JP7654279B2 - Fully closed loop smart cell culture system - Google Patents

Description

The present invention relates to a microbiology device, and in particular to a fully closed loop smart cell culture system.

With conventional cell culture technology, cells are manually extracted from the cell culture device, their status and quantity are checked, and the culture medium is added or updated based on the confirmed information. However, this manual work is time-consuming as it needs to be repeated many times, and there is also a risk of contamination of the environment in which the cells are cultured.

To solve this problem, for example, Patent Document 1 discloses a cell culture device that is configured to have a culture means that contains liquid culture medium from a culture medium supply means, an air supply means that provides the air necessary for cell growth, a pressure control means that provides the pressure environment necessary for the culture means, and a cell number detection means that detects the number of healthy living cells in the culture means.

However, this conventional technology lacks convenience because it does not have a mechanism for automatically replenishing liquid culture medium based on the detection results after the cell count detection means detects the number of live cells.

Taiwan Patent No. I732357B

Therefore, the present invention aims to provide an automated, fully closed-loop, smart cell culture system.

In view of the above problems, the present invention provides a full-closed loop smart cell culture system including a cell culture device and an integrated control means,
At least one of the cell culture devices has a culture means and a detection means,
The culture means includes a culture medium storage unit that stores a liquid culture medium not mixed with the cells to be cultured, a heating unit, a cell culture unit that contains a culture solution in which the cells to be cultured are mixed into the liquid culture medium, and a first pump that is configured to provide the culture medium from the culture medium storage unit to the heating unit to heat it and then provide it to the cell culture unit;
the detection means includes a measurement module and a second pump that supplies the culture solution from the cell culture unit to the measurement module, and is configured to output a measurement result of the culture solution by the measurement module;
the integrated control means is electrically connected to the cell culture device, receives the detection signal from the detection means, generates control parameters based on the detection signal, and transmits the control parameters to the cell culture device;
The cell culture device provides a full closed loop smart cell culture system, characterized in that the amount of culture medium provided to the cell culture section by the first pump via the heating section is controlled based on the control parameters received from the integrated control means.

The present invention drives a first pump to supply culture medium from a culture medium storage section to a cell culture section based on the state of the cells, such as the number of cells, detected by the detection means, thereby realizing the provision of an automated, fully closed-loop, smart cell culture system with reduced external intervention in the cell culture device, and reducing the increased costs and risk of contamination due to manual labor.

FIG. 1 is a perspective view showing an embodiment of a fully closed-loop smart cell culture system of the present invention. FIG. FIG. 2 is a block diagram showing the configuration of the embodiment. FIG. 2 is an explanatory diagram showing the connection relationship of the microfluidic chip in the cell culture device of the same embodiment.

Figures 1 to 3 show an embodiment of the present invention. As shown in the figures, the fully closed-loop smart cell culture system 1 of the present invention comprises three cell culture devices 2, a microinjection means 3, an integrated control means 4, and a touch panel 5. Each cell culture device 2 is configured to be capable of culturing one type of cell, and examples of cells that can be cultured by the fully closed-loop smart cell culture system 1 of the present invention include, but are not limited to, NK (natural killer) cells and T cells.

Each cell culture device 2 includes a culture means 6 and a detection means 7. The culture means 6 is electrically connected to the integrated control means 4 and includes a culture medium storage unit 61, a first pump 62, a heating unit 63, a cell culture unit 64, and an environment management module 65. The culture medium storage unit 61 includes a first container 611 that contains a liquid culture medium (not shown) that is not mixed with the cells to be cultured. The heating unit 63 is connected to the first container 611 and includes a heating container 631 that temporarily contains unheated culture medium supplied from the first container 611, and two heating modules (not shown) that are arranged, for example, on both sides of the heating container 631. The cell culture unit 64 has a cell culture vessel 641 that communicates with the heating vessel 631 and contains the heated culture medium and cells to be cultured that are supplied from the heating vessel 631, and a shaking module 642 that holds and shakes the cell culture vessel 641 to uniformly distribute the cells to be cultured contained in the cell culture vessel 641. In the following, the liquid in the cell culture vessel 641 in which the cells to be cultured are mixed in the liquid culture medium is referred to as the culture liquid.

The environmental management module 65 has a temperature sensor (not shown) disposed in the medium storage section 61 to detect the temperature around the medium storage section 61, a temperature control section (not shown) to adjust the temperature around the medium storage section 61, another temperature sensor (not shown) disposed in the cell culture section 64 to detect the temperature around the cell culture section 64, another temperature control section (not shown) to adjust the temperature around the cell culture section 64, a gas sensor (not shown) disposed in the cell culture section 64 to detect the concentration of carbon dioxide and other gases around the cell culture section 64, and a pH meter (not shown) disposed in the cell culture vessel 641 to detect the pH value of the culture solution. In this way, the environmental management module 65 monitors and adjusts the temperature around the medium storage section 61, the temperature and gas components around the cell culture section 64, and the pH value of the culture solution so that the culture environment is suitable for the cells to be cultured.

