CN208501010U - The full suspension cell culture reactor of 6000L serum-free - Google Patents
The full suspension cell culture reactor of 6000L serum-free Download PDFInfo
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- CN208501010U CN208501010U CN201820130737.9U CN201820130737U CN208501010U CN 208501010 U CN208501010 U CN 208501010U CN 201820130737 U CN201820130737 U CN 201820130737U CN 208501010 U CN208501010 U CN 208501010U
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Abstract
The utility model relates to the full suspension cell culture apparatus fields of serum-free, in particular to a kind of full suspension cell culture reactor of 6000L serum-free, including tank body；The tank interior is provided with microvesicle air-breather and agitating device；The tank skin of the tank body is internally provided with baffle；Lye injector head is provided with above the tank skin of the tank body, the lye injector head is connected by valve with storage alkali container；Surface layer air inlet is additionally provided with above the tank skin of the tank body.Cell culture reactor provided by the utility model has carried out various optimizations for the culture feature of big volume reactor, alkali system especially is mended to microvesicle aerating system, bioreactor and gas handling system is optimized, so that the reactor is very suitable to the full suspension cell culture of serum-free, efficient oxygen transfer, cell and the nutriment that can be realized under low-shearing force uniformly mix, and pH in tank is effectively adjusted, cell survival rate is high.
Description
Technical field
The utility model relates to cell cultivation equipment fields, in particular to a kind of full suspension cell of 6000L serum-free
Cultivation reactor.
Background technique
Bioreactor is the core equipment of mass cell suspension culture, can effectively increase cell-unit volume
Culture density is the mainstay of the large-scale production of such product.The biotechnologies such as the U.S., Switzerland, Germany power has been established
The bioreactor of comparatively perfect is researched and developed and industrialization technology system, can develop the high, production with production efficiency and product quality
The advantages such as at low cost, and adapt to the commercialization bioreactor of different kind organism culture.In the world, for the culture production that suspends entirely
The bioreactor research of antibody is towards high-performance, extensive, intelligent direction development.The companies such as Abec, GEA GROUP are dynamic
The market scale of object cell reactor (monomer) has reached more than 10 hundred million Euros.Switzerland dragon sand (Lonza), GlaxoSmithKline PLC (GSK)
Equal companies all establish the zooblast reactor production line of ten thousand upgradings, the bioreactor utilization rate of bio-pharmaceutical industry one after another
Rise to 80% or more.
China has been the largest production state of world's vaccine product and maximum using state, and with annual 15% rate delivery
Increase；In recent years, the development of antibody drug and the market demand also increase substantially.To improve production efficiency and product quality, effectively drop
Low production cost changes with the backwardness production technology of spinner culture cell, is badly in need of the zooblast of the above production scale of kilolitre grade
Cultivation reactor.But bioreactor scale domestic at present mostly in 1000L hereinafter, comprehensive performance and production efficiency not
Height, technical level have a long way to go compared with developed countries, and the above bioreactor manufacture of kilolitre grade scale is almost by overseas enterprise
(Switzerland Bio, U.S. NBS, France PG etc.) monopolization, forms strict technical barrier, scale amplifying technique and equipment centering
State's limitation outlet.Therefore, the research and development and industrialization for carrying out zooblast suspension cultivation reactor, to China's vaccine and antibody drug
Production has very important practical significance.
In view of this, special propose the utility model.
Utility model content
The purpose of this utility model is to provide a kind of full suspension cell culture reactors of 6000L serum-free, existing to solve
Have that cell culture reactor volume used in technology is universal (predominantly staying in 3000L or less at present) less than normal, caused by yield
It is low, energy consumption is high, labour demand is big, product quality is inhomogenous, pollution be difficult to the problems such as controlling.
In order to realize the above-mentioned purpose of the utility model, the following technical scheme is adopted:
The full suspension cell culture reactor of 6000L serum-free provided by the utility model, including tank body；
The tank interior is provided with microvesicle air-breather and agitating device；
The tank skin of the tank body is internally provided with baffle；
Lye injector head is provided with above the tank skin of the tank body, the lye injector head passes through valve and storage alkali container phase
Even；
Surface layer air inlet is additionally provided with above the tank skin of the tank body.
Compared with prior art, cell culture reactor provided by the utility model is directed to the culture of big volume reactor
Feature has carried out various optimizations, especially to be microvesicle aerating system, bioreactor mend alkali system and gas handling system into
It has gone optimization, so that the reactor is very suitable to the culture of suspension cell, can be realized efficient oxygen transfer under low-shearing force, thin
Born of the same parents and nutriment uniformly mix, and effectively adjust pH in tank, and cell survival rate is high.
