CN103305423A - Cell culture bed for bioreactor - Google Patents
Cell culture bed for bioreactor Download PDFInfo
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- CN103305423A CN103305423A CN2013102616422A CN201310261642A CN103305423A CN 103305423 A CN103305423 A CN 103305423A CN 2013102616422 A CN2013102616422 A CN 2013102616422A CN 201310261642 A CN201310261642 A CN 201310261642A CN 103305423 A CN103305423 A CN 103305423A
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Abstract
The invention discloses a cell culture bed for a bioreactor in the field of medical equipment. The cell culture bed is arranged in a tank body and comprises a guide barrel, a porous round pipe and a multistage speed adjustment stirrer, wherein the porous round pipe is concentrically connected with the guide barrel at the bottom of the guide barrel; the multistage speed adjustment stirrer is inserted into the porous round pipe from the bottom of the porous round pipe; an upper fixing plate around the circumference of the guide barrel is arranged at the top of the guide barrel; a lower fixing plate around the circumference of the porous round pipe is arranged at the bottom of the porous round pipe; the upper fixing plate and the lower fixing plate are both porous plates; filler comprising cell culture carriers is filled between the bottom surface of the upper fixing plate and the top surface of the lower fixing plate; and the height of the filler is maximally 2/3 of the distance between the bottom surface of the upper fixing plate and the top surface of the lower fixing plate. The cell culture bed has the technical effects that cells and cell culture carriers are not easily rushed out when the cell culture bed is used for cell culture, and the cell growth speed is uniform.
Description
Technical field
The present invention relates to a kind of cell cultures bed for bio-reactor in medical equipment field.
Background technology
Be used for the cell cultures bed of bio-reactor at present, mainly be divided into fluidized-bed and fixed bed.Fixed bed is the container at a mount in tank body, is filled with the filler of being made up of cell culture vector in the middle of this container and carries out cell cultures, and described cell culture vector is diameter 2-6mm particulate vector, porous ball or sheet-like fiber normally.Cell culture fluid cycles through described fixed bed, and the negative pressure that cell culture fluid relies on agitator to produce in stirring constantly flows in the cell cultures bed, to carry out the transmission of nutritive ingredient and oxygen.The cell that rigidly connects kind is grown in the surface of described cell culture vector, and along with cell proliferation, cell begins to be full of the space between cell culture vector.Fixed bed inner cell nutrient solution velocity of flow is big, cell stand density height, and the consumption of the albumen in the time of can reducing serum-free culture is applicable to the propagation suspension cell.Cell can not suffer damage because of the effect of mechanical shear stress.But, because the filler that cell culture vector constitutes is compressed by upper mounted plate and bottom plate, the meta-bolites of cell and dead cell be difficult to be discharged, and cell culture fluid is inhomogeneous simultaneously flows and cause channeling and nutrient distribution inhomogeneous, and cell speed of growth consistence is poor.
Fluidized-bed comprises tank body and agitator etc., in described tank body, produce the swirling flow of upwards flowing by described agitator, make the cell culture fluid of sustenticular cell growth be fluidization, and the cell culture vector of inner filling is stirred up and down, depend on a large amount of cells of cell culture vector growth and grow at suspended state.The good fluidity of fluidized-bed inner cell nutrient solution, the required nutrition of the growth of cell is evenly distributed in fluidized-bed, cell growth high conformity, but cell and cell culture vector are gone out, while, cell was in fluidized-bed inner suspension attitude variation along with cell increases.The scale of fluidized-bed can't be amplified simultaneously, and it only is that the cross-sectional area maximum-norm only is 1m about 2m that the degree of depth of existing fluidized-bed is to the maximum
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Summary of the invention
The objective of the invention is in order to overcome the deficiencies in the prior art, a kind of cell cultures bed for bio-reactor is provided, when it can either overcome fluidized-bed and carries out cell cultures, cell and cell culture vector are gone out easily, cause the remaining middle cell of supernatant liquor and the more defective of cell culture vector of extraction, can overcome when adopting the curing bed to carry out cell cultures the inconsistent defective of the cell speed of growth again.
