CN107904170A

CN107904170A - Media to cell culture modules and cell culture system

Info

Publication number: CN107904170A
Application number: CN201711364008.6A
Authority: CN
Inventors: 钱其军; 周进; 师传胤; 颜开冬
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 Cell Therapy Engineering Technology Research Center Group Co Ltd; Shanghai Cell Therapy Research Institute; Shanghai Baize Medical Instrument Co Ltd
Priority date: 2017-12-18
Filing date: 2017-12-18
Publication date: 2018-04-13
Also published as: WO2019120135A1

Abstract

The present invention relates to media to cell culture modules and cell culture system, the media to cell culture modules includes：Only exported by nutrient solution import, nutrient solution, the closed body that cell liquid import and cell liquid outlet are in communication with the outside；The culture sap cavity and lumen in portion are located within the body, wherein, the culture sap cavity includes the independent culture sap cavity with the culture sap cavity of nutrient solution inlet communication and with nutrient solution outlet respectively, the lumen and the cell liquid import and cell liquid outlet；Sealing block, for being separated to form the lumen and culture sap cavity；Be placed in lumen, wall have do not allow cell to pass through micropore hollow fibre filament；Wherein, the hollow fibre filament passes through the sealing block, and the both ends of hollow fibre filament are connected with the culture sap cavity of nutrient solution input end and the culture sap cavity of the nutrient solution port of export respectively.The cell culture system includes the cell cultivation chip, the culture available for various kinds of cell.

Description

Media to cell culture modules and cell culture system

Technical field

The invention belongs to biomedical field of cell culture, and in particular to media to cell culture modules and cell culture system.

Background technology

Nowadays, biological immune treatment becomes a kind of emerging means for capturing tumor and cancer, is biological therapy technical field A key technology, have well research and development and application prospect.Cell therapy in biological immune treatment is from patient's body Extraction sub-elects a certain amount of immunocyte, and in carrying out amplification cultivation in vitro, which may include specific stimulating factor addition And/or the use of transgenic technology, after cell is expanded to certain scale or quantity, according to the course for the treatment of of patient need feed back into Itself is internal.During the entire course for the treatment of, the amplification in vitro culture of immunocyte is the important component that technology is realized, rationally Cell culture apparatus be to realize the key factor that immunocyte safely, effectively expands.Existing cell therapy mechanism is for cell In vitro culture carries out static culture using common porous plate, Tissue Culture Flask or permeability type culture bag as container more, this Mode can only provide a static growth environment to cell, its cells grown environment and internal true environment difference are larger, and Culture medium is only carried out in incubation to add, is not replaced, and the product of cells grown metabolism easily accumulates, so as to influence immunocyte Growth rate, form size, cell phenotype, expression status and cell quantity and vigor etc., easily limitation and reduce immune The effect for the treatment of；And need artificial container of repeatedly opening to be added nutrient solution and the operation of other confactors in production, increase The risk for having added sample to pollute, also proposes the technology for cultivating operating personnel and requires higher.Meanwhile more people Work operation link also easily influences the uniformity of quality between immunocyte culture products batch.

Immunocyte can be divided into attached cell and suspension cell according to the growth pattern of cell, and attached cell depends on culture Grown on wall of a container, suspension cell suspension growth in the nutrient solution of culture vessel.Preferable cell culture apparatus needs energy It is enough to take into account this two classes cell at the same time, to reduce the input for instrument or consumptive material, improve utilization rate of equipment and installations.Immunocyte is in body In interior growth course, the blood of flowing can not only feed vegetative cell, can also take away the discarded object that cell secretion produces, but these The dynamic environment such as the transmission of nutriment, the discharge for secreting discarded object are difficult to simulate in common static culture apparatus.Exist at present In field of cell culture it is existing some can be used for devices and technology of dynamic cell culture, disclosure satisfy that cells grown space, The functions such as liquid, such as rolling bottle, bioreactor (bioreactor), hollow fiber conduit are changed, but the design of these equipment is complicated, operation Cumbersome, wherein rolling bottle is simply possible to use in attached cell culture, and culture space is limited, more and more thin by extensive multilayer at present Born of the same parents factory is substituted；Culture tank can meet the cell culture of large volume, but traditional stainless steel or glass tank body produce every time Be required for being cleaned and being sterilized before, can not suitable for cell therapy clinical practice individuation cell culture.Other classes The reactor that the reactor of type such as wave is waved uses medical disposable material, but it is multi-party to be limited by equipment instrument, toxigenic capacity etc. Face factor, therefore be not widely accepted.Existing doughnut training method often exclusively carries out design for certain cell, its Application range is narrow.

And the culture for developing into cell of microfluidic chip technology provides new platform, micro-fluidic chip has space body Product is small, integrated level is high, the advantage such as disposable.But current existing related micro fluidic chip developmental achievement still suffers from nutrient solution The problems such as inconvenient, cell culture area is endless totally-enclosed, gas exchanges are incomplete is replaced, and is intended for the survey of few cells mostly Examination culture and the real-time dynamic monitoring direction of cell state, are not particularly suited for the large-scale production of cell.

CN158703A discloses a kind of minicell hollow fiber reactor, which is that pre-buried to insert one small by cell In section doughnut filament, then it is placed on containing in the closed ventilative silicone tube of nutrient solution.The device is by means of hollow fibre The transmission that silk micropore carries out nutriment is tieed up, Related Drug can be can be applied to by the culture of filament inner cell to certain scale The research of thing.The device is the pharmacological research applied to few cells, for large-scale clinical cytology culture be difficult to meet with It is required that.

CN103667054A discloses a kind of integrated micro-flow control cell cultivation chip, and the chip is by chip body, with phase The accessories such as temperature control component, Micropump, micro-valve and the control circuit of pass are integrated in one.Chip body is formed by multilayer bonding, including Cell culture area, nutrient solution memory block, waste etc., are connected between different zones by microfluidic circuit.The apparatus integration Integrated, fluid passage inner cavity is closed, and suitable for the culture studies of few cells, but flow path designs are simple, for immunocyte Culture environment simulation is not comprehensive enough, and ignores the gas exchanges condition of cell culture, it is impossible to it is thin to directly apply to clinical immunization The culture of born of the same parents.

CN105754856A discloses a kind of drawer type three-dimensional class blood vessel tissue culture organ chip, which is by taking out Drawer casket, sealing plate, fixed frame, class vascularization three-dimensional tissue and its relevant inlet and outlet pipe fitting are formed.The level of integrated system is high, can Dismounting or sealing, automation monitoring in real time, simulate emphatically the stabilization adhere-wall culture of class blood vessel three-dimensional tissue, and use transparent air-permeable Formula chip material, is conducive to the growth and monitoring, but the chip is only the training for being intended for blood vessel tissue in real time of relevant cell Support, passability is not strong.

The content of the invention

The present invention with reference to doughnut and cultivates chip for the shortcoming of existing various types of cells cultural method and device Advantage, there is provided it is a kind of it is simple in structure, operation is convenient, suitable for the media to cell culture modules and its culture apparatus of immunocyte, With spatial volume is small, culture density is high, can automated control, chip culture space closing, chip assembly be easily assembled replacement, life The features such as thing security performance is high.The cell culture apparatus is by cultivating immunocyte in the abundant simulated blood vessel of built-in chip type filament Growing environment, establish fairly large 3D cell culture, the training of panimmunity cell suitable for cell therapy clinical practice Support, such as the DC cells (Dendritic Cells) of adherent type, the training of a variety of T lymphocytes (CIK, CTL, TIL etc.) of suspension type Support.

The media to cell culture modules of the present invention includes：

Only it is in communication with the outside by nutrient solution import, nutrient solution outlet, cell liquid import and cell liquid outlet closed Body；

Be located within the body the culture sap cavity and lumen in portion, wherein, the culture sap cavity include it is independent respectively with culture The culture sap cavity and culture sap cavity with nutrient solution outlet of liquid inlet communication, the lumen and the cell liquid import and Cell liquid outlet；

Sealing block, for being separated to form the lumen and culture sap cavity；With

Being placed in has the hollow fibre filament for the micropore for not allowing cell to pass through in lumen, wall；Wherein, it is described hollow Filament passes through the sealing block, and the culture sap cavity and nutrient solution of the both ends of hollow fibre filament respectively with nutrient solution input end go out The culture sap cavity at mouth end connects.

In certain embodiments, the volume of the culture sap cavity is 0.2~0.5L, is preferably 0.3~0.35L.

In certain embodiments, the volume of the lumen is 0.4~1L, is preferably 0.6~0.8L.

In certain embodiments, the length of the hollow fibre filament is 130~170mm, 20~50 μm of thickness of pipe wall, tube wall The aperture of upper micropore is less than 1 μm.

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

In certain embodiments, the culture sap cavity is located at lumen both sides, is separated by sealing block, hollow fibre filament Through lumen to connect the culture sap cavity of lumen both sides.

In certain embodiments, the culture sap cavity is all located at lumen the same side, between two culture sap cavities and Separated between culture sap cavity and lumen by sealing block.

In certain embodiments, nutrient solution import and nutrient solution outlet inner side are respectively equipped with sedimeter, to Broth filtrate, the pollution that may be introduced when avoiding nutrient solution inlet and outlet from being connected with nutrient solution module.

In certain embodiments, nutrient solution import, nutrient solution outlet are sealed using medical rubber stopper.

