This activates the corresponding solenoid valve on the parallel flow channel module at the bending angle via the drive circuit on the control circuit board. Specifically, the gas flow rate is stepped and regulated by the on / off state of the parallel flow channel module at the bending angle and the solenoid valve array. The sequential control method of the parallel flow channel module at the bending angle from low flow rate to high flow rate is as follows: the flow channel valves at the first and second angles are both open, while the flow channel valves at the other angles are closed; the flow channel valve at the first angle is open, while the flow channel valves at the other angles are closed; the flow channel valve at the second angle is open, while the flow channel valves at the other angles are closed; the flow channel valve at the third angle is open, while the flow channel valves at the other angles are closed; the flow channel valve at the fourth angle is open, while the flow channel valves at the other angles are closed.

[0035] 4) Determine whether the current gas concentration is within the predetermined range from the target concentration value under the appropriate gas flow rate replenishment. If so, switch the fan to low speed mode; otherwise, repeat step 2).

[0036] 5) Once the gas concentration value meets the deviation requirement range, the fan switches to low speed mode and then continues to continuously judge whether the current gas concentration value meets the deviation requirement range in real time.

[0037] 6) Repeat step 3 above.

[0038] Preferably, the middle layer contain a thermal insulation filler material, which is an aerogel material composed of SiO2 nanoparticles, Al2O3 nanoparticles, polyurethane and ceramic fibers.

[0039] The specific mass ratio of the precursor for the thermal insulation filling slurry material is as follows: SiO2 nanoparticles: ethanol: pure water: Al2O3 nanoparticles: polyurethane = 1.6:15:5:1:2;

[0040] Alternatively, the ratio of SiO2 nanoparticles: pure water: Al2O3 nanoparticles: polyurethane is 1.6:15:1:1.4.

[0041] The slurry precursor is degassed and soaked in ceramic fiber felt for more than 8 hours. After vacuum freeze-drying for at least 24 hours, it is sandwiched in the middle layer containment structure as a filler material.

[0042] Therefore, the present invention employs the above-mentioned integrated in-situ observation stage cell culture device and its environmental control method, which has the following beneficial effects:

[0043] (1) The surrounding enclosure adopts a three-layer enclosure structure of inner, middle and outer layers. The middle enclosure structure is made of heat insulation material, which can play a role in heat preservation and block the outward diffusion of heat from the inner layer, and also protect the external circuit. The temperature and gas concentration are homogenized through the integrated structure of internal gas mixing, internal circulation and external circulation.

[0044] (2) The cultivation and control are integrated together, and it has the characteristics of small size, high portability, low power consumption, low cost and strong resistance to biological pollution.

[0045] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the overall structure of an integrated in-situ observation stage cell culture device according to the present invention;

[0047] Figure 2 This is an exploded view of an integrated in-situ observation stage cell culture device according to the present invention;

[0048] Figure 3 Exploded view of the partial structure of the invention after removing the top cover and the U-shaped cover;

[0049] Figure 4 This is a partial structural diagram of the present invention;

[0050] Figure 5This is a schematic diagram of the control circuit board and wiring connections in an embodiment of the present invention;

[0051] Figure 6 This is a schematic diagram of gas flow control according to the present invention;

[0052] Figure 7 This is a schematic diagram showing the position of the display screen and keyboard set on the top surface in an embodiment of the present invention;

[0053] Figure 8 This is a schematic diagram of the control circuit board in an embodiment of the present invention;

[0054] Figure 9 This is a schematic diagram of the parallel flow channel module structure with bending angle in an embodiment of the present invention;

[0055] Figure 10 This is a flowchart illustrating the temperature and gas concentration control process in an embodiment of the present invention.

[0056] Figure 11 This is a schematic diagram of the connecting pipeline between the air exchange holes in an embodiment of the present invention.

