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WO2021043257A1 - 生物反应器系统及其应用 - Google Patents

生物反应器系统及其应用 Download PDF

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Publication number
WO2021043257A1
WO2021043257A1 PCT/CN2020/113460 CN2020113460W WO2021043257A1 WO 2021043257 A1 WO2021043257 A1 WO 2021043257A1 CN 2020113460 W CN2020113460 W CN 2020113460W WO 2021043257 A1 WO2021043257 A1 WO 2021043257A1
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container
bioreactor system
oscillator
eccentricity
culture
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PCT/CN2020/113460
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English (en)
French (fr)
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罗顺
胡福林
原世平
侯爵
宋金沛
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浙江金仪盛世生物工程有限公司
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Priority to EP20859828.4A priority Critical patent/EP4026894A4/en
Priority to JP2022515002A priority patent/JP7580451B2/ja
Priority to US17/640,450 priority patent/US20220333049A1/en
Priority to CN202080062546.3A priority patent/CN114341342A/zh
Publication of WO2021043257A1 publication Critical patent/WO2021043257A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/14—Bags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/28—Constructional details, e.g. recesses, hinges disposable or single use
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16—Vibrating; Shaking; Tilting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/42—Means for regulation, monitoring, measurement or control, e.g. flow regulation of agitation speed

Definitions

  • This application relates to a bioreactor system for cell culture, especially suitable for the culture of cell-free cells such as animal cells, and a method for cell culture using the bioreactor system.
  • the bioreactor system involved in this application can realize cell culture and proliferation with high cell survival rate.
  • Bioreactors as the main body is the hardware driving force that promotes the technological innovation and process improvement of large-scale animal cell culture, and is also the basis for improving the production efficiency, production scale and product quality of biological products.
  • Commercialized animal cell bioreactors at home and abroad include agitated bioreactors, hollow fiber bioreactors, airlift bioreactors and bag bioreactors.
  • the reactor is required to have high oxygen transfer efficiency, good mixing performance, and low shear effect. Therefore, as the key equipment for animal cell culture, the bioreactor mainly solves the common problem of making the reactor have low shear effect, good mass transfer and mixing effect according to the requirements of cell growth.
  • most of the existing bioreactors use bottom ventilation and mechanical stirring to realize the supply of oxygen and the mixing of the culture solution. The resulting strong shear force, high-concentration oxygen foam and the bursting of its bubbles cause great damage to animal cells. , And cause the cell culture density to be greatly reduced.
  • CFD computational fluid dynamics
  • CFD software such as FLUENT, CFD-ACE+ (CFDRC), Phoenics, CFX, Star-cd, etc.
  • CFD software for flow field simulation is well known to those skilled in the art.
  • this application relates to a bioreactor system for culturing cells, especially cell-free cell culture, which comprises:
  • the oscillator is set to make the container move eccentrically according to a certain eccentricity and speed
  • -Ventilation device set to pass oxygen-containing gas from the upper part of the container to the inside of the container
  • the oscillator is set to make the container move eccentrically, so that the ratio (S/V) of the total surface area S of the culture solution to the volume V of the culture solution in the steady state of motion (S/V) is above 5.65, and the turbulent kinetic energy is at 2.73E-03m 2 /s 2 or more, and the flow field shear rate is below 20.27/s, where the total liquid surface area is the sum of the contact area between the culture fluid and the inner wall of the container and the contact area between the culture fluid and the gas in the container.
  • the S/V value, turbulent kinetic energy, and flow field shear rate are obtained by CFD simulation.
  • CFD simulation can be realized by FLUENT software.
  • the S/V value of the culture solution in the steady state of motion is related to the shape of the container, the volume of the culture solution V, the rotation speed of the container, and the eccentricity R, and can be obtained by CFD simulation.
  • the shear rate generated in the container is related to the shape, speed and eccentricity of the container, and can be obtained by CFD simulation.
  • the turbulent kinetic energy in the container is related to the speed and eccentricity of the container, and can be obtained by CFD simulation.
  • the bioreactor system may also include a disposable culture bag arranged in the container for containing the culture solution, the disposable culture bag has a multifunctional cover plate connected to the top of the culture bag to seal the culture bag, And a plurality of connecting holes leading to the inside of the disposable culture bag are provided.
  • the disposable culture bag can be a flexible culture bag, or made of a hard material, and has a shape corresponding to the container when it is unfolded.
  • the disposable culture bag may be provided with a device for fixing it to the container.
  • the holes on the above-mentioned multifunctional cover plate have good airtightness, and detection electrodes, catheters, etc. can be connected when needed.
  • each connecting hole is sealed by a thread with good airtightness.
  • each detection electrode is connected through any connecting hole to monitor environmental parameters such as temperature, dissolved oxygen, and pH in the cell culture process in real time.
  • the catheter is connected through the connecting hole to perform various operations such as cell culture inoculation, medium addition, sampling, recovery, harvest, and ventilation, so as to further optimize the culture conditions and increase the cell culture density.
  • each connection hole of the multifunctional cover plate that can be applied to various disposable culture bags uses a uniform standard threaded interface, which has good air tightness and can be flexibly selected according to the needs of cell culture. The holes that are not needed during a specific culture process can be easily sealed.
  • D1 and D2 of the container in the bioreactor system are 400-4000mm, D3 is 40-400mm, H1 is 100-1500mm, and H2 is 40-1200mm.
  • the value of D1:D3 or D2:D3 is any value in the interval of about 5 to about 16, or any value in the interval of 5 to 16. In some embodiments, the value of D1:D3 or D2:D3 is about 5, about 7.37, about 7.89, about 8.89, about 10.27, or about 15.77. In some embodiments, the value of D1:D3 or D2:D3 is 5, 7.37, 7.89, 8.89, 10.27, or 15.77.
  • “About” in this context means ⁇ 20%, ⁇ 18%, ⁇ 15%, ⁇ 12%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ A range of 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, or ⁇ 0.5%, and the range includes the end points of the range and any value within the range.
  • the value of D1:H1 or D2:H1 is any value in the interval of about 2 to about 5, or any value in the interval of 2 to 5. In some embodiments, the value of D1:H1 or D2:H1 is about 2.68, about 3, about 3.55, about 3.74, about 3.66, or about 3.46. In some embodiments, the value of D1:H1 or D2:H1 is 2.68, 3, 3.55, 3.74, 3.66, or 3.46.
  • the value of D1:H2 or D2:H2 is any value in the interval of about 4 to about 5, or any value in the interval of 4 to 5. In some embodiments, the value of D1:H2 or D2:H2 is about 4.08, about 4.94, about 4.95, about 4.87, about 4.20, or about 4.51. In some embodiments, the value of D1:H2 or D2:H2 is 4.08, 4.94, 4.95, 4.87, 4.20, or 4.51.
  • the D1 and D2 of the vessel in the bioreactor system are about 400 to about 2997 mm, D3 is about 80 to about 190 mm, H1 is about 149 to about 867 mm, and H2 is about 98 to about 664 mm, containing about 5 -About 3000L of cell culture solution, the rotation speed of the shaker is about 55 to about 24 rpm, and the eccentricity is about 30 to about 65 mm.
  • the D1 and D2 of the container in the bioreactor system are 400-2997mm, D3 is 80-190mm, H1 is 149-867mm, H2 is 98-664mm, and contains 5-3000L cell culture medium, shaker The speed is 55-24rpm, and the eccentricity is 30-65mm.
  • the D1 and D2 of the container in the bioreactor system are about 400mm, D3 is about 80mm, H1 is about 149mm, H2 is about 98mm, contains about 5L of cell culture solution, and the rotation speed of the shaker is about 55rpm, The eccentricity is about 30mm.
  • the D1 and D2 of the container in the bioreactor system are 400mm, D3 is 80mm, H1 is 149mm, H2 is 98mm, contains about 5L of cell culture solution, the rotation speed of the shaker is 55rpm, and the eccentricity is 30mm.
  • the D1 and D2 of the container in the bioreactor system are about 840mm, D3 is about 114mm, H1 is about 280mm, and H2 is about 170mm, containing about 50L of cell culture solution, and the rotating speed of the oscillator is about 40rpm.
  • the eccentricity is about 40mm.
  • the D1 and D2 of the container in the bioreactor system are 840 mm, D3 is 114 mm, H1 is 280 mm, and H2 is 170 mm, containing 50 L of cell culture fluid, the rotating speed of the oscillator is 40 rpm, and the eccentricity is 40 mm.
  • the D1 and D2 of the container in the bioreactor system are about 840mm, D3 is about 114mm, H1 is about 280mm, and H2 is about 170mm, containing about 18L of cell culture solution, and the rotating speed of the oscillator is about 37- About 39rpm, the eccentricity is about 40mm.
  • the D1 and D2 of the container in the bioreactor system are 840mm, D3 is 114mm, H1 is 280mm, and H2 is 170mm, containing 18L cell culture medium, the rotating speed of the oscillator is 37-39rpm, and the eccentricity is 40mm .
  • the D1 and D2 of the container in the bioreactor system are about 840mm, D3 is about 114mm, H1 is about 280mm, and H2 is about 170mm, containing about 18L of cell culture solution, and the rotating speed of the oscillator is about 39rpm.
  • the eccentricity is about 40mm.
  • the D1 and D2 of the container in the bioreactor system are 840 mm, D3 is 114 mm, H1 is 280 mm, and H2 is 170 mm, containing 18 L of cell culture fluid, the rotating speed of the oscillator is 39 rpm, and the eccentricity is 40 mm.
  • the D1 and D2 of the container in the bioreactor system are about 1500mm, D3 is about 190mm, H1 is about 422mm, and H2 is about 303mm, containing about 18L of cell culture solution, and the rotating speed of the oscillator is about 37- About 39rpm, the eccentricity is about 40mm.
  • the D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 303mm, contains 18L cell culture fluid, the rotation speed of the oscillator is 37-39rpm, and the eccentricity is 40mm .
  • the D1 and D2 of the container in the bioreactor system are about 1500mm, D3 is about 190mm, H1 is about 422mm, H2 is about 203mm, containing about 200L of cell culture solution, and the rotating speed of the oscillator is about 30rpm.
  • the eccentricity is about 60mm.
  • the D1 and D2 of the container in the bioreactor system are 1500 mm, D3 is 190 mm, H1 is 422 mm, and H2 is 203 mm, containing 200 L of cell culture fluid, the rotating speed of the oscillator is 30 rpm, and the eccentricity is 60 mm.
  • the D1 and D2 of the container in the bioreactor system are about 1500mm, D3 is about 190mm, H1 is about 422mm, H2 is about 203mm, containing about 205L of cell culture solution, and the rotating speed of the oscillator is about 30rpm.
  • the eccentricity is about 60mm.
  • D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 203mm, contains 205L of cell culture solution, the rotation speed of the oscillator is 30rpm, and the eccentricity is 60mm.
  • the D1 and D2 of the container in the bioreactor system are about 1500mm, D3 is about 190mm, H1 is about 422mm, H2 is about 203mm, containing about 250L of cell culture solution, and the rotating speed of the oscillator is about 30rpm.
  • the eccentricity is about 60mm.
  • the D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 203mm, contains 250L of cell culture fluid, the rotation speed of the oscillator is 30rpm, and the eccentricity is 60mm.
  • the D1 and D2 of the container in the bioreactor system are about 1690mm, D3 is about 190mm, H1 is about 452mm, and H2 is about 347mm, containing about 500L of cell culture solution, and the rotating speed of the oscillator is about 28rpm.
  • the eccentricity is about 65mm.
  • the D1 and D2 of the container in the bioreactor system are 1690 mm, D3 is 190 mm, H1 is 452 mm, and H2 is 347 mm, containing 500 L of cell culture fluid, the rotating speed of the oscillator is 28 rpm, and the eccentricity is 65 mm.
  • the D1 and D2 of the container in the bioreactor system are about 1952mm, D3 is about 190mm, H1 is about 533mm, and H2 is about 465mm, containing about 1200L of cell culture solution, and the rotating speed of the oscillator is about 25rpm.
  • the eccentricity is about 65mm.
  • the D1 and D2 of the container in the bioreactor system are 1952mm, D3 is 190mm, H1 is 533mm, H2 is 465mm, contains 1200L cell culture solution, the rotation speed of the shaker is 25rpm, and the eccentricity is 65mm.
  • D1 and D2 of the container in the bioreactor system are about 1952mm, D3 is about 190mm, H1 is about 533mm, and H2 is about 465mm, containing about 250L of cell culture solution, and the rotating speed of the oscillator is about 30rpm.
  • the eccentricity is about 65mm.
  • the D1 and D2 of the container in the bioreactor system are 1952 mm, D3 is 190 mm, H1 is 533 mm, and H2 is 465 mm, containing 250 L of cell culture solution, the rotating speed of the shaker is 30 rpm, and the eccentricity is 65 mm.
  • the D1 and D2 of the container in the bioreactor system are about 1952mm, D3 is about 190mm, H1 is about 533mm, and H2 is about 465mm, containing about 330L of cell culture solution, and the rotating speed of the oscillator is about 30rpm.
  • the eccentricity is about 65mm.
