WO2018184568A1 - Device and method for automatically conducting cell culture metabolic experiment and online collection or monitoring - Google Patents

Abstract

The present invention discloses a device and a method for automatically conducting a cell culture metabolic experiment and online collection or monitoring. The device comprises a cell culture device, a culture fluid delivery device, an automatic injection and sampling device, and a collection or monitoring device. According to the device and method for automatically conducting a cell culture metabolic experiment and online collection or monitoring, a cell culture metabolic experiment and online collection or monitoring can be automatically conducted, reducing manual operations, and resulting in higher experiment accuracy. Conditions required for cell culture can be set and monitored such that cells can be cultured under specified environmental conditions. Microscopic observation states of the cells, components of a metabolic fluid, and the like can be collected or monitored online, thereby carrying out real-time comparative analysis on experimental states.

Description

Device and method for automatically performing cell culture metabolic experiment and online collection or detection Technical field

The present invention relates to an apparatus and method for automatically performing cell culture metabolic experiments and online collection or detection.

Background technique

In the cell culture metabolism study using a microfluidic chip, the culture solution and the drug solution are pumped from the inlet of the microfluidic channel where the cells are placed, and the reaction of the cells is observed. The metabolic fluid of the cells was recovered from the exit of the microfluidic channel and analyzed by HPLC and LCMS to investigate drug dynamics.

With this method, the culture solution and the drug solution are more easily controlled, and the consumption of the culture solution and the cells themselves can be reduced. A plurality of microfluidic channels are formed on a microfluidic chip, and the advantages of being able to simultaneously test under various conditions are made, so that the drug dynamics can be investigated more efficiently.

The patent application of the publication No. CN102637574 proposes a system in which the metabolic fluid discharged from the outlet of the microfluidic chip is dropped onto the paper by an electric immersion flow, and a high voltage is applied to cause it to be scattered, and analyzed by LCMS.

The patent application of the publication No. CN105647789 proposes to use an autosampler to sample the metabolic fluid discharged from the outlet of the microfluidic chip and perform an automatic analysis by MS.

However, in the prior art, the above-mentioned pump for transporting the culture solution and the drug solution and the microchannel inlet are connected, and/or the metabolic fluid discharged from the microchannel outlet is sampled and introduced into an analytical instrument. Manual operation by the experimenter is required, and all processes cannot be performed automatically.

Summary of the invention

Problems to be solved by the invention

It is an object of the present invention to provide an apparatus and method for automatically performing cell culture metabolic experiments and on-line collection or detection, thereby overcoming the problems of errors, low efficiency, and easy contamination of cell samples by external exposure in the prior art.

Technical means of solving problems

The apparatus for automatically performing cell culture metabolic experiments and on-line collection or detection according to the present invention is characterized by comprising a cell culture device, a culture solution delivery device, an automatic sample sampling device, and a collection or detection device, the cell culture device being placed Inside the auto-injection sampling device, and a microfluidic chip for culturing the cells is fixed inside the cell culture device, and the culture solution delivery device is connected to the inlet of the microfluidic chip through a pipeline. Transmitting a culture solution and/or a reagent into the microfluidic chip, the autosampler sampling device being connected to an inlet of the collection or detection device through a conduit for a metabolic fluid in the microfluidic chip Collection sampling is performed, and the collected sample is delivered to the collection or detection device.

According to the above device, the cell culture metabolic experiment and the online collection or detection can be automatically performed, the manual operation is reduced, the accuracy of the experiment is higher, and the metabolic fluid can be collected or detected online.

Further, in the apparatus of the present invention, the cell culture apparatus is further capable of controlling and adjusting the culture environment of the cells, thereby ensuring that the culture of the cells is performed in a designated culture environment; the culture environment includes a gas environment, temperature, and humidity. Wait.

Further, in the device of the present invention, the culture solution delivery device may be a liquid delivery device such as a peristaltic pump, a syringe pump, or a constant flow pump; and the culture solution delivery device sets the culture solution and/or reagent according to a set flow rate. Continuous delivery through the tubing into the microfluidic channel of the microfluidic chip thereby providing a defined liquid environment to cells within the microfluidic chip.

Further, in the apparatus of the present invention, the auto-injection sampling device is capable of automatically cleaning a microfluidic chip and/or a sampling port using a cleaning solution, the auto-injection sampling device having a sample bottle to be placed in the The cells in the vial are injected into the microfluidic chip.

Further, in the apparatus of the present invention, the detecting means includes, but is not limited to, a liquid chromatography, a gas chromatography, an ion chromatography or a mass spectrometry detection system. The state of the cells is confirmed by analyzing the components of the culture solution after the metabolism of the cells by the detection device.

