WO2019120135A1 - Cell culture module and cell culture system - Google Patents

Abstract

Provided are a cell culture module and a cell culture system. The cell culture module comprises: a closed body that is in communication with the outside only by means of a culture fluid inlet, a culture fluid outlet, a cell fluid inlet, and a cell fluid outlet; culture fluid cavities and a cell cavity which are located in the body, the culture fluid cavities comprising independent culture fluid cavities respectively in communication with the culture fluid inlet and in communication with the culture fluid outlet, and the cell cavity being in communication with the cell fluid inlet and the cell fluid outlet; sealing blocks used for separating the cell cavity and the culture fluid cavities; and a hollow fiber filament placed in the cell cavity and having the wall provided with micropores that do not allow cells to pass, the hollow fiber filament passing through the sealing blocks, and both ends of the hollow fiber filament being respectively in communication with the culture liquid cavities at the culture liquid inlet end and at the culture liquid outlet end. The cell culture system comprises a cell culture chip, and can be used for culturing different cells.

Description

Cell culture module and cell culture system Technical field

The invention belongs to the field of biomedical cell culture, and particularly relates to a cell culture module and a cell culture system.

Background technique

Nowadays, biological immunotherapy has become an emerging means to overcome cancer cancer. It is a key technology in the field of biotherapy technology and has good research and development and application prospects. The cell therapy in biological immunotherapy is to extract a certain amount of immune cells from the patient and perform expansion culture in vitro. The process may include the addition of specific stimulation factors and/or the use of transgenic technology, when the cells are expanded to After a certain scale or quantity, it is required to be returned to the body according to the patient's course of treatment. In vitro expansion and culture of immune cells is an important part of technical realization throughout the treatment process. A reasonable cell culture device is a key factor for achieving safe and effective amplification of immune cells. The existing cell treatment mechanism uses a conventional multi-well plate, a cell culture bottle or a ventilated culture bag as a container for static culture in vitro, which can only provide a static growth environment for the cells, and the cell growth environment and the body are real. The environmental difference is large, and only the medium is added during the cultivation process, and the product of cell growth and metabolism is easily accumulated, thereby affecting the growth rate, morphology, cell phenotype, expression state and cell number of the immune cells. In terms of vitality, it is easy to limit and reduce the effect of immunotherapy; and in the production, it is necessary to manually open the container to add the culture solution and other auxiliary factors, which increases the risk of sample contamination and also the process technology for cultivating the operator. A higher requirement was put forward. At the same time, more manual operations are also likely to affect the consistency of quality between batches of immune cell culture products.

The immune cells can be classified into adherent cells and suspension cells according to the growth mode of the cells, and the adherent cells are grown on the wall of the culture container, and the suspended cells are suspended and grown in the culture solution of the culture container. The ideal cell culture device needs to be able to combine both types of cells to reduce the investment in instruments or consumables and improve equipment utilization. In the process of growth of immune cells in the body, the flowing blood can not only supply vegetative cells, but also take away the waste produced by the cells, but the dynamic environment such as the transfer of these nutrients and the discharge of secreted waste is difficult in conventional static culture devices. simulation. At present, there are some devices and technologies that can be used for dynamic cell culture in the field of cell culture, which can satisfy the functions of cell growth and liquid exchange, such as spinner flasks, cell culture tanks (bioreactors), hollow fiber tubes, etc., but These devices are complex in design and cumbersome to operate. The rotary bottles can only be used for adherent cell culture, and the culture space is limited. Currently, more and more plants have been replaced by large-scale multi-layer cell factories; culture tanks can satisfy large volume of cells. Culture, but traditional stainless steel or glass cans need to be cleaned and sterilized before each production, and cannot be applied to individualized cell culture in cell therapy clinical applications. Other types of reactors, such as wave-swing reactors, use disposable consumables, but are not widely accepted due to factors such as instrument volume, culture cost, and the like. Existing hollow fiber culture methods are often designed specifically for a certain type of cells, and their application range is narrow.

The development of microfluidic chip technology provides a new platform for cell culture. Microfluidic chips have the advantages of small space, high integration and one-time use. However, the existing research and development of related microfluidic chips still have problems such as inconvenient replacement of culture fluid, incomplete closure of cell culture area, incomplete gas exchange, etc., and most of them are oriented to the test culture of micro-cells and the real-time dynamic monitoring direction of cell state. It is not suitable for large-scale production of cells.

CN158703A discloses a microcellular hollow fiber reactor in which a cell is pre-buried in a small section of hollow fiber filaments and placed in a closed, breathable silicone tube containing a culture solution. The device utilizes hollow fiber filament micropores for nutrient transfer, and the cells in the fiber filament can be cultured to a certain scale, and can be applied to research of related drugs. The device is a pharmacological study applied to micro cells, which is difficult to meet for large-scale clinical cell culture.

CN103667054A discloses an integrated microfluidic cell culture chip that integrates a chip body with associated temperature control components, micropumps, microvalves, and control circuits. The main body of the chip is formed by multi-layer bonding, including a cell culture area, a culture liquid storage area, a waste liquid area, and the like, and different areas are connected by a micro flow path. The device is integrated and integrated, and the inner cavity of the fluid channel is sealed, which is suitable for the cultivation of micro cells. However, the flow path design is simple, the simulation of the culture environment of the immune cells is not comprehensive, and the gas exchange conditions of the cell culture are neglected, and cannot be directly applied. Culture of clinical immune cells.

CN105754856A discloses a drawer type three-dimensional vascularized tissue culture organ chip which is composed of a drawer plaque, a sealing plate, a fixing frame, a vascularized three-dimensional tissue and related inlet and outlet tubes. The system has high integration, detachable or sealed, automatic real-time monitoring, and focuses on simulating the stable adherent culture of three-dimensional tissue of blood vessels, and adopts transparent ventilated chip material, which is beneficial to the growth and real-time monitoring of related cells, but the chip is only For the culture of vascularized tissue, the passability is not strong.

Summary of the invention

The invention aims at the deficiencies of the existing various cell culture methods and devices, and combines the advantages of hollow fibers and culture chips to provide a cell culture module and a culture device thereof which are simple in structure, convenient in operation, and suitable for immune cells, and have The utility model has the advantages of small volume, high culture density, automatic control, closed chip culture space, easy assembly and replacement of chip components, and high biosafety performance. The cell culture device fully simulates the growth environment of immune cells in blood vessels by culturing the built-in filaments of the microchip, and establishes a large-scale 3D cell culture, which is suitable for the culture of various immune cells in cell therapy clinical applications, such as adherent type DC cells. (dendritic cells), culture of various T lymphocytes (CIK, CTL, TIL, etc.) of suspension type.

The cell culture module of the present invention comprises:

a closed body that communicates with the outside only through the culture fluid inlet, the culture fluid outlet, the cell fluid inlet, and the cell fluid outlet;

a culture liquid chamber and a cell chamber located inside the body, wherein the culture liquid chamber includes independent culture liquid chambers respectively communicating with the culture liquid inlet and a culture liquid chamber communicating with the culture liquid outlet, the cell chamber and the cells The liquid inlet is connected to the cell liquid outlet;

a sealing stopper for separating the cell cavity and the culture fluid cavity; and

a hollow fiber filament placed in a cell cavity having micropores that do not allow passage of cells; wherein the hollow fiber filament passes through the sealing stopper, and both ends of the hollow fiber filament are respectively cultured with the inlet end of the culture fluid The liquid chamber and the culture solution chamber at the outlet end of the culture solution are both connected.

In certain embodiments, the volume of the culture fluid chamber is from 0.2 to 0.5 L, preferably from 0.3 to 0.35 L.

In certain embodiments, the cell lumen has a volume of from 0.4 to 1 L, preferably from 0.6 to 0.8 L.

In certain embodiments, the hollow fiber filaments have a length of from 130 to 170 mm, a tube wall thickness of from 20 to 50 μm, and a pore size of the micropores on the tube wall of less than 1 μm.

In certain embodiments, the body and sealing block are transparent biocompatible materials.

In certain embodiments, the culture fluid chamber is located on either side of the cell lumen, separated by a sealing block, and the hollow fiber filaments pass through the cell lumen to communicate with the culture fluid chambers on either side of the cell lumen.

In certain embodiments, the culture fluid chamber is located on the same side of the cell lumen, and the two culture fluid chambers are separated from each other by a sealing block between the culture fluid chamber and the cell chamber.

In some embodiments, a liquid filtration device is disposed on the inside of the culture solution inlet and the culture solution outlet for filtering the culture solution to avoid contamination that may be introduced when the culture solution inlet and outlet are connected to the culture solution module.

In certain embodiments, the culture fluid inlet and the culture fluid outlet are sealed using a medical rubber stopper.

In some embodiments, the culture fluid chamber connected to the culture fluid inlet has a culture fluid chamber inlet, and the culture fluid chamber inlet is in communication with the culture fluid inlet; the culture fluid chamber connected to the culture fluid outlet has a culture chamber a liquid chamber outlet, the culture liquid chamber outlet being in communication with the culture liquid outlet.

In certain embodiments, the culture fluid chamber inlet is disposed within the body and is in communication with the culture fluid inlet through a processing flow path within the body.

In certain embodiments, the culture fluid chamber outlet is disposed within the body, the cell culture module further comprising a processing flow path disposed inside the body and optionally a culture fluid disposed outside the body The road drives the pump line, and the culture liquid chamber outlet communicates with the culture liquid outlet through the processing flow path and the optional culture liquid path driving pump line.

