CN102199537A - Membrane bioreactor used in microgravity environment and simulated microgravity environment - Google Patents
Membrane bioreactor used in microgravity environment and simulated microgravity environment Download PDFInfo
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Abstract
本发明涉及一种悬浮生物细胞高效培养的膜生物反应器装置，特别涉及一种用于微重力和模拟微重力环境下的膜生物反应器，主要用于微重力环境和作为地面模拟微重力实验装置进行悬浮生物细胞的长时间培养和生物学效应研究以及重组蛋白的高效制备研究。包括生物反应器主体、供气系统、液体加注系统、搅拌系统、参数测量控制系统、排气系统和取样系统，其中，参数测量控制系统包括pH值参数测量控制系统、温度参数测量控制系统、溶氧参数测量控制系统和压力参数测量控制系统。本发明的膜生物反应器装置能实现微重力环境和地面模拟微重力环境下悬浮生物细胞的长时间高效培养、多参数实时检测和自动调控。
The invention relates to a membrane bioreactor device for efficiently cultivating suspended biological cells, in particular to a membrane bioreactor used in microgravity and simulated microgravity environments, mainly used in microgravity environments and as ground simulated microgravity experiments The device is used for long-term culture of suspended biological cells and research on biological effects, as well as research on the efficient preparation of recombinant proteins. Including bioreactor main body, gas supply system, liquid filling system, stirring system, parameter measurement and control system, exhaust system and sampling system, wherein the parameter measurement and control system includes pH value parameter measurement and control system, temperature parameter measurement and control system, Dissolved oxygen parameter measurement control system and pressure parameter measurement control system. The membrane bioreactor device of the invention can realize long-term and high-efficiency cultivation of suspended biological cells in a microgravity environment and a ground simulated microgravity environment, multi-parameter real-time detection and automatic regulation.
Description
Technical field
The present invention relates to the film bioreactor device that a kind of suspended biological cell high-efficient is cultivated, be particularly related to a kind of membrane bioreactor that is used under microgravity and the simulated microgravity environment, be mainly used in microgravity environment and carry out the long-time cultivation of suspended biological cell and the efficient production research of biological effect research and recombinant protein as ground simulation microgravity experiment device.
Background technology
In the space microgravity environment, flow phenomenon is disappeared in gas and the liquid, buoyancy disappears, and the component that different densities causes is separated and the sink-float phenomenon disappears, and the fluidic static pressure disappears or the like.This makes that gas-liquid-solid three-phase is difficult to separate, and has increased the supply of oxygen in the biomass cells culturing process, the discharge of waste gas and the uniform difficulty of mass transfer of nutrition.In order to carry out space microgravity environmental biomedicine effect and spatial cell biology, microbiological correlative study, must develop the biomass cells culture technique that is fit to the space microgravity environment, develop corresponding culture device.Astropowers such as the U.S., Russia, Europe, Japan carry out the research of DESIGN OF REACTOR and space life science for many years, make space biomass cells culture device and technology obtain significant progress.Some novel biomass cellss are cultivated unit hardware equipment and are come into operation, can be used for from higher organism such as animal, plant to microorganism, cell, organize level, from the space biomedicine to different levels researchs such as space biotechnologys.Up to the present, the rotation wall type has been developed in countries in the world, the continous pouring formula, the device that the microgravity environment biomass cells of number of different types such as pulsed is cultivated carried out RWV (Rotating Wall Vessel Bioreactor) reactor that biomass cells and tissue suspension cultivate 90 years last century by the microgravity environment that is used for of NASA Johnson Space Center generation exploitation just as the U.S..NASA exploitation satisfy the U.S. carries out life science research on international space station cell culture unit reactor (Cell CultureUnit Bioreactor, CCUB).The single loop cell culture reactor of development such as NASA's Ames Research Center (Single Loop for Cell Culture Bioreactor, SLCCB) etc.This serial reaction device system changes the deficiency that has solved reactor assembly cultivation biomass cells for exhaust mode and a series of measures such as installing defoaming device additional preferably by outer circulation, can better adapt to the demand that the space microgravity environment carries out biomass cells culture studies and scientific experiment.
