CN110678540B - Device and method for automatically performing cell culture metabolism experiment and online collection or detection - Google Patents
Device and method for automatically performing cell culture metabolism experiment and online collection or detection Download PDFInfo
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- CN110678540B CN110678540B CN201880020854.2A CN201880020854A CN110678540B CN 110678540 B CN110678540 B CN 110678540B CN 201880020854 A CN201880020854 A CN 201880020854A CN 110678540 B CN110678540 B CN 110678540B
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Abstract
The invention discloses a device and a method for automatically performing cell culture metabolism experiments and collecting or detecting on line. The device comprises a cell culture device, a culture solution conveying device, an automatic sample injection sampling device and a collecting or detecting device. According to the device and the method for automatically performing the cell culture metabolism experiment and the on-line collection or detection, the cell culture metabolism experiment and the on-line collection or detection can be automatically performed, so that manual operation is reduced, and the accuracy of the experiment is higher; the conditions required by cell culture can be set and monitored, so that the cells are cultured under the specified environmental conditions; the microscopic observation state of the cells, the components of the metabolic solution, and the like can be collected or detected on line, thereby performing comparative analysis on the experimental state in real time.
Description
Technical Field
The invention relates to a device and a method for automatically performing cell culture metabolism experiments and collecting or detecting on line.
Background
In the cell culture metabolism study using a microfluidic chip, a culture solution and a chemical solution were pumped from the inlet of a microchannel where cells were placed, and the reaction of the cells was observed. The metabolic solution of the cells was recovered from the outlet of the microfluidic circuit and analyzed by HPLC and LCMS methods to investigate the drug kinetics.
By using the method, the culture solution and the liquid medicine are easier to control, and the consumption of the culture solution and the liquid medicine and the cells themselves can be reduced. A plurality of micro-flow paths are formed on one micro-flow control chip, so that the method has the advantages of being capable of simultaneously carrying out experiments under various conditions and the like, and the medicine dynamics can be more efficiently investigated.
The patent application of publication No. CN102637574 proposes a system in which metabolic liquid discharged from the outlet of a microfluidic chip is dropped onto a paper sheet by an electric saturation flow, and is sprayed by applying a high voltage, and analyzed by LCMS.
The patent application of publication number CN105647789 proposes the use of an autosampler to sample the metabolic fluid discharged at the outlet of the microfluidic chip and to perform an automatic analysis by MS.
However, in the prior art, the above-described steps of connecting the pump for transporting the culture solution and the chemical solution to the inlet of the micro flow path and/or sampling the metabolic solution discharged from the outlet of the micro flow path and introducing the same into the analyzer require manual operations by an experimenter, and thus all the steps cannot be automatically performed.
Disclosure of Invention
Problems to be solved by the invention
The invention aims to provide a device and a method for automatically performing cell culture metabolism experiments and online collection or detection, so that the problems of errors, low efficiency, easiness in external pollution of cell samples and the like existing in manual operation in the prior art are solved.
Technical means for solving the problems
The device for automatically carrying out cell culture metabolism experiments and on-line collection or detection is characterized by comprising a cell culture device, a culture solution conveying device, an automatic sampling device and a collection or detection device, wherein the cell culture device is arranged in the automatic sampling device, a microfluidic chip for culturing cells is fixed in the cell culture device, the culture solution conveying device is connected with a liquid inlet of the microfluidic chip through a pipeline and is used for conveying culture solution and/or reagent into the microfluidic chip, the automatic sampling device is connected with an inlet of the collection or detection device through a pipeline and is used for collecting and sampling metabolic solution in the microfluidic chip, and the collected and sampled sample is conveyed to the collection or detection device.
According to the device, the cell culture metabolism experiment and the on-line collection or detection can be automatically carried out, so that manual operation is reduced, and the accuracy of the experiment is higher; the metabolic liquid can be collected or detected on line.
Further, in the apparatus of the present invention, the cell culture apparatus is also capable of controlling and adjusting the culture environment of cells, thereby ensuring that the culture of cells is performed under a specified culture environment; the culture environment comprises a gas environment, temperature, humidity and the like.
Further, in the device of the present invention, the culture solution delivery device may be a peristaltic pump, an injection pump, a constant flow pump, or the like; the culture solution conveying device continuously conveys culture solution and/or reagent into a micro-flow path of the micro-fluidic chip through a pipeline according to a set flow rate, thereby providing a specified liquid environment for cells in the micro-fluidic chip.
Further, in the device of the present invention, the automatic sampling device is capable of automatically cleaning a microfluidic chip and/or a sampling port using a cleaning liquid, the automatic sampling device having a sample bottle into which cells placed in the sample bottle are injected.
Further, in the device of the present invention, the detection device includes, but is not limited to, a liquid chromatography, gas chromatography, ion chromatography, or mass spectrometry detection system. The cell state is confirmed by analyzing the composition of the culture medium after the cell metabolism by the detection device.
Further, in the device of the invention, an automatic microscopic observation device is also included, and the automatic microscopic observation device is positioned above or below the microfluidic chip, so as to observe and record cells in the microfluidic chip. Thus, the microscopic observation form of the cells is detected on line, and the experimental state is compared and analyzed in real time.
Further, in the device of the invention, the automatic microscopic observation device comprises one or a plurality of microscopes, the lens of the microscope is positioned above or below the microfluidic chip and corresponds to one or a plurality of micro-flow paths in the microfluidic chip, and the automatic microscopic observation device performs on-line observation and photographing record on cells in the one or a plurality of micro-flow paths; it is further preferred that the position of the microscope is movable, whereby observation and recording of a plurality of observation positions using one microscope can be achieved.
