CN103266182A - Method and device for regulating tower plate type perfusion bioreactor - Google Patents
Method and device for regulating tower plate type perfusion bioreactor Download PDFInfo
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- CN103266182A CN103266182A CN2013102089299A CN201310208929A CN103266182A CN 103266182 A CN103266182 A CN 103266182A CN 2013102089299 A CN2013102089299 A CN 2013102089299A CN 201310208929 A CN201310208929 A CN 201310208929A CN 103266182 A CN103266182 A CN 103266182A
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000001105 regulatory effect Effects 0.000 title abstract description 14
- 230000010412 perfusion Effects 0.000 title abstract description 7
- 239000012530 fluid Substances 0.000 claims abstract description 58
- 230000000694 effects Effects 0.000 claims abstract description 16
- 241001672694 Citrus reticulata Species 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 4
- 238000005457 optimization Methods 0.000 abstract description 2
- 239000010410 layer Substances 0.000 description 41
- 235000015097 nutrients Nutrition 0.000 description 34
- 239000011229 interlayer Substances 0.000 description 12
- 230000003321 amplification Effects 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 230000002706 hydrostatic effect Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002572 peristaltic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 239000002912 waste gas Substances 0.000 description 1
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Abstract
The invention relates to the technical field of bioreactors in medical instruments, and in particular relates to a method and a device for regulating a tower plate type perfusion bioreactor. The method for regulating the tower plate type perfusion bioreactor comprises the following steps of: (1), regulating a regulating valve on each layer of tower plate; (2), cultivating cells under same fluid inlet structure parameters; (3), determining a tower plate layer number N with an optimum cultivation effect; (4), computing the flowing speed M of a fluid on the surface of an N-th layer of tower plate; (5), computing structure parameters A(i) of a fluid inlet between adjacent layers of tower plates; (6), regulating the regulating valve on each layer of tower plate; and (7), continuing cultivating the cells on the surface of each layer of regulated tower plate. According to the method and the device, the structure parameters of an inflow port in each layer of tower plate in the perfusion bioreactor can be regulated, so that the fluid shear stress between the adjacent layers of tower plates can be regulated according to a geometric structure, an optimization way is provided for distribution of the fluid shear stresses in each layer of tower plate, and the fluid shear stresses of all layers of tower plates can be optimized.
Description
Technical field
The present invention relates to the bioreactor technology field in the medicine equipment, more specifically, relate to a kind of control method and using appts thereof of column plate type filling type bioreactor.
Background technology
The structure of existing bio-reactor as shown in Figure 1, its function is environment similar in one of external manufacturing and body, to accelerate the system of culturing cell, formative tissue and even organ, can be widely used in the organ defect patient and substitute, make up, keep or strengthen its function of organization, and field such as bio-pharmaceuticals.Its principle is physiology, physics, the chemical property of the microenvironment of growing in vivo according to cell, utilize engineering, at external structure one covering device, can provide with body in close microenvironment, be beneficial to cell and in this external microenvironment, breed, break up, and formative tissue and even organ.
Fig. 1 is the structural representation of existing filling type bioreactor, among Fig. 1, nutrient solution in the nutrient solution hold-up vessel 2 is drawn into the entrance of culture tank 7 medium-height trestles 8 by peristaltic pump 6 by conduit, see through support 8, flow in the culture tank 7, nutrient solution in the culture tank 7 is extracted out from culture tank 7 by peristaltic pump 4 by conduit, by T-valve 5, is back to nutrient solution hold-up vessel 2 or flows into the discarded liquid hold-up vessel 1 from conduit 3.The gas that contains oxygen is added in the nutrient solution from airway 9, and the waste gas in the nutrient solution also can be discharged by airway 9.Support 8 mostly is porous media material, is planted in the cell in the support 8 and flows through between the nutrient solution of support to have nutritive substance and metabolic excremental exchange process.Cell can be bred in reactor on a large scale, also can and then carry out cytodifferentiation.