In certain embodiments, the cell lumen has a cell lumen inlet and a cell lumen outlet that are in communication with the cell fluid inlet and the cell fluid outlet, respectively.

In certain embodiments, the cell lumen inlet and the cell lumen outlet are both disposed inside the body, the cell culture module further comprising a cell fluid path driven pump conduit disposed outside the body, wherein The cell lumen inlet and the cell lumen outlet are respectively connected to the cell fluid inlet and the cell fluid outlet through a processing flow path disposed inside the body, and the cell fluid path driving pump is passed between the cell liquid inlet and the cell liquid outlet. The pipeline is connected.

In some embodiments, the cell culture module has a culture fluid flow path and a cell fluid flow path; wherein the culture fluid flow path includes a culture liquid inlet, a processing flow path, a culture liquid chamber inlet, and a culture liquid chamber at the inlet end, The inner wall and the outlet end of the hollow fiber filament, the culture liquid chamber outlet, the processing flow path, the optional culture liquid path drive pump line and the culture liquid outlet; the cell liquid flow path includes the cell liquid inlet, the processing flow path, and the cell in sequence. The lumen inlet, the hollow fiber extrafilament space within the cell lumen, the cell lumen outlet, the processing flow path, the cell fluid outlet, and the optional cell fluid path drive pump tubing.

In certain embodiments, the processing flow path is machined or injection molded within the body.

In certain embodiments, the body of the cell culture module consists of an upper layer, a middle layer, and a lower layer, wherein:

The upper layer and the middle layer, the middle layer and the lower layer bonding surface are tightly combined and do not leak liquid;

The upper layer is provided with an upper culture liquid inlet and an upper culture liquid outlet, respectively connected to the culture liquid chamber disposed in the middle layer; and a processing flow path connecting the upper culture liquid inlet and the middle layer culture liquid inlet, and the upper culture liquid outlet and the middle layer culture Processing flow path of the liquid outlet;

The middle layer is a central hollow frame structure, and the frame structure is provided with a middle layer culture liquid inlet connected to the upper layer culture liquid inlet, a culture liquid chamber inlet, a middle layer culture liquid inlet and a culture liquid chamber inlet processing flow path, The culture liquid chamber outlet, the middle layer culture liquid outlet connected to the upper culture liquid outlet, the processing flow path between the culture liquid chamber outlet and the middle layer culture liquid outlet, and the cell liquid inlet, the cell chamber inlet, the cell liquid inlet and the cell chamber inlet a processing flow path between the processing flow path, the cell lumen outlet, the cell fluid outlet, and the cell lumen outlet and the cell fluid outlet;

The culture fluid chamber and the cell lumen are disposed in the hollow portion of the middle layer; and

The hollow fiber filaments are placed in a cell cavity, and both ends thereof pass through the sealing stopper and are respectively connected to the culture fluid chamber.

In certain embodiments, the cell culture module further comprises:

a culture liquid path provided outside the body drives a pump line, and the culture liquid path drives a pump line to connect a processing flow path between the culture liquid chamber outlet and the middle layer culture liquid outlet; and/or

A cell fluid circuit disposed outside the body drives a pump line, and the cell fluid path drives a pump line to communicate with the cell fluid inlet and the cell fluid outlet.

In certain embodiments, the broth drive pump line and the cell fluid path drive pump line are medical hoses.

In certain embodiments, the upper layer is further provided with a socket and a chip tab, wherein the socket is for positioning a plug culture fluid module, and the chip tab is for facilitating removal of the culture fluid module.

In some embodiments, the sealing block is sealingly adhered to the contact surface of the middle layer, and the upper layer, the lower layer and the middle layer bonding surface are sealingly sealed, and the sealing block and the upper inner wall, the lower inner wall and the hollow portion are respectively sealed. The sidewall forms the culture fluid chamber and the cell lumen.

In some embodiments, a liquid filtering device is disposed between the upper culture fluid inlet and the middle culture fluid inlet and between the upper culture fluid outlet and the middle culture fluid outlet to avoid contamination introduced by the medical rubber plug; Preferably, the membrane pore size is 0.2 μm.

The culture fluid module of the present invention comprises:

a closed body that communicates with the outside only through the storage liquid inlet and the storage liquid outlet;

a storage chamber disposed within the body, wherein the storage chamber is in communication with a storage fluid inlet and a storage fluid outlet; and

A gas permeable membrane that allows gas molecules to pass through to seal the storage chamber.

In certain embodiments, the volume of the storage chamber is from 0.6 to 2 L, preferably from 0.8 to 1.5 L.

In certain embodiments, the body and the gas permeable membrane are both transparent biocompatible materials.

In some embodiments, the storage chamber inlet and the storage chamber outlet are sealed with a medical rubber stopper.

In some embodiments, the storage chamber in communication with the storage fluid inlet has a storage chamber inlet that is in communication with the storage fluid inlet through a predetermined processing line; the storage chamber in communication with the storage fluid outlet There is a storage liquid chamber outlet that communicates with the storage liquid outlet through a predetermined processing line.

In certain embodiments, a liquid filtration device is provided after the storage chamber outlet and the storage chamber inlet for filtering and blocking impurities and bacteria from entering the storage chamber.

In some embodiments, the culture fluid module is composed of an upper layer, a middle layer, a lower layer, and a gas permeable membrane, wherein the upper layer and the middle layer, the middle layer and the lower layer bonding surface are tightly combined and do not leak liquid, wherein the upper layer and the middle layer are disposed between the upper layer and the middle layer. There is a gas permeable membrane and/or a gas permeable membrane between the middle layer and the lower layer.

In some embodiments, the inlet and outlet of the storage liquid and the inlet and outlet of the storage chamber are both disposed in the middle layer, and the liquid injection needle type bidirectional interface is used for liquid supply, and the medical rubber plug directly penetrates the cell culture module and the culture liquid module at the same time. .

In some embodiments, the culture fluid module further has a filling port, and the interface is sealed by a medical rubber plug for filling the initial culture liquid; preferably, the liquid filtering device is further disposed at the filling port. .

The cell culture apparatus of the present invention comprises: the cell culture module described herein, the culture fluid module described herein, the liquid path drive module, the gas module, the temperature module, the control module, and the tubing or line connecting the modules.

In one or more embodiments, the liquid path driving module includes:

a first driving device for controlling the circulation of the culture solution between the culture fluid module and the cell culture module; and

A second driving device for controlling the circulation of the cell fluid in the cell cavity of the cell culture module.

In certain embodiments, the first drive means is disposed on the communication line of the storage fluid outlet and the culture fluid inlet, or on the communication line of the culture fluid outlet and the storage fluid inlet.

In certain embodiments, the cell culture module includes an external broth drive pump line, the first drive being disposed on the broth drive pump line.

In certain embodiments, the second drive device is disposed on a cell fluid path driven pump line.

In certain embodiments, both the first drive device and the second drive device are pinch peristaltic pumps, preferably bi-directional peristaltic pumps.

In certain embodiments, the gas module includes an intake manifold, an outlet conduit, a gas control valve, and an optional gas container and an optional gas mixer.

In certain embodiments, the gas container includes a container containing compressed air, oxygen, and carbon dioxide, each container being in communication with a gas mixer, the inlet tube being in communication with the gas mixer.

In some embodiments, a filter device is disposed in the inlet and outlet nozzles, and a gas filter is disposed in the device.

In some embodiments, a gas control valve is disposed on the line connecting the vessel containing the compressed air, oxygen, carbon dioxide, and the gas mixer, and the gas control valve is provided with a gas flow meter.

In certain embodiments, the temperature module includes a semiconductor temperature control sheet and a temperature sensing element platinum resistor mounted thereon for controlling heating or cooling.

In certain embodiments, the control module comprises:

a first control switch for controlling the first driving device;

a second control switch for controlling the second driving device;

a third control switch for controlling gas delivery;

The fourth control switch is used to control the temperature module to perform heating or cooling.

In certain embodiments, the control module further includes a control circuit for monitoring the entire cell culture process in real time to achieve automated cell culture.

In certain embodiments, the cell culture device further includes a sensor module for monitoring temperature, oxygen concentration, carbon dioxide concentration, and pH in real time.

In certain embodiments, the sensor module includes a temperature sensor, an oxygen concentration sensor, a carbon dioxide concentration sensor, and a liquid pH sensor.

In certain embodiments, the cell culture module, the culture fluid module, and the sensor module are placed in a closed chip cavity having an air inlet and an air outlet.

In some embodiments, the liquid path driving module, the gas module, the temperature module, and the control module are integrally installed in the chassis, and a sealed chip cavity in which the cell culture module, the culture fluid module, and the sensor module are placed is disposed inside the chassis, and the outside of the chassis Install the man-machine exchange interface and control buttons.

In certain embodiments, the culture fluid is a serum-free medium dedicated to immune cells;

In certain embodiments, the cell is selected from the group consisting of a peripheral blood mononuclear cell, a neural stem cell, or a tumor cell.

In certain embodiments, the materials in contact with the cells in the device are each disposable biocompatible material and are sterilized and sterilized.

The invention also provides a kit comprising the cell culture module and culture fluid module described herein, or a chip as described herein.

In certain embodiments, the kit further includes a chassis integrally mounted with the fluid circuit driving module, the gas control valve, the temperature module, and the control module described herein, and a disposable culture consumable including: The cell culture module, the culture fluid module, and all the tubing.

DRAWINGS

1 is a schematic plan view showing the structure of a chip type immune cell culture system provided by the present invention.

