Cell culture device and cell culture method using same

Abstract

Summary: Disclosed are a cell culture device capable of applying one or a plurality of physical and / or chemical stimuli to cells during culture, and a cell culture method using the same. This cell culture device is equipped with a cell culture region, a first pump connected to the cell culture region, and a second pump connected to the cell culture region. The first pump, the cell culture region, and the second pump form a line connected in this order. The first pump and the second pump can each move a fluid in the forward direction and the reverse direction independently of each other, and the pressures at which the fluid is moved are variable and independent of each other.

Description

Cell culture device and cell culture method using the same

[0001] The present invention relates to a cell culture device and a cell culture method using the same.

[0002] Improving the functionality of cells, which are the raw material for tissues and organs used in regenerative medicine, and increasing the efficiency of their production is one of the most important issues in the development of regenerative medicine. Naturally, the automated cell culture equipment used in this field is required to reduce the number of human operations and work errors, thereby reducing costs.

[0003] It has been reported that cells that form tissues and organs can rapidly grow, differentiate, and regenerate and reorganize tissues when cultured in an environment similar to that found in the living body. This has led to a strong demand for the development of equipment that can create a culture environment similar to that found in the living body, and has spurred active research in the new field of mechanobiology. Users who have taken notice of this technology have begun to strongly desire the practical application of automated culture equipment with the ability to simultaneously apply multiple physical and chemical stimuli, just like in the living body, in order to improve cell functionality and production efficiency. However, there are currently no automated culture equipment on the market that can simultaneously apply multiple physical and chemical stimuli like in the living body. As a result, they rely on expensive reagents to improve the speed of growth and differentiation, resulting in skyrocketing running costs.

[0004] Patent No. 5866663 WO 2017-111148

[0005] An object of the present invention is to provide a cell culture device capable of applying one or more physical and / or chemical stimuli to cells being cultured, and to provide cell culture cells using the same.

[0006] As a result of extensive research, the inventors of the present invention discovered that the above-mentioned objectives could be achieved by placing pumps on both sides of the cell culture area, and by using pumps that can move fluid in forward and reverse directions independently of each other and that can vary the pressures at which the fluids are moved independently of each other, thereby completing the present invention.

[0007] That is, the present invention provides the following: (1) A cell culture device comprising a cell culture area, a first pump connected to the cell culture area, and a second pump connected to the cell culture area, wherein the first pump, the cell culture area, and the second pump form a line connected in this order, the first pump and the second pump can move a fluid in the forward direction and the reverse direction, respectively, independently of each other, and the pressures at which the fluids are moved are independently variable. (2) The cell culture device according to (1), further comprising a liquid medium reservoir container incorporated in the line, wherein the liquid medium contained in the liquid medium reservoir container is supplied to the cell culture area by the first pump and / or the second pump. (3) The cell culture device according to (1) or (2), further comprising a cell culture component container arranged upstream of the first pump, wherein one or more cell culture components contained in the cell culture component container are supplied to the liquid culture medium contained in the liquid culture medium reserve container by the first pump and / or second pump. (4) The cell culture device according to (3), wherein the one or more cell culture components are supplied in gas form to the liquid culture medium contained in the liquid culture medium reserve container. (5) The cell culture device according to any of (1) to (4), further comprising a cell container arranged upstream of the cell culture region, wherein cells contained in the cell container are supplied to the cell culture region by the first pump and / or second pump. (6) The cell culture device according to any of (1) to (5), further comprising a cell recovery column incorporated in the line, wherein the first pump and / or second pump detach the cultured cells on the cell culture region and recover the detached cultured cells in the cell recovery column. (7) The cell culture device according to (6), further comprising a cell collection container connected to the cell collection column, wherein the cultured cells separated from the cell collection column are collected into the cell collection container by the first pump and / or the second pump. (8) The cell culture device according to any one of (1) to (7), wherein the cell culture region is coated with a polymer that makes it easy for the cells cultured on the cell culture region to detach from the cell culture region at low temperatures, and the cells are cultured on the polymer.(9) The cell culture device according to any one of (1) to (8), further comprising an observation camera, a pH meter, a dissolved oxygen meter, a flow meter, and a weight sensor incorporated in the line. (10) A cell culture method, comprising culturing cells in the cell culture region of the cell culture device according to any one of (1) to (9). (11) The method according to (10), wherein the cell culture device is the cell culture device according to (9), measuring the pH and dissolved oxygen of the culture medium with the pH meter and the dissolved oxygen meter, and culturing cells while appropriately adjusting the pH and dissolved oxygen concentration of the culture medium. (12) The method according to (10) or (11), wherein the cell culture device is the cell culture device according to (9), culturing cells while measuring the flow rate of the liquid culture medium with the flow meter, and culturing cells while applying shear stress to the cultured cells by appropriately adjusting the pressure of the first pump and the second pump. (13) The method according to any one of (10) to (12), wherein the cell culture device is the cell culture device according to (9), and the observation camera is capable of measuring the differentiation state, cell density, cell proliferation prediction, and the presence and size of bubbles of cells cultured in the cell culture area. (14) The method according to any one of (10) to (13), wherein the cell culture device is the cell culture device according to (9), and the weight sensor measures the weight of a cell culture component container, thereby notifying the timing of replacement of consumables by a warning function.