Further, in the device of the present invention, an automatic microscopic observation device is disposed above or below the microfluidic chip to observe cells in the microfluidic chip and recording. The on-line detection of the microscopic observation morphology of the cells was carried out, and the experimental state was compared and analyzed in real time.

Further, in the apparatus of the present invention, the automatic microscopic observation apparatus includes one or several microscopes, and the lens of the microscope is located above or below the microfluidic chip, and in the microfluidic chip Corresponding to one or more micro-flow paths, the automatic microscopic observation device performs on-line observation and photographing of cells in one or more micro-flow paths; further preferably, the position of the microscope can be moved, thereby enabling use A microscope observes and records multiple viewing positions.

Further, in the apparatus of the present invention, the microfluidic chip has: a cell injection hole; and a micro flow path communicating with the cell injection hole, one end of the micro flow path having a flow path inlet for the culture The input of the culture solution and/or the reagent of the liquid transport device, the other end of the microchannel having a flow path outlet for discharging the metabolic liquid of the cell, wherein the microchannel has a cell culture chamber in the middle, and the cell passes through the cell An injection hole is injected into the cell culture chamber of the micro flow path.

Further, a gasket is disposed on the microfluidic chip, and the gasket covers the cell injection hole, and the gasket can be penetrated by a sharp needle at the tip of the cell injection, and the culture solution can be prevented And / or reagent leakage.

Further, the gasket is further disposed between the microfluidic chip and the gasket, the center of the gasket has a through hole, and a portion of the through hole adjacent to the gasket is disposed away from the The tapered shape of the gasket having a smaller diameter, the other portion of the through hole away from the gasket is disposed to have a cylindrical shape having the same diameter as the smallest diameter of the portion, and the minimum diameter of the portion is smaller than the injection The minimum diameter of the needle. Thereby, the needle can be easily inserted due to the guiding action of the conical portion of the through hole; the positioning of the needle can be easily performed due to the fixing action of the cylindrical portion of the through hole.

Further, the gasket may also have a through hole at the center, and 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 flow path opening and closing valve that opens and closes a cell input channel that communicates with the cell injection hole. Further, the flow path opening and closing valve is a check valve that can supply liquid in the direction of the cell culture chamber and cannot supply liquid in the opposite direction.

Further, in the apparatus of the present invention, on the microfluidic chip, a sampling and draining member is mounted, the sampling and draining member having a peripheral wall, one end of which is sealed with the microfluidic chip Connecting, the other end extending away from the microfluidic chip for preventing the metabolic liquid from overflowing; the sampling port, the sampling port is located in the peripheral wall, and the flow path outlet of the microfluidic channel of the microfluidic chip Connected to store a certain volume of the metabolic fluid; and a discharge port disposed adjacent to the sampling port for discharging the metabolic liquid overflowing from the sampling port.

Further, the microfluidic chip has a plurality of micro flow paths, and the sampling and draining member is an integrated member that forms a sampling port and a liquid discharge port at the outlets of the plurality of micro flow paths.

Further, a cover is mounted on the peripheral wall of the sampling and draining member, and the cover can be penetrated by the sampling needle and maintain a constant airtightness after the penetration.

Further, in the apparatus 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 barcode. Thereby, the experimenter can clearly distinguish the type and model of the microfluidic chip used to accurately complete the experiment.

In addition, the inventors of the present invention found in the research that in the current microfluidic chip cell culture device, there is no fixed microfluidic chip clamping device, and the microfluidic chip is basically fixed by manual placement and manual operation. In the cell culture apparatus, the requirement of all automatic culture cannot be achieved.

Therefore, in the apparatus for automatically performing cell culture metabolic experiments and in-line collection or detection of the present invention, the cell culture apparatus further includes a gripping device that grips the microfluidic chip, the gripping device having a gripping chamber An upper side plate and a lower side plate of the microfluidic chip, wherein a cavity for placing the microfluidic chip is disposed between the upper side plate and the lower side plate, wherein the cavity height is greater than that of the microfluidic chip The overall height is provided with a positioning spring for positioning the microfluidic chip on the lower side plate.

According to the above clamping device, the clamping problem of the microfluidic chip in the device of the present invention can be solved, and the microfluidic chip can be accurately positioned to further realize the automatic operation.

Further, in the holding device, there is provided: an intake port; and a gas passage, one end of the gas passage is in communication with the intake port, and the other end is in communication with the cavity. The gas passage becomes a preheating passage of the gas, which can ensure that the gas is preheated to the ambient temperature before reaching the microfluidic chip, and the influence of the airflow on the temperature of the microfluidic chip is reduced.

Further, in the holding device, the upper side plate has a liquid inlet for a culture solution and a reagent to enter the microfluidic chip, and a cell inlet for the liquid outlet A metabolic fluid that discharges cells for injecting cells into the microfluidic chip.

