Automated modular cell culture apparatus having perfusion-based culture medium transfer function

Abstract

Disclosed is a cell culture apparatus comprising: a cell culture module including a cell culture unit in which a well plate forming at least one culture space is mounted on a cartridge; a culture medium supply module including a pump unit for forming a supply and discharge flow of a culture medium to the cell culture unit, and a culture medium storage compartment accommodating at least one culture medium chamber; and a pair of culture medium tubes that are made of a flexible material and connect the culture medium supply module and the cell culture module, which are spatially separated from each other, to each other to form a conduit for the supply and discharge flow of the culture medium, wherein the cell culture module is mounted in a CO2 incubator that creates a thermal environment, the culture medium storage compartment creates a low-temperature environment less than the room temperature, and the pair of culture medium tubes exposed between the culture medium supply module and the cell culture module are in a room-temperature environment.

Description

Automated modular cell culture device with perfusion-based culture medium transfer function

[0001] The present invention relates to an automated cell culture device, wherein a cell culture module and a culture medium supply module are connected by a tubing network so that a culture medium is automatically supplied and discharged, the cell culture module can be miniaturized to the extent that only the cell culture module can be placed in a commercial CO2 incubator, and a modular cell culture device capable of precisely supplying a very small amount of culture agent without deterioration during the transfer process through perfusion-based culture medium transfer.

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2022-0042443, filed April 5, 2024, and Korean Patent Application No. 10-2022-0042443, filed April 5, 2024, the entire contents of which are incorporated herein by reference.

[0003] Cell culture is an essential technology for studying and applying cell functions, characteristics, diseases, and treatments in various fields. However, cell culture generally requires significant time and effort, and the process itself is particularly prone to risk factors such as contamination and mutation, requiring a highly sophisticated culture environment. Furthermore, the quality and reproducibility of cell cultures depend heavily on the skill and manipulation techniques of the operator, making it difficult to ensure the reliability and consistency of experimental results. Against this backdrop, the need for automated cell culture devices has grown significantly, and a significant number of companies are investing human and material resources in the development of technologies to automate the cell culture process.

[0004] The present invention was also conceived to address these issues, and its primary feature is the automation of an environment capable of automatically replacing the culture medium necessary for cell growth in a cell culture space. Furthermore, in addition to the aforementioned technical concepts, the present invention seeks to provide additional technical elements that would not be readily apparent to those skilled in the art.

[0005] For long-term cell culture processes, the cell culture module must be installed in a space with a thermal environment suitable for cell growth (usually around 37°C), and the culture medium supply module must be installed in a space with a low-temperature environment (usually 4-8°C) to prevent the stored culture medium from deteriorating.

[0006] When considering a means to maintain two spaces at different temperatures, secondary problems such as energy waste and moisture condensation due to heat transfer between the two spaces may occur. Therefore, the present invention has a main purpose of physically separating a heated space for cell culture and a low-temperature space for storing culture medium, and implementing perfusion-based culture medium transport using a thin and long culture medium tube and a gas-liquid interface transport mechanism, thereby enabling an accurate amount of culture medium to be transported between a cell culture module and a culture medium supply module without waste or deterioration.

[0007] In addition, the most important issue in the cell culture process is the cross-contamination problem where contaminants are introduced from the outside. Therefore, the cell culture device must be able to block the possibility of contamination at the source during the process of using it, and additionally, in order to improve productivity and process efficiency, all surfaces that can come into contact with the culture medium and cells must be implemented to be detachable from the equipment so that a pre-washed and sterilized unit can be used during the culture work. Another object of the present invention is to omit the washing and sterilization process during the culture process by focusing on this problem. For reference, examples of surfaces that can come into contact with the culture medium and cells may include the wall surface of the culture space where the cells are cultured, the wall surface of the storage space where the culture medium is stored, the wall surface of the pipe through which the culture medium moves, and the pump and valve that generate the flow.

[0008] In addition, the present invention aims to provide an automated cell culture device in which a cell culture module and a culture solution supply module are connected by a tubing network so that a culture solution is automatically supplied and discharged, and the cell culture module alone can be miniaturized to the extent that it can be placed in a commercial CO2 incubator.

[0009] In addition, the present invention aims to solve the problem of culture medium waste and deterioration by implementing perfusion-based culture medium transport using a thin and long culture medium tube and a gas-liquid interface transport mechanism, and to enable an accurate amount of culture medium to be supplied without waste or deterioration.

[0010] In addition, the present invention has another purpose of providing an automated cell culture device that can eliminate the possibility of contamination and omit the washing and sterilization process by implementing a practical disposable pump unit, thereby improving productivity and process efficiency and simplifying the verification process.

[0011] The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0012] The cell culture device provided by the present invention, in one embodiment, comprises a cell culture module including a cell culture unit in which a well plate forming at least one culture space is mounted on a cartridge, a culture medium supply module including a pump unit forming a flow of supply and discharge of a culture medium to the cell culture unit, and a culture medium storage room accommodating at least one culture medium chamber, and a pair of culture medium tubes made of a flexible material connecting the culture medium supply module and the cell culture module, which are spatially separated, to form a conduit for the flow of supply and discharge of the culture medium, wherein the cell culture module is placed in a CO2 incubator that creates a warm environment, the culture medium storage room creates a low-temperature environment below room temperature, and the pair of culture medium tubes exposed between the culture medium supply module and the cell culture module are in a room temperature environment.

[0013] A cell culture module includes a valve module for controlling the supply and discharge of a culture medium for each culture space of a well plate, and a rotatable lid having a plurality of culture medium channels corresponding to each culture space, wherein a pair of culture medium tubes extending through the valve module are coupled thereto, and the rotatable lid and the culture medium channels may be arranged at the top of the well plate so that a culture medium flowing from the culture medium channels may fall from the air toward the bottom surface. In a specific embodiment, the plurality of culture medium channels may be composed of one or more supply channels and one or more discharge channels, wherein the supply channel may extend from the lower surface of the lid to the middle height of the culture space, and the discharge channel may extend from the lower surface of the lid to the bottom height of the culture space.

[0014] The valve module includes a plurality of valve units that control the opening and closing of a pair of culture solution tubes allocated to each culture space, and the valve unit includes a movable clamp having a tube slot through which the culture solution tube passes, and the tube slot can close the culture solution tube by squeezing it by linear movement of the movable clamp.