In certain embodiments, it is described that there is culture sap cavity import, the training with the culture sap cavity of nutrient solution inlet communication The import of nutrient solution chamber and the nutrient solution inlet communication；It is described with the culture sap cavity of nutrient solution outlet to there is culture sap cavity to go out Mouthful, the culture sap cavity outlet and the nutrient solution outlet.

In certain embodiments, the culture sap cavity import is located in the body, passes through intrinsic processing flow path With the nutrient solution inlet communication.

In certain embodiments, the culture sap cavity outlet is located in the body, and the media to cell culture modules also wraps The processing flow path for being located at the body interior and the optional culture fluid path transfer tube pipeline for being located at the body exterior are included, it is described Sap cavity outlet is cultivated by the processing flow path and the optional culture fluid path transfer tube pipeline with nutrient solution outlet to connect It is logical.

In certain embodiments, there is the lumen lumen import and lumen to export, respectively with cell liquid into Mouth and cell liquid outlet.

In certain embodiments, the lumen import is each provided at the body interior with lumen outlet, described thin Born of the same parents cultivate module and further include the cell fluid path transfer tube pipeline for being located at the body exterior, wherein, the lumen import with it is thin Cell outlet is described thin respectively by being located at processing flow path and the cell liquid import and cell liquid outlet of the body interior Pass through the cell fluid path transfer tube pipeline connection between cytosol import and cell liquid outlet.

In certain embodiments, the media to cell culture modules has nutrient solution flow path and cell liquid flow path；Wherein, cultivate Liquid stream road is successively including in nutrient solution import, processing flow path, the import of culture sap cavity, the culture sap cavity of input end, hollow fibre filament Portion, the culture sap cavity of the port of export, the outlet of culture sap cavity, processing flow path, optional culture fluid path transfer tube pipeline and nutrient solution go out Mouthful；Cell liquid flow path is successively including the hollow fibre filament outer space in cell liquid import, processing flow path, lumen import, lumen Between, lumen outlet, processing flow path, cell liquid outlet and optional cell fluid path transfer tube pipeline.

In certain embodiments, the processing flow path is machined or injection is arranged on the body interior.

In certain embodiments, the body of the media to cell culture modules is made of the upper, middle and lower, wherein：

Combine closely between upper strata and middle level, middle level and lower floor's binding face, no leakage；

The upper strata is equipped with culture supernatants import and culture supernatants and exports, respectively with being arranged at the culture sap cavity in middle level Connection；And connection culture supernatants import is exported with the processing flow path of middle level nutrient solution import with culture supernatants are connected The processing flow path of layer nutrient solution outlet；

The middle level is the frame structure of a middle part hollow out, which is equipped with and culture supernatants inlet communication Processing flow path, nutrient solution between middle level nutrient solution import, the import of culture sap cavity, middle level nutrient solution import and culture sap cavity import Chamber is exported between middle level nutrient solution outlet, the outlet of culture sap cavity and the outlet of middle level nutrient solution of culture supernatants outlet Process processing flow path, the cell between flow path, and cell liquid import, lumen import, cell liquid import and lumen import Processing flow path between chamber outlet, cell liquid outlet and lumen outlet and cell liquid outlet；

The culture sap cavity and lumen are arranged on the hollow-out part in middle level；With

The hollow fibre filament is placed in lumen, its both ends passes through the sealing block, is connected respectively with culture sap cavity.

In certain embodiments, the media to cell culture modules further includes：

It is arranged on the culture fluid path transfer tube pipeline of the body exterior, the culture fluid path transfer tube pipeline connection culture Sap cavity is exported exports separated processing flow path with middle level nutrient solution；And/or

The cell fluid path transfer tube pipeline of the body exterior is arranged on, described in the cell fluid path transfer tube pipeline connection Cell liquid import is exported with cell liquid.

In certain embodiments, the culture fluid path transfer tube pipeline and cell fluid path transfer tube pipeline are medical soft Pipe.

In certain embodiments, the upper strata is additionally provided with slot and chip pulling mouth, wherein, the slot is used to position slotting Enter nutrient solution module, the chip pulling mouth is used for convenient taking-up nutrient solution module.

In certain embodiments, sealing block is bonded with the contact surface leakproofness in middle level, and upper strata, lower floor are bonded with middle level Equal leakproofness fitting, the training is formed by the side wall of sealing block and upper strata inner wall, lower floor's inner wall and hollow-out part between face Nutrient solution chamber and lumen.

In certain embodiments, trained between the culture supernatants import and middle level nutrient solution import and on upper strata Nutrient solution is exported sets sedimeter between the outlet of middle level nutrient solution, with the pollution introduced to avoid puncture medical rubber stopper； Preferably, filter sizes are 0.2 μm.

The nutrient solution module of the present invention includes：

Closed body only by storing liquid import, the outlet of storage liquid is in communication with the outside；

Intrinsic storage chamber is located at, wherein the storage chamber and storage liquid import and storage liquid outlet；With

The ventilated membrane for allowing gas molecule to pass through, for sealed storage chamber.

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

In certain embodiments, the body and ventilated membrane are transparent biocompatible materials.

In certain embodiments, storage chamber import, storage chamber outlet are sealed using medical rubber stopper.

In certain embodiments, the storage chamber with storage liquid inlet communication has storage chamber import, the storage chamber Import passes through default processing pipeline and the storage liquid inlet communication；The storage chamber with storage liquid outlet has storage Sap cavity exports, which exports by default processing pipeline with storing liquid outlet with described.

In certain embodiments, storage chamber outlet after storage chamber import with being equipped with sedimeter, for filtering And obstruct impurity and bacterium and enter in storage chamber.

In certain embodiments, the nutrient solution module is made of upper strata, middle level, lower floor and ventilated membrane, it is at the middle and upper levels Combine closely between middle level, middle level and lower floor's binding face, no leakage, its at the middle and upper levels between middle level be equipped with ventilated membrane and/or Ventilated membrane is equipped between middle level and lower floor.

In certain embodiments, the storage liquid inlet and outlet and storage chamber inlet and outlet are each provided in middle level, and when feed flow adopts With hollow injection pin type bidirectional interface, the direct medical rubber stopper of penetration cell culture module and nutrient solution module at the same time.

In certain embodiments, also there is filling exit in nutrient solution module, which is sealed using medical rubber stopper, For the filling of initial incubation liquid；Preferably, sedimeter is further respectively had at filling exit.

The cell culture apparatus of the present invention includes：Media to cell culture modules as described herein, nutrient solution module as described herein, Fluid path drive module, gas module, thermal module, control module and the pipeline or circuit that connect each module.

In one or more embodiments, the fluid path drive module includes：

First driving means, for controlling the circulation of nutrient solution between nutrient solution module and media to cell culture modules；With

Second driving device, the circulation of the lumen inner cell liquid for controlling media to cell culture modules.

In certain embodiments, the first driving means are arranged on the communicating pipe of the outlet of storage liquid and nutrient solution import Lu Shang, or be arranged on connecting pipeline of the nutrient solution outlet with storing liquid import.

In certain embodiments, the media to cell culture modules includes external culture fluid path transfer tube pipeline, and described One driving device is arranged on the culture fluid path transfer tube pipeline.

In certain embodiments, second driving device is arranged on cell fluid path transfer tube pipeline.

In certain embodiments, the first driving means and the second driving device are clamp peristaltic pump, are preferably Two-way peristaltic pump.

In certain embodiments, the gas module includes：Air inlet pipe, air outlet pipe, gas control valve and optional gas Body container and optional gas vortex mixer.

In certain embodiments, the gas container includes the container for accommodating compressed air, oxygen and carbon dioxide, respectively Container is connected with gas vortex mixer respectively, and the air inlet pipe is connected with the gas vortex mixer.

In certain embodiments, filtration apparatus is equipped with air inlet pipe and the air outlet pipe mouth of pipe, gas is equipped with device Filter membrane.

In certain embodiments, connection accommodates the pipe of compressed air, oxygen, the container of carbon dioxide and gas vortex mixer Gas control valve is respectively equipped with road, gas control valve is equipped with gas flowmeter.

In certain embodiments, the thermal module includes semiconductor temperature-control piece and the temperature sense being mounted thereon member Part platinum resistance, for controlling heating or refrigeration.

In certain embodiments, the control module includes：

First controlling switch, for controlling the first driving means；

Second controlling switch, for controlling second driving device；

3rd controlling switch, for controlling gas to convey；With

4th controlling switch, for controlling thermal module to perform heating or refrigeration.

In certain embodiments, the control module further include for the whole cell cultivation process of real-time inspection and control, Realize the control circuit of automatic cytological culture.

In certain embodiments, the cell culture apparatus further includes sensor assembly, to real-time monitoring temperature, oxygen Gas concentration, gas concentration lwevel and pH value.

In certain embodiments, the sensor assembly includes temperature sensor, oxygen concentration sensor, dense carbon dioxide Spend sensor and liquid pH value sensor.

In certain embodiments, the media to cell culture modules, nutrient solution module and sensor assembly be positioned over equipped with into The closed chip intracavitary of stomata and venthole.

In certain embodiments, fluid path drive module, gas module, thermal module and the control module integrate installation In in cabinet, cabinet inside is equipped with the closed chip chamber for placing media to cell culture modules, nutrient solution module and sensor assembly, cabinet Outside is provided with alternating interface between man and computer, control button.