  • the D1 and D2 of the container in the bioreactor system are 1952mm, D3 is 190mm, H1 is 533mm, H2 is 465mm, contains 330L cell culture solution, the rotation speed of the oscillator is 30rpm, and the eccentricity is 65mm.
  • a bioreactor with D1 and D2 of about 2997mm, D3 of about 190mm, H1 of about 867mm, and H2 of about 664mm, containing about 3000L of cell culture fluid for cell culture is used for cell culture, and the oscillator speed is set to about 24rpm , The eccentricity is set to about 65mm.
  • a bioreactor with D1 and D2 of 2997mm, D3 of 190mm, H1 of 867mm, and H2 of 664mm, containing 3000L of cell culture fluid is used for cell culture, the oscillator speed is set to 24rpm, and the eccentricity is set to 65mm .
  • D1 and D2 of the container in the bioreactor system are 400mm, D3 is 80mm, H1 is 149mm, H2 is 98mm, contains 5L of cell culture fluid, the speed of the shaker is 55rpm, the eccentricity is 30mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 50.48
  • the turbulent kinetic energy is 2.73E-03m 2 /s 2
  • the flow field shear rate is 10.18/ s.
  • the D1 and D2 of the container in the bioreactor system are 840mm, D3 is 114mm, H1 is 280mm, H2 is 170mm, contains 50L of cell culture fluid, the rotational speed of the oscillator is 40rpm, the eccentricity is 40mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 26.61
  • the turbulent kinetic energy is 5.29E-03m 2 /s 2
  • the flow field shear rate is 7.02 s.
  • the D1 and D2 of the container in the bioreactor system are 840mm, D3 is 114mm, H1 is 280mm, H2 is 170mm, contains 18L cell culture fluid, the rotation speed of the oscillator is 37-39rpm, and the eccentricity is 40mm ,
  • the steady-state S/V value of culture fluid motion obtained by CFD simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the rotational speed of the oscillator in the bioreactor system is 37 rpm
  • the steady-state S/V value of motion obtained by CFD simulation is 43.34
  • the turbulent kinetic energy is 4.71E-03m 2 /s 2
  • the flow field shear The cut rate is 6.8056/s.
  • the D1 and D2 of the container in the bioreactor system are 840mm, D3 is 114mm, H1 is 280mm, H2 is 170mm, contains 18L of cell culture fluid, the rotational speed of the oscillator is 39rpm, the eccentricity is 40mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 47.89
  • the turbulent kinetic energy is 5.35E-03m 2 /s 2
  • the flow field shear rate is 7.1149/ s.
  • the D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 303mm, and contains 200L of cell culture fluid, the rotation speed of the oscillator is 30rpm, the eccentricity is 60mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 18.46
  • the turbulent kinetic energy is 9.91E-03m 2 /s 2
  • the flow field shear rate is 5.9707/ s.
  • the D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 303mm, contains 205L cell culture fluid, the rotational speed of the oscillator is 30rpm, the eccentricity is 60mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 18.24
  • the turbulent kinetic energy is 9.89E-03m 2 /s 2
  • the flow field shear rate is 5.7476/ s.
  • the D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 303mm, contains 250L of cell culture fluid, the rotational speed of the oscillator is 30rpm, the eccentricity is 60mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 15.98
  • the turbulent kinetic energy is 9.30E-03m 2 /s 2
  • the flow field shear rate is 5.4623/ s.
  • the D1 and D2 of the container in the bioreactor system are 1690mm, D3 is 190mm, H1 is 452mm, H2 is 347mm, contains 500L of cell culture fluid, the rotational speed of the oscillator is 28rpm, the eccentricity is 65mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 10.61
  • the turbulent kinetic energy is 7.59E-03m 2 /s 2
  • the flow field shear rate is 4.66/ s.
  • the D1 and D2 of the container in the bioreactor system are 1952mm, D3 is 190mm, H1 is 533mm, H2 is 465mm, contains 1200L cell culture fluid, the rotational speed of the shaker is 25rpm, the eccentricity is 65mm, CFD
  • the simulated steady-state S/V value of the culture fluid movement is 6.60, the turbulent kinetic energy is 9.30E-03m 2 /s 2 , and the flow field shear rate is 4.42/s.
  • the D1 and D2 of the container in the bioreactor system are 1952mm, D3 is 190mm, H1 is 533mm, H2 is 465mm, contains 250L of cell culture fluid, the rotational speed of the oscillator is 30rpm, the eccentricity is 65mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 20.96
  • the turbulent kinetic energy is 1.46E-02m 2 /s 2
  • the flow field shear rate is 6.0922/ s.
  • the D1 and D2 of the container in the bioreactor system are 1952mm, D3 is 190mm, H1 is 533mm, H2 is 465mm, contains 330L cell culture fluid, the rotation speed of the oscillator is 30rpm, the eccentricity is 65mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 18.09
  • the turbulent kinetic energy is 1.52E-02m 2 /s 2
  • the flow field shear rate is 6.3173/ s.
  • the D1 and D2 of the container in the bioreactor system are 2997mm, D3 is 190mm, H1 is 867mm, H2 is 664mm, contains 3000L of cell culture fluid, the rotational speed of the oscillator is 24rpm, the eccentricity is 65mm, CFD
  • the steady-state S/V value of the culture fluid motion obtained by the simulation is X
  • the turbulent kinetic energy is X m 2 /s 2
  • the flow field shear rate is X/s.
  • the steady-state S/V value of the culture fluid movement obtained by the CFD simulation of the bioreactor system is 5.65
  • the turbulent kinetic energy is 2.48E-02m 2 /s 2
  • the flow field shear rate is 3.98/ s.
  • the present application relates to a method for culturing cells, particularly cell-wall-free cells, using a bioreactor system, the bioreactor system comprising:
  • the oscillator can be set to make the container move eccentrically according to a certain eccentricity and rotation speed
  • -Ventilation device set to pass oxygen-containing gas from the upper part of the container to the inside of the container
  • Cell culture methods include:
  • the ratio of the total surface area S of the culture fluid to the volume V of the culture fluid V is calculated by CFD simulation when the eccentric motion reaches a steady state, S/V is above 5.65, and the turbulent kinetic energy is 2.73 E-03
  • the total surface area of the culture fluid is the contact area of the culture fluid and the reactor wall and the culture fluid and gas The sum of the contact area; and
  • the rotational speed of the oscillator decreases as the volume of the container increases.
  • the eccentricity of the oscillator increases as the volume of the container increases.
  • a bioreactor system with a maximum working volume of 5L can set the oscillator speed in the range of 45-70rpm, and the eccentricity can be set to about 30mm;
  • a bioreactor system with a maximum working volume of 50L can set the oscillator speed at Within the range of 40-60rpm, the eccentricity can be set to about 40mm;
  • the bioreactor system with a maximum working volume of 500L can set the oscillator speed in the range of 25-30rpm, and the eccentricity can be set to about 65mm;
  • the maximum working volume is 1200L
  • the bioreactor system can set the oscillator speed in the range of 20-30rpm, and the eccentricity can be set to about 65mm;
  • the bioreactor system with a maximum working volume of 3000L can set the oscillator speed in the range of 24-26rpm, eccentric The distance can be set to about 65mm.
  • the bioreactor system may also include a disposable culture bag arranged in the container for containing the culture solution, the disposable culture bag has a multifunctional cover plate connected to the top of the culture bag to seal the culture bag, And a plurality of connecting holes leading to the inside of the disposable culture bag are provided.
  • the disposable culture bag can be a flexible culture bag, or made of a hard material, and has a shape corresponding to the container when it is unfolded.
  • the disposable culture bag may be provided with a device for fixing it to the container.
  • the holes on the above-mentioned multifunctional cover plate have good airtightness, and detection electrodes, catheters, etc. can be connected when needed.
  • each connecting hole is sealed by a thread with good airtightness.
  • each detection electrode is connected through any connecting hole to monitor environmental parameters such as temperature, dissolved oxygen, and pH in the cell culture process in real time.
  • the catheter is connected through the connecting hole to perform various operations such as cell culture inoculation, medium addition, sampling, recovery, harvest, and ventilation, so as to further optimize the culture conditions and increase the cell culture density.
  • each connection hole of the multifunctional cover plate that can be applied to various disposable culture bags uses a uniform standard threaded interface, which has good air tightness and can be flexibly selected according to the needs of cell culture. The holes that are not needed during a specific culture process can be easily sealed.
  • a bioreactor with D1 and D2 being 400mm, D3 being 80mm, H1 being 149mm, H2 being 98mm, and containing 5L cell culture solution is used for cell culture, the oscillator speed is set to 55rpm, and the eccentricity is set to 30mm.
  • a bioreactor with D1 and D2 of 840mm, D3 of 114mm, H1 of 280mm, and H2 of 170mm, and containing 50L of cell culture solution is used for cell culture, the oscillator speed is set to 40rpm, and the eccentricity is set to 40mm.
  • the D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 303mm, contains 18L of cell culture fluid, the rotation speed of the oscillator is 37-39rpm, and the eccentricity is 40mm .
  • the D1 and D2 of the container in the bioreactor system are 1500 mm, D3 is 190 mm, H1 is 422 mm, and H2 is 203 mm, containing 200 L of cell culture fluid, the rotating speed of the oscillator is 30 rpm, and the eccentricity is 60 mm.
  • D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 203mm, contains 205L of cell culture fluid, the rotation speed of the oscillator is 30rpm, and the eccentricity is 60mm.
  • D1 and D2 of the container in the bioreactor system are 1500mm, D3 is 190mm, H1 is 422mm, H2 is 203mm, contains 250L of cell culture solution, the rotational speed of the oscillator is 30rpm, and the eccentricity is 60mm.
  • a bioreactor with D1 and D2 of 1690mm, D3 of 190mm, H1 of 452mm, H2 of 347mm, and 500L of cell culture medium is used for cell culture, the oscillator speed is set to 28rpm, and the eccentricity is set to 65mm.
  • a bioreactor with D1 and D2 of 1952mm, D3 of 190mm, H1 of 533mm, and H2 of 465mm, containing 1200L of cell culture fluid, is used for cell culture, the oscillator speed is set to 25rpm, and the eccentricity is set to 65mm .
  • the D1 and D2 of the container in the bioreactor system are 1952 mm, D3 is 190 mm, H1 is 533 mm, and H2 is 465 mm, containing 250 L of cell culture solution, the rotating speed of the oscillator is 30 rpm, and the eccentricity is 65 mm.
  • the D1 and D2 of the container in the bioreactor system are 1952mm, D3 is 190mm, H1 is 533mm, H2 is 465mm, contains 330L cell culture solution, the rotation speed of the oscillator is 30rpm, and the eccentricity is 65mm.
  • a bioreactor with D1 and D2 of 2997mm, D3 of 190mm, H1 of 867mm, and H2 of 664mm, containing 3000L of cell culture fluid, is used for cell culture, the oscillator speed is set to 24rpm, and the eccentricity is set to 65mm .
  • the culture solution produces lower shear force when performing eccentric movement, and causes less damage to cells. It is especially suitable for the cultivation of cells without cell walls, such as animal cells; and when eccentric movement
  • the steady-state S/V value and turbulent kinetic energy of the culture fluid are controlled within a certain range, the efficiency of dissolved oxygen and oxygen expansion is high, and the uneven oxygen distribution will not cause the oxygen toxicity when the oxygen concentration is too high or the oxygen concentration is too low.
  • Cell growth and proliferation are restricted, which can better support high-density cell growth.
  • the cell growth and proliferation efficiency is high, and the cell survival rate is maintained at 90.0%, 95.0%, or even 99.0% or more.
  • the disposable culture bag is used in conjunction, cross-contamination can be avoided, the inter-batch processing cycle can be shortened, no complicated pipelines and other auxiliary facilities, no cleaning, disinfection, and verification are required, which greatly improves work efficiency.
  • Fig. 1 shows a schematic diagram of the structure of the container in the bioreactor system of the present application.
  • Figure 2 shows a schematic diagram of the movement trajectory of any point on the container during eccentric movement.
  • Figure 3 shows a simulation diagram of the free liquid surface change of the CUR300L when the working volume is 300L and the rotating speed is 28rpm.
  • Fig. 4 shows a simulation diagram of the free liquid surface change of CUR300L when the working volume is 300L and the rotating speed is 30rpm.
  • Figure 5 shows a simulation diagram of the free liquid surface change of CUR300L when the working volume is 175L and the rotating speed is 28rpm.
  • Fig. 6 shows a simulation diagram of the free liquid surface change of CUR300L when the working volume is 175L and the rotating speed is 30rpm.
  • the ideal situation is that the reactor causes little physical damage to the cells, and the reactor can provide enough oxygen in time to support cell growth and proliferation. .
  • the purpose of this application is to provide a bioreactor system with the above-mentioned effects.
  • the inventor of the present application simulates bioreactors under various scales of working conditions through computational fluid dynamics (CFD) simulation, focusing on five types of reactors with different volumes, including CUR2.5L, CUR50L, CUR300L, CUR1200L, and CUR3000L.
  • CFD computational fluid dynamics
  • the bioreactor system in this application mainly includes a container and an oscillator.
  • the container is composed of a hollow cylinder on the upper part and a hollow truncated cone on the lower part.
  • the container moves in a constant velocity circle around an eccentricity of equal radius.