Further, in the clamping device, the upper side plate has a viewing port opposite to the microfluidic chip, which facilitates observation of the cell state by the automatic microscopic observation device.

Further, the clamping device is made of a material having good thermal conductivity, for example, aluminum, thereby ensuring that an external temperature such as an external heating source can be quickly transmitted to the microfluidic chip to ensure the temperature of the microfluidic chip and the outside world. Be consistent and improve the credibility of the actual analysis.

In addition, the inventors of the present invention found in the prior art that in the prior art, the connection between the microfluidic chip and the pipeline generally adopts the following methods: 1) bonding the joint to the microfluidic chip by using glue, and then using The PEEK joint tightens the pipe to the joint; 2) insert one end of the stainless steel pipe directly into the hole of the microfluidic chip, insert the other end into the pipe; 3) connect the pipe to the fixture, and then rigid The clamp is pressed onto the surface of the microfluidic chip.

However, if the PEEK connector is used, first of all, it is inconvenient to use, and requires more preparation before use. Secondly, the glue has the possibility of being mixed into the microfluidic chip, which leads to experimental error; finally, when the microfluidic chip is scrapped The joints bonded to it also need to be scrapped at the same time, resulting in a large waste.

If the stainless steel joint is used, firstly, the installation is inconvenient. Secondly, when the stainless steel joint is inserted into the microfluidic chip, the microfluidic chip is easily damaged, resulting in leakage; finally, the inner diameter of the pipeline and the aperture of the microfluidic chip are affected. Limitations of stainless steel fittings.

If a rigid fixture is used: the contact force of the clamp and the microfluidic chip is controlled by the tightening force of the bolt or other rigid fixing means. This pressing method makes it difficult to effectively control the contact force between the two. Excessive pressure will cause the microfluidic chip to be damaged by force, while too small a force will cause leakage.

Therefore, in the clamping device of the present invention, there is further provided a connecting device for connecting the microfluidic chip and the pipeline, the connecting device comprising an elastic member and a connecting joint, one end of the connecting joint and the clamping device Abutting, the other end abutting the microfluidic chip, the connecting joint has: a threaded hole or a through hole connecting the pipe joint; and a passage connecting the threaded hole or the through hole and the microfluidic chip, One end of the elastic member abuts against the clamping device, and the other end of the elastic member abuts against the connection joint.

According to the above connecting device, since the pressing force of the connecting joint and the microfluidic chip is provided by the elastic member, it is substantially constant, and the microfluidic chip is not damaged or deformed excessively, and the pressing force is not too small. Leakage. At the same time, the pipe connection can be easily completed by pressing the holding device provided with the connecting device of the present invention on the microfluidic chip.

Further, the connection joint further has a cylindrical body and a flange portion protruding from the body, and the other end of the elastic member abuts against the flange portion.

The method for automatically performing cell culture metabolic experiments and on-line detection according to the present invention is characterized in that it comprises the following steps:

Controlling the culture fluid delivery device and the automatic injection sampling device, cleaning one or more microfluidic channels of the microfluidic chip and delivering the culture fluid and/or reagent;

Controlling the automatic sample sampling device to take cells from the sample bottle and inject them into the microfluidic chip, and perform cell culture in the cell culture chamber in the micro flow path of the microfluidic chip;

Control the automatic microscopic observation device to perform automatic scanning to observe and record the state of the cells in the micro flow path;

Controlling the automatic sample sampling device to collect and sample the metabolic fluid of the cells from the microfluidic chip, and send it to a collection or detection device for online collection or detection;

The experimental conditions, the image data observed by the automatic microscopic observation apparatus, the detection method of the metabolic liquid, and the detection result of the metabolic liquid are collectively stored.

According to the method of the invention, the 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 the user to grasp the current state of the cells, and can store the current image, and can adjust the current image. Read the previous images for comparative analysis; automate cell culture experiments and online detection, reducing the amount of manual work, and the accuracy of the experiment is higher than manual operation.

Further, in the method of the present invention, the method further includes the steps of: automatically analyzing the image data, calculating and saving the number of cells in the image data, and generating a history chart, according to the calculated cell In the process of cell injection, it is judged whether the number of cell injections is accurate. When the number of cells is insufficient, the automatic sample sampling device is controlled to suck cells from the sample bottle and inject into the microfluidic chip, when the number of cells exceeds When the number is specified, an alarm is given by a screen or a sound, and when the number of cells grows more than a predetermined number in the cell culture process, an alarm is given by a screen or a sound.

Further, in the method of the present invention, the method further includes the following steps: identifying the identification code on the microfluidic chip to distinguish the type and model of the microfluidic chip. Thereby, the experimenter can clearly distinguish the type and model of the microfluidic chip used to accurately complete the experiment.