In certain embodiments, the nutrient solution is the special serum free medium of immunocyte；

In certain embodiments, the cell is selected from peripheral blood mononuclear cells, neural stem cell or tumour cell.

In certain embodiments, the material contacted in described device with cell uses disposable biological compatibility material Material, and by disinfection and sterilization treatment.

The present invention also provides a kind of box set, which includes media to cell culture modules as described herein and nutrient solution module, or Contain chip as described herein.

In certain embodiments, the box set, which further includes integrated installation, fluid path drive module described herein, gas control The cabinet of valve processed, thermal module and control module, and batch cultur consumptive material, the medical disposable material include：It is as described herein Media to cell culture modules, nutrient solution module and all pipelines.

Brief description of the drawings

Fig. 1 is the planar structure schematic diagram of chip type immunocyte culture systems provided by the invention.

Fig. 2 is the structure diagram of immunocyte culture chip provided by the invention.

Fig. 3 is the structure diagram of the cell culture unit of present invention culture chip.

Fig. 4 is the structural blast schematic diagram of the cell culture unit of present invention culture chip.

Fig. 5 is the structure diagram of a doughnut chunking example of the invention.

Fig. 6 is the structural blast schematic diagram of the nutrient solution unit of present invention culture chip.

Fig. 7 is the overall structure diagram of cell culture apparatus of the present invention.

Fig. 8 is the overall structure diagram that culture chip is put into cell culture apparatus of the present invention.

Embodiment

The present invention is illustrated below in conjunction with embodiment.It is to be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the invention.Other aspects of the present invention are due to this disclosure, to those skilled in the art It is obvious for member.

The cell culture system of the present invention is chip type immunocyte culture systems (device).The cell culture dress of the present invention Put a kind of achievable new immunocyte culture scheme, can every clinical patients sample of independent process cell culture.By from Patient's body takes out a certain amount of cell, especially immunocyte, is put into the culture systems of the present invention and cultivates, can finally obtain The aim cell of respective numbers, as DC cells Antigen culture cell scale 0.5~2 × 10⁸Sum on, T is thin The cell scale of the quantity amplification cultivation of born of the same parents is 0.5~2 × 10⁹Sum.Whole immunocyte incubation can automate control System, it is only necessary to regularly replace nutrient solution module, cell culture carried out all the time in the environment of closing (no open-sky technique, effectively Third party is avoided to pollute), the immunocyte finally recycled may be directly applied to clinic, feeds back patient's body and carries out immunocyte Treatment.Whole incubation builds the training method of class blood vessel by hollow fibre filament, and filament is built in lumen, cultivates Liquid flows into opposite side culture chamber by filament from side culture sap cavity, it, which is flowed, ensures that cell intracavity liquid also has certain stream Dynamic property, 3D cultivate the biotic environment for effectively simulating immune cell growth, also improve the efficiency of nutrient solution exchange.

This cell culture apparatus includes media to cell culture modules, nutrient solution module, fluid path drive module, gas module, temperature Six big main modular of module and control module, and connected between nutrient solution module, media to cell culture modules, fluid path drive module Pipeline.In certain embodiments, this cell culture apparatus further includes sensor assembly.Fig. 1 shows the one of present system A structure diagram, including chip chamber 1, media to cell culture modules 2, nutrient solution module 3, fluid path drive module 4, gas module 5, temperature Spend module 6, sensor assembly 7 and control module 8.

Media to cell culture modules

Media to cell culture modules is the place of cell culture, including culture two big structure of sap cavity and lumen, is led between the two Sealing block is crossed to be separated by.Sap cavity is cultivated to accommodate the place of fresh medium or the nutrient solution through exchange, and lumen is cell Grow place.Hollow fibre filament is provided with lumen, filament inner spout both ends connect culture sap cavity.Cultivate in sap cavity Fresh medium is through passing through lumen in hollow fiber conduit pipe.By the micropore on hollow fibre filament, lumen and culture sap cavity Between can carry out small molecule exchange, take away cell growth generation metabolic waste thing, supplement cell needed for nutriment.

More specifically, media to cell culture modules includes：Only by nutrient solution import, nutrient solution export, cell liquid import and The closed body that cell liquid outlet is in communication with the outside；The culture sap cavity and lumen in portion are located within the body, wherein, cultivate sap cavity Including the independent culture sap cavity with the culture sap cavity of nutrient solution inlet communication and with nutrient solution outlet respectively, lumen with Cell liquid import and cell liquid outlet；Sealing block, for being separated to form lumen and culture sap cavity；Be placed in lumen Hollow fibre filament that is interior, having the micropore for not allowing cell to pass through on wall；Wherein, hollow fibre filament passes through sealing block, hollow Filametntary both ends respectively the culture sap cavity with nutrient solution input end, connect with the culture sap cavity of the nutrient solution port of export.Cell All building blocks of module are cultivated, including the material of body and sealing block are all biocompatible materials, it is preferably transparent Material.

Nutrient solution import and nutrient solution outlet are be located on media to cell culture modules two openings, for nutrient solution module Connection, the inlet and outlet flowed in and out for nutrient solution.Culture sap cavity can be located at lumen both sides, be separated by sealing block, Hollow fibre filament passes through lumen to connect the culture sap cavity of lumen both sides；Alternatively, culture sap cavity is located at lumen the same side, Separated between two culture sap cavities and between culture sap cavity and lumen by sealing block.With the training of nutrient solution inlet communication Nutrient solution chamber itself is equipped with culture sap cavity import, the culture sap cavity import and the nutrient solution inlet communication.Export and connect with nutrient solution Logical culture sap cavity itself is equipped with the outlet of culture sap cavity, culture sap cavity outlet and nutrient solution outlet.Cultivate sap cavity import It is each provided in body with the outlet of culture sap cavity, is connected respectively with nutrient solution import and nutrient solution outlet by intrinsic processing flow path It is logical.Nutrient solution import and nutrient solution outlet can be sealed using medical rubber stopper, rear side (i.e. inner side, close to the one of culture sap cavity Side) sedimeter can be respectively equipped with, can when avoiding nutrient solution inlet and outlet from being connected with nutrient solution module to broth filtrate The pollution that can be introduced.In general, the total measurement (volume) of culture sap cavity is 0.2~0.5L, it is preferably 0.3~0.35L.

In certain embodiments, media to cell culture modules further includes the culture fluid path transfer tube pipeline for being located at body exterior, Cultivate and disconnected among the processing flow path between sap cavity outlet and nutrient solution outlet, by the culture fluid path transfer tube pipeline connection. In this case, the processing flow path of the disconnection forms two interfaces on body, connects to connect culture fluid path transfer tube pipeline It is logical.

Cell liquid import and cell liquid outlet are the another two openings that are located on media to cell culture modules, flowed into for cell liquid and The inlet and outlet of outflow.There is lumen itself lumen import and lumen to export, respectively with cell liquid import and cell Liquid outlet.The outlet of lumen import and lumen is each provided at body interior, can be by being located at the processing flow path of body interior Respectively with cell liquid import and cell liquid outlet.Media to cell culture modules further includes the cell fluid path driving for being located at body exterior Pump conduit, the cell fluid path transfer tube pipeline are used for connecting cell liquid import and cell liquid outlet.The volume of lumen for 0.4~ 1L, is preferably 0.6~0.8L.

Hollow fibre filament is intermediate throughholes filament, and the length of 130~170mm, 20~50 μm of thickness of pipe wall, is covered with hole on wall Footpath is less than 1 μm of micropore, to prevent cell from passing through.Small-molecule substance or dissolved oxygen can pass through the micropore and fiber on filament wall Lumen outside silk carries out material and gas exchanges, keeps the validity of nutrient solution in lumen, realizes and cell is continued Nutrient.The material of hollow fibre filament can be biocompatible materials commonly used in the art, be preferably biocompatibility compared with Good polypropylene, polysulfones, polypropylene be fine or polyethylene etc..The quantity of hollow fibre filament in lumen is had no it is specifically limited, but Under normal conditions, the space (volume) in lumen outside hollow fibre filament should be made in the range of 0.2~0.6L.

In general, the media to cell culture modules of the present invention has nutrient solution flow path and cell liquid flow path.Nutrient solution flow path is culture The path that liquid stream is moved, successively including nutrient solution import, processing flow path, the import of culture sap cavity, the culture sap cavity of input end, hollow fibre Tie up inside silk, the culture sap cavity of the port of export, culture sap cavity export, process flow path, optional culture fluid path transfer tube pipeline and training Nutrient solution exports.Cell liquid flow path be cell liquid flowing path, successively including cell liquid import, processing flow path, lumen import, The hollow fibre filament external space, lumen outlet, processing flow path, cell liquid outlet and cell fluid path transfer tube pipe in lumen Road.Nutrient solution flow path can form circulation loop with nutrient solution module as described herein, and in cell liquid flow path, the driving of cell fluid path Pump conduit is with after cell liquid inlet communication, forming circulation loop.

For example, when Cyclic culture liquid, nutrient solution is flowed out from nutrient solution module, enters cell culture mould from nutrient solution import Block, flows to culture sap cavity import via processing flow path, flows into the culture sap cavity of nutrient solution input end, flow through in hollow fibre filament Empty inner cavity, reaches the culture sap cavity of the nutrient solution port of export, goes out via culture sap cavity outlet stream, into processing flow path, is then out Nutrient solution exports, and flows back into nutrient solution module, is consequently formed a circulation loop, herein also referred to as nutrient solution circulation loop.At certain In a little embodiments, disconnected among the processing flow path between the outlet of culture sap cavity and nutrient solution outlet, by being placed outside cell culture Culture fluid path transfer tube pipeline connection outside module body.And cell liquid then the hollow fibre filament external space in lumen, Circulated between cell liquid import and cell liquid outlet, form cell liquid circulation loop.

