CN109825436A - Totally closed cell culture system - Google Patents
Totally closed cell culture system Download PDFInfo
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- CN109825436A CN109825436A CN201910184785.5A CN201910184785A CN109825436A CN 109825436 A CN109825436 A CN 109825436A CN 201910184785 A CN201910184785 A CN 201910184785A CN 109825436 A CN109825436 A CN 109825436A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
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Abstract
The invention provides a totally-enclosed cell culture system, which at least comprises the following parts: the system comprises an incubator, a culture tank, an airflow component, a liquid flow component, a temperature regulation component and a central controller. The system of the invention realizes a constant temperature culture environment, adopts a perfusion mode to culture cells, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The cells and the culture solution are axially stirred in the tank body, so that the radial shearing force is reduced as much as possible, the cells can be effectively protected, and the cell yield is improved. The system separately admits air, guarantees that each gaseous component content is stable among the culture process, can guarantee that the change of external gas can not directly cause the influence to cell culture among the cell culture process, guarantees that gaseous each component content is unchangeable, accomplishes the control to cultivateing temperature, liquid measure and gas concentration at the culture in-process, keeps the cultivation environment stable, reduces manual operation simultaneously, reduce cost, reduces the risk of cultivateing in-process misoperation, improves cultivation efficiency.
Description
Technical field
The present invention relates to technical field of cell culture, more particularly to a kind of totally-enclosed cell culture system.
Background technique
In recent years, CAR-T cellular immunotherapy is considered as most being hopeful to capture one of therapy of cancer.It has it is many its
The incomparable advantage of his therapy improves the accuracy of oncotherapy if CAR-T cell can have multiple target sites, and
Mechanism is not limited by MHC (major histocompatibility complex)；CAR-T cell kills tumor range more
Extensively, effect is more longlasting；Technical attributes are strong, and reproducibility is strong etc..2018, FDA had approved two CD19CAR-T cell drug
(respectively Kymriah and Yescarta), this two CAR-T cell drug achieve good in Hematological malignancies treatment
Effect.However there are still very various limitations for CAR-T cellular immunotherapy, such as the preparation of CAR-T cell.It is treated in CAR-T
In the process, the T cell Jing Guo technological transformation need to be subjected in vitro culture, reaches and meets the cell quantity (general one that treatment requires
A patient's needs several hundred million or even tens CAR-T cells) after, then they are fed back to targeting in patient body and kills cancer cell,
However be limited to current technology means, the Time in Vitro of CAR-T cell account for it is long, thus extend clinical treatment week
Phase.
Cell culture (cell culture) refers to simulated in vivo environment (sterile, preference temperature, pH value and one in vitro
Determine nutritional condition etc.), it is allowed to survive, grow, breed and maintain a kind of method of primary structure and function.Cell culture technology is
Important and common technology in RESEARCH ON CELL-BIOLOGY method, being cultivated the cells by cell culture technology can both obtain greatly
Cell is measured, and the growing multiplication etc. of the signal transduction of cell, the anabolism of cell, cell can be studied whereby.
Current cell culture is manual operation culture mostly, when needing mass propgation cell towards industrialization, is needed
It spends a large amount of human cost and time cost, while as the burden of operator increases, causes the risk of fault also significantly
Increase；In addition, artificial culture can not accurately control the environment of cell growth, it is unfavorable for the growth of cell.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of totally-enclosed cell culture systems
System.
In order to achieve the above objects and other related objects, first aspect present invention provides a kind of totally-enclosed cell culture system
System includes at least following part:
Incubator；
Culture tank is set in incubator for carrying out cell culture；The culture tank is equipped with blender, the blender
In culture tank；
Air-flow component is connected to the incubator, for adjusting oxygen and gas concentration lwevel in the incubator；
Liquid stream component is connected to the culture tank, and for adjusting the fluid flow in culture tank, filtration cell culture is generated
Metabolite and recycling finished product cell；
Temperature adjustment component, for adjusting the temperature in the incubator；
Central controller connects and controls the air-flow component, liquid stream component and temperature adjustment component.
Second aspect of the present invention provides the purposes of totally-enclosed cell culture system above-mentioned, to train for totally-enclosed cell
It supports.
Third aspect present invention provides the method for carrying out totally-enclosed cell culture using aforementioned totally-enclosed cell culture system,
Using following steps:
1) feed liquor process: setting liquid inlet volume injects culture medium to culture tank；
2) build culture environment: the concentration value of default oxygen and carbon dioxide is injected separately into oxygen and two into incubator
Carbonoxide, the gas concentration value of oxygen and carbon dioxide in the real time measure incubator, respectively compared with setting value, when in incubator
Oxygen and carbon dioxide values it is all up to standard after, the gas in incubator is injected into culture tank；Incubator temperature is set, and right
Incubator preheating；
3) lasting culture: injection cell injects the factor, starts blender, in lasting culture to culture tank injection culture medium,
Filter metabolite and discharge waste liquid；
4) it replaces and is concentrated: after the completion of cell culture, first using physiological saline replacement medium, carried out after being replaced dense
Waste liquid is persistently discharged in contracting, reduces liquid volume in culture tank；
5) finished product recycles: stopping blender, recycles the finished product cell in culture tank.
As described above, totally-enclosed cell culture system of the invention, has the advantages that
System of the present invention realizes constant temperature incubation environment, carries out cell culture using perfusion mode, realizes from cell
The Full enclosed integrated process of activation, infection, amplification to finished product recycling.System of the present invention uses perfusion mode, rather than
Waste liquid can be discharged in reperfusion mode during the cultivation process, prevent the accumulation of Toxic Metabolites, be conducive to reach higher cell training
Support density, it is possible to reduce subsequent processing step can carry out finished product cell recycling without operations such as centrifugations, simplify operation, can
To improve culture efficiency, it is easy to industrialization, realizes the Full enclosed integrated mistake recycled from cell-stimulating, infection, amplification to finished product
Journey.By axially stirring cell and culture solution in tank body, radial shearing force is minimized, cell is can effectively protect, is improved
Cell yield.The system separates air inlet, guarantees each gas component stable content in incubation, it can be ensured that cell culture
Ambient atmos variation will not directly impact cell culture in journey, guarantee that gas each component content is constant, in incubation
In accomplish control to cultivation temperature, liquid measure and gas concentration, keep culture environment to stablize, while reducing manual operation, reduce
Cost reduces the risk of operation error in incubation, improves culture efficiency.
