automating the cell culture sampling process

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


• 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)


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



septum

septum

Flame


• Very little risk of contamination



• 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


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



Mechanical arm

Pure air inlet


Test tube rack with collecting tubes


• Reduces the risk of contamination


• 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


Final DesignCombination of Design Ideas #3 and #4 (uses flame sterilization with the movable door feature)

septum

Track



Mechanical arm

Pure air inlet


Test tube rack with collecting tubes

Movable dividing door

Advantages Disadvantages

Final Design

• Reduces the risk of contamination



• 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


Mechanical arm

Pure air inlet


Test tube rack with collecting tubes flame

Modification of Final Design

• Addition of a second door


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>

automating the cell culture sampling process

Documents

design ideasopen flame

cultureinsert syringe

sampling processdraw

sampling line

syringe switchdoes

hood advantages

multiple bioreactorsidea

alcohol wash