Effect of the invention

According to the apparatus and method for automatically performing cell culture metabolic experiment and on-line detection according to the present invention, cell culture metabolic experiments and on-line detection can be automatically performed, the manual operation is reduced, the accuracy of the experiment is higher, and the cell culture can be required. The conditions are set and monitored so that the cells can be cultured under the specified environmental conditions; the microscopic observation state of the cells can be detected online, thereby real-time comparative analysis of the experimental state, and online detection of the metabolic fluid Or collect.

DRAWINGS

1 is a schematic diagram of an apparatus for automatically performing cell culture metabolic experiments and online collection or detection according to the present invention.

2 is a perspective view of a microfluidic chip according to an embodiment of the present invention.

Figure 3 is a perspective view of a microfluidic chip having a gasket on a cell injection well.

Figure 4 is a cross-sectional view taken along line A-A of Figure 3.

Figure 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 showing a microfluidic chip equipped with a sampling and draining member.

Fig. 7 is a cross-sectional view showing a microfluidic chip on which a sampling and draining member having a lid is mounted.

Figure 8 is a schematic diagram of a microfluidic chip with an identification code.

Figure 9 is a cross-sectional view of a clamp device according to the present invention.

Fig. 10 is a cross-sectional view showing a connecting device according to the present invention.

detailed description

Hereinafter, specific embodiments of the present invention will be described with reference to the accompanying drawings.

1 is a schematic diagram of an apparatus for automatically performing cell culture metabolic experiments and online collection or detection according to the present invention.

As shown in FIG. 1 , the apparatus for automatically performing cell culture metabolic experiments and on-line detection according to the present invention includes a cell culture device 1, a culture solution delivery device 2, an automatic sample sampling device 3, a collection or detection device 4, and an automatic display. a micro-observation device 5; the cell culture device 1 is placed inside the auto-injection sampling device 3, and a microfluidic chip 6 for culturing cells is fixed inside the cell culture device 1, the culture solution delivery device 2 is connected to the liquid inlet of the microfluidic chip 6 through a line 21 to deliver a culture solution and/or a reagent to the microfluidic chip 6, the autosampler device 3 having a sample vial 7, The cells placed in the vial 7 are injected into the microfluidic chip 6, the autosampler sampling device 3 being connected to the inlet of the collection or detection device 4 via a line 31, to the micro The metabolic fluid in the flow control chip 6 is collected and sampled, and the collected sample is sent to the collection or detection device 4, and the automatic microscopic observation device 5 is located above or below the microfluidic chip 6, Microfluidic core The cells in slice 6 were observed and recorded.

In the apparatus of the present invention, the cell culture apparatus 1 is also capable of controlling and adjusting the culture environment of the cells, thereby ensuring that the cell culture is carried out in a designated culture environment; the culture environment includes a gas environment (for example, a CO ₂ concentration, etc.) ), temperature, humidity, etc.

In the device of the present invention, the culture solution delivery device 2 may be a liquid delivery device such as a peristaltic pump, a syringe pump, or a constant flow pump; the culture solution delivery device 2 passes the culture solution and/or reagent at a set flow rate. The line 21 is continuously delivered into the microchannel 68 of the microfluidic chip 6, thereby providing a designated liquid environment to the cells within the microfluidic chip 6.

Here, the cell may also refer to a mixture of a cell individual as an experimental subject and a liquid existing around the cell individual.

In the apparatus of the present invention, the autosampler sampling device 3 can automatically clean the microfluidic chip 6 and/or the sampling port using a cleaning solution.

In the apparatus of the present invention, the collection or detection device 4 includes, but is not limited to, a liquid chromatography, gas chromatography, ion chromatography or mass spectrometry detection system. The state of the cells is confirmed by analyzing the components of the culture solution after the metabolism of the cells by the detecting device 4.

Further, in the apparatus of the present invention, it is also possible to have a collecting device which is connected to the automatic sample sampling device 3 via a line 31 to collect the cell metabolic fluid sampled by the automatic sample sampling device 3.

In the apparatus of the present invention, the automatic microscopic observation device 5 contains one or several microscopes, the lens of which is located above or below the microfluidic chip 6, and in the microfluidic chip 6 One or more microfluidic channels 68 correspond to the on-line observation and photographing of cells in one or more microfluidic channels 68. It is further preferred that the position of the microscope can be moved, thereby enabling observation and recording of a plurality of observation positions using one microscope.

In the apparatus of the present invention, the cell culture device 1, the culture solution delivery device 2, the collection or detection device 4, the automatic sample sampling device 3, and the automatic microscopy are controlled by a control unit 9. The operation of the device 5 is observed. The control unit 9 can be a personal computer or other device having a storage unit, an input unit, a display unit, and a processing unit.

2 is a perspective view of a microfluidic chip according to an embodiment of the present invention.