Further, in the device of the present invention, the microfluidic chip has: a cell injection hole; and a micro flow path communicating with the cell injection hole, wherein one end of the micro flow path is provided with a flow path inlet for inputting culture solution and/or reagent from the culture solution conveying device, the other end of the micro flow path is provided with a flow path outlet for discharging metabolic solution of cells, a cell culture chamber is arranged in the middle of the micro flow path, and cells are injected into the cell culture chamber of the micro flow path through the cell injection hole.
Further, a gasket is provided on the microfluidic chip, the gasket covers the cell injection hole, the gasket can be penetrated by a sharp-tipped injection needle for cell injection, and leakage of the culture solution and/or the reagent can be prevented.
Further, the gasket is further arranged between the microfluidic chip and the sealing gasket, a through hole is formed in the center of the gasket, a part of the through hole, which is close to the sealing gasket, is arranged in a conical shape with a smaller diameter farther away from the sealing gasket, and the other part of the through hole, which is far away from the sealing gasket, is arranged in a cylindrical shape with the same diameter as the smallest diameter of the part, and the smallest diameter of the part is smaller than the smallest diameter of the injection needle. Thus, the sample injection needle can be conveniently inserted due to the guiding function of the conical part of the through hole; due to the fixing effect of the cylindrical part of the through hole, the sample injection needle can be simply positioned.
Further, the gasket may have a through hole in the center, the diameter of the through hole is kept uniform, and the diameter is larger than the maximum diameter of the injection needle.
Further, the microfluidic chip is provided with a channel opening/closing valve for opening/closing a cell input channel communicating with the cell injection hole. Further, the flow path opening/closing valve is a check valve capable of feeding liquid in the direction of the cell culture chamber but incapable of feeding liquid in the opposite direction.
Further, in the device of the present invention, a sampling and draining means is mounted on the microfluidic chip, the sampling and draining means having: one end of the peripheral wall is connected with the microfluidic chip in a sealing way, and the other end of the peripheral wall extends in a direction away from the microfluidic chip and is used for preventing the metabolic liquid from overflowing; a sampling port located in the peripheral wall and communicating with a flow path outlet of a microfluidic path of the microfluidic chip, the sampling port being configured to store the metabolic liquid in a predetermined volume; and a discharge port provided adjacent to the sampling port and configured to discharge the metabolic solution overflowed from the sampling port.
Further, the microfluidic chip has a plurality of micro-channels, and the sampling and liquid discharging member is an integrated member in which sampling ports and liquid discharging ports are formed at the outlets of the plurality of micro-channels.
Further, a cover is mounted on the peripheral wall of the sampling and drain member, the cover being capable of being penetrated by a sampling needle and maintaining a certain air tightness after penetration.
Further, in the device of the present invention, an identification code that can be identified is provided on the microfluidic chip. The identification code can be a two-dimensional code or a bar code. Therefore, an experimenter can clearly distinguish the type and model of the used microfluidic chip so as to accurately complete the experiment.
In addition, the inventor of the invention discovers that in the conventional cell culture device with the microfluidic chip, a fixed microfluidic chip clamping device is not available, and the microfluidic chip is basically fixed in the cell culture device by adopting a manual placement and manual operation mode, so that the requirement of full automatic culture cannot be met.
Therefore, in the device for automatically performing cell culture metabolism experiments and on-line collection or detection, the cell culture device further comprises a clamping device for clamping the microfluidic chip, the clamping device is provided with an upper side plate and a lower side plate for clamping the microfluidic chip, a cavity for placing the microfluidic chip is arranged between the upper side plate and the lower side plate, the height of the cavity is larger than the whole height of the microfluidic chip, and a positioning spring for positioning the microfluidic chip is arranged on the lower side plate.
According to the clamping device, the clamping problem of the microfluidic chip in the device can be solved, the microfluidic chip can be accurately positioned, and further automatic operation is realized.
Further, the clamping device includes: an air inlet; and one end of the gas passage is communicated with the gas inlet, and the other end of the gas passage is communicated with the cavity. The gas passage becomes a preheating passage of gas, so that the gas can be preheated to the ambient temperature before reaching the microfluidic chip, and the influence of the gas flow on the temperature of the microfluidic chip is reduced.
Further, in the clamping device, the upper side plate is provided with a liquid inlet, a liquid outlet and a cell injection port, wherein the liquid inlet is used for allowing culture solution and reagent to enter the microfluidic chip, the liquid outlet is used for discharging metabolic solution of cells, and the cell injection port is used for injecting cells into the microfluidic chip.
Further, in the clamping device, the upper side plate is provided with an observation port, and the observation port is opposite to the microfluidic chip, so that the automatic microscopic observation device can observe the cell state conveniently.
Furthermore, the clamping device is made of a material with good heat conductivity, such as aluminum, so that external temperature such as an external heating source can be rapidly transmitted to the microfluidic chip, the temperature of the microfluidic chip is kept consistent with that of the outside, and the reliability of actual analysis is improved.
In addition, the inventors of the present invention have found in the study that in the prior art, the connection of a microfluidic chip to a pipeline is generally performed in the following manner: 1) Bonding the connecting joint to the microfluidic chip by adopting glue, and screwing the pipeline to the connecting joint by using the PEEK joint; 2) One end of the stainless steel tube is directly inserted into a hole of the microfluidic chip, and the other end of the stainless steel tube is inserted into a pipeline; 3) The pipeline is connected to the clamp, and then is pressed to the surface of the microfluidic chip by the rigid clamp.