[0057] Figure Labels

[0058] 1. Surrounding containment mechanism; 2. Cell culture chamber; 3. Cell carrier; 4. Vacuum pump; 5. Pressure sensing module; 6. Display screen; 7. Buttons;

[0059] 11. Transparent top cover; 12. Top U-shaped cover; 13. Inner enclosing structure; 14. Middle enclosing structure; 41. Oxygen sensor; 42. Carbon dioxide sensor; 15. Outer enclosing structure; 16. Base plate; 17. Glass slide; 18. Bottom metal plate; 19. Glass slide gasket; 20. Flange; 21. Flange connecting pipe; 22. Variable diameter flexible hose; 23. Objective lens;

[0060] 131. Inner front panel; 1310. Fan; 132. Inner rear panel; 133. Inner left panel; 134. Inner right panel; 141. Middle front panel; 1411. Trapezoidal groove; 142. Middle rear panel; 143. Middle left panel; 144. Middle right panel; 151. Outer front panel; 152. Outer rear panel; 153. Outer left panel; 154. Outer right panel; 161. First recess; 162. Second recess;

[0061] 1311. Air filter membrane; 1321. Regulating gas inlet; 1322. Air exchange port; 163. Temperature sensor; 164. Heat-conducting block; 165. Temperature sensor mounting slot;

[0062] 31. Control circuit board; 310. Power supply harness; 311. Side panel mounting bolts; 312. Front panel mounting bolts; 313. Drive circuit output interface; 314. Sensor signal connection interface; 1421. First gas inlet; 1422. Second gas inlet; 1423. Air exchange port; 1440. Return air vent; 41. Oxygen sensor; 42. Carbon dioxide sensor;

[0063] 411. Oxygen inlet pipe; 421. Carbon dioxide inlet pipe; 301. Oxygen solenoid valve; 302. Carbon dioxide solenoid valve; 1540. Outer layer surrounding structure conduit inlet; 50. Thermal insulation filling material;

[0064] 800. Parallel flow channel module with curved angle; 81. Curved flow channel; 80. Straight flow channel; 811. First angle curved flow channel; 812. Second angle curved flow channel; 813. Third angle curved flow channel; 814. Fourth angle curved flow channel; 70. First gas inlet selector valve; 71. Second gas inlet selector valve; 72. Straight flow channel valve; 73. First angle curved flow channel valve; 74. Second angle curved flow channel valve; 75. Third angle curved flow channel valve; 76. Fourth angle curved flow channel valve; In the figure, α1 < α2 < α3 < α4;

[0065] 901. Reducing diameter copper pipe; 902. Winding type copper pipe. Detailed Implementation

[0066] Example

[0067] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of this invention is usually placed when in use. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0068] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," and "connect" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. Specific model specifications need to be selected and determined according to the actual specifications of the device, etc. The specific selection and calculation methods adopt existing technology in the art, and therefore will not be described in detail.

[0069] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0070] An integrated in-situ observation stage cell culture device includes a surrounding containment mechanism 1, a cell carrier 3, a stress loading mechanism, and a control circuit board 31, with an overall size of 270×240×75mm.

[0071] The surrounding enclosure 1 includes a transparent top cover 11, a top U-shaped cover 12, an inner enclosure structure 13, a middle enclosure structure 14, an outer enclosure structure 15, and a base plate 16.

[0072] The inner enclosing structure 13, the middle enclosing structure 14, and the outer enclosing structure 15 are sequentially nested from the inside to the outside to form an enclosing structure. A transparent top cover plate 11 and a top U-shaped cover 12 are sequentially arranged on the top of the enclosing structure from top to bottom. The transparent top cover plate 11 and the top U-shaped cover 12 are perpendicular to the front and rear plates and left and right plates of each enclosing structure. The outer edge of the top U-shaped cover 12 is aligned with the outer edge of the outer enclosing structure 15, and the inner edge of the top U-shaped cover 12 is aligned with the inner edge of the inner enclosing structure 13. The transparent top cover plate 11 is connected to the middle enclosing structure 14 by studs and nuts and is tightly pressed onto the upper surface of the top U-shaped cover 12. The top U-shaped cover 12 is connected to the outer enclosing structure 15 by studs and nuts.