[0015] An optical microscope system may be additionally installed at the bottom of the cell culture module to monitor the condition of the cells. The optical microscope system includes a lens and a camera for magnifying and capturing images, and the lens and camera can move vertically to focus. The optical microscope system may include a motorized stage for moving the lens and camera.

[0016] Additionally, a light source of an optical microscope system may be placed on the top of the cell culture module, and the light source may be a white LED, or a red or yellow LED to minimize the impact on the cells.

[0017] In order to observe the entire culture space, the lens, camera, and light source of the microscope system can move horizontally, and in another embodiment, a motorized stage that enables horizontal movement is attached to the top of the microscope system, so that the lens and camera can move only vertically, and the cell culture module installed on the top of the microscope system can move horizontally.

[0018] The above pair of culture medium tubes is a supply tube and a discharge tube, and the pump unit sequentially pressurizes and supplies gas after injecting a predetermined amount of culture medium into the supply tube to move the culture medium to the culture space, and the inner diameter of the supply tube must have a diameter that prevents the culture medium from retreating or advancing due to gravity.

[0019] The above pump unit can apply negative pressure to one end of the discharge tube to suck the culture medium from the culture space and recover it into a recovery chamber contained in the culture medium storage room.

[0020] In addition, the cell culture device provided by the present invention may include a plurality of cell culture modules connected in parallel to one culture medium supply module, and by closing all valve modules provided in each of the plurality of cell culture modules and opening only the culture medium channel corresponding to the target culture space, a one-to-one correspondence of the culture medium supply module to the cell culture module serving as the transfer target can be achieved while the flow of the culture medium being supplied and / or withdrawn occurs.

[0021] Meanwhile, the present invention provides a culture solution supply module capable of supplying a culture solution to at least one culture space that is spatially separated and recovering the culture solution from the culture space, wherein the culture solution supply module includes a pump unit that forms a supply and discharge flow of the culture solution to the cell culture unit, and a culture solution storage room that accommodates a recovery chamber and at least one culture solution chamber, wherein the culture solution storage room creates a low-temperature environment below room temperature, and the pump unit can supply the culture solution stored in the culture solution chamber to each culture space through a supply tube and also discharge the culture solution to the recovery chamber through a discharge tube.

[0022] In one embodiment, the pump unit comprises a plurality of syringe pumps, wherein the plurality of syringe pumps may include a gas syringe, at least one culture solution syringe, and a recovery syringe.

[0023] The above gas syringe and at least one culture solution syringe are connected to the middle of the tube through a T- or Y-shaped branch connector, and a check valve is installed upstream and downstream of the branch connector to which the syringe is connected, so as to limit the direction of the flow generated by the operation of the syringe to one direction. The syringe for supplying the culture solution is connected to the culture solution chamber at the upstream side and to the culture solution supply tube at the downstream side, so as to allow only one-way flow from the culture solution chamber toward the culture space. The assembly including the culture solution syringe and the check valve can be connected in parallel to the supply tube.

[0024] The above gas syringe is connected in parallel with the culture solution syringe and is placed at the uppermost point of the supply tube, and like the culture solution syringe, is connected to the supply tube through a branch connector, and a check valve is connected upstream and downstream of the branch connector so that the flow of gas generated by the operation of the gas syringe can be restricted in one direction toward the culture space.

[0025] The above gas syringe may be equipped with an air filter upstream of the upstream check valve.

[0026] A predetermined amount of culture medium is introduced into the supply tube by the suction and discharge strokes of the culture medium syringe, and the gas introduced into the supply tube by the suction and discharge strokes of the gas syringe can transport the culture medium introduced into the supply tube to the culture space.

[0027] According to an embodiment, the plurality of syringe pumps include a gas syringe and a plurality of culture solution syringes, and the culture solution transfer between one of the culture solution syringes and the gas syringe is performed with a time difference, so that the plurality of culture solutions to be transferred can be transferred to the culture space in a state where they are isolated by gas.

[0028] The above recovery syringe is connected to the most downstream of the discharge tube, and check valves are installed upstream and downstream of the confluence of the recovery syringe with the discharge tube to allow only one-way flow toward the recovery chamber.

[0029] The above pump unit may be provided with a plurality of pump actuators that linearly move the plunger of each syringe pump to perform suction and discharge strokes, and since the direction of flow is limited by a check valve linked to the syringe, a volume of liquid exceeding the capacity of the syringe can be transported by repeatedly moving the plunger of the syringe pump.

[0030] Meanwhile, the present invention provides a culture medium supply module capable of supplying a culture medium to at least one culture space that is spatially separated, wherein the culture medium supply module includes a pump unit having a plurality of syringe pumps that form a supply flow of a culture medium to the cell culture section, and a culture medium storage room that accommodates at least one culture medium chamber, wherein the plurality of syringe pumps include a gas syringe connected to the most upstream of the supply tube, and at least one culture medium syringe connected in parallel downstream of the gas syringe, and check valves are respectively installed upstream and downstream of the confluence of the gas syringe and each culture medium syringe with respect to the supply tube, so that only one-way flow toward the culture space is permitted, and a predetermined amount of culture medium is introduced into the supply tube by a suction and discharge stroke of the culture medium syringe, and the gas introduced into the supply tube by a suction and discharge stroke of the gas syringe can transport the culture medium introduced into the supply tube to the culture space.

[0031] The above plurality of syringe pumps include a gas syringe and a plurality of culture solution syringes, and the culture solution transfer between one of the culture solution syringes and the gas syringe is performed with a time difference, so that the plurality of culture solutions being transferred can be transferred to the culture space in a state where they are isolated by gas.

[0032] In addition, the culture solution supply module may include a function capable of cooling the culture solution storage room to prevent deterioration of the culture solution, and may cool the entire tube assembly including the syringe and check valve to prevent deterioration of the culture solution already introduced into the supply tube by the syringe. In another embodiment, the culture solution supply module may be manufactured in a compact size and installed inside a commercial refrigerator or refrigeration facility to maintain the entire culture solution supply module at a low temperature.

[0033] In addition, since the above-mentioned culture medium supply module applies a gas-liquid interface transport mechanism to transport the culture medium, even if the flow rate of the fluid flowing inside the tube is not directly measured, by using a tube made of a transparent or translucent material, it is possible to determine whether the equipment is operating normally by determining whether the tube is installed and whether gas or liquid is contained inside the tube using an infrared LED, and when a command to supply or discharge the culture medium is input, it is possible to determine whether the state inside the supply tube and discharge tube changes from gas-liquid-gas, thereby confirming whether the culture medium is actually transported normally to the culture medium supply tube and discharge tube.