Detailed description of the invention
Fig. 1 is shown as totally-enclosed cell culture system signal transmission figure of the invention；
Fig. 2 is shown as totally-enclosed cell culture system Facad structure figure of the invention；
Fig. 3 is shown as totally-enclosed cell culture system backside structure figure of the invention；
Fig. 4 is shown as each component distribution figure of the totally-enclosed cell culture system incubator space of a whole page of the invention；
Fig. 5 is shown as the culture tank of totally-enclosed cell culture system of the invention and the connected relation of liquid stream component is illustrated
Figure.
Fig. 6 is shown as the culture tank internal structure of totally-enclosed cell culture system of the invention.
Fig. 7 is shown as the overlooking structure figure of the culture jar agitator of totally-enclosed cell culture system of the invention.
Component label instructions
1 incubator
2 culture tanks
2.1 inlet
2.2 first circulation mouths
2.3 second circulation mouths
2.4 recovery port
2.5 blender
2.51 axis
2.52 blade
2.53 closing accommodating chamber
2.6 culture lids
2.61 lid ontology
2.62 exhaust portion
2.63 intake section
2.7 inner fovea part
3 air-flow components
3.1 air flue
3.1.1 air filter
3.1.2 air pipe line
3.1.3 blow vent
3.2 carbon dioxide accesses
3.2.1 carbon dioxide plant is stored up
3.2.2 carbon dioxide pressure reducing valve
3.2.3 carbon dioxide channel selector
3.2.4 carbon dioxide pipeline
3.2.5 carbon dioxide access incubator import
3.3 oxygen accesses
3.3.1 oxygen unit is stored up
3.3.2 oxygen pressure reducing valve
3.3.3 oxygen channel selector
3.3.4 oxygen pipeline
3.3.5 oxygen access incubator import
3.4 mixed gas suction passage
3.4.1 mixed gas suction pump
3.4.2 mixed gas suction line
3.4.3 mixed gas access incubator exports
3.5 exhaust gas vent pathways
3.6 gas concentration induction modules
3.6.1 oxygen gas concentration sensor
3.6.2 density of carbon dioxide gas sensor
3.7 air discharge passage
3.8 fan
4 liquid stream components
4.1 feed liquor accesses
4.1.1 liquid storing bag
4.1.2 inlet pipe
4.1.3 liquid feeding pump
4.1.5 tank gage
4.2 circulation path
4.2.1 circulation line
4.2.2 circulating pump
4.2.3 filter
4.3 waste liquid accesses
4.3.1 waste drains pump
4.3.2 waste liquid barrel
4.3.3 waste-solution line
4.4 recovery passage
4.4.1 recovery pipe
4.4.2 recovery pump
4.4.3 collection bag
4.5 weighing sensor
5 temperature adjustment components
5.1 heating device
5.2 temperature sensor
6 central controllers
7 sterilamps
8 stirring drivers
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily.
Please refer to Fig. 1-Fig. 7.It should be clear that this specification structure depicted in this specification institute accompanying drawings, ratio, size etc., only to
Cooperate the revealed content of specification, so that those skilled in the art understands and reads, being not intended to limit the invention can be real
The qualifications applied, therefore do not have technical essential meaning, the tune of the modification of any structure, the change of proportionate relationship or size
It is whole, in the case where not influencing the effect of present invention can be generated and the purpose that can reach, it should all still fall in disclosed skill
In the range of art content can cover.Meanwhile in this specification it is cited as "upper", "lower", "left", "right", " centre " and
The term of " one " etc. is merely convenient to being illustrated for narration, rather than to limit the scope of the invention, relativeness
It is altered or modified, under the content of no substantial changes in technology, when being also considered as the enforceable scope of the present invention.
It should be clear that central controller of the present invention can be located at any position of culture box outer wall, or it is located at outside incubator
Console on etc., as long as can be connect with other component in totally-enclosed cell culture system, therefore, not in attached drawing 2-
Central controller position is shown on 4.
As Figure 1-Figure 4, totally-enclosed cell culture system provided by the invention includes at least following part:
Incubator 1.The incubator is for providing stable culture environment, including stable gaseous environment and temperature ring
Border.
Culture tank 2 is set in incubator for carrying out cell culture；The culture tank 2 is equipped with blender, the stirring
Device is set in culture tank；
Air-flow component 3 is connected to the incubator 1, dense for adjusting oxygen in the incubator 1 and carbon dioxide
Degree；
Liquid stream component 4 is connected to the culture tank 2, for adjusting the fluid flow in culture tank 2, filtration cell culture
The metabolite and recycling finished product cell of generation；
Temperature adjustment component 5, for adjusting the temperature in the incubator；
Central controller 6 connects and controls the air-flow component, liquid stream component and temperature adjustment component.
The culture systems use perfusion mode, and waste liquid can be discharged during the cultivation process, prevented for rather than reperfusion mode
The accumulation of evil metabolin, is conducive to reach higher cell culture density, it is possible to reduce subsequent processing step, without centrifugation etc.
Operation can carry out finished product cell recycling, simplify operation, facilitate the recycling of cell, culture efficiency can be improved, be easy to industrialization.
Further, the air-flow component 3 includes:
Isolated air flue 3.1, carbon dioxide access 3.2 and oxygen access 3.3, is connected to the incubator 1 respectively,
For delivering gas in the incubator, mixed gas is formed.
Mixed gas suction passage 3.4 is connected to the incubator 1 and culture tank 2, for by the gaseous mixture in incubator 1
Body inputs culture tank 2.
Exhaust gas vent pathway 3.5 is connected to, for discharging the exhaust gas generated in cell cultivation process with the culture tank 2.