A kind of technical scheme that realizes above-mentioned purpose is: a kind of cell cultures bed for bio-reactor, this cell cultures bed places in the tank body, comprise guide shell, stephanoporate round tube and multistep speed regulation agitator, described stephanoporate round tube joins with described guide shell is concentric in the bottom of described guide shell, and described multistep speed regulation agitator inserts described stephanoporate round tube from the bottom of described stephanoporate round tube;
The top of described guide shell is provided with the upper mounted plate around described guide shell circumference, the bottom of described stephanoporate round tube is provided with the bottom plate around described stephanoporate round tube circumference, the excircle of described bottom plate and the inwall of described tank body are fixed, described upper mounted plate and described bottom plate are porous plate, fill the filler of being made up of cell culture vector between the bottom surface of described upper mounted plate and the end face of described bottom plate, the height of described filler mostly is 2/3rds of distance between described upper mounted plate bottom surface and the described bottom plate end face most.
Further, also comprise in this cell cultures bed around the even number root thrust-augmenting nozzle of described guide shell and described stephanoporate round tube circumference uniform distribution, described even number root thrust-augmenting nozzle runs through described upper mounted plate and described bottom plate, is plugged with first ventpipe spaced reciprocally in the described even number root thrust-augmenting nozzle.
Further again, all be provided with even column first radial direction through hole around described thrust-augmenting nozzle circumference uniform distribution on the tube wall of any described thrust-augmenting nozzle.
Further, on the tube wall of any described thrust-augmenting nozzle, any described first radial direction through hole of the adjacent two row layout that all interlaces.
Further again, be plugged with second ventpipe of being close to described guide shell inwall in described guide shell and the described stephanoporate round tube, the height of the described second ventpipe end face is lower than the height of the described first ventpipe end face.
Further, described second ventpipe is fixed by the filter screen that is positioned at described guide shell end face.
Further, the top of described stephanoporate round tube is up-small and down-big toroidal.
Further, also comprise overhead gage and lower baffle plate between described upper mounted plate and described bottom plate in this cell cultures bed, the diameter of described overhead gage and described lower baffle plate is less than the diameter of described bottom plate, described overhead gage and described lower baffle plate are porous plate, the end face of described filler is lower than the bottom surface of described overhead gage, is higher than the end face of described lower baffle plate.
Adopted the technical scheme of a kind of cell cultures bed for bio-reactor of the present invention, namely the height of the filler in the cell cultures bed of fixed bed class mostly is 2/3rds technical scheme of this cell cultures bed height most.Its technique effect is: when it can either overcome fluidized-bed and carries out cell cultures, cell and cell culture vector are gone out easily, cause remaining cell and the more defective of cell culture vector of supernatant liquor of extraction, can overcome when adopting the curing bed to carry out cell cultures the inconsistent defective of the cell speed of growth again.
Description of drawings
Fig. 1 is the structural representation of a kind of cell cultures bed for bio-reactor of the present invention.
Embodiment
See also Fig. 1, the present inventor be in order to understand technical scheme of the present invention better, below by embodiment particularly, and is described in detail by reference to the accompanying drawings:
See also Fig. 1, a kind of cell cultures bed for bio-reactor of the present invention belongs to fixed bed, place in the tank body 1, comprise: guide shell 21, stephanoporate round tube 22, multistep speed regulation agitator 3, upper mounted plate 41, overhead gage 42, lower baffle plate 43, bottom plate 44, thrust-augmenting nozzle 51, the first ventpipes 52, second ventpipe 23 and filter screen 24.
Second ventpipe 23 is close to the inwall of guide shell 21, stretches into guide shell 21 from the top of guide shell 21, passes whole guide shell 21, stretches into stephanoporate round tube 22.Second ventpipe 23 is fixing by filter screen 24, and filter screen 24 is fixed on the end face of guide shell 21.