Detailed description of the invention
It, below will be right in order to illustrate more clearly of specific embodiment of the present invention or technical solution in the prior art
Specific embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, it is described below
In attached drawing be that some embodiments of the utility model are not paying creativeness for those of ordinary skill in the art
Under the premise of labour, it is also possible to obtain other drawings based on these drawings.
Fig. 1 is the knot of the full suspension cell culture reactor of 6000L serum-free provided by the utility model one embodiment
Structure schematic diagram；
Fig. 2 is the schematic diagram that alkali system is mended provided by the utility model one embodiment；
Fig. 3 is the structural schematic diagram of microvesicle air-breather provided by the utility model one embodiment；
Fig. 4 is the schematic diagram of connecting rod provided by the utility model one embodiment；
Fig. 5 is the schematic diagram of the amplification of microvesicle air-breather inlet port provided by the utility model one embodiment；
Fig. 6 is the schematic diagram of the amplification of microvesicle air-breather notch end provided by the utility model one embodiment；
Fig. 7 is bowing for the full suspension cell culture reactor of 6000L serum-free provided by the utility model one embodiment
View；
Fig. 8 is the full suspension cell culture chamber lid of 6000L serum-free provided by the utility model one embodiment
Structural schematic diagram；
Fig. 9 is the schematic diagram for amplifying culture systems provided by the utility model one embodiment step by step.
Appended drawing reference:
Bioreactor tank body A；Agitation Tank B；Higher level's tank body C；
Microvesicle air-breather 1；Air inlet 101；Micropore 102；Attachment device 103；Sticking department 1031；Protruding portion 1032；Even
Extension bar 104；Plug 105；
Agitating device 2；Agitating shaft 201；Agitating paddle 202；
Power device 3；Baffle 4；
Lye injector head 5；Valve 501；
Surface layer air inlet 6；Exhaust outlet 7；For air assembly 8；For carbon dioxide plant 9；Apparatus of oxygen supply 10；
Collet temperature-controlling system 11；
Mend alkali mouth 12；The malicious access port 13 of kind；Kind cell entry 14；First spare mouth 15；Culture medium adds mouth 16；CIP
17-1；CIP 17-2；Second spare mouth 18；Lamp mirror mouth 19；Gauge port 20；Mirror mouth 21 and manhole 22.
Specific embodiment
The technical solution of the utility model is clearly and completely retouched below in conjunction with the drawings and specific embodiments
It states, it will be appreciated by those skilled in the art that following, the embodiments are a part of the embodiments of the present invention, without
It is whole embodiments, is merely to illustrate the utility model, and is not construed as limitation the scope of the utility model.It is practical based on this
Embodiment in novel, every other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, is fallen within the protection scope of the utility model.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer
It is recommended that condition carry out.Reagents or instruments used without specified manufacturer is the routine that can be obtained by commercially available purchase
Product.
It is in the description of the present invention, it should be noted that term " center ", "upper", "lower", "left", "right", " perpendicular
Directly ", the orientation or positional relationship of the instructions such as "horizontal", "inner", "outside" is to be based on the orientation or positional relationship shown in the drawings, and is only
For ease of description the utility model and simplify description, rather than the device or element of indication or suggestion meaning must have it is specific
Orientation, be constructed and operated in a specific orientation, therefore should not be understood as limiting the present invention.In addition, term " the
One ", " second ", " third " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " is pacified
Dress ", " connected ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integrally
Connection；It can be mechanical connection, be also possible to be electrically connected；Can be directly connected, can also indirectly connected through an intermediary,
It can be the connection inside two elements.For the ordinary skill in the art, above-mentioned art can be understood with concrete condition
The concrete meaning of language in the present invention.
The utility model relates to a kind of full suspension cell culture reactors of 6000L serum-free, as shown in Figure 1, including tank body
A；
The tank body A is internally provided with microvesicle air-breather 1 and agitating device 2；
The tank skin of the tank body A is internally provided with baffle 4；
As shown in Fig. 2, being provided with lye injector head 5 above the tank skin of the tank body A, the lye injector head 5 passes through valve
Door 501 is connected with storage alkali container；
Surface layer air inlet 6 is additionally provided with above the tank skin of the tank body A.