However, if the PEEK joint is used, firstly, the use is inconvenient, and more preparation work is required before the use; secondly, glue is possibly mixed into the microfluidic chip, so that experimental errors are caused; finally, when the microfluidic chip is scrapped, the joints adhered to the microfluidic chip are scrapped simultaneously, so that great waste is caused.
If the stainless steel joint is used, firstly, the installation is inconvenient; secondly, when the stainless steel connector is inserted into the micro-fluidic chip, the micro-fluidic chip is easily damaged, so that liquid leakage is caused; finally, the inner diameter of the pipeline and the aperture of the microfluidic chip are limited by the stainless steel joint.
If a rigid clamp is used: the contact force between the clamp and the microfluidic chip is controlled by the tightening force of the bolts or other rigid fixing means. The contact force of the two is difficult to effectively control by the pressing mode, the micro-fluidic chip is stressed and damaged by the excessive pressure, and the leakage is caused by the too small force.
Therefore, in the clamping device of the present invention, there is further provided a connecting device for connecting a microfluidic chip and a pipeline, the connecting device including an elastic member and a connection joint, one end of the connection joint being in abutment with the clamping device, the other end being in abutment with the microfluidic chip, the connection joint having: threaded holes or through holes for connecting the pipeline joints; and a channel for communicating the threaded hole or the through hole with the microfluidic chip, wherein one end of the elastic element is abutted to the clamping device, and the other end of the elastic element is abutted to the connecting joint.
According to the connecting device, the compression force between the connecting joint and the microfluidic chip is provided by the elastic element, so that the connecting device is basically constant, the microfluidic chip is not damaged or deformed due to overlarge compression force, and liquid leakage is not caused due to overlarge compression force. Meanwhile, when the connecting device is used, the clamping device provided with the connecting device is pressed on the micro-fluidic chip, so that the pipeline connection can be simply and conveniently completed.
Further, the connection joint further has: a cylindrical main body; and a flange portion protruding from the main body, the other end of the elastic element being in contact with the flange portion.
The method for automatically carrying out the cell culture metabolism experiment and the on-line detection is characterized by comprising the following steps:
controlling a culture solution conveying device and an automatic sample feeding sampling device, cleaning one or more micro-flow paths of the micro-fluidic chip and conveying culture solution and/or reagent;
controlling an automatic sample sampling device to absorb cells from a sample bottle and inject the cells into a microfluidic chip, and culturing the cells in a cell culture chamber in a micro-flow path of the microfluidic chip;
controlling an automatic microscopic observation device to automatically scan, and observing and recording the state of cells in the micro-flow path;
controlling an automatic sampling device to collect and sample the metabolic liquid of the cells from the microfluidic chip, and conveying the metabolic liquid to a collecting or detecting device for online collection or detection; and
and uniformly storing experimental conditions, image data observed by the automatic microscopic observation device, a detection method of the metabolic solution and a detection result of the metabolic solution.
According to the method, liquid feeding and cleaning of one or more flow paths can be automatically performed, and the working efficiency is improved; the image provided by the automatic microscopic observation device is convenient for a user to grasp the current cell condition, can store the current image, and can review the previous image, thereby being convenient for comparison and analysis; cell culture experiments and online detection can be automatically carried out, the workload of manual operation is reduced, and compared with manual operation, the accuracy of the experiments is higher.
Further, in the method of the present invention, the method further comprises the steps of: automatically analyzing the image data, calculating and storing the cell number in the image data, generating a history chart, judging whether the cell injection number is accurate or not in the cell injection process according to the calculated cell number, controlling the automatic sample injection sampling device to suck cells from a sample bottle and inject the cells into a micro-fluidic chip when the cell number is insufficient, alarming through a picture or a prompt tone when the cell number exceeds a specified number, and alarming through the picture or the prompt tone when the cell number is increased beyond the specified number in the cell culture process.
Further, the method of the present invention further comprises a step of identifying the identification code on the microfluidic chip to distinguish the type and model of the microfluidic chip. Therefore, an experimenter can clearly distinguish the type and model of the used microfluidic chip so as to accurately complete the experiment.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the device and the method for automatically performing the cell culture metabolism experiment and the online detection, which are related by the invention, the cell culture metabolism experiment and the online detection can be automatically performed, so that the manual operation is reduced, and the accuracy of the experiment is higher; the conditions required by cell culture can be set and monitored, so that the cells are cultured under the specified environmental conditions; the microscopic observation state of the cells can be detected on line, so that the experimental state can be compared and analyzed in real time, and the metabolic solution can be detected or collected on line.
Drawings
FIG. 1 is a schematic diagram of an apparatus for automated cell culture metabolism experiments and on-line collection or detection in accordance with the present invention.
Fig. 2 is a perspective view of a microfluidic chip according to an embodiment of the present invention.
Fig. 3 is a perspective view of a microfluidic chip having a gasket on a cell injection hole.
Fig. 4 is a cross-sectional view of the A-A plane of fig. 3.
Fig. 5 is a cross-sectional view of a microfluidic chip having a gasket and a gasket on a cell injection hole.
Fig. 6 is a cross-sectional view of a microfluidic chip with a sampling and drain member mounted.
Fig. 7 is a cross-sectional view of a microfluidic chip mounted with a sampling and drain member having a cover.
Fig. 8 is a schematic diagram of a microfluidic chip with identification codes.
Fig. 9 is a cross-sectional view of a clamping device according to the present invention.