2 is a schematic view showing the structure of an immunocyte culture chip provided by the present invention.

Fig. 3 is a schematic view showing the structure of a cell culture unit of the culture chip of the present invention.

Fig. 4 is a schematic exploded view showing the structure of a cell culture unit of the culture chip of the present invention.

Figure 5 is a schematic view showing the structure of an example of a hollow fiber block of the present invention.

Fig. 6 is a schematic exploded view showing the structure of a culture liquid unit of the culture chip of the present invention.

Fig. 7 is a schematic view showing the overall structure of a cell culture apparatus of the present invention.

Fig. 8 is a view showing the overall structure of a culture chip placed in the cell culture apparatus of the present invention.

Detailed ways

The invention will be elucidated below in conjunction with specific embodiments. It is to be understood that the invention is not intended to limit the scope of the invention. Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

The cell culture system of the present invention is a chip type immune cell culture system (device). The cell culture device of the present invention can realize a new immune cell culture protocol, which can independently process the cell culture of each clinical patient sample. By taking a certain amount of cells, especially immune cells, from the patient's body and culturing them in the culture system of the present invention, a corresponding number of cells of interest can be obtained, for example, the cell-loaded antigen-cultured cells of the DC cells are 0.5 to 2×10. ^On the total number of ⁸ , the number of T cells is expanded to a total of 0.5 to 2 × 10 ⁹ cells. The whole immune cell culture process can be automatically controlled, and only the culture fluid module needs to be replaced regularly. The cell culture is always carried out in a closed environment (no open operation, effectively avoiding third-party pollution), and finally the recovered immune cells can be directly used in clinical practice. , return to the patient for immune cell therapy. Throughout the culture process, a vascular culture method is constructed by hollow fiber filaments, and the fiber filaments are built in the cell cavity, and the culture fluid flows from one side of the culture liquid chamber to the other side culture chamber, and the flow ensures that the liquid in the cell cavity is also certain. The fluidity, 3D culture effectively mimics the biological environment in which immune cells grow, and also improves the efficiency of medium exchange.

The cell culture device comprises six main modules of a cell culture module, a culture liquid module, a liquid path driving module, a gas module, a temperature module and a control module, and a pipeline connecting the culture liquid module, the cell culture module and the liquid path driving module. . In certain embodiments, the present cell culture device further comprises a sensor module. 1 shows a schematic structural view of a system of the present invention, including a chip cavity 1, a cell culture module 2, a culture fluid module 3, a liquid path driving module 4, a gas module 5, a temperature module 6, a sensor module 7, and a control module 8.

Cell culture module

The cell culture module is a place for cell culture, including two structures of a culture liquid chamber and a cell chamber, which are separated by a sealing stopper. The culture chamber is a place for accommodating a fresh culture solution or an exchanged culture solution, and the cell cavity is a cell growth site. A hollow fiber filament is arranged in the cell cavity, and both ends of the fiber tube are connected to the culture liquid cavity. The fresh culture medium in the culture chamber passes through the cell cavity through the hollow fiber tube. Through the micropores on the hollow fiber filaments, small molecules can be exchanged between the cell cavity and the culture fluid cavity, taking away the metabolic waste generated by cell growth and supplementing the nutrients required by the cells.

More specifically, the cell culture module comprises: a closed body that communicates with the outside only through the broth inlet, the broth outlet, the cell fluid inlet, and the cell fluid outlet; a culture solution chamber and a cell chamber located inside the body, wherein the culture solution The chamber comprises independent culture liquid chambers respectively communicating with the culture liquid inlet and a culture liquid chamber communicating with the culture liquid outlet, the cell chamber is connected with the cell liquid inlet and the cell liquid outlet; and the sealing block is used for separating the cell cavity and the culture medium. a hollow fiber filament placed in the cell cavity and having pores on the wall that do not allow passage of cells; wherein the hollow fiber filament passes through the sealing stopper, and the two ends of the hollow fiber filament and the culture solution at the inlet end of the culture fluid respectively The chamber is connected to the culture liquid chamber at the outlet end of the culture solution. All of the components of the cell culture module, including the material of the body and the sealing stop, are biocompatible materials, preferably transparent materials.

The culture solution inlet and the culture solution outlet are two openings provided on the cell culture module for communicating with the culture solution module, and are inlets and outlets for the inflow and outflow of the culture solution. The culture fluid chamber can be located on both sides of the cell cavity, separated by a sealing block, and the hollow fiber filaments pass through the cell cavity to connect the culture liquid chambers on both sides of the cell cavity; or, the culture liquid cavity is located on the same side of the cell cavity, and the two culture fluids The chambers are separated from each other by a sealing block between the chamber and the cell chamber. The culture liquid chamber connected to the inlet of the culture liquid itself is provided with a culture liquid chamber inlet, and the culture liquid chamber inlet is in communication with the culture liquid inlet. The culture liquid chamber connected to the outlet of the culture solution is provided with a culture liquid chamber outlet, and the culture liquid chamber outlet is in communication with the culture liquid outlet. The culture liquid chamber inlet and the culture liquid chamber outlet are both disposed in the body, and are respectively connected to the culture liquid inlet and the culture liquid outlet through the processing flow path in the body. The culture liquid inlet and the culture liquid outlet may be sealed by a medical rubber stopper, and the rear side (ie, the inner side, the side close to the culture liquid chamber) may be respectively provided with a liquid filtering device for filtering the culture liquid to avoid the import and export of the culture liquid and the culture. Contamination that may be introduced when the liquid module is connected. Usually, the total volume of the culture solution chamber is 0.2 to 0.5 L, preferably 0.3 to 0.35 L.

In some embodiments, the cell culture module further comprises a culture liquid path driving pump line disposed outside the body, wherein the processing flow path between the culture liquid chamber outlet and the culture liquid outlet is disconnected, and the pump is driven by the culture liquid path. The pipeline is connected. In this case, the disconnected processing flow path forms two interfaces on the body for communicating the communication circuit to drive the pump line communication.

The cell fluid inlet and the cell fluid outlet are the other two openings provided on the cell culture module, which are the inlet and outlet of the influx and outflow of the cell fluid. The cell cavity itself has a cell lumen inlet and a cell lumen outlet, which are in communication with the cell fluid inlet and the cell fluid outlet, respectively. The cell cavity inlet and the cell cavity outlet are both disposed inside the body, and can be connected to the cell liquid inlet and the cell liquid outlet through a processing flow path disposed inside the body. The cell culture module further includes a cell liquid path driving pump line disposed outside the body, the cell circuit driving the pump line for connecting the cell liquid inlet and the cell liquid outlet. The volume of the cell chamber is 0.4 to 1 L, preferably 0.6 to 0.8 L.

The hollow fiber filament is an intermediate through-hole filament having a length of 130 to 170 mm, a tube wall thickness of 20 to 50 μm, and a wall covered with micropores having a pore diameter of less than 1 μm to prevent passage of cells. The small molecular substance or dissolved oxygen can exchange material and gas through the micropores on the fiber wall and the cell cavity outside the filament to maintain the effectiveness of the culture medium in the cell cavity, and achieve continuous nutrient supply to the cells. The material of the hollow fiber filaments may be a biocompatible material commonly used in the art, preferably polypropylene, polysulfone, polypropylene or polyethylene which is more biocompatible. There is no particular limitation on the number of hollow fiber filaments in the cell cavity, but in general, the space (volume) outside the hollow fiber filaments in the cell cavity should be in the range of 0.2 to 0.6 L.

Generally, the cell culture module of the present invention has a culture fluid flow path and a cell fluid flow path. The culture liquid flow path is a passage for the culture liquid to flow, and includes a culture liquid inlet, a processing flow path, a culture liquid chamber inlet, a culture liquid chamber at the inlet end, a hollow fiber filament inside, a culture liquid chamber at the outlet end, a culture liquid chamber outlet, and a processing flow. The road, optional culture circuit drives the pump line and the culture fluid outlet. The cell fluid flow path is a passage for cell fluid flow, which in turn includes cell fluid inlet, processing flow path, cell cavity inlet, hollow fiber filament space in cell cavity, cell cavity outlet, processing flow path, cell liquid outlet, and cell fluid path. Drive the pump line. The culture fluid flow path can form a circulation loop with the culture fluid module described herein, and in the cell fluid flow path, the cell fluid pathway drives the pump pipeline to communicate with the cell fluid inlet to form a circulation loop.

For example, when circulating the culture solution, the culture solution flows out from the culture solution module, enters the cell culture module from the culture solution inlet, flows through the processing flow path to the inlet of the culture liquid chamber, flows into the culture liquid chamber at the inlet end of the culture liquid, and flows through the hollow fiber filament. The hollow inner cavity reaches the culture liquid chamber at the outlet end of the culture liquid, flows out through the outlet of the culture liquid chamber, enters the processing flow path, and then flows out of the culture liquid outlet, and flows back to the culture liquid module, thereby forming a circulation loop, which is also referred to herein as Culture medium circulation loop. In some embodiments, the processing fluid flow path between the culture fluid chamber outlet and the culture fluid outlet is disconnected, and the pump circuit is driven by a culture fluid circuit externally disposed outside the body of the cell culture module. The cell fluid circulates between the hollow fiber filament space, the cell fluid inlet and the cell fluid outlet in the cell cavity to form a cell fluid circulation loop.

Herein, the processing flow path may be disposed inside the body through a process such as injection molding or machining; the culture liquid path driving pump line and the cell liquid path driving pump line may be conventional medical hoses.