As shown in FIG. 2, in the apparatus 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, and the micro flow path 68 has a flow at one end thereof. The road inlet is provided with an input of the culture solution and the reagent from the culture solution delivery device 2, and the other end of the microchannel 68 has a flow path outlet for discharging the metabolic liquid of the cells, and the microchannel 68 has a middle of the flow path 68 In the cell culture chamber 61, cells are injected into the cell culture chamber 61 of the microchannel 68 through the cell injection hole 62.

As shown in FIG. 3 and FIG. 4, a gasket 64 may be disposed on the microfluidic chip 6, and the gasket 64 covers the cell injection hole 62, and the gasket 64 can be topped by a cell for injection. The sharp needle 31 is penetrated and the culture solution and/or the reagent can be prevented from leaking.

As shown in FIG. 5, a gasket 65 may be disposed between the microfluidic chip 6 and the gasket 64. The center of the gasket 65 has a through hole, and the through hole is adjacent to the gasket 64. a portion of the through hole that is smaller in diameter from the gasket 64, and another portion of the through hole away from the gasket 64 is disposed to have a cylindrical shape having the same diameter as the smallest diameter of the portion, The minimum diameter of a portion is smaller than the smallest diameter of the syringe 31. The gasket 65 may also have a through hole at the center, the diameter of the through hole being kept uniform, the diameter being larger than the maximum diameter of the syringe 31.

The microfluidic chip 6 may be provided with a flow path opening and closing valve that opens and closes the cell input channel that communicates with the cell injection hole 62. Further, the flow path opening and closing valve is a check valve that can supply liquid in the direction of the cell culture chamber 61 and cannot supply liquid in the opposite direction. Thereby, automatic injection of cells is achieved by opening and closing the control valve.

As shown in FIG. 6, in the apparatus of the present invention, a sampling and draining member 66 may be mounted on the microfluidic chip 6, and the sampling and draining member 66 has a peripheral wall having one end of the peripheral wall. The microfluidic chip 6 is sealingly connected, and the other end extends away from the microfluidic chip 6 for preventing the metabolic fluid from overflowing; the sampling port is located in the peripheral wall, and the microflow is The flow path outlet of the micro flow path 68 of the control chip 6 (ie, the micro flow path outlet 63) communicates with the metabolic fluid for a certain capacity; and the discharge port is disposed adjacent to the sampling port for supply The metabolic fluid overflowing from the sampling port is discharged. Thereby, sampling of the metabolic liquid accumulated in the sampling port becomes easy, and the metabolic liquid which is not required for analysis can be automatically discharged by gravity. In addition, the collected metabolic fluid can be collected using a collecting device.

When the microfluidic chip 6 has a plurality of microchannels 68, the sampling and draining member 66 may be an integrated member that forms a sampling port and a liquid discharge port at the outlets of the plurality of microchannels 68.

As shown in FIG. 7, a cover 67 can be mounted on the peripheral wall of the sampling and draining member 66, and the cover 67 can be penetrated by the sampling needle and maintain a constant airtightness after penetration. The cover 67 may be composed of, for example, silicone rubber.

As shown in FIG. 8, in the apparatus of the present invention, an identification code 69 that can be recognized may be provided on the microfluidic chip 6. Thereby, the experimenter can clearly distinguish the type and model of the microfluidic chip 6 used to accurately complete the experiment.

The identification code 69 may be a two-dimensional code or a barcode, and may be attached to the microfluidic chip 6 using a label, or may be engraved on the surface of the microfluidic chip 6 by direct lithography.

When the device of the present invention is in use, the cells to be tested can be injected into the microfluidic chip 6 through the autosampler sampling device 3. The culture solution delivery device 2 delivers the culture solution and/or the reagent in the culture solution and the 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 solution and the reagent change. Become a metabolic fluid containing cellular metabolites. The reacted cell metabolic fluid is automatically sampled by the autosampler sampling device 3. The metabolic fluid can be stored in the collection device and also directly sent to the detection device for detection. The state of the cells is confirmed by detecting the components and changes of the metabolic fluid. Further, at the time of the metabolic reaction process, the image of the cells can be observed by the automatic microscopic observation device 5 at any time.

Further, 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 gripping device 11 that clamps 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 sandwiching the microfluidic chip 6, and the upper side plate 1107 and the lower side plate 1109 are placed between the upper side plate 1107 and the lower side plate 1109. The cavity of the microfluidic chip 6 has a cavity height greater 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 disposed on the lower side plate. The upper side plate 1107 and the lower side plate 1109 are positioned by the positioning pins 1108.

According to the above-mentioned clamping device 11, the clamping problem of the microfluidic chip 6 can be solved, and the microfluidic chip 6 can be accurately positioned to further realize the automatic operation.