Preferably, the tank skin of the tank body A is long tubular, and tank base is arc-shaped to arch upward outward.
Preferably, the bottom of the tank body A is provided with fluid distributing hole, and the leakage fluid dram is for discharging and/or blowdown.
Preferably, the tank body A is additionally provided with temperature-controlling system；It is furthermore preferred that the temperature-controlling system is collet temperature-controlling system
11。
The several systems being arranged out of tank separately below illustrate that 6000L serum-free provided by the utility model hangs entirely respectively
The structure of floating cell culture reactor.
Preferably, culture medium inlet and/or culture transferring mouth are provided with above the tank skin of the tank body A；
The culture medium inlet is connected with Agitation Tank B, and the culture transferring mouth is connected with higher level's tank body C.
As shown in figure 9, the cell culture process of the utility model is amplification technique step by step.It, preferably can be from when cell culture
5L → 25L → 125L → 600L → 1000L → 3000L → 6000L reactor is amplified step by step.
Thus higher level's tank body of 6000L reactor is preferably 3000L tank body.
It is needing it is noted that in the present invention, " the full suspension cell culture reactor of 6000L serum-free " is only prominent
The utility model reactor is suitable for big volume culture cell, the especially culture of suspension cell out, but " 6000L " is not represented pair
The protection scope of reactor has restriction effect.In fact, the utility model design of the utility model and content are suitable for 600L
Bioreactor within the scope of~8000L volume, preferably 3000L~6000L.
Lid is provided at the top of the cell culture reactor；Preferably, exhaust outlet 7 is provided on the lid；
Preferably, as shown in figure 8, the lid Shanghai is provided with one of under shed/device or a variety of:
It mends alkali mouth 12, plant malicious access port 13 (being used for virus inoculation), kind cell entry 14, the first spare mouth 15, culture medium
Add mouth 16, CIP 17-1 and CIP 17-2, the second spare mouth 18, lamp mirror mouth 19, gauge port 20, mirror mouth 21 and manhole
22。
CIP 17-1 and CIP 17-2 is a part of CIP cleaning system, and CIP cleaning system is commonly called as cleaning system on the spot,
Also known as cleaning positioning or positioning cleaning (cleaning in place).Cleaning, which refers to, on the spot does not have to dismantle or mobile device, that is, adopts
With high temperature, the cleaning solution of high concentration, forceful action is subject to apparatus, the contact surface with product is cleaned up, for defending
Raw rank requires the cleaning of stringenter production equipment, purification.
One, microvesicle aerating system
Microvesicle aerating system mainly includes microvesicle air-breather 1, agitating device 2 and baffle 4；Preferably, the tank body A
Inside it is additionally provided with dissolved oxygen amount detecting device；Preferably, there are two the dissolved oxygen amount detecting devices.
Double dissolved oxygen amount detecting devices ensure the accuracy of DO in tank by the way of 2 DO electrodes, even if an and dissolved oxygen
Amount detecting device, which goes wrong, will not influence the operating of equipment when needing to overhaul.
Wherein, the switching of ventilated body in the microvesicle air-breather 1, agitating device 2 revolving speed can be by an electronics
Control device control, the control device control the operating of above-mentioned apparatus by reading dissolved oxygen amount detecting device and analyzing.
As shown in figure 3, the microvesicle air-breather 1 is with not closed circle ring jaggy；The microvesicle air-breather 1
Ring wall on be provided with an air inlet 101 and several equally distributed micropores 102；The ring body of the microvesicle air-breather
Inner hollow, the micropore 102 are communicated through ring wall with the hollow cavity of the ring body.
It is carried out in the microvesicle air-breather 1 in use, being injected gas such as oxygen by the air inlet 101, one
Under fixed air pressure, oxygen is full of entire microvesicle air-breather by the ring body of inner hollow, and is discharged by the micropore 102.The dress
It is simple to set structure, thus micropore is not easy to plug, and is safeguarded also more convenient.The structure of annular increases gas distribution area, can more
It is even that oxygen is released in cell culture fluid.
Preferably, the diameter of the micropore 102 is 0.05mm~0.2mm.
For the residence time using oxygen when the device in culture solution can be increased as much as possible, it is preferred that described micro-
The side of plane where hole 102 is distributed in the circular rings, in this way in use, can be by setting increases down where micropore 102
The advanced distance of oxygen floating.