[0008] 1 is a schematic diagram showing a preferred embodiment of the cell culture device of the present invention. FIG. 2 is a schematic diagram showing an example in which AI and IoT are applied to the culture method of the present invention.

[0009] A preferred embodiment of the present invention will be described below with reference to Fig. 1. In the following description, since the flow of liquid medium originates in the liquid medium reservoir container, the side where the first pump 12 is located in Fig. 1 may be referred to as "upstream" and the side where the second pump 14 is located may be referred to as "downstream."

[0010] The cell culture device shown in Fig. 1 includes a cell culture region 10. A first pump 12 is connected upstream of the cell culture region 10. A second pump 14 is connected downstream of the cell culture region 10. The first pump 12, the cell culture region 10, and the second pump 14 form a line connected in this order. The first pump 12 and the second pump 14 can move fluid in the forward and reverse directions, respectively, independently of each other, and the pressures at which the fluids are moved can be varied independently of each other.

[0011] In the embodiment shown in FIG. 1, a liquid medium reservoir container 16 for storing the medium is installed in the line between the first pump 12 and the cell culture region 10 .

[0012] The specific example shown in FIG. 1 further includes a cell culture component container located upstream of the first pump 12. In the illustrated example, three cell culture component containers 18a, 18b, and 18c are included. The three cell culture component containers 18a, 18b, and 18c contain, for example, a stimulant solution, a buffer solution (PBS), and a liquid medium, respectively. By bubbling gas from a gas mixer 20 into each of the cell culture component containers 18a, 18b, and 18c, one or more cell culture components are supplied in gaseous form to the liquid medium contained in the liquid medium reservoir containers 18a, 18b, and 18c. The three cell culture component containers 18a, 18b, and 18c are preferably stored in a refrigerator 22.

[0013] In the specific example shown in FIG. 1, a cell container 24 is disposed upstream of the cell culture region 10 via a three-way cock 23 and an electromagnetic valve 21 .

[0014] In the embodiment shown in FIG. 1, a cell recovery column 26 is further incorporated in the line between the cell culture region 10 and the second pump 14 .

[0015] 1, a cell collection container 40 is connected upstream of the cell collection column 26 via a three-way cock 36 and a solenoid valve 38. The solenoid valve 38 is connected to the cell culture region 10 via a three-way cock 42 and a solenoid valve 44 downstream of the cell culture region 10.

[0016] 1, two pH meters 28, 29, two dissolved oxygen meters 30, 31, two flow meters 32, 34, and two pressure sensors 46, 48 are incorporated into pressure chambers 47, 50 in the lines. The pressure chambers 47, 50 have the effect of suppressing pulsation of fluids such as liquid culture medium sent from the first pump 12 due to the damping effect caused by gas expansion and compression within the chambers, and by installing dampers in two locations, upstream and downstream, it is possible to suppress pulsation to a greater extent than by installing dampers in one location.

[0017] The cell culture region 10 may be a flat surface covered with a solid medium such as agar or agarose gel, but is preferably one whose surface is coated with a polymer that facilitates detachment from the cell culture region at low temperatures. Such polymers are known and are described, for example, in Patent Document 2. When the polymer described in Patent Document 2 is coated on a flat surface, the polymer becomes hydrophilic at temperatures below 20°C, making it easy for cells to detach naturally, and becomes hydrophobic at temperatures above 25°C, allowing cells to adhere and proliferate.