Gas concentration induction module 3.6, including oxygen gas concentration sensor 3.6.1 and density of carbon dioxide gas sensing
Device 3.6.2；Oxygen gas concentration value and real-time density of carbon dioxide gas value in real time, the gas are respectively used in measurement incubator
Bulk concentration induction module provides detection information to central controller 6.
Further, the air flue 3.1 includes air pipe line 3.1.2；The air pipe line 3.1.2 and the culture
Case 1 is connected to.
In one embodiment, the air flue 3.1 further includes air filter 3.1.1, for filtering extraneous sky
Gas makes the air cleaning entered in incubator 1.
The carbon dioxide access 3.2 includes storage carbon dioxide plant 3.2.1, carbon dioxide pressure reducing valve 3.2.2, titanium dioxide
Carbon channel selector 3.2.3 and carbon dioxide pipeline 3.2.4；The storage carbon dioxide plant 3.2.1 and the carbon dioxide pipeline
3.2.4 it connects, the carbon dioxide pipeline 3.2.4 is driven by carbon dioxide pressure reducing valve, the carbon dioxide pipeline 3.2.4 and institute
State the connection of incubator 1；The carbon dioxide access is equipped with carbon dioxide channel selector 3.2.3, and the carbon dioxide access is opened
3.2.3 is closed to be controlled by the central controller 6.
In one embodiment, the carbon dioxide channel selector is selected from storage carbon dioxide plant switch, carbon dioxide
One of pipeline switch, carbon dioxide decompression threshold switch are a variety of.It can be solenoid valve.
The oxygen access 3.3 includes storage oxygen unit 3.3.1, oxygen pressure reducing valve 3.3.2, oxygen channel selector 3.3.3
With oxygen pipeline 3.3.4；The storage oxygen unit 3.3.1 is connect with the oxygen pipeline 3.3.4, the oxygen pipeline 3.3.4
It is driven by oxygen pressure reducing valve 3.3.2, the oxygen pipeline 3.3.4 is connected to the incubator 1；The oxygen access is equipped with oxygen
Gas channel selector 3.3.3, the oxygen channel selector 3.3.3 are controlled by the central controller 6.
In one embodiment, the oxygen channel selector is selected from storage oxygen unit switch, oxygen pipeline switch, oxygen
Depressurize one of threshold switch or a variety of.It can be solenoid valve.
The mixed gas suction passage 3.4 includes mixed gas suction pump 3.4.1 and mixed gas suction line
3.4.2；The mixed gas suction line 3.4.2 is driven by mixed gas suction pump 3.4.1, the mixed gas suction line
3.4.2 for mixed gas to be injected into culture tank 2 from incubator 1；The mixed gas suction pump 3.4.1 is controlled by center
Device 6 processed controls.
In one embodiment, the mixed gas suction line 3.4.2 is equipped with pneumatic filter.For to entrance
The gas of culture tank is filtered.
Further, the exhaust gas vent pathway 3.5 includes exhaust pipe road.The exhaust pipe road is equipped with list
To valve, prevent outside air from entering in tank by exhaust pipe road.
The exhaust gas vent pathway directly discharges the exhaust to outside system, is not connected to the incubator 4.
Further, it is equipped with air discharge passage 3.7 in the incubator, for gas in case to be discharged, keeps gas in case
Pressure is stablized.
Further, the air discharge passage 3.7 includes gas exhaust manifold, for gas in case to be discharged, keeps case
Interior stable gas pressure.
In one embodiment, the air discharge passage 3.7 and the air flue 3.1 are same access.
In one embodiment, the carbon dioxide access 3.2 and oxygen access 3.3 are equipped with titanium dioxide on incubator
Carbon access incubator import 3.2.5 and oxygen access incubator import 3.3.5, the carbon dioxide access incubator import
3.2.5 the top in incubator is set to oxygen access incubator import 3.3.5.Cold air is easy to sink to the bottom, and hot-air rises,
It is consistent to reach entire environmental gas concentration after top can faster allow gas mixing.
In one embodiment, mixed gas access is equipped with mixed gas access incubator on incubator and exports
3.4.3, the mixed gas access incubator outlet 3.4.3, oxygen gas concentration sensor 3.6.1 and carbon dioxide gas are dense
Spend the lower part that sensor 3.6.2 is set in incubator.The concentration that more can accurately reflect mixed gas makes the mixing into culture tank
Gas index is truer.
The air flue 3.1 and/or the air discharge passage 3.7 are equipped with blow vent 3.1.3 on incubator, described
Blow vent 3.1.3 is far from the carbon dioxide access incubator import 3.2.5, oxygen access incubator import 3.3.5, gaseous mixture
Body access incubator exports 3.4.3, oxygen gas concentration sensor 3.6.1 and density of carbon dioxide gas sensor 3.6.2.It is anti-
Only gas escapes too fast.
In one embodiment, it is equipped with fan 3.8 in the incubator, for stirring air-flow, accelerates mixing, makes gas
Mixing more evenly, and accelerates the heat exchange inside incubator.
In one embodiment, the fan 3.8 is set to the top in incubator.
Further, the central controller 6 includes following part:
Gas concentration comparing unit, the real-time oxygen gas concentration value for sending gas concentration induction moduleWith
Real-time density of carbon dioxide gas valueWith default oxygen gas concentration valueWith default carbon dioxide concentration value
It is compared respectively, the difference of required concentration, i.e. concentration difference is obtained according to public formula (I) and (II)With
Gas concentration switch control unit, for controlling oxygen access, carbon dioxide access, mixed gas suction passage
Opening and closing:
According toValue, adjust oxygen access make-and-break time；
According toValue, the make-and-break time of regulation of carbon dioxide access；
WhenAndWhen being all satisfied the threshold range of setting, mixed gas suction passage is opened, it will be in incubator
Gas sucks culture tank.
WhenAndIn at least one be unsatisfactory for setting threshold range when, close mixed gas suction passage.