[0073] The enclosing structure is mounted on the base plate 16. The cell carrier 3 is placed inside the enclosing structure and mounted on the base plate 16. The inner enclosing structure 13 has gas exchange holes and is equipped with a surrounding heating device and a fan 1310 on its outer side. A detection device and a serpentine gas inlet pipe are provided between the inner enclosing structure 13 and the middle enclosing structure 14 to facilitate gas preheating. The gas inlet pipe is provided with a first gas inlet pipe (oxygen inlet pipe 411) and a second gas inlet pipe (carbon dioxide inlet pipe 421). A first solenoid valve 301 and a second solenoid valve 302 are respectively provided on the first gas inlet pipe and the second gas inlet pipe to control the on / off of constant pressure oxygen and carbon dioxide, respectively.

[0074] A bottom metal sheet 18 with through holes is installed on the lower side of the base plate 16. In this embodiment, a thin metal sheet is used. A flange 20 is installed on the bottom metal sheet 18. A flange connecting pipe 21 and a variable diameter hose 22 are connected sequentially to the lower end of the flange 20. An objective lens 23 is provided at the lower end of the variable diameter hose 22. The cell carrier 3 is made of non-metallic material and a glass slide 17 is attached to its lower surface. In this embodiment, an ultra-thin glass slide is used. A glass slide gasket 19 is provided between the glass slide 17 and the flange 20. An elastic membrane is installed in the lower part of the hole of the cell carrier 3.

[0075] The stress loading mechanism includes a pressure sensing module 5, a vacuum pump 4, a detection connection pipe and a connecting pipe. The vacuum pump 4 is a WeChat vacuum pump and is connected to the variable diameter hose 22 through the connecting pipe. The variable diameter hose 22 is connected to the pressure sensing module 5 through the detection connection pipe.

[0076] The inner layer enclosure structure 13 is made of metal and is mounted on the base plate 16 via the first recess 161 of the base plate 16. The inner layer enclosure structure 13 includes an inner front plate 131, an inner rear plate 132, an inner left plate 133, and an inner right plate 134. A fan 1310 is provided on the inner front plate 131, and an air filter membrane 1311 is provided on the side of the fan 1310 facing inward. An air inlet 1321 (including carbon dioxide and oxygen) and an air exchange pore 1322 are provided on the inner rear plate 132. The inner left plate 133 and the inner right plate 134 both include a gas exchange section and a sealing section. The sealing section is connected to the inner front plate, and the gas exchange section is connected to the inner rear plate. The gas exchange section is provided with a single or array-arranged gas exchange pore. In this embodiment, an array-arranged gas exchange pore is used.

[0077] The detection device in this embodiment includes an oxygen sensor 41, a carbon dioxide sensor 42, and a temperature sensor 163. The temperature sensor 163 is installed in a temperature sensor mounting groove 165 on the base plate 16 between the inner layer enclosure structure and the middle layer enclosure structure 14. A heat-conducting block 164 is also provided on the temperature sensor 163.

[0078] The middle layer enclosure structure 14 is made of heat-insulating material and is mounted on the base plate 16 via a second recess 162. The second recess 162 is deeper than the first recess 161. The middle layer enclosure structure 14 includes a middle layer front plate 141, a middle layer rear plate 142, a middle layer left plate 143, and a middle layer right plate 144. The middle layer front plate 141 has mounting holes for mounting oxygen sensor 41 and carbon dioxide sensor 42. The two mounting holes are respectively positioned opposite to the inner front plates on both sides of the fan 1310. The inner side of the middle layer front plate opposite the air outlet side of the fan 1310 has a trapezoidal groove 1411, and the inner wall of the trapezoidal groove 1411 has smooth horizontal stripes for local airflow guidance. The middle layer enclosure structure 14 has a return air vent 1440. The middle layer enclosure structure 14 also has inlets and outlets for wires and pipes (including inlets for temperature sensors, various heating elements, heating films, and fans, as well as insertion holes for carbon dioxide or oxygen intake pipes). The middle layer rear plate 142 has a first gas inlet 1421, a second gas inlet 1422, and an air exchange port 1423. The air exchange port 1423 on the middle layer rear plate and the air exchange port 1322 on the inner layer rear plate are sealed together by a variable diameter copper pipe 901 or a meandering copper pipe 902, and the bent copper pipe contains filter fiber material (see...). Figure 11The middle layer enclosing structure 14 is filled with a thermal insulation filler material 50. The thermal insulation filler material 50 is prepared by impregnating a slurry containing SiO2 nanoparticles, Al2O3 nanoparticles, liquid polyurethane, and pure water (after removing air bubbles) with ceramic fiber felt, followed by vacuum drying. The thermal insulation filler material 50 contained in the middle layer enclosing structure 14 is an aerogel material composed of SiO2 nanoparticles, Al2O3 nanoparticles, polyurethane, and ceramic fibers.