In certain embodiments, the cell culture module consists of three layers of an upper layer, a middle layer, and a lower layer. The upper layer and the middle layer, the middle layer and the lower layer bonding surface are sealed and sealed, and no leakage is caused.

The upper layer is provided with an upper culture liquid inlet and an upper culture liquid outlet, and is a culture liquid inlet and an outlet for the culture liquid to enter and exit the cell culture module; and a processing flow path connecting the upper culture liquid inlet and the middle culture liquid inlet and the upper culture liquid outlet And the processing flow path of the middle layer culture liquid outlet. The upper layer can also be provided with slots and chip tabs. The slot can be used to position the inserted culture fluid module, and the chip pull can be used to facilitate the removal of the culture fluid module. In some embodiments, the upper culture fluid inlet and the upper culture fluid outlet can be sealed with a medical rubber stopper.

The middle layer has a certain thickness, usually 5-15 mm, preferably 8-10 mm, which is a central hollow frame structure. The frame structure is provided with a middle layer culture liquid inlet, a culture liquid chamber inlet, and a middle layer which are connected with the upper culture liquid inlet. a processing flow path between the culture liquid inlet and the culture liquid chamber inlet, a culture liquid chamber outlet, a middle layer culture liquid outlet connected to the upper culture liquid outlet, a processing flow path between the culture liquid chamber outlet and the middle layer culture liquid outlet, and a cell liquid Processing flow path between the inlet, the cell cavity inlet, the cell fluid inlet and the cell lumen inlet, the cell lumen outlet, the cell fluid outlet, and the cell lumen outlet and the cell fluid outlet. A filtering device may be respectively disposed between the upper culture liquid inlet and the middle culture liquid inlet, and between the upper culture liquid outlet and the middle culture liquid outlet, for filtering the culture liquid, and avoiding the possibility that the culture liquid chamber inlet and outlet are connected with the culture liquid module Imported pollution. The membrane pore size is preferably 0.2 μm pore size or less.

The hollowed out portion of the middle layer is used to accommodate the hollow fiber block. Herein, the hollow fiber block is composed of a hollow fiber filament and a sealing stopper disposed at both ends of the hollow fiber filament. The hollow fiber filaments pass through the sealing block, and are respectively communicated with the culture liquid chamber at the inlet end of the culture liquid and the culture liquid chamber at the outlet end of the culture liquid. Typically, the ends of the hollow fiber filaments do not extend beyond the outside of the sealing block, respectively, preferably flush with the outside of the sealing block (i.e., the side facing the culture fluid chamber). The sealing block is sealingly fitted to the outer wall of the hollow fiber filament passing therethrough. At the same time, the contact surface of the sealing block and the middle layer is also sealingly fitted. After the sealing between the upper layer, the lower layer and the intermediate layer bonding surface, the culture liquid chamber and the cell cavity are formed by the sealing stopper and the inner wall of the upper layer, the inner wall of the lower layer, and the side wall of the hollow portion.

The middle layer culture solution inlet can be connected to the upper layer of the culture liquid inlet through a processing flow path built in the upper layer, and the middle layer culture liquid outlet can communicate with the upper layer of the culture liquid outlet through a processing flow path built in the upper layer. The middle layer culture liquid inlet and the middle layer culture liquid outlet may be respectively connected to the culture liquid chambers at the inlet end and the outlet end through a processing flow path provided inside the body. In some embodiments, the processing flow path between the culture liquid chamber outlet and the middle layer culture liquid outlet is disconnected in the middle, and two interfaces are formed on the middle layer, and the pump line is driven by the culture liquid path placed outside the cell culture module body. Connected.

The cell liquid inlet is connected to the cell cavity inlet through a processing flow path disposed inside the middle layer, and the cell liquid outlet communicates with the cell cavity outlet through a processing flow path disposed inside the middle layer, and finally communicates with the outer space of the hollow fiber filament of the cell cavity, and the cell The liquid inlet and the cell liquid outlet are connected to each other through the cell fluid path to drive the pump line.

When the culture liquid is circulated, the culture liquid is introduced from the upper culture liquid inlet, flows into the middle culture liquid inlet through the filtration device built in the upper layer, and then enters the culture liquid chamber at the inlet end of the culture liquid chamber through the middle processing flow path. Flowing through the hollow inner cavity of the hollow fiber filament, entering the culture liquid chamber at the outlet end, passing through the processing flow path from the outlet of the culture liquid chamber, sequentially flowing out the outlet of the middle layer culture liquid and the outlet of the upper culture liquid, and flowing back to the culture liquid module, thereby forming a closed Culture medium circulation loop. When the cell culture module is provided with the culture liquid path driving pump pipeline, the culture liquid flows out of the outlet of the culture liquid chamber, and after leaving a section of the processing flow path, enters the culture liquid path to drive the pump pipeline, and then flows through the processing flow path, and sequentially enters The middle layer culture solution outlet and the upper layer culture solution outlet are returned to the culture liquid module, thereby forming a closed culture liquid circulation loop. As the culture fluid flows through the hollow fiber mass, nutrients flow through the micropores in the hollow fiber filament wall to the outside of the hollow fiber filament to nourish cells outside the hollow fiber. The cell fluid is in the processing flow path between the cell liquid inlet, the cell liquid inlet and the cell cavity inlet, the cell cavity inlet, the hollow fiber filament space of the cell cavity, the cell cavity outlet, the cell cavity outlet and the cell liquid outlet. The circuit, the cell fluid outlet, and the cell fluid circuit drive the circulation in the pump line.

2, 3, 4 and 5 respectively show the overall structure and the structure of each part of a cell culture module example (cell culture module 2) of the present invention. The cell culture module 2 includes an upper layer 201, a middle layer 203 and a lower layer 204, all made of a biocompatible material. The three layers of the bonding surfaces are tightly bonded and the bonding sites are bonded together using an effective adhesive or ultrasonic welding to prevent leakage and/or gas leakage.

The upper layer 201 includes an upper culture fluid inlet 205, an upper culture fluid outlet 206, a socket 207, and a chip pull 208. The upper culture solution inlet 205 and the upper culture solution outlet 206 are the inlet and outlet of the culture medium into and out of the cell culture module 2. The slot 207 is used to position the insertion of the culture fluid module. Filter means 205b and 206b may be provided in the culture solution inlet 205 and the culture solution outlet 206, respectively. The filtering device is a liquid filtering device with a built-in filter membrane, which can be used to avoid contamination that may be introduced by puncture of the medical rubber stopper. The chip pull 208 can be used to facilitate the removal of the culture fluid module.

The middle layer 203 is a central hollow frame structure having a certain thickness, and the frame structure is provided with a middle layer culture liquid inlet 211, a culture liquid chamber inlet 211a, a middle layer culture liquid outlet 212, a culture liquid chamber outlet 212a, and a culture liquid chamber outlet 212a. The processing flow path 9a between the middle layer culture solution outlet 212, the processing flow path 10a between the cell liquid inlet 215 and the cell chamber inlet 215a, the culture liquid path driving pump line 213, the cell liquid path driving pump line 214, and the cell chamber inlet 215a , a cell fluid inlet 215, a cell lumen outlet 216a, and a cell fluid outlet 216. In the middle layer 203 shown in FIG. 4, the processing flow path between the culture liquid chamber outlet 212a and the middle layer culture liquid outlet 212 is disconnected, and two openings are formed in the outer wall of the middle layer for connecting the culture liquid path driving pump line. Both ends of the 213.

The cell culture module 2 also includes a hollow fiber block 202 embedded in the hollow portion of the middle layer 203. Figures 3 and 4 show an exemplary hollow fiber block 202 structure. The hollow fiber block 202 includes a plurality of hollow fiber filaments 209 placed side by side and a seal stop 210 at both ends of the hollow fiber filaments 209. Sealing stop 210 can be made of any suitable biocompatible material. Each of the hollow fiber filaments 209 passes through the sealing stopper 210 at both ends, and the passing portion is sealingly fitted to the sealing stopper. Typically, the portion of the hollow fiber filament that passes through the seal stop is flush with the outside of the seal block. Each of the sealing blocks is in sealing contact with the contact surface of the intermediate layer 203. A space is left between the two ends of the sealing block 210 and the middle layer 203 to form the culture liquid chamber 12 and the culture liquid chamber 14. Located between the culture fluid chambers 12 and 14, the cell chamber 13 housing the hollow fiber filaments 209.

FIG. 5 shows the structure of another hollow fiber block 202a, including hollow fiber filaments 209 and sealing stops 210. The hollow fiber filaments 209 are bent at 180° U-shape so that the two culture fluid chambers are located on the same side of the cell lumen. Similarly, the two culture fluid chambers are still separated by a sealing stopper, which may be integrally formed with the sealing stopper 210 or may be a separate sealing stopper, but it is required to separate the two culture liquid chambers. No leakage. When the hollow fiber block shown in Fig. 5 is used, other structures of the cell culture module, including the respective inlet and outlet and each processing flow path, can be provided as described above.

Generally, taking FIG. 4 as an example, the culture solution enters from the upper culture solution inlet 205, and passes through the processing flow path built in the upper layer 201 to communicate with the middle layer culture liquid inlet 211 of the middle layer 203, and enters the culture through the culture liquid chamber inlet 211a through the processing flow path. The liquid chamber 12 flows through the hollow inner cavity of the hollow fiber filament 209, enters the culture liquid chamber 14, flows from the culture liquid chamber outlet 212a through the culture liquid passage to drive the pump line 213, enters the processing flow path 9a, and finally passes through the middle layer culture liquid outlet. The 212 and upper culture solution outlets 206 are returned to the culture fluid module 3, thereby forming a closed culture fluid circulation circuit 9. As the culture fluid flows through the hollow fiber filaments 209, the nutrients flow out of the hollow fiber filaments through the micropores in the hollow fiber filament wall to nourish the cells located outside the hollow fiber filaments.