Specifically, for example, the environmental temperature of the culture medium storage unit 61 is preferably 4°C±2°C, the environmental temperature of the cell culture unit 64 is preferably 37°C±1°C, the _CO2 concentration around the cell culture unit 64 is preferably 5%±1%, and the pH value of the culture solution is preferably set to pH 7.0 to pH 7.6. The environmental management module 65 transmits data such as the detected temperatures, gas components, and pH value of the culture medium storage unit 61 and the cell culture unit 64 to the integrated control means 4, and the integrated control means 4 controls the operation of the environmental management module 65 and other necessary devices based on the detection results.

In this embodiment, a tube pump is used as the first pump 62, and culture medium is supplied from the first container 611 to the heating container 631. The heating module heats the culture medium contained in the heating container 631, and the first pump 62 supplies the heated culture medium from the heating container 631 to the cell culture container 641 of the cell culture section 64. The shaking module 642 shakes the cell culture container 641 to uniformly mix the newly supplied culture medium with the specimen cells that were in the cell culture container 641, and to uniformly distribute the cells to be cultured contained in the cell culture container 641.

As shown in Figures 2 to 4, the detection means 7 is electrically connected to the integrated control means 4, and includes a second pump 71, a measurement module 72, a buffer solution storage section 73, a measurement valve module 74, and a waste liquid storage tank 75.

In this embodiment, a tube pump is used as the second pump 71, and supplies the culture fluid from the cell culture container 641 to the measurement module 72. The measurement module 72 has a microfluidic chip 76 and an optical member 77. The microfluidic chip 76 surrounds a micropassage 764 through which the culture fluid can pass, and the micropassage 764 communicates with the second pump 71 via the first inlet 761, with the microinjection means 3 via the second inlet 762 and the third inlet 763, and with the waste liquid storage tank 75 via the outlet 765.

The optical member 77 has a microscope 771, a camera 772, and an image processor 773 electrically connected to the camera 772.

The buffer storage section 73 is connected to the second pump 71 and has a buffer container 731 that contains a buffer solution (not shown).

The detection valve module 74 is configured to control the communication state between the buffer solution container 731 and the second pump 71, and the communication state between the second pump 71 and the microfluidic chip 76.

In this embodiment, the first container 611, the heating container 631, the cell culture container 641, and the buffer container 731 of each cell culture device 2 can be configured in a bag shape, a case shape, or a dish shape. In other words, there are no limitations on their shape as long as they can appropriately contain the liquid to be contained.

The microinjection means 3 is electrically connected to the integrated control means 4 and includes a first microinjector 31 that is connected to the second inlet 762 of the microfluidic chip 76 of each cell culture device 2, a buffer tank 32 that is connected to the first microinjector 31 and contains a buffer solution (not shown), a second microinjector 33 that is connected to the third inlet 763 of the microfluidic chip 76 of each cell culture device 2, a staining agent storage tank 34 that is connected to the second microinjector 33 and contains a staining agent (not shown), and a drive motor 35 that is electrically connected to the integrated control means 4. The drive motor 35 uses a screw mechanism to simultaneously drive the first microinjector 31 and the second microinjector 33, so that the first microinjector 31 and the second microinjector 33 simultaneously supply the buffer solution from the buffer tank 32 and the staining agent from the staining agent storage tank 34 into the micropassage 764.

For each cell culture device 2, the integrated control means 4 starts the second pump 71 to supply a portion of the culture fluid from the cell culture container 641 into the micropassage 764 via the first inlet 761 of the microfluidic chip 76, then drives the first microsyringe 31 using the drive motor 35 to supply the buffer solution from the buffer tank 32 into the micropassage 764 via the second inlet 762, and sends the culture fluid into the micropassage 764. In addition, the second microsyringe 33, which is simultaneously driven by the drive motor 35, supplies the dye from the dye storage tank 34 into the micropassage 764 via the third inlet 763, thereby dyeing the cells to be cultured contained in the culture fluid and making them easier to observe.

The camera 772 photographs the culture fluid in the micropassage 764 via the microscope 771, generates several pieces of cell image data, and outputs them to the image processor 773. The image processor 773 generates detection results by performing image recognition on these cell image data, and outputs detection signals reflecting these detection results to the integrated control means 4. Here, the image recognition that the image processor 773 performs on these cell image data is to observe the state and number of the cells to be cultured contained in the culture fluid in the micropassage 764, and generate detection results including the survival rate, density, and number of live cells of the cells to be cultured.