[0034] According to the cell culture device of the present invention having the above configuration, the cell culture module and the culture solution supply module are connected by a tubing network so that the culture solution is automatically supplied and discharged, and the cell culture module can be miniaturized to the extent that it can be placed in a commercial CO2 incubator, so that the convenience of operating the automated cell culture device is excellent.

[0035] In addition, the present invention implements perfusion-based culture medium transfer using a thin and long culture medium tube and a gas-liquid interface transfer mechanism, thereby resolving the problems of culture medium waste and deterioration, and enabling the supply of an accurate amount of culture medium without waste or deterioration.

[0036] In addition, the present invention can provide an automated cell culture device that can prevent contamination at the source by implementing a practical disposable pump unit and omit washing and sterilization processes, thereby improving productivity and process efficiency and simplifying the verification process.

[0037] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0038] FIG. 1 is a drawing showing the overall configuration of a modular cell culture device according to one embodiment of the present invention.

[0039] Figure 2 is a drawing illustrating a cell culture module.

[0040] Figure 3 is a drawing showing the configuration of a cartridge.

[0041] Figure 4 is a drawing showing the structure and operating principle of the valve unit.

[0042] Figure 5 is an enlarged view of the well plate and lid mounted on the cartridge.

[0043] Figure 6 is a cross-sectional view showing one culture space.

[0044] Figure 7 is a drawing illustrating a culture solution supply module.

[0045] Figure 8 is a drawing for explaining a culture medium transport mechanism using a gas-liquid interface.

[0046] Figure 9 is a drawing for explaining the concept of a culture medium transfer mechanism using multiple syringe pumps.

[0047] Figure 10 is a diagram illustrating the concept of a syringe pump-based tubing network for quantitatively transporting and recovering various liquids to a culture space.

[0048] Figure 11 is a drawing illustrating the priming process performed in a culture syringe.

[0049] Fig. 12 is a drawing showing one embodiment of a pump unit.

[0050] Figure 13 is a drawing showing one embodiment of an automated pump unit.

[0051] Figure 14 is a drawing showing the inside of the culture solution supply module.

[0052] Figure 15 is a drawing showing the culture medium temperature environment in a modular cell culture device according to the present invention.

[0053] Figure 16 is a diagram illustrating a configuration in which multiple cell culture modules are connected to one culture solution supply module.

[0054]

[0055] [Explanation of symbols]

[0056] 10: Cell culture device 100: Cell culture module

[0057] 110: Cell culture unit 120: Cartridge

[0058] 122: Supply tube connection 124: Discharge tube connection

[0059] 130: Valve module 132: Valve unit

[0060] 132-1: Supply valve unit 132-2: Discharge valve unit

[0061] 134: Movable clamp 136; Tube slot

[0062] 137: Spring 138: Clamp actuator

[0063] 140: Well plate 142: Culture space

[0064] 144: Insert 146: Membrane

[0065] 150: Lead 152: Culture channel

[0066] 154: Supply channel 156: Discharge channel

[0067] 160: Optical monitoring unit 162: Optical microscope system

[0068] 200: Culture solution supply module 210: Culture solution storage room

[0069] 212: Cooling unit 220: Culture chamber

[0070] 222: Recovery chamber 230: Pump unit

[0071] 232: Syringe pump 234: Check valve

[0072] 236: Branch connector 238: Air filter

[0073] 240: Plunger 242: Pump actuator

[0074] 250: Gas syringe 252: Culture solution syringe

[0075] 254: Recovery syringe 260: Culture tube

[0076] 260-M: Main tube 260-B: Branch tube

[0077] 262: Supply tube 264: Discharge tube

[0078] 300: Incubator

[0079] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. The advantages and features of the present invention, and methods for achieving them, will become clear with reference to the embodiments described in detail below together with the attached drawings. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

[0080] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, singular forms also include plural forms, unless specifically stated otherwise.

[0081] The terms "comprises" and / or "comprising" as used in the specification do not exclude the presence or addition of one or more other components, steps, operations and / or elements.

[0082] Also, for reference, it is noted that this invention was produced with the support of the research project below.

[0083] Assignment ID: 1415187257 (20023762)

[0084] Ministry Name: Ministry of Trade, Industry and Energy

[0085] Name of specialized organization: Korea Institute of Industrial Technology Planning and Evaluation

[0086] Research Project Name: Machinery and Equipment Industry Technology Development - Manufacturing-Based Production System

[0087] Research Project Title: Development of a 3D bioreactor automated culture system for in situ uniform cell aggregation, proliferation, and differentiation using a material-permeable thin-film scaffold with a three-dimensional roughened structure and a permeable microperfusion-based system.

[0088] Contribution rate: 100

[0089] Project execution organization name: Celloid Co., Ltd.

[0090] Research period: April 1, 2023 - December 31, 2026

[0091]

[0092] FIG. 1 is a drawing illustrating the overall configuration of a modular cell culture device (10) (hereinafter, simply referred to as “cell culture device”) according to one embodiment of the present invention. The cell culture device (10) according to the present invention includes a cell culture module (100) and a culture solution supply module (200). The cell culture module (100) and the culture solution supply module (200) are connected by a pair of tubes so that culture solution can be supplied and discharged to the cell culture module (100). Culture solution is supplied through one tube and recovered through the other tube. By this tubing network, perfusion-based culture solution transport is achieved.

[0093] The tubing network configured for perfusion-based culture medium transfer is broadly divided into a supply tube (262) through which the culture medium is supplied to the culture space (142), and a discharge tube (264) through which the culture medium is recovered from the culture space (142). That is, the culture medium tube (260) is composed of a pair of a supply tube (262) and a discharge tube (264). According to the tubing network, the culture medium stored in the culture medium chamber (220) is supplied to the culture space (142), discharged from the culture space (142), and returned to the recovery chamber (222), thereby forming a one-way flow of the culture medium. Based on the module of the cell culture device (10), the culture medium is supplied from the culture medium supply module (200) to the cell culture module (100), and the culture medium supplied to the cell culture module (100) is recovered back to the culture medium supply module (200). The culture tube (260) may not be formed as a single tube, such as by branching out from time to time or extending through fittings, but is clearly distinguished as a supply tube (262) and a discharge tube (264) in terms of the overall flow. For a clear understanding of the invention, unless it is necessary to explain it in detail, the culture tube (260) will be collectively referred to as a supply tube (262) and a discharge tube (264).