[0079] The specific mass ratio of the precursor of the thermal insulation filling material is as follows: SiO2 nanoparticles: ethanol: pure water: Al2O3 nanoparticles: polyurethane = 1.6:15:5:1:2.

[0080] Alternatively, the ratio of SiO2 nanoparticles: pure water: Al2O3 nanoparticles: polyurethane is 1.6:15:1:1.4.

[0081] After degassing and freeze-drying, the precursor is sandwiched as a filler material in the middle layer containment structure. This reduces heat loss on the inner side while protecting the outer circuit structure.

[0082] The outer enclosure structure 15 is made of metal for easy heat dissipation and includes an outer front plate 151, an outer rear plate 152, and outer side plates. The outer side plates include an outer left plate 153 and an outer right plate 154. In this embodiment, the outer right plate 154 has inlet and outlet holes for wiring harnesses and pipes. Air exchange pores are also provided on the outer right plate 154, and an air filter membrane is installed on these pores. A display screen 6 and buttons 7 are located on the outer surface of the outer enclosure structure 15, greatly improving the portability of the incubator, reducing power consumption and cost, and enhancing resistance to biofouling. Both the display screen 6 and buttons 7 are electrically connected to the control circuit board 31. A thermally conductive insulating layer is provided on the inner side of the outer enclosure structure 15.

[0083] The surrounding heating device is a heating element or a heating film, the thickness of which is less than or equal to the thickness of the gap between the inner surrounding structure 13 and the middle surrounding structure 14. In this embodiment, 2-4 heating elements are used, employing micro-ceramic heating plates and closely attached to the outer surface of the inner surrounding structure 13.

[0084] A bottom heating device is also provided, which includes a first heating film and a second heating film. The first heating film is installed on the inside or outside of the variable diameter hose sheath; the second heating film is attached to the lower surface of the bottom metal sheet, or is a 2-3 mm thick nano-alumina and aluminum silicate composite fiber felt. A transparent heating film is provided on the lower surface of the glass slide. The transparent heating film, the first heating film, and the second heating film are all electrically connected to the control circuit board 31.

[0085] A curved angle parallel flow channel module 800 is provided on the gas inlet pipe. The curved angle parallel flow channel module 800 includes a straight flow channel 80 and several parallel curved flow channels 81. In this embodiment, four curved flow channels 81 are provided with progressively increasing angles and each is equipped with a curved flow channel valve. A straight flow channel valve is provided on the straight flow channel. The first solenoid valve, the second solenoid valve, the curved flow channel valve, and the straight flow channel valve are all electrically connected to the control circuit board 31. The curved angle parallel flow channel module 800 is controlled sequentially from low flow rate to high flow rate (see...). Figure 9 The following are the configurations: First angle flow channel valve 73 and second angle flow channel valve 74 are both open, and the other angle flow channel valves are closed; First angle flow channel valve 73 is open, and the other angle flow channel valves are closed; Second angle flow channel valve 74 is open, and the other angle flow channel valves are closed; Third angle flow channel valve 75 is open, and the other angle flow channel valves are closed; Fourth angle flow channel valve 76 is open, and the other angle flow channel valves are closed.