  • the “eccentric movement” of the oscillator means that the motor in the oscillator drives a pulley to rotate at the same time through a belt.
  • the pulley and three cranks are connected together to drive the moving platform to move. Every point on the moving platform is eccentric with the same radius. Constant velocity circular motion, and the motion trajectory of any point on the moving platform is the same, and the speed and acceleration of each point at any time are the same.
  • the container of the bioreactor system is fixed on the moving platform of the oscillator, and the movement of the container is translational on the horizontal surface. You only need to measure the movement trajectory of any point on the container to determine the movement trajectory of other parts, as shown in Figure 2. .
  • Dissolved oxygen means that the top surface of the culture liquid is in contact with oxygen-containing gas to bring the gas into the culture liquid, and when the oscillator drives the container of the bioreactor system to move eccentrically, the culture liquid produces slow motion and continuously washes the container. On the surface, nano-scale soluble micro-bubbles are produced, which drag the gas into the culture solution.
  • Oxygen expansion refers to the transfer of oxygen-containing gas brought into the liquid to the inside of the culture liquid through the turbulent diffusion of the culture liquid.
  • total liquid surface area of the culture solution refers to the sum of the contact area between the culture solution and the reactor wall and the contact area between the culture solution and the gas, abbreviated as S, and its ratio to the volume of the culture solution is called the S/V ratio.
  • S the contact area between the culture fluid and the gas
  • A the contact area between the culture fluid and the gas
  • the top surface of the culture medium is in direct contact with oxygen, which will dissolve part of the oxygen.
  • the gas renewal speed of the liquid film adhered to the inner wall of the reactor vessel may be different from that of the liquid main body, and the contact between the liquid and the liquid film so far may affect the main gas composition.
  • the culture solution When the bioreactor system is in eccentric motion, the culture solution will deviate from the central axis of the reactor vessel to varying degrees. The higher the speed, the greater the centrifugal force on the liquid, the greater the degree of deviation, and the thinner the liquid is squeezed. The more conspicuous the contact interface is. Therefore, during eccentric movement, the contact area between the culture fluid and the wall of the bioreactor and the contact area between the top surface of the culture fluid and the gas will increase to varying degrees, so that the total surface area of the culture fluid and the S/ V ratio increases.
  • the shear force received by cells in the bioreactor is divided into three categories: the flow field shear force generated by the relative movement inside the culture fluid, the size of the flow field shear force is proportional to the velocity gradient, and the kinematic viscosity of the culture fluid; The shear force generated when bubbles rise in the culture medium and the surface bursts; the shear force generated by the collision between cells and the inner wall of the reactor, and the shear force generated by the collision between cells.
  • the bioreactor system of the present application is selected, since the surface oxygen transfer method is adopted, there are very few bubbles generated without the bubble distributor, so the shear force damage caused by the bubbles can be ignored.
  • the bioreactor system of the present application is a hollow cylinder and a hollow truncated cone structure. When it moves eccentrically, the reactor wall that the culture fluid in the bioreactor and the cells in it touches is very smooth, resulting in collision shear The shear force is also smaller.
  • the shear force in the bioreactor of the present application is mainly the flow field shear force.
  • the destruction of cells by the shear force of the flow field is closely related to the smallest vortex size generated by the turbulence of the fluid.
  • the culture fluid rotates against the wall in the tank to produce turbulent vortices. Small vortex, along with the transfer of energy in this process, the mechanical energy of the culture fluid turns into thermal energy, causing its temperature to rise. In the process of the gradual change of the vortex, when the size of the vortex is close to the size of the cell, the vortex transfers energy to the cell, causing the cell to rupture.
  • the average shear force or average shear rate increases with the increase in speed, and the volume of the culture fluid has a small effect on it; while the larger bioreactor produces The shear force may be smaller.
  • the bioreactor system of the present application is selected, regardless of the shape and specification of the container, the volume of the culture medium, the rotation speed of the oscillator, and the eccentricity, the average shear rate produced is very low, and the underwater shear that kills Chinese hamster cancer cells (CHO)
  • the cut rate threshold (391.41s - 1 ) differs by an order of magnitude. Therefore, when setting up the bioreactor system, the shear force parameter can be omitted.
  • the dissolved oxygen and oxygen diffusion efficiency of the culture fluid are related to the surface area, turbulent kinetic energy, and turbulence intensity of the culture fluid.
  • efficiency of dissolved oxygen and oxygen diffusion is high enough to support cell growth and proliferation, there is no need to use a gas with a particularly high oxygen concentration.
  • gases with a particularly high oxygen concentration, especially pure oxygen are likely to cause oxygen toxicity.
  • the dissolved oxygen efficiency of the culture solution is mainly related to the surface area of the culture medium that contacts the oxygen-containing gas.
  • the container of the bioreactor system When the container of the bioreactor system is in eccentric movement, the top surface of the culture solution and the contact surface with the container wall may contact the oxygen-containing gas. Therefore, compared to the total surface area or S/V ratio in static state, the total surface area or S/V ratio in steady state motion has more reference value.
  • the CFD simulation it is found that different container specifications, culture medium volume, and oscillator speed will affect the S/V ratio at steady state of motion. Generally speaking, the larger the S/V ratio, the higher the dissolved oxygen efficiency.
  • the container constructed in the present application under the condition of a low S/V ratio, the effect of stable cell growth and proliferation and a high cell survival rate can still be achieved.
  • the oxygen diffusion efficiency of the culture fluid is mainly related to the turbulent kinetic energy and turbulence intensity of the culture fluid.
  • the movement of the fluid clusters can be expressed as the superposition of the average velocity that flows with the body and the pulsating velocity that fluctuates randomly.
  • Random fluctuations of the micelles cause turbulent flow.
  • the input power increases with the increase in speed, which increases the proportion of fluid clusters that carry higher energy, and the number of micro vortices that can produce effective collision and transfer energy increases, that is, the increase in the average value of turbulent kinetic energy. From the CFD simulation point of view, under the condition of constant working volume, the higher the speed, the higher the turbulent kinetic energy, so the material transfer and mixing effect in the container is better.
  • random fluctuations of the micelles cause turbulent diffusion. The velocity of the micelles fluctuates violently, and the stronger the interaction, the more significant the energy transfer and dispersion.
  • Turbulent kinetic energy and turbulence intensity are mainly related to the average fluid velocity and turbulent pulsation velocity, and have a certain positive correlation with the oscillator speed.
  • the appropriate culture fluid volume, oscillator speed, and eccentricity can be selected for a certain bioreactor to achieve the effect of small shearing effect, high dissolved oxygen and high oxygen diffusion efficiency.
  • the eccentricity needs to be selected according to the volume of the bioreactor. Generally speaking, the eccentricity of the oscillator increases as the volume of the container increases.
  • a bioreactor system with a maximum working volume of 5L can set the oscillator speed in the range of 45-70rpm, and the eccentricity can be set to about 30mm;
  • a bioreactor system with a maximum working volume of 50L can set the oscillator speed at In the range of 45-60rpm, the eccentricity can be set to about 40mm;
  • the bioreactor system with a maximum working volume of 500L can set the oscillator speed in the range of 25-30rpm, and the eccentricity can be set to about 65mm;
  • the maximum working volume is 1200L
  • the bioreactor system can set the oscillator speed in the range of 20-30rpm, and the eccentricity can be set to about 65mm.
  • the CFD software-FLUENT software simulates the characteristics of the flow field in each reactor with different working volumes at different speeds.
  • the focus is on the changes in the relative position of the culture solution and the area of the gas-liquid interface when the oscillators move eccentrically along the vertical axis of five types of reactor vessels with different volumes, including CUR2.5L, CUR50L, CUR300L, CUR1200L, and CUR3000L.
  • the total surface area of the liquid, the turbulent kinetic energy of the liquid, the turbulent energy dissipation rate, the turbulence intensity and the change of the shear force, the specific inventive steps, methods and results are as follows:
  • the container of the bioreactor system is a rigid body with no wrinkles on the surface and no deformation during movement;
  • the physical properties of the culture medium in the container are the same as those of water.
  • This experiment uses an unstructured tetrahedral grid, and the grid information is shown in Table 3.
  • the VOF model 2-phase flow is used to track the interface between gas and liquid, and the RNG k- ⁇ model is selected for the turbulence model.
  • the trajectory of the bioreactor is eccentric.
  • the eccentric distances of the CUR2.5L, CUR50L, CUR300L, CUR1200L and CUR3000L reactors are 30mm, 40mm, 60mm, 65mm and 65mm, respectively.
  • compile UDF to define the movement of the reactor.
  • the factors that affect the movement of the tank are the angular velocity ⁇ and the eccentricity R.
  • the motion equation of a point on the tank body is:
  • V x the linear velocity of the point in the x direction
  • V y the linear velocity of the point in the y direction
  • t time; ——The initial phase angle of this point (in order to simplify the calculation, this point is taken as the origin coordinate [0,0,0] when the reactor is modeled).
  • This simulation does not consider the change of the liquid in the initial stage, and only examines the quasi-steady state process when the shape of the culture solution in the reactor reaches a relatively stable state.
  • Table 4 The total liquid surface area, gas-liquid interface area and the ratio of the working volume to the working volume in each working volume reactor change with the speed
  • Turbulent kinetic energy k is used to characterize the energy contained in the pulsation process of fluid clusters, and the formula is as follows:
  • I turbulent intensity
  • the magnitude is equal to the ratio of the root mean square of the turbulent pulsating velocity v'to the average velocity vave.
  • the turbulent energy dissipation rate ⁇ is used to measure the energy loss rate caused by the collision of micelles and viscous dissipation, and the formula is as follows:
  • ⁇ , ⁇ , ⁇ 1 -fluid density, viscosity, turbulent viscosity ⁇ , ⁇ , ⁇ 1 -fluid density, viscosity, turbulent viscosity.
  • the movement of the fluid clusters can be expressed as the superposition of the average velocity that flows with the body and the pulsating velocity that fluctuates randomly. Random fluctuations of the micelles cause turbulent flow. It can be seen from Table 5 that when the working volume is constant, the higher the rotation speed, the higher the turbulent kinetic energy, so the material transfer and mixing effect in the reactor is better, and ⁇ also increases to varying degrees.
  • the input power increases with the increase of the rotation speed, which increases the proportion of fluid micelles carrying higher energy, and the number of micro vortices capable of generating effective collision transfer energy increases, that is, the increase in the average value of k.
  • the area of the liquid sweeping the inner wall of the container per unit time increases with the increase of the speed, the chance of gas-liquid contact and exchange increases, and the wall adhesion and the internal viscous friction of the liquid also increase.
  • the increase in the proportion of high-energy micelles causes more micelle collisions and energy transfer, and the decay rate of the transmission frequency of k increases.
  • the turbulent energy dissipation rate ⁇ increases as the rotation speed increases.
  • the above parameters are constant under the experimental conditions, and the turbulent energy dissipation rate ⁇ can be used to characterize the strength of the gas-liquid transfer process.
  • the gas-liquid transfer becomes stronger with the increase of ⁇ .
  • turbulent diffusion plays a leading role.
  • the movement of the fluid clusters can be expressed as the superposition of the average velocity that flows with the body and the pulsating velocity that fluctuates randomly. Random fluctuations of the micelles cause turbulent diffusion.
  • the velocity between the micelles fluctuates violently, and the interaction is stronger.
  • the transfer and dispersion of energy are also more pronounced. Therefore, the greater the intensity of turbulence, the better the effect of mass transfer and mixing in the liquid phase.
  • turbulence intensity I can be used to characterize the intensity of fluid micelle velocity fluctuations and interactions.
  • the velocity of the micelles fluctuates violently, and the stronger the interaction, the more significant the energy transfer and dispersion. Therefore, the greater the intensity of turbulence, the better the effect of mass transfer and mixing in the liquid phase.
  • the formula of turbulence intensity I is:
  • v' is the root mean square of the turbulent pulsating velocity
  • v avg is the average flow velocity of the fluid.
  • the movement of the fluid clusters can be expressed as the superposition of the average velocity that flows with the body and the pulsating velocity that fluctuates randomly. Random fluctuations of micelles cause turbulent flow.
  • the shear force received by cells in the bioreactor is divided into three categories: the flow field shear force generated by the relative movement inside the culture fluid, the size of the flow field shear force is proportional to the velocity gradient, and the kinematic viscosity of the culture fluid; The shear force generated when bubbles rise in the culture medium and the surface bursts; the shear force generated by the collision between cells and the inner wall of the reactor, and the shear force generated by the collision between cells.
  • the destruction of cells by the shear force of the flow field is closely related to the smallest vortex size generated by the turbulence of the fluid.
  • the culture fluid rotates against the wall in the container to produce turbulent vortices.
  • the large vortex gradually develops into a small vortex, and the small vortex develops into a larger vortex.
  • Small vortex along with the transfer of energy in this process, the mechanical energy of the culture fluid turns into thermal energy, causing its temperature to rise.
  • the vortex transfers energy to the cell, causing the cell to rupture.
  • the bioreactor of the present application due to the use of surface oxygen transfer, there are very few bubbles generated without a bubble distributor, so the shear force damage caused by the bubbles can be neglected. Because the inner wall surface of the tank in the bioreactor is relatively smooth, the collision shear force is also relatively small.