[0018] A cell culture method using the specific example of the cell culture device shown in Figure 1 will be described below. First, when seeding cells, only the second pump 14 is operated to draw the cells from the cell container 24 into the cell culture region 10. At this time, the solenoid valve 21 is set so that the liquid flows from the cell container 24 to the cell culture region 10.

[0019] During cell culture, the first pump 12 and the second pump 14 are driven sequentially (flowing liquid from upstream to downstream) to send culture medium, stimulants, etc. from the cell culture component containers 18a, 18b, and 18c to the cell culture region 10 via the liquid culture medium reservoir container 16. At this time, the solenoid valve 21 is set so that liquid flows from the liquid culture medium reservoir container 16 to the cell culture region 10. A weight sensor 51 is preferably provided below the cell culture component containers 18a, 18b, and 18c, and by measuring the weight, it is possible to provide a warning function to indicate when it is time to replace consumables. During cell culture, the pH and dissolved oxygen of the culture medium are preferably measured using pH meters 28 and 29 and the dissolved oxygen meters 30 and 31, and the culture medium pH and dissolved oxygen concentration are preferably adjusted as needed during cell culture. At this time, the cells can be cultured while measuring the flow rate of the liquid medium with flow meters 32, 34, and the pressure from first pump 12 and second pump 14 can be appropriately adjusted to apply shear stress to the cultured cells. It is further preferable to provide an observation camera 53 above and / or below the cell culture area to determine the differentiation state of the cultured cells, to predict the cell density and cell proliferation, and to measure the presence and size of air bubbles.

[0020] When recovering cells, for example, cold phosphate buffered saline (PBS) stored in a refrigerator 22 is pumped into the cell culture area 10. At this time, the solenoid valves 21 and 44 are switched so that the flow path connects to the cell collection column 26. As described above, the cell culture area 10 is coated with a polymer that causes cells to detach at temperatures below 20°C, and the detached cells are accumulated in the cell collection column 26. If detachment is difficult, the first pump 12 and the second pump 14 are repeatedly pumped forward and backward to promote cell detachment by the water flow.

[0021] The cells accumulated in the cell collection column 26 are sent to the cell collection container 40 by reversely flowing the cell collection liquid from the second pump 14. At this time, the electromagnetic valve 38 is set so that the liquid flows from the cell collection column 26 to the cell collection container 40.

[0022] According to the cell culture device of the present invention described above, by varying the liquid delivery rates (liquid delivery pressures) of the first pump 12 and the second pump 14, it is possible to apply physical stimuli such as shear stress to the cultured cells. Furthermore, by adjusting the desired gas concentration and bubbling it into the cell culture component containers 18b and 18c, it is possible to add one or more medium components, such as gas stimuli and stimulant drugs, to the culture medium. This allows multiple chemical and / or physical stimuli to be applied to the cultured cells simultaneously, making it possible to more closely resemble the in vivo environment. Furthermore, it is possible to automate the process from cell seeding to cell recovery after culture.

[0023] The method of the present invention described above can be easily optimized by utilizing AI and IoT. More specifically, this is as follows.

[0024] 1. Main functions of AI (1) Data integration and analysis: Integrates data such as pressure, shear stress, pH, oxygen concentration, cell cycle, cell proliferation, and cell differentiation, and analyzes associations and correlations. Statistical methods and machine learning algorithms are used to extract patterns and trends from massive amounts of data. This allows the status of the cell culture process to be monitored and stored in the cloud.

[0025] (2) Pattern prediction and optimization: Learning from accumulated data, the system builds predictive models for cell culture processes, enabling prediction and automatic control to optimize parameters such as pressure, shear stress, chemicals, gas stimulation, pH, oxygen concentration, cell cycle, cell proliferation, and cell differentiation.

[0026] (3) Automatic control and feedback loop: By linking sensor information and control systems, data can be collected and analyzed in real time to automatically control cell culture conditions. Various stimuli, pH, and oxygen concentration can be adjusted and controlled to effectively promote cell cycle, cell proliferation, and cell differentiation.

[0027] (4) Camera-based differentiation status assessment, cell density calculation, cell proliferation prediction, and bubble presence / size measurement functions: Once correlations with pH, ​​gas, and pressure sensors are determined, the correlation coefficients can be used as triggers to further optimize culture conditions.

[0028] 2. Main functions of IoT (1) Real-time monitoring and remote control: Cell culture processes can be monitored in real time and data and control commands can be accessed remotely, allowing for cell culture processes to be monitored from a remote location and controlled or adjusted as needed.