According to ventilation demand needed for, the micropore 102 can according to S-shaped or it is Z-shaped arranged, may be set to be more
Column.In order to reduce technology difficulty, it is preferred that the micropore 102 is arranged along the ring wall at a column；It is furthermore preferred that the micropore
102 distance apart from 1 center of circle of microvesicle air-breather is equal.
Preferably, microvesicle air-breather as described above, the air inlet 101 are oppositely arranged with the notch, it is described into
Port 101 is fixed on bioreactor bottom by attachment device 103.
Preferably, microvesicle air-breather as described above, the air inlet 101 pass through the attachment device 103 connection one
A hollow connecting rod 104 is fixed on bioreactor bottom；
Preferably, as shown in figure 4, the connecting rod 104 is the L-shaped structure of middle bent；It is furthermore preferred that the connecting rod
The angle of 104 middle bents is 120 °~160 °；Also 130 °~150 ° or 140 ° be can choose.
The angle of 104 middle bent of connecting rod is 120 °~160 °, i.e., when one section is connected with the microvesicle air-breather,
The other end can horizontal plane upwarp in 20 °~60 ° angles, effect is by microvesicle air-breather as below agitating paddle, but not with stir
Mixing paddle has scraping collision.104 other side of connecting rod and reactor can silk mouth nut be connected, convenient disassembly.
The microvesicle air-breather 1 is cleaned for convenience, it is preferred that the ring body of the microvesicle air-breather 1 can
Disassembly is two semi-rings；Cleaning way can be ultrasonic cleaning.
In order to preferably simplify structure, it is preferred that the ring body disassembly of the microvesicle air-breather 1 is two semi-rings
Detachment point is set near the air inlet；It is furthermore preferred that as shown in figure 5, the attachment device 103 is three-way device, one end
It is communicated with the connecting rod 104, in addition both ends are then communicated with two semi-rings respectively, and movable at least one of two semi-rings
Connection.Active connection place can be attached by a connector, as shown in figure 5, the connector is by sticking department 1031 and protrudes
Portion 1032 forms；The two can be connected by way of lock, may be alternatively provided as magnetic connection.
Preferably, microvesicle air-breather as described above, the ring body end of 1 indentation, there of microvesicle air-breather are that can beat
The structure opened；It is furthermore preferred that as shown in fig. 6, the ring body end of the microvesicle air-breather indentation, there is provided with dismountable block up
First 105.In an embodiment of the utility model, the circle diameter of the microvesicle air-breather 1 is 570mm, indentation, there
Length be 80mm, 102 spacing of micropore has a 2.5mm, and 102 diameter of micropore is 0.05mm~0.2mm.
As shown in Figure 1, the air inlet 101 of the microvesicle air-breather 1 is respectively by valve and apparatus of oxygen supply 10, for dioxy
Change carbon device 9 and is connected for air assembly 8；
Wherein, preferably above-mentioned gas is sterile compressed gas state when being stored in above-mentioned feeder.Oxygen and
Carbon dioxide is the common gas in cell culture.
Preferably, the agitating device 2 includes agitating shaft 201 and agitating paddle 202；
The agitating shaft 201 is perpendicular to horizontal plane and is fixed on tank bottom, and the agitating paddle 202 is set to the agitating shaft
On 201, the bottom of the agitating shaft 201 is connected with power device 3；
The microvesicle air-breather 1 is set to the underface of the agitating paddle 202, around the agitating shaft 201；It is preferred that
, the micropore 102 of the microvesicle air-breather 1 is arranged downward.
Preferably, the power device 3 is magnetic stirring apparatus.
Zooblast reactor is very strict to sterility requirements, operations and the mechanically-sealing apparatus such as charging, sterilizing and inoculation
Deng may all bring microbiological contamination risk, once leading to inside reactor microbiological contamination, huge economic loss is caused.The utility model exploitation
There is the magnetic drives stirring system of completely isolated function, power for the bioreactor of 6000L animal cell culture
Transmitting fully relies on the electromagnetic force of magnetic cylinder generation, microbiological contamination caused by thoroughly having prevented because of sealing problem.
The microvesicle air-breather 1 is inserted in around the agitating shaft 201 by its notch.
Preferably, there are three the agitating paddles 202, the first agitating paddle, second are from top to bottom followed successively by from the agitating shaft
Agitating paddle and third agitating paddle, the second stirring paddle stirring liquid move from top to bottom；The first and third stirring paddle stirring
Liquid moves from bottom to top.