Biological scientific experiment has suitable challenge under the space microgravity environment, these experiments need a large amount of time to go to prepare and hardware technology support reliably, therefore study microgravity environment and the influence of biomass cells is had very big restricted, comprise electric energy, weight, volume, loading restriction; Cosmonaut's restriction; Flying machine can limit; The commercial value restriction; Also need special experimental installation.Thereby need an energy on ground, to finish the equipment of simulated microgravity experiment.Simulated microgravity is to utilize simulated microgravity devices such as gyrator to make the successive randomly changing of the gravity direction that acts on the object on the ground, rotate a week the vector and i.e. " zero gravity " phenomenon that equals zero that (360 °) produce, cell and biomacromolecule are in a kind of simulation free falling body state, i.e. simulated microgravity state.Under the simulated microgravity environment, shearing force and turbulent property reduce in the fluid, and the settling ratio of organism changes.
Can simulate the device that produces microgravity on the ground has: the machine that falls certainly, aerostat, sounding rocket, the tower that falls, parabolic flight machine etc.But the shortcoming of these devices is to produce microgravity by successive, and is also inapplicable for the biological culture that needs long-time continuous generation microgravity effect.The simulated microgravity bio-reactor is that US National Aeronautics and Space Administration (NASA) develops for carrying out the spatial cell culture experiment the earliest, develops into the culture apparatus of studying biological effect under the simulated microgravity condition such as animal and plant cells, microorganism under surface condition gradually.Simultaneously, the filling type cultured continuously of widespread use in face of its type is also cultivated by incipient simple batch.Its function also develops into present multiparameter on-line monitoring regulation and control etc. by the initial rotating speed of can only simply regulating and control.Along with the fast development of space science technology, China had also developed the culture apparatus of cultivating animal and plant cells, tissue and various algae, microorganism etc. in the last few years.
But all kinds of bio-reactors of world's exploitation at present can not realize fully during biomass cells is cultivated under microgravity environment and the ground simulation microgravity environment gas-liquid mass transferring, technical barrier such as separate, realize the high-efficient culture of biomass cells and the monitoring and the regulation and control of correlation parameter.
Summary of the invention
The objective of the invention is to satisfy in order to solve biomass cells culture apparatus under microgravity environment that the suspended biological cell is long-time, the technical problem of high-efficient culture research, provide a kind of simple in structure, culture efficiency is high, can realize that the culture system multiparameter detects in real time and auto-control, long-time high-efficient culture, the multiparameter that can be used for carrying out under microgravity and the ground simulation microgravity environment suspended biological cell detects and the multifunctional membrane bio-reactor of auto-control in real time.
The objective of the invention is to be achieved through the following technical solutions.
Membrane bioreactor under a kind of microgravity of the present invention and the simulated microgravity environment, comprise bio-reactor main body, airing system, liquid filling system, stirring system, parameter measurement Controlling System, exhaust system and sampling system, wherein, parameter measurement Controlling System comprises pH value parameter Measurement and Control System, temperature parameter Measurement and Control System, dissolved oxygen parameter measurement Controlling System and pressure parameter Measurement and Control System.
The aspect ratio of bio-reactor main body is 1.5～2; The accumbency of bio-reactor main body is fixedlyed connected on the bio-reactor base for supporting and with it; Bio-reactor main body top is provided with the inlet mouth and first venting port, and the left side wall body is provided with fluid inlet, and the right side wall body is provided with the leakage fluid dram and second venting port from top to bottom, and outer side surface is provided with heating jacket; The air feed membrane module is coiled in the inwall side of bio-reactor main body by the support slot of bio-reactor main body inner wall; The cylinder spiral stirring rake is positioned on the intravital central horizontal axis of bio-reactor master and is tightly connected with bio-reactor main body two side; Central row air film device is positioned at cylinder spiral stirring rake and coaxial with it, and the cylinder spiral stirring rake rotates around central row air film device under power wheel drive.
