automating the cell culture sampling process
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Automating the Cell Culture Sampling Process. Mike Phipps Tara Ryan BME 273 April 5, 2002. Problem/Background. Cell cultures maintained in bioreactors for Research and Development purposes in pharmaceutical companies must be sampled regularly - PowerPoint PPT PresentationTRANSCRIPT
Automating the Cell Culture Sampling Process
Mike Phipps
Tara Ryan
BME 273
April 5, 2002
Problem/Background• Cell cultures maintained in bioreactors for Research and Development purposes in pharmaceutical companies must be sampled regularly
• Samples (10-15mL) are typically taken once most days, and twice every three days or so when the culture is split
• methods of manually withdrawing a sample from the bioreactor can be reliable but still come with risks of culture contamination
• lab workers must be trained and experienced in sterile technique
• lab workers must come into the lab on weekends or during vacations if they cannot find someone they can trust to sample their cultures
Existing Sampling Methods
Hot plate to maintain temperature
Sampling syringe
ethanol
DO sparger Temperatureprobe
pH probeAgitator
Existing Sampling Methods
ethanol
Sam
plin
g po
rt
Sampling syringe
3-way valve
Water gasket for temperature control
Water inWater out
DO spargerTemperature probe
pH probe agitator
Flowchart of the Sampling Process
Obtain new syringe
Ensure sterility of syringe tip
Make sure tip is okay to enter
culture
Draw sample from culture
Insert syringe tip into culture
Pull sample into syringe
tube
Remove syringe tip
from culture
Move syringe to collecting
tube
Deposit sample into collecting
tube
Move collecting tube to analysis
machines
Dispose of used
syringe
Activate sampling
system
Project Goals
• reduce the risk of contamination that occurs due to sampling
• reduce the time it takes a lab worker to draw a sample from a culture
• reduce the skill and training required by a lab worker
Design IdeasIdea #1
• Continuous flow of medium and cells through tubing loop
• switch 3-way valve to the sampling line in order to draw a sample
Assessment of Design Idea #1
• does not avoid the “syringe switch”
• does not reduce the time or labor needed to sample
• simple
• inexpensive
• easy setup
Advantages: Disadvantages:
Design IdeasIdea #2
• Ethanol and wash sterilize the syringe tip (needle)
• Use of septum
• Expand to multiple bioreactors
Design Ideas
Idea #2
Mechanical arm
Track
EthanolWashReservoir of new
syringes
Syringe disposal container
Autoclavable, contained environment
septum
septum
Assessment of Design Idea #2
• Very little risk of contamination
• Can enclose/sample many bioreactors
• Reduces the labor/time needed to sample
• Ethanol and wash supplies must be changed frequently
• Expensive
• Chance of alcohol residue on syringe tip (can kill cells and influence viability counts)
Advantages: Disadvantages:
Design IdeasIdea #3
• Open flame sterilizes the syringe tip (needle)• Use of septum• Water-gasket bioreactor system for better maintenance of the
culture’s temperature• Expand to a multiple bioreactors
Design Ideas
Idea #3
Mechanical arm
Track
Reservoir of new syringes
Syringe disposal container
Autoclavable, contained environment
septum
septum
Flame
Assessment of Design Idea #3
• Very little risk of contamination
• Can enclose/sample many bioreactors
• Reduces the labor/time needed to sample
• Once cooled, syringe tip is safe to enter the culture (you can calculate how long the tip needs to cool off after submergence in the flame, but in #2, there is no easy way of making sure all the alcohol wash is gone)
• Expensive
• Heat from flame may influence temperature of hood environment or of culture
• No flammable materials/chemicals should be
in the hood
Advantages: Disadvantages:
Design IdeasIdea #4
• Simpler (fewer steps for mechanical arm)
• Reliance on hood to provide sterility
• Expand to multiple bioreactors
Design Ideas
septum
Idea #4
Track
Reservoir of new syringes
Autoclavable, contained environment
Mechanical arm
Pure air inlet
Syringe disposal container
Test tube rack with collecting tubes
Assessment of Design Idea #4
• Reduces the risk of contamination
• Can enclose/sample many bioreactors
• Reduce the labor/time needed to sample
• The hood air source only blowing when the door flap is open
• Expensive
• Reservoir of new syringes is briefly exposed to outside environment each time a sample is transferred to a collecting tube
Advantages: Disadvantages:
Final DesignCombination of Design Ideas #3 and #4 (uses flame sterilization with the movable door feature)
septum
Track
Reservoir of new syringes
Autoclavable, contained environment
Mechanical arm
Pure air inlet
Syringe disposal container
Test tube rack with collecting tubes
Movable dividing door
Advantages Disadvantages
Final Design
• Reduces the risk of contamination
