CN107400633A

CN107400633A - Doughnut exchanger and doughnut switch type culture systems

Info

Publication number: CN107400633A
Application number: CN201710708156.9A
Authority: CN
Inventors: 钱其军; 周进; 师传胤; 颜开冬; 朱星宇; 张琴; 王子龙
Original assignee: Shanghai Cell Therapy Engineering Technology Research Center Group Co Ltd; Shanghai Cell Therapy Research Institute; Shanghai Baize Medical Instrument Co Ltd
Current assignee: Shanghai Baize Medical Instrument Co ltd; Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2017-08-17
Filing date: 2017-08-17
Publication date: 2017-11-28
Anticipated expiration: 2037-08-17
Also published as: CN107400633B

Abstract

The present invention relates to doughnut exchanger and doughnut switch type culture systems.The doughnut exchanger of the present invention includes outer wall pipe, the hollow fibre filament being arranged on inside outer wall pipe, the sealing fixture block and end cap and interface for being arranged on outer wall pipe both ends；Sealing fixture block is arranged on inside at the port at outer wall pipe both ends, fills between all fibres silk and the gap between filament and outer wall pipe, is bonded with the inner tight of outer wall pipe and plays a part of sealing；End cap is used for outer enclosed wall pipe both ends, and filament internal buffer is formed between sealing fixture block and end cap；Flow path space in filament is formed by the inner space of hollow fibre filament, forms flow path space outside filament between hollow fibre filament outer wall, outer wall inside pipe wall and sealing fixture block, flow path space is connected with filament internal buffer in filament.The culture systems of the present invention meet cell large scale and high density culture, and operation automation, stability are good, security reliability is high, pollution risk is small.

Description

Hollow fiber exchanger and hollow fiber exchange type culture system

Technical Field

The invention belongs to the field of cell culture, and particularly relates to a hollow fiber exchanger and a hollow fiber exchange type culture system.

Background

The biological treatment technology is a new molecular and cell biological technology emerging at the end of the last century, and has good effect and application prospect when being applied to biological immunotherapy. In recent years, with the rapid expansion of the technical contents related to the biological immunotherapy, part of the techniques in which immune cells are used for therapy are starting to be put into practical use gradually, and some of the existing cell therapy products in the countries of the united states, europe, japan, and the like have been successfully put into clinical use through pharmaceutical approval. The biological immunotherapy is a treatment method for resisting tumors by greatly enhancing the tumor recognition and killing functions by infusing immune cells collected and extracted from blood in a patient body into the patient body after being modified by a specific antigen loading or transgenic technology and being amplified and cultured in vitro. According to the current research, the biological immunotherapy needs to realize a certain treatment effect, and a certain amount of cells need to be returned in the treatment course, but the prior art needs to invest a large amount of manpower and time for the expansion and culture of immune cells, and has higher requirements on the operation environment and the proficiency of operators.

The cell mass culture technique is a technique for producing a biological product by artificially providing a cell culture expanded carrier and a growth environment thereof in a cell bioreactor or a cell culture factory to perform high-density mass culture of cells. The technology is widely applied to the fields of cytology, genetics, immunology, experiments, clinical medicine and the like. The principle of the method is that the method provides necessary conditions for large-scale in-vitro culture and amplification of cells, including space conditions, liquid conditions, gas conditions, temperature conditions and clean conditions for cell growth, and specifically comprises specific cell culture containers, basic cell culture solution, cell growth factors, specific gas concentration, pH value of the culture solution, environmental temperature and other conditions.

The current cell culture methods can be divided into static culture and dynamic culture. Traditional static cell culture generally adopts cell culture dishes, cell culture bottles or multi-hole culture plates and the like to culture in a sterile culture room or an incubator. Static culture is difficult to realize high-density cell culture because cell growth metabolic waste is easy to accumulate, and a small-scale culture container can only meet the requirement of small-scale low-density cell culture due to space limitation. The dynamic cell culture can replace basic culture solution and add cell growth factors and the like in a culture container, mainly adopts a shake flask, a cell culture tank or hollow fibers and the like to culture in an aseptic environment, is mostly applied to large-scale production of engineering cell strains to obtain antibodies or other recombinant proteins expressed by the engineering cell strains, has a larger difference with the requirement of cells in clinical level, for example, the shake flask culture has a wider container opening, the pipeline needs to be cleaned and sterilized before each production in the ordinary cell tank culture, and has higher cross contamination risk, moreover, the dynamic culture mode is more suitable for the engineering cell strains enduring a high-speed shaking table and a stirring paddle, and the primary cells require milder culture conditions, different from the collection of expression products of the engineering cell strains, and the immune cell culture collection cells are used as final products, it is therefore desirable that the cells are always in an optimal state.

At present, the types of immune cells can be divided into two types, namely suspension cell culture and adherent cell culture according to the mode and condition of cell growth culture. The existing hollow fiber culture system is designed specially and integrally aiming at certain type of cells, the application range is narrow, the universality is not strong, and a plurality of limitations are placed on the culture mode, and the scale of the cell culture can not be further increased, so that the culture system suitable for immune cell culture in the market is rare. And with the standardization of the national immune cell therapy regulation, higher requirements are necessarily put on an automatic closed immune cell culture system.

CN100337698C relates to an isolated artificial liver cell supporting device, which is specially used for specific detoxification metabolism and biological action of blood, and comprises a culture solution circulating and flowing mechanism and a patient plasma circulating and flowing mechanism, wherein the device detoxifies and purifies the patient plasma through high-activity liver cells in a reactor under the condition that a small amount of normal human plasma leaves a human body. However, the design of the biological support device is only to ensure the detoxification and purification effects of high-activity hepatocytes on blood, and the flow path design is incomplete to exchange waste generated by metabolism of large-scale hepatocytes during operation, so that the activity of the hepatocytes is greatly influenced.

CN158703A relates to a micro cell hollow fiber reactor, which is characterized in that cells are embedded in hollow fiber filaments by collagen gel, then the hollow fiber filaments are put into a silica gel tube made of permeable silica gel material, and two ends of the hollow fiber filament are sealed by permeable silica gel plugs. The device can culture cells to a certain scale by means of the oxygen transfer and mass transfer capabilities of the fiber yarns, and has good significance for research of medicines. The device is only suitable for small-scale cell culture, and the requirement of large-scale high-density cell culture is difficult to meet.

CN104321419A relates to a cell culture system and culture method, the system is composed of a cell culture tank, a composition adjusting liquid storage tank, a culture liquid composition adjusting component, a perfusion component and its related connecting pipelines. The system transfers the culture solution in the composition adjusting solution storage tank to the cell culture tank through the perfusion component for culture solution exchange. In the system, the importance of the culture solution is considered when cells are cultured in a large scale and a high density, and the conveying and replacement mode of the culture solution is mainly designed, but the type of cell culture is not designed in the system, and other partial conditions such as gas exchange and the like during culture are not mentioned.

CN104017727A relates to a disposable aseptic cell culture bag culture system, and the device comprises stirring unit, support pipeline, temperature control unit, gas circuit control unit and controller unit. The system is mainly designed aiming at the gas control part in cell culture, the stirring unit is provided with the stirring blade which is integrally designed with the hollow gas distributor, so that the dissolved oxygen in the culture solution can be effectively improved, but the shearing force of the stirring system is large, and the stirring system is mainly used for industrial large-scale production of stable cell lines.

Disclosure of Invention

Provided herein is a hollow fiber exchange culture system for solving the above-described problems, which is capable of satisfying large-scale high-density culture of multiple types of cells, particularly immune cells. The hollow fiber exchange culture system aims at five environmental elements of container, liquid, gas, temperature and sterility required by cell growth and proliferation, and comprises a culture container, a sealed flow path system and complete liquid path replacement, gas path replacement and temperature control. Because the cell growth and proliferation are all carried out in the culture solution, the liquid path replacement is more important to realize large-scale high-density culture of cells and solve the problem that the metabolic waste generated during cell growth in the culture solution is easy to accumulate. The liquid path replacement of the system of the invention mainly adopts hollow fibers to continuously replace the culture solution in the culture container. Besides liquid path replacement, the system also has a good gas replacement, temperature control and automatic control system, carries out real-time monitoring, data acquisition and effective feedback on the whole culture process, ensures the stability of the culture environment, can meet the requirement of large-scale high-density culture of cells, and also has the advantages of automatic operation, good stability, high safety and reliability and small pollution risk. Another flow path switching hollow fiber exchanger proposed herein can provide a larger volume of fluid path displacement, higher efficiency, and wider application.