[0094] Fig. 2 is a drawing illustrating a cell culture module (100). The cell culture module (100) includes a cell culture unit (110) and an optical monitoring unit (160) arranged below the cell culture unit (110). The cell culture unit (110) includes a well plate (140) made of a light-transmitting material forming a culture space (142), and the optical monitoring unit (160) includes an optical microscope system (162) that acquires images of cells cultured in the well plate (140). Through the images acquired by the optical microscope system (162), cell culture can be remotely observed and management such as adjusting the culture environment can be easily performed.

[0095] In the embodiment of FIG. 2, the well plate (140) is mounted on a cartridge (120) provided in the cell culture unit (110). To facilitate various tasks such as replacing the well plate (140) and installing the culture medium tube (260), the cartridge (120) may be configured to be drawer-type and withdrawable. The cartridge (120) is equipped with a replaceable well plate (140), and is provided with a valve module (130) for controlling the input and output of the culture medium, and a lid (150) having a hinged structure for sealing the well plate (140).

[0096] Fig. 3 is a drawing illustrating the configuration of a cartridge (120). Referring to Fig. 3, a valve module (130) is fixedly installed in the cartridge (120). The valve module (130) controls the flow of the culture medium supplied to the culture space (142) provided in the well plate (140) and the flow of the culture medium discharged from the culture space (142). If a plurality of culture spaces (142) are provided in the well plate (140), the valve module (130) is provided with a corresponding number of valve units (132). In the illustrated embodiment, six culture spaces (142) are formed in the well plate (140), and correspondingly, the valve module (130) is provided with seven supply valve units (132-1) and seven discharge valve units (132-2). As will be described later, the cell culture device (10) of the present invention employs a culture medium transport mechanism utilizing an air-liquid interface. Accordingly, the seven valve units (132) are composed of six valve units (132) for controlling the flow of culture medium and one valve unit (132) for controlling the flow of gas.

[0097] A supply tube connection (122) and a discharge tube connection (124) are provided on one side of the front of the cartridge (120). A tubing network is connected between the cell culture module (100) and the culture medium supply module (200) through the supply tube connection (122) and the discharge tube connection (124). A valve module (130) is arranged between the supply tube / discharge tube connection (122, 124) and the well plate (140). A valve unit (132) is assigned to each culture space (142), through which the culture medium is supplied and discharged independently to each culture space (142).

[0098] FIG. 4 is a drawing illustrating the structure and operating principle of the valve unit (132). As illustrated in (a) of FIG. 4, the valve unit (132) is provided with a movable clamp (134) that can be elastically moved up and down linearly by a spring (137). A tube slot (136) having a through-hole shape is provided at the distal end of the movable clamp (134), and the culture medium tube (260) extends toward the well plate (140) after passing through the tube slot (136). Referring to (b) of FIG. 4, in a neutral state where no external force is applied to the movable clamp (134), the movable clamp (134) pulls and compresses the culture medium tube (260) upward by the elastic force of the spring (137). That is, the culture medium tube (260) is occluded, thereby prohibiting the flow of the culture medium.

[0099] Above the valve module (130), one clamp actuator (138) is arranged to correspond to each valve unit (132). In the illustrated embodiment, the valve module (130) includes a total of 14 (2×7) valve units (132), and correspondingly, a total of 14 clamp actuators (138) are provided. The clamp actuators (138) are fixedly installed on the main body of the cell culture module (100). Each clamp actuator (138) moves linearly up and down, and when moving downward, it compresses a spring (137) and presses a movable clamp (134). As shown in (b) of Fig. 4, the culture medium tube (260) that was compressed in the tube slot (136) by the downward movement of the movable clamp (134) returns to a free state, and the culture medium tube (260) is restored by its own elasticity and becomes an open state in which the flow of the culture medium is possible.

[0100] The cartridge (120) is drawn out in a drawer-like manner from the main body of the cell culture module (100). If any of the clamp actuators (138) is lowered while the cartridge (120) is removed, interference occurs when attempting to store the cartridge (120). To prevent malfunction of the clamp actuator (138), a home sensor (not shown) may be installed. This problem can be prevented by allowing the downward movement of the clamp actuator (138) only when the home sensor confirms that the cartridge (120) has been stored.

[0101] Figure 5 is an enlarged view of a well plate (140) and a lid (150) mounted on a cartridge (120). In the illustrated embodiment, one well plate (140) may have six independent culture spaces (142) arranged in a 2×3 arrangement. This is merely an example, and the number, shape, size, arrangement, etc. of the culture spaces (142) may be varied.

[0102] And, a lid (150) having a hinged structure that seals the well plate (140) is provided with a plurality of culture medium channels (152), one pair of which is allocated to each culture space (142). Each pair of culture medium channels (152) is divided into a supply channel (154) and a discharge channel (156). Fig. 6 is a cross-sectional view illustrating one culture space (142). An insert (144) is inserted into the culture space (142), and the bottom surface of the insert (144) is composed of a membrane (146). The membrane (146) ensures smooth movement and diffusion of substances and provides an environment in which cells can grow stably. Since cells cultured on the membrane (146) cannot pass through the membrane (146), and the culture medium can pass through the membrane (146) and move to the discharge channel (156), cell loss during the culture medium replacement process can be fundamentally prevented. Unlike the embodiment of the culture space (142) exemplarily illustrated in Fig. 6, cell culture can be performed in the same manner even when cells are attached to the bottom using a hydrogel or the like without using a membrane (146).

[0103] Referring to Fig. 6, the supply channel (154) extends downward along the inner surface of the insert (144) to the middle depth, and the discharge channel (156) extends downward along the outer surface of the insert (144) to the bottom depth. That is, the extension length of the discharge channel (156) is longer than that of the supply channel (154). Since the supply channel (154) is adjacent to the inner surface of the insert (144), the culture medium is slowly supplied by dropping or dripping, thereby preventing the culture medium from scattering in all directions or causing excessive flow rate to affect the growing cells. The discharge channel (156) extends long enough to be adjacent to the bottom of the culture space (142) outside the insert (144) where cells are growing, thereby allowing the culture medium to be sucked and discharged without leaving any residue.