By above-mentioned set-up mode, the oxygen discharged in microvesicle air-breather 1 can be confined between the first and third agitating paddle
(it is considered that foring spiral liquid stream between three agitating paddles), so that it is resident in culture solution to extend bubbles of oxygen
Time increases the utilization rate of oxygen.
Preferably, the agitating paddle 202 is turbine type agitating paddle or pusher agitating paddle；
The blade of pusher agitating paddle has certain radian, and culture solution is enabled to form axial flowing, stirs to mixing
It is more advantageous with suspension effect.
It is furthermore preferred that the agitating paddle 202 is four leaf push type agitating paddles.
Preferably, the rake angle degree of the agitating paddle is 40 °~60 °；More preferably 50 °；Since each agitating paddle makes
The direction of motion of liquid stream is different, thus the deflection of the deflection angle and first and third agitating paddle of preferably second agitating paddle
Angle mirror symmetry.
Preferably, the ratio of the paddle diameter of the agitating paddle and tank diameter is 0.2~0.7, more preferably 0.3~0.5.
Preferably, the revolving speed of the agitating paddle is 30~70rpm.
Preferably, it is additionally provided with baffle 4 inside the tank skin of the tank body A, the baffle 4 is perpendicular to horizontal plane；
It is furthermore preferred that the bottom of the baffle 4 is concordant with the third agitating paddle, top is higher than the liquid level of cell culture fluid
Highly.
Preferably, the baffle 4 has 3~5, more preferably 4, and is uniformly distributed on the tank skin of the tank body；
Preferably, as shown in fig. 7, the baffle 4 deviate its with 30 ° of Grain lines where the tank body A tie point~
40 °, and each baffle deflection direction is consistent；
Preferably, the width of the baffle is 1/13~1/10 tank diameter.
It is furthermore preferred that each baffle deflection direction is consistent with the direction of rotation of agitating paddle；Such as the direction of rotation of agitating paddle is
Counterclockwise, then the deflection direction of the baffle 4 just as shown in fig. 7, being deflected in obtuse angle along water flow counterclockwise.
Due to second agitating paddle and first and third agitating paddle by the direction of motion of aqueous agitation on the contrary, culture solution
When being stirred liquid flow direction can than three agitating paddles equidirectional (upwards agitation) it is more chaotic, therefore, this agitation
Although mode can greatly increase dissolved oxygen amount, it is also easier in turn result in thin because the chaotic impact of liquid stream forms turbulent flow simultaneously
The broken death of born of the same parents.Based on this reason, the utility model is provided with baffle 4, and the direction of rotation one of its deflection angle and agitating paddle
Cause, baffle 4 can play the role of water conservancy diversion, due to cell culture fluid bioreactor to close on flow velocity at tank skin maximum, thus
Baffle 4 on tank skin can slow down flow rate of liquid, guidance liquid flow direction herein, and then effectively avoid the generation of turbulent flow.
For example, in one embodiment, bioreactor tank body is 6000L tank body, microvesicle air-breather used is Fig. 3
Shown in annular shape air-breather.As shown in Figure 1, bottom is provided with agitating device in tank, the agitating device includes stirring
Axis and agitating paddle, the agitating shaft is perpendicular to horizontal plane and is fixed on tank bottom, and the agitating paddle is set on the agitating shaft, institute
The bottom for stating agitating shaft is connected with power device；On agitating shaft be arranged three agitating paddles, be followed successively by from bottom to top the first agitating paddle,
Second agitating paddle and third agitating paddle.Revolving speed is 40 turns/min, is to rotate counterclockwise.The second stirring paddle stirring liquid
It moves from top to bottom；The first and third stirring paddle stirring liquid moves from bottom to top.The rake angle degree of agitating paddle is 50 °,
The ratio of paddle diameter and tank diameter is 0.4.On tank skin be arranged four baffles, the deflection direction of baffle be deviate counterclockwise its with it is described
35 ° of Grain lines where tank body tie point, the width of baffle are 1/12 tank diameter.Experimental group is set by this embodiment, in this base
Comparative example is set on plinth；
Comparative example 1: it is only that with experimental group difference, removes baffle.
Comparative example 2: it is only that baffle is arranged perpendicular to tank body with experimental group difference.
Comparative example 3: it is only that three agitating paddles stir culture solution upwards with experimental group difference, and removes baffle.
Comparative example 4: being only that with experimental group difference, which is conventional cylindrical body, and inside has porous
Reticular structure.