Accordingly, a first aspect herein provides a hollow fiber exchanger comprising an outer wall tube, hollow fiber filaments disposed inside the outer wall tube, seal cartridges and end caps disposed at both ends of the outer wall tube, and a port;

the sealing fixture blocks are arranged in the ports at the two ends of the outer wall pipe, fill all gaps among the fiber yarns and between the fiber yarns and the outer wall pipe, are tightly attached to the inside of the outer wall pipe and play a role in sealing;

the end covers are used for sealing two ends of the outer wall pipe, and a fiber yarn inner buffer area is formed between the sealing fixture block and the end covers;

in the hollow fiber exchanger, a fiber yarn inner flow path space is formed by an inner space of a hollow fiber yarn, a fiber yarn outer flow path space is formed among an outer wall of the hollow fiber yarn, an inner wall of an outer wall pipe and a sealing clamping block, and the fiber yarn inner flow path space is communicated with a fiber yarn inner buffer area.

In one or more embodiments, the hollow fiber filaments are hollow filaments having walls with micropores that are not capable of passing through cells.

In one or more embodiments, the hollow fiber filaments are co-axially disposed with the outer wall tube, not beyond the outside of the sealing cartridge, and preferably flush with the outside of the sealing cartridge.

In one or more embodiments, the sealing block is disposed not to exceed the end ports of the outer wall pipe, and preferably, the distance from the end ports of the outer wall pipe is in the range of 2-20 mm.

In one or more embodiments, the interfaces include two interfaces disposed on the end caps at both ends, and two interfaces disposed on the outer wall tube near both ends.

In one or more embodiments, the hollow fiber exchanger further comprises a flow path switching cartridge disposed between the sealing cartridge and the end cap.

In one or more embodiments, the flow path switching cartridge is in close fitting and sealing engagement with the outer wall tube.

In one or more embodiments, a separation region is disposed on the flow path conversion fixture block, and the separation region divides a buffer region between the flow path conversion fixture block and the sealing fixture block into a filament inner buffer region and a filament outer buffer region.

In one or more embodiments, the inner fiber buffer area is communicated with the inner fiber flow path space, and the outer fiber buffer area is communicated with the outer fiber flow path space through a through hole formed in the sealing fixture block.

In one or more embodiments, the flow path conversion block is provided with two through holes arranged at 180 ° and respectively corresponding to the inner fiber buffer area or the outer fiber buffer area for communicating the inner fiber buffer area or the outer fiber buffer area.

In one or more embodiments, the flow path switching block is provided with a flow path switching groove which is a 360 ° circular ring space inner groove.

In one or more embodiments, in the hollow fiber exchanger provided with the flow path conversion fixture block, 2 connectors are respectively arranged on two side end covers, each connector is respectively provided with an outer side port and an inner side port, the outer side port is used for connecting an external pipe, and the inner side port is used for penetrating through the through hole on the flow path conversion fixture block and communicating the fiber yarn inner buffer area or the fiber yarn outer buffer area.

In one or more embodiments, the inner port edge has a raised transition edge that rotates 180 ° in the flow path transition slot during flow path transition to interchange the filament buffer regions with which the existing ports communicate.

In one or more embodiments, a compression spring installed in a spring hole is further arranged between the end cover and the flow path conversion clamping block, and when an inner side port of the interface penetrates through a through hole of the flow path clamping block and is communicated with an inner fiber buffer area and an outer fiber buffer area, the compression spring can provide pre-tightening force, so that the conversion edge is tightly attached to the flow path conversion clamping block, and liquid leakage is prevented; the rear side edge of the flow path conversion clamping block is also provided with an elastic buckle which is used for being matched with a clamping position arranged on the end cover.

A second aspect of the present disclosure provides a hollow fiber exchange culture system comprising: the device comprises a cell culture unit, a hollow fiber unit, a culture solution displacement unit, a gas displacement unit, a pipeline unit and a system master control unit, wherein the pipeline unit and the system master control unit are communicated with the cell culture unit, the hollow fiber unit, the culture solution displacement unit and the gas displacement unit.

In one or more embodiments, the hollow fiber unit comprises a hollow fiber converter as described in any of the embodiments herein.

In one or more embodiments, the cell culture unit comprises: the culture container is provided with an outlet and an inlet which are communicated with the hollow fiber unit, namely a culture container outlet and a culture container inlet; an outlet and an inlet which are communicated with the gas replacement unit, namely a gas replacement outlet and a gas replacement inlet; and optionally a sensor mounting hole, an alternative interface and/or a filter element disposed at the outlet and inlet.

In one or more embodiments, the cell culture unit further comprises a liquid path driving device disposed on the communication line between the culture vessel outlet and the hollow fiber unit, the culture vessel inlet and the hollow fiber unit.

In one or more embodiments, the cell culture unit further comprises a mixing vessel and its connections and tubing in communication with the culture vessel, and/or a harvesting vessel and its connections and tubing in communication with the culture vessel.

In one or more embodiments, the cell culture unit further comprises a fluid path driving device disposed on the communication line between the culture vessel and the mixing vessel, the culture vessel and the harvest vessel.

In one or more embodiments, the culture vessel is placed on a support that can swing through a range of angles.

In one or more embodiments, the culture solution replacement unit includes: a replacement container, wherein an outlet and an inlet which are communicated with the hollow fiber unit, namely the outlet and the inlet of the replacement container, and an optional spare interface and/or a filter element arranged at the outlet and the inlet are arranged on the replacement container.

In one or more embodiments, the culture solution replacement unit further comprises a solution path driving device arranged on a communication pipeline between the outlet of the replacement container and the hollow fiber unit, and between the outlet of the replacement container and the hollow fiber unit.

In one or more embodiments, the culture solution replacement unit further comprises a fresh culture solution container and/or a waste solution container, which are in communication with the replacement container through a connection part and a pipe.

In one or more embodiments, the culture solution replacement unit further comprises a solution path driving device disposed on a communication line between the replacement vessel and the fresh culture solution vessel, and between the replacement vessel and the waste solution vessel.

In one or more embodiments, the gas replacement unit comprises a gas source, a mixer, a gas outlet pipe orifice and a filter, a gas pipeline, a flow meter and a control valve; wherein, the blending device is directly communicated with the gas replacement inlet of the culture container.

In one or more embodiments, the hollow fiber exchange culture system further comprises a temperature control unit disposed in a fixed jacket capable of accommodating the entire culture system, for presetting, regulating and maintaining the temperature during the culture process.

In one or more embodiments, the system general control unit is used for controlling the hollow fiber exchange culture system to operate according to a preset scheme, and performing data acquisition, data analysis and data recording functions, and comprises a control circuit and user software.

In one or more embodiments, the system general control unit includes a display for controlling the operation of the hollow fiber exchange culture system according to a preset scheme and displaying the operation state of the hollow fiber exchange culture system in real time.

A third aspect of the present disclosure provides the use of the hollow fiber exchanger and hollow fiber exchange culture system described herein in adherent cell culture, suspension cell culture, and mixed culture of adherent cells and suspension cells.

Drawings

FIG. 1 is a schematic view showing a cell culture system of the present invention.

FIG. 2 is a schematic view showing a cell culture system of the present invention in which a flow path switching type hollow fiber exchanger is used.

FIG. 3 is a sectional view of a culture vessel 5 showing the cell culture unit of the present invention.

Fig. 4 is a schematic view showing the structure (partial sectional view 1/4) of a conventional hollow fiber exchanger according to the present invention.

Fig. 5 is a schematic view showing the structure of a flow path switching type hollow fiber exchanger according to the present invention (partially cut-away section 1/4).

Fig. 6 is an enlarged partial sectional view showing an end cap of a flow path switching hollow fiber exchanger according to the present invention.

Fig. 7 is a schematic sectional view showing the structure 1/4 of the flow path switching block 37 of the flow path switching type hollow fiber exchanger according to the present invention.