Upper mounted plate 41, overhead gage 42, lower baffle plate 43, bottom plate 44 set gradually from top to bottom along vertical direction.Wherein upper mounted plate 41 is to arrange around the top of guide shell 21, and overhead gage 42 is that the middle part around guide shell 21 arranges, and lower baffle plate 43 is that the bottom around guide shell 21 arranges, and bottom plate 44 is that the bottom around stephanoporate round tube 22 arranges.Upper mounted plate 41 bottom surfaces are these cell cultures bed end faces, and bottom plate 44 end faces are this cell cultures bed bottom surface, and the distance between upper mounted plate 41 bottom surfaces and bottom plate 44 end faces is the high h of this cell cultures bed.Wherein, the distance between overhead gage 42 end faces and upper mounted plate 41 bottom surfaces is smaller or equal to 1/3rd of the high h of this cell cultures bed, and the distance between lower baffle plate 43 bottom surfaces and bottom plate 44 end faces is smaller or equal to 1/2nd of the high h of this cell cultures bed.The diameter of upper mounted plate 41, overhead gage 42 and lower baffle plate 44 is less than the diameter of bottom plate 44 simultaneously.Therefore, the inwall of the excircle of bottom plate 44 and tank body 1 is fixed, and all leaves the gap around its excircle between upper mounted plate 41, overhead gage 42 and lower baffle plate 43 and tank body 1 inwall.Form back cavity 11 between bottom plate 44 bottom surfaces and tank body 1 bottom surface.
In the present embodiment, upper mounted plate 41, overhead gage 42, lower baffle plate 43, bottom plate 44 all be usefulness all be porous plate, select for use stainless steel or plastics to make.Upper mounted plate 41, overhead gage 42, lower baffle plate 43, bottom plate 44 preferable material are porous polyethylene plate, porous polypropylene plate and porous EFTE(polyethylene-polytetrafluoroethyl-ne alkene copolymer) plate.
Thrust-augmenting nozzle 51 is around the circumference uniform distribution of guide shell 21 and stephanoporate round tube 22, and the quantity of thrust-augmenting nozzle 51 is even number.All thrust-augmenting nozzles 51 all run through upper mounted plate 41, overhead gage 42, lower baffle plate 43 and bottom plate 44 successively.First ventpipe 52 inserts thrust-augmenting nozzle 51 spaced reciprocally, and the thrust-augmenting nozzle 51 that namely is inserted with the thrust-augmenting nozzle 51 of first ventpipe 52 and is not inserted with first ventpipe 52 is spaced apart.
Wherein the height of first ventpipe, 51 end faces is higher than the height of second ventpipe, 23 end faces.
On the tube wall of all thrust-augmenting nozzles 51, be evenly distributed in some row first radial direction through hole (not shown), adjacent described first radial direction through hole of the two row layout that interlaces arbitrarily around thrust-augmenting nozzle 51.Therefore, the columns of first radial direction through hole on every thrust-augmenting nozzle 51 is even column.
Also be filled with the filler 6 that cell culture vector is formed in this cell cultures bed.In the present embodiment, what cell culture vector adopted is sheet-like fiber carrier.The height d of filler 6 is 1/2nd to 2/3rds of this cell cultures bed height h, be that bottom plate 44 end faces are to 1/2nd to 2/3rds of upper mounted plate 41 bottom surface distances, be that the end face of filler 6 will be higher than the height of lower baffle plate 43 end faces and be lower than the height of overhead gage 42 bottom surfaces, so that the space in the cell cultures bed is utilized fully.