Fig. 10 is a cross-sectional view of a connection device according to the present invention.
Detailed Description
Hereinafter, a specific embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic diagram of an apparatus for automated cell culture metabolism experiments and on-line collection or detection in accordance with the present invention.
As shown in fig. 1, the device for automatically performing cell culture metabolism experiments and online detection according to the present invention comprises a cell culture device 1, a culture solution conveying device 2, an automatic sample sampling device 3, a collecting or detecting device 4, and an automatic microscopic observation device 5; the cell culture device 1 is placed in the automatic sample sampling device 3, a microfluidic chip 6 for culturing cells is fixed in the cell culture device 1, the culture solution conveying device 2 is connected with a liquid inlet of the microfluidic chip 6 through a pipeline 21, culture solution and/or reagent are conveyed into the microfluidic chip 6, the automatic sample sampling device 3 is provided with a sample bottle 7, cells placed in the sample bottle 7 are injected into the microfluidic chip 6, the automatic sample sampling device 3 is connected with an inlet of the collecting or detecting device 4 through a pipeline 31, metabolic solution in the microfluidic chip 6 is collected and sampled, the collected and sampled sample is conveyed to the collecting or detecting device 4, the automatic microscopic observation device 5 is positioned above or below the microfluidic chip 6, and cells in the microfluidic chip 6 are observed and recorded.
In the device of the present invention, the cell culture device 1 is also capable of controlling and adjusting the culture environment of cells, thereby ensuring that cell culture is performed under a specified culture environment; the culture environment includes a gaseous environment (e.g., CO 2 Concentration, etc.), temperature, humidity, etc.
In the device of the present invention, the culture solution conveying device 2 may be a peristaltic pump, an injection pump, a constant flow pump, or the like; the culture solution transporting device 2 continuously transports the culture solution and/or the reagent into the micro flow path 68 of the micro flow control chip 6 through the pipeline 21 at a set flow rate, thereby providing a specified liquid environment to the cells in the micro flow control chip 6.
The cells may also be a mixture of an individual cell as a subject and a liquid present around the individual cell.
In the device of the present invention, the automatic sampling device 3 may automatically clean the microfluidic chip 6 and/or the sampling port using a cleaning solution.
In the device of the present invention, the collection or detection means 4 includes, but is not limited to, a liquid chromatography, gas chromatography, ion chromatography or mass spectrometry detection system. The detection device 4 analyzes the components of the culture medium after the metabolism of the cells, and confirms the state of the cells.
The apparatus of the present invention may further include a collection device connected to the autosampler 3 via a line 31 to collect the cell metabolic solution sampled by the autosampler 3.
In the device of the present invention, the automatic microscopic observation device 5 comprises one or several microscopes, the lens of each microscope is located above or below the microfluidic chip 6 and corresponds to one or several micro-channels 68 in the microfluidic chip 6, and the automatic microscopic observation device 5 performs on-line observation and photographing recording on cells in one or several micro-channels 68. It is further preferred that the position of the microscope is movable, whereby observation and recording of a plurality of observation positions using one microscope can be achieved.
In the apparatus of the present invention, the operations of the cell culture apparatus 1, the culture medium transport apparatus 2, the collection or detection apparatus 4, the autosampler 3, and the automatic microscopic observation apparatus 5 are controlled by a control unit 9. The control unit 9 may be a personal computer or other device having a storage unit, an input unit, a display unit and a processing unit.
Fig. 2 is a perspective view of a microfluidic chip according to an embodiment of the present invention.
As shown in fig. 2, in the device of the present invention, the microfluidic chip 6 has: a cell injection hole 62; and a micro flow path 68 communicating with the cell injection hole 62, wherein one end of the micro flow path 68 has a flow path inlet for inputting the culture solution and the reagent from the culture solution transporting device 2, the other end of the micro flow path 68 has a flow path outlet for discharging the metabolic solution of the cell, the micro flow path 68 has a cell culture chamber 61 in the middle, and the cell is injected into the cell culture chamber 61 of the micro flow path 68 through the cell injection hole 62.
As shown in fig. 3 and 4, a gasket 64 may be provided on the microfluidic chip 6, the gasket 64 covering the cell injection hole 62, the gasket 64 being capable of being penetrated by the injection needle 31 having a sharp tip for cell injection, and preventing leakage of the culture solution and/or the reagent.
As shown in fig. 5, a gasket 65 may be further disposed between the microfluidic chip 6 and the gasket 64, the gasket 65 having a through hole at its center, a portion of the through hole near the gasket 64 being disposed in a conical shape having a smaller diameter as it is farther from the gasket 64, and another portion of the through hole far from the gasket 64 being disposed in a cylindrical shape having the same diameter as a minimum diameter of the portion, which is smaller than a minimum diameter of the injection needle 31. The washer 65 may have a through hole in the center, and the diameter of the through hole is uniform, and is larger than the maximum diameter of the injection needle 31.
The microfluidic chip 6 may be provided with a flow path opening/closing valve for opening and closing a cell input flow path communicating with the cell injection hole 62. Further, the flow path opening/closing valve is a check valve capable of feeding liquid in the direction of the cell culture chamber 61 but incapable of feeding liquid in the opposite direction. Thus, automatic injection of cells is achieved by controlling the opening and closing of the valve.