Oxygen gas concentration valueCarbon dioxide concentration valueAnd threshold range can cell be cultivated as needed
Demand setting.In a preferred manner,AndThreshold range can be selected from -0.1%~0.1%.
In one embodiment, whenAndWhen being all satisfied the threshold range of setting, timing controlled opens gaseous mixture
Gas in incubator is sucked culture tank by body suction passage, and timing is according to the gas capacity and gaseous mixture in culture tank
The flow velocity of body suction pump codetermines.Timing controlled refers to, whenAndWhen being all satisfied the threshold range of setting, not immediately
Mixed gas suction passage is opened, but according to the time of default, to control the opening and closing of mixed gas suction passage.
Further, the opening and closing of oxygen access is controlled by the opening and closing of control oxygen channel selector；By controlling dioxy
Change the opening and closing of carbon channel selector to control the opening and closing of carbon dioxide access, is controlled by controlling the opening and closing of mixed gas suction pump
The opening and closing of mixed gas suction passage.
In one embodiment, programmed algorithm can be used to control oxygen channel selector, carbon dioxide channel selector, mix
Close the opening and closing of gas suction passage.According to current gas concentration measurement, different lead to is switched by process control gas passage
The disconnected time, so that the real gas concentration value in incubator is close or equal to setting value.
In one embodiment, mixed gas suction passage is adjusted by the opening and closing of regulation mixed gas suction pump
Opening and closing.
It, can basis in a kind of feasible embodimentValue, using classification regulation by the way of regulation of carbon dioxide
Access make-and-break time.Such as basisValue classification,It is smaller, compartment carbon dioxide access is detected twice opens duration
It is shorter.
It, can basis in a kind of feasible embodimentValue, adjust oxygen access by the way of classification regulation and open
The make-and-break time of pass.Such as basisValue classification,It is smaller, it is shorter that compartment oxygen access duration is detected twice.
Hierarchical level andHierarchical level can flexible design.Hierarchical level generally can be 1-10 grades.Example
1 grade, 2 grades, 3 grades, 4 grades, 5 grades, 6 grades, 7 grades, 8 grades, 9 grades, 10 grades can be such as divided into.
Carbon dioxide access or oxygen access gas flow rate in ventilation are constant, and the control access switch on and off time is adjustable
Ventilatory capacity.Opening time is longer, and ventilatory capacity is bigger.Such mode controls that ventilatory capacity is simply accurate, and accessory cost is controllable.
Further, to guarantee that gas mixing is uniform, oxygen or carbon dioxide access can be disconnected, is filling gas mixing
After point, then by the progress gas concentration value measurement of gas concentration induction module.I.e. when disconnecting one section of access mixing after injecting gas
Between after, then by gas concentration induction module carry out gas concentration value measurement.In general, ventilation duration is shorter, required open circuit is mixed
It is shorter to close the time.The gas refers to oxygen or carbon dioxide.Access refers to oxygen access or carbon dioxide access.
By taking incubator is having a size of 373mm × 330mm × 250mm as an example:
In a specific embodiment,Value be divided into seven grades, Keep logical
When gaseity, gas flow rate is constant；WhenWhen more than or equal to 2%, control carbon dioxide channel selector closes after opening 1.5 seconds
It closes, then waits 12 seconds so as to read the concentration value of density of carbon dioxide gas sensor after gas mixing, continue and set
Value compares；WhenWhen, control carbon dioxide channel selector is closed after opening 1 second, after then waiting 9 seconds, is read
The concentration value of density of carbon dioxide gas sensor is taken, continues and setting value compares；WhenWhen, control two
Carbonoxide channel selector is closed after opening 0.8 second, after then waiting 3 seconds, reads the dense of density of carbon dioxide gas sensor
Angle value, continues and setting value compares；WhenWhen, control carbon dioxide channel selector closes after opening 0.6 second
It closes, directly reads the concentration value of density of carbon dioxide gas sensor, continue and setting value compares；When
When, control carbon dioxide channel selector is closed after opening 0.5 second, directly reads the concentration of density of carbon dioxide gas sensor
Value, continues and setting value compares；WhenWhen, control carbon dioxide channel selector closes after opening 0.3 second
It closes, directly reads the concentration value of density of carbon dioxide gas sensor, continue and setting value compares；WhenWhen, two
Carbonoxide channel selector remains off.
Value be equally divided into seven grades, When keeping aeration status, gas flow rate is constant；When
When more than or equal to 2%, control oxygen channel selector is closed after opening 1.5 seconds, and after then waiting 12 seconds, it is dense to read oxygen gas
The concentration value of sensor is spent, continues and setting value compares；WhenWhen, after control oxygen channel selector is opened 1 second
It closes, after then waiting 9 seconds, reads the concentration value of oxygen gas concentration sensor, continue and setting value compares；When When, control oxygen channel selector is closed after opening 0.8 second, after then waiting 3 seconds, reads oxygen gas concentration
The concentration value of sensor, continues and setting value compares；WhenWhen, control oxygen channel selector opens 0.6
It is closed after second, directly reads the concentration value of oxygen gas concentration sensor, continued and setting value compares；When
When, control oxygen channel selector is closed after opening 0.5 second, directly reads the concentration value of oxygen gas concentration sensor, continue and
Setting value compares；WhenWhen, control oxygen channel selector is closed after opening 0.3 second, directly reads oxygen
The concentration value of gas concentration sensor, continues and setting value compares；WhenWhen, oxygen channel selector remains turned-off shape
State.
The liquid stream component includes feed liquor access 4.1, circulation path 4.2, waste liquid access 4.3 and recovery passage 4.4；It is described
Feed liquor access 4.1 and circulation path 4.2 are connected to the culture tank 2 respectively, the feed liquor access 4.1 be used for culture tank 2 into
Liquid, the circulation path 4.2 are used for the metabolite that filtration cell generates；The waste liquid access 4.3 and the circulation path 4.2
Connection, for the metabolite to be discharged, the recovery passage 4.4 is connected to the culture tank 2, for recycling finished product cell；
The flow control component of the feed liquor access 4.1, circulation path 4.2, waste liquid access 4.3 and recovery passage 4.4 is by the center
Controller 6 controls.