FIG. 8 is a graph showing the cell density change of the mixed culture of DC-CIK cells in the examples.

FIG. 9 is a graph showing the change in cell viability of the mixed culture of DC-CIK cells in the examples.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) can be combined with each other to constitute a preferred technical solution.

The hollow fiber exchange culture system of the present invention comprises: the device comprises a cell culture unit, a hollow fiber unit, a culture solution displacement unit, a gas displacement unit, a pipeline unit and a system master control unit, wherein the pipeline unit and the system master control unit are communicated among the cell culture unit, the hollow fiber unit, the culture solution displacement unit and the gas displacement unit.

Cell culture unit

The cell culture unit comprises a culture vessel. The culture vessel is used for mixing primary cells, cell growth factors, a primary cell culture solution, and the like, and can be used as a space for cell culture proliferation. Thus, a culture vessel suitable for use in the present invention may be a disposable cell culture bag or flask or the like as is common in the art. Typically, culture vessels are used for the culture of suspension cells.

The culture vessel is provided with an outlet and an inlet (referred to as a culture vessel outlet and a culture vessel inlet) communicating with the hollow fiber unit. The liquid in the culture vessel flows into the hollow fiber unit through the culture vessel outlet, and flows back into the culture vessel through the culture vessel inlet after the hollow fiber unit exchanges with the fresh culture solution. The culture vessel is also provided with an outlet and an inlet (referred to as a gas replacement outlet and a gas replacement inlet) which communicate with the gas replacement means. Gas from the gas replacement unit enters the culture container through the gas replacement inlet to replace the gas in the culture container and adjust the gas composition; the gas replacement outlet is a passage through which gas is discharged from the culture vessel.

The outlet and inlet of the culture container and the gas replacement outlet and inlet of the culture container may be provided with filter members. The filter may be used to block the passage of cells, but allow the passage of culture fluid and/or gas. Generally, the filter members provided at the outlet and inlet of the culture vessel allow the culture solution and gas to pass therethrough, while the filter members provided at the gas replacement outlet and inlet allow only the gas to pass therethrough. The filter element can be directly and fixedly arranged on the corresponding interface, or flexibly arranged or not arranged according to the experimental requirements of users in a connecting buckle or thread mode. The filter piece can be made of materials which can block cells and are not easy to adhere to the surfaces of the filter piece, such as polyethylene, polyether sulfone, nylon, cellulose and the like, and the average pore diameter of the filter piece can be in the range of 1-10 mu m.

Sensor mounting holes and/or alternative interfaces may also be provided on the culture vessel. The sensor mounting hole can be used for mounting related monitoring sensors, such as a pH sensor, a dissolved oxygen sensor and the like, and is used for detecting the environmental index conditions such as the pH value, the dissolved oxygen value and the like of the culture solution in the culture container. Other specific types of sensors can be installed according to actual needs. Alternative interfaces may be used to interface with other associated containers or add liquid reagents.

The culture vessel may be placed on a support. The support can swing within a certain angle range to provide the condition that accords with suspension cell growth, guarantee the composition homogeneity of culture solution in the culture container, avoid the concentration of local position discarded object too high, improve the area of contact of gas and culture solution in the culture container.

The cell culture unit may further comprise a mixing vessel and its connecting parts and conduits communicating with the culture vessel, and/or a harvesting vessel and its connecting parts and conduits communicating with the culture vessel. The mixing vessel is used for storing the starting cells, the cell growth factors and the starting cell culture solution to be supplied to the culture vessel separately, or is used for mixing the starting cells, the cell growth factors and the starting cell culture solution, etc. and then supplying them to the culture vessel. The harvest container is used to collect and hold the cells that have been cultured last or to detect samples of the cells during the culturing process. A liquid path driving device can be arranged between the culture container and the mixing container and between the culture container and the harvesting container to ensure the normal circulation of the liquid path.

Culture solution replacement unit

The culture solution replacement unit includes a replacement vessel for containing and storing a quantity of fresh culture solution. The replacement container may be a disposable cell culture bag or flask, etc. as is common in the art. The displacement vessel is provided with an outlet and an inlet which are communicated with the hollow fiber unit and are respectively called as an outlet and an inlet of the displacement vessel. Fresh culture solution in the replacement container enters the hollow fiber unit through the outlet of the replacement container, replacement occurs in the hollow fiber unit, and replaced culture solution flows back to the replacement container through the inlet of the replacement container. The replacement container may also be provided with a spare interface for docking with other associated containers or for adding liquid reagents. A filter element may be provided at the outlet and inlet of the displacement vessel to block the passage of cells but allow the passage of culture fluid and/or gas. The filter element can be directly and fixedly arranged on the corresponding interface, or flexibly arranged or not arranged according to the experimental requirements of users in a connecting buckle or thread mode. The filter piece can be made of materials which can block cells and are not easy to adhere to the surfaces of the filter piece, such as polyethylene, polyether sulfone, nylon, cellulose and the like, and the average pore diameter of the filter piece can be in the range of 1-10 mu m.

The culture solution replacement unit may further include a fresh culture solution container and/or a waste solution container, which may be communicated with the replacement container through respective connecting members and pipes. The fresh culture solution container is used for storing fresh cell culture solution; the waste container is used for recovering and storing the spent culture solution in the replacement container. After a part of the volume of the liquid in the replacement container is periodically recovered to the waste liquid container, the same volume of culture solution can be supplemented from the fresh culture solution container to the replacement container so as to ensure the concentration of the effective components of the culture solution in the replacement container. The arrangement of the fresh cell culture solution container is beneficial to increasing the replacement volume of the cell culture solution, realizing multiple and staged replacement and also facilitating the establishment of full-closed and full-automatic culture (namely, the cell culture solution is only added once on the premise of calculating the required cell culture solution in advance). Liquid path driving devices can be arranged between the replacement container and the fresh culture solution container and between the replacement container and the waste liquid container to ensure the normal circulation of the liquid path.

An optional spare interface can also be provided on the replacement container for docking with other associated containers or adding liquid reagents.

Hollow fiber unit

The hollow fiber unit is a place where the exchange of culture solution is completed, is a core component of liquid path replacement, and is also a place for culturing adherent cells. The hollow fiber unit comprises a hollow fiber exchanger, and the hollow fiber exchanger comprises an outer wall pipe, hollow fiber filaments arranged inside the outer wall pipe, and sealing clamping blocks and end covers arranged at two ends of the outer wall pipe. The end caps, which are herein provided at both ends of the outer wall pipe, may be open caps having a certain depth, and may be hermetically connected to the outer wall pipe by means of screw threads or the like. The material of the outer wall tube may be a biocompatible material commonly used in the art for cell culture. The hollow fiber filament may be polypropylene, polysulfone, polyacrylonitrile, polyethylene, etc. with high biocompatibility.

The hollow fiber filaments are parallel and in the same direction with the outer wall tube and are hollow filaments, and micropores with the aperture smaller than 1 mu m are distributed on the wall of the hollow fiber filaments. The molecular weight cut-off of the micropores can be in the range of 10KDa to 1500KDa, exchange of small molecular substances (such as ions, small molecular nutrients and gas molecules) can be carried out, but the micropores can not pass through cells. The hollow fiber has a length of 50-300 mm, a fiber wall thickness of 30-50 μm, and a membrane inner diameter of 100-500 μm. The number of the hollow fiber filaments is not limited, and may be determined according to the actual production scale, for example, may be in the range of 100 to 1000.

Sealing fixture blocks are respectively arranged in the port parts at the two ends of the outer wall pipe, and the sealing fixture blocks are filled in gaps among all the fiber yarns and between the fiber yarns and the outer wall pipe and are tightly attached to the inside of the outer wall pipe to play a role in sealing. The sealing clip may be formed using a sealant commonly used in the art, including but not limited to epoxy, polyurethane, and the like. Generally, the thickness of the sealing block is not particularly limited, and may be in the range of 2-10 mm, and the sealing block is tightly attached to the outer wall pipe and the outer wall of the hollow fiber filament to prevent liquid or gas from infiltrating.