As shown in fig. 6, in the device of the present invention, a sampling and draining means 66 may be mounted on the microfluidic chip 6, and the sampling and draining means 66 may include: a peripheral wall, one end of which is connected with the microfluidic chip 6 in a sealing manner, and the other end extends in a direction away from the microfluidic chip 6, for preventing the metabolic liquid from overflowing; a sampling port located in the peripheral wall and communicating with a channel outlet (i.e., a microchannel outlet 63) of a microchannel 68 of the microfluidic chip 6, the sampling port being configured to store a predetermined volume of the metabolic solution; and a discharge port provided adjacent to the sampling port and configured to discharge the metabolic solution overflowed from the sampling port. This facilitates sampling of the metabolic solution stored in the sampling port, and allows the metabolic solution unnecessary for analysis to be automatically discharged by gravity. In addition, the discharged metabolic liquid may be collected using a collection device.
When the microfluidic chip 6 has a plurality of micro flow channels 68, the sampling and drain member 66 may be an integrated member in which sampling ports and drain ports are formed at the outlets of the plurality of micro flow channels 68.
As shown in fig. 7, a cover 67 may be mounted on the peripheral wall of the sampling and drain member 66, and the cover 67 may be penetrated by a sampling needle and maintain a certain air tightness after penetration. The cover 67 may be composed of, for example, silicone rubber.
As shown in fig. 8, in the device of the present invention, an identification code 69 that can be identified may be further provided on the microfluidic chip 6. Therefore, an experimenter can clearly distinguish the type and model of the used microfluidic chip 6 so as to accurately complete the experiment.
The identification code 69 may be a two-dimensional code or a bar code, and may be attached to the microfluidic chip 6 by using a label, or may be engraved on the surface of the microfluidic chip 6 by using a direct photolithography method.
When the device is used, cells to be tested can be injected into the microfluidic chip 6 through the automatic sample injection sampling device 3. The culture medium transporting device 2 transports the culture medium and/or the reagent in the culture medium and reagent bottle 8 to the microfluidic chip 6, and after the metabolic reaction of the cells in the microfluidic chip 6, the components of the culture medium and reagent change to become a metabolic solution containing cell metabolites. The reacted cell metabolic solution is automatically sampled by an automatic sampling device 3. The metabolic liquid can be stored in the collecting device and also directly conveyed to the detecting device for detection. The state of the cells is confirmed by detecting the composition and change of the metabolic solution. In addition, during the metabolic reaction process, the image of the cells can be observed by the automatic microscopic observation device 5 at any time.
In addition, in the apparatus for automatically performing a cell culture metabolism experiment according to another embodiment of the present invention, the cell culture apparatus 1 further includes a clamping device 11 for clamping the microfluidic chip 6. As shown in fig. 9, the holding device 11 has an upper side plate 1107 and a lower side plate 1109 for holding the microfluidic chip 6, and a cavity for placing the microfluidic chip 6 is provided between the upper side plate 1107 and the lower side plate 1109, and the height of the cavity is larger than the overall height of the microfluidic chip 6, thereby ensuring that the introduced gas can reach the periphery of the microfluidic chip 6. A positioning spring 1101 for positioning the microfluidic chip is provided on the lower plate. The upper side plate 1107 and the lower side plate 1109 are positioned by positioning pins 1108.
According to the clamping device 11, the clamping problem of the microfluidic chip 6 can be solved, the microfluidic chip 6 can be accurately positioned, and further automatic operation is realized.
In the clamping device 11, the lower side plate 1109 includes: an air inlet 1102; and a gas passage 1110, one end of the gas passage 1110 communicates with the gas inlet 1102, and the other end communicates with the cavity. The long and narrow gas channel 1110 becomes a preheating channel of gas, so that the gas can be preheated to the ambient temperature before reaching the microfluidic chip 6, and the influence of the gas flow on the temperature of the microfluidic chip 6 is reduced. The clamping device 11 is designed to be as small as possible, so that the gas consumption can be reduced, and the analysis cost can be reduced.
The upper plate 1107 has a liquid inlet 1103, a liquid outlet 1106, and a cell injection port 1104, wherein the liquid inlet 1103 is used for allowing a culture solution and a reagent to enter the microfluidic chip 6, the liquid outlet 1106 is used for discharging a metabolic solution of cells, and the cell injection port 1104 is used for injecting cells into the microfluidic chip 6.
The upper plate 1107 has a viewing port 1105 covered with a transparent material, and the viewing port 1105 is opposite to the microfluidic chip 6, so that the automatic microscopic observation device 5 can observe the cell state.
The clamping device 11 is made of a material with good heat conductivity, for example, aluminum, so that the external temperature of the external heating source 12 and the like can be rapidly transmitted to the microfluidic chip 6, the temperature of the microfluidic chip 6 is ensured to be consistent with that of the outside, and the reliability in actual analysis is improved.
In addition, in another embodiment of the present invention, in the clamping device 11, a connection device for connecting the microfluidic chip 6 and the pipeline 21 is further provided. As shown in fig. 10, the connection device includes an elastic element 1111 and a connection joint 1112, one end of the connection joint 1112 abuts against the clamping device 11, the other end abuts against the microfluidic chip 6, and the connection joint 1112 includes: threaded holes or through holes connecting the pipe fittings 22; and a channel for communicating the threaded hole or the through hole with the microfluidic chip 6, wherein one end of the elastic element 1111 is abutted against the clamping device 11, and the other end of the elastic element 1111 is abutted against the connection joint 1112. The elastic element 1111 may be a spring, a leaf spring, etc.