In this cell cultures bed, cell culture fluid adds this cell cultures bed from thrust-augmenting nozzle 51 and guide shell 21, and cell culture fluid forms the swirling flow that flows downward in each thrust-augmenting nozzle 51, and this swirling flow that flows downward is gone out from the bottom of each thrust-augmenting nozzle 51.Cell culture fluid also forms the swirling flow that flows downward in guide shell 21, this swirling flow that flows downward flow through guide shell 21 and stephanoporate round tube 22, go out from the bottom of stephanoporate round tube 22, from each thrust-augmenting nozzle 51 and the swirling flow that flows downward of going out from stephanoporate round tube 22 bottoms, under the effect of multistep speed regulation agitator 3, in back cavity 11, formed the swirling flow of upwards flowing.
Because upper mounted plate 41, overhead gage 42, lower baffle plate 43, bottom plate 44 all are porous plate, the swirling flow that should upwards flow enters filler 6 from the bottom of bottom plate 44, for the cell in the cell culture vector in the filler 6 provides nutrition and dissolved oxygen.The continuation of the swirling flow that should upwards flow is upwards flowed, flow through lower baffle plate 43, overhead gage 42 and upper mounted plate 41, perhaps around the gap of lower baffle plate 43, overhead gage 42 and upper mounted plate 41, flow out this cell cultures bed, finish the once circulation of cell culture fluid in the cell cultures bed.Be owing to the diameter of lower baffle plate 43, overhead gage 42 and upper mounted plate 41 diameter less than bottom plate 44 forms around the gap of lower baffle plate 43, overhead gage 42 and upper mounted plate 41.
In the present embodiment, multistep speed regulation agitator 3 has the function of multistep speed regulation, by changing the stirring velocity of multistep speed regulation agitator 3, can change the movement velocity that this swirling flow of upwards flowing makes progress in filler 6.When multistep speed regulation agitator 3 is in zero span, can't form the swirling flow of upwards flowing in the cell cultures bed.When the speed of rotation of multistep speed regulation agitator 3 is in a grade of lowest speed, the speed that the swirling flow that should upwards flow is upwards flowed is lower than filler 6 inner cells and cultivates going out a little of carrier, can in the cell cultures bed, settle down in the cell culture vector in the filler 6, form the training method that similar fixed bed is cultivated.There is the gap between the cell culture vector in the filler 6 at this moment, and the cell culture vector that can observe in the filler 6 is carrying out small rocking, this rocking helps to make cell culture fluid evenly to distribute at filler 6, and be more superior than existing fixed bed training method.
The speed of rotation of multistep speed regulation agitator 3 rises, and when namely the speed of rotation of multistep speed regulation agitator 3 was in the most at a high speed N shelves, the swirling flow that should upwards flow was higher than the settling velocity of the cell culture vector in the filler 6 to the speed at upper reaches.Because the cell culture vector in the filler 6 is in suspended state, forms and be similar to the training method that fluidized-bed is cultivated.Because the porous plate that upper mounted plate 41, overhead gage 42, lower baffle plate 43 and bottom plate 44 adopt, its aperture is all less than the diameter of the cell culture vector in the filler 6, cell culture vector in the filler 6 is subjected to the two-layer obstruct of overhead gage 42 and upper mounted plate 41, and can not go out this cell cultures bed, guaranteed that the top of upper mounted plate 41 end faces is clear liquids.
This cell cultures bed fixed bed training method is main, intermittently change the speed of rotation of multistep speed regulation agitator 3 momently, make the cell culture vector in the filler 6 be in suspended state, form and be similar to the fluidized-bed training method, cell culture fluid and meta-bolites in the cell reactor are homogenized.This cell cultures bed preferably program of cultivating can be set at: the fixed bed training method was cultivated 1-3 hour, the fluidized-bed training method was cultivated 1-3 minute, multistep speed regulation agitator 3 stopped to stir 0.5-1 minute, and recycling fixed bed training method was carried out cell cultures 1-3 hour.