In the holding device 11, the lower side plate 1109 has: an air inlet 1102; and a gas passage 1110, one end of which is in communication with the air inlet 1102, and the other end is empty The cavity is connected. The narrow gas passage 1110 serves as a preheating passage for the gas, which ensures that the gas is preheated to the ambient temperature before reaching the microfluidic chip 6, reducing the influence of the gas flow on the temperature of the microfluidic chip 6. The design of the clamping device 11 which is miniaturized as much as possible can reduce the consumption of gas and reduce the analysis cost.

The upper side plate 1107 has a liquid inlet 1103 for a culture solution and a reagent into the microfluidic chip 6, and a cell inlet 1106 for discharging cells. A metabolic fluid, the cell injection port 1104 is used to inject cells into the microfluidic chip 6.

The upper side plate 1107 has a viewing port 1105 covered with a transparent material, and the viewing port 1105 is opposite to the microfluidic chip 6, facilitating the observation of the cell state by the automatic microscopic observation device 5.

The clamping device 11 is made of a material having good thermal conductivity, for example, made of aluminum, thereby ensuring that the external temperature of the external heating source 12 and the like can be quickly transmitted to the microfluidic chip 6, thereby ensuring the microfluidic chip 6 and the outside world. The temperature is consistent and the credibility of the actual analysis is improved.

Further, in another embodiment of the present invention, in the holding device 11, a connecting device for connecting the microfluidic chip 6 and the line 21 is further provided. As shown in FIG. 10, the connecting device includes an elastic member 1111 and a connecting joint 1112. One end of the connecting joint 1112 is in contact with the clamping device 11, and the other end is in contact with the microfluidic chip 6. The connecting joint 1112 has: a threaded hole or a through hole connecting the pipe joint 22; and a passage connecting the threaded hole or the through hole and the microfluidic chip 6, and one end of the elastic member 1111 abuts against the clamping In the device 11, the other end of the elastic member 1111 abuts against the connection joint 1112. The elastic member 1111 may be a spring, a spring piece or the like.

According to the above connecting device, since the pressing force of the connecting joint 1112 and the microfluidic chip 6 is provided by the elastic member 1111, it is substantially constant, and the microfluidic chip 6 is not damaged or deformed, and the pressing force is not caused by the pressing force. Too small to cause leakage. At the same time, as long as the holding device 11 provided with the connecting device of the present invention is pressed against the microfluidic chip 6, the pipe connection can be easily completed.

The connection joint 1112 may further include: a main body having a cylindrical shape; and a flange portion protruding from the main body, and the other end of the elastic member 1111 abuts against the flange portion.

Further, the material in contact with the liquid can be made into an inert material, thereby avoiding an influence on the liquid in the flow path.

The method for automatically performing cell culture metabolic experiments and on-line detection according to the present invention comprises the following steps:

Controlling the culture solution delivery device 2 and the automatic sample sampling device 3, cleaning one or more microchannels 68 of the microfluidic chip 6 and delivering the culture solution and/or reagent;

Controlling the automatic sample sampling device 3 to suck cells from the sample vial 7 and inject them into the microfluidic chip 6, and perform cell culture in the cell culture chamber 61 in the micro flow path 68 of the microfluidic chip 6;

The automatic microscopic observation device 5 is controlled to perform automatic scanning to observe and record the state of the cells in the micro flow path 68;

Controlling the automatic sample sampling device 3 to collect and sample the metabolic fluid of the cells from the microfluidic chip 6 and transport it to the collection or detection device 4 for online collection or detection;

The experimental conditions, the image data observed by the automatic microscopic observation apparatus 5, the detection method of the metabolic liquid, and the detection result of the metabolic liquid are collectively stored.

According to the method of the present invention, the 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 the user to grasp the current state of the cells and can store the current image. The previous images are accessed for comparative analysis; the cell culture experiments and online tests can be performed automatically, which reduces the workload of manual operations, and the accuracy of the experiments is higher than that of manual operations.

Further, in the method of the present invention, a plurality of flow paths can be selected for unified control, for example, uniform setting of flow path parameters, unified start or stop of liquid feeding, and the like.

Further, 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:

Performing automatic analysis on the image data, calculating and storing the number of cells in the image data, and generating a history chart, and determining whether the cell injection number is accurate during the cell injection process according to the calculated number of the cells When the number of cells is insufficient, the automatic sample sampling device 3 is controlled to suck cells from the sample vial 7 and inject into the microfluidic chip 6, and when the number of cells exceeds a prescribed number, an alarm is issued through a screen or a prompt tone; Further, in the cell culture process, when the number of cells grows more than a predetermined number, an alarm is given by a screen or a sound.