According to the above-described connection device, since the pressing force of the connection joint 1112 to the microfluidic chip 6 is provided by the elastic member 1111, it is substantially constant, and it does not cause damage or deformation of the microfluidic chip 6 due to an excessive amount, nor leakage due to an excessive pressing force. Meanwhile, when in use, the clamping device 11 provided with the connecting device can simply complete pipeline connection by pressing the clamping device on the micro-fluidic chip 6.
The connection joint 1112 may further have: a cylindrical main body; and a flange portion protruding from the main body, the other end of the elastic element 1111 being in abutment with the flange portion.
In addition, the material in contact with the liquid can be made of an inert material, thereby avoiding the influence on the liquid in the flow path.
The method for automatically carrying out cell culture metabolism experiments and on-line detection comprises the following steps:
controlling the culture solution conveying device 2 and the automatic sample feeding and sampling device 3, and cleaning one or more micro-flow paths 68 of the micro-fluidic chip 6 and conveying culture solution and/or reagent;
controlling the automatic sample sampling device 3 to suck cells from the sample bottle 7 and inject the cells into the microfluidic chip 6, and culturing the cells in the cell culture chamber 61 in the microfluidic channel 68 of the microfluidic chip 6;
the automatic microscopic observation device 5 is controlled to automatically scan, and the state of the cells in the micro flow path 68 is observed and recorded;
controlling an automatic sample sampling device 3 to collect and sample the metabolic liquid of the cells from the microfluidic chip 6, and conveying the metabolic liquid to a collecting or detecting device 4 for online collection or detection; and
and uniformly storing experimental conditions, image data observed by the automatic microscopic observation device 5, a detection method of the metabolic solution and a detection result of the metabolic solution.
According to the method, liquid feeding and cleaning of one or more flow paths can be automatically performed, and the working efficiency is improved; the image provided by the automatic microscopic observation device 5 is convenient for a user to grasp the current cell condition, can store the current image, and can review the previous image, thereby being convenient for comparison and analysis; cell culture experiments and online detection can be automatically carried out, the workload of manual operation is reduced, and compared with manual operation, the accuracy of the experiments is higher.
In the method of the present invention, a plurality of channels can be selected for unified control, for example, unified setting of channel parameters, unified start or stop of liquid feeding, and the like.
In the method of the present invention, the approximate volume of the cells can be calculated by analyzing the image data, and the position of the automatic microscopic observation device 5 can be corrected by adjusting the sharpness of the image.
In another embodiment of the method of the present invention, the method further comprises the steps of:
automatically analyzing the image data, calculating and storing the cell number in the image data, generating a history chart, and judging whether the cell injection number is accurate or not in the cell injection process according to the calculated cell number; when the number of the cells is insufficient, controlling the automatic sampling device 3 to suck the cells from the sample bottle 7 and inject the cells into the microfluidic chip 6, and when the number of the cells exceeds the specified number, alarming through pictures or prompt tones; and when the number of cells increases beyond a predetermined number during the cell culture, an alarm is given by a screen or a warning sound.
In another embodiment of the method of the present invention, the method further comprises the step of identifying the identification code 69 on the microfluidic chip 6 to distinguish the type and model of the microfluidic chip 6. Therefore, an experimenter can clearly distinguish the type and model of the used microfluidic chip 6 so as to accurately complete the experiment.
In the embodiment of the present invention, a plurality of observation positions can be observed by moving the automatic microscopic observation device 5. However, the present invention is not limited to this, and for example, observation at different observation positions may be achieved by moving the cell culture apparatus 1.
The clamping device of the present invention is not limited to use in the device of the present invention. The clamping device can be used as long as the inlet and outlet of the microfluidic chip and the cell injection position are ensured to be consistent with the clamping device during design of the microfluidic chip.
The embodiments of the present invention have been described above, but the embodiments are merely illustrative and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, changes, and combinations can be made without departing from the spirit of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are included in the invention described in the claims and the scope equivalent thereto.
Symbol description
1 cell culture device, 2 culture solution conveying device, 3 autosampler, 4 collection or detection device, 5 automatic microscopic observation device, 6 microfluidic chip, 7 sample bottle, 8 culture solution and reagent bottle, 9 control unit, 11 clamping device, 12 external heating source, 1101 positioning spring, 1102 air inlet, 1103 liquid inlet, 1104 cell injection port, 1105 observation port, 1106 liquid outlet, 1107 upper side plate, 1108 positioning pin, 1109 lower side plate, 1110 gas passage, 1111 elastic element, 1112 connection joint, 21 pipeline, 22 pipeline joint, 31 sample needle, 61 cell culture chamber, 62 cell injection hole, 63 microchannel outlet, 64 sealing gasket, 65 gasket, 66 sampling and draining component, 67 cover, 68 microchannel, 69 identification code.
Claims (25)
1. An automatic cell culture metabolism experiment and on-line collection or detection device is characterized in that,
comprises a cell culture device, a culture solution conveying device, an automatic sample feeding and sampling device and a collecting or detecting device,
the cell culture device is arranged in the automatic sampling device, a micro-fluidic chip for culturing cells is fixed in the cell culture device,
the culture solution conveying device is connected with the liquid inlet of the micro-fluidic chip through a pipeline to convey culture solution and/or reagent into the micro-fluidic chip,
the automatic sampling device is connected with the inlet of the collecting or detecting device through a pipeline, collects and samples the metabolic liquid in the micro-fluidic chip, and conveys the collected and sampled sample to the collecting or detecting device,
the microfluidic chip has: a cell injection hole; a microfluidic circuit communicating with the cell injection hole,
one end of the micro-channel is provided with a channel inlet for inputting culture solution and/or reagent from the culture solution conveying device,
the other end of the micro-flow path is provided with a flow path outlet for discharging the metabolic liquid of the cells,
a cell culture chamber is provided in the middle of the microchannel,
cells are injected into the cell culture chamber of the microfluidic circuit through the cell injection hole,
and a sealing gasket is arranged on the microfluidic chip, covers the cell injection hole, can be penetrated by a sample injection needle with a sharp top end for cell injection, and can prevent culture solution and/or reagent from leaking.