Airing system is made up of air feed bottle, reducing valve and gas meter; By pilot-gas flow velocity and gaseous tension, realize that oxygen is to the still supply of nutrient solution in the oxygen supply membrane module, the oxygen supply membrane module is the poly tetrafluoroethylene pipe.
The liquid filling system is made up of deformable Liquid storage bag, peristaltic pump and TT﹠C system; Wherein, the deformable Liquid storage bag comprises acid solution bag, alkali lye bag, nutrient solution bag and low temperature seed liquor bag, and peristaltic pump comprises acid solution conveying peristaltic pump, alkali lye conveying peristaltic pump, nutrient solution conveying peristaltic pump and seed liquor conveying peristaltic pump; Acid solution is carried the conveying of liquid in the peristaltic pump control acid solution bag, alkali lye is carried the conveying of liquid in the peristaltic pump control alkali lye bag, nutrient solution is carried the conveying of nutritive medium in the peristaltic pump control nutrient solution bag, and seed liquor is carried the conveying of liquid spawn in the peristaltic pump control low temperature seed liquor bag; Working hour by controlling each peristaltic pump and flow are realized the accurate pulse filling of liquid; The temperature parameter Measurement and Control System is made up of micro temperature sensor probe, heating jacket and TT﹠C system, and when temperature was lower than preset temp in the bio-reactor main body, the heating jacket follow procedure was started working, and realizes accuracy control over temperature; Minisize pH sensor probe, micro temperature sensor probe, miniature dissolved oxygen sensor probe and the sealing of micro pressure sensor probe are installed on the inwall of bio-reactor main body upper and lower.
Stirring system is made up of magnetic force driving motor and cylinder spiral stirring rake, and wherein, the cylinder spiral stirring rake comprises cylinder spiral stirring rake base, magnetic sheet and cylinder spiral stirrer paddle, and drum surface is evenly distributed with sieve aperture.Utilize the rotation of magnetic force driving electric motor driving cylinder spiral stirring rake, and be used for the fragmentation that culturing micro-organisms, cell and tissue can not cause cell walls on the one hand, realize the solid thorough mixing of gas-liquid; The rotation of cylinder spiral stirrer paddle under the normal gravity of ground on the other hand is for the cultivation of biomass cells provides the simulated microgravity environment; The power system of stirring system can also be motor direct-drive.
Exhaust system is made up of central row air film device, vacuum pump and gas sampling bag.
Sampling system is made up of sampling peristaltic pump and sample collection pond.
Beneficial effect
Film bioreactor device of the present invention can realize that long-time high-efficient culture, the multiparameter of microgravity environment and ground simulation microgravity environment low suspension biomass cells detect and auto-control in real time.
Description of drawings
Fig. 1 is a film bioreactor device embodiment synoptic diagram of the present invention;
Fig. 2 is a bio-reactor main body embodiment diagrammatic cross-section of the present invention;
Fig. 3 is a cylinder spiral stirring rake embodiment diagrammatic cross-section of the present invention;
Wherein, 1-bio-reactor main body; 2-oxygen supply membrane module; 3-cylinder spiral stirring rake; 4-central row air film device; The 5-heating jacket; 6-bio-reactor base for supporting; The 7-air feed bottle; The 8-reducing valve; The 9-gas meter; 10-acid solution bag; 11-alkali lye bag; 12-nutrient solution bag; 13-low temperature seed liquor bag; The 14-acid solution is carried peristaltic pump; 15-alkali lye is carried peristaltic pump; The 16-nutrient solution is carried peristaltic pump; The 17-seed liquor is carried peristaltic pump; 18-minisize pH sensor probe; 19-micro temperature sensor probe; 20-magnetic force driving motor; The miniature dissolved oxygen sensor probe of 21-; 22-micro pressure sensor probe; The 23-peristaltic pump of taking a sample; 24-sample collection pond; The 25-vacuum pump; 26-gas sampling bag; The 101-fluid inlet; The 102-inlet mouth; 103-first venting port; The 104-leakage fluid dram; 105-second venting port; 301-cylinder spiral stirring rake base; The 302-magnetic sheet; 303-cylinder spiral stirrer paddle; The 304-sieve aperture.