[0104] Meanwhile, looking at FIG. 5, the culture medium tube (260) extended from the valve module (130) is connected to the culture medium channel (152) provided in the lid (150), and has a connection structure that sequentially bends upward, forward, and downward in three places. The culture medium tube (260) is a soft tube made of a polymer compound such as polyethylene (PE), polypropylene (PP), polystyrene (PS), or silicone. Since the culture medium tube (260) is made of such a flexible material, in the valve module (130), the opening and closing of the flow path is performed through compression and restoration by the tube slot (136), and in the lid (150) of the opening / closing structure, a force is applied in the direction of closing the lid (150) by bending the culture medium tube (260) in three places. In this way, by applying force in the direction in which the flexible culture medium tube (260) always closes the lid (150), the blocking of contaminants into the culture space (142) is effectively suppressed.

[0105] Figure 7 is a drawing illustrating a culture solution supply module (200). The culture solution supply module (200) includes a culture solution storage room (210) at the bottom and a pump unit (230) positioned above the culture solution storage room (210). The pump unit (230) can be coupled to the culture solution storage room (210) with a hinged structure that rotates up and down, and the culture solution storage room (210) can be accessed through the rotation opening of the pump unit (230) to perform tasks such as replenishment and replacement of the culture solution chamber (220).

[0106] The pump unit (230) serves to generate a flow that supplies the culture medium stored in at least one culture medium chamber (220) accommodated in the culture medium storage room (210) to the cell culture module (100) and a flow that recovers the culture medium from the culture space (142) of the cell culture module (100). In particular, in the present invention, the pump unit (230) adopts a culture medium transport mechanism using an air-liquid interface. FIG. 8 is a drawing for explaining a culture medium transport mechanism using an air-liquid interface.

[0107] Figure 8 (a) illustrates a conventional culture medium transfer mechanism, which transfers the culture medium by pressure through a tube to the target area. Since the culture medium itself is transferred by pressure, the longer the tube, the greater the consumption of the culture medium. Furthermore, even after a single transfer is complete, the culture medium remains in the tube, increasing the likelihood of problems with the culture medium quality due to temperature (approximately 15-25°C based on room temperature). To address this problem of culture medium quality degradation, refrigeration of the entire tube may be required.

[0108] Figure 8 (b) illustrates the concept of a culture medium transport mechanism utilizing a gas-liquid interface adopted by the present invention. The culture medium transport mechanism utilizing a gas-liquid interface sequentially injects culture medium and gas, such as air, and allows the culture medium to reach the target area by the force of pressurizing the air. The sequential injection of culture medium and gas can be repeated multiple times, and the culture medium with a higher priority can be sequentially reached to the target area by the gas injected later in time. Since the culture medium is transported by gas pressure, when a single transport is completed, only the gas remains in the tube, leaving no culture medium behind. Therefore, even in a room temperature environment, there is little concern about the quality of the culture medium deteriorating due to temperature influence.

[0109] The effective diameter of the culture tube (260) affects the maximum and minimum volumes of the culture medium that can be transferred at one time. Specifically, in the process of transferring the culture medium, if the culture medium does not fill the cross-section of the culture tube (260), the gas (air) can no longer push the culture medium and can simply pass through the culture tube (260). Therefore, the culture medium to be transferred must form a liquid column within the culture tube (260), and the length of the liquid column needs to be at least 1.5 times the inner diameter of the culture tube (260). For example, the volume of the culture medium that can be transferred by the method according to the present invention in a circular pipe having an inner diameter of D is at least It needs to be ideal.

[0110] Likewise, the maximum volume of the culture medium that can be transported by the method according to the present invention is also determined by the inner diameter of the culture medium tube (260). In order to prevent the culture medium from retreating due to gravity when the culture medium tube (260) is placed vertically, the surface tension between the culture medium and the culture medium tube (260) must be in balance with the gravity acting on the culture medium, and the inner diameter of the culture medium tube (260) that satisfies this condition is determined by the density and surface tension of the culture medium, and the contact angle, which is a surface characteristic of the culture medium tube (260). In addition, in the case of a culture medium tube (260) with an excessively small inner diameter, the pressure drop phenomenon due to the resistance generated on the inner wall of the culture medium tube (260) when the culture medium passes through the inside becomes excessively large, making it difficult to smoothly supply the culture medium. Therefore, it is necessary to limit the minimum inner diameter in consideration of this.

[0111] The range of values ​​of the inner diameter of the culture solution tube (260) will be examined through several formulas below.

[0112] When the culture tube (260) is placed vertically, in order to prevent the liquid from sinking downward due to gravity, the surface tension between the liquid and the culture tube (260) must be in balance with the gravity acting on the liquid. Therefore, the following inequality holds for the length of the liquid (liquid section, liquid column) that can be moved within the culture tube (260).

[0113] [Formula 1]

[0114]

[0115] In the above inequality, the density (ρ) and surface tension (Τ) of the liquid can be determined depending on the type of liquid being moved, and the contact angle (θ) can be determined by the interaction between the liquid and the inner wall of the culture tube (260). Consequently, the condition for the maximum movable liquid length can be applied together with the condition for the minimum moving flow rate of the present invention mentioned above, and can be organized into the following inequality.

[0116] [Formula 2]

[0117]

[0118] At this time, the minimum length of the movable liquid is proportional to the inner diameter of the culture tube (260), and the maximum length is inversely proportional to the inner diameter. Therefore, in order to utilize the present invention as a means for moving various amounts of liquid, it is advantageous to utilize the above conditions to expand the range of the allowable movement amount. Accordingly, in the present invention, the inner diameter of the culture tube (260) was designed to be sufficiently small, and in particular, in order for the above inequality to be established and the technical idea proposed in the present invention to be implemented, at least 1.5D, which is the minimum condition for the length of the moving liquid, is the maximum condition. It is desirable to have a smaller value. According to this condition, in order for the present invention to be implemented, the diameter of the pipe must meet the conditions disclosed in [Formula 3] below.

[0119] [Formula 3]

[0120]

[0121] As a result of various considerations including this method, it was determined that it is desirable for the culture solution tube (260) to have a length of 15 cm or more for long-distance transport, and for the inner diameter of the culture solution tube (260) to be 10 μm or more and 5 mm or less for smooth liquid transport without loss.

[0122] In one embodiment of the present invention, the pump unit (230) provided in the culture solution supply module (200) of FIG. 7 includes a plurality of syringe pumps (232). A tubing network is formed by connecting culture solution tubes (260) to the plurality of syringe pumps (232) through a plurality of check valves (234) and a plurality of branch connectors (236). In the present invention, all of the syringe pumps (232), check valves (234), branch connectors (236), and culture solution tubes (260) are configured to be capable of applying commercially available plastic ready-made products, thereby enabling the implementation of a practical disposable pump unit (230).