[0086] The control circuit board 31 is disposed between the middle enclosure structure 14 and the outer enclosure structure 15. The surrounding heating device, fan 1310, detection device, pressure sensing module, and vacuum pump are all electrically connected to the control circuit board 31. The control circuit board 31 is fixed to the outer enclosure structure 15 by side panel fixing bolts 311 and front panel fixing bolts 312. The drive circuit output interface 313 on the control circuit board 31 is connected to the heating element and the solenoid valve, and controls the actual power of the heating element and the real-time on / off state of the solenoid valve through a PID algorithm.

[0087] The sensor signal connection interface 314 is connected to the signal ports of the oxygen sensor 41 and the carbon dioxide sensor 42. The display screen 6 is used for setting and real-time display of the current temperature, carbon dioxide concentration, and oxygen concentration. The buttons 7 are used to set the target temperature, target carbon dioxide concentration, and target oxygen concentration. The carbon dioxide sensor 42 and the oxygen sensor 41 achieve real-time detection by contacting the gas environment within the space between openings at corresponding positions on the middle layer front panel. The circuit diagram of the control circuit board 31 is shown below. Figure 8 As shown, it includes a microcontroller, a button module, a display interface, and a program debugging and download interface.

[0088] The display screen 6 and buttons 7 can also be mounted on top of the enclosure structure, such as... Figure 6 As shown, the two are connected to the control circuit board 31 via flexible flat cables. The circuit board in the control circuit can be installed on the lower surface of the top cover 1212, or it can be installed on... Figure 5 The location shown.

[0089] An environmental control method based on the above-mentioned integrated in-situ observation stage cell culture device.

[0090] The homogenization process is as follows:

[0091] Carbon dioxide, oxygen, and air from the cell culture chamber 2 are drawn into the area between the inner front plate 131 and the middle front plate 141 by the fan 1310 on the inner front plate 131 through the air filter membrane 1311.

[0092] Part of the airflow flows along the slit area between the inner enclosing structure 13 and the middle enclosing structure 14 to the gas exchange section of the inner left plate 133, and the airflow re-enters the cell culture chamber 2 through the matrix-arranged gas exchange holes.

[0093] Another airflow flows to the gas exchange section of the inner right plate 134, and the airflow re-enters the cell culture chamber 2 through the matrix-arranged gas exchange holes.

[0094] Meanwhile, a small portion of the airflow passes through the return air inlet of the middle enclosing structure 14 and then through the gap between the middle enclosing structure 14 and the outer enclosing structure 15 back to the gas exchange holes on the outer side plate of the outer enclosing structure 15, thus achieving gas circulation and stabilizing the pressure inside the cell culture chamber 2.

[0095] Temperature control process: A composite control method is used, which controls the on / off state of two heating elements and simultaneously modulates the input voltage pulse width (PWM) of the other two heating elements or heating films. First, coarse adjustment is performed through the two heating elements, and then intermediate frequency voltage PWM and high frequency voltage PWM are performed one at a time to gradually reduce the deviation between the temperature and the set value.

[0096] Set the desired temperature value, coarse temperature difference value, fine temperature difference value, and interval time.

[0097] The actual temperature value is detected at intervals. When the difference between the actual temperature value and the set temperature value is greater than or equal to the coarse adjustment temperature difference value, the coarse adjustment heating mode is activated, which means that the heating power is controlled by switching the heating elements or heating film in the surrounding heating device on and off.

[0098] When the difference between the actual temperature value and the set temperature value is less than the coarse adjustment temperature difference value but greater than or equal to the fine adjustment temperature difference value, the medium frequency PWM mode in the fine adjustment heating working mode is activated.

[0099] When the difference between the actual temperature value and the set temperature value is less than the fine-tuning temperature difference value, the high-frequency PWM mode in the fine-tuning heating working mode is activated.