The hollow fiber filaments do not extend beyond the outer side of the sealing jaw in the length direction, and are preferably flush with the outer side of the sealing jaw. The outer side of the sealing fixture block is arranged not to exceed the ports at the two ends of the outer wall pipe, and the distance between the sealing fixture block and the ports at the two ends of the outer wall pipe is preferably within the range of 2-20 mm. When the flow path switching block is provided in the hollow fiber exchanger as described below, the distance between the outer side of the sealing block and the end of the outer wall tube may be deeper, for example, up to 30mm, so as to leave a buffer height of 2-20 mm between the sealing block and the flow path switching block. After the end cover is installed on the outer wall pipe, a fiber filament inner buffer area is formed between the outer side of the sealing fixture block and the inner wall of the cover surface of the end cover, and the buffer area is a buffer area with uniform liquid flowing pressure, so that the flowing pressure in each hollow fiber filament is ensured to be the same, and the liquid replacement efficiency and uniformity of the exchanger assembly are improved. In certain embodiments, after the end cap is installed on the outer wall tube, its facing inner wall is flush with the outer wall tube port, thereby forming a filament inner buffer zone from the facing inner wall, the outer wall tube inner wall, and the outside of the sealing cartridge together. Under the condition that the outer side of the sealing fixture block is flush with the port of the outer wall pipe, the inner buffer area of the fiber filament can be formed by the end cover and the outer side of the sealing fixture block. Of course, in certain embodiments, the formed inner buffer region of the filament may be comprised of a portion of the interior space on the port side of the outer wall tube and a portion of the interior space of the end cap.

The hollow fiber exchanger is divided into a fiber yarn inner flow path space and a fiber yarn outer flow path space according to the fiber yarn inner and outer spaces, and the fiber yarn inner flow path space and the fiber yarn outer flow path space are communicated only through micropores on the fiber yarn wall. The internal flow path space of the fiber yarn is communicated with the internal buffer area of the fiber yarn.

The end covers at both ends can be respectively provided with an interface communicated with the outlet (inlet) of the culture container of the cell culture unit, and the positions of the outer wall pipe close to both ends are respectively provided with an interface communicated with the outlet (inlet) of the replacement container of the culture solution replacement unit. The angle between the two interfaces on the outer wall tube, as seen from a top view of the outer wall tube, may be between 0 ° and 180 °. The interface communicated with the culture container outlet of the cell culture unit is connected with the culture container outlet of the cell culture unit through a connecting part and a pipeline; the interface communicated with the culture container inlet of the cell culture unit is connected with the culture container inlet of the cell culture unit through a connecting part and a pipeline; the interface communicated with the replacement container outlet of the culture solution replacement unit is connected with the replacement container outlet of the culture solution replacement unit through a connecting part and a pipeline; the port communicating with the inlet of the replacement vessel of the culture solution replacement unit is connected to the inlet of the replacement vessel of the culture solution replacement unit through a connecting member and a pipe.

The cell culture unit is communicated with the hollow fiber unit to form a circulating cell culture loop; the culture solution replacement unit is communicated with the hollow fiber unit to form a circulating replacement solution loop. In the cell culture circuit, the mixed solution in the culture container flows through the space of the fiber internal flow path of the hollow fiber exchanger and then flows back into the culture container. In the replacement liquid circuit, the fresh culture liquid in the replacement container flows through the space of the hollow fiber exchanger fiber outer flow path and then flows back into the replacement container. The cell culture loop and the replacement liquid loop are replaced by the culture liquid through the micropores in the hollow fiber filaments, and cell metabolites in the mixed liquid are taken away, wherein the flow path direction of the cell culture loop and the replacement liquid loop in the hollow fiber exchanger forms an included angle of 0-180 degrees, preferably an included angle of 0 or 180 degrees (namely the same direction or the opposite direction), and more preferably 180 degrees, so that the replacement efficiency of the culture liquid is higher. The connection between the port on the end cap of the hollow fiber exchanger and the inlet/outlet of the culture vessel and the connection between the port on the outer wall tube and the inlet/outlet of the replacement vessel can be determined according to the flow path direction between the cell culture circuit and the replacement fluid circuit. For example, when the flow path has an included angle of 180 degrees, the port on the end cap communicates with the outlet of the culture vessel, and the port on the outer wall tube at the end of the hollow fiber exchanger communicates with the inlet of the replacement vessel; the interface on the other end cover is communicated with the inlet of the culture container, and the interface on the outer wall pipe of the end is communicated with the outlet of the replacement container. The flow rate of the cell culture circuit and the replacement liquid circuit is 0.2 to 5ml/min, and the flow rate of the replacement liquid circuit is usually 0.8 to 1.2 times of the flow rate of the cell culture circuit, so that effective replacement can be realized.

The hollow fiber exchanger herein may be provided as a flow-switching hollow fiber exchanger. In this type of hollow fiber exchanger, the flow path inside the exchanger can be switched as needed to increase the volume of single liquid path replacement, improve the efficiency of liquid path replacement, quickly adjust the composition of the culture solution in the culture vessel, and cope with sudden situations during culture. This type of exchanger enables cell culture, in particular when adherent cell culture is carried out, either inside or outside the fibres; even cell culture is carried out in the cellosilk and outside the cellosilk simultaneously, the space for adherent cell growth is improved, and the conversion of flow paths is often carried out during culture, the direction of a liquid flow path in each space is changed, the liquid path replacement efficiency is improved, the uniformity of cell growth in the hollow fiber is ensured, and the problem that adherent cell growth possibly blocks the micropore of the cellosilk is solved.

The flow path conversion type hollow fiber exchanger comprises hollow fiber wires, an outer wall pipe, sealing clamping blocks at the left end and the right end, flow path conversion clamping blocks and end covers. Two interfaces are respectively arranged on the end covers at the two sides, and are respectively an interface communicated with the outlet or the inlet of a culture container of the cell culture unit and an interface communicated with the outlet or the inlet of a replacement container of the culture solution replacement unit.

The flow path conversion clamping block is arranged between the end cover and the sealing clamping block, and is tightly attached to and sealed with the outer wall pipe. Typically, the exterior side of the flow path transition block may be flush with the exterior wall tube port. The flow path conversion fixture block and the sealing fixture block have a certain distance to form a buffer area. The height of the buffer zone is not particularly limited, and may be, for example, 2 to 20 mm. The flow path conversion clamping block is provided with a separation area which divides the space between the flow path conversion clamping block and the sealing clamping block into a fiber filament inner buffer area and a fiber filament outer buffer area. The fiber yarn inner buffer area is communicated with the fiber yarn inner flow path space; the outer buffer area of the fiber is communicated with the outer flow path space of the fiber through a through hole arranged on the sealing fixture block. The flow path conversion clamping block is provided with two through holes which are arranged in 180 degrees, the positions of the through holes respectively correspond to the inner fiber wire buffer area or the outer fiber wire buffer area, and the through holes are used for communicating the inner fiber wire buffer area or the outer fiber wire buffer area. The flow path clamping block is also provided with a flow path conversion groove which is a 360-degree circular space inner groove. The end covers on two sides are respectively provided with interfaces, each interface is respectively provided with an outer side port and an inner side port, the outer side ports are used for connecting external pipe fittings, and the inner side ports are used for penetrating through holes in the flow path conversion clamping block and communicating with a fiber yarn inner buffer area or a fiber yarn outer buffer area. The edge of the inner port can be provided with a convex conversion edge, and when the flow path is converted, the conversion edge rotates 180 degrees in the flow path conversion groove to exchange the fiber silk buffer area communicated with the existing interface. And a compression spring arranged in the spring hole is arranged between the end cover and the flow path conversion clamping block. In addition, the rear side edge of the flow path conversion clamping block can be also provided with an elastic buckle for matching with a clamping position on the end cover. When the through hole of the flow path conversion clamping block is penetrated through by the inner side port of the interface to be communicated with the inner buffer area of the fiber or the outer buffer area of the fiber, the compression spring can provide certain pretightening force, and meanwhile, the elastic buckle is buckled with the clamping position of the end cover, so that the position of the end cover relative to the outer wall is fixed, the conversion edge can be ensured to be tightly attached to the flow path conversion clamping block, and the liquid leakage is prevented. When the flow path conversion is needed, the cell culture loop and the replacement liquid loop are suspended, the clamping position is pressed, the elastic buckle springs out the clamping position, the end covers on the two sides are pulled outwards, the conversion edge retracts into the flow path conversion groove and then rotates 180 degrees, and the fiber silk buffer area communicated with the existing interface is exchanged.

