authors: vasily bankovsky, igor bankovsky, pavel bankovsky
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Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky. Part I Introduction. RTS-1C, Reverse Tube Spinner — Real Time Cell Growth Logger. - PowerPoint PPT PresentationTRANSCRIPT
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Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky
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Part IIntroduction
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RTS-1C, Reverse Tube Spinner — Real Time Cell Growth Logger
Specifications: Temperature setting range +4°C ... + 70°C Measurement periodicity till 60 measurements per hour Rotation Speed 250–2000 rpm Software Controlled Experiments 10 units simultaneouslyContinuous Operating Time n/d Warranty 35 000 hrs
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RTS-1 — 4 Instruments in 1
Measuring Thermostating
Mixing
SoftwareLogging
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Introduction: Noninvasive Mixing Principles
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Reverse Spinning vs Orbital Shaking
• Ability to measure OD non invasively• Cell Growth in single use reactors • Natural centric auto-balancing • Simplicity • No power consumption for contra-
balancing • Do not need to take aliquot for OD
measurement
• Proportionality between orbital diameter and the diameter of the moving vessel
• Artificial hula-hoop auto-balancing• Larger size• Heavy• Extra power consumption for
contra-balancing
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Orbital Shaking vs Reverse Spinning
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Part IIPrinciples of Reverse Spinning
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Growth medium behavior inside the bioreactor
Rotation Intensity Rotation around axis
Spread of the broth media inside of rotation tube as a function of rotation intensity
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Vortex behavior inside the tube
Video demonstration:
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Reverse Spinning Lifecycle
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Centrifugal Forces as a Mechanical Defoamer
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Principle of OD measurement
Automatic(A) -
Wavelength of light source: 850nm
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Principle of absorption measurement and measurement modes H-L-A
Video demonstration:
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Fig. 1 The effect of media volume on layer thickness(mm) during 2000 rpm rotation intensity
Fig. 2
Video demonstration:
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Recommended bioreactor tube: TPP plastic 50mL Tubespin bioreactor
• working volume 1 – 30 ml • conical form • 5 openings (A, B, C, D, E) of different size
above the gas permeable, sterile PTFE filter of the screw cap
• openings can be sealed and by this, exchange adjusted to need
• sterile gas exchange is guaranteed by the 0.22 µm filter membrane
• even with a high cell density the supply of oxygen through the openings is sufficient
• tube fits in a standard 50 ml centrifuge rotor• Single use
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Part IIIExperimental Results
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Reverse Spin longitude (RSL) effect on E.coli BL21 growth kinetics and growth rate [ µ ( 1/h ) vs time (hr)]
Fig. 1 RSL effect on E.coli growth kinetics over time
Fig. 2 RSL effect on E.coli growth rate over time
Fig. 3 3D representation of Fig. 1
Fig. 4 3D representation of Fig. 1
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Growth media volume effect on E.coli BL21 growth kinetics [ 0D(860 nm) vs time (hr)] and growth rate [ µ ( 1/h ) vs time (hr)]
Fig. 1 Growth media volume effect on E.coli growth kinetics over time
Fig. 2 Growth media volume on E.coli growth rate over time
Fig. Growth media volume on E.coli growth kinetics over time in 3D
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LB x 2
LB x 1
LB x 1/2
LB x 1/4
LB x 1/8
LB x 4
Influence of LB concentration on E.Coli BL21 growth kinetics [ OD(860 nm) vs time (hr)] and growth rate [ µ ( 1/h ) vs time (hr)]
Fig. 1 LB conc. effect on E.coli growth kinetics over time
Fig. 2 LB conc. effect on E.coli growth rate over time