On the other hand, outside the hollow fiber filament, a cell-liquid circulation circuit 10 is formed. This circulation circuit is composed of a space outside the hollow fiber filament of the cell chamber 13, a processing flow path 10a communicating with the space, and a cell liquid path driving pump line 214. The cytosol enters from the cell fluid inlet 215, flows through the processing channel 10a, enters the hollow space of the cell cavity 13 through the cell cavity inlet 215a, and then flows out from the cell cavity outlet 216a, and flows out of the cell liquid outlet 216 through the processing flow path to enter. The cell fluid circuit drives the pump line 214 and then flows into the cell fluid inlet 215 to form the circuit.

The whole culture process is constructed by vascular culture of hollow fiber filaments, and the filaments 209 are built in the cell chamber 13, and the culture solution flows from the one culture fluid chamber 12 to the other culture chamber 14 by the filaments 209, and the flow is administered to the cells. A certain shearing force, and ensure that the liquid in the cell cavity also has a certain fluidity, effectively mimics the biological environment in which the immune cells grow, and also improves the efficiency of the exchange of the culture fluid.

Culture fluid module

The culture fluid module mainly comprises a storage chamber structure for storing fresh cell culture fluid, and may be a disposable bioreactor container, such as a disposable bioreactor bag or a bio memory chip. The culture fluid module is mainly used for providing the culture medium to the cell culture module, and has two interfaces, wherein the first interface is a storage liquid outlet for communicating with the culture liquid inlet of the cell culture module; the second interface is a storage liquid inlet for It is connected to the culture solution outlet of the cell culture module. The volume of the storage chamber is 0.6 to 2 L, preferably 0.8 to 1.5 L.

In certain embodiments, the culture fluid module has a gas permeable membrane that is gas impermeable and allows the extramembranous gas molecules to pass through the membrane in the culture fluid in the storage chamber to ensure the dissolved oxygen content of the culture fluid.

In some embodiments, the culture fluid module is composed of an upper layer, a middle layer and a lower layer, and the upper layer and the middle layer, the middle layer and the lower layer bonding surface are hermetically sealed, and no liquid leakage is caused. A gas permeable membrane is sandwiched between the upper layer and the middle layer and/or a gas permeable membrane is sandwiched between the middle layer and the lower layer. Preferably, the upper, middle and lower layers and the gas permeable membrane are all made of a transparent biocompatible material. The inlet and outlet of the storage liquid are all disposed in the middle layer, and are respectively connected to the inlet and outlet of the storage chamber of the middle layer through the preset processing pipeline. The storage liquid inlet and the storage liquid outlet are sealed by a medical rubber stopper. The liquid injection needle type bidirectional interface is used for liquid supply, and the medical rubber plug of the cell culture module and the culture liquid module is directly penetrated at the same time. In certain embodiments, a liquid filtration device is also provided at the outlet of the storage chamber and the inlet of the storage chamber, respectively.

In certain embodiments, the culture fluid module also has a filling port that can be sealed with a medical rubber stopper for use in the filling of the initial culture fluid. Preferably, a liquid filtering device is also provided at the filling opening.

The gas can enter the cell culture medium through the gas permeable membrane on the culture fluid module. The gas permeable membrane can provide a large area of gas-liquid contact and can provide a high oxygen transfer coefficient at a lower gas flow rate.

2 and 6 show the overall structure and the structure of each part of a culture liquid module example (culture liquid module 3) of the present invention, respectively. The culture fluid module 3 includes an upper layer 301, a middle layer 303, and a lower layer 304, all made of a biocompatible material. A gas permeable membrane 302 is disposed between the upper layer 301 and the middle layer 303, and between the middle layer 303 and the lower layer 304. The storage chamber 305 is formed between the three layers and between the layer block and the gas permeable membrane, and an effective adhesive or ultrasonic wave is used at the joint portion. The welding is combined and there is no leakage or/or air leakage. The culture fluid module 3 has a storage liquid outlet 306 and a storage liquid inlet 307, and is connected to the storage chamber outlet 306a and the storage chamber inlet 307a of the middle layer 303 through a predetermined processing line. When the liquid is supplied, the hollow injection needle type bidirectional interface 311 is used to directly penetrate the sealed medical rubber plug connecting the storage liquid outlet 306 and the culture liquid inlet 205, the storage liquid inlet 307 and the culture liquid outlet 206, respectively, for rehydration, and at the same time, Guarantee good sealing. A liquid filtering device 309 is provided at the storage chamber outlet 306a and the storage chamber inlet 307a. The layer 303 of the culture fluid module 3 further includes a filling port 310 for initial filling of the culture liquid, and a liquid filtering device 309 is also provided at the filling port 310.

The culture fluid module can be placed above the cell culture module or inserted into the module 207 of the cell culture module to secure the module. The chip tab 308 facilitates the installation or removal of the module.

Liquid circuit drive module

The liquid path driving module is used to drive the liquid to continuously flow into and out of the cell culture module and/or the culture liquid module at a constant flow rate, thereby ensuring circulation of the flow path in the entire system. The liquid path drive module typically contains means for driving the flow of liquid, preferably a pinch peristaltic pump. More preferably, the peristaltic pump is a bi-directional peristaltic pump. Generally, the liquid path driving module includes two driving devices respectively located on the culture liquid circulation circuit and the cell liquid circulation circuit for driving the flow of the culture liquid and the flow of the cell liquid.

As shown in FIG. 1, the liquid path driving module 4 includes a first driving device 401 and a second driving device 402. The first driving device 401 is located on the culture fluid circulation circuit 9, and the second driving device 402 is located on the cell fluid circulation circuit 10.

As shown in FIG. 8, the first driving device peristaltic pump 401 is fixedly fixed to the culture liquid path driving pump line 213 for controlling the circulation of the culture liquid between the cell culture module and the culture liquid module; the second driving device peristaltic pump 402 The clamping is fixed to the cell liquid path driving pump line 214 for circulating the cell liquid.

Generally, the rotational speed of the peristaltic pump is usually controlled in the range of 10 to 50 r/min, for example, 20 to 30 r/min.

Gas module

The gas module is mainly used to transport a mixed gas into the chip cavity 1, and the mixed gas usually contains air, oxygen, carbon dioxide and the like. Among them, oxygen is necessary for cell metabolism, and the ventilation needs to increase with the increase of the volume and cell concentration of the bioreactor (ie cell culture module), but the higher oxygen content will also be toxic to the cells, so the oxygen concentration It is precisely controlled by sensors.

Generally, the gas module includes an intake pipe and an air outlet pipe, one end of the intake pipe is for communicating with the air inlet of the culture device, and the other end is for communicating with the gas container; the air outlet pipe is for communicating with the air outlet of the culture device. A gas control valve is arranged on the intake pipe to control the delivery of gas to the culture device through the opening or closing of the gas control valve. A filter device may also be disposed on the intake pipe and the outlet pipe, and a gas filter is disposed therein. A filter device on the intake manifold is typically disposed between the gas control valve and the inlet end of the culture device.

When gas is required to be delivered, one end of the inlet pipe is connected to the gas container, and a gas control valve is opened to deliver the desired gas.

The gas can be a premixed gas. In this case, one end of the intake pipe may be directly connected to a vessel containing the premixed gas.

In certain embodiments, the gas is not premixed. In this case, the gas module also includes a mixer. The mixer is disposed between the gas control valve of the intake pipe and the filtering device (if any), and is connected to a gas container respectively containing a gas to be mixed (such as compressed air, oxygen, and carbon dioxide). Gas control valves are respectively disposed in the passages of the gas containers and the mixer, and the gas in the gas containers is controlled to be conveyed to the mixer by the opening or closing of the gas control valves. Therefore, the number of gas control valves may be 1 to 5, for example, 1 to 3.

In certain embodiments, the gas module further comprises one or several gas containers, such as 1-5 or 1-5 gas containers.

Figures 1 and 8 show an example of a gas module of the present invention. Specifically, the gas module 5 includes an intake pipe 501 and an air outlet pipe 507, wherein one end of the intake pipe 501 communicates with the intake hole of the chip cavity 1, and the other end communicates with the mixer 502, and the compressed air 504, the oxygen 505, and the carbon dioxide 506 are both Connected into the gas mixer 502. A filter device 508 is installed in both the intake pipe 501 and the outlet pipe 507, and a gas filter is disposed in the device. The control module 8 controls the opening and closing of the third control switch, that is, the compressed air, oxygen, carbon dioxide gas control valve 503, so that each gas is mixed in the gas mixer 502 and enters the chip cavity. When a plurality of gas control valves 503 are present, a corresponding number of third control switches can be provided.

Temperature module

The temperature module is used to maintain a suitable cell culture temperature throughout the chip cavity. The main body of the temperature module is a semiconductor temperature control sheet, which can be heated or cooled, and continuously supplied with a stable heat dissipation. Preferably, a temperature sensing element platinum resistor is mounted on the semiconductor temperature control sheet. As shown in Figures 1 and 8, the temperature module 6 is mounted on the inside of the chip cavity 1 and is controlled by a fourth control switch of the control module 8.