• Can enclose/sample many bioreactors
• Reduces the labor/time needed to sample
• Once cooled, syringe tip is safe to enter the culture
• Heat from flame may influence temperature of hood environment or of culture
• No flammable materials/chemicals should be
in the hood
• Reservoir of new syringes is briefly exposed to outside environment each time a sample is transferred to a collecting tube
Conveyor Belt Idea• Conveyor belt would transport multiple bioreactors
to a stationary mechanical arm so that arm will not require a track along which it can move
• Cost of a 12-feet long conveyor belt with a diameter/width of 30 inches is estimated to be $6000*
• Air inlets (nitrogen, oxygen, etc.) come from pipes running down from the ceiling; can’t easily move these with the bioreactor
* according to http://www.matche.com/EquipCost/Conveyor.htm
septum
Track
Autoclavable, contained environment
Mechanical arm
Pure air inlet
Syringe disposal container
Test tube rack with collecting tubes flame
Modification of Final Design
• Addition of a second door
Reservoir of new syringes
Sequence:Arm gets new syringe
Syringe is sterilized in flame
Insulator door opens; flame is extinguishedSample is drawn from reactor
Air source turns on
Second door opens
Sample is deposited in tube
Arm disposes of syringe
Arm moves back; air source turns off
Arm moves back; second door closes
Arm moves back; insulator door closes
Parts Information
• Bioreactor type used– New Brunswick Scientific - BioFlo3000 Universal Fermentor
– glass tube reactor with stainless-steel dished jacketed bottom, stainless-steel head plate with 11 penetrations including septum port for inoculation, harvest tube, sampling system, (2) addition tubes, multiorifice ring sparger, exhaust condenser, thermowell and (2) 6-blade Rushton impellers
– 1.25L working volume, 1.6L total capacity, and working minimum volume is 0.6L vessel dimensions: height=19" (48cm), diameter=9.5" (24cm); overall dimensions: height=30" (76cm), width=25.5" (65cm), front-to-back=24.75" (63cm)
– price: call for specific quote (~$30,000 per bioreactor)
Parts Information
Part Manufacturer PriceMaterial
Composition Dimensions ReferenceOther Notes
Test TubesGlobe Scientific
Inc.500 for $138
15mL polypropylene with attached
screw cap, 25/bag, sterile
Test Tube RackGlobe Scientific
Inc. 7.6high density
polypropylene16mm
diameter
http://www.globescientific.com/cpage4
0.html
autoclavable; nonfloating in
water bath; holds 60
tubes
Disposable Syringes With
NeedlesMedPlus
Corporation$65/1000
pieces plastic 20mL volume
http://www.medpluscorp.c
om/price-list.htm
sterilized with ethylene
oxide, non toxic,
pyrogen-free
Syringe Disposal Bag
GRP Medical Services: Biohazard Disposal Supplies
$8 (or 12 for $90) 3 gallon
http://sharpssupply.com/m
cart/
Parts InformationPart Manufacturer Price
Material Composition Dimensions Reference Other Notes
Mechanical ArmTrack
Bunsen BurnerHome Training
Tools $25
fuel is butane or butane-propane
mix
8 inches tall when on
cartridge; flame is 3-6 inches high
http://www.httstore.com/store/prodinfo.asp?number=
CE-BURNSPL&variation=&aitem=21&mi
tem=109
portable, lightweight, reaches max
temperature of 3000F
Metal Dividers negligible1m X 1m (2
of these)
price estimate is based on
determining the cost per
additional m^3 of volume, and scaling up from an existing base
product
Enclosed Environment and
Air Inlet Allometrics, Inc. $23,274.66
includes motor and blower
(115V) 10m^3
http://www.allometrics.com/hoods.htm
Cost of Resources• Natural Gas Supply
– $1.186/therm*
• Electricity– $0.06178/kWhr (first 2000 kWhr/month)**
– $0.06817/kWhr (over 2000 kWhr/month)**
• Labor– mean hourly wage for ChE is $32.29***
– mean hourly wage for Chemical Technicians is $17.83***
* Based on Nashville Gas personal charges; 1 therm = 100,000 Btu
** Based on Nashville Electric Service personal charges
*** Based on Occupational Employment Statistics, http://www.bls.gov/oes/home.htm
Economic Analysis
• Current system vs. Proposed System– Equipment costs– Production costs/year– Labor costs/year
• Single vs. Multiple (4 or 8) Bioreactors
• Effects of Contamination on Cost of Current Systems
AutoCAD Drawing
Future Work
• More specifics regarding the system’s design and operation
• Complete economic analysis
• Complete AutoCAD drawing
Suggestions
• Run the sampling machine on a timer
• Investigate the reliability of the use of septa
References
• ABEC Website, <http://www.abec.com>
• B. Braun Biotech Website, <http://www.bbraunbiotech.com>
• Bailey, James E., and Ollis, David F. Biochemical Engineering Fundamentals. McGraw-Hill Inc.: St. Louis, 1986.
• Balcarcel, R. Robert. Associate Professor of Chemical Engineering, Vanderbilt University.
• New Brunswick Scientific Website, <http://www.nbsc.com>
• Todar, Kenneth. “The Control of Microbial Growth.” 21 September 2000 <http://www.bact.wisc.edu/microtextbook/ControlGrowth/sterilization.html>