[0029] (2) Data sharing: Users can choose to share the optimal parameters stored in the cloud server with other users, groups, or limited stakeholders around the world.

[0030] (3) Real-time alerts and remote monitoring: The status of cell culture can be monitored remotely, and abnormalities and important events in the cell culture process can be detected from various sensor information. Alerts can be sent in real time to provide early warning of problems, allowing appropriate measures to be taken as necessary.

[0031] (4) Consumables (culture media and reagents) replacement / maintenance warning function: By adding a consumables replacement and warning function using weight sensors for culture media and reagents, the information obtained through IoT will be further enhanced.

[0032] Figure 2 shows a schematic diagram of how the above-mentioned AI and IoT can be utilized.

[0033] 10 Cultivation area 12 First pump 14 Second pump 16 Liquid medium reservoir container 18a, 18b, 18c Cell culture component container 20 Gas mixer 21, 38, 44 Solenoid valve 22 Refrigerator 23, 36, 42 Three-way cock 24 Cell container 26 Cell recovery column 28, 29 pH meter 30, 31 Dissolved oxygen meter 32, 34 Flow meter 46, 48 Pressure sensor 47, 50 Pressure chamber 51 Weight sensor 52 Bubble sensor 53 Observation camera

Claims

1. A cell culture device comprising a cell culture area, a first pump connected to the cell culture area, and a second pump connected to the cell culture area, wherein the first pump, the cell culture area, and the second pump form a line connected in this order, and the first pump and the second pump can move fluid in forward and reverse directions, respectively, independently of each other, and the pressures at which the fluids are moved can be varied independently of each other.

2. The cell culture device according to claim 1, further comprising a liquid culture medium reservoir container incorporated in the line, wherein the liquid culture medium contained in the liquid culture medium reservoir container is supplied to the cell culture area by the first pump and / or the second pump.

3. A cell culture device according to claim 1 or 2, further comprising a cell culture component container arranged upstream of the first pump, wherein one or more cell culture components contained in the cell culture component container are supplied to the liquid culture medium contained in the liquid culture medium reserve container by the first pump and / or the second pump.

4. The cell culture device according to claim 3, wherein the one or more cell culture components are supplied in gaseous form to the liquid culture medium contained in the liquid culture medium reservoir container.

5. The cell culture device according to claim 3, further comprising a cell container arranged upstream of the cell culture region, wherein cells contained in the cell container are supplied to the cell culture region by the first pump and / or the second pump.

6. The cell culture device according to claim 3, further comprising a cell recovery column incorporated in the line, wherein the first pump and / or the second pump detach the cultured cells on the cell culture area and recover the detached cultured cells in the cell recovery column.

7. The cell culture device according to claim 6, further comprising a cell collection container connected to the cell collection column, wherein cultured cells separated from the cell collection column are collected into the cell collection container by the first pump and / or the second pump.

8. The cell culture device according to claim 6, wherein the cell culture area is coated with a polymer that makes it easier for cells cultured on the cell culture area to detach from the cell culture area at low temperatures, and the cells are cultured on the polymer.

9. The cell culture device according to claim 3, further comprising an observation camera, a pH meter, a dissolved oxygen meter, a flow meter and a weight sensor incorporated in said line.

10. A method for culturing cells, comprising culturing cells on the cell culture area of the cell culture device according to claim 1 or 2.

11. The method according to claim 10, wherein the cell culture device is the cell culture device according to claim 9, the pH meter and the dissolved oxygen meter measure the pH and dissolved oxygen concentration of the culture medium, and the cells are cultured while appropriately adjusting the pH and dissolved oxygen concentration of the culture medium.

12. The method according to claim 10, wherein the cell culture device is the cell culture device according to claim 9, and the method comprises culturing cells while measuring the flow rate of the liquid medium with the flow meter, and culturing the cells while applying pressure or shear stress, or both, to the cultured cells by appropriately adjusting the pressures applied by the first pump and the second pump.

13. The method according to claim 10, wherein the cell culture device is the cell culture device according to claim 9, and the observation camera is capable of determining the differentiation state of cells cultured in the cell culture area, measuring cell density, predicting cell proliferation, and measuring the presence and size of bubbles.

14. The method according to claim 10, wherein the cell culture device is the cell culture device according to claim 9, and includes a function of notifying the timing of replacement of consumables by a warning function by measuring the weight of the cell culture component container with the weight sensor.

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