The flow control component can be flow pump or flow control switch.
The feed liquor access 4.1 includes liquid storing bag 4.1.1, inlet pipe 4.1.2 and liquid feeding pump 4.1.3, the liquid storing bag
4.1.1 it is connected to the inlet pipe 4.1.2；The inlet pipe 4.1.2 is driven by the liquid feeding pump 4.1.3, the feed liquor
Pipeline 4.1.2 is connected to the culture tank 2；The liquid feeding pump 4.1.3 is controlled by central controller 6；Liquid in the liquid storing bag
Body, which can according to need, to be replaced, such as can be culture medium or physiological saline.The culture medium can be fluid nutrient medium.
Further, the inlet pipe is equipped with cell branch pipe, cell can be injected into culture tank by the branch pipe.
It injects after the completion of cell, branch pipe is in close state, such as can be set branch pipe lid and be closed branch pipe.
In one embodiment, the feed liquor access 4.1 further includes tank gage 4.1.5.The tank gage 4.1.5 is set to
It is connect on the inlet pipe 4.1.2 and with the central controller, the liquid level for detecting inlet pipe changes.By inlet tube
Liquid level information in road is sent to central controller 6 in real time.
The circulation path 4.2 includes circulation line 4.2.1, circulating pump 4.2.2 and filter 4.2.3, the circulation pipe
Road 4.2.1 is connected to the culture tank 2, the filter 4.2.3 be set to the circulation line 4.2.1 on and with the circulation pipe
Road 4.2.1 connection, the circulation line 4.2.1 are driven by the circulating pump 4.2.2, and the circulating pump 4.2.2 is by the center
Controller 6 controls.
In one embodiment, the filter 4.2.3 can be hollow fiber column.
In one embodiment, the waste liquid access 4.3 includes waste drains pump 4.3.1, waste liquid barrel 4.3.2 and waste-solution line
4.3.3, the waste-solution line 4.3.1 is connected to the circulation path 4.2, the waste liquid barrel 4.3.2 and the waste-solution line
4.3.1 it connects, the waste-solution line 4.3.1 is driven by the waste drains pump 4.3.1, and the waste drains pump 4.3.1 is controlled by the center
Device 6 processed controls.
In one embodiment, the recovery passage 4.4 includes recovery pipe 4.4.1, recovery pump 4.4.2 and collection bag
4.4.3, the recovery pipe 4.4.1 is connected to the culture tank 2, and the recovery pipe 4.4.1 is driven by the recovery pump 4.4.2
Dynamic, the collection bag 4.4.3 is connected to the recovery pipe 4.4.1, and the recovery pump 4.4.2 is controlled by the central controller 6
System.
In one embodiment, the culture systems are equipped with collection bag and put disk, for placing collection bag.Described time
Closing bag puts disk equipped with anti-skidding muscle.
Further, as shown in figure 5, the side wall of the culture tank 2 is arranged, there are four mouths:
Inlet 2.1, for being connected to feed liquor access 4.1；
First circulation mouth 2.2 and second circulation mouth 2.3, for being connected to circulation path 4.2；
Recovery port 2.4, for being connected to recovery passage 4.4.
As shown in figure 5, circulation path in the open state, under the driving of circulating pump, is trained in cell cultivation process
The mixture for supporting cell culture medium and cell in tank is discharged into the filter of circulation path 4.2 from first circulation mouth 2.2
4.2.3 in, the filter 4.2.3 membrane aperture can be 0.2-1 microns, can produce through cell culture in water and culture medium
The ingredient of raw metabolic waste, and cell itself can not then penetrate, therefore, partial medium and metabolic waste form waste liquid by mistake
Filter, and remaining partial medium and cell are entered in culture tank 2 by second circulation mouth 2.3 again.
In incubation, the circulation path can be opened or closed according to user.
Further, the filter 4.2.3 is equipped with waste liquid chamber, for keeping in waste liquid.It is set on the filter 4.2.3
There are first outlet and second outlet, the first outlet is used to the remaining partial medium and cell being re-delivered to culture
In tank.The second outlet is for being connected to waste liquid chamber and the waste-solution line 4.3.1, for waste liquid to be discharged.
The first circulation mouth 2.2, recovery port 2.4 are all set in the bottom position of culture pot sidewall, sufficiently to pump out
Cell and liquid in culture tank 2.
In one embodiment, the liquid stream component 4 includes weighing sensor 4.5, and the weighing sensor 4.5 is used for
Real-time culture tank weight is measured, the culture pot bottom is located at and provides detection information to central controller 6.
The central controller contains fluid control module: the central controller can receive user instruction, and according to
User instruction controls the on-off of feed liquor access, circulation path, waste liquid access and recovery passage.
During the cultivation process, user can instruct, and liquid inlet volume, waste liquid discharge rate is set as needed.
In one embodiment, liquid volume can be converted into weight according to fluid density by the central controller 6, so
The control of liquid inlet volume, waste liquid discharge rate is carried out according to weighing sensor afterwards.When cultivating just beginning and end liquid feeding, carries out culture tank and go
Skin, then in incubation, culture tank weight is then the weight of culture tank content.
In one embodiment, the central controller 6 includes:
Weight comparing unit, when feed liquor, the culture tank weight M of the real time measure for sending weighing sensort, with
The culture tank weight M of family instructionin0It is compared, weight difference M is obtained according to public formula (III)in:
Min=M0-Mt (III)
Feed liquor access on-off control unit, for according to MinControl the on-off of the feed liquor access.
Further, described according to MinControl the on-off of the feed liquor access are as follows:
MinGreater than 0, feed liquor access, M are openedinFeed liquor access is disconnected when equal to 0 or less than 0.
The central controller further includes waste liquid access on-off control unit.When drain, the weight comparing unit will claim
Retransmit the culture tank weight M for the real time measure that sensor is sentt, culture tank weight M with user instructionout0It is compared according to public affairs
Formula (IV) obtains weight difference Mout:
Mout=Mt-Mout0(Ⅳ)
Waste liquid access on-off control unit, according to MoutControl the on-off of the waste liquid access.