  • the shear force in the rapid flow bioreactor is mainly the shear force of the flow field. Therefore, detecting the average value of the internal shear rate of the reactor through simulation has important reference value for the design of the bioreactor.
  • the average shear rate is the volume integration of the shear rate of the entire flow field area, and then the average value.
  • the calculation formula is,
  • the area A of the gas-liquid interface does not increase linearly with the increase of the rotation speed.
  • the degree of influence of the increase of the rotation speed on the total surface area S of the liquid changes with the working volume;
  • the rated turbulence parameters of the liquid phase are both affected by the speed and the working volume. According to the relationship between k L and ⁇ , ⁇ can be used to approximate the strength of the gas-liquid transfer process;
  • the average shear rate increases with the increase of speed. Although the average shear rate becomes smaller, the damage to the cells will be smaller, but the shear rate and the mixing effect are mutually restricted, and the smaller the shear rate means the worsening of the mixing effect.
  • the average shear rate in the reactor is much lower than the shear rate that damages the cells.
  • Testing instrument Mettler pH electrode.
  • Measurement content After adding NaOH reagent to the bioreactor, measure the uniform mixing time with a pH electrode to determine the time it takes for the materials added to the container to mix uniformly during the shaking process of the reactor. Use the length of time to simulate the actual application process of the bioreactor, and the added materials can actually affect the ability of culturing cells.
  • This experiment simulates the conditions of normal cell culture in the reactor, and sets three rotational speeds of 50rpm, 55rpm, and 60rpm, and three different working volumes, including a minimum working volume of 2L, an optimal working volume of 3.5L, and a maximum working volume of 5L.
  • the pH sensor is placed at the top interface of the reactor, and the mixing efficiency is reflected by measuring the pH of the liquid.
  • the steps are basically the same as 3.1, except that the PBS buffer added into the reactor is 3.5L and the NaOH solution with a concentration of 1mol/L is injected into the reactor each time is 5mL.
  • the steps are basically the same as 3.1, except that the PBS buffer added to the reactor is 5L and the NaOH solution with a concentration of 1mol/L is injected into the reactor each time is 10mL.
  • the speed is set to 38rpm, 39rpm, 40rpm three speed values and the minimum working volume is 15L, the most suitable work
  • 30L and 50L maximum working volume the eccentricity is set to 40mm
  • the temperature is controlled at 25-27°C.
  • the test method is basically the same as above.
  • the working volume is 15L
  • the working volume is 30L
  • the working volume is 50L
  • the speed is set to 25rpm, 27rpm, 29rpm three speed values and the minimum working volume is 100L, the most suitable work
  • 300L and 500L maximum working volume the eccentricity is set to 65mm, and the temperature is controlled at 25-27°C.
  • the test method is basically the same as above.
  • the working volume is 100L
  • the working volume is 300L
  • the working volume is 500L
  • the test method is basically the same as above.
  • the working volume is 300L
  • the working volume is 800L
  • the working volume is 1200L
  • the mixing efficiency of this device can fully meet the mixing and mass transfer requirements in the large-scale cell culture process.
  • the CUR5L, CUR50L, CUR500L and CUR1200L bioreactors used in Example 2 were used for the cultivation of MDCK cells.
  • the cell seeding density is 1.5 ⁇ 10 6 cells/ml
  • the growth medium is CD MDCK SFM (DP304, Jianshun Biological)
  • the culture temperature is 37°C
  • the dissolved oxygen value DO% is 35-65.
  • the CUR5L reactor has a working volume of 5L, a rotating speed of 55rpm, an eccentricity of 30mm, and the introduction rates of oxygen and air at 100mL/min and 200mL/min, respectively.
  • the CUR50L reactor is set with a working volume of 50L, a rotating speed of 40rpm, an eccentricity of 40mm, and the introduction rates of oxygen and air at 500mL/min and 500mL/min, respectively.
  • the CUR500L reactor is set with a working volume of 500L, a rotating speed of 28rpm, an eccentricity of 65mm, and the introduction rates of oxygen and air at 900mL/min and 1000mL/min, respectively.
  • the CUR1200L reactor has a working volume of 1200L, a rotation speed of 25rpm, an eccentricity of 65mm, and the introduction rates of oxygen and air at 1500mL/min and 1500mL/min, respectively.
  • the concentration of the supplemented glucose solution was 200g/L.
  • the pH value of the bioreactor is set within the range of 7.0 to 7.4. When the system detects that the pH value is lower than 7.0, the system will automatically add NaHCO 3 with a concentration of 7.5%.
  • the reactor speed is 30rpm
  • the eccentricity is 60mm
  • the initial volume of the culture medium is 200L, 205L, 250L
  • the temperature is controlled at 37°C
  • the oxygen and air introduction rates are set to 800mL/min and 800mL/min, respectively.
  • the initial medium is the special medium for CHO cells of Gansu Jianshun Biological Co., Ltd.
  • the supplemented medium is CD Feed002 from Gansu Jianshun Biological Co., Ltd.
  • the seeding density of CHO cells is about 1.0 ⁇ 10 6 /mL. Add 5%, 2%, 5%, and 2% of the current working volume of CD Feed002 on the 4th, 6th, 8th, and 10th days, and the glucose is lower than 2.0g When /L, add glucose solution to 8.0g/L, and set the pH value in the range of 7.0-7.2. When the pH value is lower than 7.0, the system will automatically add NaHCO 3 with a concentration of 7.5%. The culture lasted for 14 days, and the cell growth density, viability, osmotic pressure, oxygen partial pressure, carbon dioxide partial pressure, and protein expression were observed every day. The cell density and viability during the culture process are specifically described in Table 13.
  • the culture volume at the time of pot harvest reached 247L, 256L, and 312L, and the final protein expression reached 1.133g/L, 1.594g/L, 1.311g/L.
  • the cell density reached 15.49 ⁇ 10 6 cells/mL when the cell density was the highest, and the cells were always maintained at a high viability rate and maintained in the period of high protein expression. The result was ideal.
  • CHO cell culture was carried out by batch feeding.
  • the speed of the reactor is set to 30rpm
  • the working volume is 250L and 330L
  • the eccentricity is set to 65mm
  • the temperature is controlled at 37°C.
  • the introduction rates of oxygen and air were set at 1500 mL/min and 1500 mL/min, respectively.
  • the initial medium is the special medium for CHO cells of Gansu Jianshun Biological Co., Ltd.
  • the supplemented medium is CD Feed002 from Gansu Jianshun Biological Co., Ltd.
  • the inoculation density is about 1 ⁇ 10 6 /mL.
  • the pH value is set in the range of 7.0 to 7.2.