[0123] Industries requiring stringent hygiene and quality control standards, such as the biotechnology, pharmaceutical, and food and beverage industries, utilize a variety of pump technologies (piston pumps, diaphragm pumps, peristaltic pumps, etc.) to accurately transport liquid samples or solutions. However, these reusable pumps must be cleaned, sterilized, and validated after use, which complicates the production process, increases costs, and leads to cross-contamination issues due to insufficient cleaning.

[0124] Single-use pump technology emerged to address these issues. Single-use pumps provide the pump's core components, including the fluid contact parts, in a single-use configuration. By using a new pump head for each batch, the possibility of contamination is eliminated and cleaning and sterilization processes are eliminated. This simplifies the production process, improves productivity and process efficiency, and streamlines the validation process.

[0125] However, currently commercialized disposable pump solutions often focus primarily on maintaining a constant flow rate and preventing contamination. This often fails to meet the requirements for precise quantitative transfer. In other words, existing disposable pump technologies focus on flow stability and basic pumping functions, while lacking accurate volume measurement and quantitative control capabilities. The present invention overcomes these limitations of existing disposable pumps and implements a pump unit (230) capable of preventing contamination and achieving high quantitative accuracy.

[0126] Fig. 9 is a drawing for explaining the concept of a culture medium transport mechanism by a plurality of syringe pumps (232). Fig. 9 is the most basic configuration, and includes one gas syringe (250) and one culture medium syringe (252). The arrow indicates the direction of supply of the culture medium, and the gas syringe (250) is arranged at the uppermost point based on the flow of the culture medium, and the culture medium syringe (252) is arranged at the downstream point. In the description of Fig. 9, if the culture medium tube (260) is divided into a main tube (260-M) and a branch tube (260-B), the main tube (260-M) is connected to the supply tube connection (122) of the cell culture module (100), and the branch tube (260-B) joins the gas syringe (250) and the culture medium syringe (252) to the main tube (260-M) via a branch connector (236), respectively. In addition, a check valve (234) is connected to the upstream and downstream sides of the branch connector (236). By the check valve (234), the gas (air) and the culture medium can only flow in one direction indicated by the arrow, and flow in the reverse direction is prohibited.

[0127] The upstream side of the gas syringe (250) is open to the atmosphere, and an air filter (236) for filtering out foreign substances may be provided at the inlet of the check valve (234). The upstream side of the culture solution syringe (252) is in communication with the culture solution chamber (220), and an air filter (236) may also be provided in a vent hole formed open to allow atmospheric pressure to act inside the culture solution chamber (220).

[0128] Figure 9 (a) illustrates a process of aspirating the culture solution stored in the culture solution chamber (220) with a culture solution syringe (252). By the aspiration stroke of the culture solution syringe (252), the culture solution stored in the culture solution chamber (220) passes through the upstream check valve (234) and is filled into the branch tube (260-B) and the culture solution syringe (252). However, by the downstream check valve (234), the gas in the main tube (260-M) is not aspirated into the culture solution syringe (252).

[0129] Figure 9 (b) illustrates a process in which the culture solution filled in the branch tube (260-B) and the culture solution syringe (252) is quantitatively discharged into the main tube (260-M) by the discharge stroke of the culture solution syringe (252). In contrast to the case of Figure 9 (a), the culture solution is not discharged toward the culture solution chamber (220) by the operation of the check valve (234), and thus a predetermined amount of culture solution corresponding to the length of the discharge stroke of the culture solution syringe (252) is discharged into the main tube (260-M).

[0130] Figure 9 (c) illustrates a process in which gas is introduced into the main tube (260-M) by the suction and discharge strokes of the gas syringe (250), and the culture medium inside the main tube (260-M) is transferred in the direction of the arrow by the pressurized gas. The process in which gas is introduced into the main tube (260-M) by the suction and discharge strokes of the gas syringe (250) is substantially the same as the processes of Figures 9 (a) and (b). The culture medium can be transferred to a distance as much as the volume of gas sucked and discharged by the gas syringe (250) fills the main tube (260-M).

[0131] Fig. 10 illustrates a concept of a tubing network based on a syringe pump (232) that quantitatively transfers various liquids (such as culture medium) to a culture space (142) and recovers the culture medium within the culture space (142). The culture medium tube (260) forms a two-branched main stem consisting of a supply tube (262) and a discharge tube (264), and a gas syringe (250) and a plurality of culture medium syringes (252) are connected in parallel to the supply tube (262) (main tube) through a branch tube (260-B), a branch connector (236), and a check valve (234), respectively. As in Fig. 9, the gas syringe (250) is positioned at the uppermost end of the supply tube (262), and the various liquids introduced into the supply tube (262) from each culture syringe (252) are transferred toward the cell culture module (100) by the suction and discharge strokes of the gas syringe (250). As described in Fig. 8 (b), the transfer of the culture liquid between one culture syringe (252) and the gas syringe (250) can be performed with a time difference, so that the various liquids can be transferred sequentially toward the cell culture module (100) in a state separated by a gas column without being mixed with each other. Therefore, the risk of cross-contamination can be significantly reduced, and the various liquids can be transferred through a single supply tube (262).

[0132] Also, FIG. 10 illustrates a recovery syringe (254) for recovering the culture medium. A recovery chamber (222) is connected to the most downstream side of the discharge tube (264), and the recovery syringe (254) is connected in parallel to the discharge tube (264) adjacent to the recovery chamber (222). The configuration of the branch tube (260-B), the branch connector (236), the check valve (234), and the air filter (236) is the same as the connection structure of the culture medium syringe (252), but the difference is that the check valve (234) of the recovery syringe (254) only allows one-way flow from the cell culture module (100) to the recovery chamber (222). In this tubing network, the culture medium in the culture space (142) can be collected into the recovery chamber (222) without backflow from the discharge tube (264) by the suction and discharge strokes of the recovery syringe (254).

[0133] Figure 11 is a drawing illustrating a priming process performed in a culture syringe (252). Priming refers to the process of completely filling a transfer tube with a fluid without air during fluid transfer. In the present invention, for accurate quantitative transfer of a culture medium, the culture medium must be filled without any gaps from the culture medium chamber (220) to the end of the branch tube (260-B) (just before joining the main tube). To this end, a priming process as shown in Figure 11 can be performed.