MoutGreater than 0, waste liquid access, M are openedoutFeed liquor access is disconnected when equal to 0 or less than 0.
The temperature adjustment component 5 includes heating device 5.1 and temperature sensor 5.2；The heating device 5.1 and the temperature
Sensor 5.2 is set in the incubator 1, is connect respectively with the central controller 6, and the heating device 5.1 is used for training
Feeding box cavity is heated；The temperature sensor 5.2 is for measuring real-time temperature values in incubator and controlling to the center
Device 6 provides detection information；Control of the heating device 5.1 by the central controller 6.The temperature adjustment component can make to be located at
Culture tank in incubator is in isoperibol, and guarantees that the gas temperature entered in culture tank is constant.
In one embodiment, preset temperature value T0, the temperature value can be the temperature value of suitable cell culture；Institute
It states temperature sensor and provides real-time temperature values T in incubator to the central controllert, with T0Compare, works as TtLess than T0When, in
Entreat controller control heating device starting；Work as TtMore than or equal to T0When, the operation of central controller controls heater stop.
The heating device 5.1 can be heating plate.It is attached on the culture chamber interior wall.
The heating plate and temperature sensor are commercial product.
As shown in Figure 6 and Figure 7, the blender 2.5 includes: axis 2.51；And with the axis of the axis in rotation
At least two panels blade 2.52 that is symmetrical and connecting the axis, the every blade includes blade body, and the blade body is in
Helical form extends, and the axial length of the blade body accounts for the 20% to 35% of the length of the axis, the rotation of blade body
Angle is 15 ° to 50 °, and maximum radial length is 20mm to 54mm, and radical length from bottom to top gradually becomes smaller.Using upper
The blade of shape is stated, the shearing force for T cell can be reduced while T cell and cultivating system are stirred evenly.
Further, from the bottom of the blade body to top, radical length with axial height with linear relationship by
Gradual change is small.
Further, the helical form is to be retained in the circular cone after being cut off on the basis of a right helicoid by circular conical surface
Part within face is formed, and the semi-cone angle of the circular conical surface is 20-45 degree.Can reach be conducive to axial rolling cell up and down with
It while the technical effect of culture solution, reduces blade and tank wall forms the space of radial whirl, to reduce whipping process production
Raw shearing force improves its yield to protect T cell.
The culture tank bottom surface includes inner fovea part 2.7 at its center, for fixing culture tank.
Stirring driver 8 is equipped in the culture systems, for driving the blender.The stirring driver is by described
Central controller 6 controls.
In one embodiment, the blade further includes the closing accommodating chamber at blade body maximum radial length
2.53, for accommodating the magnetite of driving blender rotation.At this point, the stirring driver 8 is magnetic driver, the magnetic force is driven
Dynamic device generates magneticaction to the magnetite, and then drives blender rotation.In a non-contact manner using magnetic agitation principle
Blender is driven, is compared compared to by the way of the shaft of motor direct-drive blender, it is ensured that inside culture tank
Cell culture environment it is clean and easy to maintain.The reason is that motor is logical using the shaft of motor direct-drive blender
It often needs to be placed in except culture tank, then the shaft of blender must extend culture tank, therefore the shaft and culture of blender
Needing to guarantee the stringent sealing for a long time under culture environment between tank, the complexity for so increasing system is difficult to safeguard,
And the abrasion of sealing element itself may also pollute cultivating system.
The blade of blender of the present invention uses helicoid and specific axial dimension, radial dimension of design,
Reach in cultivating system and axially stir cell and culture solution in tank body, minimize radial shearing force, to protect cell, mentions
The effect of high cell yield.
Culture lid 2.6 is additionally provided in the culture tank 2, culture lid is in close state during the cultivation process.
Further, as shown in fig. 6, the culture lid 2.6 of culture tank 2 includes lid ontology 2.61 and is set to the lid
Ontology is used to convey and be discharged to tank interior space the intake section 2.63 of gas, exhaust portion 2.62, and intake section 2.63 is hanging down
Directly the length in the bottom surface direction of lid ontology is greater than exhaust portion 2.62；In cell culture, intake section is used for bioreactor
Interior conveying gas, when carrying out cell culture, intake section conveys gas, the i.e. structure of intake section by the way of non-contact conveying
Itself is contactless with the liquid level of culture solution, and such setting is beneficial in that, is protruded into liquid level with the air inlet pipe of the prior art, is led
It causes to generate bubble in cultivating system and damage T cell, contactless conveying can prevent the generation of bubble, improve T cell
Yield.Meanwhile intake section is greater than exhaust portion perpendicular to the length in the bottom surface direction of lid ontology, the beneficial effect is that, compared to
The equal length of intake and exhaust or shorter setting can adjust oxygen and carbon dioxide in cultivating system more quickly
Concentration.The gas componant of air inlet is deployed with the different phase of culture, and the ratio of the carbon dioxide of initial stage air inlet is appropriate
Improve, and with the progress of culture, cellular respiration also generates a certain amount of carbon dioxide, thus in air inlet carbon dioxide ratio
It can reduce.
In one embodiment, it is equipped with ultraviolet sterilization lamp in the incubator 4, for sterilizing to incubator.
In one embodiment, the culture systems are equipped with alarm module, and are driven by the central controller 6.In advance
First be arranged liquid stream component, air-flow component, temperature adjustment component alarm critical value, when the central controller receive liquid stream component,
Detection information when transfiniting of air-flow component, temperature adjustment component, controls the alarm module and sounds an alarm.
The central controller can be single-chip microcontroller, and single-chip microcontroller can be 8 minimum systems.The central controller
Different brand and model, or the controller or processor of more seniority top digit can also be selected.The central controller can be used for
Associated control procedures are installed.After installing associated control procedures, the central controller can receive liquid stream component, air-flow component, adjust
The signal of warm component and the instruction of user, and the Parameters of The Parts in component is adjusted as needed, so that system is steadily transported
Row.