  • the system will automatically add NaHCO 3 with a concentration of 7.5%.
  • the cell culture lasted for 14 days, and the cell growth density, viability, osmotic pressure, oxygen partial pressure, carbon dioxide partial pressure, and protein expression were observed every day.
  • the cell density and viability during the culture process are specifically described in Table 14.
  • the culture volume at harvest time reached 314L and 417L, and the final protein expression reached 1.481g/L and 1.308g/L.
  • the cell density reached 18.51 ⁇ 10 6 /mL when the cell density was the highest, and the cells were always maintained at a high viability rate and maintained in the period of high protein expression. The result was ideal.
  • the rotational speed of the bioreactor is 37 rpm (days 1-8) to 39 rpm (days 9-20), the working volume is 18L, the eccentricity is 40mm, and the temperature is controlled at 37°C.
  • the introduction rates of oxygen and air were set to 500 mL/min and 500 mL/min, respectively.
  • the initial medium is the special medium for CHO cells of Gansu Jianshun Biological Co., Ltd.
  • the supplemented medium is CD Feed from Ganshun Biological Co., Ltd.
  • the inoculation density is about 1 ⁇ 10 6 /mL.
  • the glucose concentration in the culture system is maintained not less than 3.0g/L, and the pH value is set at Within the range of 7.0 ⁇ 7.2, when the pH value is lower than 7.0, the system will automatically add NaHCO 3 with a concentration of 7.5%.
  • the cell culture lasted for 20 days, and the density, viability, diameter, osmotic pressure, residual amount of lactic acid, and protein expression of the cell growth were observed every day.
  • the cell density and viability during the culture process are specifically described in Table 15.
  • the culture volume when harvested in the pot was 14L, and the final protein expression reached 27g. This result exceeded expectations.
  • the cell density reached 90.1 ⁇ 10 6 /mL at the highest time, and the cell viability was always maintained at a high rate (above 97%).

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Abstract

本申请涉及一种用于培养细胞，特别是无细胞壁细胞，的生物反应器系统，其包含：容器，该容器含有直径D1且高度H1的中空圆柱，以及上直径D2、下直径D3且高度H2的中空圆台，其中该中空圆柱与中空圆台的顶面连接，D1＝D2；振荡器，设置成使容器按照一定的偏心距和转速做偏心运动；通气设备，设置成从容器上部向容器内通入含氧气体，和填充在容器中的培养液，其顶面暴露于含氧气体，其中，振荡器设置成使容器处于偏心运动，使运动稳态时的培养液液体总表面积与体积的比值(S/V)处于5.65以上，湍动能处于2.73E-03m 2/s 2以上，且流场剪切率处于20.27/s以下，其中液体总表面积为培养液与反应器壁面接触面积和顶面与气体接触面积之和。

Description

生物反应器系统及其应用 技术领域
本申请涉及一种用于细胞培养，特别是适用于无细胞壁细胞如动物细胞的培养的生物反应器系统，以及使用该生物反应器系统进行细胞培养的方法。本申请中涉及的生物反应器系统能够实现高细胞存活率的细胞培养和增殖。
背景技术
哺乳动物细胞表达的产品已经在生物制药产业中占有绝对优势，成为生物制药的主流和趋势。而以生物反应器为主体的生产线及细胞培养工艺则是推动动物细胞大规模培养技术革新和工艺改进的硬件推动力，也是提升生物制品的生产效率、生产规模、产品质量的基础。国内外现已商品化的动物细胞生物反应器有搅拌式生物反应器、中空纤维式生物反应器、气升式生物反应器以及袋式生物反应器。
一方面，由于动物细胞没有细胞壁，对培养环境如剪切力、渗透压等比较敏感，要求反应器的传氧效率高、混合性能好、剪切效应低。因此，作为动物细胞培养关键设备的生物反应器，主要解决的共性问题是要按细胞生长的要求，使反应器具备低的剪切效应、良好的传质和混合效果。但现有的生物反应器大多采用底部通气和机械搅拌的方式实现氧气的供给和培养液混合，由此产生的强剪切力、高浓度氧气泡沫以及其气泡破裂，给动物细胞造成较大损伤，并导致细胞培养密度的大大降低。
另一方面，生物反应器的设备开发和生物反应过程工艺研究，需要先在小型设备中进行摸索和试验，再逐渐放大到较大规模的设备中进行工艺放大试验和商业化生产。然而实践中往往是在小型反应器中进行科学实验所取得的数据、工艺和规律，并不能完全在大型反应器中取得同样或更好的效果。因此，生物反应器尺度设计无法通过简单的等比例放大来满足动物细胞的工业化规模培养需要。目前，依据对已有生物反应器的操作经验所建立起的一些规律而进行放大的方法，多半是定性的，仅有一些简单的、粗糙的定量概念，放大比例通常较小，并且不够精确。为此，在未获得完整的理论体系解析前，对生物反应器进行尺度放大设计，还需根据经验和实用的原则进行反应器的设计和放大。
发明内容
鉴于上述问题，本申请的发明人采用计算流体力学(CFD)来仿真模拟工作状态下的生物反应器，探索可以满足不同规模培养所需的生物反应器理论模型，并在实际细胞培养中进行验证。利用该理论模型，可以得到满足不同规模下动物细胞培养和增殖的生物反应器系统，并使动物细胞存活率保持在85.0％以上。CFD指使用数值方法在计算机 中对流体力学的控制方程进行求解，从而可预测流场的流动。目前有多种商业CFD软件问世，比如FLUENT、CFD-ACE+(CFDRC)、Phoenics、CFX、Star-cd等。使用CFD软件进行流场模拟是本领域技术人员所熟知的。
一方面，本申请涉及一种用于培养细胞，特别是无细胞壁细胞培养的生物反应器系统，其包含：
-容器，该容器含有直径D1且高度H1的中空圆柱，以及上直径D2、下直径D3且高度H2的中空圆台，其中该中空圆柱与中空圆台的顶面连接，D1＝D2；
-振荡器，设置成使容器按照一定的偏心距和转速做偏心运动；
-通气装置，设置成从容器上部向容器内通入含氧气体，和
-填充在容器中的培养液，其顶面暴露于含氧气体，
其中，振荡器设置成使容器处于偏心运动，使运动稳态时的培养液液体总表面积S与培养液体积V的比值(S/V)处于5.65以上，湍动能处于2.73E-03m 2/s 2以上，且流场剪切率处于20.27/s以下，其中液体总表面积为培养液与容器内壁面接触面积和培养液与容器内气体接触面积之和。在一些实施方式中，S/V值、湍动能和流场剪切率经CFD模拟得出。
CFD模拟可以经FLUENT软件实现。运动稳态时的培养液S/V值与容器形状、培养液体积V、容器转速以及偏心距R相关，可通过CFD模拟得到。容器内产生的剪切率与容器形状、转速和偏心距相关，可通过CFD模拟得到。容器内的湍动能与容器转速和偏心距相关，可通过CFD模拟得到。
生物反应器系统还可以包含设置在容器中的用于容纳培养液的一次性培养袋，该一次性培养袋具有多功能盖板，该多功能盖板连接到培养袋的顶部以密封培养袋，并且设置有多个通向一次性培养袋内部的接孔。该一次性培养袋可以是柔性培养袋，或由硬质材料制成，且在展开时具有与容器相对应的形状。该一次性培养袋可以设置有将其固定于容器的装置。上述多功能盖板上的接孔具有良好的密封性，可以在需要时接入探测电极、导管等。在一些实施方式中，各接孔通过气密性好的螺纹进行密封。在一些实施方式中，通过任意接孔接入各探测电极，对细胞培养过程的温度、溶氧、pH等环境参数进行实时监测。在一些实施方式中，通过接孔接入导管，进行细胞培养接种以及培养液添加、取样、回收、丰收、换气等各种操作，以便进一步的优化培养条件，提高细胞培养密度。同时，可应用于各种一次性培养袋的多功能盖板的各接孔均使用统一标准的螺纹接口，气密性好，可根据细胞培养的需要进行灵活的取舍操作。特定培养过程中不需要的接孔可以容易地密封。
在一些实施方式中，生物反应器系统中容器的D1和D2为400-4000mm，D3为40-400mm,H1为100-1500mm,H2为40-1200mm。
在一些实施方式中，D1:D3或D2:D3的值为约5至约16区间内的任一数值，或为5至16区间内的任一数值。在一些实施方式中，D1:D3或D2:D3的值为约5、约7.37、约7.89、约8.89、约10.27或约15.77。在一些实施方式中，D1:D3或D2:D3的值 为5、7.37、7.89、8.89、10.27或15.77。“约”在本文中的意思为所述数值的±20％、±18％、±15％、±12％、±10％、±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％、±1％或±0.5％的范围，所述范围包扩所述范围的端点及所述范围内的任一数值。
在一些实施方式中，D1:H1或D2:H1的值为约2至约5区间内的任一数值，或为2至5区间内的任一数值。在一些实施方式中，D1:H1或D2:H1的值为约2.68、约3、约3.55、约3.74、约3.66或约3.46。在一些实施方式中，D1:H1或D2:H1的值为2.68、3、3.55、3.74、3.66或3.46。
在一些实施方式中，D1:H2或D2:H2的值为约4至约5区间内的任一数值，或4至5区间内的任一数值。在一些实施方式中，D1:H2或D2:H2的值为约4.08、约4.94、约4.95、约4.87、约4.20或约4.51。在一些实施方式中，D1:H2或D2:H2的值为4.08、4.94、4.95、4.87、4.20或4.51。
在一些实施方式中，生物反应器系统中容器的D1和D2为约400-约2997mm，D3为约80-约190mm，H1为约149-约867mm，H2为约98-约664mm，含有约5-约3000L的细胞培养液，振荡器的转速为约55-约24rpm，偏心距为约30-约65mm。在一些实施方式中，生物反应器系统中容器的D1和D2为400-2997mm，D3为80-190mm，H1为149-867mm，H2为98-664mm，含有5-3000L的细胞培养液，振荡器的转速为55-24rpm，偏心距为30-65mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约400mm，D3为约80mm，H1为约149mm，H2为约98mm，含有约5L细胞培养液，振荡器的转速为约55rpm，偏心距为约30mm。在一些实施方式中，生物反应器系统中容器的D1和D2为400mm，D3为80mm，H1为149mm，H2为98mm，含有约5L细胞培养液，振荡器的转速为55rpm，偏心距为30mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约840mm，D3为约114mm，H1为约280mm，H2为约170mm，含有约50L细胞培养液，振荡器的转速为约40rpm，偏心距为约40mm。在一些实施方式中，生物反应器系统中容器的D1和D2为840mm，D3为114mm，H1为280mm，H2为170mm，含有50L细胞培养液，振荡器的转速为40rpm，偏心距为40mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约840mm，D3为约114mm，H1为约280mm，H2为约170mm，含有约18L细胞培养液，振荡器的转速为约37–约39rpm，偏心距为约40mm。在一些实施方式中，生物反应器系统中容器的D1和D2为840mm，D3为114mm，H1为280mm，H2为170mm，含有18L细胞培养液，振荡器的转速为37–39rpm，偏心距为40mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约840mm，D3为约114mm，H1为约280mm，H2为约170mm，含有约18L细胞培养液，振荡器的转速为约39rpm，偏心距为约40mm。在一个实施方式中，生物反应器系统中容器的D1和D2 为840mm，D3为114mm，H1为280mm，H2为170mm，含有18L细胞培养液，振荡器的转速为39rpm，偏心距为40mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1500mm，D3为约190mm，H1为约422mm，H2为约303mm，含有约18L细胞培养液，振荡器的转速为约37-约39rpm，偏心距为约40mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1为422mm，H2为303mm，含有18L细胞培养液，振荡器的转速为37-39rpm，偏心距为40mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1500mm，D3为约190mm，H1为约422mm，H2为约203mm，含有约200L细胞培养液，振荡器的转速为约30rpm，偏心距为约60mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1为422mm，H2为203mm，含有200L细胞培养液，振荡器的转速为30rpm，偏心距为60mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1500mm，D3为约190mm，H1为约422mm，H2为约203mm，含有约205L细胞培养液，振荡器的转速为约30rpm，偏心距为约60mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1为422mm，H2为203mm，含有205L细胞培养液，振荡器的转速为30rpm，偏心距为60mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1500mm，D3为约190mm，H1为约422mm，H2为约203mm，含有约250L细胞培养液，振荡器的转速为约30rpm，偏心距为约60mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是203mm，含有250L细胞培养液，振荡器的转速为30rpm，偏心距为60mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1690mm，D3为约190mm，H1为约452mm，H2为约347mm，含有约500L细胞培养液，振荡器的转速为约28rpm，偏心距为约65mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1690mm，D3为190mm，H1为452mm，H2为347mm，含有500L细胞培养液，振荡器的转速为28rpm，偏心距为65mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1952mm，D3为约190mm，H1为约533mm，H2为约465mm，含有约1200L细胞培养液，振荡器的转速为约25rpm，偏心距为约65mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有1200L细胞培养液，振荡器的转速为25rpm，偏心距为65mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1952mm，D3为约190mm，H1为约533mm，H2为约465mm，含有约250L细胞培养液，振荡器的转速为约30rpm，偏心距为约65mm。在一些实施方式中，生物反应器系统中容器的D1 和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有250L细胞培养液，振荡器的转速为30rpm，偏心距为65mm。