Embodiment
Below in conjunction with drawings and Examples the present invention is elaborated.
Embodiment
As shown in Figure 1, membrane bioreactor comprises bio-reactor main body, airing system, liquid filling system, stirring system, parameter measurement Controlling System, exhaust system and sampling system, wherein, parameter measurement Controlling System comprises pH value parameter Measurement and Control System, temperature parameter Measurement and Control System, dissolved oxygen parameter measurement Controlling System and pressure parameter Measurement and Control System.
Bio-reactor main body 1 adopts polycarbonate material to make, and aspect ratio is 1.5, and volume is 100ml.1 accumbency of bio-reactor main body is fixedlyed connected on bio-reactor base for supporting 6 and with it; Bio-reactor main body 1 top is provided with the inlet mouth 102 and first venting port 103 as shown in Figure 2, and the left side wall body is provided with fluid inlet 101, and the right side wall body is provided with the leakage fluid dram 104 and second venting port 105 from top to bottom, and outer side surface is provided with heating jacket 5; Air feed membrane module 2 is coiled in the inwall side of bio-reactor main body 1 by the support slot of bio-reactor main body inner wall; Cylinder spiral stirring rake 3 is positioned on the central horizontal axis of bio-reactor main body 1 and is tightly connected with bio-reactor main body 1 two side; Central row air film device 4 is positioned at cylinder spiral stirring rake 3 and coaxial with it, and cylinder spiral stirring rake 3 rotates around central row air film device 4 under the driving of magnetic force driving motor 20;
Airing system is made up of air feed bottle 7, reducing valve 8 and gas meter 9, and the gas in the air feed bottle 7 is air; By pilot-gas flow velocity 10-30ml/min and gaseous tension, realize that oxygen is to the still supply of nutrient solution in the oxygen supply membrane module 2, oxygen supply membrane module 2 is the poly tetrafluoroethylene pipe.
The liquid filling system is made up of deformable Liquid storage bag, peristaltic pump and TT﹠C system; Wherein, the deformable Liquid storage bag comprises acid solution bag 10, alkali lye bag 11, nutrient solution bag 12 and low temperature seed liquor bag 13, and peristaltic pump comprises acid solution conveying peristaltic pump 14, alkali lye conveying peristaltic pump 15, nutrient solution conveying peristaltic pump 16 and seed liquor conveying peristaltic pump 17; Acid solution carry liquid in the peristaltic pump 14 control acid solution bags 10 conveying, alkali lye carry liquid in the peristaltic pump 15 control alkali lye bags 11 conveying, nutrient solution is carried the conveying of nutritive medium in the peristaltic pump 16 control nutrient solution bags 12, and seed liquor is carried the conveying of liquid spawn in the peristaltic pump 17 control low temperature seed liquor bags 13; Working hour by controlling each peristaltic pump and flow are realized the accurate pulse filling of liquid, and the power of peristaltic pump is 6W, and flow is 0.1-7.5ml/min; The temperature parameter Measurement and Control System is made up of micro temperature sensor probe 19, heating jacket 5 and TT﹠C system, when temperature is lower than preset temp in the bio-reactor main body 1, heating jacket 5 follow procedures are started working, and realize accuracy control over temperature, and heating jacket 5 power are 15W; Minisize pH sensor probe 18, micro temperature sensor probe 19, miniature dissolved oxygen sensor probe 21 and micro pressure sensor probe 22 seal on the inwall that is installed in bio-reactor main body 1 upper and lower and with bio-reactor main body 1 bottom surface and are an angle of 90 degrees.