[0134] The priming of the culture syringe (252) is performed through a total of two suction and discharge (pumping) steps. In the first pumping process illustrated in (a) to (c) of FIG. 11, the culture is filled from the culture chamber (220) to the middle of the branch connector (236). In the second pumping process illustrated in (d) to (e) of FIG. 11 that follows, the culture is additionally suctioned to fill the culture up to the end of the branch tube (260-B). If more culture is pumped than this, some of the culture is discharged into the main tube (260-M) and is wasted, and if less culture is pumped than this, gas remains, which causes an error in the transport amount.

[0135] FIG. 12 is a drawing illustrating one embodiment of a pump unit (230) in which the tubing network conceptually illustrated in FIG. 10 is actually implemented. The exemplary embodiment of FIG. 12 illustrates, for convenience of explanation, one gas syringe (250), two culture solution syringes (252), and one recovery syringe (254), and also illustrates a branch connector (236), a check valve (234), and an air filter (236) connected to the gas syringe (250). For convenience of understanding, FIG. 12 schematically illustrates a culture solution chamber (220) and a recovery chamber (222), which are housed in a culture solution storage room (210) (see FIG. 7). Arrows indicate the flow of culture solution supplied and recovered. Since a large amount of gas is required to transport the culture medium to the remote culture space (142), the gas syringe (250) may be larger than the culture medium syringe (252). In addition, in the case of the recovery syringe (254), since one syringe is used without distinguishing between a syringe for sucking liquid and a syringe for sucking gas, the recovery syringe (254) may be as large as the gas syringe (250), and since the recovery chamber (222) sucks various types of culture medium, a chamber with a larger capacity than the culture medium chamber (220) may be used.

[0136] Fig. 13 is a drawing illustrating one embodiment of a pump unit (230), and corresponds to an embodiment including a pump actuator (242) that automatically drives each syringe pump (232) included in the pump unit (230). In Fig. 13, for the sake of understanding the invention, the configuration of the branch connector (236) and the check valve (234) is omitted, and the structure including the branch connector (236) and the check valve (234) can be referred to Fig. 12.

[0137] In the embodiment of Fig. 13, the pump unit (230) includes one gas syringe (250), five culture solution syringes (252), and one recovery syringe (254). A total of seven syringe pumps (232) are provided, and correspondingly, seven pump actuators (242) are provided (see Fig. 7). The pump actuator (242) is a linear actuator that holds the plunger (240) end of the syringe pump (232) and moves linearly, thereby automatically performing the suction and discharge strokes of the syringe pump (232).

[0138] To facilitate the detachment between the pump actuator (242) and the plunger (240) end of the syringe pump (232), an additional detachment clamp (244) is attached to the end of the plunger (240), and the plunger (240) and the pump actuator (242) can be coupled in such a way that a shaft extending from the pump actuator (242) passes through the detachment clamp (244).

[0139] Fig. 14 is a drawing showing the inside of a culture solution supply module (200). The inside is shown by removing a part of the culture solution supply module (200), and a culture solution storage room (210) that accommodates a plurality of culture solution chambers (220) and a pump unit (230) that opens with a rotating door structure above the culture solution storage room (210) are arranged. In addition to the plurality of culture solution chambers (220), the culture solution storage room (210) can accommodate a large-capacity recovery chamber (222). At least one cooling unit (212) (e.g., a Peltier unit) is installed in the culture solution storage room (210), and the culture solution storage room (210) maintains a low-temperature environment of 4 to 8°C by the cooling unit (212). Alternatively, the culture solution supply module (200) can be installed in a separate refrigerator that creates a low-temperature environment, so that the culture solution storage room (210) can maintain a low-temperature environment.

[0140] Due to the low-temperature environment of the culture medium storage room (210), the culture medium stored in the culture medium chamber (220) maintains good quality for a long period of time. In addition, it is preferable that the culture medium tube (260), branch connector (236), and check valve (234) forming the tubing network are also placed in the culture medium storage room (210). This is because, for quantitative transfer of the culture medium, after the priming process of FIG. 11 is performed, the culture medium is also filled in the culture medium tube (260) on the upstream side of the culture medium syringe (252). In addition, it may be preferable that cold air be transmitted to the pump unit (230), which is partially connected to the culture medium storage room (210), to form an appropriate low-temperature environment. For this purpose, the pump unit (230), which opens with a rotating opening structure, may have an insulating structure that seals the culture medium storage room (210).

[0141] Fig. 15 is a diagram illustrating the culture medium temperature environment in the cell culture device (10). In the culture medium supply module (200), the temperature of the culture medium is maintained at 4 to 8°C due to the low-temperature environment of the culture medium storage room (210). The culture medium tube (260) connected from the culture medium supply module (200) to the cell culture module (100) is exposed to a room temperature of about 15 to 25°C, and the cell culture module (100), which is usually placed in a CO2 incubator, maintains a temperature range of about 36 to 37°C. Therefore, during the process of supplying the culture medium, the culture medium undergoes a natural temperature control in which the temperature increases from low temperature → room temperature → culture temperature, thereby preventing the supply of culture medium at an excessively low temperature to the culture space (142), thereby preventing the cells from being subjected to thermal shock and resulting in functional deterioration or damage. On the other hand, if the culture medium is stored in a room temperature environment for an excessively long period of time, various substances such as proteins in the culture medium may be decomposed, which may cause a problem in that it becomes difficult to create an environment necessary for cell culture. However, in the present invention, this problem of temperature increase in the culture medium can be solved by controlling the transport speed of the culture medium being pumped.

[0142] Fig. 16 is a conceptual diagram illustrating a configuration in which a plurality of cell culture modules (100) are connected to a single culture medium supply module (200). A supply tube (262) and a discharge tube (264) extended from the culture medium supply module (200) are connected in parallel to a plurality of cell culture modules (100). A one-to-one correspondence of the culture medium supply module (200) to the cell culture module (100) serving as the transfer target, i.e., the transfer of the culture medium by the culture medium supply module (200) to a single cell culture module (100) serving as the transfer target, is controlled by a valve module (130) provided in each cell culture module (100).

[0143] That is, each valve module (130) provided in the remaining cell culture modules (100) except for the cell culture module (100) that is the transfer target closes the flow of the culture medium. Accordingly, although it is configured as a one-to-many tubing network in which a plurality of cell culture modules (100) are connected to a single culture medium supply module (200), during actual transfer of the culture medium, a one-to-one correspondence is achieved by the linked opening and closing operation of the plurality of valve modules (130).