Then, the integrated control means 4 continues to control the drive motor 35 to drive the first micro-injector 31 and the second micro-injector 33, respectively supplying the buffer solution and the staining agent into the micro-passage 764 simultaneously, and further feeding the culture solution into the micro-passage 764 until the culture solution enters the waste liquid storage tank 75 from the outlet 765.

Here, by adjusting the driving force from the drive motor 35 to adjust the speed at which the first micro-injector 31 and the second micro-injector 33 supply the buffer solution and the staining agent into the micro-passage 764, the speed at which the culture medium advances through the micro-passage 764 can be controlled, making it easier for the camera 772 to capture images of the micro-passage 764.

The integrated control means 4 receives a detection signal from the cell culture device 2, generates control parameters based on the detection results contained in the detection signal and pre-set processing rules, and transmits a control signal containing these control parameters to the corresponding cell culture device 2. The cell culture device 2 drives the first pump 62 based on these control parameters, and adjusts the additional amount of culture medium supplied from the first container 611 to the cell culture container 641 via the heating container 631.

Regarding the processing rule here, several control modes are selectable, and for example, the additional amount of culture medium to be added to the cell culture vessel 641 can be calculated based on a countermeasure table set in advance, or can be set according to the user's judgment. As an example of the countermeasure table, for example, the following Table 1 can be referred to. That is, when the volume of the culture medium in the cell culture vessel 641 is 10 ml and the number of cells to be cultured is unknown, a part of the culture medium is supplied from the cell culture vessel 641 to the measurement module 72 by the second pump 71 and measurement is performed, and when a detection result is obtained that the culture medium in the cell culture vessel 641 contains 2,000,000 cells to be cultured, the integrated control means 4 calculates 10 ml as the additional amount of culture medium to be added from the culture medium storage unit 61 to the cell culture vessel 641 in order to return the amount of culture medium in the cell culture vessel 641 to 20 ml, and accordingly controls the first pump 62 to provide 10 ml of culture medium from the first vessel 611 to the cell culture vessel 641 and mix it.

When the integrated control means 4 receives a detection signal from a cell culture device 2, it drives the second pump 71 of the corresponding cell culture device 2 to inject the buffer solution from the buffer solution container 731 via the detection valve module 74 into the first inlet 761 of the microfluidic chip 76, and causes the buffer solution to flow into the micropassage 764 until it is discharged from the outlet 765 into the waste liquid storage tank 75. Therefore, each cell culture device 2 can clean the detection valve module 74 and the micropassage 764 by sending the buffer solution from the buffer solution container 731 into the micropassage 764 and discharging the culture solution remaining in both, thereby preventing them from affecting the next detection.

After receiving a detection signal from the cell culture device 2, the integrated control means 4 can automatically add culture medium to the cell culture device 2 and simultaneously perform cleaning operations for the detection valve module 74 and the micropassage 764.

In this embodiment, the three cell culture devices 2 of the fully closed-loop smart cell culture system 1 are integrated into the same housing, and the medium storage section 61 and cell culture section 64 of each of these cell culture devices 2 are grouped together in the same location.

The cells cultured by each of the three cell culture devices 2 in the fully closed-loop smart cell culture system 1 may be of the same type or different types. The same type of cells can also be cultured under different culture conditions. The control status for each cell culture device 2 does not need to be the same. For example, when detecting the number of cells in one cell culture device 2, it is also possible to add culture medium and clean the detection valve module 74 and micropassage 764 in the other cell culture devices 2.

The touch panel 5 is electrically connected to the integrated control means 4, and displays information such as the environmental temperature of the medium storage section 61 in each cell culture device 2, the environmental temperature of the cell culture section 64, the concentration of _CO2 and other gases, and the pH value of the culture solution contained in the cell culture vessel 641, or image data of the culture solution in the micro passage 764 captured by the camera 772. This allows the user to monitor the cell culture status. In addition, the user can set processing rules corresponding to each cell culture device 2 and parameters of the culture environment to the integrated control means 4 via the touch panel 5. In addition, the integrated control means 4 can also support remote control through communication means such as the Internet. This allows the user to take appropriate action through a mobile device such as a smartphone, even if he or she cannot access the touch panel 5.