Cultivated cell is mdck cell, is after 1,800,000/ml is cultivated 5 days, to detect following data by cell culture density:
Note: cell survival rate=(total number of cells-dead cell number) ÷ total number of cells × 100%, platform phenol indigo plant decoration method meter
It calculates.
From comparative example 1 and 2 it is found that baffle plate setting mode it is incorrect or removal baffle, although flow of oxygen can be reduced,
It will form more violent turbulent flow, Cell viability caused to be greatly lowered.By comparative example 3 it is found that three agitating paddles will culture
When liquid stirs upwards, it is resident and is shortened in culture solution due to oxygen, thus oxygen utilization rate declines, oxygen consumption total amount is soaring obvious；
It is uniformly dispersed in addition, the change of aqueous agitation mode is also unfavorable for oxygen, it is possible to which the decline for causing local oxygen concentration causes
Cell viability reduces.By comparative example 4 it is found that routine microvesicle air-breather in the prior art, since gas distribution is uneven, same meeting
Cell viability is reduced, and since the decline of oxygen dissolved efficiency leads to the raising of flow of oxygen.Further, since the prior art is adopted
Conventional microvesicle air-breather not easy cleaning, can be blocked in the normal time using rear micropore, if increasing bleed pressure at this time,
It is too fast that the ventilation gas flow hole rate of outflow for causing not block is returned again, gas effciency is caused to reduce, and is especially reacted in 6000L
When device culture cell, microvesicle air inlet can only supply the dissolved oxygen of culture 0-20% under the maximum mixing speed for not damaging cell
Amount, the dissolved oxygen amount of 30-50% needed for being unable to reach cell growth.
In conclusion becoming larger with overall volume, cell cloth oxygen difficulty skyrockets.Due to always not used before the country
Large capacity bioreactor (6000L) cultivates the precedent of cell, thus lacks be directed to large capacity bioreactor in the prior art
The optimization and adjustment of equipment.Present utility model application people adapts to transformation stirring system by adjusting air-breather, to realize
The uniform gas distribution of big volume bioreactor, also reduces the dosage of oxygen, improves the survival rate of cell.
Two, bioreactor mends alkali system
Mending alkali system mainly includes lye injector head 5, and agitating device 2 as described above and baffle 4.Preferably,
PH meter is additionally provided in the tank body A；Preferably, there are two the pH meters.
Double pH meters ensure the accuracy of pH in tank by the way of 2 pH electrodes, even and if a pH meter go wrong need
The operating of equipment will not be influenced when overhauling.
Wherein, the revolving speed of the opening and closing of the injection of lye injector head 5 and the control of the amount of injection, agitating device 2 can be by one
A electronic control unit control, the control device control the operating of above-mentioned apparatus by reading pH meter and analyzing.
As shown in Fig. 2, lye injector head 5 is spurted into lye in the culture medium of tank body with pulsed, it is fast in agitating device 2
Speed stirs and evenly mixs, can to avoid local pH it is excessively high caused by cellular damage.
It is intermittent to open the valve 501 when carrying out mending alkali operation using equipment as described above, so that the lye
5 pulsed of injector head sprays lye, opens simultaneously the agitating device 2 so that the lye of supplement mixes rapidly completely.
Preferably, the lye is the NaHCO of 0.8mol/L~1.2mol/L3Solution；The more preferably NaHCO of 1mol/L3
Solution.
Preferably, the valve 501 is opened primary at interval of 4.5s~5.5s, opens 50ms~200ms every time；More preferably
, the valve 501 is opened once at interval of 5s, opens 100ms~150ms every time.
Preferably, when spraying lye, the fluid pressure in the lye injector head 5 is 0.08MPa~0.12MPa；It is more excellent
Choosing, the fluid pressure in the lye injector head 5 is 0.10MPa.
Preferably, when carrying out mending alkali operation, the revolving speed of the agitating device 2 is 30rpm~70rpm；It is furthermore preferred that institute
The revolving speed for stating agitating device 2 is 50rpm.
The switch time of concentration of lye, valve 501, the revolving speed of agitating device 2 and the pressure in lye injector head 5 (are matched
The outlet diameter of injector head is closed, what is actually limited is the lye amount sprayed every time) it is that applicant combines the utility model
Bioreactor tank mends what alkali device structure was especially set, to can quickly mix lye, to reduce its injury to cell.