Herein, the body interior can be arranged on through the technique such as being molded or machining by processing flow path；Cultivate fluid path driving Pump conduit and cell fluid path transfer tube pipeline can be conventional flexible pipe for medical purpose.

In certain embodiments, media to cell culture modules is by the upper, middle and lower up of three layers.Upper strata and middle level, in Leakproofness is bonded between layer and lower floor binding face, and no leakage is air tight.

Upper strata is equipped with culture supernatants import and culture supernatants export, and media to cell culture modules is flowed into and out for nutrient solution Nutrient solution inlet and outlet；And the processing flow path of connection culture supernatants import and middle level nutrient solution import with connect upper strata Nutrient solution exports and the processing flow path of middle level nutrient solution outlet.Upper strata can also be equipped with slot and chip pulling mouth.Slot can be used for fixed Position insertion nutrient solution module, chip pulling mouth can be used for convenient taking-up nutrient solution module.In certain embodiments, culture supernatants Import, culture supernatants outlet can be sealed using medical rubber stopper.

Middle level has certain thickness, and usual thickness is 5~15mm, is preferably 8~10mm, is the frame knot of a middle part hollow out Structure, the frame structure are equipped with middle level nutrient solution import, the import of culture sap cavity, middle level culture with culture supernatants inlet communication Train in the middle level of processing flow path, the outlet of culture sap cavity and culture supernatants outlet between liquid import and culture sap cavity import Nutrient solution outlet, culture sap cavity outlet middle level nutrient solution export between processing flow path, and cell liquid import, lumen import, Processing flow path, lumen outlet, cell liquid outlet and lumen outlet and cell between cell liquid import and lumen import Processing flow path between liquid outlet.Between culture supernatants import and middle level nutrient solution import and culture supernatants outlet and Middle level can be respectively equipped with filtration apparatus between nutrient solution outlet, to broth filtrate, avoid culture sap cavity inlet and outlet and culture The pollution that liquid module may introduce when connecting.Filter sizes are preferably 0.2 μm of aperture or following.

The part of hollow out is used to accommodate doughnut chunking in middle level.Herein, doughnut chunking is by hollow fibre filament Sealing block with being arranged on hollow fibre filament both ends forms.Hollow fibre filament passes through the sealing block, respectively with nutrient solution The culture sap cavity connection of the culture sap cavity and the nutrient solution port of export of input end.In general, the both ends of hollow fibre filament do not surpass respectively Go out the outside of sealing block, preferably flushed with the outside of sealing block (i.e. towards the side of culture sap cavity).Sealing block with The hollow fibre filament outer wall leakproofness fitting passed through.Meanwhile also leakproofness is bonded the contact surface in sealing block and middle level.When After leakproofness is bonded between upper strata, lower floor and middle level binding face, by sealing block and upper strata inner wall, lower floor's inner wall and hollow-out parts The side wall divided forms the culture sap cavity and lumen.

Middle level nutrient solution import can be by being built in the processing flow path on upper strata and the nutrient solution inlet communication on upper strata, middle level training Nutrient solution outlet can be by being built in the processing flow path on upper strata and the nutrient solution outlet on upper strata.Middle level nutrient solution import and middle level Nutrient solution outlet can be connected with the culture sap cavity of inlet end and an outlet end respectively by the processing flow path arranged on body interior.At certain In a little embodiments, disconnect among the processing flow path between the outlet of culture sap cavity and the outlet of middle level nutrient solution, formed on middle level Two interfaces, the culture fluid path transfer tube pipeline connection external by being placed in media to cell culture modules sheet.

Cell liquid import is via the processing flow path that is located inside middle level and lumen inlet communication, and cell liquid outlet is via setting Processing flow path and lumen outlet inside middle level, finally connect with the hollow fibre filament external space of lumen, and thin Pass through cell fluid path transfer tube pipeline connection between cytosol import and cell liquid outlet.

When carrying out nutrient solution circulation, nutrient solution is entered by culture supernatants import, passes through the processing being built in upper strata The filtered device of flow path flows into middle level nutrient solution import, then processes flow path into the nutrient solution of culture sap cavity input end by middle level Chamber, flows through the hollow cavity of hollow fibre filament, and into the culture sap cavity of the port of export, processed flow path is exported by culture sap cavity, according to The secondary outlet of outflow middle level nutrient solution and culture supernatants outlet, flow back to nutrient solution module, are consequently formed the nutrient solution circulation of closing Circuit.When media to cell culture modules is equipped with culture fluid path transfer tube pipeline, the outlet of nutrient solution outflow culture sap cavity, stayed one section to add After work flow path, into the culture fluid path transfer tube pipeline, processing flow path is passed through, sequentially enters middle level nutrient solution outlet and upper strata Nutrient solution exports, and flows back to nutrient solution module, is consequently formed the nutrient solution circulation loop of closing.Doughnut group is flowed through in nutrient solution During block, nutritional ingredient is flowed to outside hollow fibre filament by the micropore on hollow fibre filament wall, is located to nourish outside doughnut Cell.And cell liquid then the processing flow path between cell liquid import, cell liquid import and lumen import, lumen import, Processing flow path, the cell liquid that the hollow fibre filament external space of lumen, lumen export, lumen is exported between cell liquid outlet Circulation in outlet and cell fluid path transfer tube pipeline.

Fig. 2, Fig. 3, Fig. 4 and Fig. 5 respectively show one media to cell culture modules example (media to cell culture modules 2) of the present invention Overall structure and Each part.Media to cell culture modules 2 includes upper strata 201, middle level 203 and lower floor 204, all uses bio-compatible Property material manufacture.Combine closely between three layers of binding face, binding site is combined using effective binding agent or ultrasonic bonding Together, with liquid-leakage preventing and/or gas leakage.

Upper strata 201 includes culture supernatants import 205, culture supernatants outlet 206, slot 207 and chip pulling mouth 208. Culture supernatants import 205 and culture supernatants outlet 206 pass in and out the inlet and outlet of media to cell culture modules 2 for nutrient solution.Slot 207 Nutrient solution module is inserted into for positioning.Nutrient solution import 205 and nutrient solution outlet 206 in can be respectively equipped with filtration apparatus 205b and 206b.The filtration apparatus is sedimeter, built-in filter membrane, available for the pollution that avoids puncturing medical rubber stopper and may introduce. Chip pulling mouth 208 can be used for convenient taking-up nutrient solution module.

Middle level 203 is the frame structure of the middle part hollow out with certain thickness, which cultivates equipped with middle level Liquid import 211, culture sap cavity import 211a, middle level nutrient solution outlet 212, culture sap cavity outlet 212a, the outlet of culture sap cavity The processing stream between processing flow path 9a, cell liquid import 215 and lumen import 215a between 212a and middle level nutrient solution outlet 212 Road 10a, culture fluid path transfer tube pipeline 213, cell fluid path transfer tube pipeline 214, lumen import 215a, cell liquid import 215th, lumen outlet 216a and cell liquid outlet 216.In middle level 203 shown in Fig. 4, culture sap cavity outlet 212a is trained with middle level Processing flow path between nutrient solution outlet 212 is disconnected, and two openings are formed on the outer wall in middle level, is driven for connecting culture fluid path The both ends of dynamic pump conduit 213.

Media to cell culture modules 2 further includes the doughnut chunking 202 of embedded 203 hollow-out part of middle level.Fig. 3 and 4 is shown Exemplary 202 structure of doughnut chunking.The doughnut chunking 202 includes more hollow fibre filaments 209 placed side by side With the sealing block 210 positioned at 209 both ends of hollow fibre filament.Sealing block 210 can be by any appropriate biocompatible materials It is made.Every 209 both ends of hollow fibre filament are each passed through sealing block 210, are bonded through part with sealing block leakproofness.It is logical Often, hollow fibre filament passes through the part of sealing block with being flushed on the outside of sealing block.Every piece of sealing block connects with middle level 203 Contacting surface leakproofness is bonded.Certain space is left in the both ends of sealing block 210 between middle level 203 respectively, to form culture sap cavity 12 and culture sap cavity 14.The lumen 13 of the hollow fibre filament 209 is as accommodated between culture sap cavity 12 and 14.

Fig. 5 shows the structure of another doughnut chunking 202a, including hollow fibre filament 209 and sealing block 210. Hollow fibre filament 209 is in 180 ° of U-bends, therefore two culture sap cavities are located at the same side of lumen.Similarly, two cultures Sap cavity still has sealing block to separate, which can be integrally formed with sealing block 210, or independent sealing gear Block, but need two culture sap cavities can be separated, ne-leakage., can be as mentioned before when using the doughnut chunking shown in Fig. 5 The other structures of media to cell culture modules, including each inlet and outlet and each processing flow path are set.