在一些实施方式中，生物反应器系统中容器的D1和D2为约1952mm，D3为约190mm，H1为约533mm，H2为约465mm，含有约330L细胞培养液，振荡器的转速为约30rpm，偏心距为约65mm。在一些实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有330L细胞培养液，振荡器的转速为30rpm，偏心距为65mm。
在一些实施方式中，使用D1和D2为约2997mm、D3为约190mm、H1为约867mm、H2为约664mm、含有约3000L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为约24rpm，偏心距设置为约65mm。在一些实施方式中，使用D1和D2为2997mm、D3为190mm、H1为867mm、H2为664mm、含有3000L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为24rpm，偏心距设置为65mm。
在一个实施方式中，生物反应器系统中容器的D1和D2为400mm，D3为80mm，H1为149mm，H2为98mm，含有5L细胞培养液，振荡器的转速为55rpm，偏心距为30mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为50.48，湍动能为2.73E-03m 2/s 2，流场剪切率为10.18/s。
在一个实施方式中，生物反应器系统中容器的D1和D2为840mm，D3为114mm，H1为280mm，H2为170mm，含有50L细胞培养液，振荡器的转速为40rpm，偏心距为40mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为26.61，湍动能为5.29E-03m 2/s 2，流场剪切率为7.02/s。
在一个实施方式中，生物反应器系统中容器的D1和D2为840mm，D3为114mm，H1为280mm，H2为170mm，含有18L细胞培养液，振荡器的转速为37-39rpm，偏心距为40mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统中振荡器的转速为37rpm，CFD模拟得出的运动稳态S/V值为43.34，湍动能为4.71E-03m 2/s 2，流场剪切率为6.8056/s。在一个实施方式中，生物反应器系统中容器的D1和D2为840mm，D3为114mm，H1为280mm，H2为170mm，含有18L细胞培养液，振荡器的转速为39rpm，偏心距为40mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为47.89，湍动能为5.35E-03m 2/s 2，流场剪切率为7.1149/s。
在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是303mm，含有200L细胞培养液，振荡器的转速为30rpm，偏心距为60mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运 动稳态S/V值为18.46，湍动能为9.91E-03m 2/s 2，流场剪切率为5.9707/s。在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是303mm，含有205L细胞培养液，振荡器的转速为30rpm，偏心距为60mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为18.24，湍动能为9.89E-03m 2/s 2，流场剪切率为5.7476/s。在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是303mm，含有250L细胞培养液，振荡器的转速为30rpm，偏心距为60mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为15.98，湍动能为9.30E-03m 2/s 2，流场剪切率为5.4623/s。
在一个实施方式中，生物反应器系统中容器的D1和D2为1690mm，D3为190mm，H1是452mm，H2是347mm，含有500L细胞培养液，振荡器的转速为28rpm，偏心距为65mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为10.61，湍动能为7.59E-03m 2/s 2，流场剪切率为4.66/s。
在一个实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有1200L细胞培养液，振荡器的转速为25rpm，偏心距为65mm，CFD模拟得出的培养液运动稳态S/V值为6.60，湍动能为9.30E-03m 2/s 2，流场剪切率为4.42/s。在一个实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有250L细胞培养液，振荡器的转速为30rpm，偏心距为65mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为20.96，湍动能为1.46E-02m 2/s 2，流场剪切率为6.0922/s。在一个实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有330L细胞培养液，振荡器的转速为30rpm，偏心距为65mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为18.09，湍动能为1.52E-02m 2/s 2，流场剪切率为6.3173/s。
在一个实施方式中，生物反应器系统中容器的D1和D2为2997mm，D3为190mm，H1为867mm，H2为664mm，含有3000L细胞培养液，振荡器的转速为24rpm，偏心距为65mm，CFD模拟得出的培养液运动稳态S/V值为X，湍动能为X m 2/s 2，流场剪切率为X/s。在一个实施方式中，所述生物反应器系统经CFD模拟得出的培养液运动稳态S/V值为5.65，湍动能为2.48E-02m 2/s 2，流场剪切率为3.98/s。
另一方面，本申请涉及一种使用生物反应器系统培养细胞，特别是无细胞壁细胞，的方法，该生物反应器系统包含：
-容器，该容器含有直径D1且高度H1的中空圆柱，以及上直径D2、下直径D3且高度H2的中空圆台，其中该中空圆柱与中空圆台的顶面连接，D1＝D2；
-振荡器，能够设置成使容器按照一定的偏心距和转速做偏心运动；
-通气装置，设置成从容器上部向容器内通入含氧气体，和
-填充在容器中的培养液，其顶面暴露于含氧气体，
细胞培养方法包括：
-根据生物反应器系统的形状和培养液体积，经CFD模拟算出培养液在偏心运动达稳态时培养液液体总表面积S与培养液体积V的比值S/V在5.65以上、湍动能在2.73E-03m 2/s 2以上、且流场剪切率在20.27/秒以下所需的振荡器转速和偏心距，其中培养液液体总表面积为培养液与反应器壁面接触面积和培养液与气体接触面积之和；以及
-在生物反应器系统中加入培养液和接种细胞，并根据计算出的振荡器转速和偏心距来设定振荡器，进行细胞培养。
可以使用FLUENT软件进行CFD模拟。
根据生物反应器系统的工作总功率，振荡器的转速随容器体积增大而减小。根据生物反应器系统的工作效率，振荡器的偏心距随容器体积增大而增大。
例如，最大工作体积为5L的生物反应器系统可以将振荡器转速设置在45-70rpm范围内，偏心距可以设置为约30mm；最大工作体积为50L的生物反应器系统可以将振荡器转速设置在40-60rpm范围内，偏心距可以设置为约40mm；最大工作体积为500L的生物反应器系统可以将振荡器转速设置在25-30rpm范围内，偏心距可以设置为约65mm；最大工作体积为1200L的生物反应器系统可以将振荡器转速设置在20-30rpm范围内，偏心距可以设置为约65mm；最大工作体积为3000L的生物反应器系统可以将振荡器转速设置在24-26rpm范围内，偏心距可以设置为约65mm。
生物反应器系统还可以包含设置在容器中的用于容纳培养液的一次性培养袋，该一次性培养袋具有多功能盖板，该多功能盖板连接到培养袋的顶部以密封培养袋，并且设置有多个通向一次性培养袋内部的接孔。该一次性培养袋可以是柔性培养袋，或由硬质材料制成，且在展开时具有与容器相对应的形状。该一次性培养袋可以设置有将其固定于容器的装置。上述多功能盖板上的接孔具有良好的密封性，可以在需要时接入探测电极、导管等。在一些实施方式中，各接孔通过气密性好的螺纹进行密封。在一些实施方式中，通过任意接孔接入各探测电极，对细胞培养过程的温度、溶氧、pH等环境参数进行实时监测。在一些实施方式中，通过接孔接入导管，进行细胞培养接种以及培养液添加、取样、回收、丰收、换气等各种操作，以便进一步的优化培养条件，提高细胞培养密度。同时，可应用于各种一次性培养袋的多功能盖板的各接孔均使用统一标准的螺纹接口，气密性好，可根据细胞培养的需要进行灵活的取舍操作。特定培养过程中不需要的接孔可以容易地密封。
在一个实施方式中，使用D1和D2为400mm、D3为80mm、H1为149mm、H2为98mm、且含有5L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为55rpm，偏心距设置为30mm。
在一个实施方式中，使用D1和D2为840mm、D3为114mm、H1为280mm、H2为170mm、且含有50L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为40rpm，偏心距设置为40mm。
在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1为422mm，H2为303mm，含有18L细胞培养液，振荡器的转速为37-39rpm，偏心距为40mm。
在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是203mm，含有200L细胞培养液，振荡器的转速为30rpm，偏心距为60mm。在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是203mm，含有205L细胞培养液，振荡器的转速为30rpm，偏心距为60mm。在一个实施方式中，生物反应器系统中容器的D1和D2为1500mm，D3为190mm，H1是422mm，H2是203mm，含有250L细胞培养液，振荡器的转速为30rpm，偏心距为60mm。
在一个实施方式中，使用D1和D2为1690mm、D3为190mm、H1为452mm、H2为347mm、且含有500L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为28rpm，偏心距设置为65mm。
在一个实施方式中，使用D1和D2为1952mm、D3为190mm、H1为533mm、H2为465mm、含有1200L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为25rpm，偏心距设置为65mm。
在一个实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有250L细胞培养液，振荡器的转速为30rpm，偏心距为65mm。
在一个实施方式中，生物反应器系统中容器的D1和D2为1952mm，D3为190mm，H1为533mm，H2为465mm，含有330L细胞培养液，振荡器的转速为30rpm，偏心距为65mm。
在一个实施方式中，使用D1和D2为2997mm、D3为190mm、H1为867mm、H2为664mm、含有3000L细胞培养液的生物反应器进行细胞培养，振荡器转速设置为24rpm，偏心距设置为65mm。
在本申请的生物反应器系统中，培养液做偏心运动时产生较低的剪切力，对细胞的损伤较小，特别适用于无细胞壁的细胞，例如动物细胞的培养；而且当偏心运动时培养液的稳态S/V值和湍动能控制在一定范围内时，溶氧和扩氧效率较高，不会因为氧气分布不均造成氧浓度过高时的氧毒性或者氧浓度过低时细胞生长和增殖受限，可以较好地支持细胞高密度生长。当使用本申请中的方法培养细胞时，不管是较小规模，还是大规 模的培养，细胞生长和增殖效率均较高，且细胞成活率保持在90.0％、95.0％甚至99.0％以上。特别当配合使用一次性培养袋时，可避免交叉污染、缩短批间处理周期，无需复杂管道等附属设施、无需清洗、消毒、验证，极大地提高了工作效率。
附图说明
图1示出本申请生物反应器系统中容器的结构示意图。
图2示出偏心运动时容器上任一点的运动轨迹示意图。
图3示出CUR300L在工作体积300L、转速28rpm时转动一周的自由液面变化模拟图。
图4示出CUR300L在工作体积300L、转速30rpm时转动一周的自由液面变化模拟图。
图5示出CUR300L在工作体积175L、转速28rpm时转动一周的自由液面变化模拟图。
图6示出CUR300L在工作体积175L且转速30rpm时转动一周的自由液面变化模拟图。
具体实施方式
当进行细胞，特别是无细胞壁细胞，例如动物细胞的培养时，最理想的状况是反应器对细胞造成的物理损伤很小，同时反应器能够及时地提供足够的氧气来支持细胞的生长和增殖。这需要生物反应器系统产生较小的剪切力，且提供较高的溶氧和扩氧效率。而本申请的目的就是提供一种有如上所述效果的生物反应器系统。
而在生物反应器的设备开发和生物反应过程工艺研究中，往往需要先在小型设备中进行摸索和试验，再逐渐放大到较大规模的设备中进行工艺放大试验和商业化生产。然而实践中往往是在小型反应器中进行科学实验所取得的数据、工艺和规律，并不能完全在大型反应器中取得同样或更好的效果，生物反应器尺度设计无法通过简单的等比例放大来满足动物细胞的工业化规模培养需要。
因此，本申请的发明人通过计算流体力学(CFD)仿真模拟各种规模的工作状态下的生物反应器，重点考察了CUR2.5L、CUR50L、CUR300L、CUR1200L、CUR3000L等5种型号不同体积反应器在振荡器沿着垂直轴线作偏心运动的工况下，培养液的相对位置变化、气液交界面面积、液体总表面积、液体湍动能、湍动能耗散率、湍流强度和剪切力变化等。当综合选择其中的一些参数组合并将其应用于实际细胞培养时，发现不管是采用5L还是1200L的生物反应器系统，均可以实现细胞的稳定增长，且细胞成活率在90.0％、95.0％甚至99％以上。
本申请中的生物反应器系统主要包含容器和振荡器。容器由上部的中空圆柱加下部的中空圆台构成，容器围绕着相等半径的偏心距作等速圆周运动。通过将偏心运动稳态 时培养液液体总表面积与体积的比值S/V、湍动能以及流场剪切率控制在一定范围内，可以实现细胞的稳定增长，且将细胞成活率保持在高水平。