For in bio-reactor, realizing amplification culture, existing feasible program is that a kind of tower tray device of support 8 usefulness among Fig. 1 is realized, as shown in Figure 2, one group of flat board is fitted together abreast by the thrust-augmenting nozzle 12 that passes dull and stereotyped center, profile is turriform, is called column plate 13.Column plate 13 is placed in the reactor 10 that is full of nutrient solution vertically, nutrient solution flows in the reactor 10 from nutrient solution entrance 11, hole in again from the interflow of column plate 13 to column plate on thrust-augmenting nozzle 12 sidewalls, flow to nutrient solution outlet 14 again in the column plate in the pipe of thrust-augmenting nozzle 12, thereby each aspect at column plate 13 forms the perfusion stream that flows, the speed of fluid flow on column plate 13 each aspect, the effect that the column plate inner cell is cultivated has material impact.
Vertically be placed on the column plate 13 in the reactor 10 that is full of nutrient solution, increase along with the degree of depth under the liquid level, suffered hydrostatic pressure also can increase, if among Fig. 2 in the column plate 13 on thrust-augmenting nozzle 12 sidewalls geometrical structure parameter in hole all identical, then the fluid on column plate 13 different aspects produces different flow velocitys because of the difference of hydrostatic pressure, thereby the effect difference that causes cell cultures on different column plate 13 aspects, the cell cultures effect that the amplification culture mode of feasible employing column plate type obtains is inconsistent.Namely existing column plate type filling type bioreactor mainly has the following disadvantages:
(1) existing column plate type filling type bioreactor can obtain inconsistent hydrodynamic shear action effect when adopting tower tray device to carry out amplification culture.
(2) existing column plate type filling type bioreactor can not be regulated its crucial structural parameter, is difficult to obtain the hydrodynamic shear that conforming rate of flow of fluid produces.
(3) existing column plate type filling type bioreactor lacks structure parameter optimizing method in amplification culture, is difficult to be implemented in the hydrodynamic shear that all obtains between each layer column plate the cell cultures optimum.
Summary of the invention
The present invention is for overcoming the described at least a defective of above-mentioned prior art, a kind of control method of column plate type filling type bioreactor is provided, it can solve the consistency problem of the cell hydrodynamic shear that fluid flow rate produces in the amplification culture process, further, its using appts is provided, this apparatus structure is simple, is easy to install rotation and regulates.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of control method of column plate type filling type bioreactor, comprising following steps:
S1. regulate the variable valve on each layer column plate, make to have identical fluid intake structural parameter between each layer column plate; Column plate vertically is placed in the culture tank that is full of nutrient solution, and the cell that is planted on the column plate is cultivated in circulating the nutrient solution of perfusion;
S2. culturing cell under same fluid entrance structure parameter; The structural parameter of each layer column plate are identical because nutrient solution is flowed through, make the flow velocity of nutrient solution on each layer column plate only be subjected to each layer column plate subsurface effect of depth in nutrient solution, namely the different liqs static pressure that has of the nutrient solution of different depths is as unique factor of nutrient solution velocity of flow on the different column plate aspects of influence;
S3. observe the cultivation situation of cell on each layer column plate, determine the column plate number of plies N of culture effect optimum;
S4. use the CFD of fluid simulation software, calculate flow rate of fluid M on the N layer tower plate surface; The CFD(computational fluid design of fluid simulation software Fluid Computation design is a kind of common software);
S5. using the CFD of fluid simulation software, serves as to calculate target with flow velocity M, calculates the structural parameter A (i) of fluid intake between each layer column plate, and i is the number of plies of column plate;
S6. according to the fluid intake structural parameter A (i) between each layer column plate that calculates, regulate the variable valve on each layer column plate, the geometrical structure parameter that fluid on each layer column plate is become a mandarin mouthful meets parameter A (i); Described structural parameter A (i) is the sectional area that becomes a mandarin on the flow-guiding mouth on the thrust-augmenting nozzle outside wall surface in the column plate between i layer column plate.