[0100] The gas concentration control process is as follows:

[0101] The connection method of the parallel flow channel module 800 with the setting bending angle includes two methods:

[0102] ① Each gas path passes through a curved angle parallel flow channel module 800 before entering valves 301 and 302, meaning that each gas path can be individually speed-regulated (at this time, valves 70 and 71 in the curved angle parallel flow channel module 800 are not necessary). Two curved angle parallel flow channel modules 800 are required.

[0103] ② Only valve 301 and gas inlet 1421 are retained (valve 302 and gas inlet 1422 are not needed). Then, before the gas enters valve 301, a curved angle parallel flow channel module 800 is connected. At this time, the curved angle parallel flow channel module 800 completes the selection of multiple gas channels and the stepped speed regulation of gas flow. Only one curved angle parallel flow channel module 800 is needed.

[0104] 2) Based on the current concentration values ​​of the two gases, output their respective continuous control quantities. When the second connection method is used, carbon dioxide and oxygen are selected through solenoid valves; after selecting a specific gas, the gas flow rate is steppedly regulated by the parallel flow channel module with bending angle and the on / off status of the corresponding solenoid valves.

[0105] 3) Every 2-5 seconds, based on the continuous control quantity output by the corresponding PID algorithm, the closest value of the graded discretized digital control quantity is selected and output. This output activates the corresponding solenoid valve on the bending angle parallel flow channel module 800 via the drive circuit on the control circuit board 31. Specifically, the gas flow rate is steppedly regulated by the on / off state of the bending angle parallel flow channel module 800 and the solenoid valve array. The sequential control method of the bending angle parallel flow channel module 800 from low flow rate to high flow rate is as follows (see...). Figure 9 ): First angle curve flow channel valve 73 and second angle curve flow channel valve 74 are both open, and the other angle flow channel valves are closed; First angle curve flow channel valve 73 is open, and the other angle flow channel valves are closed; Second angle curve flow channel valve 74 is open, and the other angle flow channel valves are closed; Third angle curve flow channel valve 75 is open, and the other angle flow channel valves are closed; Fourth angle curve flow channel valve 76 is open, and the other angle flow channel valves are closed.

[0106] 4) Determine whether the current gas concentration, with a suitable gas supply flow, deviates from the target concentration value within a predetermined range. If so, switch the fan to low speed mode; otherwise, repeat step 2.

[0107] 5) Once the gas concentration value meets the deviation requirement range, the fan switches to low speed mode and then continues to judge in real time whether the current gas concentration value meets the deviation requirement range.

[0108] 6) Repeat step 3 above.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An integrated in-situ observation stage cell culture apparatus, characterized by: This includes the surrounding containment mechanism, cell carrier, stress loading mechanism, and control circuit board; The surrounding enclosure mechanism includes a transparent top cover, a top U-shaped cover, an inner enclosure structure, a middle enclosure structure, an outer enclosure structure, and a base plate. The inner, middle, and outer enclosure structures are sequentially nested from the inside out to form the enclosure structure. The transparent top cover and the top U-shaped cover are sequentially arranged on the top of the enclosure structure from top to bottom. The enclosure structure is mounted on the base plate. The cell carrier is disposed within the enclosure structure and mounted on the base plate. The inner enclosure structure has gas exchange holes and an external heating device and a fan are provided. A detection device and a serpentine gas inlet pipe are provided between the inner and middle enclosure structures. The gas inlet pipe is provided with a first gas inlet pipe and a second gas inlet pipe. The gas inlet pipe is equipped with an oxygen solenoid valve and a carbon dioxide solenoid valve, respectively, on the first and second gas inlet pipes. The gas inlet pipes are equipped with a parallel flow channel module with curved angles, comprising a straight flow channel and several parallel curved flow channels. The angles of the curved flow channels increase sequentially, and each is equipped with a curved flow channel valve. A straight flow channel valve is installed on the straight flow channel. The oxygen solenoid valve, carbon dioxide solenoid valve, curved flow channel valve, and straight flow channel valve are all electrically connected to a control circuit board. A bottom metal plate with a through hole is installed on the lower side of the base plate. A flange is installed on the bottom metal plate, and a flange connecting pipe and a variable diameter hose are sequentially connected to the lower end of the flange. An objective lens is installed at the lower end of the variable diameter hose. The stress loading mechanism includes a pressure sensing module, a vacuum pump, a detection connection pipe, and a connecting pipe. The vacuum pump is connected to the variable diameter hose through the connecting pipe, and the variable diameter hose is connected to the pressure sensing module through the detection connection pipe. The middle layer enclosure structure includes a middle layer front plate, a middle layer rear plate, a middle layer left plate, and a middle layer right plate. The middle layer front plate has mounting holes for installing oxygen sensors and carbon dioxide sensors. The two mounting holes are respectively set opposite to the inner layer front plates on both sides of the fan. The inner side of the middle layer front plate opposite to the fan exhaust side has a trapezoidal groove, and the inner wall of the trapezoidal groove has horizontal stripes. The control circuit board is disposed between the middle layer enclosure structure and the outer layer enclosure structure; the ambient heating device, the fan, the detection device, the pressure sensing module, and the vacuum pump are all electrically connected to the control circuit board.