Further, the culture tank is made of air-impermeable material.
The culture tank and/or its attachment only pass through individual channel and extraneous progress gas or fluid exchange.
Further include culture chamber door on the incubator, incubator and external environment are separated, makes to form one in incubator
Relatively independent environment.
Full-closed structure refers to, during the entire process of cell culture is recycled from activation, infection, amplification to finished product, entirely
Culture environment (including tank body, filter, pipeline etc.) is in the state of relative closure, only by aseptic gas or fluid path and
The external world communicates, and the incubator is relatively independent with external environment, is in the environment in incubator in controllable range.
Totally-enclosed cell culture system provided by the invention can be used for totally-enclosed cell culture.
The method provided by the invention that totally-enclosed cell culture is carried out using aforementioned totally-enclosed cell culture system, using such as
Lower step:
1) feed liquor process: setting liquid inlet volume injects culture medium to culture tank；
2) build culture environment: the concentration value of default oxygen and carbon dioxide is injected separately into oxygen and two into incubator
Carbonoxide, the gas concentration value of oxygen and carbon dioxide in the real time measure incubator, respectively compared with setting value, when in incubator
Oxygen and carbon dioxide values it is all up to standard after, the gas in incubator is injected into culture tank；Incubator temperature is set, and right
Incubator preheating；
3) lasting culture: injection cell injects the factor, starts blender, in lasting culture to culture tank injection culture medium,
Metabolite and discharge waste liquid are filtered,；
4) it replaces and is concentrated: after the completion of cell culture, first using physiological saline replacement medium, carried out after being replaced dense
Waste liquid is persistently discharged in contracting, reduces liquid volume in culture tank；
5) finished product recycles: stopping blender, recycles the finished product cell in culture tank.
In step 1), culture medium is injected to culture tank using feed liquor access.Specifically, setting liquid inlet volume, according to liquid inlet volume
The culture tank weight M setin0, start liquid feeding pump, the culture medium in liquid storing bag made to be injected into culture tank through inlet pipe
In, the amount for needing that culture medium is injected into culture tank is determined using weight sensor the real time measure culture tank weight；It will weighing
The culture tank weight M for the real time measure that sensor is sentt, culture tank weight M with settingin0It is compared, according to formula
(III) weight difference M is obtainedin:
Min=Min0-Mt (III)
According to MinControl the on-off of the feed liquor access.
Further, MinGreater than 0, feed liquor access, M are opened1Feed liquor access is disconnected when equal to 0 or less than 0.
In step 2), oxygen and carbon dioxide are injected separately into incubator using oxygen access and carbon dioxide access,
The gas in incubator is injected into culture tank using mixed gas suction passage.It is surveyed using density of carbon dioxide gas sensor
The gas concentration value for determining carbon dioxide in incubator utilizes oxygen gas concentration in oxygen gas concentration sensor measurement incubator
Value；By real-time oxygen gas concentration valueWith real-time density of carbon dioxide gas valueRespectively with default oxygen gas concentration
ValueWith default carbon dioxide concentration valueIt is compared, the difference of required concentration is obtained according to public formula (I) and (II), i.e.,
Concentration difference With
According toValue, adjust oxygen access make-and-break time；
According toValue, the make-and-break time of regulation of carbon dioxide access；
WhenAndWhen being all satisfied the threshold range of setting, mixed gas suction passage is opened, it will be in incubator
Gas sucks culture tank；
WhenAndIn at least one be unsatisfactory for setting threshold range when, close mixed gas suction passage.
Further, according toValue, adjust the make-and-break time of oxygen access by the way of classification regulation, and/or,
According toValue, using classification regulation by the way of regulation of carbon dioxide channel selector make-and-break time.
In one embodiment, air-flow is stirred using fan, accelerates mixing, makes gas mixing more evenly.
In step 3), cell is injected using feed liquor access, filters metabolite using circulation path.On the circulation path
Equipped with filter and circulating pump.After starting blender, starts to cultivate, needed to open circulation path at this time according to culture, work as circulation
In the open state, under the driving of circulating pump, the mixture of cell culture medium and cell in culture tank is from culture for access
First circulation mouth on tank is discharged into the filter of circulation path, and the filter, membrane aperture can be 0.2~1 micron,
The ingredient for the metabolic waste that can be generated through cell culture in water and culture medium, and cell itself can not then penetrate, therefore,
Partial medium and metabolic waste form waste liquid and are filtered, the waste liquid chamber being temporarily stored on filter；And remaining partial medium with
Cell is entered in culture tank by the second circulation mouth in culture tank again.The filter is equipped with first outlet and second and goes out
Mouthful, the remaining partial medium and cell are re-delivered in culture tank by first outlet.When needing that waste liquid is discharged, beat
Open waste liquid access, the culture tank weight M that setting waste liquid discharge rate is setout0, waste liquid is drained by second outlet useless
In liquid pipe, and then it is drained into waste liquid barrel.The culture tank weight M for the real time measure that weighing sensor is sentt, with preset training
Support tank weight Mout0It is compared, weight difference M is obtained according to public formula (IV)out:
Mout=Mt-Mout0 (IV)
According to MoutControl the on-off of the waste liquid access.
Further, MoutGreater than 0, waste liquid access, M are openedoutWaste liquid access is disconnected when equal to 0 or less than 0.
When needing feed liquor, liquid inlet volume, the culture tank weight M set according to liquid inlet volume are setin0, start liquid feeding pump, make
Culture medium in liquid storing bag is injected into culture tank through inlet pipe, using weight sensor the real time measure culture tank weight come really
Need to inject the amount of culture medium calmly into culture tank；The culture tank weight M for the real time measure that weighing sensor is sentt, and set
Fixed culture tank weight Min0It is compared, weight difference M is obtained according to public formula (III)in:
Min=Min0-Mt (III)
According to MinControl the on-off of the feed liquor access.
Further, MinGreater than 0, feed liquor access, M are opened1Feed liquor access is disconnected when equal to 0 or less than 0.