Gas replacement unit

The gas replacement unit can comprise a gas source, a blending machine, a gas outlet pipe orifice, a filter element, a related gas pipeline, a flow meter, a control valve and the like. The gas source is used to provide air, oxygen and carbon dioxide or other suitable gases. The gas source can be one or a plurality of containers for storing required gases, and each container can store a single gas or a mixed gas with a pre-prepared proportion. The mixer is a gas buffering mixing area and is used for the place where air, oxygen, carbon dioxide and the like are independently fed or mixed and then mixed. The mixer may be a conventional gas mixing device.

The mixer of the gas replacement unit is directly communicated with the culture container, and external gas is directly input into the culture container and is provided for a gas environment required by cell culture. The gas replacement is performed above the liquid surface of the culture medium in the culture container. Gas enters the culture container from a gas replacement inlet on one side of the culture container, stays above the liquid level of the culture solution, replaces the gas in the original container, stabilizes the components of the gas in the culture container, and provides and ensures a gas environment required by cells. Displaced gas flows out of the gas displacement outlet. Flow meters and control valves are common gas flow meter and valve components in the art.

Pipeline unit

The pipeline unit of the hollow fiber exchange culture system respectively connects the cell culture unit, the hollow fiber unit, the culture solution replacement unit, the liquid path driving device and the gas replacement unit of the system, and is used for providing gas and liquid channels required by the system.

Liquid path driving device

The liquid path driving device is used for driving the flow of liquid in the pipeline, for example, for driving the circulation flow of liquid in the cell culture circuit A and the replacement liquid circuit B. The drive unit in the liquid path drive device may be a peristaltic pump, more preferably a bidirectional peristaltic pump. In this context, a bi-directional peristaltic pump means that the pump can drive fluid in a forward direction and can also drive fluid in a reverse direction. The method can be implemented by adopting a peristaltic pump commonly used in the field, and generally, the peristaltic pump with the flow rate of 1-100 ml/min can be used for implementing the method.

System general control unit

The system master control unit is used for controlling the system to operate according to a preset scheme and comprises data acquisition, data analysis, data recording, a control circuit and user software, the system monitors the cell culture process in real time through various sensors and acquires technical index data such as flow rate, pH value of culture solution, dissolved oxygen amount, temperature and the like in a circulation loop in the whole culture process. The user can adjust the executive component of the liquid path replacement, the gas replacement and the temperature control system according to the culture state of the cells, and the stability of the cell culture environment is ensured. The control system of the system master control unit can also be set to preset standard technical index numerical values and corresponding processing schemes for users, and the system can automatically select and execute the processing schemes according to real-time data fed back by the sensors, so that the culture is automatic and intelligent. In some embodiments, the system general control unit further comprises a display for controlling the operation of the culture system according to a preset scheme and displaying the operation state of the culture system in real time. The display may be a touch screen display.

The hollow fiber exchange culture system can be integrally placed in a specially-made fixed jacket, a temperature control unit which can be used for presetting, regulating and maintaining the proper temperature in the culture process is arranged in the fixed jacket, and a temperature control device of the temperature control unit can be used for heating or refrigerating through a control system according to the real-time data of a temperature sensor. The fixing jacket can be various cell culture boxes commonly used in the field, and the cell culture boxes are generally provided with temperature control units. In specific implementation, the temperature control unit can also adopt a semiconductor temperature chip system or a water circulation temperature control system.

Thus, in certain embodiments, the hollow fiber exchange culture system herein comprises a fixing jacket, a cell culture unit, a hollow fiber unit, a culture solution replacement unit, and a gas replacement unit, as well as a piping unit, a system general control unit, and a temperature control unit communicating the above units.

FIG. 1 shows an example of the hollow fiber exchange culture system herein. As shown in FIG. 1, the hollow fiber exchange culture system of the present invention comprises a cell culture unit 1, a culture medium replacement unit 2, a hollow fiber unit 3, a gas replacement unit 4, a pipeline unit and a system master control unit for communicating the above units. These units may be arranged, mounted or housed within a housing or on a support frame forming part of the system, which may be mounted and secured using conventional techniques.

The cell culture unit 1 comprises a culture vessel 5 for providing a vessel for culturing cells of the suspension type. As shown in fig. 1, 2 and 3, the culture vessel 5 may be provided with: an outlet 6 of the culture vessel, an inlet 7 of the culture vessel, an outlet 8 for gas replacement, and an inlet 9 for gas replacement. The culture vessel outlet 6 and the culture vessel inlet 7 are respectively communicated with the hollow fiber unit 3 through pipelines to form a cell culture loop A. The gas replacement inlet 9 is communicated with the blending device 32 of the gas replacement unit 4, gas in the blending device 32 enters the culture container 5 through the gas replacement inlet 9, stays above the liquid level of the culture solution, replaces the gas existing in the culture container 5, and provides and ensures a gas environment required by cells; the displaced gas then flows out of the gas displacement outlet 8. A fluid path driving device 6a and/or 7a may be provided in a communication line between the cell culture unit 1 and the hollow fiber unit 3 for driving circulation of the cell culture fluid between the cell culture unit 1 and the hollow fiber unit 3. The culture vessel 5 may be further provided with a sensor mounting hole 10 for mounting a sensor for performing related monitoring. For example, the sensor mounting hole 10 may be generally mounted with a pH sensor for monitoring the pH value of the liquid environment in the culture vessel 5 in real time and a dissolved oxygen sensor for monitoring the dissolved oxygen value of the liquid environment in the culture vessel 5 in real time. Other specific types of sensors can be installed according to actual needs. The culture container 5 can be arranged on the support 501, particularly the support 501 can swing within a certain angle range, the growth condition of suspension cells is met, the uniformity of components of the culture solution in the culture container 5 is guaranteed, the excessive high concentration of waste at local positions is avoided, and the contact area between gas and the culture solution in the culture container can be increased.

The cell culture unit 1 may further comprise: an optional mixing vessel 11 for receiving a mixed feed of the sample and the basal broth; an optional harvest vessel 12 for collecting and holding cells that have completed culturing or a test sample of cells during culturing; and an optional back-up port 5' for docking other associated containers or adding liquid reagents. The mixing container 11 and the harvesting container 12 can be respectively communicated to the culture container 5 through a connecting part and a pipeline, and liquid path driving devices 11a and 12a can be respectively arranged on the communicating pipeline to ensure the normal circulation of the liquid paths.

The cell culture unit 1 may also comprise a filter b which allows the passage of culture liquid or gas, but not the passage of cells. The filter piece b is made of a material with an average pore diameter of 1-10 mu m, which can block cells and is not easy to adhere to the surfaces of the filter piece b, such as polyethylene, polyether sulfone, nylon, cellulose and the like, and two materials of liquid passing and air passing can be selected. The filter b is arranged in the culture container 5 and comprises filter elements 6b and 7b which are respectively arranged on the culture container outlet 6 and the culture container inlet 7 and can pass liquid and gas; and filter members 8b and 9b provided on the gas replacement outlet 8 and the gas replacement inlet 9, the filter members being capable of passing only gas. The filter element b can be directly and fixedly arranged on the corresponding interface, or flexibly arranged or not arranged according to the experimental requirements of users in a connecting buckle or thread mode.

The culture solution replacement unit 2 includes a replacement vessel 13 for accommodating and storing a fresh culture solution for liquid path replacement. The replacement vessel 13 is provided with a replacement vessel outlet 14 and a replacement vessel inlet 15, which are communicated with the hollow fiber unit 3 through pipes, respectively, to form a replacement liquid circuit B. The communicating pipe may be provided with a liquid path driving device 14a and/or 15a, respectively, for driving liquid to circulate between the culture liquid substitution unit 2 and the hollow fiber unit 3. The replacement container 13 may be a disposable biological cell culture bag, a culture bottle, or the like.