In another embodiment of the method of the present invention, the method further includes the step of identifying the identification code 69 on the microfluidic chip 6 to distinguish the type and model of the microfluidic chip 6. Thereby, the experimenter can clearly distinguish the type and model of the microfluidic chip 6 used 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 thereto, and observation of different observation positions can be realized by, for example, moving the cell culture device 1.

Further, the holding device of the present invention is not limited to use only in the device of the present invention. The clamping device can be used as long as the microfluidic chip is designed to ensure that the inlet and outlet of the microfluidic chip and the cell injection position are consistent with the clamping device.

The embodiments of the present invention have been described above, but the embodiments are merely illustrative and not intended to limit the scope of the invention. The embodiments can be implemented in various other forms, and various omissions, substitutions, changes and combinations are possible without departing from the scope of the invention. The invention and its modifications are intended to be included within the scope of the invention and the scope of the invention.

Symbol Description

1 cell culture device, 2 culture solution delivery device, 3 automatic sample sampling device, 4 collection or detection device, 5 automatic microscopic observation device, 6 microfluidic chip, 7 sample vial, 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 inlet, 1105 viewing port, 1106 liquid outlet, 1107 upper side plate, 1108 positioning pin, 1109 lower side Plate, 1110 gas path, 1111 elastic element, 1112 connection joint, 21 line, 22 line joint, 31 injection needle, 61 cell culture chamber, 62 cell injection hole, 63 micro flow path outlet, 64 seal, 65 washer , 66 sampling and draining components, 67 covers, 68 micro flow paths, 69 identification codes.

Claims (27)

1. A device for automatically performing cell culture metabolic experiments and online collection or detection, characterized in that

   Including a cell culture device, a culture solution delivery device, an automatic sample sampling device, and a collection or detection device,

   The cell culture device is placed inside the autosampler sampling device, and a microfluidic chip for culturing the cells is fixed inside the cell culture device.

   The culture fluid delivery device is connected to the liquid inlet of the microfluidic chip through a pipeline, and the culture fluid and/or the reagent is delivered to the microfluidic chip.

   The automatic sample sampling device is connected to the inlet of the collecting or detecting device through a pipeline, collects and samples the metabolic fluid in the microfluidic chip, and transports the collected sample to the collecting or detecting device. .
2. The device of claim 1 wherein:

   The cell culture device is capable of controlling and adjusting the culture environment of the cells.
3. The device of claim 1 wherein:

   The culture fluid delivery device is one or more of a peristaltic pump, a syringe pump, and a constant flow pump.
4. The device of claim 1 wherein:

   The automatic sample sampling device can automatically clean the microfluidic chip and/or the sampling port by using the cleaning liquid.

   The autosampler sampling device has a sample vial into which cells placed in the vial are injected.
5. The device of claim 1 wherein:

   The detection device includes a liquid chromatography, a gas chromatography, an ion chromatography or a mass spectrometry detection system.
6. The device of claim 1 wherein:

   Also included is an automated microscopic viewing device positioned above or below the microfluidic chip to view and record cells in the microfluidic chip.
7. The device of claim 6 wherein:

   The automated microscopic viewing device comprises one or several microscopes, the lens of the microscope being located above or below the microfluidic chip, corresponding to one or more microfluidic channels in the microfluidic chip, The automated microscopic viewing device performs on-line observation and photographing of cells in one or more microfluidic channels.
8. The device of claim 7 wherein:

   The position of the microscope can be moved.
9. The device of claim 1 wherein:

   The microfluidic chip has: a cell injection hole; and a micro flow path connected to the cell injection hole,

   One end of the micro flow path has a flow path inlet for input of a culture liquid and/or a reagent from the culture liquid delivery device,

   The other end of the micro flow path has a flow path outlet for discharging the metabolic liquid of the cell.

   In the middle of the micro flow path, there is a cell culture chamber.

   Cells are injected through the cell injection wells into the cell culture chamber of the microfluidic channel.
10. The device of claim 9 wherein:

    A sealing pad is disposed on the microfluidic chip, the sealing pad covers the cell injection hole, and the sealing pad can be penetrated by a sharp needle at the tip end for cell injection, and can prevent the culture solution and/or The reagent leaked.
11. The device of claim 10 wherein:

    Further disposed between the microfluidic chip and the gasket, a gasket having a through hole at a center thereof, a portion of the through hole adjacent to the gasket being disposed farther away from the gasket diameter A small conical shape, the other portion of the through hole remote from the gasket is disposed to have a cylindrical shape having the same diameter as the smallest diameter of the portion, the minimum diameter of the portion being smaller than the smallest diameter of the injection needle.
12. The device of claim 10 wherein:

    A gasket is further disposed between the microfluidic chip and the gasket, the gasket having a through hole at a center thereof, the diameter of the through hole being uniform, the diameter being larger than a maximum diameter of the needle.
13. The device of claim 9 wherein:

    The microfluidic chip is provided with a flow path opening and closing valve that opens and closes a cell input channel that communicates with the cell injection hole.
14. The device of claim 13 wherein:

    The flow path opening and closing valve is a check valve that can supply liquid in the direction of the cell culture chamber and cannot supply liquid in the opposite direction.
15. The device of claim 1 wherein:

    A sampling and draining member is mounted on the microfluidic chip.