Further, in incubation, the circulation path can be opened or closed according to user.
In step 4), after the completion of cell culture, physiological saline replacement medium is first used, concrete operations are: being discharged quantitative
After waste liquid, the physiological saline of equivalent is filled into, then repeatedly aforesaid operations are displaced completely until culture medium.In culture tank
Original liquid amount is 400 milliliters, 200 milliliters of waste liquids is first discharged, then fill into 200 milliliters of physiological saline, culture medium concentration is reduced to
Originally 50%, after operating ten times repeatedly, culture medium concentration is reduced to original (1/2)10, it will be recognized that it is replaced.Then into
Enter and link is concentrated, waste liquid is persistently discharged, reduces liquid volume in culture tank.
In concentration process, liquid volume in culture tank is controlled, control method are as follows: set target culture tank weight as
M, the culture tank weight M for the real time measure that weighing sensor is sentt, it is compared with target culture tank weight M, according to formula
(V) weight difference M is obtained1:
M1=Mt-M (Ⅴ)
According to M1Control the on-off of the waste liquid access.
Further, M1Greater than 0, waste liquid access, M are opened1Waste liquid access is disconnected when equal to 0 or less than 0.
In step 5), finished product cell is recycled using cell recovery passage.Specifically, under the driving of recovery pump, culture tank
In mixed liquor containing cell entered in collection bag by the recovered pipeline of recovery port.
It further, further include inverted running circulating pump in the step 5), it is thin to recycle the finished product in circulation path
Born of the same parents.
In one embodiment, the step 2) includes pre-processing to the filter；
In one embodiment, the feed liquor access further includes tank gage.Utilize the liquid of tank gage monitoring inlet pipe
Position variation.
In one embodiment, the culture systems are equipped with alarm module.Preset liquid level in system, oxygen gas
Bulk concentration, density of carbon dioxide gas, culture tank weight and temperature alarm critical value, the alarm critical value includes that alarm is faced
Boundary's upper limit and the critical lower limit of alarm, when each detection information in system is higher than alarm critical upper limit or is lower than the critical lower limit of alarm
When, it sounds an alarm.
In one embodiment, ultraviolet sterilization lamp is equipped in the incubator, using ultraviolet sterilization lamp to incubator into
Row sterilizing.
The opening and closing of feed liquor access, circulation path, waste liquid access in the liquid stream component, liquid inlet volume, waste liquid discharge rate
Parameter, can during the cultivation process, and user carries out live setting according to culture situation and is immediately performed corresponding operating；It can also mention
Before set, operated by the way of start by set date using system.
In conclusion system of the present invention realizes constant temperature incubation environment, cell culture is carried out using perfusion mode,
Waste liquid can be discharged in incubation, prevent the accumulation of Toxic Metabolites, be conducive to reach higher cell culture density, it can be with
Subsequent processing step is reduced, finished product cell recycling can be carried out without operations such as centrifugations, simplify operation, culture effect can be improved
Rate is easy to industrialization, realizes the Full enclosed integrated process recycled from cell-stimulating, infection, amplification to finished product.By in tank body
Middle axial agitation cell and culture solution, minimize radial shearing force, can effectively protect cell, improve cell yield.This hair
The bright system separates air inlet, guarantees each gas component stable content in incubation, it can be ensured that in cell cultivation process
Ambient atmos variation will not directly impact cell culture, guarantee that gas each component content is constant, while reduce artificial behaviour
Make, reduce cost, reduce the risk of operation error in incubation, improves culture efficiency.So the present invention effectively overcomes now
There is the various shortcoming in technology and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (15)
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010167455.8A CN111334427B (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
| CN201910184785.5A CN109825436A (en) | 2019-03-12 | 2019-03-12 | Totally closed cell culture system |
| PCT/CN2019/098474 WO2020181709A1 (en) | 2019-03-12 | 2019-07-31 | Fully enclosed cell incubation system |
| CN202010168036.6A CN111218403B (en) | 2019-03-12 | 2020-03-11 | Totally enclosed cell culture method |
| US17/216,699 US12297415B2 (en) | 2019-03-12 | 2021-03-30 | Fully enclosed cell culture system |
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| CN201910184785.5A CN109825436A (en) | 2019-03-12 | 2019-03-12 | Totally closed cell culture system |
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| CN201910184785.5A Pending CN109825436A (en) | 2019-03-12 | 2019-03-12 | Totally closed cell culture system |
| CN202010168036.6A Active CN111218403B (en) | 2019-03-12 | 2020-03-11 | Totally enclosed cell culture method |
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| CN114907971A (en) * | 2021-02-08 | 2022-08-16 | 中国石油天然气股份有限公司 | A kind of closed microbial culture device and microbial culture method |
| CN112662631A (en) * | 2021-03-16 | 2021-04-16 | 合源生物科技(天津)有限公司 | CAR-T cell perfusion culture method |
| CN112662631B (en) * | 2021-03-16 | 2021-06-29 | 合源生物科技(天津)有限公司 | CAR-T cell perfusion culture method |
| CN115521872A (en) * | 2021-06-24 | 2022-12-27 | 清华大学 | Tubular cultivation equipment |
| CN115521873A (en) * | 2021-06-24 | 2022-12-27 | 清华大学 | A perfusion device for cell culture |
| CN114317270A (en) * | 2022-03-12 | 2022-04-12 | 广州赛太特生物医学科技有限公司 | Cell culture device for biological gene research |
| CN114317270B (en) * | 2022-03-12 | 2022-06-17 | 广州赛太特生物医学科技有限公司 | Cell culture device for biological gene research |
| CN115386488A (en) * | 2022-04-21 | 2022-11-25 | 山东威高瑞生医疗器械有限公司 | A gas supply system for a cell culture box and a method for dynamic control of gas concentration |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111334427A (en) | 2020-06-26 |
| CN111218403B (en) | 2021-01-29 |
| CN111218403A (en) | 2020-06-02 |
| CN111334427B (en) | 2023-05-05 |
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