This cell cultures bed can be realized highdensity cell cultures, and cell density can reach 10
8Individual/more than the ml, and can carry out the magnificent cultivation of long-time continuous stream, namely by supplying with cell culture fluid and dissolved oxygen continuously in this cell cultures bed, this cell cultures bed is exported supernatant liquor simultaneously, thus the cultured continuously of realization cell.Because the aperture of second radial direction through hole on the stephanoporate round tube 22 is also less than the diameter of the cell culture vector in the filler 6, therefore upper mounted plate 41, bottom plate 42 and the stephanoporate round tube 22 in this cell cultures bed all has iris action, cell culture vector in the filler 6 is limited in the space of a sealing, makes that the extraction of supernatant liquid is more convenient.
In this cell cultures bed, the effect of first radial direction through hole is on the tube wall of thrust-augmenting nozzle 51: in the vertical direction guarantees the homogeneity of filler 6 inner cell nutrient solutions, any first radial direction through hole of adjacent two row layouts that interlace on the tube wall of thrust-augmenting nozzle 51 can further improve the homogeneity that in the vertical direction guarantees flour filler 6 interior dissolved oxygens and cell culture fluid.In all thrust-augmenting nozzles 51, have in half the thrust-augmenting nozzle 51 and be inserted with first ventpipe 52, it can carry out deep ventilation to the cell cultures bed with external oxygen on the one hand, one side is from the oxygen of first ventpipe, 52 inputs in addition, go out first ventpipe 52 and thrust-augmenting nozzle 51, bubble wherein, after the stirring through multistep speed regulation agitator 3, be dissolved in the cell culture fluid, form dissolved oxygen, by the bottom plate 44 to filler 6 bottoms, enter filler 6, carry out oxygen supply in the cell culture vector of filler 6, carrying out cultured cells, rather than directly enter filler 6 by first radial direction through hole on the thrust-augmenting nozzle 51.Because the bubble of the oxygen that produces in the deep ventilation process under the stirring of multistep speed regulation agitator 3, is dissolved in the cell culture fluid, has reduced bubble like this to carry out the damage of culturing cell in the cell culture vector of filler 6.First radial direction through hole on the thrust-augmenting nozzle 51 limited from the oxygen of first ventpipe 52 input bubble directly first radial direction through hole from the thrust-augmenting nozzle 51 enter filler 6.
The effect of stephanoporate round tube 22 also is: the cell culture vector multistep speed regulation agitator 3 in the filler 6 is separated, with the mechanical shear stress that prevents multistep speed regulation agitator 3 cell in the cell culture vector in the filler 6 is produced damage.Cell has good population effect, difficult drop-off in the cell culture vector growth inside simultaneously.In addition on the one hand, when this cell cultures bed was cultivated to solidify the bed mode, products of cellular metabolism and dead cell can be under the effects of multistep speed regulation agitator 3, and second radial direction through hole from stephanoporate round tube 22 is discharged.
Therefore, in the filler 6 cell culture fluid to enter the path of discharging with products of cellular metabolism be different.
The height of the end face of second ventpipe 23 is lower than the height of tank body 1 top supernatant liquid liquid level, so that the dissolved oxygen in the supernatant liquid is carried out deep ventilation to this bio-reactor, further guarantees the supply of the oxygen that cell cultures is required.When filter screen 24 is fixed second ventpipe 23, also filter in the supernatant liquid because cellular respiration produces, gush out from the gap between upper mounted plate 41 excircles and tank body 1 inwall, contain the carbonic acid gas viscous foam, prevent from containing the bottom that the carbonic acid gas viscous foam is back to the cell cultures bed, thereby influence is to carrying out the oxygen supply of culturing cell in the cell culture vector of filler 6.
Because this cell cultures bed also by insert first ventpipe 52 in first thrust-augmenting nozzle 51, has solved the problem of deep ventilation, so its amplification potential is good, and can realize highdensity cell cultures in this cell cultures bed, and cell density can reach 10
8Individual/more than the ml; Can also carry out the cell cultures of long-time continuous.This cell cultures bed is particularly suitable for serum-free culture, long-time continuous perfusion, long-time harvested cell meta-bolites.Use a kind of cell cultures bed for bio-reactor of the present invention, the volume of bio-reactor can reach 500L at least.