    The sampling and draining member has:

    a peripheral wall, one end of the peripheral wall is sealingly connected to the microfluidic chip, and the other end extends away from the microfluidic chip for preventing the metabolic liquid from overflowing;

    a sampling port, the sampling port is located in the peripheral wall, and communicates with a flow path outlet of the microfluidic channel of the microfluidic chip, wherein the metabolic fluid accumulates a certain capacity;

    a discharge port disposed adjacent to the sampling port for discharging the metabolic liquid overflowing from the sampling port.
16. The device of claim 15 wherein:

    The microfluidic chip has a plurality of microchannels, and the sampling and draining member is an integrated member that forms a sampling port and a liquid discharge port at the outlets of the plurality of microchannels.
17. The device of claim 15 wherein:

    A cover is mounted on the peripheral wall of the sampling and draining member, and the cover can be penetrated by the sampling needle and maintain a constant airtightness after the penetration.
18. The device of claim 1 wherein:

    On the microfluidic chip, an identification code that can be identified is provided.
19. The device of claim 1 wherein

    The cell culture device further includes a clamping device that clamps the microfluidic chip, the clamping device having an upper side plate and a lower side plate that clamp the microfluidic chip, the upper side plate and the lower side There is a cavity between the side plates for placing the microfluidic chip, the cavity height is greater than the overall height of the microfluidic chip, and the lower side plate is provided with a positioning spring for positioning the microfluidic chip.
20. The device of claim 19, wherein

    The clamping device has an air inlet, and a gas passage, one end of which is in communication with the air inlet and the other end is in communication with the cavity.
21. The device of claim 20 wherein:

    The upper side plate has a liquid inlet for a culture solution and a reagent to enter the microfluidic chip, and a cell inlet for discharging a metabolic fluid of the cell, the cell injection The inlet is for injecting cells into the microfluidic chip.
22. The device of claim 19, wherein

    The upper side plate has a viewing port opposite to the microfluidic chip.
23. The device of claim 19, wherein

    In the clamping device, a connecting device for connecting the microfluidic chip and the pipeline is further provided,

    The connecting device includes a resilient member and a connecting joint,

    One end of the connecting joint abuts the clamping device, and the other end abuts the microfluidic chip,

    The connecting joint has: a threaded hole or a through hole connecting the pipe joint; and a passage connecting the threaded hole or the through hole and the microfluidic chip,

    One end of the elastic member abuts against the clamping device, and the other end of the elastic member abuts against the connecting joint.
24. The device of claim 23, wherein

    The joint joint further has: a body having a cylindrical shape; and a flange portion protruding from the body,

    The other end of the elastic member abuts against the flange portion.
25. A method for automatically performing cell culture metabolic experiments and online collection or detection, comprising the steps of:

    Controlling the culture fluid delivery device and the automatic injection sampling device, cleaning one or more microfluidic channels of the microfluidic chip and delivering the culture fluid and/or reagent;

    Controlling the automatic sample sampling device to take cells from the sample bottle and inject them into the microfluidic chip, and perform cell culture in the cell culture chamber in the micro flow path of the microfluidic chip;

    Control the automatic microscopic observation device to perform automatic scanning to observe and record the state of the cells in the micro flow path;

    Controlling the automatic sample sampling device to collect and sample the metabolic fluid of the cells from the microfluidic chip, and send it to a collection or detection device for online collection or detection;

    The experimental conditions, the image data observed by the automatic microscopic observation apparatus, the detection method of the metabolic liquid, and the detection result of the metabolic liquid are collectively stored.
26. The method of claim 25 wherein:

    It also includes the following steps:

    Performing automatic analysis on the image data, calculating and saving the number of cells in the image data, and generating a history chart,

    According to the calculated number of cells, during the cell injection process, it is determined whether the cell injection number is accurate. When the number of cells is insufficient, the automatic sample sampling device is controlled to suck cells from the sample bottle and inject into the microfluidic chip. In the case where the number of cells exceeds a predetermined number, an alarm is issued through a screen or a sound.

    Further, in the cell culture process, when the number of cells grows more than a predetermined number, an alarm is given by a screen or a sound.
27. A method according to claim 25 or 26, wherein

    It also includes the following steps:

    Identifying the identification code on the microfluidic chip to distinguish the type and model of the microfluidic chip.

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