振荡器进行“偏心运动”，是指振荡器中的电机通过皮带带动一个皮带轮同时转动，皮带轮和三个曲柄连接在一起，带动动平台运动，动平台上每一点都绕着相等半径的偏心做等速圆周运动，且动平台上任何一点的运动轨迹相同，在任一时刻每个点的速度和加速度都相同。生物反应器系统的容器固定在振荡器的动平台上，容器的运动是在水平面上的平动，只需测容器上任一点的运动轨迹，即可以确定其他部分的运动轨迹，如图2所示。
“溶氧”是指培养液顶面与含氧气体接触，将气体带入培养液中，以及当振荡器带动生物反应器系统的容器作偏心运动时，使培养液产生缓慢运动，不断冲刷容器表面，制造具有纳米级可溶微气泡，拖带气体进入培养液中。“扩氧”是指通过培养液的湍流扩散等将带入液体的含氧气体传递到培养液内部。
培养液的“液体总表面积”是指培养液与反应器壁面接触面积和培养液与气体接触面积之和，简称为S，其与培养液体积之比称为S/V比。在本文中，培养液与气体接触面积简称为A。培养液的顶面与氧气直接接触，会溶解一部分氧。同时，反应器容器内壁黏附的液膜的气体更新速度可能异于液相主体，旋转至此的液体与液膜接触可能影响主体气体成分。当生物反应器系统处于偏心运动时，培养液会不同程度地偏离反应器容器的中心轴，转速越高，液体受到的离心力越大，偏离程度越明显，液体被挤压得越薄，与空气接触的界面凹陷得越明显。从而，处于偏心运动时，培养液与生物反应器壁面接触的面积、以及培养液顶面与气体接触面积均会有不同程度的增加，从而使得运动稳态时培养液的液体总表面积以及S/V比增大。
在无细胞壁细胞，如动物细胞的培养中，由于细胞壁的缺失，细胞对流场内的力学环境比较敏感，外力对细胞造成的机械损伤是动物细胞生物反应器设计和使用过程中一个不可忽略的因素。在生物反应器中细胞受到的剪切力分为三大类：培养液内部的相对运动产生的流场剪切力，流场剪切力的大小和速度梯度、培养液的运动粘度成正比；气泡在培养液中上升及表面破裂时产生的剪切力；细胞与反应器内壁的碰撞，以及细胞间的相互碰撞所产生的剪切力。
当选择本申请的生物反应器系统时，由于采用表面传氧方式，没有气泡分布器因而产生的气泡极少，因此气泡产生的剪切力伤害可以忽略不计。而且，本申请的生物反应器系统为中空圆柱加中空圆台结构，当其做偏心运动时，生物反应器中培养液以及其中的细胞所接触到的反应器壁十分地平滑，因而产生的碰撞剪切力也较小。
因此，本申请生物反应器内剪切力主要是流场剪切力。流场剪切力对细胞的破坏跟流体湍动所产生的最小涡流尺寸密切相关，培养液在罐体内贴着壁面转动，产生湍动漩涡，大漩涡逐渐发展成小漩涡，小漩涡发展成更小的漩涡，这个过程中伴随着能量的传递，培养液机械能变为热能，使其温度升高。在漩涡逐渐变化的过程中，当漩涡的尺寸与细胞的尺寸接近的时候，漩涡把能量传递给细胞，从而导致细胞的破裂。在CFD模 拟中，发现在同一生物反应器内，平均剪切力或平均剪切率随转速增大而增大，培养液体积对其影响较小；而体积较大的生物反应器中产生的剪切力可能会更小。当选用本申请的生物反应器系统时，不论容器形状规格、培养液体积、振荡器转速和偏心距如何，产生的平均剪切率均很低，和杀伤中华仓鼠癌细胞(CHO)的水中剪切率阈值(391.41s - 1)相差一个数量级。因此，在设置生物反应器系统时，可以不用特别考虑剪切力这一参数。
培养液的溶氧和扩氧效率与培养液的表面积、湍动能、湍流强度等有关。当溶氧和扩氧效率较高、足以支持细胞的生长和增殖时，就无需使用氧浓度特别高的气体。根据以往经验，氧浓度特别高的气体，尤其是纯氧，容易造成氧毒性。
培养液的溶氧效率主要和培养基接触含氧气体的表面积有关。当生物反应器系统的容器处于偏心运动时，培养液的顶面以及与容器壁的接触面都可能接触含氧气体。因此，相比静态时的总表面积或S/V比，运动稳态时的总表面积或S/V比更具有参考价值。在CFD模拟中，发现不同容器规格、培养液体积和振荡器转速等均会影响运动稳态时的S/V比。通常而言，S/V比越大，溶氧效率会越高。在使用本申请构造的容器时，在S/V比值较低的情况下，仍能达到细胞稳定生长和增殖且细胞成活率较高的效果。
培养液的扩氧效率主要和培养液的湍动能、湍流强度等有关。
流体微团的运动可以表示为随主体流动的平均速度与随机波动的脉动速度的叠加。微团的随机波动造成了湍流流动。输入功率随转速升高而增加，使得携带较高能量地流体微团比例增大，能够产生有效碰撞传递能量的微观漩涡数量增加，即表现为湍动能平均值的增大。从CFD模拟来看，在工作体积不变的情况下，转速越高，湍动能越高，因此容器内物质传递和混合效果越好。此外，微团的随机波动造成了湍流扩散。微团间的速度波动剧烈，相互作用越强烈，能量的传递和分散作用也越显著。因此，湍流强度越大，液相内物质传递与混合的效果也越好。随着转速增加，湍流强度呈增大趋势，转速提高使得输入流体内的能量增加，造成了流体微团的动能增加，脉动速度加快，湍流强度也相应增加。湍动能、湍流强度主要与流体平均速度以及湍流脉动速度相关，且和振荡器转速呈一定的正相关。在考虑培养液的扩氧效率时，可以只考虑其中之一。
通过CFD模拟，可以针对一定的生物反应器选择合适的培养液体积、振荡器转速和偏心距，以达到剪切效应小、溶氧和扩氧效率高的效果。
由于生物反应器的自重随体积增大而增大，在考虑生物反应器系统的工作总功率的前提下，可以针对大反应器选择较小的转速。在另一方面，从生物反应器的工作效率方面考虑，需要根据生物反应器的体积来选择偏心距。通常而言，振荡器的偏心距随容器体积增大而增大。
例如，最大工作体积为5L的生物反应器系统可以将振荡器转速设置在45-70rpm范围内，偏心距可以设置为约30mm；最大工作体积为50L的生物反应器系统可以将振荡器转速设置在45-60rpm范围内，偏心距可以设置为约40mm；最大工作体积为500L的生物反应器系统可以将振荡器转速设置在25-30rpm范围内，偏心距可以设置为约 65mm；最大工作体积为1200L的生物反应器系统可以将振荡器转速设置在20-30rpm范围内，偏心距可以设置为约65mm。
在下文中，将通过具体的实施例来进一步阐述本申请的内容，但是本申请并不限于以下实施例。
实施例1 生物反应器的计算机模拟仿真实验
通过CFD软件——FLUENT软件模拟各总不同工作体积反应器在不同转速下容器内流场的特性。重点考察了CUR2.5L、CUR50L、CUR300L、CUR1200L、CUR3000L等5种型号不同体积反应器容器在振荡器沿着垂直轴线作偏心运动的工况下，培养液的相对位置变化、气液交界面面积、液体总表面积、液体湍动能、湍动能耗散率、湍流强度和剪切力变化，具体发明步骤、方法和结果如下：
一、生物反应器系统的仿真模型的建立
1.提出假设
为了实验能够顺利进行，更好地控制变量，在建立模型前对实验对象及初始条件进行了简化，假设以下几点：
1.1生物反应器系统的容器为刚体，表面无褶皱且在运动中不产生形变；
1.2工作环境为标准大气压、常温；
1.3容器内培养液的物理特性与水一致。
2.建立模型
2.1几何参数
生物反应器系统中容器的结构示意图如图1所示，几何参数列于表1中。
表1 各生物反应器系统中容器的几何参数
Figure PCTCN2020113460-appb-000001
2.2转速、工作体积及对应的初始液面高度
根据各规格的最佳转速范围及工作体积，确定了各规格反应器的3种转速和3种工作体积(其中CUR3000L确定了满载工况下的2种转速)，共计38组数据，具体数值如表2所示。
表2 各规格体积反应器对应的工况示意图
Figure PCTCN2020113460-appb-000002
2.3网格划分
本次实验采用的是非结构化四面体网格，网格信息如表3所示。
表3 各规格反应器非结构化四面体网格信息
Figure PCTCN2020113460-appb-000003
2.4流场初始条件的设置
采用VOF模型2相流来追踪气体和液体的交界面，湍流模型选择的是RNG k-ε模型。生物反应器的运动轨迹是偏心运动，CUR2.5L、CUR50L、CUR300L、CUR1200L和CUR3000L反应器运动的偏心距分别为30mm、40mm、60mm、65mm和65mm。在FLUENT软件中利用smoothing和layering动网格技术，编译UDF以定义反应器的运动，影响罐体运动的因素是角速度ω和偏心距R。罐体上某点的运动方程为：
Figure PCTCN2020113460-appb-000004
Figure PCTCN2020113460-appb-000005
其中，
V x——该点在x方向上的线速度；V y——该点在y方向上的线速度；
t——时间；
Figure PCTCN2020113460-appb-000006
——该点的初始相位角(为了简化计算，该点取为反应器建模时的原点坐标[0,0,0])。
以CUR300L在转速为25rpm的情况下为例，UDF文件具体内容如下：
Figure PCTCN2020113460-appb-000007
二、实验结果
1.计算机模拟工作状态下，液体在容器中相对位置和形态变化
本模拟不考虑初始阶段液体的变化情况，只考察培养液在反应器内形状达到相对稳定时的准稳态过程。
从CUR300L达到准稳态时反应器转动一周的自由液面变化模拟图(图3、图4、图5、图6)来看，液体均不同程度地偏离了反应器容器的中心轴。相同体积下，转速越高，液体受到的离心力越大，偏离程度越明显；转速越高，液体被挤压得越薄，与气体接触的界面凹陷得越明显。300L和175L工作体积具有相同的变化趋势。同时，通过观察转动过程中反应器内的自由液面还可以发现，在达到准稳态时，反应器内自由液面的形态变化过程不再发生明显的改变。
2.计算机模拟工作状态下，液体总表面积、气液交界面积及其与工作体积的比值
由两相传质的双膜理论假设，实际过程中，反应器容器内壁黏附的液膜的气体更新速度可能异于液相主体，旋转至此的液体与液膜接触可能影响主体气体成分。因此,将液体与反应器容器内壁面接触面积(W)与气液界面面积(A)之和表述为液体总表面积(S，S＝W+A)。
表4 各工作体积反应器中液体总表面积、气液交界面积及其与工作体积比值随转速变化情况
Figure PCTCN2020113460-appb-000008
Figure PCTCN2020113460-appb-000009
从表4可以看出，转速提高对S的影响程度随工作体积的不同而改变。通过对比各型号反应器不同工作体积反应器的液体总表面积S和气液交界面积A的变化趋势，发现两者的变化趋势基本上保持一致。转速增加，液体在反应器内旋转的频率随之加快，单位时间内液体扫掠面积增加，使得气液更新速度加快。因此可以简单地用转速大小来粗略衡量气液更新速度。
3.计算机模拟工作状态下，反应器内的流体湍动能和湍动能耗散率
(1)湍动能k用于表征流体微团脉动过程中所蕴含的能量，公式如下：
Figure PCTCN2020113460-appb-000010
其中，
v ave——流体的平均速度；
I——湍流强度，大小等于湍流脉动速度均方根v'与平均速度vave的比值。
(2)湍动能耗散率ε用于衡量微团碰撞和粘性耗散造成的能量损失速度大小，公式如下：
Figure PCTCN2020113460-appb-000011
其中，ρ、μ、μ 1——流体的密度、黏度、湍流黏度。
流体微团的运动可以表示为随主体流动的平均速度与随机波动的脉动速度的叠加。微团的随机波动造成了湍流流动。从表5可以看出，在工作体积不变的情况下，转速越高，湍动能越高，因此反应器内物质传递和混合效果越好，同时ε也有不同程度的增大。
输入功率随转速升高而增加，使得携带较高能量的流体微团比例增大，能够产生有效碰撞传递能量的微观漩涡数量增加，即表现为k平均值的增大。同时，单位时间内 液体扫过容器内壁的面积随转速的增加而变大，气液接触交换的机会增大，壁面黏附作用和液体内部黏性摩擦也随之增加。高能微团的比例升高造成了更多的微团碰撞和能量传递，k的传递频率衰减速度加快。由此，湍动能耗散率ε随着转速升高而加快。
氧气液膜传递系数k L与湍动能耗散率ε的关系式如下：
Figure PCTCN2020113460-appb-000012
其中，
Figure PCTCN2020113460-appb-000013
表示氧气扩散系数，ρ L和μ L分别表示液相的密度和黏度。以上参数在本次实验条件下均为常数，可以用湍动能耗散率ε表征气液传递过程的强弱。气液传递随着ε的增大而变得强烈，近似地，气液传质系数k La越大。
表5 各工作体积反应器中的湍动能k和湍动能耗散率ε随转速变化情况
Figure PCTCN2020113460-appb-000014
Figure PCTCN2020113460-appb-000015
4.计算机模拟工作状态下，反应器内的湍流强度
从微环境角度来说，湍流扩散起主导作用。流体微团的运动可以表示为随主体流动的平均速度与随机波动的脉动速度的叠加。微团的随机波动造成了湍流扩散。微团间的速度波动剧烈，相互作用越强烈。能量的传递和分散作用也越显著。因此，湍流强度越大，液相内物质传递与混合的效果也越好。
可以用I来表征流体微团速度波动和相互作用的强烈程度。微团间的速度波动剧烈，相互作用越强烈，能量的传递和分散作用也越显著。因此，湍流强度越大，液相内物质传递与混合的效果也越好。湍流强度I的公式为：
Figure PCTCN2020113460-appb-000016
其中，v'为湍流脉动速度的均方根，v avg为流体的平均流速。流体微团的运动可以表示为随主体流动的平均速度与随机波动的脉动速度的叠加。微团的随机波动造成了湍流流动。
从表6可以得出，在工作体积不变的情况下，随着转速增加，I呈增大趋势，转速提高使得输入流体内的能量增加，造成了流体微团的动能增加，脉动速度加快，湍流强度也相应增加。
表6 各工作体积反应器，湍流强度I随转速变化情况
Figure PCTCN2020113460-appb-000017
Figure PCTCN2020113460-appb-000018
5.平均剪切率
动物细胞膜外没有细胞壁，对流场内的力学环境比较敏感，外力对细胞造成的机械损伤是动物细胞生物反应器设计和使用过程中一个不可忽略的因素。
在生物反应器中细胞受到的剪切力分为三大类：培养液内部的相对运动产生的流场剪切力，流场剪切力的大小和速度梯度、培养液的运动粘度成正比；气泡在培养液中上升及表面破裂时产生的剪切力；细胞与反应器内壁的碰撞，以及细胞间的相互碰撞所产生的剪切力。流场剪切力对细胞的破坏跟流体湍动所产生的最小涡流尺寸密切相关，培养液在容器内贴着壁面转动，产生湍动漩涡，大漩涡逐渐发展成小漩涡，小漩涡发展成更小的漩涡，这个过程中伴随着能量的传递，培养液机械能变为热能，使其温度升高。在漩涡逐渐变化的过程中，当漩涡的尺寸与细胞的尺寸接近的时候，漩涡把能量传递给细胞，从而导致细胞的破裂。对于本申请的生物反应器来说由于采用表面传氧，没有气泡分布器因而产生的气泡极少，因此气泡产生的剪切力伤害可以忽略不计。由于生物反应器内罐体内侧壁面比较光滑碰撞剪切力也比较小。通过上述分析可以发激流式生物反应器内剪切力主要是流场剪切力。因此通过仿真检测反应器内部剪切率的平均值对生物反应器的设计具有重要参考价值。
表7 各工作体积反应器，平均剪切率随转速变化情况
Figure PCTCN2020113460-appb-000019
Figure PCTCN2020113460-appb-000020
从表7可以得出，运动稳态时的培养液液体流场平均剪切率处于20.27/s以下。
平均剪切率是对整个流场区域的剪切率进行体积积分，然后求平均值。其计算公式为，
Figure PCTCN2020113460-appb-000021
从表7可以看出，对于CUR2.5L，在工作体积不变的情况下，平均剪切率随着振荡器转速的增大而增大，且工作体积的变化对平均剪切率的影响相对较小。CUR1200L受到的平均剪切率要比CUR2.5L小得多。虽然平均剪切率变小，对细胞的伤害会更小，但是剪切率和混合效果是相互制约的，剪切率变小也就意味着混合效果的变差。
以中华仓鼠癌细胞(CHO)为例，当剪切力大于0.392Pa，即在水中剪切率大于391.41s -1时，细胞就会受到破坏。而从表7中了解到的反应器内平均剪切率要远远低于对细胞构成伤害的剪切率。
三、小结
本实验以CUR2.5L、CUR50L、CUR300L、CUR1200L和CUR3000L作为模拟对象，采用动网格模型、RNG k-ε湍流模型结合VOF模型分别对14种转速、13种工作体积(共38种工况)下容器内液体的流动行为进行了数值分析。结果表明：
1.达到准稳态时液相形状受转速影响明显，液面随着转速增加弯曲程度加重；