Claims (8)
1. cell cultures bed that is used for bio-reactor, this cell cultures bed places in the tank body (1), it is characterized in that: comprise guide shell (21), stephanoporate round tube (22) and multistep speed regulation agitator (3), described stephanoporate round tube (22) is in the bottom and concentric joining of described guide shell (21) of described guide shell (21), and described multistep speed regulation agitator (3) inserts described stephanoporate round tube (22) from the bottom of described stephanoporate round tube (22);
The top of described guide shell (21) is provided with the upper mounted plate (41) around described guide shell (21) circumference, the bottom of described stephanoporate round tube (22) is provided with the bottom plate (44) around described stephanoporate round tube (22) circumference, the inwall of the excircle of described bottom plate (44) and described tank body (1) is fixed, described upper mounted plate (41) and described bottom plate (44) are porous plate, fill the filler of being made up of cell culture vector (6) between the end face of the bottom surface of described upper mounted plate (41) and described bottom plate (44), the height of described filler (6) mostly is 2/3rds of distance between described upper mounted plate (41) bottom surface and described bottom plate (44) end face most.
2. a kind of cell cultures bed for bio-reactor according to claim 1, it is characterized in that: also comprise in this cell cultures bed around the even number root thrust-augmenting nozzle (51) of described guide shell (21) and described stephanoporate round tube (22) circumference uniform distribution, described even number root thrust-augmenting nozzle (51) runs through described upper mounted plate (41) and described bottom plate (44), is plugged with first ventpipe (52) in the described even number root thrust-augmenting nozzle (51) spaced reciprocally.
3. a kind of cell cultures bed for bio-reactor according to claim 2 is characterized in that: all be provided with even column first radial direction through hole around described thrust-augmenting nozzle (51) circumference uniform distribution on the tube wall of any described thrust-augmenting nozzle (51).
4. a kind of cell cultures bed for bio-reactor according to claim 3 is characterized in that: on the tube wall of any described thrust-augmenting nozzle (51), and described first radial direction through hole of the adjacent two row layout that all interlaces arbitrarily.
5. a kind of cell cultures bed for bio-reactor according to claim 2, it is characterized in that: be plugged with second ventpipe (23) of being close to described guide shell (21) inwall in described guide shell (21) and the described stephanoporate round tube (22), the height of described second ventpipe (23) end face is lower than the height of described first ventpipe (52) end face.
6. according to the described a kind of cell cultures bed for bio-reactor of claim 5, it is characterized in that: described second ventpipe (23) is fixing by the filter screen (24) that is positioned at described guide shell (21) end face.
7. according to the described a kind of cell cultures bed for bio-reactor of claim 1, it is characterized in that: the top of described stephanoporate round tube (22) is up-small and down-big toroidal.
8. a kind of cell cultures bed for bio-reactor according to claim 1, it is characterized in that: also comprise the overhead gage (42) and the lower baffle plate (43) that are positioned between described upper mounted plate (41) and the described bottom plate (44) in this cell cultures bed, the diameter of described overhead gage (42) and described lower baffle plate (43) is less than the diameter of described bottom plate (44), described overhead gage (42) and described lower baffle plate (43) are porous plate, the end face of described filler (6) is lower than the bottom surface of described overhead gage (42), is higher than the end face of described lower baffle plate (43).
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| CN106701578A (en) * | 2017-03-15 | 2017-05-24 | 上海戈洛思生物科技有限公司 | Ventilation stirring system for bioreactor |
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| CN114276927A (en) * | 2021-12-29 | 2022-04-05 | 上海日泰医药设备工程有限公司 | Folding carrier column for bioreactor |
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