S7. each layer tower plate surface of cell continuation after adjusting cultivated.On after the adjusting each layer tower plate surface, will have identical nutrient solution velocity of flow, thereby have identical optimum hydrodynamic shear, realize unanimity and the optimal adjustment of tower plate structure parameter.
By by step S6, overcome the discordance of rate of flow of fluid between each layer column plate that the influence of the column plate inter-layer fluid flow velocity under dark to different liquid of hydrostatic pressure among the step S2 causes, thereby obtain conforming hydrodynamic shear at different column plate interlayers.With the reference configuration of optimum culture effect in the step S3 acquisition amplification culture, the optimum structure parameter by step S5 obtains each layer column plate goes out the conforming optimum hydrodynamic shear of each interlayer of column plate according to the optimum structure parameter regulation in step S6.
In the tower plate structure of column plate type filling type bioreactor, the method for design that the present invention proposes makes its crucial geometrical structure parameter adjustable, thereby makes that flow rate of fluid is adjustable in the column plate, and then regulates the hydrodynamic shear of fluid on the tower plate surface.The culture effect of the cell of planting on the tower plate surface is subjected to the influence of its hydrodynamic shear remarkable.
A kind of setting device of column plate type filling type bioreactor, comprise thrust-augmenting nozzle, the outer wall of thrust-augmenting nozzle is provided with a plurality of flow-guiding mouths, wherein, also comprise variable valve and column plate, column plate is located on the variable valve, and variable valve is located on the outer wall of thrust-augmenting nozzle, and described variable valve is provided with a plurality of be used to the controllable register that blocks flow-guiding mouth.Crucial geometrical structure parameter is the size of flow-guiding mouth, and the nutrient solution that these structural parameter can influence the column plate interlayer flows to the interior flow velocity of column plate thrust-augmenting nozzle.Among the present invention, rotatable variable valve, controllable register blocks flow-guiding mouth, thereby can change the sectional area size that becomes a mandarin of this flow-guiding mouth, and then regulates the velocity of flow that nutrient solution flows into this flow-guiding mouth.
Further, variable valve is provided with inner ring surface, and inner ring surface is close to the outer wall of thrust-augmenting nozzle; Have fixing threaded hole on the controllable register, screw passes fixing threaded hole variable valve and thrust-augmenting nozzle is fixed.Variable valve is inserted in the thrust-augmenting nozzle outside, the rotatable adjusting of variable valve, and then block flow-guiding mouth by controllable register, when adjusting position is suitable, passes fixing threaded hole with screw variable valve and thrust-augmenting nozzle are fixed.
Further, described variable valve main body is circular ring, and controllable register is located at the upper surface of variable valve main body.Described variable valve also is provided with and supports the bottom surface, supports bottom surface support tower plate.During assembling, variable valve is inserted in the thrust-augmenting nozzle outside, and the rotatable adjusting of variable valve is supported the bottom surface and can be supported column plate, and the variable valve main body is that the shape of circular ring and column plate is complementary, and is easy for installation.
Concrete, described controllable register is rectangular configuration, and controllable register quantity is 6, and it is along the circumference setting of inner ring surface, and the central angle between the adjacent controllable register is 60 degree.The height of controllable register determines the height between column plate, rotating regulation valve, make in the controllable register partial occlusion column plate flow-guiding mouth on the thrust-augmenting nozzle outer wall, thereby can regulate the sectional area size that fluid enters this flow-guiding mouth, this sectional area size is the key structure parameter of tower plate structure, regulates the adjusting that this parameter can realize the column plate inner fluid speed.
Further, the bottom of described thrust-augmenting nozzle also is provided with the mouth that confluxes.Nutrient solution from the interflow of column plate to column plate in flow-guiding mouth on the thrust-augmenting nozzle outer wall, be directed downwards by the caliber in the thrust-augmenting nozzle again and flow from mouthful flowing out confluxing of thrust-augmenting nozzle.