Stirring system is made up of magnetic force driving motor 20 and cylinder spiral stirring rake 3, wherein, as shown in Figure 3, cylinder spiral stirring rake 3 comprises cylinder spiral stirring rake base 301, magnetic sheet 302 and cylinder spiral stirrer paddle 303, its spiral number of turns is 2 circles, and drum surface is evenly distributed with sieve aperture 304.Utilize magnetic force driving motor 20 to drive 3 rotations of cylinder spiral stirring rake, the power of magnetic force driving motor 20 is 12W, maximum stirring velocity is 60rpm, and is used for the fragmentation that culturing micro-organisms, cell and tissue can not cause cell walls on the one hand, realizes the solid thorough mixing of gas-liquid; The rotation of cylinder spiral stirrer paddle 3 under the normal gravity of ground on the other hand is for the cultivation of biomass cells provides the simulated microgravity environment.
Exhaust system is made up of central row air film device 4, vacuum pump 25 and gas sampling bag 26.
Sampling system is made up of sampling peristaltic pump 23 and sample collection pond 24.
Working process:
Before carrying out the suspended biological cell cultures under microgravity and the simulated microgravity environment, need utilize alcohol immersion, uv irradiating, modes such as sterilized water are carried out cold sterilization to reactor assembly and are handled and clean.Carry out biomass cells then and cultivate operation.At first, nutrient solution carries peristaltic pump 16 that the aseptic culture fluid in the nutrient solution bag 12 is annotated in bio-reactor main body 1 with the speed of 7.5ml/min, carry 16 working hour of peristaltic pump 12min to make liquid charge into bio-reactor main body 1 by the control nutrient solution, the liquid that charges into accounts for 90% of bio-reactor main body 1 volume; Gas enters along in the oxygen supply membrane module 2 of bio-reactor main body 1 inwall coiling through air feed bottle 7, reducing valve 8 and gas meter 9, by pilot-gas flow velocity 10-30ml/min and gaseous tension, the still supply of realization oxygen in bio-reactor main body 1 improves oxygen utilization efficient; When bio-reactor main body 1 culture system is inoculated, 4 ℃ low temperature seed liquor bag 13 carries peristaltic pump 17 in pulsed filling in bio-reactor main body 1 under the control of supervisory system by seed liquor, working hour 80s by control seed liquor conveying peristaltic pump 17 realizes that the seed liquor filling rate is 10ml; Utilize power to rotate with the speed of 30rpm around central row air film device 4 for the cylinder spiral stirring rake 3 in the magnetic force driving motor 20 driving bio-reactor main bodys 1 of 12W, by rotatablely moving of cylinder spiral stirrer paddle 303 on the cylinder spiral stirring rake 3, realize the uniform distribution (under the normal gravity environment of ground, carry out biomass cells when cultivating the high-density culture that rotatablely moves to cell of cylinder spiral stirring rake 3 the simulated microgravity environment is provided) of gas-liquid-solid three-phase in the bio-reactor main body 1; Utilize temperature parameter Measurement and Control System control bio-reactor main body 1 inner suspension biomass cells to cultivate the stable of required microenvironment, when temperature is lower than preset temp 37 and spends in temperature sensor probe 19 detects bio-reactor main body 1, supervisory system is opened the switch of heating jacket 5 automatically, heating jacket 5 begins heating, when reaching preset temp, heating jacket 5 switches cut out automatically, realize the accurate monitoring of temperature (± 0.5 degree); Utilize the minisize pH sensor probe 18 that is provided with on the bio-reactor main body 1 upper and lower inwall, miniature dissolved oxygen sensor probe 21 and micro pressure sensor probe 22 are respectively to the pH value (2-12) of bio-reactor main body 1 inner suspension biomass cells fermentation culture system, oxygen dissolving value (20-150%) and force value (70-90KPa) carry out that real-time online detects and by predefined value, carry the working hour of peristaltic pump 16 and alkali lye conveying peristaltic pump 17 by the control acid solution, realization is to the accurate control of culture system pH value, enter the flow and the pressure of bio-reactor main body 1 by control oxygen, realize accurate monitoring the culture system oxygen dissolving value.