2. The apparatus of claim 1, wherein,
the cell culture apparatus is capable of controlling and adjusting the culture environment of cells.
3. The apparatus of claim 1, wherein,
the culture solution conveying device is one or more of a peristaltic pump, an injection pump and a constant flow pump.
4. The apparatus of claim 1, wherein,
the automatic sample injection sampling device can automatically clean the microfluidic chip and/or the sampling port by using cleaning liquid,
the automatic sample sampling device is provided with a sample bottle, and cells placed in the sample bottle are injected into the microfluidic chip.
5. The apparatus of claim 1, wherein,
the detection device comprises a liquid chromatography, gas chromatography, ion chromatography or mass spectrometry detection system.
6. The apparatus of claim 1, wherein,
the device also comprises an automatic microscopic observation device which is positioned above or below the microfluidic chip and used for observing and recording cells in the microfluidic chip.
7. The apparatus of claim 6, wherein,
the automatic microscopic observation device comprises one or a plurality of microscopes, the lens of each microscope is positioned above or below the microfluidic chip and corresponds to one or a plurality of micro-flow paths in the microfluidic chip, and the automatic microscopic observation device performs on-line observation and photographing record on cells in the one or a plurality of micro-flow paths.
8. The apparatus of claim 7, wherein,
the position of the microscope can be moved.
9. The apparatus of claim 1, wherein,
a gasket is further arranged between the microfluidic chip and the sealing gasket, a through hole is formed in the center of the gasket, a part of the through hole, which is close to the sealing gasket, is arranged in a conical shape with the smaller diameter, which is farther away from the sealing gasket, another part of the through hole, which is far away from the sealing gasket, is arranged in a cylindrical shape with the same diameter as the smallest diameter of the part, and the smallest diameter of the part is smaller than the smallest diameter of the sample injection needle.
10. The apparatus of claim 1, wherein,
and a gasket is further arranged between the microfluidic chip and the sealing gasket, the center of the gasket is provided with a through hole, the diameters of the through holes are kept consistent, and the diameters are larger than the maximum diameter of the sample injection needle.
11. The apparatus of claim 1, wherein,
the microfluidic chip is provided with a channel opening/closing valve for opening/closing a cell input channel communicating with the cell injection hole.
12. The apparatus of claim 11, wherein the device comprises a plurality of sensors,
the flow path opening/closing valve is a check valve capable of feeding liquid in the direction of the cell culture chamber but incapable of feeding liquid in the opposite direction.
13. The apparatus of claim 1, wherein,
the microfluidic chip is provided with a sampling and liquid discharging component,
the sampling and draining member has:
one end of the peripheral wall is connected with the microfluidic chip in a sealing way, and the other end of the peripheral wall extends in a direction away from the microfluidic chip and is used for preventing the metabolic liquid from overflowing;
a sampling port located in the peripheral wall and communicating with a flow path outlet of a microfluidic path of the microfluidic chip, the sampling port being configured to store the metabolic liquid in a predetermined volume; and
and a discharge port provided adjacent to the sampling port and configured to discharge the metabolic solution overflowed from the sampling port.
14. The apparatus of claim 13, wherein the device comprises a plurality of sensors,
the microfluidic chip is provided with a plurality of micro-flow paths, and the sampling and liquid draining component is an integrated component for forming sampling ports and liquid draining ports at the outlets of the micro-flow paths.
15. The apparatus of claim 13, wherein the device comprises a plurality of sensors,
the peripheral wall of the sampling and drain member is provided with a cover that can be penetrated by a sampling needle and maintains a certain air tightness after penetration.
16. The apparatus of claim 1, wherein,
on the microfluidic chip, an identification code capable of being identified is provided.
17. The apparatus of claim 1, wherein,
the cell culture device further comprises a clamping device for clamping the microfluidic chip, the clamping device is provided with an upper side plate and a lower side plate for clamping the microfluidic chip, a cavity for placing the microfluidic chip is arranged between the upper side plate and the lower side plate, the height of the cavity is larger than the whole height of the microfluidic chip, and a positioning spring for positioning the microfluidic chip is arranged on the lower side plate.
18. The apparatus of claim 17, wherein the device comprises a plurality of sensors,
the clamping device has: an air inlet; and one end of the gas passage is communicated with the gas inlet, and the other end of the gas passage is communicated with the cavity.
19. The apparatus of claim 18, wherein the device comprises a plurality of sensors,
the upper side plate is provided with a liquid inlet, a liquid outlet and a cell injection opening, wherein the liquid inlet is used for allowing culture solution and reagents to enter the microfluidic chip, the liquid outlet is used for discharging metabolic solution of cells, and the cell injection opening is used for injecting the cells into the microfluidic chip.
20. The apparatus of claim 17, wherein the device comprises a plurality of sensors,
the upper side plate is provided with an observation port, and the observation port is opposite to the microfluidic chip.