Compared with prior art, beneficial effect is:
1), make column plate type filling type bioreactor when carrying out amplification culture, can obtain conforming hydrodynamic shear effect.
Overcome the employing tower plate structure and carried out in the amplification culture method, the otherness influence of the column plate inter-layer fluid flow velocity that causes because of different hydrostatic pressures on the different column plate aspects makes that flow rate of fluid arrives consistence between each layer column plate.
2), realized that column plate key structure parameter can conveniently adjust accent, conforming hydrodynamic shear provides approach in the column plate in order to obtain by the adjustment structure parameter.
Existing tower plate structure is because the head piece sectional area of going into of column plate inter-layer fluid immobilizes, and makes the difference of the column plate inter-layer fluid flow velocity that causes because of the different liqs static pressure between the different layers column plate be difficult to adjust.The head piece sectional area of going into of the column plate inter-layer fluid that the present invention proposes is adjustable, thereby can adjust flow rate of fluid between each layer column plate, for the fluid trend consistence of fluid between each layer column plate provides approach.
The optimization of the hydrodynamic shear when 3), having realized carrying out amplification culture with column plate type filling type bioreactor is regulated.
The control method that the present invention proposes is to serve as with reference to regulating target with the hydrodynamic shear on one deck column plate of cell cultures effect optimum in the column plate, by regulating the structural parameter of each layer column plate, make that the hydrodynamic shear on all column plates is all consistent with the hydrodynamic shear of this optimum layer, thereby make the hydrodynamic shear on each layer column plate all reach optimum, therefore, be a kind of optimized control method.
Description of drawings
Fig. 1 is existing filling type bioreactor overall schematic.
Fig. 2 is reactor and tower plate structure synoptic diagram in the existing column plate type filling type bioreactor.
Fig. 3 is the schematic flow sheet of control method of the present invention.
Fig. 4 is thrust-augmenting nozzle structural representation of the present invention.
Fig. 5 is variable valve structural representation of the present invention.
Fig. 6 is one-piece construction synoptic diagram of the present invention.
Embodiment
Accompanying drawing only is used for exemplary illustration, can not be interpreted as the restriction to this patent; For better explanation present embodiment, some parts of accompanying drawing have omission, amplify or dwindle, and do not represent the size of actual product; To those skilled in the art, in the accompanying drawing some known features and explanation thereof may to omit be understandable.
As shown in Figure 3, a kind of control method of column plate type filling type bioreactor, comprising following steps:
S1. regulate the variable valve on each layer column plate, make to have identical fluid intake structural parameter between each layer column plate; Column plate vertically is placed in the culture tank that is full of nutrient solution, and the cell that is planted on the column plate is cultivated in circulating the nutrient solution of perfusion;
S2. culturing cell under same fluid entrance structure parameter; The structural parameter of each layer column plate are identical because nutrient solution is flowed through, make the flow velocity of nutrient solution on each layer column plate only be subjected to each layer column plate subsurface effect of depth in nutrient solution, namely the different liqs static pressure that has of the nutrient solution of different depths is as unique factor of nutrient solution velocity of flow on the different column plate aspects of influence;
S3. observe the cultivation situation of cell on each layer column plate, determine the column plate number of plies N of culture effect optimum;
S4. use the CFD of fluid simulation software, calculate flow rate of fluid M on the N layer tower plate surface; The CFD(computational fluid design of fluid simulation software Fluid Computation design is a kind of common software);
S5. using the CFD of fluid simulation software, serves as to calculate target with flow velocity M, calculates the structural parameter A (i) of fluid intake between each layer column plate, and i is the number of plies of column plate;
S6. according to the fluid intake structural parameter A (i) between each layer column plate that calculates, regulate the variable valve on each layer column plate, the geometrical structure parameter that fluid on each layer column plate is become a mandarin mouthful meets parameter A (i); Described structural parameter A (i) is the sectional area that becomes a mandarin on the flow-guiding mouth on the thrust-augmenting nozzle outside wall surface in the column plate between i layer column plate.
