Three-dimensional microfabricated bioreactor and closed-loop control system Alex Makowski Michael Hwang Jenny Lu Sam Cassady Dr. John Wikswo

Ultimate Project Goals Computer modulated growth of tissue structures within microfluidic devices. Possible uses include drug testing and dose determination, bypassing several stages of FDA trials.

Problem Statement- Design progress is blocked on three distinct fronts: Sensors are required to provide computer with necessary information. Bioreactor design limits the quantity and quality of cell morphology within the device. Previously used cells (primary human fibroblasts) do not easily form tissue-like structures.

Subsequent Requirements: Choose or design an appropriate sensor for pH measurements (most needed to determine cell metabolism and health). Choose a cell line that will exhibit observable morphological change under successful conditions. Redesign the bioreactor to incorporate new cell line and maximize efficiency of pH sensors.

Resultant Main Chip Design Channel Layer - PDMS Glass Slide Plexiglas Matrigel Layer Seeded with Cells Access Ports Top View: Side View:

MCF10A Human breast epithelial cells (MCF10A) Growth- 2 to 3 days to grow to ~90% confluence in culture dish. Acinar morphology-3D hollow cell ball.

Acinar morphology of MCF10A cells Matrigel experiment shows it takes 20 days to form into acinar morphology. (Debnath and Brugge 2005)

Investigating cell concentration Matrigel experiment on chamber slide with different cell concentrations: 5000 cells/chamber 1000 cells/chamber 500 cells/chamber 250 cells/chamber Illustration courtesy of Cassio Lynm, JAMA

Getting the right concentration Show what concentration gives the best images under the confocal microscope. Correlate the optimal concentration in chamber slide with what we will put in the bioreactor.

Cell Media for Infusion into Bioreactor and for Differential pH Reference Fresh Media Acidified Media Bioreactor Iridium Oxide pH-Sensing Electrode Iridium Oxide Quasi-Reference Electrode Cavro® XLP 6000 Syringe Pump pH 6 Calibration Solution pH 8 Calibration Solution Diagram of the Entire Fluidic System

Cell Media for Infusion into Bioreactor and for Differential pH Reference Fresh Media Acidified Media Bioreactor Iridium Oxide pH-Sensing Electrode Iridium Oxide Quasi-Reference Electrode Cavro® XLP 6000 Syringe Pump pH 6 Calibration Solution pH 8 Calibration Solution Why do we need a reference electrode?

Junction Potentials Exist at Heterogeneous Metal/Metal, Liquid/Liquid, or Liquid/Metal Interfaces For example, at a metal/liquid interface metal cations enter solution while electrons remain restricted to the metal phase. Hence, a potential develops. Charges are confined to interfaces in electrostatic conditions. Junction potentials vary among interfaces between materials of different compositions and are often difficult to determine — — — — — — — Metal Solution

Absolute Reference Electrodes Remain at a Known, Constant Potential Relative to Sample Solutions Examples include the Ag/AgCl liquid junction reference electrode or Hg/Hg 2 Cl 2 (saturated calomel electrode). Ag/AgCl or Hg/Hg 2 Cl 2 wire remains at a constant potential relative to an internal saturated KCl solution whose composition does not change. Liquid junction potential between internal electrolyte and sample solution at the high resistance porous frit is very small and invariant –High concentration of salt in internal electrolyte dominates the exchange current across the porous frit. –Similar mobility of the cation and anion of the salt causes both charges to cross the frit at an equal rate and thus create virtually no potential difference at the interface.

A Quasi-Reference Electrode is Suitable for Our Measurements The quasi-reference iridium oxide exhibits a relatively constant potential relative to sample solutions for short time scales. –The solution bathing the quasi-reference electrode remains unchanged, causing the electrode to do the same despite its pH-sensitivity. –If the ionic composition of sample solutions do not vary too much, the liquid junction potential between the solution immersing the quasi-reference and the sample solutions will also be nearly constant. –The iridium oxide quasi-reference therefore behaves like an absolute liquid junction reference electrode, except that its potential does slowly drift. An absolute reference electrode that remains at a constant potential for longer periods of time is superfluous. –The pH-sensing electrode drifts. –The quasi-reference electrode is an identical type of electrode and therefore drifts at the same rate. –Recalibration simultaneously compensates for the drift of both electrodes. –Hence, for example, measuring a series of solutions of known pH followed by a solution of unknown pH can yield the unknown pH. A 125-μm-diameter iridium oxide quasi-reference electrode is cheaper (produced in lab) and easier to integrate into the fluidic system. `

Another look at the fluidic system Cell Media for Infusion into Bioreactor and for Differential pH Reference Fresh Media Acidified Media Bioreactor Iridium Oxide pH-Sensing Electrode Iridium Oxide Quasi-Reference Electrode Cavro® XLP 6000 Syringe Pump pH 6 Calibration Solution pH 8 Calibration Solution

Change in location of quasi-reference electrode Shorter distance between iridium oxide electrodes reduces: inter-electrode impedance number of liquid junction potentials between the sensors

Bioreactor pH 6 Calibration Solution pH 8 Calibration Solution Iridium Oxide pH-Sensing Electrode Iridium Oxide Quasi- Reference Electrode Acidified Media Fresh Media Waste or Recirculates Back to Media Pool Faraday Cage Cavro® XLP 6000 Syringe Pump Further Protection against Noise Cell Media for Infusion into Bioreactor and for Differential pH Reference

Disproportionate Investment in Pumps and Controls for the pH Measurement System Compared to the Control(s) for the Actual System of Interest? DAQ Board + - System of interest

DAQ Board + - Extra valves in syringe pumps allow recalibration of a second bioreactor running in parallel