In general, by taking Fig. 4 as an example, nutrient solution is entered by culture supernatants import 205, by being built in adding in upper strata 201 The middle level nutrient solution import 211 in work fluid communication middle level 203, enters culture by processing flow path via culture sap cavity import 211a Sap cavity 12, flows through the hollow cavity of hollow fibre filament 209, and into culture sap cavity 14, culture is flowed through from culture sap cavity outlet 212a Fluid path drives pump conduit 213, into processing flow path 9a, finally by middle level nutrient solution outlet 212 and culture supernatants outlet 206 Nutrient solution module 3 is flowed back to, is consequently formed the nutrient solution circulation loop 9 of a closing.Hollow fibre filament 209 is flowed through in nutrient solution When, nutritional ingredient is flowed to outside hollow fibre filament by the micropore on hollow fibre filament wall, is located to nourish outside hollow fibre filament Cell.

And cell liquid circulation loop 10 is then formed outside hollow fibre filament, this circulation loop is by 13 doughnut of lumen Space, the processing flow path 10a that is connected with the space and cell fluid path transfer tube pipeline 214 outside silk form.Cell liquid is by thin Cytosol import 215 enters, and flows through and processes after flow path 10a through entering 13 hollow fibre filament outer space of lumen by lumen import 215a Between, then by lumen outlet 216a outflows, via processing flow path outflow cell liquid outlet 216, into cell fluid path transfer tube Pipeline 214, then cell liquid import 215 is flowed into, form the circulation loop 10.

Whole incubation builds the training method of class blood vessel by hollow fibre filament, and filament 209 is built in lumen In 13, nutrient solution flows into opposite side culture chamber 14 by filament 209 from side culture sap cavity 12, and it is certain that cell is given in its flowing Shearing force, and ensure cell intracavity liquid also have certain mobility, effectively simulate the biocycle of immune cell growth Border, also improves the efficiency of nutrient solution exchange.

Nutrient solution module

Nutrient solution module mainly includes storage cavity configuration, accommodates fresh cell culture fluid for storing, can be once The biological reaction container of property, such as disposable biological reaction bag or biometric storage chip.Nutrient solution module is mainly used for training to cell Support module and nutrient solution is provided, there are two interfaces, wherein first interface is the outlet of storage liquid, for the training with media to cell culture modules Nutrient solution inlet communication；Second interface is storage liquid import, for the nutrient solution outlet with media to cell culture modules.Storage chamber Volume is 0.6~2L, is preferably 0.8~1.5L.

In certain embodiments, there is ventilated membrane, the ventilated membrane gas-permeable, liquid-impermeable, it is allowed to film outer gas in nutrient solution module Body molecule is dissolved in the nutrient solution in storage chamber by the film, to ensure the dissolved oxygen amount of nutrient solution.

In certain embodiments, the nutrient solution module is made of the upper, middle and lower three, its upper strata is with Leakproofness is bonded between layer, middle level and lower floor binding face, no leakage.Gripped between upper strata and middle level ventilated membrane and/or middle level with Ventilated membrane is gripped between lower floor.Preferably, upper, middle and lower-ranking and ventilated membrane all use transparent biocompatible materials system Make.Storage liquid inlet and outlet are arranged in middle level, are connected respectively by default processing pipeline with the storage chamber inlet and outlet in middle level.Deposit Liquid storage import, the outlet of storage liquid are sealed using medical rubber stopper.Hollow injection pin type bidirectional interface is used during feed flow, directly The medical rubber stopper of penetration cell culture module and nutrient solution module at the same time.In certain embodiments, storage chamber outlet and storage Chamber entrance further respectively has sedimeter.

In certain embodiments, also there is filling exit, it is close which can use medical rubber stopper to carry out in nutrient solution module Envelope, available for the filling of initial incubation liquid.Preferably, sedimeter is further respectively had at filling exit.

Gas can be entered in cell culture fluid by the ventilated membrane in nutrient solution module, and ventilated membrane can provide the gas of large area Liquid contacts, and higher oxygen mass transfer coefficient can be provided under relatively low gas flow rate.

Fig. 2 and Fig. 6 shows the overall structure of one nutrient solution module example (nutrient solution module 3) of the present invention and each portion respectively Separation structure.Nutrient solution module 3 includes upper strata 301, middle level 303 and lower floor 304, is all manufactured with biocompatible materials.Upper strata Ventilated membrane 302 is designed between 301 and middle level 303, middle level 303 and lower floor 304, between three layers and between layer block and ventilated membrane Combine closely to form storage chamber 305, binding site is combined together using effective binding agent or ultrasonic bonding, no leakage And/or the situation of gas leakage.Nutrient solution module 3 has storage liquid outlet 306 and storage liquid import 307, passes through default processing pipeline Connection is arranged at the storage chamber outlet 306a and storage chamber import 307a in middle level 303.It is two-way using hollow injection pin type during feed flow Interface 311, directly penetrates connection storage liquid outlet 306 and nutrient solution import 205, storage liquid import 307 and culture at the same time respectively The sealing medical rubber stopper of liquid outlet 206, carries out fluid infusion, while also ensure good closure.Storage chamber outlet 306a, storage Sedimeter 309 is equipped with chamber import 307a.3 middle level 303 of nutrient solution module further includes filling exit 310, for initially training Nutrient solution it is filling, sedimeter 309 also is provided with filling exit 310.

Nutrient solution module can be positioned over the top of media to cell culture modules or is inserted into by the slot 207 of media to cell culture modules solid This fixed module, chip pulling mouth 308 are easy to the installation or taking-up of module.

Fluid path drive module

Fluid path drive module is used to drive liquid to continue to flow into flowing out media to cell culture modules and/or training with stable flow velocity Nutrient solution module, so as to ensure the circulation of flow path in whole system.Fluid path drive module usually contains the device of driving liquid flowing, Preferably clamp peristaltic pump.It is further preferred that peristaltic pump is two-way peristaltic pump.In general, fluid path drive module includes two drivings Device, respectively on nutrient solution circulation loop and cell liquid circulation loop, for driving flowing and the cell liquid of nutrient solution Flowing.

As shown in Figure 1, fluid path drive module 4 includes 401 and second driving device 402 of first driving means.First driving Device 401 is located on nutrient solution circulation loop 9, and the second driving device 402 is located on cell liquid circulation loop 10.

As shown in figure 8,401 clamping of first driving means peristaltic pump is fixed on culture fluid path transfer tube pipeline 213, for controlling The circulation of nutrient solution between media to cell culture modules and nutrient solution module processed；Second driving device peristaltic pump, 402 clamping is fixed on carefully Cytosol road drives pump conduit 214, is used for realization the circulation of cell liquid.

In general, the rotating speed of peristaltic pump is normally controlled in the range of 10~50r/min, such as 20~30r/min.

Gas module

Gas module is mainly used for conveying mixed gas into chip chamber 1, the mixed gas usually contain air, oxygen and Carbon dioxide etc..Necessary to wherein oxygen is cell metabolism, and throughput is needed with bioreactor (i.e. cell culture mould Block) volume and cell concentration raising and increase, but higher oxygen content can also produce cell toxicity, therefore oxygen concentration meeting Accurate control is carried out by sensor.

In general, gas module includes air inlet pipe and air outlet pipe, air inlet pipe one end is used to connect with the air admission hole of culture apparatus, The other end is then used to connect with gas container；Air outlet pipe is used to connect with the venthole of culture apparatus.Air inlet pipe is equipped with gas Control valve, control conveying of the gas to culture apparatus is turned on and off by gas control valve.In air inlet pipe and air outlet pipe also Filtration apparatus can be equipped with, it is interior to be equipped with gas filter membrane.Filtration apparatus in air inlet pipe is generally arranged at gas control valve and is filled with culture Put between air inlet nose end.

When needing to convey gas, air inlet pipe one end is connected with gas container, gas control valve is opened, with needed for conveying Gas.

Gas can be the gas being pre-mixed.In this case, can be by air inlet pipe one end directly with being mixed in advance containing this The container connection of the gas got togather.

In certain embodiments, gas is not pre-mixed.In this case, gas module further includes vortex mixer.Mix Device is located between the gas control valve of air inlet pipe and filtration apparatus (if any), with being respectively provided with gas to be mixed (such as compressed air, oxygen Gas and carbon dioxide) gas container be connected.Gas control valve is set respectively on the path of each gas container and vortex mixer, passes through gas Body control valve is turned on and off controlling conveying of the gas to vortex mixer in each gas container.Therefore, the quantity of gas control valve Can be 1~5, such as 1~3.

In certain embodiments, gas module further includes 1 or several gas containers, and such as 1~5 or 1~5 gas holds Device.

Fig. 1 and Fig. 8 displays gas module example of the invention.Specifically, gas module 5 includes 501 He of air inlet pipe Air outlet pipe 507, wherein 501 one end of air inlet pipe connect the air admission hole into chip chamber 1, and the other end is connected with vortex mixer 502, and compression is empty Gas 504, oxygen 505 are connected in gas vortex mixer 502 with carbon dioxide 506.Air inlet pipe 501 and 507 mouth of pipe of air outlet pipe are equal Equipped with filtration apparatus 508, gas filter membrane is housed in device.Control module 8 controls three controlling switches, that is, compressed air, oxygen respectively Gas, carbon dioxide gas control valve 503 open and close, each gas is entered core after being mixed in gas vortex mixer 502 Piece chamber.When there are the 3rd controlling switch that during multiple gas control valves 503, can set respective numbers.