Control module

The control module includes a first control switch, a second control switch, a third control switch, and a fourth control switch. The first control switch is configured to control a driving device between the culture liquid module and the cell culture module, and the cell culture module, the driving module and the culture fluid module form or not form a loop through the opening and closing of the first control switch. Or controlling the flow rate or flow rate of the culture solution; the second control switch is for controlling the second driving device between the cell liquid inlet and the cell liquid outlet of the cell culture module, and the cell culture is performed by opening and closing the second control switch a loop that forms or does not form a circulation between the inlet and the outlet, or controls the flow rate or flow rate of the cell liquid; a third control switch for controlling the gas delivery to the device chip cavity; and a fourth control switch for controlling the temperature module The temperature rise or temperature drop of the semiconductor chip.

The control module may further include a corresponding control circuit for controlling the first control switch, the second control switch, the third control switch, and the fourth control switch. Generally, the first control switch is used to control the first driving device 401, the second control switch is used to control the second driving device 402, the third control switch is used to control the gas control valve 503, and the fourth control switch is used to control the temperature. Module semiconductor chip 6. The control module further includes a display for displaying the operation state of the device according to a predetermined program operation condition and an environmental value in the real-time acquisition device. The display can be a touch screen display. Alternatively, the control module may include a control panel including a control button for controlling opening and closing of the first control switch, the second control switch, the third control switch, and the fourth control switch. Or the system control module can include both a display and a control button. Through the control module, the device of the invention can precisely control the whole cell culture process and realize fully automated immune cell culture.

Sensor module

In certain embodiments, the cell culture system herein further comprises a sensor module. More specifically, the sensor module is used to detect environmental parameters in a cell culture system including, but not limited to, temperature, oxygen concentration, carbon dioxide concentration, and liquid pH.

1 and 8 exemplarily show a sensor module 7 of the cell culture system herein, including a temperature sensor 701, an oxygen concentration sensor 702, a carbon dioxide concentration sensor 703, and a liquid pH sensor 704. The temperature sensor 701 is for detecting the temperature at the position of the culture chip; the oxygen concentration sensor 702 is for monitoring the concentration of the oxygen gas in the chip cavity 1; the carbon dioxide concentration sensor 703 is for monitoring the concentration of the carbon dioxide gas in the chip cavity 1; the liquid pH sensor 704 It is a non-contact sensor for monitoring the pH of the liquid in the culture chip.

Culture chip

The present invention also provides a culture chip consisting of the cell culture module and the culture fluid module described herein, both of which are connected by respective interfaces. Specifically, the culture solution inlet and the culture solution outlet of the cell culture module are respectively connected to the storage solution outlet and the storage solution inlet of the culture solution module, for example, through a hose or a hollow needle-type bidirectional interface.

For example, FIG. 2 shows a schematic structural view of a culture chip of the present invention, comprising a cell culture module 2 and a culture solution module 3, and the culture solution module 3 is inserted into the cell culture module 2 via a slot on the cell culture module 2. The storage liquid outlet of the culture liquid module is connected to the culture liquid inlet of the cell culture module, and the storage liquid inlet is connected with the culture liquid outlet of the cell culture module to form a complete culture chip.

Cell culture chassis

Also provided herein is a cell culture chassis comprising: a chassis housing, a fluid circuit drive module, a control module, and a chip cavity as described herein integrated in or on the chassis. In certain embodiments, the cell culture housing further includes a temperature module and a sensor module integrated into the interior of the chassis.

The chassis can be any suitable size and shape and can be made of any suitable material.

The liquid path driving module includes a first driving device and a second driving device. The drive means is disposed on the liquid circulation circuit described herein and may be a pinch peristaltic pump, preferably a bi-directional peristaltic pump.

The control module may include: a first control switch for controlling the first driving device; a second control switch for controlling the second driving device; a third control switch for controlling gas delivery; and a fourth control switch for Controls the temperature rise or temperature drop of the temperature module. The third control switch may be provided according to the number of gas control valves, for example, 1 to 5 or 1 to 3 third control switches.

The chip cavity may be a container independent of the chassis, made of a metal material such as stainless steel or transparent plexiglass, or a space in the chassis. When the chassis is closed, the space is only provided through the chassis. The air inlet and the air outlet are in communication with the outside. A suitable fixing structure can be arranged in the chip cavity to facilitate fixing the modules disposed in the cavity, such as a cell culture module, a culture fluid module, a temperature module, and a sensor module. When the chip cavity is a separate container, a suitable fixing structure can be provided outside the chip cavity and/or in the chassis to fix the chip cavity in the chassis. When the chip cavity is a separate container, the container may be provided with an air inlet hole and an air outlet hole for communicating with the air inlet hole and the air outlet hole of the chassis to communicate with the gas module. When the chip cavity is a sealable space in the chassis, the temperature module and sensor module are typically integrated into the chassis.

The temperature module includes a semiconductor temperature control sheet and a temperature sensing element platinum resistor mounted thereon for controlling heating or cooling. The sensor module includes a temperature sensor, an oxygen concentration sensor, a carbon dioxide concentration sensor, and a liquid pH sensor.

A positive panel can also be arranged on the chassis, and a display screen and a control button can be installed thereon for real-time display of the working operation of the instrument and the control operation of the instrument.

Intake and vents may also be provided in the chassis for communication with the gas modules described herein. In some embodiments, one or more (e.g., 1-5 or 1-3) gas control valves may also be integrated on the chassis, the opening or closing of which is controlled by a corresponding number of third control switches.

Figure 7 is a schematic view showing the structure of a portion of the cell culture cabinet of the present invention. The cell culture cabinet 101 of FIG. 7 includes a chip cavity 1, a see-through window 102, an incubator flap 103, a heat dissipation hole 105, a first driving device 401, a second driving device 402, a display screen 801, and a control button 802.

Cell culture device

The cell culture device of the present invention comprises the cell culture module, the culture liquid module, the liquid path driving module, the gas module, the temperature module and the control module described herein, and the connection between the culture liquid module, the cell culture module, and the liquid path driving module. Pipeline. In certain embodiments, the cell culture device further comprises a sensor module. In the device, the cell culture module and the culture solution module are connected through respective interfaces, for example, the culture solution inlet and the culture solution outlet of the cell culture module are respectively connected with the storage liquid outlet and the storage liquid inlet of the culture liquid module, for example, through a hose or a hollow. The needle-type two-way interface is connected.

Generally, the liquid path driving module, the gas control valve, the temperature module, the sensor module and the control module in the gas module described herein can be integrally installed in the casing of the cell culture device, and the display and the control button are externally mounted on the chassis. The cell culture module and the culture fluid module can be placed in the chip cavity of the chassis.

In certain embodiments, the cell culture module, the culture fluid module, the temperature module, and the sensor module are placed within the chip cavity. In these embodiments, the fluid circuit drive module, the gas control valve of the gas module, and the control module are integrated into the chassis.

The chip cavity is a small environmental space for cell culture, which is used to construct a closed environment for culturing the chip, and is convenient for controlling the temperature and gas composition conditions of the cultured chip environment. The cell culture module, the culture fluid module and its associated communication lines can be placed in the chip cavity. The temperature module and sensor module can also be mounted in the chip cavity. An air inlet hole and an air outlet hole may be disposed on the chip cavity for communicating with the gas module.

The chip cavity may be a separate container made of a metal material such as stainless steel or a transparent plexiglass; or a space in the cell of the cell culture device, which only passes through the air inlet and the vent when the case is closed Externally connected. A suitable fixing structure can be arranged in the chip cavity to facilitate fixing the modules disposed in the cavity, such as a cell culture module, a culture fluid module, a temperature module, and a sensor module. When the chip cavity is a separate container, a suitable fixing structure can also be provided outside the chip cavity and/or in the chassis to secure it in the chassis. When the chip cavity is a separate container, the container may be provided with an air inlet hole and an air outlet hole for communicating with the gas module through the air inlet hole and the air outlet hole in the chassis.

1 shows a schematic structural view of a cell culture apparatus of the present invention, including a chip cavity 1, a cell culture module 2, a culture fluid module 3, a liquid path driving module 4, a gas module 5, a temperature module 6, a sensor module 7, and a control module 8.

Figure 8 shows the overall structure and internal structure of the cell culture device. The culture chip is inserted into the cell culture device 101 of FIG. 8, and includes a chip cavity 1, a cell culture module 2, a culture solution module 3, a gas module 5, a temperature module 6, a control module 8, sensor components 701-704, and a first driving device. 401. The second driving device 402, the display screen 801 and the control button 802, and the flip switch 104. The gas module 5 includes an intake pipe 501, an air outlet pipe 507, a compressed air container 504, an oxygen container 505, a carbon dioxide container 506, a gas mixer 502, a gas control valve 503, and a filtering device 508. Figure 8 also shows the circulation loops 9 and 10. When the incubator flap door 103 is opened, the culture chip is placed or taken out, and when closed, a sealed chip cavity space for accommodating the culture chip is formed. The flap switch 104 is used to control the opening or closing of the incubator flap door 103. The heat dissipation holes 105 are used for heat dissipation of components inside the cell culture incubator. The display 801 and the control button 802 installed on the front panel of the chassis can display the working status of the instrument and the control operation of the instrument in real time.

Set of boxes

The invention also provides a kit comprising the cell culture module and culture fluid module described herein, or a culture chip as described herein. The kit may also include a connecting tube, such as a connecting tube that forms a broth drive pump line and a cell fluid drive pump line. The cell culture module, the culture solution module or the culture chip and the connection tube may all be disposable consumables. The connecting tube is detachable from the cell culture module and the culture fluid module.