2. The integrated in situ observation stage cell culture apparatus according to claim 1, wherein: The inner enclosing structure is made of metal and is mounted on the base plate via a first recess in the base plate. The inner enclosing structure includes an inner front plate, an inner rear plate, an inner left plate, and an inner right plate. The fan is mounted on the inner front plate, and an air filter membrane is mounted on the side of the fan facing inward. The inner rear plate has an adjustable gas inlet and an air exchange port. Both the inner left and inner right plates include a gas exchange section and a sealing section. The sealing section is connected to the inner front plate, and the gas exchange section is connected to the inner rear plate. The gas exchange section has single or arrayed gas exchange ports.

3. The integrated in situ observation stage cell culture apparatus according to claim 2, wherein: The detection device includes at least one oxygen sensor, at least one carbon dioxide sensor, and at least one temperature sensor, wherein the temperature sensor is mounted on a base plate between the inner layer enclosure structure and the middle layer enclosure structure.

4. The integrated in situ observation stage cell culture apparatus according to claim 3, wherein: The middle layer surrounding structure is made of heat insulation material and is installed on the base plate through the second recess of the base plate. The middle layer surrounding structure has a return air vent, and the middle layer rear plate has a first gas inlet, a second gas inlet, and an air exchange vent.

5. The integrated in situ observation stage cell culture apparatus according to claim 4, wherein: The outer enclosure structure is made of metal and includes an outer front plate, an outer rear plate, and outer side plates. The outer side plates include an outer left plate and an outer right plate. The outer left plate or the outer right plate is provided with inlet and outlet holes for wire harnesses and pipes, or air exchange pores are provided on the outer left plate or the outer right plate, and air filter membranes are installed on these air exchange pores. A display screen and buttons are installed on the outer side or transparent top cover of the outer enclosure structure. The display screen and buttons are electrically connected to the control circuit board. A thermally conductive electrical insulation layer is provided on the inner side of the outer enclosure structure.

6. The integrated in-situ observation stage cell culture device according to claim 5, characterized in that: The cell carrier is made of non-metallic material and its bottom surface is a glass slide. A glass slide gasket is provided between the glass slide and the flange. An elastic film is installed in the lower part of the hole of the cell carrier.

7. The integrated in-situ observation stage cell culture device according to claim 6, characterized in that: The surrounding heating device is a heating element or a heating film, and a bottom heating device is also provided. The bottom heating device includes a first heating film and a second heating film. The first heating film is installed on the inner or outer side of the variable diameter hose wrapping layer or wrapped around the objective lens surface. The second heating film is attached to the lower surface of the bottom metal sheet, and the lower edge of the second heating film is coated with a heat-insulating coating. A transparent heating film is provided on the lower surface of the glass slide. The transparent heating film, the first heating film, and the second heating film are all electrically connected to the control circuit board.