Thermal module

Thermal module is used to ensure that whole chip intracavitary maintains suitable cell culture temperature.The main body of thermal module is Semiconductor temperature-control piece, can be heated or be freezed, and persistently be confessed with stable heat dissipation capacity.Preferably, semiconductor temperature-control on piece Temperature sensitive member platinum resistance is installed.As illustrated in figures 1 and 8, thermal module 6 is installed on the inner side of chip chamber 1, by control module 8 the 4th controlling switches control.

Control module

Control module includes the first controlling switch, the second controlling switch, the 3rd controlling switch and the 4th controlling switch.First Controlling switch is used to control driving device between nutrient solution module and media to cell culture modules, via the first controlling switch open with Close the circuit for making the media to cell culture modules, drive module and nutrient solution module be formed with or without circulation, or control culture The flow velocity or flow of liquid；Second controlling switch be used to controlling the import of media to cell culture modules cell liquid and cell liquid export between the Two driving devices, make to be formed with or without between the cell culture import and outlet via the Push And Release of the second controlling switch The circuit of circulation, or the flow velocity or flow of control cell liquid；3rd controlling switch, for controlling gas module to device chip chamber The conveying of gas；4th controlling switch, temperature rise or temperature drop for the semiconductor chip that controls thermal module.

Control module may also include corresponding control circuit, for controlling the first above-mentioned controlling switch, the second control to open Pass, the 3rd controlling switch and the 4th controlling switch.In general, the first controlling switch is used to control first driving means 401, the second control System switch is used to control the second driving device 402；3rd controlling switch, for controlling gas control valve 503；4th control is opened Close, for controlling the semiconductor chip 6 of thermal module.Control module further includes display, and driver is used to show described device According to the environment numerical value in predetermined program operating condition and real-time acquisition device, the operating status of device is shown.Display can To be touch screen displays.Alternatively, control module may include control panel, including control button, to control the first control The open and close of switch, the second controlling switch, the 3rd controlling switch and the 4th controlling switch.Or system control module may include Both display and control button.By control module, apparatus of the present invention accurately control whole cell cultivation process, realize complete Automate immunocyte culture.

Sensor assembly

In certain embodiments, the cell culture system of this paper further includes sensor assembly.More specifically, the sensing Device module is used to detect the environmental parameter in cell culture system, includes but not limited to temperature, oxygen concentration, gas concentration lwevel With liquid pH value etc..

The exemplary sensor assemblies 7 for having put on display this paper cell culture systems of Fig. 1 and Fig. 8, including temperature sensor 701, Oxygen concentration sensor 702, gas concentration lwevel sensor 703 and liquid pH value sensor 704.Temperature sensor 701 is used for Temperature at detection culture chip position；Oxygen concentration sensor 702 is used for the concentration for monitoring oxygen gas in chip chamber 1；Two Carbonoxide concentration sensor 703 is used for the concentration for monitoring carbon dioxide in chip chamber 1；Liquid pH value sensor 704 is non- Touch sensor, for monitoring liquid pH value in culture chip.

Cultivate chip

The present invention also provides one kind to cultivate chip, and the culture chip is by media to cell culture modules as described herein and nutrient solution mould Block forms, both pass through respective orifice.Specifically, the nutrient solution import of media to cell culture modules and nutrient solution outlet point Not the storage liquid outlet with nutrient solution module and storage liquid inlet communication, such as pass through hose or hollow injection pin type bidirectional interface Connection.

For example, Fig. 2 shows the structure diagram of present invention culture chip, including media to cell culture modules 2 and nutrient solution mould Block 3, nutrient solution module 3 are inserted into media to cell culture modules 2 via the slot on media to cell culture modules 2.The storage liquid of nutrient solution module Outlet and the nutrient solution inlet communication of media to cell culture modules, store the nutrient solution outlet of liquid import and media to cell culture modules, Form a complete culture chip.

Cell culture cabinet

A kind of cell culture cabinet is also provided herein, including：Casing, is integrated in the cabinet or this paper institutes above Fluid path drive module, control module and the chip chamber stated.In certain embodiments, which, which further includes, is integrated in The thermal module and sensor assembly of cabinet inside.

Cabinet can be the size and dimension of any suitable size, can be made of any suitable material.

Fluid path drive module includes first driving means and the second driving device.Driving device is arranged on liquid as described herein Can be clamp peristaltic pump on body circulation circuit, preferably bidirectional peristaltic pump.

Control module may include：First controlling switch, for controlling first driving means；Second controlling switch, for controlling Make the second driving device；3rd controlling switch, for controlling gas to convey；With the 4th controlling switch, for controlling thermal module Heating or cooling.3rd controlling switch can be set according to the quantity of gas control valve, be, for example, 1~5 or 1~3 the 3rd control System switch.

Chip chamber can be independently of a container outside cabinet, by stainless steel and other metal materials or transparent organic glass It is made Deng material；Can also be a space in cabinet, the air inlet that the space is only equipped with by cabinet after cabinet is closed Hole and venthole are connected with outside.Chip intracavitary can be equipped with suitable fixed structure, easy to be fixed at the module of intracavitary, such as Media to cell culture modules, nutrient solution module, thermal module and sensor assembly etc.., can be in core when chip chamber is independent container Suitable fixed structure is set outside piece chamber and/or in cabinet, which is fixed in cabinet.When chip chamber is independent Container when, may be provided with air admission hole and venthole on the container, for being connected with the air admission hole on cabinet with venthole, with Gas module connects.When the space that one that chip chamber is cabinet can be sealed, thermal module and sensor assembly usually collect Into in cabinet.

Thermal module includes semiconductor temperature-control piece and the temperature sensitive member platinum resistance being mounted thereon, and is heated for controlling Or refrigeration.Sensor assembly includes temperature sensor, oxygen concentration sensor, gas concentration lwevel sensor and liquid pH value sensing Device.

Front plate can be also equipped with cabinet, display screen and control button can be installed thereon, the work for real-time display instrument Make operating condition and the control operation to instrument.

It can also be set on cabinet with air admission hole and venthole, for being connected with gas module as described herein.In some implementations Can also be integrated in scheme, on cabinet with one or several (such as 1~5 or 1~3) gas control valve, its be turned on and off by The 3rd controlling switch control of respective numbers.

Fig. 7 shows the schematic diagram of cell raise machine case portion separation structure of the present invention.The cell culture cabinet 101 of Fig. 7 includes It is chip chamber 1, window 102, incubator overhead door 103, heat emission hole 105, first driving means 401, the second driving device 402, aobvious Display screen 801 and control button 802.

Cell culture apparatus

The cell culture apparatus of the present invention includes media to cell culture modules as described herein, nutrient solution module, fluid path driving mould Connect between block, gas module, thermal module and control module, and nutrient solution module, media to cell culture modules, fluid path drive module The pipeline connect.In certain embodiments, cell culture apparatus further includes sensor assembly.In the device, media to cell culture modules With nutrient solution module by respective orifice, for example, the nutrient solution import of media to cell culture modules and nutrient solution outlet respectively with The storage liquid outlet of nutrient solution module and storage liquid inlet communication, such as connected by hose or hollow injection pin type bidirectional interface It is logical.

In general, fluid path drive module as described herein, the gas control valve in gas module, thermal module, sensor die Block and control module can integrated installation in the cabinet of cell culture apparatus, installation display, control button outside cabinet.Carefully Born of the same parents, which cultivate module and nutrient solution module, can then be placed in the chip intracavitary of cabinet.

In certain embodiments, media to cell culture modules, nutrient solution module, thermal module and sensor assembly are placed in core Piece intracavitary.In these embodiments, fluid path drive module, the gas control valve of gas module and control module are integrated in cabinet It is interior.

Chip chamber is the subenvironment space for carrying out cell culture, for building the enclosed environment of culture chip, easy to control Cultivate the temperature and gas member condition of chip environments.Media to cell culture modules, nutrient solution module and its phase can be put into chip chamber The connecting pipeline of pass.Thermal module and sensor assembly are also mountable to chip intracavitary.Air admission hole can be set on chip chamber with going out Stomata, for being connected with gas module.

Chip chamber can be an independent container, by material systems such as stainless steel and other metal materials or transparent organic glass Into；Can also be cell culture apparatus cabinet in a space, after cabinet is closed only by air admission hole and venthole with Outside connection.Chip intracavitary can be equipped with suitable fixed structure, easy to be fixed at the module of intracavitary, such as cell culture mould Block, nutrient solution module, thermal module and sensor assembly etc.., also can be outside chip chamber when chip chamber is independent container And/or suitable fixed structure is set in cabinet, to be fixed in cabinet.When chip chamber is independent container, the appearance Air admission hole and venthole are may be provided with device, for being connected by the air admission hole on cabinet with venthole with gas module.

Fig. 1 shows the structure diagram of cell culture apparatus of the present invention, including chip chamber 1, media to cell culture modules 2, training Nutrient solution module 3, fluid path drive module 4, gas module 5, thermal module 6, sensor assembly 7 and control module 8.

Fig. 8 illustrates the overall structure and internal structure of cell culture apparatus.It is inserted into the cell culture apparatus 101 of Fig. 8 Culture chip, including chip chamber 1, media to cell culture modules 2, nutrient solution module 3, gas module 5, thermal module 6, control module 8th, sensor component 701-704, first driving means 401, the second driving device 402, display screen 801 and control button 802 with And overhead door operator 104.Gas module 5 includes air inlet pipe 501, air outlet pipe 507, compressed air container 504, oxygen container 505, two Aoxidize carbon vessel 506, gas vortex mixer 502, gas control valve 503 and filtration apparatus 508.Fig. 8 also show circulation loop 9 With 10.Incubator overhead door 103 is put into or takes out culture chip when opening, accommodating for sealing is formed during closing and cultivates chip Chip cavity space.Overhead door operator 104 is used to control opening or closing for incubator overhead door 103.Heat emission hole 105 is used for cell culture The heat dissipation of case internal component.The display screen 801 and control button 802 installed in cabinet front plate, can real-time display instrument The operating condition that works and the control operation to instrument.