The kit may also include a cell culture chassis as described herein that may integrate all of the other components than the cell culture module and the culture fluid module described herein, including but not limited to liquid circuit drive modules, gas modules. Gas control valve, temperature module, sensor module and control module. If necessary, the kit can also be equipped with a connecting tube (snorkel) and an optional gas mixer. The gas tube can be provided with a filtering device with a gas filter.

However, in some embodiments, when the chip cavity is a container independent of the chassis, preferably, only the liquid path driving module, the gas control valve and the control module in the gas module are integrated in the chassis, wherein the cell culture module The culture fluid module, the temperature module, and the sensor module are preferably disposed within the separate chip cavity. In these aspects, the kit may include:

a chassis integrated with a liquid path driving module, a gas control valve, and a control module;

Chip cavity

a cell culture module, a culture fluid module, a temperature module, and a sensor module;

Connecting pipe.

In some embodiments, a temperature module and a sensor module can be integrated into a separate chip cavity. Therefore, the kits in this article include:

a chassis integrated with a liquid path driving module, a gas control valve, and a control module;

a chip cavity integrated with a temperature module and a sensor module;

a cell culture module and a culture fluid module, or a culture chip described herein;

Connecting pipe.

Upon receipt of the kit of the present invention, those skilled in the art can mount the components of the kit to the chassis in accordance with the instructions described herein or in accordance with the instructions provided with the kit, in conjunction with the cell culture apparatus described herein.

All of the cell-contacting materials of the cell culture devices described herein use disposable biocompatible materials and are specially sterilized and sterilized. As used herein, when referring to a hermetic fit, meaning airtight and/or leaking, it is possible to achieve airtightness and/or leakage by conventional means in the art, for example, using a suitable binder. .

Training method

The method of performing cell culture using the cell culture apparatus herein includes the following steps:

(1) providing a culture solution and a culture chip previously added to the cell suspension to be cultured;

(2) turning on the first control switch, the second control switch, the third control switch and the fourth control switch to form a culture liquid circulation circuit and a cell liquid circulation circuit; controlling temperature and gas component concentration of the chip cavity sealing space;

(3) After the culture is completed, the cell resuspension reagent is added, the cell culture module 2 cell liquid outlet is opened, the first control switch, the third control switch and the fourth control switch are turned off, and the second control switch is turned on to open the cell control chamber 13 Mature cells are harvested into a recovered container.

Specifically, a certain amount of cell culture fluid is preliminarily contained in the culture chamber 12, the cell chamber 13, and the culture chamber 14. Before starting the culture, the cell suspension to be cultured and the cell growth factor were added to the cell chamber 13 and thoroughly mixed. The culture medium module 3 is inserted from the slot 207 of the cell culture module 2, and the storage chamber outlet 306 and the storage chamber inlet 307 are directly penetrated by the hollow injection needle type bidirectional interface 311 to directly penetrate the upper layer of the culture solution inlet 205 and the upper layer of the culture solution outlet 206. Plug and replenish the culture solution. At the same time, the control module 8 is activated to control the peristaltic pump 401 to be in a forward open state, and the culture fluid module 3 and the cell culture module 2 form a culture fluid circulation loop. The culture solution in the storage chamber 305 of the culture medium module 3 flows out through the storage chamber outlet 306, enters the culture liquid chamber 12 through the upper culture medium inlet 205 and the middle layer culture liquid inlet 211, and flows into the culture solution chamber through the hollow fiber filament 209 tube. , flowing through the culture liquid path to drive the pump line 213. The culture fluid is driven by the peristaltic pump 401 of the liquid path driving module 4, and the culture liquid flows back into the storage chamber 305 via the processing flow path 9a, the intermediate layer culture solution outlet 212, the upper layer culture solution outlet 206, and the storage chamber inlet 307. This process is carried out cyclically, and fresh culture fluid in the storage chamber 305 is continuously passed through the cell cavity 13 through the hollow fiber filaments 209 to participate in the exchange of the culture liquid. The control module 8 controls the peristaltic pump 402 to be in a forward open state, and the cell fluid inlet 215 and the cell fluid outlet 216 of the cell chamber 13 communicate with the processing flow path 10a in the form of a circulating circuit to ensure that the cells in the culture have a certain fluidity. At this time, the sensor module 7 turns on the sensors 701, 702, 703, and 704, and monitors the temperature, oxygen concentration, carbon dioxide concentration, and pH value in the chip cavity 1 in real time. During cell culture, the control system 8 collects temperature data within the chip cavity 1 to control the temperature rise or temperature drop of the semiconductor chip of the temperature module 6. The control system 8 controls the opening/closing states of the respective gas control valves 503 of oxygen, carbon dioxide, and air based on the data collection of the oxygen and carbon dioxide concentrations in the chip cavity 1. The control system 8 adjusts the flow rates of the peristaltic pumps 401 and 402 or prompts the replacement of the new culture fluid module 3 based on the feedback of the pH value. When the culture liquid module 3 needs to be replaced, the incubator flap 103 is opened, and when the culture chip is taken out, the grasping chip pull port 308 removes the existing culture liquid module 3 from the slot 207 of the cell culture module 2, and then wipes with medical alcohol. After the surface of the medical rubber plug of the cell culture module is disinfected, the new culture liquid module 3 can be reinstalled. After the completion of the culture, the cells are resuspended, and the cell culture solution 2 cell outlet is opened to harvest the mature cells in the cell chamber 13.

The present apparatus and the present method can be used to culture various cells known in the art, preferably various immune cells. Cells include, but are not limited to, DC cells, various T cells such as CIK cells, CTL cells, TIL cells, CAR-T cells, and the like.

Claims (23)

1. A cell culture module comprising:

   a closed body that communicates with the outside only through the culture fluid inlet, the culture fluid outlet, the cell fluid inlet, and the cell fluid outlet;

   a culture liquid chamber and a cell chamber located inside the body, wherein the culture liquid chamber includes independent culture liquid chambers respectively communicating with the culture liquid inlet and a culture liquid chamber communicating with the culture liquid outlet, the cell chamber and the cells The liquid inlet is connected to the cell liquid outlet;

   a sealing stopper for separating the cell cavity and the culture fluid cavity; and

   a hollow fiber filament placed in a cell cavity having micropores that do not allow passage of cells; wherein the hollow fiber filament passes through the sealing stopper, and both ends of the hollow fiber filament are respectively cultured with the inlet end of the culture fluid The liquid chamber and the culture solution chamber at the outlet end of the culture solution are both connected.
2. The cell culture module according to claim 1, wherein

   The culture fluid chamber is located on both sides of the cell cavity, separated by a sealing block, and the hollow fiber filaments pass through the cell cavity to connect the culture liquid chambers on both sides of the cell cavity; or the culture liquid cavity is located on the same side of the cell cavity, two The culture fluid chambers and between the culture fluid chamber and the cell chamber are separated by a sealing stopper.
3. The cell culture module according to claim 1 or 2, wherein

   The culture liquid chamber communicating with the inlet of the culture liquid has a culture liquid chamber inlet, and the culture liquid chamber inlet is in communication with the culture liquid inlet; the culture liquid chamber communicating with the culture liquid outlet has a culture liquid chamber outlet, and the culture The liquid chamber outlet is in communication with the culture fluid outlet;

   Wherein, the culture liquid chamber inlet is disposed in the body, and is connected to the culture liquid inlet through a processing flow path in the body; the culture liquid chamber outlet is disposed in the body, and the cell culture module further includes a processing flow path disposed inside the body and optionally a culture liquid path provided outside the body to drive a pump line, the culture liquid chamber outlet passing through the processing flow path and the optional culture liquid path A drive pump line is in communication with the culture fluid outlet.
4. The cell culture module according to any one of claims 1 to 3, wherein

   The cell chamber has a cell lumen inlet and a cell lumen outlet, respectively connected to the cell fluid inlet and the cell fluid outlet; wherein the cell lumen inlet and the cell lumen outlet are both disposed inside the body, and the cell culture module further comprises a cell liquid path driving pump circuit disposed outside the body, wherein the cell cavity inlet and the cell cavity outlet are respectively connected to the cell liquid inlet and the cell liquid outlet through a processing flow path disposed inside the body, The cell fluid inlet is connected to the cell fluid outlet through the cell fluid path to drive the pump line.
5. The cell culture module according to claim 1, wherein

   The cell culture module has a culture liquid flow path and a cell liquid flow path; wherein the culture liquid flow path includes a culture liquid inlet, a culture liquid chamber at the inlet end, a hollow fiber filament inside, a culture liquid chamber at the outlet end, and a culture liquid outlet; The cell fluid flow path in turn includes a cell fluid inlet, a hollow fiber extrafilament space in the cell cavity, and a cell fluid outlet.
6. The cell culture module according to claim 4, wherein

   The cell culture module has a culture liquid flow path and a cell liquid flow path; wherein the culture liquid flow path includes a culture liquid inlet, a processing flow path, a culture liquid chamber inlet, a culture liquid chamber at the inlet end, a hollow fiber filament inner portion, and an outlet end. The culture liquid chamber, the culture liquid chamber outlet, the processing flow path, the optional culture liquid path drive pump line and the culture liquid outlet; the cell liquid flow path includes the cell liquid inlet, the processing flow path, the cell cavity inlet, and the cell cavity in sequence. The hollow fiber outer space, the cell cavity outlet, the processing flow path, the cell liquid outlet, and the cell liquid path drive pump line.
7. The cell culture module according to claim 1, wherein the body of the cell culture module is composed of an upper layer, a middle layer, and a lower layer, wherein:

