WO2025164695A1 - Cell culture device and cell culture method using same - Google Patents

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

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

Increasing 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 culture equipment used in this field is expected to reduce the number of human operations and operational errors, thereby reducing costs.

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 led to active research in the new field of mechanobiology. Users who have taken an interest in this technology have begun to strongly desire the practical application of a function that can simultaneously apply multiple physical and chemical stimuli to automated culture equipment, 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, which has led to skyrocketing running costs.

Patent No. 5866663 WO 2017-111148

The 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.

After extensive research, the inventors discovered that the above objectives could be achieved by placing pumps on both sides of the cell culture area, and by using pumps that can move fluid in the forward and reverse directions independently of each other and that can vary the pressures independently of each other when moving the fluid, and thus completed the present invention.

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, 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 (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) 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 the second pump.
(4) The cell culture device according to (3), wherein the one or more cell culture components are supplied in the form of a gas to the liquid culture medium contained in the liquid culture medium reservoir container.
(5) A cell culture device according to any one 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 the second pump.
(6) A cell culture device according to any one of (1) to (5), further comprising a cell collection column incorporated in the line, wherein the first pump and/or the second pump detach the cultured cells on the cell culture area, and the detached cultured cells are collected in the cell collection column.
(7) The cell culture device according to (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 any one of (1) to (7), wherein the cell culture area is coated with a polymer that makes it easy 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 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 method for culturing cells, comprising culturing cells on 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), the pH meter and the dissolved oxygen meter are used to 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 (10) or (11), wherein the cell culture device is the cell culture device according to (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 shear stress to the cultured cells by appropriately adjusting the pressures 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 can be used to determine the differentiation state, cell density, and cell proliferation prediction of the cells cultured in the cell culture area, and measure the presence and size of bubbles.
(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 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.

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

A preferred embodiment of the present invention will now be described with reference to Figure 1. In the following description, the flow of liquid culture medium originates in the liquid culture medium reservoir container, and therefore, in Figure 1, the side where the first pump 12 is located will sometimes be referred to as "upstream," and the side where the second pump 14 is located will sometimes be referred to as "downstream."

The cell culture device shown in Figure 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, cell culture region 10, and second pump 14 form a line connected in this order. The first pump 12 and 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.

In the specific example shown in Figure 1, a liquid culture medium reservoir container 16 for storing culture medium is installed in the line between the first pump 12 and the cell culture area 10.

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 culture medium, respectively. By bubbling gas from a gas mixer 20 into each cell culture component container 18a, 18b, and 18c, one or more cell culture components are supplied in gaseous form to the liquid culture medium contained in the liquid culture medium reservoir containers 18a, 18b, and 18c. The three cell culture component containers 18a, 18b, and 18c are preferably stored in a refrigerator 22.

In addition, in the specific example shown in Figure 1, a cell container 24 is placed upstream of the cell culture area 10 via a three-way cock 23 and a solenoid valve 21.

In addition, in the specific example shown in Figure 1, a cell recovery column 26 is further incorporated into the line between the cell culture region 10 and the second pump 14.

In the specific example shown in Figure 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.

Furthermore, in the specific example shown in Figure 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 line. The pressure chambers 47, 50 have a damping effect caused by the expansion and compression of gas within the chamber, which serves to suppress pulsation of fluids such as liquid culture medium pumped from the first pump 12. By installing dampers in two locations, upstream and downstream, it is possible to suppress pulsation to a greater extent than installing them in one location.

The cell culture area 10 may be a flat surface covered with a solid medium such as agar or agarose gel, but it is preferable that the surface of the flat surface is coated with a polymer that facilitates detachment from the cell culture area at low temperatures. Such polymers are well 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 easier for cells to detach naturally, and becomes hydrophobic at temperatures above 25°C, allowing cells to adhere and proliferate.

The following describes a cell culture method using the specific example of the cell culture device shown in Figure 1. First, when seeding cells, only the second pump 14 is operated to draw the cells from the cell container 24 into the cell culture area 10. At this time, the solenoid valve 21 is set so that liquid flows from the cell container 24 to the cell culture area 10.