In summary, the full-closed loop smart cell culture system 1 of the present invention uses each cell culture device 2 to match the culture means 6 and the detection means 7, making it possible to culture cells in the cell culture container 641 of the culture means 6, and the second pump 71 of the detection means 7 provides a part of the culture solution in the cell culture container 641 to the microfluidic chip 76, and the first micro-injector 31 and the second micro-injector 33 control the movement of the culture solution in the micro-passage 764. In addition, the camera 772 takes an image of the culture solution, and the image processor 773 generates a detection result corresponding to the data of the cells contained in the culture solution. Furthermore, the integrated control means 4 is used to judge the detection result and generate a control parameter based on the processing rule, so that the first pump 62 can be controlled based on this control parameter to adjust the additional amount added from the first container 611 to the cell culture container 641. Therefore, the full-closed loop smart cell culture system 1 of the present invention reduces external intervention in the cell culture device and realizes the provision of an automated full-closed loop smart cell culture system, thereby suppressing the increase in costs and the risk of contamination due to human labor.

The above describes an embodiment of the present invention, but the present invention is not limited to this, and various modifications are possible without departing from the spirit of the invention.

1. Fully closed loop smart cell culture system
2 Cell culture device 3 Microinjection means 31 First microinjector 32 Buffer tank 33 Second microinjector 34 Dye storage tank 35 Drive motor 4 Integrated control means 5 Touch panel 6 Culture means 61 Culture medium storage section 611 First container 62 First pump 63 Heating section 631 Heating container 64 Cell culture section 641 Cell culture container 642 Shaking module 65 Environmental control module 7 Detection means 71 Second pump 72 Inspection module 73 Buffer storage section 731 Buffer container 74 Inspection valve module 75 Waste liquid storage tank 76 Microfluidic chip 761 First inlet 762 Second inlet 763 Third inlet 764 Micro passage 765 Discharge port 77 Optical member 771 Microscope 772 Camera 773 Image processor

Claims (7)

A fully closed loop smart cell culture system comprising a cell culture device and an integrated control means,
At least one of the cell culture devices has a culture means and a detection means,
The culture means includes a culture medium storage unit that stores a liquid culture medium not mixed with the cells to be cultured, a heating unit, a cell culture unit that contains a culture solution in which the cells to be cultured are mixed into the liquid culture medium, and a first pump that is configured to supply the culture medium from the culture medium storage unit to the heating unit, heat the culture medium, and then supply the culture medium to the cell culture unit;
the detection means includes a measurement module and a second pump that supplies the culture solution from the cell culture unit to the measurement module, and is configured to output a measurement result of the culture solution by the measurement module;
the integrated control means is electrically connected to the cell culture device, receives a detection signal from the detection means, generates control parameters based on the detection signal, and transmits the control parameters to the cell culture device;
the cell culture device controls the amount of the culture medium to be provided to the cell culture unit via the heating unit by the first pump based on the control parameters received from the integrated control means;
a micro-injection means electrically connected to said integrated control means;
The detection module of the cell culture device includes a microfluidic chip having a microchannel through which the culture solution can pass, and an optical member that calculates the number of the cells in the culture solution by observing the culture solution passing through the microchannel;
The second pump supplies the culture medium from the cell culture unit to the microfluidic chip;
A fully closed loop smart cell culture system , characterized in that the microinjection means has a first microinjector communicating with the micropassage and feeding the culture medium into the micropassage . 2. The full-closed-loop smart cell culture system according to claim 1, wherein the microinjection means comprises: a staining agent storage tank for storing a staining agent; and a second microinjector communicating with the staining agent storage tank and the micropassage and supplying the staining agent from the staining agent storage tank to the micropassage , thereby staining the culture solution in the micropassage. The detection means of the cell culture device
a buffer storage unit for storing a buffer and communicating with the second pump; and a detection valve module for controlling a communication state between the buffer storage unit and the second pump and a communication state between the second pump and the microfluidic chip;
the integrated control means controls operation of the second pump to provide the buffer solution from the buffer solution reservoir to the microfluidic chip via the detection valve module;
The full-closed-loop smart cell culture system according to claim 1 , characterized in that the buffer solution is pumped into the micro-passage to wash the detection valve module and the micro-passage of the microfluidic chip. The full-closed loop smart cell culture system according to claim 1, characterized in that the culture means of the cell culture device further comprises an environmental management module electrically connected to the integrated control means, and configured to detect and control the ambient temperature of the medium storage unit, the ambient temperature and _CO2 concentration of the cell culture unit, and the pH value of the culture solution. The fully closed loop smart cell culture system according to claim 1, characterized in that the cell culture unit of the cell culture device has a cell culture vessel that contains the culture medium, and a shaking module that shakes the cell culture vessel to uniformly distribute the cells in the culture medium contained in the cell culture vessel. The fully closed loop smart cell culture system according to claim 1, characterized in that the culture medium storage unit of the cell culture device has a first container that contains the culture medium, and the heating unit has a heating container that contains and heats the culture medium provided from the first container. The fully closed loop smart cell culture system according to claim 1, characterized in that the first pump and the second pump are both tube pumps.