   The upper layer and the middle layer, the middle layer and the lower layer bonding surface are tightly combined and do not leak liquid;

   The upper layer is provided with an upper culture liquid inlet and an upper culture liquid outlet, respectively connected to the culture liquid chamber disposed in the middle layer; and a processing flow path connecting the upper culture liquid inlet and the middle layer culture liquid inlet, and the upper culture liquid outlet and the middle layer culture Processing flow path of the liquid outlet;

   The middle layer is a central hollow frame structure, and the frame structure is provided with a middle layer culture liquid inlet connected to the upper layer culture liquid inlet, a culture liquid chamber inlet, a middle layer culture liquid inlet and a culture liquid chamber inlet processing flow path, The culture liquid chamber outlet, the middle layer culture liquid outlet connected to the upper culture liquid outlet, the processing flow path between the culture liquid chamber outlet and the middle layer culture liquid outlet, and the cell liquid inlet, the cell chamber inlet, the cell liquid inlet and the cell chamber inlet a processing flow path between the processing flow path, the cell lumen outlet, the cell fluid outlet, and the cell lumen outlet and the cell fluid outlet;

   The culture fluid chamber and the cell lumen are disposed in the hollow portion of the middle layer; and

   The hollow fiber filament is placed in a cell cavity, and both ends thereof pass through the sealing stopper and are respectively connected to the culture fluid chamber; and

   Optionally, a culture liquid path disposed outside the body drives a pump line, and the culture liquid path drives a pump line to connect a processing flow path disconnected between the culture liquid chamber outlet and the middle layer culture liquid outlet; and/or

   Optionally, a cell fluid circuit disposed outside the body drives a pump line that drives the pump line to communicate with the cell fluid inlet and the cell fluid outlet.
8. The cell culture module according to claim 7, wherein

   The culturing fluid path driving pump line and the cell liquid path driving pump line are medical hoses; and/or

   The upper layer is further provided with a slot and a chip tab, wherein the slot is used for positioning and inserting a culture fluid module, and the chip tab is for conveniently taking out the culture fluid module; and/or

   The sealing block is sealingly adhered to the contact surface of the middle layer, and the upper layer, the lower layer and the middle layer bonding surface are hermetically sealed, and the culture liquid is formed by the sealing block and the inner wall of the upper layer, the inner wall of the lower layer and the side wall of the hollow portion. Cavity and cell cavity; and/or

   Providing a liquid filtering device between the upper culture liquid inlet and the middle culture liquid inlet and between the upper culture liquid outlet and the middle culture liquid outlet to avoid contamination introduced by the puncture medical rubber plug; preferably, the filter pore diameter is 0.2 μm.
9. The cell culture module according to claim 1, wherein

   The volume of the culture solution chamber is 0.2 to 0.5 L, preferably 0.3 to 0.35 L; and/or

   The cell chamber has a volume of 0.4 to 1 L, preferably 0.6 to 0.8 L; and/or

   The hollow fiber filament has a length of 130-170 mm, a tube wall thickness of 20-50 μm, and a pore diameter of the micropores on the tube wall is less than 1 μm; and/or

   The body and the sealing block are transparent biocompatible materials; and/or

   The culture solution inlet and the culture solution outlet are sealed by a medical rubber stopper.
10. A culture fluid module comprising:

    a closed body that communicates with the outside only through the storage liquid inlet and the storage liquid outlet;

    a storage chamber disposed within the body, wherein the storage chamber is in communication with a storage fluid inlet and a storage fluid outlet; and

    A gas permeable membrane that allows gas molecules to pass through to seal the storage chamber.
11. The culture fluid module according to claim 10, wherein

    The volume of the storage chamber is 0.6 to 2 L, preferably 0.8 to 1.5 L; and/or

    The body and the gas permeable membrane are both transparent biocompatible materials; and/or

    The storage chamber inlet and the storage chamber outlet are sealed by a medical rubber plug; and/or

    The storage chamber communicating with the storage liquid inlet has a storage chamber inlet, and the storage chamber inlet is connected to the storage liquid inlet through a preset processing pipeline; the storage chamber communicating with the storage liquid outlet has a storage liquid chamber outlet, The storage chamber outlet is in communication with the storage liquid outlet through a predetermined processing line; and/or

    A liquid filtering device is provided on the inlet and outlet of the storage chamber for filtering and blocking impurities and bacteria from entering the storage chamber.
12. The culture fluid module according to claim 10, wherein

    The culture fluid module is composed of an upper layer, a middle layer, a lower layer and a gas permeable membrane, wherein the upper layer and the middle layer, the middle layer and the lower layer bonding surface are tightly combined and do not leak liquid, wherein a gas permeable membrane and/or a middle layer are disposed between the upper layer and the middle layer. a gas permeable membrane is provided between the lower layer and the lower layer;

    Preferably, the inlet and outlet of the storage liquid and the inlet and outlet of the storage chamber are both disposed in the middle layer, and the liquid injection needle type bidirectional interface is used for liquid supply, and the medical rubber plug of the cell culture module and the culture liquid module is directly penetrated at the same time.

    Preferably, the culture liquid module further has a filling port, and the interface is sealed by a medical rubber plug for filling the initial culture liquid; preferably, the liquid filling device is further disposed at the filling port.
13. A cell culture chip comprising the cell culture module according to any one of claims 1 to 9 and the culture solution module according to any one of claims 10 to 12.
14. A cell culture device comprising:

    The cell culture module according to any one of claims 1-9;

    The culture fluid module according to any one of claims 10-12;

    Liquid circuit drive module;

    Gas module

    Temperature module

    Control module; and

    Connect the pipes or lines of each module.
15. The cell culture apparatus according to claim 14, wherein the liquid path driving module comprises:

    a first driving device for controlling the circulation of the culture solution between the culture fluid module and the cell culture module; and

    a second driving device for controlling the circulation of the cell liquid in the cell cavity of the cell culture module;

    Preferably, the first driving device is disposed on the communication line of the storage liquid outlet and the culture liquid inlet, or is disposed on the communication line of the culture liquid outlet and the storage liquid inlet; or the cell culture module includes an external culture The liquid circuit drives the pump line, and the first driving device is disposed on the culturing liquid path driving pump pipeline;

    Preferably, the second driving device is disposed on the cell liquid path driving pump pipeline;

    Preferably, the first drive means and the second drive means are both pinch peristaltic pumps, preferably bi-directional peristaltic pumps.
16. The cell culture apparatus according to claim 14 or 15, wherein the gas module comprises: an intake pipe, an outlet pipe, a gas control valve, and an optional gas container and an optional gas mixer;

    Preferably, the gas container comprises a container for containing compressed air, oxygen and carbon dioxide, each container being in communication with a gas mixer, the inlet pipe being in communication with the gas mixer;

    Preferably, a filter device is arranged in the inlet pipe of the intake pipe and the outlet pipe, and a gas filter membrane is arranged in the device;

    Preferably, a gas control valve is respectively disposed on the pipeline connecting the container for containing compressed air, oxygen, carbon dioxide and the gas mixer, and the gas control valve is provided with a gas flow meter.
17. The cell culture apparatus according to claim 14 or 15, wherein said temperature module comprises a semiconductor temperature control sheet and a temperature sensing element platinum resistor mounted thereon for controlling heating or cooling.
18. The cell culture apparatus according to claim 14 or 15, wherein the control module comprises:

    a first control switch for controlling the first driving device;

    a second control switch for controlling the second driving device;

    a third control switch for controlling gas delivery;

    a fourth control switch for controlling the temperature module to perform heating or cooling;

    Preferably, the control module further comprises a control circuit for real-time monitoring and control of the entire cell culture process to achieve automated cell culture.
19. The cell culture apparatus according to claim 14, wherein said cell culture apparatus further comprises a sensor module for monitoring temperature, oxygen concentration, carbon dioxide concentration, and pH value in real time; preferably, said sensor module includes a temperature sensor , oxygen concentration sensor, carbon dioxide concentration sensor and liquid pH sensor.
20. The cell culture apparatus according to claim 14, wherein

    The cell culture module, the culture fluid module, and the sensor module are placed in a sealable chip cavity having an air inlet and an air outlet; and/or

    The liquid path driving module, the gas control valve, the temperature module and the control module in the gas module are integrally installed in the chassis, and the air inlet hole and the air outlet hole are arranged in the chassis, and the cell culture module and the culture liquid module are arranged inside the chassis. The sensor module has a sealable chip cavity, and a man-machine exchange interface and control buttons are installed outside the chassis.
21. A culture apparatus according to claim 14, wherein

    The culture solution is a serum-free medium dedicated to immune cells; and/or

    The cell is selected from the group consisting of peripheral blood mononuclear cells, neural stem cells or tumor cells; and/or

    The materials in contact with the cells in the device are all disposable biocompatible materials and are sterilized and sterilized.
22. A kit comprising the cell culture module according to any one of claims 1 to 9 and the culture solution module according to any one of claims 10 to 12, or the cell of claim 13. Train the chip.
23. A kit according to claim 22, further comprising a fluid path driving module according to claim 15, a gas control valve, the temperature module according to claim 17, and claim 18 The chassis of the control module and the disposable culture consumables include pipes or lines connecting the modules.

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