21. The apparatus of claim 17, wherein the device comprises a plurality of sensors,
in the clamping device, a connecting device for connecting the micro-fluidic chip and the pipeline is also arranged,
the connecting means comprise an elastic element and a connecting joint,
one end of the connecting joint is abutted with the clamping device, the other end is abutted with the micro-fluidic chip,
the connection joint has: threaded holes or through holes for connecting the pipeline joints; and a channel communicating the threaded hole or through hole and the microfluidic chip,
one end of the elastic element is abutted to the clamping device, and the other end of the elastic element is abutted to the connecting joint.
22. The apparatus of claim 21, wherein the device comprises a plurality of sensors,
the connection joint further has: a cylindrical main body; and a flange portion protruding from the main body,
the other end of the elastic element is abutted against the flange part.
23. A method for automatically performing a cell culture metabolism assay and on-line collection or detection, comprising the steps of:
controlling a culture solution conveying device and an automatic sample feeding sampling device, cleaning one or more micro-flow paths of the micro-fluidic chip and conveying culture solution and/or reagent;
controlling an automatic sample sampling device to absorb cells from a sample bottle and inject the cells into a microfluidic chip, and culturing the cells in a cell culture chamber in a micro-flow path of the microfluidic chip;
controlling an automatic microscopic observation device to automatically scan, and observing and recording the state of cells in the micro-flow path;
controlling an automatic sampling device to collect and sample the metabolic liquid of the cells from the microfluidic chip, and conveying the metabolic liquid to a collecting or detecting device for online collection or detection; and
uniformly storing experimental conditions, image data observed by the automatic microscopic observation device, a detection method of the metabolic liquid and a detection result of the metabolic liquid,
the microfluidic chip has: a cell injection hole; a microfluidic circuit communicating with the cell injection hole,
one end of the micro-channel is provided with a channel inlet for inputting culture solution and/or reagent from the culture solution conveying device,
the other end of the micro-flow path is provided with a flow path outlet for discharging the metabolic liquid of the cells,
a cell culture chamber is provided in the middle of the microchannel,
cells are injected into the cell culture chamber of the microfluidic circuit through the cell injection hole,
and a sealing gasket is arranged on the microfluidic chip, covers the cell injection hole, can be penetrated by a sample injection needle with a sharp top end for cell injection, and can prevent culture solution and/or reagent from leaking.
24. The method of claim 23, wherein,
the method also comprises the following steps:
automatically analyzing the image data, calculating and storing the cell number in the image data, generating a history chart,
judging whether the cell injection number is accurate or not in the cell injection process according to the calculated cell number, controlling the automatic sampling device to suck cells from a sample bottle and inject the cells into a micro-fluidic chip when the cell number is insufficient, alarming through a picture or a prompt tone when the cell number exceeds the specified number,
and when the number of cells increases beyond a predetermined number during the cell culture, an alarm is given by a screen or a warning sound.
25. The method of claim 23 or 24, wherein,
the method also comprises the following steps:
and identifying the identification code on the microfluidic chip to distinguish the type and the model of the microfluidic chip.
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| CN201710224713.XA CN108690805A (en) | 2017-04-07 | 2017-04-07 | A kind of automatic carry out cell culture metabolism and the online device and method collected or detect |
| CN201710224713X | 2017-04-07 | ||
| PCT/CN2018/081924 WO2018184568A1 (en) | 2017-04-07 | 2018-04-04 | Device and method for automatically conducting cell culture metabolic experiment and online collection or monitoring |
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| CN110724635B (en) * | 2019-10-31 | 2021-08-17 | 中国人民解放军总医院 | Stomach cancer cell analysis equipment for general surgery clinical use |
| AU2022279500B1 (en) * | 2022-12-01 | 2023-11-23 | Provectus IP Pty Ltd | Automated cell analyser and method |
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| CN108690805A (en) | 2018-10-23 |
| WO2018184568A1 (en) | 2018-10-11 |
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Inventor after: Lin Jinming Inventor after: Xu Bo Inventor after: Zhu Peng Inventor after: Chen Wenliang Inventor after: Fan Xueting Inventor after: Tanaka Shinji Inventor after: Iwata Yosuke Inventor before: Xu Bo Inventor before: Zhu Peng Inventor before: Chen Wenliang Inventor before: Fan Xueting Inventor before: Tanaka Shinji Inventor before: Iwata Yosuke Inventor before: Lin Jinming |
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Address after: Building C801, Huaxin Tiandi Phase II, No. 180 Yizhou Road, Xuhui District, Shanghai Patentee after: TSINGHUA University Country or region after: China Patentee after: SHIMADZU ENTERPRISE MANAGEMENT (CHINA) Co.,Ltd. Patentee after: SHIMADZU Corp. Country or region after: Japan Address before: 200233 Building C801, Phase II, Huaxin Tiandi, No. 180 Yizhou Road, Xuhui District, Shanghai Patentee before: SHIMADZU ENTERPRISE MANAGEMENT (CHINA) Co.,Ltd. Country or region before: China Patentee before: TSINGHUA University Patentee before: SHIMADZU Corp. Country or region before: Japan |
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Address after: Tsinghua Yuan, Haidian District, Beijing Patentee after: TSINGHUA University Country or region after: China Patentee after: SHIMADZU ENTERPRISE MANAGEMENT (CHINA) Co.,Ltd. Patentee after: SHIMADZU Corp. Country or region after: Japan Address before: 200233 Building C801, Phase II, Huaxin Tiandi, No. 180 Yizhou Road, Xuhui District, Shanghai Patentee before: TSINGHUA University Country or region before: China Patentee before: SHIMADZU ENTERPRISE MANAGEMENT (CHINA) Co.,Ltd. Patentee before: SHIMADZU Corp. Country or region before: Japan |