In one embodiment, bioreactor tank body is 6000L tank body, cultivates floating type mdck cell.On tank skin
Side is provided with lye injector head, and lye injector head passes through valve and storage 1.0mol/LNaHCO3The container of solution is connected, valve
It is opened once at interval of 5s, opens 120ms every time.When spraying lye, the fluid pressure in the lye injector head is 1.0MPa,
The jet port diameter of lye injector head is 2mm.Bottom is provided with microvesicle air-breather for leading to oxygen in tank, and bottom is also set in tank
It is equipped with agitating device, the agitating device includes agitating shaft and agitating paddle, and the agitating shaft is perpendicular to horizontal plane and is fixed on tank
Bottom, the agitating paddle are set on the agitating shaft, and the bottom of the agitating shaft is connected with power device；Three are arranged on agitating shaft
A agitating paddle is followed successively by the first agitating paddle, the second agitating paddle and third agitating paddle from top to bottom, is to rotate clockwise；It is described
Second stirring paddle stirring liquid moves from top to bottom；The first and third stirring paddle stirring liquid moves from bottom to top.Agitating paddle
Rake angle degree is 50 °, and the ratio of paddle diameter and tank diameter is 0.4.When lye is added dropwise, rotating speed of agitator is 50 turns/min；Just
When often culture cell leads to oxygen, rotating speed of agitator is 40 turns/min.Four baffles are set on tank skin, and the deflection direction of baffle is suitable
Hour hands deviate itself and 35 ° of Grain lines where the tank body tie point, and the width of baffle is 1/12 tank diameter.This embodiment is arranged
For experimental group, comparative example is set on this basis；
Comparative example 1: it is only that with experimental group difference, removes baffle.
Comparative example 2: lye injector head is removed, and add alkaline methods replace with side wall in the prior art and formula is added dropwise；Agitating paddle
Revolving speed is always 40 turns/min.
Comparative example 3: it is only that baffle is arranged perpendicular to tank body with experimental group difference.
Comparative example 4: it is only that three agitating paddles stir culture solution upwards with experimental group difference, and removes baffle.
It is that 1,800,000/ml is cultivated with batch mdck cell 5 days by cell culture density, by adding alkali to maintain, pH is constant to be
6.8-7.2 detecting following data:
From comparative example 1 and 2 it is found that mode, which is added dropwise, in different lye has large effect to Cell viability.Adherent dropwise addition drop
The mode added is also continuously, it is excessive to often result in concentration of lye at dropwise addition, thus will cause more cell death.If accelerating
Revolving speed then also results in the increase that mechanical damage causes cell mortality；Also can not in addition, dosage is added dropwise in adherent dropwise addition every time
Control, can not precisely adjust pH.From comparative example 1 and 3 it is found that baffle plate setting mode it is incorrect or removal baffle, although can reduce
Flow of oxygen, but will form more violent turbulent flow, cause Cell viability to be greatly lowered.By comparative example 4 it is found that three stirrings
When paddle stirs culture solution upwards, it is resident and is shortened in culture solution due to oxygen, thus oxygen utilization rate declines, oxygen consumption total amount
It is soaring obvious；It is uniformly dispersed in addition, the change of aqueous agitation mode is also unfavorable for oxygen, since the utility model uses big volume
Bioreactor, the mixing difficulty of oxygen is consequently increased, it is possible to which the decline for causing local oxygen concentration causes cell living
Rate reduces.
In conclusion lye additional way of the utility model by using pulsed, the stirring dress being especially arranged of arranging in pairs or groups
Set and baffle, can effectively less stirring when turbulent flow generation, quickly mix lye, to reduce because of mechanical damage and local alkali
Cell death caused by liquid excessive concentration.In addition, surprisingly, unique agitating paddle rotation mode can also extend oxygen
Residence time, so that the consumption of oxygen be effectively reduced.
Three, gas handling system
Gas handling system mainly includes surface layer gas handling system and deep layer gas handling system.As shown in Figure 1, deep layer gas handling system is main
Including for air assembly 8, microvesicle air-breather 1, exhaust outlet 7；Surface layer gas handling system mainly include for air assembly 8, surface layer into
Port 6, exhaust outlet 7.
The air that microvesicle air-breather 1 is passed through constitutes the deep layer gas handling system of cell culture reactor, the compressed air
The mode of being passed through can extend the flow path of air in the medium, increase the transfer amount of carbon dioxide and ammonia.