When the suspended biological cell cultures was carried out sampling analysis, sampling place peristaltic pump 23 was started working and is pumped into cell cultures liquid until being full of collecting tank in sample collection pond 24.
In the middle and later periods of suspended biological cell cultures, the oxygen that the consumption of cell growth metabolism is a large amount of, producing a large amount of refuse oxygen dissolving values this moment also decreases, under the control of supervisory system, the vacuum pump 25 of exhaust system is started working, to the pipeline that connects central row air film device 4 take out negative pressure (≤0.08MPa), realize the discharging rapidly and efficiently of biomass cells metabolic waste carbon dioxide, reduce it to the metabolic restraining effect of Growth of Biologic Cell.
Above-described specific descriptions; purpose, technical scheme and beneficial effect to invention further describe; institute is understood that; the above only is specific embodiments of the invention; and be not intended to limit the scope of the invention; within the spirit and principles in the present invention all, any modification of being made, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
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| CN102703313B (en) * | 2012-05-14 | 2013-11-13 | 上海交通大学 | Batch type bioreactor |
| CN102703313A (en) * | 2012-05-14 | 2012-10-03 | 上海交通大学 | Batch type bioreactor |
| CN104531766B (en) * | 2014-12-16 | 2018-10-12 | 中国科学院成都生物研究所 | A kind of method and device of stalk micro-aerobe fermentation production hydrogen |
| CN104531766A (en) * | 2014-12-16 | 2015-04-22 | 中国科学院成都生物研究所 | Method and device for producing hydrogen by microaerobic fermentation of straw |
| CN106683534A (en) * | 2016-11-11 | 2017-05-17 | 李树增 | A device that creates a microgravity environment |
| CN106683534B (en) * | 2016-11-11 | 2019-04-12 | 李树增 | A device that creates a microgravity environment |
| CN107151625A (en) * | 2017-06-09 | 2017-09-12 | 杭州泰林精密仪器有限公司 | A kind of liquid microbe trap setting |
| CN107151625B (en) * | 2017-06-09 | 2023-08-22 | 中国航天员科研训练中心 | A liquid microbial capture device |
| CN107739713A (en) * | 2017-10-13 | 2018-02-27 | 中国科学院上海技术物理研究所 | It is a kind of to change liquid system suitable for what spatial cell was cultivated automatically |
| CN108380148A (en) * | 2018-03-07 | 2018-08-10 | 中国工程物理研究院激光聚变研究中心 | A kind of drop revolution curing reaction system of simulated microgravity |
| CN108380148B (en) * | 2018-03-07 | 2019-12-13 | 中国工程物理研究院激光聚变研究中心 | droplet gyration solidification reaction system of simulation microgravity |
| CN110244612A (en) * | 2019-06-06 | 2019-09-17 | 西安交通大学 | A Binary Digital Communication System Based on Chemical Diffusion Mechanism |
| CN111084158A (en) * | 2019-12-26 | 2020-05-01 | 重庆大学 | Ecological circle culture method suitable for extraterrestrial space |
| CN111518693A (en) * | 2020-04-30 | 2020-08-11 | 王家文 | Rotary radial spreading axial multi-sheet adherent cell culture device |
| CN112675929A (en) * | 2020-11-23 | 2021-04-20 | 宁波大学 | All-in-one machine and method for providing simulated microgravity effect for organisms |
| CN112675929B (en) * | 2020-11-23 | 2024-05-24 | 宁波大学 | Integrated machine and method for providing simulated microgravity effect for organisms |
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