Box set

The present invention also provides a kind of box set, which includes media to cell culture modules as described herein and nutrient solution module, or Including culture chip as described herein.It may also include connecting tube in box set, such as form culture fluid path transfer tube pipeline and cell liquid Road drives the connecting tube of pump conduit.The media to cell culture modules, nutrient solution module or the culture chip and connecting tube can all be Medical disposable material.It is dismountable between connecting tube and media to cell culture modules, nutrient solution module.

It may also include cell culture cabinet as described herein in box set, which can be integrated with except cell as described herein is trained All other component outside module and nutrient solution module is supported, includes but not limited to the gas in fluid path drive module, gas module Control valve, thermal module, sensor assembly and control module.If it is desired, be also provided with box set connecting tube (snorkel) and Optional gas vortex mixer, can be equipped with filtration apparatus on tracheae, inside set gas filter membrane.

But in certain embodiments, when chip chamber is independently of a container outside cabinet, it is preferable that in cabinet The gas control valve and control module being only integrated with fluid path drive module, gas module, wherein media to cell culture modules, nutrient solution Module, thermal module and sensor assembly are preferably provided at the independent chip intracavitary.In these described schemes, the box set It may include：

It is integrated with the cabinet of fluid path drive module, gas control valve and control module；

Chip chamber；

Media to cell culture modules, nutrient solution module, thermal module and sensor assembly；And

Connecting tube.

In certain embodiments, independent chip intracavitary can be integrated with thermal module and sensor assembly.Therefore, herein Box set include：

It is integrated with the chip chamber of thermal module and sensor assembly；

Media to cell culture modules and nutrient solution module, or culture chip as described herein；And

Connecting tube.

After the box set for taking the present invention, those skilled in the art can be according to the specification enclosed herein or according to box set Each component in box set is installed on cabinet, is made into cell culture apparatus as described herein.

Whole cells contact class material of cell culture apparatus as described herein uses disposable biological compatibility material, And pass through special disinfection and sterilization treatment.Herein, when referring to leakproofness fitting, it is intended that air tight and/or leakage, can use The means of this area routine realize air tight and/or leakage, for example, suitable binding agent can be used to realize.

Cultural method

The method that cell culture is carried out using the cell culture apparatus of this paper is comprised the following steps：

(1) provide nutrient solution and be previously added the culture chip of cell suspension to be cultivated；

(2) the first controlling switch, the second controlling switch, the 3rd controlling switch and the 4th controlling switch are opened, forms culture Liquid circulation loop and cell liquid circulation loop；The temperature and gas composition concentration of control chip chamber sealing space；

(3) after cultivating, add cell and reagent is resuspended, open the outlet of 2 cell liquid of media to cell culture modules, close the first control System switch, the 3rd controlling switch and the 4th controlling switch, open the second controlling switch, and the mature cell in lumen 13 is harvested Into the container of recycling.

Specifically, it is preinstalled with a certain amount of cell culture fluid in culture chamber 12, lumen 13, culture chamber 14.Start Before culture, cell suspension and Porcine HGF to be cultivated are added into lumen 13, is sufficiently mixed.Nutrient solution module 3 from The slot 207 of media to cell culture modules 2 is inserted into, and storage chamber outlet 306, storage chamber import 307 are by hollow injection pin type bidirectional interface 311 are directed through culture supernatants import 205, the rubber rubber plug of culture supernatants outlet 206 respectively, carry out supplement nutrient solution. Start control module 8 at the same time, control peristaltic pump 401 is in positive open mode, nutrient solution module 3 and 2 shape of media to cell culture modules Into nutrient solution circulation loop.Stored 306 outflow of chamber outlet of nutrient solution in 3 storage chamber 305 of nutrient solution module, is cultivated through upper strata Liquid import 205 and middle level nutrient solution import 211 enter culture sap cavity 12, then manage interior flow path to nutrient solution through hollow fibre filament 209 Chamber 14, flows through culture fluid path transfer tube pipeline 213.Pass through the power drive of the peristaltic pump 401 of fluid path drive module 4, nutrient solution Storage chamber is flow back into via processing flow path 9a, middle level nutrient solution outlet 212, culture supernatants outlet 206 and storage chamber import 307 In 305.The circulation of this process carries out, and the fresh medium in storage chamber 305 is continuously passed through through hollow fibre filament 209 Lumen 13 participates in nutrient solution fluid path and exchanges.Control module 8 controls peristaltic pump 402 to be in positive open mode, lumen 13 Cell liquid import 215 connects processing flow path 10a with cell liquid outlet 216, and the circuit of one circulation of form, ensures thin in culture Born of the same parents possess certain mobility.7 turn on sensor 701,702,703 and 704 of sensor assembly at this time, monitors chip in real time respectively Temperature, oxygen concentration, gas concentration lwevel and pH value in chamber 1.In cell cultivation process, control system 8 is according in chip chamber 1 Temperature data acquisition, control thermal module 6 semiconductor chip temperature rise or temperature drop.Control system 8 is according to oxygen in chip chamber 1 The data acquisition of gas and gas concentration lwevel, control oxygen, carbon dioxide and each gas control valve 503 of air beats ON/OFF Closed state.Control system 8 adjusts the flow velocity of peristaltic pump 401 and 402 or is prompted what is more renewed according to the feedback of pH value Nutrient solution module 3.When needing replacing nutrient solution module 3, incubator overhead door 103 is opened, when taking out culture chip, grab chips are drawn Existing nutrient solution module 3 after the slot 207 of media to cell culture modules 2 is removed, is used medicinal alcohol to wipe cell culture by mouth 308 After the medical rubber stopper surface of module carries out disinfection, new nutrient solution module 3 is reinstalled.After the completion of culture, cell is added Reagent is resuspended, opens the outlet of 2 cell liquid of media to cell culture modules, just the mature cell in lumen 13 can be harvested.

The present apparatus and this method culture various cells known in the art can be used, preferably various immunocytes.Cell Including but not limited to DC cells, a variety of T cells such as CIK cell, CTL cells, til cell, CAR-T cells etc..

Claims

1. a kind of media to cell culture modules, including：

Only exported by nutrient solution import, nutrient solution, the closed body that cell liquid import and cell liquid outlet are in communication with the outside；

Be located within the body the culture sap cavity and lumen in portion, wherein, the culture sap cavity include it is independent respectively with nutrient solution into The culture sap cavity and the culture sap cavity with nutrient solution outlet of mouth connection, the lumen and the cell liquid import and cell Liquid outlet；

Being placed in has the hollow fibre filament for the micropore for not allowing cell to pass through in lumen, wall；Wherein, the doughnut Silk passes through the sealing block, the both ends of the hollow fibre filament culture sap cavity and the nutrient solution port of export with nutrient solution input end respectively Culture sap cavity connect.

2. media to cell culture modules as claimed in claim 1, it is characterised in that

The culture sap cavity is located at lumen both sides, is separated by sealing block, hollow fibre filament passes through lumen to connect cell The culture sap cavity of chamber both sides；Or the culture sap cavity be located at lumen the same side, between two culture sap cavities and sap cavity is cultivated Separated between lumen by sealing block.

3. media to cell culture modules as claimed in claim 1 or 2, it is characterised in that

It is described that there is culture sap cavity import, the culture sap cavity import and the nutrient solution with the culture sap cavity of nutrient solution inlet communication Inlet communication；It is described that there is the outlet of culture sap cavity, the culture sap cavity outlet and institute with the culture sap cavity of nutrient solution outlet State nutrient solution outlet；

Wherein, the culture sap cavity import is located in the body, passes through intrinsic processing flow path and the nutrient solution import Connection；The culture sap cavity outlet is located in the body, and the media to cell culture modules, which further includes, is located at the body interior Flow path and the optional culture fluid path transfer tube pipeline for being located at the body exterior are processed, the culture sap cavity outlet passes through described Process flow path and the optional culture fluid path transfer tube pipeline and the nutrient solution outlet.

4. such as the media to cell culture modules any one of claim 1-3, it is characterised in that

There is the lumen lumen import and lumen to export, respectively with cell liquid import and cell liquid outlet；Its In, the lumen import is each provided at the body interior with lumen outlet, and the media to cell culture modules, which further includes, is located at institute The cell fluid path transfer tube pipeline of body exterior is stated, wherein, the lumen import is exported respectively by being located at lumen The processing flow path and cell liquid import and cell liquid outlet, the cell liquid import for stating body interior go out with the cell liquid Pass through the cell fluid path transfer tube pipeline connection between mouthful.

5. media to cell culture modules as claimed in claim 1, it is characterised in that

The media to cell culture modules has nutrient solution flow path and cell liquid flow path；Wherein, nutrient solution flow path includes nutrient solution successively Import, the culture sap cavity of input end, inside hollow fibre filament, the culture sap cavity of the port of export and nutrient solution outlet；Cell liquid flow path Include cell liquid import, the hollow fibre filament external space in lumen and cell liquid outlet successively.