Method described in the utility model is also passed through compressed air on liquid phase surface layer, can avoid lower compression air and is flowing over
It is dissolved in journey in the medium, increases the spilling of lower compression air.Preferably, the setting of surface layer air inlet 6 is in tank skin side height
In with 40~60 centimeters air inlet of liquid level.In side into compressed air, top over there is vented, and makes compressed air on liquid level
The distance that side is walked is elongated, can carry out more carbon dioxide；Higher than 40~60 centimeters of liquid level can guarantee surface layer into
Directly culture solution is not arrived in piping and druming to gas, and then prevents and generate foam in cell cultivation process.
Preferably, surface layer air inlet and when deep layer air inlet, the pressure of compressed air is 0.02~0.06MPa, more preferably
0.04MPa, the compressed air pass through the purification of two-stage air filter.
Gas handling system used by the utility model combines surface layer gas handling system and deep layer gas handling system, can effectively drive
The ammonia and excessive carbon dioxide in culture solution are caught up with, culture environment stable in cell cultivation process is kept.
Finally, it should be noted that the above various embodiments is only to illustrate the technical solution of the utility model, rather than it is limited
System；Although the present invention has been described in detail with reference to the aforementioned embodiments, but those skilled in the art answer
Work as understanding: it is still possible to modify the technical solutions described in the foregoing embodiments, either to part of or complete
Portion's technical characteristic is equivalently replaced；And these are modified or replaceed, it does not separate the essence of the corresponding technical solution, and this is practical
The range of novel each embodiment technical solution.
Claims (10)
1. a kind of full suspension cell culture reactor of 6000L serum-free, which is characterized in that including tank body；
The tank interior is provided with microvesicle air-breather and agitating device；
The tank skin of the tank body is internally provided with baffle；
Lye injector head is provided with above the tank skin of the tank body, the lye injector head is connected by valve with storage alkali container；
Surface layer air inlet is additionally provided with above the tank skin of the tank body.
2. the full suspension cell culture reactor of 6000L serum-free according to claim 1, which is characterized in that the microvesicle
Air-breather is to be provided with an air inlet on ring wall and several are equally distributed micro- with not closed circle ring jaggy
Hole；The ring body inner hollow of the microvesicle air-breather, the micropore are communicated through ring wall with the hollow cavity of the ring body.
3. the full suspension cell culture reactor of 6000L serum-free according to claim 2, which is characterized in that the air inlet
Mouth is connected by valve with apparatus of oxygen supply, for carbon dioxide plant and for air assembly respectively.
4. the full suspension cell culture reactor of 6000L serum-free according to claim 1, which is characterized in that the stirring
Device includes agitating shaft and agitating paddle；
The agitating shaft is perpendicular to horizontal plane and is fixed on tank bottom, and the agitating paddle is set on the agitating shaft, the stirring
The bottom of axis is connected with power device.
5. the full suspension cell culture reactor of 6000L serum-free according to claim 4, which is characterized in that the stirring
There are three paddles, is from top to bottom followed successively by the first agitating paddle, the second agitating paddle and third agitating paddle from the agitating shaft；Described
Two stirring paddle stirring liquid move from top to bottom；The first and third stirring paddle stirring liquid moves from bottom to top.
6. the full suspension cell culture reactor of 6000L serum-free according to claim 4, which is characterized in that the stirring
The bottom of axis is connected with power device.
7. the full suspension cell culture reactor of 6000L serum-free according to claim 1, which is characterized in that the baffle
There are 3~5, and is uniformly distributed on the tank skin of the tank body.
8. the full suspension cell culture reactor of 6000L serum-free according to claim 1, which is characterized in that the tank body
Tank skin above be additionally provided with surface layer air inlet, be provided with exhaust outlet on the lid of the top of the tank.
9. the full suspension cell culture reactor of 6000L serum-free according to claim 1, which is characterized in that the tank body
Inside it is additionally provided with pH meter and dissolved oxygen amount detecting device.
10. the full suspension cell culture reactor of 6000L serum-free according to claim 9, which is characterized in that the pH meter
There are two；
There are two the dissolved oxygen amount detecting devices.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107988074A (en) * | 2018-01-25 | 2018-05-04 | 吉林冠界生物技术有限公司 | The full suspension cell culture reactor of 6000L serum-frees |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988074A (en) * | 2018-01-25 | 2018-05-04 | 吉林冠界生物技术有限公司 | The full suspension cell culture reactor of 6000L serum-frees |
| CN107988074B (en) * | 2018-01-25 | 2024-02-06 | 吉林冠界生物技术有限公司 | 6000L serum-free full-suspension cell culture reactor |
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