[0144] During operation of the cell culture device (10), the absolute time for transfer of the culture medium is short, so the culture medium supply module (200) can be operated more efficiently by configuring a one-to-many tubing network in which multiple cell culture modules (100) are connected to one culture medium supply module (200).

[0145] On the other hand, since the culture solution supply module (200) applies a gas-liquid interface transport mechanism to transport the culture solution, even if the flow rate of the flow inside the tube is not directly measured, by using a tube made of a transparent or translucent material, it is possible to determine whether the culture solution supply module (200) is operating normally by determining whether the presence of liquid inside the tube is detected. As an example of abnormality detection for the culture solution supply module (200), it is possible to determine whether the tube is normally installed and whether gas or liquid is contained inside the tube using an infrared LED or other monitoring means, and when a command to supply or discharge the culture solution is input, it is possible to determine whether the state inside the supply tube and the discharge tube changes from gas-liquid-gas, thereby confirming whether the culture solution is actually transported normally to the culture solution supply tube and the discharge tube. Although not shown separately, the infrared LED or other monitoring means may be provided in direct contact with the tube (an infrared LED or other monitoring means may be provided on a clipper capable of clipping to the tube to irradiate or photograph the tube from a close distance), or may be provided so as to irradiate or photograph light toward the tube from a distance away from the tube.

[0146] Although embodiments of the present invention have been described with reference to the attached drawings, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without altering the technical concept or essential features thereof. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

Claims

1. A cell culture module including a cell culture unit in which a well plate forming at least one culture space is mounted on a cartridge; A culture solution supply module including a pump unit that forms a supply and discharge flow of culture solution to the cell culture section, and a culture solution storage room that accommodates at least one culture solution chamber; and A pair of flexible culture tubes that connect the culture supply module and the cell culture module, which are spatially separated, to form a conduit for supplying and discharging the culture medium; Including, A cell culture device wherein the above cell culture module is placed in a CO2 incubator that creates a warm environment, the above culture medium storage room creates a low-temperature environment below room temperature, and a pair of culture medium tubes exposed between the culture medium supply module and the cell culture module are in a room-temperature environment.

2. In paragraph 1, The above pair of culture tubes are a supply tube and a discharge tube, The above pump unit sequentially pressurizes and supplies a predetermined amount of culture medium and gas to the culture space through the supply tube, A cell culture device, wherein the inner diameter of the above supply tube has a diameter at which the culture medium does not retreat due to gravity.

3. In paragraph 2, A cell culture device in which the pump unit applies negative pressure to the discharge tube to suck the culture medium from the culture space and recover it into a recovery chamber contained in the culture medium storage room.

4. In paragraph 1, Comprising a plurality of cell culture modules connected in parallel to one culture medium supply module, A cell culture device in which a one-to-one correspondence of a culture solution supply module to a cell culture module serving as a transfer target occurs while a flow of culture solution is supplied and / or withdrawn by controlling the opening and closing of a valve module provided for each of a plurality of cell culture modules.

5. In a culture solution supply module capable of supplying culture solution to at least one culture space that is spatially separated and recovering culture solution from the culture space, A pump unit that forms a supply and discharge flow of culture medium to the cell culture section, and a culture medium storage room that accommodates a recovery chamber and at least one culture medium chamber, The above culture medium storage room creates a low-temperature environment below room temperature. The above pump unit is a culture solution supply module that supplies the culture solution stored in the culture solution chamber to each culture space through a supply tube and also discharges it to the recovery chamber through a discharge tube.

6. In paragraph 5, The above pump unit comprises a plurality of syringe pumps, The above plurality of syringe pumps, A culture fluid supply module comprising a gas syringe, at least one culture fluid syringe, and a recovery syringe.

7. In paragraph 6, The above gas syringe and at least one culture solution syringe are connected in parallel to the supply tube, A culture medium supply module in which check valves are installed upstream and downstream of the confluence point for the above supply tubes.

8. In paragraph 7, The above gas syringe is placed at the uppermost end of the above supply tube, A culture solution supply module in which the check valve installed in the above gas syringe and culture solution syringe only allows one-way flow toward the culture space.

9. In paragraph 8, The above gas syringe, A culture medium supply module having an air filter upstream of the upstream check valve.

10. In paragraph 8, A predetermined amount of culture medium is introduced into the supply tube by the suction and discharge stroke of the culture medium syringe, A culture medium supply module in which gas introduced into the supply tube by the suction and discharge strokes of the gas syringe is transported to the culture medium introduced into the supply tube to the culture space.

11. In paragraph 10, The above plurality of syringe pumps include a gas syringe and a plurality of culture solution syringes, A culture solution supply module in which the culture solution transfer between one culture solution syringe and the gas syringe is performed at a time interval, and thus the plurality of culture solutions transferred are transferred to the culture space in a state of being isolated by gas.

12. In paragraph 6, The above recovery syringe is connected to the most downstream of the above discharge tube, A culture medium supply module, wherein check valves are installed upstream and downstream of the confluence of the recovery syringe and the discharge tube, respectively, to allow only one-way flow toward the recovery chamber.

13. In paragraph 6, The above pump unit, A culture medium supply module having a plurality of pump actuators that linearly move the plunger of each syringe pump to perform suction and discharge strokes.

14. In a culture medium supply module capable of supplying culture medium to at least one culture space that is spatially separated, A pump unit having a plurality of syringe pumps for forming a supply flow of culture medium to the cell culture section, and a culture medium storage room accommodating at least one culture medium chamber, The plurality of syringe pumps include a gas syringe connected to the uppermost portion of the supply tube and at least one culture solution syringe connected in parallel downstream of the gas syringe, Check valves are installed upstream and downstream of the confluence of the gas syringe and each culture syringe for the above supply tube, so that only one-way flow toward the culture space is allowed. A culture solution supply module in which a predetermined amount of culture solution is introduced into the supply tube by the suction and discharge strokes of the culture solution syringe, and the culture solution introduced into the supply tube is transported to the culture space by the gas introduced into the supply tube by the suction and discharge strokes of the gas syringe.

15. In paragraph 14, The above plurality of syringe pumps include a gas syringe and a plurality of culture solution syringes, A culture solution supply module in which the culture solution transfer between one culture solution syringe and the gas syringe is performed at a time interval, and thus the plurality of culture solutions transferred are transferred to the culture space in a state of being isolated by gas.

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