Automating the Cell Culture Sampling Process Mike Phipps Tara Ryan BME 273 March 18, 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

Existing Sampling Methods

Existing Sampling Methods

Flowchart of the Sampling Process Make sure tip is okay to enter culture Ensure sterility of syringe tip Obtain new syringe Draw sample from culture Insert syringe tip into culture Dispose of used syringe Pull sample into syringe tube Remove syringe tip from culture Move collecting tube to analysis machines Deposit sample into collecting tube Move syringe to collecting tube

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 Ideas Idea #1 Continuous flow of medium and cells through tubing loop switch 3-way valve to the sampling line in order to draw a sample simple does not avoid the traditional syringe switch

Assessment of Design Idea #1 simple inexpensive easy setup Advantages: Disadvantages: does not avoid the “syringe switch” does not reduce the time or labor needed to sample

Design Ideas Idea #2 Ethanol and wash sterilize the syringe tip (needle) Use of septa Expand to a set of 4 bioreactors

Assessment of Design Idea #2 Advantages: Disadvantages: 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 Ideas Idea #3 Open flame sterilizes the syringe tip (needle) Use of septa Water-gasket bioreactor system for better maintenance of the culture’s temperature Expand to a set of 4 bioreactors

Assessment of Design Idea #3 Advantages: Disadvantages: 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

Design Ideas Idea #4 Simpler (fewer steps for mechanical arm) Reliance on hood to provide sterility Expand to a set of 4 bioreactors

Assessment of Design Idea #4 Advantages: Disadvantages: 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

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

Final Design Advantages: Disadvantages: Combination of Design Ideas #3 and #4 (uses flame sterilization with the movable door feature) Advantages: Disadvantages: 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 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) 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

Current Work Components of the Device: test tubes, test tube rack, disposable syringes, syringe disposal bag, bioreactor system(s) with septa embedded in top, hood device with movable divider, mechanical arm, track, air purifier, Bunsen burner, natural gas line Determine the manufacturers, material composition, price, dimensions, and weight of each component

Future Work Calculations (heat transfers, air flows, etc.) and research of parts manufacturers to determine specifications of the final design Draw final design using AutoCAD Production of a prototype?

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>