The culture solution replacing unit 2 may further include: a fresh broth container 16 and a waste liquid container 17. After a part of the volume of the liquid in the replacement vessel 13 is periodically recovered to the waste liquid vessel 17, the fresh culture liquid vessel 16 is supplemented with the same volume of culture liquid in the replacement vessel 13. The fresh culture solution container 16 stores fresh cell culture solution for periodic replacement, which is beneficial to increasing the replacement volume of the culture solution, thereby realizing replacement of the culture solution by stages for many times and being convenient for establishing a fully-closed and fully-automatic culture system. The waste liquid container 17 is used for recovering and storing the spent culture liquid in the replacement container 13. The fresh culture solution container 16 and the waste solution container 17 are communicated to the replacement container 13 through connecting parts and pipelines, and corresponding solution path driving devices 16a and/or 17a can be arranged on the communicating pipelines so as to ensure the normal circulation of the solution paths. The replacement container 2 may also be provided with an optional spare interface 13' for docking with other associated containers or for adding liquid reagents.

The culture solution replacement unit 2 can also comprise a filter member b which is arranged in the replacement container 13 and comprises filter members 14b and 15b which are respectively arranged on an outlet 14 and an inlet 15 of the replacement container and can pass liquid and gas; the filter element b can be directly and fixedly arranged on the corresponding interface, or flexibly arranged or not arranged according to the experimental requirements of users in a connecting buckle or thread mode.

The hollow fiber unit 3 is a place for exchanging a culture solution, is a core component of a liquid path replacement system, and is also a place for culturing adherent cells. The hollow fiber unit 3 includes a hollow fiber exchanger 18. The hollow fiber exchanger 18 is provided with ports 19 and 20 for communicating the outlet 6 and the inlet 7 of the culture vessel, respectively, and ports 21 and 22 for communicating the outlet 14 and the inlet 15 of the replacement vessel, respectively. As shown in fig. 4, the hollow fiber exchanger 18 includes hollow fiber filaments 33, an outer wall tube 34, and a sealing block 36 and end cap 35 at both ends. The connectors 19 and 20 are respectively disposed on the two side end caps 35, and the connectors 21 and 22 are respectively disposed on the two end walls of the outer-wall pipe 34. Ports 19, 20, 21 and 22 may be provided as an outlet and an inlet, respectively, and ports at the same end are provided as an outlet and an inlet, respectively, e.g., port 19 is provided as an inlet, port 20 is provided as an outlet, port 21 is provided as an inlet, and port 22 is provided as an outlet.

The hollow fiber filaments 33 are parallel to the outer wall tube 34. The pore diameter of the micropores on the fiber is less than 1 mu m, the cut-off molecular weight is 10 KDa-1500 KDa, and the exchange of small molecular substances (such as ions, small molecular nutrient substances and gas molecules) can be carried out. The hollow fiber 33 has a length of 50 to 300mm, a fiber wall thickness of 30 to 50 μm, and a membrane inner diameter of 100 to 500 μm. The number of the hollow fibers 33 is not limited, and may be determined according to the actual production scale, and may be, for example, in the range of 100 to 1000. The volume of the hollow fiber exchanger 18 is not particularly limited, and may be determined according to the actual production scale, and may be, for example, in the range of 10 to 100 ml. The material of the fiber can be polypropylene, polysulfone, polyacrylonitrile or polyethylene with good biocompatibility and the like. Sealing blocks 36 are provided at both ends of the outer-wall pipe 34 for filling gaps between the hollow fiber wires 33 and the outer-wall pipe 34. The material of the sealing fixture block 36 may be epoxy resin, polyurethane, or other sealing glue. Typically, the hollow fiber filaments 33 are flush with the outside of the sealing dogs 36. The hollow fiber 33 is provided with a space for a fiber inner flow path. The space between the sealing fixture block 36 and the end cover 35 is a fiber filament inner buffer area 38 which is communicated with the fiber filament inner flow path space and is a buffer area of liquid flow rate, so as to ensure that the flow pressure in each hollow fiber filament is the same, and improve the efficiency and uniformity of liquid replacement of the exchanger assembly. The size of the inner buffer 38 is 1-50 ml. The space of the fiber strand inner flow path in the hollow fiber strand 33 and the inner buffer region 38 communicate with the inlet port 20 via the outlet ports 19 of the both-side end caps 35, and the cell culture circuit a. The space of the outer flow path of the hollow fiber 33 is formed between the outer wall tube 34 and the sealing block 36, and the replacement fluid circuit B is connected to the inlet 22 and the outlet 21 of the outer wall tube 34.

When the liquid path is replaced in the cell culture process, the mixed liquid in the culture container 5 flows through the fiber yarn internal flow path space of the hollow fiber exchanger 18 from the cell culture loop A and then flows back to the culture container 5; the fresh culture solution in the replacement vessel 13 flows through the space of the fiber outer channel of the hollow fiber exchanger 18 from the replacement solution circuit B and then flows back to the replacement vessel 13; the mixed solution in the space of the fiber inner flow path and the fresh culture solution in the space of the fiber outer flow path are exchanged through the micropores of the hollow fiber 33, the initial culture solution in the mixed solution is replaced by the fresh culture solution rich in cell nutrients, and the waste generated by the culture is taken out. The cell culture circuit A and the replacement fluid circuit B have flow paths in the hollow fiber exchanger 18 in opposite directions.

The gas replacement unit 4 comprises a gas source (air 26, oxygen 28, carbon dioxide 30), a mixer 32, a gas outlet nozzle and filter 24, and associated gas lines, flow meters and control valves 23, 25, 27, 29 and 31. The mixer 32 is a gas buffer mixing area and is used for the place where air, oxygen and carbon dioxide are independently fed or mixed and then mixed. The mixer can be a common gas mixing device or a metal gas tank. Gas replacement is carried out above the liquid level of the culture solution of the culture container 5, gas enters the container from a gas replacement inlet 9 at one side of the culture container 5, the gas stays above the liquid level, the gas in the original container is replaced, and a gas environment required by cell growth is provided and ensured; the gas flows out from the gas replacement outlet 8. Flow meters and control valves are common gas flow meter and valve components in the art.

FIG. 2 shows another embodiment of the present hollow fiber exchange culture system, in which the hollow fiber unit 3 employs a flow path switching hollow fiber exchanger 18', and the volume of the liquid path replacement is larger, the replacement efficiency is higher, and especially the pH value of the culture liquid environment can be rapidly adjusted, which can satisfy various conditions during the cell culture process.

As shown in FIGS. 5, 6 and 7, in this flow path switching type hollow fiber exchanger 18', two ports are provided in each side end cap 35, namely, ports 19 and 20 for communicating the outlet and inlet of the culture vessel 5, respectively, and ports 21 and 22 for communicating the outlet and inlet of the culture medium replacement unit, respectively. Unlike the flow path switching type hollow fiber exchanger 18, the flow path switching type hollow fiber exchanger 18' includes a flow path switching block 37 in addition to the hollow fiber filaments 33, the outer wall tube 34, the sealing blocks 36 at both ends, and the end cap 35. The flow path switching block 37 is disposed between the end cap 35 and the sealing block 36, closely attached to and sealed with the outer wall tube 34, and spaced from the sealing block 36 to form a buffer area. The flow path in the hollow fiber 33 pipe is a fiber flow path space; the flow paths of the outer tube, the outer wall tube 34 and the sealing block 36 of the hollow fiber 33 form a fiber outer flow path space.

The one-sided flow path switching block 37 is provided with a partition, which divides the space between the flow path switching block 37 and the sealing block 36 into two fluid regions, i.e., a filament inner buffer region 38 and a filament outer buffer region 39. The filament inner buffer region 38 is communicated with the filament inner flow path space; the fiber outer buffer area 39 is communicated with the fiber outer flow path space through the through hole 40 on the sealing fixture block 36.