S7. each layer tower plate surface of cell continuation after adjusting cultivated.On after the adjusting each layer tower plate surface, will have identical nutrient solution velocity of flow, thereby have identical optimum hydrodynamic shear, realize unanimity and the optimal adjustment of tower plate structure parameter.
By by step S6, overcome the discordance of rate of flow of fluid between each layer column plate that the influence of the column plate inter-layer fluid flow velocity under dark to different liquid of hydrostatic pressure among the step S2 causes, thereby obtain conforming hydrodynamic shear at different column plate interlayers.With the reference configuration of optimum culture effect in the step S3 acquisition amplification culture, the optimum structure parameter by step S5 obtains each layer column plate goes out the conforming optimum hydrodynamic shear of each interlayer of column plate according to the optimum structure parameter regulation in step S6.
In the tower plate structure of column plate type filling type bioreactor, the method for design that the present invention proposes makes its crucial geometrical structure parameter adjustable, thereby makes that flow rate of fluid is adjustable in the column plate, and then regulates the hydrodynamic shear of fluid on the tower plate surface.The culture effect of the cell of planting on the tower plate surface is subjected to the influence of its hydrodynamic shear remarkable.
Shown in Fig. 4-6, a kind of setting device of using described method, comprise thrust-augmenting nozzle 20, the outer wall of thrust-augmenting nozzle 20 is provided with a plurality of flow-guiding mouths 21, wherein, also comprise variable valve 30 and column plate 40, column plate 40 is located on the variable valve 30, variable valve 30 is located on the outer wall of thrust-augmenting nozzle 20, and variable valve 30 is provided with a plurality of be used to the controllable register 32 that blocks flow-guiding mouth 21.Crucial geometrical structure parameter is the size of flow-guiding mouth 21, and the nutrient solution that these structural parameter can influence the column plate interlayer flows to the flow velocity in column plate 40 thrust-augmenting nozzles 20.Among the present invention, rotatable variable valve 30, controllable register 32 blocks flow-guiding mouth 21, thereby can change the sectional area size that becomes a mandarin of this flow-guiding mouth 21, and then regulates the velocity of flow that nutrient solution flows into this flow-guiding mouth 21.
Variable valve is provided with inner ring surface 33, and inner ring surface 33 is close to the outer wall of thrust-augmenting nozzle 20; Have fixing threaded hole 31 on the controllable register 32, it is fixing with thrust-augmenting nozzle 20 with variable valve 30 that screw passes fixing threaded hole 31.Variable valve 30 is inserted in thrust-augmenting nozzle 20 outsides, variable valve 30 rotatable adjustings, and then block flow-guiding mouth 21 by controllable register 32, when adjusting position was suitable, it was fixing with thrust-augmenting nozzle 20 with variable valve 30 to pass fixing threaded hole 31 with screw.
In the present embodiment, the variable valve main body is circular ring, and controllable register 32 is located at the upper surface of variable valve main body.Described variable valve also is provided with and supports bottom surface 34, supports bottom surface 34 and supports column plate 40.During assembling, variable valve is inserted in the thrust-augmenting nozzle outside, and variable valve 30 rotatable adjustings are supported bottom surface 34 and can be supported column plate 40, and the variable valve main body is that the shape of circular ring and column plate is complementary, and is easy for installation.
In the present embodiment, controllable register 32 is rectangular configuration, and controllable register 32 quantity are 6, and it is along the circumference setting of inner ring surface 33, and the central angle between the adjacent controllable register 32 is 60 degree.The height of controllable register 32 determines the height of 40 of every layer of column plates, rotating regulation valve 30, make in the controllable register 32 partial occlusion column plates flow-guiding mouth 21 on the thrust-augmenting nozzle outer wall, thereby can regulate the sectional area size that fluid enters this flow-guiding mouth 21, this sectional area size is the key structure parameter of tower plate structure, regulates the adjusting that this parameter can realize the column plate inner fluid speed.