6. media to cell culture modules as claimed in claim 4, it is characterised in that

The media to cell culture modules has nutrient solution flow path and cell liquid flow path；Wherein, nutrient solution flow path includes nutrient solution successively Import, processing flow path, culture sap cavity import, the culture sap cavity of input end, inside hollow fibre filament, the culture sap cavity of the port of export, Cultivate sap cavity outlet, processing flow path, optional culture fluid path transfer tube pipeline and nutrient solution outlet；Cell liquid flow path includes successively Cell liquid import, processing flow path, lumen import, the hollow fibre filament external space in lumen, lumen outlet, processing stream Road, cell liquid outlet and cell fluid path transfer tube pipeline.

7. media to cell culture modules as claimed in claim 1, it is characterised in that the body of the media to cell culture modules by upper strata, Middle level and lower floor's composition, wherein：

The upper strata is equipped with culture supernatants import and culture supernatants export, and connects respectively with being arranged at the culture sap cavity in middle level It is logical；And connection culture supernatants import and middle level nutrient solution import processing flow path and connect culture supernatants and export and middle level The processing flow path of nutrient solution outlet；

The middle level is the frame structure of a middle part hollow out, which is equipped with the middle level with culture supernatants inlet communication Processing flow path, culture sap cavity between nutrient solution import, the import of culture sap cavity, middle level nutrient solution import and culture sap cavity import go out The middle level nutrient solution outlet, the outlet of culture sap cavity and the processing between the outlet of middle level nutrient solution of mouth and culture supernatants outlet Processing flow path, lumen between flow path, and cell liquid import, lumen import, cell liquid import and lumen import go out Mouth, cell liquid outlet and the processing flow path between lumen outlet and cell liquid outlet；

The hollow fibre filament is placed in lumen, its both ends passes through the sealing block, is connected respectively with culture sap cavity；With

The optional culture fluid path transfer tube pipeline for being arranged on the body exterior, the culture fluid path transfer tube pipeline connection training Nutrient solution chamber is exported exports separated processing flow path with middle level nutrient solution；And/or

The optional cell fluid path transfer tube pipeline for being arranged on the body exterior, the cell fluid path transfer tube pipeline connection institute Cell liquid import is stated to export with cell liquid.

8. media to cell culture modules as claimed in claim 7, it is characterised in that

The culture fluid path transfer tube pipeline and cell fluid path transfer tube pipeline are flexible pipe for medical purpose；And/or

The upper strata is additionally provided with slot and chip pulling mouth, wherein, the slot is used to position insertion nutrient solution module, the chip Pulling mouth is used for convenient taking-up nutrient solution module；And/or

Sealing block is bonded with the contact surface leakproofness in middle level, and equal leakproofness is bonded between upper strata, lower floor and middle level binding face, by The side wall of sealing block and upper strata inner wall, lower floor's inner wall and hollow-out part forms the culture sap cavity and lumen；And/or

Between the culture supernatants import and middle level nutrient solution import and in culture supernatants outlet and middle level nutrient solution Sedimeter is set between outlet, with the pollution introduced to avoid puncture medical rubber stopper；Preferably, filter sizes are 0.2 μ m。

9. media to cell culture modules as claimed in claim 1, it is characterised in that

The volume of the culture sap cavity is 0.2~0.5L, is preferably 0.3~0.35L；And/or

The volume of the lumen is 0.4~1L, is preferably 0.6~0.8L；And/or

The length of the hollow fibre filament is 130~170mm, and 20~50 μm of thickness of pipe wall, the aperture of micropore is less than 1 μm on tube wall； And/or

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

Nutrient solution import, nutrient solution outlet are sealed using medical rubber stopper.

10. a kind of nutrient solution module, including：

11. nutrient solution module as claimed in claim 10, it is characterised in that

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

The body and ventilated membrane are transparent biocompatible materials；And/or

Storage chamber import, storage chamber outlet are sealed using medical rubber stopper；And/or

The storage chamber with storage liquid inlet communication has a storage chamber import, the storage chamber import by default processing pipeline and The storage liquid inlet communication；The storage chamber with storage liquid outlet has the outlet of storage sap cavity, storage chamber outlet By default processing pipeline with storing liquid outlet with described；And/or

The inlet and outlet of storage chamber are equipped with sedimeter, enter for filtering and obstructing impurity and bacterium in storage chamber.

12. nutrient solution module as claimed in claim 10, it is characterised in that

The nutrient solution module is made of upper strata, middle level, lower floor and ventilated membrane, it is bonded with middle level, middle level with lower floor at the middle and upper levels Combine closely between face, no leakage, it is equipped with to be equipped between ventilated membrane and/or middle level and lower floor at the middle and upper levels between middle level and breathes freely Film；

Preferably, the storage liquid inlet and outlet and storage chamber inlet and outlet are each provided in middle level, and when feed flow uses hollow injection pin type Bidirectional interface, the direct medical rubber stopper of penetration cell culture module and nutrient solution module at the same time.

Preferably, filling exit is additionally provided with the nutrient solution module, which is sealed using medical rubber stopper, for initially training Nutrient solution it is filling；Preferably, sedimeter is further respectively had at filling exit.

13. a kind of cell cultivation chip, media to cell culture modules and claim any one of 1-9 containing claim Nutrient solution module any one of 10-12.

14. a kind of cell culture apparatus, including：

Media to cell culture modules any one of claim 1-9；

Nutrient solution module any one of claim 10-12；

Fluid path drive module；

Gas module；

Thermal module；

Control module；With

Connect the pipeline or circuit of each module.

15. cell culture apparatus as claimed in claim 14, it is characterised in that the fluid path drive module includes：

Second driving device, the circulation of the lumen inner cell liquid for controlling media to cell culture modules；

Preferably, the first driving means are arranged on the connecting pipeline of the outlet of storage liquid and nutrient solution import, or are arranged on Nutrient solution is exported on the connecting pipeline with storing liquid import；Driven alternatively, the media to cell culture modules includes external culture fluid path Dynamic pump conduit, the first driving means are arranged on the culture fluid path transfer tube pipeline；

Preferably, second driving device is arranged on cell fluid path transfer tube pipeline；

Preferably, the first driving means and the second driving device are clamp peristaltic pump, preferably bidirectional peristaltic pump.

16. the cell culture apparatus as described in claims 14 or 15, it is characterised in that the gas module includes：Air inlet pipe, Air outlet pipe, gas control valve and optional gas container and optional gas vortex mixer；

Preferably, the gas container includes accommodating compressed air, the container of oxygen and carbon dioxide, each container respectively with gas Vortex mixer connects, and the air inlet pipe is connected with the gas vortex mixer；

Preferably, filtration apparatus is equipped with air inlet pipe and the air outlet pipe mouth of pipe, gas filter membrane is equipped with device；

Preferably, connection accommodates compressed air, oxygen, the container of carbon dioxide with being respectively equipped with gas on the pipeline of gas vortex mixer Body control valve, gas control valve are equipped with gas flowmeter.

17. the cell culture apparatus as described in claims 14 or 15, it is characterised in that the thermal module includes semiconductor temperature Control wafer and the temperature sensitive member platinum resistance being mounted thereon, for controlling heating or refrigeration.

18. the cell culture apparatus as described in claims 14 or 15, it is characterised in that the control module includes：

First controlling switch, for controlling the first driving means；

Second controlling switch, for controlling second driving device；

3rd controlling switch, for controlling gas to convey；With

4th controlling switch, for controlling thermal module to perform heating or refrigeration；

Preferably, the control module further includes for the whole cell cultivation process of real-time inspection and control, realizes automatic cytological The control circuit of culture.

19. cell culture apparatus as claimed in claim 14, it is characterised in that the cell culture apparatus further includes sensor Module, to real-time monitoring temperature, oxygen concentration, gas concentration lwevel and pH value；Preferably, the sensor assembly includes temperature Spend sensor, oxygen concentration sensor, gas concentration lwevel sensor and liquid pH value sensor.

20. cell culture apparatus as claimed in claim 14, it is characterised in that

The media to cell culture modules, nutrient solution module and sensor assembly are positioned over sealable equipped with air admission hole and venthole Chip intracavitary；And/or

The fluid path drive module, the gas control valve in gas module, thermal module and control module integrate and are installed on cabinet Interior, cabinet is equipped with air admission hole and venthole, and cabinet inside, which is equipped with, places media to cell culture modules, nutrient solution module and sensor die The salable chip chamber of block, is provided with alternating interface between man and computer and control button outside cabinet.

21. culture apparatus as claimed in claim 14, it is characterised in that

The nutrient solution is the special serum free medium of immunocyte；And/or

The cell is selected from peripheral blood mononuclear cells, neural stem cell or tumour cell；And/or

The material contacted in described device with cell uses disposable biological compatibility material, and passes through at disinfection and sterilizing Reason.

22. a kind of box set, which includes the media to cell culture modules and claim any one of claim 1-9 Nutrient solution module any one of 10-12, or contain the cell cultivation chip described in claim 13.

23. box set as claimed in claim 22, it is characterised in that the box set further includes integrated installation and has the right the institute of requirement 15 The fluid path drive module stated, gas control valve, the thermal module described in claim 17 and the control mould described in claim 18 The cabinet of block, and batch cultur consumptive material, the medical disposable material include the pipeline or circuit for connecting each module.