The flow path conversion fixture block 37 is provided with two through holes 391 and 390 arranged in 180 degrees, the positions of the through holes correspond to the fiber yarn inner buffer area 38 or the fiber yarn outer buffer area 39 respectively, and the through holes are used for communicating the fiber yarn inner buffer area 38 or the fiber yarn outer buffer area 39. The flow path changing block 37 is provided with a flow path changing groove 42, and the flow path changing groove 42 is an inner groove of a 360-degree circular space. The ports 22 and 19 are provided on the end cap 35. Each interface is divided into an outer port and an inner port, the outer port is used for connecting an external pipe fitting, and the inner port is used for passing through a through hole 391 or 390 on the flow path conversion fixture block 37 and directly communicating with the inner fiber buffer area 38 or the outer fiber buffer area 39. The inner side port edge has a transition edge 41 projecting toward the buffer area and rotatable in a flow path transition groove 42. A pressing spring 43 is arranged between the end cover 35 and the flow path conversion block 37, 4 spring holes 392 are uniformly arranged on the flow path conversion block 37, and the pressing spring 43 is arranged in the spring holes 392. The rear edge of the flow path switching block 37 has an elastic clip 44, which is engaged with a clip 45 on the end cap 35. When the inner ports of the connectors 22 and 19 pass through the through holes of the flow path switching block 37 to communicate with the inner fiber buffer area 38 or the outer fiber buffer area 39, the pressing spring 43 will provide a certain pre-tightening force, and the elastic buckle 44 is buckled with the clamp 45 and is fixed relative to the end cover 35 of the outer wall pipe 34. At this time, the switching edge 41 is tightly attached to the flow path switching block 37, and leakage is prevented. When the flow path conversion is needed, the two sides of the end covers are required to be adjusted simultaneously, the cell culture loop A and the replacement liquid loop B are suspended, the clamping position 45 is pressed, the elastic clamping buckle 44 is bounced open, the end covers at the two sides are pulled outwards, the conversion edge 41 returns to the flow path conversion groove 42 and rotates 180 degrees, and the fiber silk buffer areas communicated with the existing interfaces are exchanged.

The materials of the above-described units, containers and pipes of the system herein are not particularly limited. Typically, pipes, containers, and the like are typically biocompatible plastics; others may be metals (e.g., stainless steel) or plastics. Each container herein may be in the form of a chamber, bottle, bag. The size of each container is not particularly limited, and may be varied depending on the object to be accommodated, and is usually in the range of 0.5 to 10L, for example, 1 to 5L.

The pipeline, the containers and all parts in each unit can be disposable consumables, and all consumable components form a closed culture system, so that fluid leakage is prevented, and the sterility of the system is guaranteed. Each container is detachable from each branch conduit, and each component between each branch conduit and each main flow conduit and in each unit. For example, all the parts in the hollow fiber unit are detachable, the end cover can be detached from the outer wall pipe, the two ends of the hollow fiber can be fixed by the sealing fixture blocks, and the fixed structure can also be detached from the outer wall pipe. Alternatively, the branch conduits may be integral with the main flow conduit and the containers may be detachable from the conduits.

The culture system comprises an automatic control culture system capable of meeting requirements of various cells, and is based on five environmental factors including a container, liquid, gas, temperature and sterility required by large-scale high-density culture of various immune cells. The hollow fiber exchange type culture system can improve the cell treatment efficiency, reduce the operation risk, enhance the cell culture effect and improve the application curative effect.

The hollow fiber exchange type culture system can be suitable for adherent cell culture or suspension cell culture, and can also be used for mixed culture of adherent cells and suspension cells, so that the problem that only a single type of cells can be cultured at present is solved, co-culture of multiple types of cells can be realized, and the hollow fiber exchange type culture system is suitable for different cell therapy applications. The cells that can be cultured using the present system can be various cells known in the art, preferably cells of mammalian origin, and more preferably human immune cells. Suitable cells include, but are not limited to, peripheral blood cells, hematopoietic cells, neural stem cells, and more specifically, include, but are not limited to, erythrocytes, neutrophils, eosinophils, basophils, peripheral blood mononuclear cells, antigen presenting cells or lymphocytes, and the like. In certain embodiments, the cells include, but are not limited to, DC-CIK (adherent DC cells versus suspension CIK cells), DC-CTL (adherent DC cells versus suspension CTL cells), DC, TIL (suspension TIL cells), and CAR-T (suspension CAR-T cells).

In the embodiment shown in FIG. 1, when only suspension cells are cultured, the cells, the starting culture medium and the cell growth factors are placed in the culture vessel 5, the outlet 6 of the culture vessel is connected to the filter members 6b and 7b of the inlet 7 of the culture vessel to block the suspension cells from flowing out of the culture vessel 5, the suspension cells are grown and proliferated in the culture vessel 5, and the culture medium replacement unit 2 is filled with a fresh culture medium; when the culture medium in the culture container 5 needs to be replaced, the cell culture circuit A and the replacement liquid circuit B are started, the culture medium in the flow path space in the fiber tube in the hollow fiber unit 3 exchanges with the fresh culture medium in the flow path space outside the fiber tube through the micropores, wastes are released, nutrient substances are obtained, and the culture state of the cells in the culture container 5 is ensured. When only adherent cells are cultured, the filtering pieces 6b and 7b are not communicated, the initial cell mixed solution is added into the hollow fiber unit 3 from the culture container 5, and the filtering pieces 6b and 7b are communicated; then placing the initial culture solution and the cell growth factors in a culture container 5, adding a fresh culture solution into the culture solution replacement unit 2, starting a cell culture loop A, and adhering adherent cells in the hollow fiber filaments for growth and proliferation; meanwhile, a replacement liquid loop B is started, and the fresh culture solution replaces the waste and the nutritive cells in the hollow fiber tube. When suspension cells and adherent cells are cultured in a mixed way, firstly, the adherent cell mixed solution is placed in the hollow fiber exchanger 18, and the filtering pieces 6b and 7b are communicated; then placing the suspension cells, the initial culture solution and the cell growth factors into a culture container 5, adding a fresh culture solution into the culture solution replacement unit 2, starting a cell culture loop A and a replacement solution loop B, and simultaneously starting temperature control and gas replacement.

In the embodiment shown in fig. 2, the same scheme as that of the above embodiment is used for the culture of only suspension cells or only adherent cells or the mixed culture of suspension cells and adherent cells, but in the scheme, the hollow fiber exchanger 18' can perform flow path switching once at regular intervals during the culture process, so that the volume of single liquid replacement is increased, and the liquid path replacement efficiency is improved.

The two different types of cells are cultured in different functional areas of the same system at the same time, so that the cell treatment efficiency is greatly improved, the operation risk is reduced, and the growth stimulating factors secreted from different cells are also beneficial to the joint growth of immune cells.

A cell culture solution suitable for use herein can be a solution that is at least capable of providing various solution components and environments for cell growth. The specific composition of the culture medium is matched with the characteristics of cultured cells. The cell culture solution can be prepared by adding components according to the types of cells, and can also be directly selected from culture media on the market in the field.

Thus, in certain aspects, also included herein is a method of culturing suspension cells using the hollow fiber exchange culture system described herein, the method comprising the steps of: providing suspension cells to be cultured and a culture solution thereof in a cell culture unit of the hollow fiber exchange culture system; a filter member for providing an outlet of the culture container and an inlet of the culture container to prevent the cells from passing therethrough; setting a fresh culture solution in the culture solution replacement unit; the cell culture circuit A and the replacement liquid circuit B are started to replace the culture liquid in the cell culture unit.

Also included herein is a method of culturing adherent cells using the hollow fiber exchange culture system described herein, the method comprising the steps of: providing adherent cells to be cultured and a culture solution thereof in a hollow fiber unit; a filter member for providing an outlet of the culture container and an inlet of the culture container to prevent the cells from passing therethrough; setting a fresh culture solution in the culture solution replacement unit; and starting the cell culture circuit A and the replacement liquid circuit B to replace the culture liquid in the cell culture unit.

In other aspects, also included herein are methods of culturing suspension cells and adherent cells using the hollow fiber exchange culture systems described herein, the methods comprising the steps of: providing adherent cells to be cultured and a culture solution thereof in a hollow fiber unit; a filter member for providing an outlet of the culture container and an inlet of the culture container to prevent the cells from passing therethrough; providing suspension cells and a culture solution thereof in a cell culture unit; setting a fresh culture solution in the culture solution replacement unit; and starting the cell culture circuit A and the replacement liquid circuit B to replace the culture liquid in the cell culture unit.

The present invention will be illustrated below by way of specific examples. It should be understood that this example is illustrative only and is not intended to limit the scope of the present invention.