2.气液交界面面积A不随转速增加而线性增加，转速提高对液体总表面积S的影响程度随工作体积的不同而改变；
3.液相额定湍流参数均受到转速和工作体积的双重影响，根据k L与ε关系式，可以用ε近似地表征气液传递过程的强弱；
4.平均剪切率随转速的增加而增大。虽然平均剪切率变小，对细胞的伤害会更小，但是剪切率和混合效果是相互制约的，剪切率变小也就意味着混合效果的变差。反应器内的平均剪切率要远远低于对细胞构成伤害的剪切率。
实施例2 生物反应器系统的混合性能测试
一、CUR5L生物反应器混合性能试验
1、实验设计
模拟生物反应器细胞培养环境，测定不同工作体积、不同转速下，生物反应器内物料的混合均一度，验证反应器的混合性能。
设备型号：CUR5L生物反应器(金仪盛世，D1＝400mm、D3＝80mm、H1＝149mm、H2＝98mm)，工作体积5L；混合所用溶液为PBS缓冲液，加入的试剂为1mol/L的NaOH。检测仪器：梅特勒pH电极。
测定内容：向生物反应器中加入NaOH试剂后，用pH电极测定混合均匀的时间，以确定反应器振荡过程中，加入容器的物料混合均匀所用时长。以时间长短来模拟生物反应器在实际应用过程中，加入的物料能实际影响培养细胞的能力。
本实验模拟反应器正常培养细胞的条件，分别设定50rpm、55rpm、60rpm三个转速值以及最小工作体积2L、最适工作体积3.5L、最大工作体积5L等三个不同工作体积。
2、实验准备
(1)在符合GMP标准的环境下，配置15L PBS缓冲液待用。
(2)在符合GMP标准的环境下，配置500mL浓度为1mol/L的NaOH溶液作为实验标准液，配置完之后，密闭在500mL储液桶中，以防止溶液变质，影响实验准确。
(3)pH传感器置于反应器顶部接口，通过测量液体的pH来反映混合效率。
3、实验步骤
3.1生物反应器CUR5L工作体积2L，在50、55、60rpm转速条件下的混合实验
(1)向反应器内先加入2L PBS缓冲液，温度控制在25-27℃，将反应器转速设定为50rpm，通过振荡器振荡运动，使反应器运行偏心距为30mm。
(2)待反应器进入稳态运转状态后，开始从反应器正上方圆心处，向反应器内注入3mL浓度为1mol/L的NaOH溶液。每间隔5分钟注入1次，一共注入3次。
(3)将反应器转速设定为55rpm，重复步骤(2)。
(4)将反应器转速设定为60rpm，重复步骤(2)。
3.2生物反应器CUR5L工作体积3.5L，在50、55、60rpm转速条件下的混合实验
步骤基本同3.1，除向反应器内加入的PBS缓冲液为3.5L且每次向反应器内注入的浓度为1mol/L的NaOH溶液为5mL外。
3.3生物反应器CUR5L工作体积5L，在50、55、60rpm转速条件下的混合实验
步骤基本同3.1，除向反应器内加入的PBS缓冲液为5L且每次向反应器内注入的浓度为1mol/L的NaOH溶液为10mL外。
利用pH传感器实时监测生物反应器内pH变化情况，以及观察两个传感器达到相同pH值所用时间，导出传感器图像数据和报表数据，分析两组传感器检测得出的混合均匀所用时长，得出反应器的混合效率。
4、实验结果
溶液pH稳定的标准：连续10s，pH稳定在某一个数值，误差范围在±0.01之间，则认为pH值达到稳定。在各条件下pH值达到稳定所用的时间总结在以下表8中。
表8 CUR5L不同工作体积和转速下达到pH稳定的时间
Figure PCTCN2020113460-appb-000022
二、CUR50L生物反应器混合性能试验
采用CUR50L生物反应器(金仪盛世，D1＝840mm、D3＝114mm、H1＝280mm、H2＝170mm)，转速分别设定为38rpm、39rpm、40rpm三个转速值以及最小工作体积15L、最适工作体积30L、最大工作体积50L等三个不同工作体积，偏心距设为40mm，温度控制在25-27℃。
测试方法基本同上。在15L工作体积时，向反应器内加入15L PBS缓冲液且每次向反应器内注入10mL浓度为1mol/L的NaOH溶液。在30L工作体积时，向反应器内加入30L PBS缓冲液且每次向反应器内注入30mL浓度为1mol/L的NaOH溶液。在50L工作体积时，向反应器内加入50L PBS缓冲液且每次向反应器内注入50mL浓度为1
mol/L的NaOH溶液。结果总结在表9中。
表9 CUR50L不同工作体积和转速下达到pH稳定的时间
Figure PCTCN2020113460-appb-000023
Figure PCTCN2020113460-appb-000024
三、CUR500L生物反应器混合性能试验
采用CUR500L生物反应器(金仪盛世，D1＝1690mm、D3＝190mm、H1＝452mm、H2＝347mm)，转速分别设定为25rpm、27rpm、29rpm三个转速值以及最小工作体积100L、最适工作体积300L、最大工作体积500L等三个不同工作体积，偏心距设为65mm，温度控制在25-27℃。
测试方法基本同上。在100L工作体积时，向反应器内加入100L PBS缓冲液且每次向反应器内注入50mL浓度为1mol/L的NaOH溶液。在300L工作体积时，向反应器内加入300L PBS缓冲液且每次向反应器内注入100mL浓度为1mol/L的NaOH溶液。在500L工作体积时，向反应器内加入500L PBS缓冲液且每次向反应器内注入100mL浓度为1mol/L的NaOH溶液。结果总结在表10中。
表10 CUR500L不同工作体积和转速下达到pH稳定的时间
Figure PCTCN2020113460-appb-000025
四、CUR1200L生物反应器混合性能试验
采用CUR1200L生物反应器(金仪盛世，D1＝1952mm、D3＝190mm、H1＝533mm、H2＝465mm)，转速分别设定为23rpm、25rpm、27rpm三个转速值以及最小工作体积300L、最适工作体积800L、最大工作体积1200L等三个不同工作体积，偏心距设为65mm，温度控制在25-27℃。
测试方法基本同上。在300L工作体积时，向反应器内加入300L PBS缓冲液且每次向反应器内注入100mL浓度为1mol/L的NaOH溶液。在800L工作体积时，向反应器内加入800L PBS缓冲液且每次向反应器内注入800mL浓度为1mol/L的NaOH溶液。在1200L工作体积时，向反应器内加入1200L PBS缓冲液且每次向反应器内注入500mL浓度为2mol/L的NaOH溶液。结果总结在表11中。
表11 CUR1200L不同工作体积和转速下达到pH稳定的时间
Figure PCTCN2020113460-appb-000026
Figure PCTCN2020113460-appb-000027
如表8-表11数据所示，本装置的混合效率是完全可以满足大规模细胞培养过程中的混合与传质要求的。
实施例3 MDCK细胞培养
使用实施例2中使用的CUR5L、CUR50L、CUR500L和CUR1200L生物反应器进行MDCK细胞的培养。细胞的接种密度为1.5×10 6细胞/ml，生长培养基为CD MDCK SFM(DP304，健顺生物)，培养温度为37℃，溶氧值DO％为35-65。
CUR5L反应器设置工作体积5L，转速55rpm，偏心距30mm，氧气和空气的导入速率分别设为100mL/min和200mL/min。CUR50L反应器设置工作体积50L，转速40rpm，偏心距40mm，氧气和空气的导入速率分别设为500mL/min和500mL/min。CUR500L反应器设置工作体积500L，转速28rpm，偏心距65mm，氧气和空气的导入速率分别设为900mL/min和1000mL/min。CUR1200L反应器设置工作体积1200L，转速25rpm，偏心距65mm，氧气和空气的导入速率分别设为1500mL/min和1500mL/min。
在细胞培养至48h时往反应系统中一次性补加2g/L的葡萄糖，补加葡萄糖溶液的浓度为200g/L。另外，生物反应器pH值设定在7.0～7.4范围内，当系统检测到pH值低于7.0时，系统会自动补加浓度为7.5％的NaHCO 3。
细胞培养数据总结在以下表12中。
表12 MDCK细胞培养数据
Figure PCTCN2020113460-appb-000028
Figure PCTCN2020113460-appb-000029
从表12可以看出，细胞培养过程中，尽管细胞密度不断增加，细胞活率一直保持在99.0％以上。
实施例4 CHO细胞培养
1.CUR300L生物反应器培养CHO细胞
采用本申请构造的CUR300L生物反应器(金仪盛世，D1＝1500mm、D3＝190mm、H1＝422mm、H2＝303mm)，采用批次流加(Fed-batch)细胞培养工艺，进行CHO细胞的培养。其中，反应器转速30rpm，偏心距60mm，培养基起始体积分别为200L、205L、250L，温度控制在37℃，氧气和空气的导入速率分别设为800mL/min和800mL/min。初始培养基为甘肃健顺生物有限公司CHO细胞专用培养基，在培养的第4天，补加的培养基为甘肃健顺生物有限公司的CD Feed002。
CHO细胞的接种密度为约1.0×10 6/mL，在第4、6、8、10天分别加入当前工作体积的5％、2％、5％、2％的CD Feed002，葡萄糖低于2.0g/L时补加葡萄糖溶液至8.0g/L，pH值设定在7.0～7.2范围内，当pH值低于偏离7.0时，系统会自动补加浓度为7.5％的NaHCO 3。培养共持续14天，每天观察细胞生长的密度、活率、渗透压、氧分压、二氧化碳分压、蛋白表达量等情况。培养过程中的细胞密度和活率具体记载于表13。
表13 CHO细胞培养数据
Figure PCTCN2020113460-appb-000030
Figure PCTCN2020113460-appb-000031
第14天下罐收获时的培养体积分别达到247L、256L、和312L，最终蛋白表达量达到1.133g/L、1.594g/L、1.311g/L。整个培养过程中，细胞的密度最高时达到15.49×10 6细胞/mL，细胞始终维持在高活率，保持在蛋白的高表达期，结果较理想。
2.CUR1200L生物反应器培养CHO细胞
采用CUR1200L生物反应器(金仪盛世，D1＝1952mm、D3＝190mm、H1＝533mm、H2＝465mm)，以批次流加方式进行CHO细胞培养。反应器转速设定为30rpm，工作体积取250L和330L，偏心距设为65mm，温度控制在37℃。氧气和空气的导入速率分别设为1500mL/min和1500mL/min。初始培养基为甘肃健顺生物有限公司CHO细胞专用培养基，在培养的第4天，补加的培养基为甘肃健顺生物有限公司的CD Feed002。
接种密度约1×10 6/mL，在第4、6、8、10天分别加入当前工作体积的5％、2％、5％、2％的CD Feed002，葡萄糖低于2.0g/L时补至葡萄糖溶液至8.0g/L，pH值设定在7.0～7.2范围内，当pH值低于7.0时，系统会自动补加浓度为7.5％的NaHCO 3。细胞培养持续14天，每天观察细胞生长的密度、活率、渗透压、氧分压、二氧化碳分压、蛋白表达量等情况。培养过程中的细胞密度和活率具体记载于表14。
第14天下罐收获时的培养体积分别达到314L、417L，最终蛋白表达量达到1.481g/L、1.308g/L。整个培养过程中，细胞的密度最高时达到18.51×10 6/mL，细胞始终维持在高活率，保持在蛋白的高表达期，结果较为理想。
表14 CHO细胞培养数据
Figure PCTCN2020113460-appb-000032
3.CUR50L生物反应器培养CHO细胞
采用本申请构造的CUR50L生物反应器(金仪盛世，D1＝1500mm、D3＝190mm、H1＝422mm、H2＝303mm)，采用ATF灌流(Perfusion)细胞培养工艺，进行CHO细胞的培养。其中，生物反应器的转速为37rpm(第1-8天)转39rpm(第9-20天)，工作体积为18L、偏心距为40mm，温度控制在37℃。氧气和空气的导入速率分别设为500mL/min和500mL/min。初始培养基为甘肃健顺生物有限公司CHO细胞专用培养基，在培养的第11天，补加的培养基为甘肃健顺生物有限公司的CD Feed。
表15 CHO细胞培养数据
Figure PCTCN2020113460-appb-000033
接种密度约1×10 6/mL，第12-20天通过ATF-4取出部分培养液，并添加同样体积培养液，培养系统中维持葡萄糖浓度不低于3.0g/L，pH值设定在7.0～7.2范围内，当pH值低于7.0时，系统会自动补加浓度为7.5％的NaHCO 3。细胞培养持续20天，每天观察细胞生长的密度、活率、直径、渗透压、乳酸残余量、蛋白表达量等情况。培养过程中的细胞密度和活率具体记载于表15。
第20天下罐收获时的培养体积位14L，最终蛋白表达量达到27g，这个结果超出预期。整个培养过程中，细胞的密度最高时达到90.1×10 6/mL，细胞始终维持在高活率(97％以上)。
从以上可以看出，在使用本申请构造的生物反应器并选取合适的转速、偏心距等时，细胞培养顺利进行，细胞密度逐渐增高，细胞存活率一直保持在较高水平如90.0％以上。可见，本申请的生物反应器系统适用于高密度动物细胞培养。

Claims (10)

  1. 一种培养无细胞壁细胞的生物反应器系统，其包含：
    -容器，该容器含有直径D1且高度H1的中空圆柱，以及上直径D2、下直径D3且高度H2的中空圆台，其中该中空圆柱与中空圆台的顶面连接，D1＝D2；
    -振荡器，设置成使容器按照一定的偏心距和转速做偏心运动；
    -通气装置，设置成从容器上部向容器内通入含氧气体，和
    -填充在容器中的培养液，其顶面暴露于含氧气体，
    其中，振荡器设置成使容器处于偏心运动，使运动稳态时的培养液液体总表面积与体积的比值S/V处于5.65以上，湍动能处于2.73E-03m 2/s 2以上，且流场剪切率处于20.27/s以下，其中培养液液体总表面积为培养液与反应器壁面接触面积和培养液与气体接触面积之和。
  2. 如权利要求1所述的生物反应器系统，其中CFD模拟经FLUENT软件实现。
  3. 如权利要求1所述的生物反应器系统，还包含设置在容器中的用于容纳培养液的一次性培养袋，在展开时具有与容器相对应的形状。
  4. 如权利要求3所述的生物反应器系统，所述一次性培养袋包含多功能盖板，所述多功能盖板设置有多个通向一次性培养袋内部的接孔。
  5. 如权利要求1所述的生物反应器系统，其中容器的D1和D2为400-2997mm，D3为80-190mm，H1为149-867mm，H2为98-664mm，含有5-3000L的细胞培养液，振荡器的转速为55-24rpm，偏心距为30-65mm。
  6. 如权利要求1所述的生物反应器系统，其中容器的D1和D2为约400mm，D3为约80mm，H1为约149mm，H2为约98mm，含有约5L细胞培养液，振荡器的转速为约55rpm，偏心距为约30mm，其中“约”指所述数值的±20％的范围。
  7. 如权利要求1所述的生物反应器系统，其中容器的D1和D2为约840mm，D3为约114mm，H1为约280mm，H2为约170mm，含有约50L细胞培养液，振荡器的转速为约40rpm，偏心距为约40mm，其中“约”指所述数值的±20％的范围。
  8. 如权利要求1所述的生物反应器系统，其中容器的D1和D2为约1690mm，D3为约190mm，H1是约452mm，H2是约347mm，含有约500L细胞培养液，振荡器的转速为约28rpm，偏心距为约65mm，其中“约”指所述数值的±20％的范围。
  9. 如权利要求1所述的生物反应器系统，其中容器的D1和D2为约1952mm，D3为约190mm，H1为约533mm，H2为约465mm，含有约1200L细胞培养液，振荡器的转速为约25rpm，偏心距为约65mm，其中“约”指所述数值的±20％的范围。
  10. 一种使用权利要求1所述的生物反应器系统培养无细胞壁细胞的方法，包括：
    -根据生物反应器系统的形状和培养液体积，经CFD模拟算出培养液在偏心运动达稳态时液体总表面积与体积的比值(S/V)在5.65以上、湍动能在2.73E-03m 2/s 2以上、且流场剪切率在20.27/s以下所需的振荡器转速和偏心距，其中培养液液体总表面积为培养液与反应器壁面接触面积和培养液与气体接触面积之和；以及
    -在生物反应器系统中加入培养液和接种细胞，并根据计算出的振荡器转速和偏心距来设定振荡器，进行细胞培养。
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