Fig. 3 3D representation of Fig. 1
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Lactobacillus acidophilus sp. growth kinetics and growth rate [ µ ( 1/h ) vs time (hr)]
• Nitrogen was purged into the bioreactor in sterile conditions to eliminate O2 presence
Fig. 1 LB conc. effect on E.coli growth kinetics over time
Fig. 2 LB conc. effect on E.coli growth rate over time
Fig. 3 3D representation of Fig. 1
Fig. 4 3D representation of Fig. 2
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Effect of T(c) on E.coli BL21 growth kinetics [ OD(860 nm) vs time (hr)] and growth rate [µ ( 1/h ) vs time (hr)]
Fig. 1 T(c) effect on E.coli growth kinetics over time
Fig. 2 T(c) on E.coli growth rate over time
Fig. 3 3D Graph representation of Fig. 1
Fig. 4 3D Graph representation of Fig. 2
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Effect of different T-shock (42-70°C) on E.coli BL21 growth kinetics of
Fig. 1 T-shock effect on E.coli growth kinetics over time
Fig. 2 T-shock effect on E.coli growth rate over time
Fig. 3 3D graph of Fig.1
Fig. 4 Temperature in bioreactor over time
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Effect of T-shock (63…69°C) on E.coli BL21 growth kinetics
Fig. 1 T-shock effect on E.coli growth kinetics over time
Fig. 3 T-shock effect on E.coli growth rate over time
Fig. 4 3D graph of Fig.1
Fig. 2 Temperature in bioreactor over time
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Coarse approximation delta 5 (C) increments from 37(C) to 70(C)
Fine approximation with a delta 1(C) increments from 63(C) to 69(C)
At 70 (C) irreversible inactivation of 30S subunit of E.coli ribosome take place. ( J.Lee at all.,Appl.Env.Micr. 2002 5379 – 5386 )
Process of inactivation in range 62 (C) till 69 (C) is reversible
Heat treatment optimization by E.coli BL21 growth kinetics investigation of T(C) value pretreatment
Fig. 1 3D graph representation of T-shock effect on E.coli growth kinetics over time (42C-70C) Fig. 2 3D graph representation of T-shock effect on E.coli
growth kinetics over time (63C-69C)
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Fig. 3 cycling type temperature profiling over time
Possibility to apply different profiling modes: A) Periodic (cycling type) B) Stochastic(random values)C) Mixed type (A+B)
Effect of temperature fluctuation (delta 10°C from 37°C) on growth kinetics and growth rate of E.coli BL21
Fig. 1 t(C) fluctuation effect on E.coli growth kinetics over time
Fig. 2 t(C) fluctuation effect on E.coli growth rate over time
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Cultivation of CHO-K1 Cell Culture using Reverse-Spin method
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Part IVThe Future Developments
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Multi-option in 6 Channel Reverse Spinner
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RS-4
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Color of Phenol Red vs pH after E.coli BL 21 growth on LB and LB+Glucose media
pH= 6 pH= 7 pH=8 LB+Glu LBOptical density vs pH(normalized)
y = 0,976x + 0,0933R2 = 0,9999
0
0,2
0,4
0,6
0,8
1
1,2
0 0,2 0,4 0,6 0,8 1 1,2
570 nm
Linear (570 nm)
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pH Indicator Dyes and Micro Color Sensors
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Thermophilus sp. cultivation using different non invasive mixing principles
Growth conditions
RS -1 Falcon……...……. ..50 mlTY(0,2%NaCl) ……25 mlSpeed of rotation 2000 rpmReverse ………… 10 secTemperature ….. 60 C
Orbital shaker ES20/60
Falcon …………… 50 mlTY(0,2%NaCl) ……25 mlOrbit ……………….20 mmSpeed of rotation .240 rpmTemperature ……. 60 C
Rocker Falcon……...……. ..50 ml TY(0,2%NaCl) ……25 mlAngle of rocking ….7 gradOscillation freq. 120 min-1Temperature …….60 C
Thermophillus sp extremelyDifficult to control cells growth in real time because of high optimum
of growth temperature ( T(C) is more than 60 C )
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New projects with Personal bioreactor RTS-1
• optical pH and Oxygen measurement • Yeast cultivation optimization• Cultivation of microbes associates• Viruses vs cells investigations• Recombinants growth optimization• Environmental models research• Fluorescence detectors for non intrusive analyses of
goal fermentation products
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Thank you for your attention