Example 1: hollow fiber reactor for DC-CIK mixed culture

This embodiment is implemented using the system shown in fig. 1. The hollow fiber exchanger 18 had a volume of 50ml and a buffer of 10ml, wherein the fiber material was polypropylene, a length of 100mm, a fiber wall thickness of 40 μm, an inner diameter of 300 μm, and a molecular weight cut-off of 300 kDa.

The total amount of blood collected from the patient's periphery was about 1 × 10⁹The Peripheral Blood Mononuclear Cells (PBMC) of (4) were subjected to density gradient centrifugation, excess red blood cells and plasma were removed, and PBMC was mixed with GibcoAIM-V serum-free medium and diluted to 4 × 10⁶And (3) placing the cells at a density of one cell/ml in a culture flask, culturing for 2 hours, separating the suspension cells from the adherent cells, wherein the suspension CIK cells are firstly placed at-80 ℃ for cryopreservation, and the DC cells are subjected to transfection treatment by adenovirus (cycle is 8 days).

Recovering suspension CIK cells stored at-80 deg.C, and mixed culturing with DC cells successfully transfected to obtain antigen load according to initial cell density of 1 × 10⁶Preparing Gibco culture medium with corresponding volume per ml of cells, placing the DC cell mixed solution in a hollow fiber exchanger 18 according to adherent cells, and connecting filter elements 6b and 7 b; then, the suspension CIK cells are placed in a culture container 5, 1000ml of Gibco culture medium is added, and stimulation factors such as interferon-gamma and the like are also added. 1000ml of Gibco medium was added to the displacement vessel 13 and 1000ml of medium was placed in the fresh medium vessel 16. The culture temperature is 37 ℃, 5% CO₂Culturing in the incubator, the bracket 501 supports the culture container 5 to swing slowly, and the cell culture loop A and the replacement liquid loop B are started. After 24 hours of co-cultivation, the cells were transferred to a culture vessel5, adding a CD3 antibody, and culturing for 6 days, wherein the total number of the finally cultured cells can reach 0.5-2 × 10¹⁰Final cell density in this example 7.36 × 10⁶Individual cells/ml.

Claims

1. A hollow fiber exchanger comprises an outer wall pipe, hollow fiber filaments arranged inside the outer wall pipe, sealing clamping blocks and end covers arranged at two ends of the outer wall pipe, and interfaces;

2. The hollow fiber exchanger of claim 1,

the hollow fiber filament is a hollow filament, and micropores which can not pass through cells are arranged on the wall of the hollow fiber filament;

the hollow fiber filaments and the outer wall pipe are arranged in the same direction, do not exceed the outer side of the sealing fixture block, and are preferably flush with the outer side of the sealing fixture block; and

the sealing fixture blocks are arranged not to exceed the ports at the two ends of the outer wall pipe, and the distance between the sealing fixture blocks and the ports at the two ends of the outer wall pipe is preferably within the range of 2-20 mm.

3. The hollow fiber exchanger of claim 1 or 2, wherein the ports comprise two ports provided on both end caps, and two ports provided on the outer wall tube at positions near both ends.

4. The hollow fiber exchanger according to claim 1 or 2, further comprising a flow path switching cartridge provided between the sealing cartridge and the end cap;

wherein, the flow path conversion fixture block is closely attached and sealed with the outer wall pipe, is equipped with on it:

the separation area divides a buffer area between the flow path conversion clamping block and the sealing clamping block into a fiber yarn inner buffer area and a fiber yarn outer buffer area; the inner fiber buffer area is communicated with the inner fiber flow path space, and the outer fiber buffer area is communicated with the outer fiber flow path space through a through hole arranged on the sealing fixture block;

the two through holes are arranged in an angle of 180 degrees, correspond to the inner fiber silk buffer area or the outer fiber silk buffer area respectively, and are used for communicating the inner fiber silk buffer area or the outer fiber silk buffer area; and

the flow path conversion groove is a 360-degree circular ring space inner groove.

5. The hollow fiber converter according to claim 4, wherein each of the two side end caps has 2 ports, each port has an outer port for connecting an external pipe and an inner port for passing through the through hole of the flow path conversion block to communicate with the filament inner buffer region or the filament outer buffer region;

preferably, the edge of the inner port is provided with a convex switching edge, the switching edge rotates 180 degrees in the flow path switching groove during flow path switching, and the fiber yarn buffer areas communicated with the existing interfaces are exchanged;

preferably, a compression spring arranged in a spring hole is further arranged between the end cover and the flow path conversion clamping block, and when an inner side port of the interface penetrates through a through hole of the flow path conversion clamping block and is communicated with a fiber filament inner buffer area and a fiber filament outer buffer area, the compression spring can provide pretightening force, so that the conversion edge is tightly attached to the flow path conversion clamping block, and liquid leakage is prevented; the rear side edge of the flow path conversion clamping block is also provided with an elastic buckle which is used for being matched with a clamping position arranged on the end cover.

6. A hollow fiber exchange culture system comprising: the system comprises a cell culture unit, a hollow fiber unit, a culture solution displacement unit, a gas displacement unit, a pipeline unit and a system master control unit, wherein the pipeline unit and the system master control unit are communicated with the cell culture unit, the hollow fiber unit, the culture solution displacement unit and the gas displacement unit;

preferably, the hollow fiber unit comprises the hollow fiber converter of any one of claims 1-5.

7. The hollow fiber exchange culture system of claim 6, wherein the cell culture unit comprises:

the culture container is provided with an outlet and an inlet which are communicated with the hollow fiber unit, namely a culture container outlet and a culture container inlet; an outlet and an inlet which are communicated with the gas replacement unit, namely a gas replacement outlet and a gas replacement inlet; and optionally a sensor mounting hole, an alternative interface and/or a filter element disposed at the outlet and inlet; and

optionally a liquid path driving device arranged on a communication pipeline between the outlet of the culture container and the hollow fiber unit and between the inlet of the culture container and the hollow fiber unit;

optionally a mixing vessel and its connecting parts and piping in communication with the culture vessel, and/or optionally a harvesting vessel and its connecting parts and piping in communication with the culture vessel; and

optionally a liquid path driving device arranged on a communication pipeline between the culture container and the mixing container and between the culture container and the harvesting container;

preferably, the culture vessel is placed on a support, which can swing within a certain angle range.

8. The hollow fiber exchange cultivation system according to claim 6 or 7, wherein the culture solution replacement unit comprises:

a replacement container, wherein an outlet and an inlet which are communicated with the hollow fiber unit are arranged on the replacement container, namely the outlet and the inlet of the replacement container, and an optional spare interface and/or a filter element arranged at the outlet and the inlet; and

optional liquid path driving device arranged on the communication pipeline between the outlet of the displacement container and the hollow fiber unit and between the inlet of the displacement container and the hollow fiber unit;

optionally a fresh broth container and/or a waste liquid container, which are in communication with the displacement vessel via connecting means and tubing; and

and a liquid path driving device which is optionally arranged on a communication pipeline between the replacement container and the fresh culture liquid container and between the replacement container and the waste liquid container.

9. The hollow fiber exchange cultivation system according to any one of claims 6 to 8, wherein the gas replacement unit comprises a gas source, a homogenizer, a gas outlet pipe orifice and a filter, and a gas pipeline, a flow meter and a control valve; wherein, the blending device is directly communicated with the gas replacement inlet of the culture container.

10. The hollow fiber exchange culture system according to any one of claims 6-9, further comprising a temperature control unit disposed in a fixed jacket capable of accommodating the entire culture system for presetting, regulating and maintaining the temperature of the culture process.

11. The hollow fiber exchange culture system according to any one of claims 6 to 10, wherein the system general control unit is configured to control the hollow fiber exchange culture system to operate according to a preset scheme, perform data acquisition, data analysis and data recording functions, and comprises a control circuit and user software;

preferably, the system general control unit comprises a display for controlling the operation of the hollow fiber exchange type culture system according to a preset scheme and displaying the operation state of the hollow fiber exchange type culture system in real time.

12. Use of the hollow fiber exchanger of any one of claims 1-5 and the hollow fiber exchange culture system of any one of claims 6-11 in adherent cell culture, suspension cell culture, and mixed culture of adherent cells and suspension cells.