The bottom of thrust-augmenting nozzle 20 also is provided with the mouth 22 that confluxes.Nutrient solution from the interflow of column plate 40 to column plate in flow-guiding mouth 21 on the thrust-augmenting nozzle outer wall, be directed downwards by the caliber in the thrust-augmenting nozzle again and flow from mouthfuls 22 flowing out confluxing of thrust-augmenting nozzle 20.
Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give all embodiments exhaustive.All any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., all should be included within the protection domain of claim of the present invention.
Claims (7)
1. the control method of a column plate type filling type bioreactor is characterized in that may further comprise the steps:
S1. regulate the variable valve on each layer column plate, make to have identical fluid intake structural parameter between each layer column plate;
S2. culturing cell under same fluid entrance structure parameter;
S3. observe the cultivation situation of cell on each layer column plate, determine the column plate number of plies N of culture effect optimum;
S4. use the CFD of fluid simulation software, calculate flow rate of fluid M on the N layer tower plate surface;
S5. using the CFD of fluid simulation software, serves as to calculate target with flow velocity M, calculates the structural parameter A (i) of fluid intake between each layer column plate, and i is the number of plies of column plate;
S6. according to the fluid intake structural parameter A (i) between each layer column plate that calculates, regulate the variable valve on each layer column plate, the geometrical structure parameter that fluid on each layer column plate is become a mandarin mouthful meets parameter A (i);
S7. each layer tower plate surface of cell continuation after adjusting cultivated.
2. a right to use requires the setting device of 1 described method, comprise thrust-augmenting nozzle (20), the outer wall of thrust-augmenting nozzle (20) is provided with a plurality of flow-guiding mouths (21), it is characterized in that, also comprise variable valve (30) and column plate (40), column plate (40) is located on the variable valve (30), and variable valve (30) is located on the outer wall of thrust-augmenting nozzle (20), and described variable valve (30) is provided with a plurality of be used to the controllable register that blocks flow-guiding mouth (21) (32).
3. the setting device of a kind of column plate type filling type bioreactor according to claim 2 is characterized in that, variable valve (30) is provided with inner ring surface (33), and inner ring surface (33) is close to the outer wall of thrust-augmenting nozzle (20); Controllable register has fixing threaded hole on (32), and it is fixing with variable valve (30) and thrust-augmenting nozzle (20) that screw passes fixing threaded hole.
4. the setting device of a kind of column plate type filling type bioreactor according to claim 3 is characterized in that, described variable valve (30) main body is circular ring, and controllable register (32) is located at the upper surface of variable valve (30) main body.
5. the setting device of a kind of column plate type filling type bioreactor according to claim 4, it is characterized in that, described controllable register (32) is rectangular configuration, controllable register (32) quantity is 6, it is along the circumference setting of inner ring surface (33), and the central angle between the adjacent controllable register (32) is 60 degree.
6. the setting device of a kind of column plate type filling type bioreactor according to claim 5 is characterized in that, described variable valve (30) also is provided with and supports bottom surface (34), supports bottom surface (34) and supports column plate (40).
7. the setting device of a kind of column plate type filling type bioreactor according to claim 2 is characterized in that, the bottom of described thrust-augmenting nozzle (20) also is provided with conflux mouthful (22).
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| CN114426926A (en) * | 2022-01-16 | 2022-05-03 | 重庆理工大学 | Assembled plate tower type adherent cell reactor and culture method of hepatic cells thereof |
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| WO2017143640A1 (en) * | 2016-02-26 | 2017-08-31 | 广州洁特生物过滤股份有限公司 | Cell culture apparatus and method |
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