During cell culture, the first pump 12 and second pump 14 are operated in sequence (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 area 10 via the liquid culture medium reserve container 16. At this time, the solenoid valve 21 is set so that liquid flows from the liquid culture medium reserve container 16 to the cell culture area 10. A weight sensor 51 is provided below the cell culture component containers 18a, 18b, and 18c, and it is preferable that it has a function to measure weight and issue a warning when it is time to replace consumables. During cell culture, it is preferable to measure the pH and dissolved oxygen of the culture medium using pH meters 28 and 29 and the dissolved oxygen meters 30 and 31, and to culture the cells while appropriately adjusting the pH and dissolved oxygen concentration of the culture medium. Furthermore, during this process, cells can be cultured while measuring the flow rate of the liquid medium using flow meters 32, 34, and the pressure from first pump 12 and second pump 14 can be adjusted appropriately to apply shear stress to the cultured cells. It is also preferable to provide an observation camera 53 above and/or below the cell culture area to determine the differentiation state of the cultured cells, predict cell density and cell proliferation, and measure the presence and size of air bubbles.

When recovering cells, for example, cold phosphate buffered saline (PBS) stored in refrigerator 22 is pumped into cell culture area 10. At this time, solenoid valves 21 and 44 are switched so that the flow path connects to cell collection column 26. As described above, 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 cell collection column 26. If detachment is difficult, the first pump 12 and second pump 14 are repeatedly operated in forward and reverse directions to promote cell detachment by the water flow.

The cells accumulated in the cell collection column 26 are sent to the cell collection container 40 by reversely pumping 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.

With the cell culture device of the present invention described above, by varying the liquid delivery volume (liquid delivery pressure) 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 include one or more medium components, such as gas stimuli or stimulant drugs, in 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.

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

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

(2) Pattern prediction and optimization:
It learns from accumulated data and builds predictive models for the cell culture process, 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.

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

(4) Camera-based differentiation status assessment, cell density calculation, cell proliferation prediction, and bubble presence/size measurement functions:
Once the correlation with the pH, gas, and pressure sensors is determined, the correlation coefficient can be used as a trigger to further optimize the culture conditions.

2. Main functions of IoT
(1) Real-time monitoring and remote control:
Real-time monitoring of cell culture processes and remote access to data and control commands allows for cell culture processes to be monitored from a remote location and controlled or adjusted as needed.

(2) Data Sharing:
Users can select optimal parameters that are stored on a cloud server, allowing data to be shared among users, groups, or limited stakeholders around the world.

(3) Real-time alerts and remote monitoring:
The system can monitor the status of cell culture from a remote location, detect abnormalities in the cell culture process and important events from various sensor information, and provide early warning of problems through real-time alert notifications, allowing appropriate measures to be taken as necessary.

(4) Consumables (culture media and reagents) replacement/maintenance warning function:
By adding functions to replace consumables and issue warnings using weight sensors for culture media and reagents, the information obtained through IoT will be further enhanced.

Figure 2 shows a schematic summary of the above-mentioned ways in which AI and IoT can be utilized.

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 (14)

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 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. The cell culture device of claim 1 further includes a liquid culture medium reservoir container incorporated in the line, and 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. The 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. 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. The cell culture device according to claim 3, further comprising a cell container disposed 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. The cell culture device of claim 3, further comprising a cell collection column incorporated in the line, wherein the first pump and/or the second pump detach the cultured cells on the cell culture area and collect the detached cultured cells in the cell collection column. The cell culture device of 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. The cell culture device according to claim 6, wherein the cell culture area is coated with a polymer that allows cells cultured on the cell culture area to easily detach from the cell culture area at low temperatures, and the cells are cultured on the polymer. The cell culture device of claim 3, further comprising an observation camera, a pH meter, a dissolved oxygen meter, a flow meter, and a weight sensor incorporated into the line. A cell culture method comprising culturing cells on the cell culture region of the cell culture device described in claim 1 or 2. The method of claim 10, wherein the cell culture device is the cell culture device of claim 9, the pH meter and the dissolved oxygen meter measure the pH and dissolved oxygen of the culture medium, and the cells are cultured while appropriately adjusting the pH and dissolved oxygen concentration of the culture medium. 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 cells while applying pressure, shear stress, or both to the cultured cells by appropriately adjusting the pressures applied by the first pump and the second pump. The method of claim 10, wherein the cell culture device is the cell culture device of 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 air bubbles. The method of claim 10, wherein the cell culture device is the cell culture device of claim 9 and includes a function for notifying the user of the timing for replacing consumables by using a warning function by measuring the weight of the cell culture component container with the weight sensor.