8. An environmental control method for an integrated in-situ observation stage cell culture device based on any one of claims 1-7, characterized in that: The homogenization process is as follows: The fan on the inner front plate draws carbon dioxide, oxygen, and air from the cell culture chamber through an air filter membrane into the area between the inner and middle front plates. Part of the airflow flows along the narrow gap between the inner and middle enclosing structures to the gas exchange section of the inner left plate, and the airflow re-enters the cell culture chamber through a single or arrayed gas exchange pore. Another airflow flows to the gas exchange section of the inner right plate, and the airflow re-enters the cell culture chamber through a single or arrayed gas exchange pore. Meanwhile, a small portion of the airflow passes through the return air inlet of the middle layer enclosure structure and then through the gap between the middle and outer layers enclosure structures back to the gas exchange holes on the outer side plate of the outer layer enclosure structure, thus achieving gas circulation and stabilizing the pressure inside the cell culture chamber. Temperature control process: Set the desired temperature value, coarse temperature difference value, fine temperature difference value, and interval time; The actual temperature value is detected at intervals. When the difference between the actual temperature value and the set temperature value is greater than or equal to the coarse adjustment temperature difference value, the coarse adjustment heating mode is activated, that is, the heating power is controlled by switching the heating elements or heating film in the surrounding heating device on and off. When the difference between the actual temperature value and the set temperature value is less than the coarse adjustment temperature difference value but greater than or equal to the fine adjustment temperature difference value, the medium frequency PWM mode in the fine adjustment heating working mode is activated. When the difference between the actual temperature value and the set temperature value is less than the fine-tuning temperature difference value, the high-frequency PWM mode in the fine-tuning heating working mode is activated; The gas concentration control methods and processes are as follows: Step 1) Set the connection method of the parallel flow channel module with bending angle, which includes two methods: The first type: Each gas path passes through a parallel flow channel module with a bending angle before entering the oxygen solenoid valve and the carbon dioxide solenoid valve, meaning that each gas path can be individually speed-regulated in stages. The second method involves setting a curved angle parallel flow channel module before the on / off valve solenoid valve of one of the gas channels. This curved angle parallel flow channel module is used to complete the selection of multiple gas channels and the stepped speed regulation of gas flow. Step 2) Based on the current concentration values ​​of the two gases, output their respective continuous control quantities. When the first connection method is used, the gas flow rate is steppedly regulated by controlling the solenoid valves of the two parallel flow channel modules with different bending angles. When the second connection method is used, carbon dioxide gas and oxygen gas are selected through the on / off valve solenoid valve; after the specific gas is selected, the gas flow rate is steppedly regulated by the parallel flow channel module with bending angle and the on / off status of the corresponding solenoid valve. Step 3) Every 2-5 seconds, based on the continuous control quantity output by the corresponding PID algorithm, select the closest digital control quantity value after hierarchical discretization and output it. Then, start the corresponding solenoid valve on the parallel flow channel module of the bending angle through the drive circuit on the control circuit board. Step 4) Determine whether the current gas concentration, with a suitable gas supply flow, deviates from the target concentration value within a predetermined range. If so, switch the fan to low speed mode; otherwise, repeat step 2). Step 5) Once the gas concentration value meets the deviation requirement range, the fan switches to low speed mode and then continues to continuously judge whether the current gas concentration value meets the deviation requirement range in real time. Step 6) Repeat step 3 above.

9. The environmental control method according to claim 8, characterized in that: The middle layer surrounding structure contains a thermal insulation filler material, which is an aerogel material composed of SiO2 nanoparticles, Al2O3 nanoparticles, polyurethane and ceramic fibers; the specific mass ratio of the precursor of the thermal insulation filler slurry material is as follows: SiO2 nanoparticles: ethanol: pure water: Al2O3 nanoparticles: polyurethane = 1.6: 15: 5: 1:

2. Alternatively, the ratio of SiO2 nanoparticles: pure water: Al2O3 nanoparticles: polyurethane is 1.6:15:1:1.

4. The slurry precursor is degassed and soaked in ceramic fiber felt for more than 8 hours. After vacuum freeze-drying for at least 24 hours, it is sandwiched in the middle layer surrounding structure as a filler material.

Images