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

Ultimate Project Goals Computer modulated growth of tissue structures within microfluidic devices. Possible uses include drug testing and dose determination, toxicity levels, and biocompatibility issues.

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 Top View: Side View: Access Ports Plexiglas Channel Layer - PDMS Matrigel Layer with Cells (8mm diam.) 500 micron Glass Slide

Piecewise Breakdown- Bottom 8mm 1.8 in. 500 micron -1mm Matrigel Layer with Cells (8mm diam.) Glass Slide 1.9 in.

Piecewise Breakdown- Top Filter layer 10 micron thick x 1.9 inches square (4.7 micron pores) Channel Layer Dimensions (1.9 inch square 3-5mm thickness) Channels: 60-100 microns deep 6mm across 15 micron wide channels Plexiglas Layer (machine shop made) Dimensions (1.9 inch square 5-7mm thick) Port Assemblies-Upchurch Scientific Interface with Tygon tubing (500micron) Note: Thickness of upper layers is due to pressure generated by fluidic system. Layer thickness prevents deformation. Plexiglas Channel Layer - PDMS

Ultimate Assembly Final Bioreactor assemblies include: Brass clamp Acrylic and Port Assemblies PDMS Layers (containing cells) Viibre Repository: Schaffer

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. Matrigel experiment: 20 days to form acinar morphology Debnath and Brugge (2005) 9

Side view of PDMS-layer bioreactor in cell culturing dish Growth Media PDMS layer Glass slide Bottom Matrigel layer Matrigel and cells layer Cells in 8-chamber slide Top View Magnified Side View Matrigel and cells layer Growth Media Bottom Matrigel layer Parallel experiments run in chamber slide and PDMS-layer bioreactor to ensure the normal physiological change. Difference in well size requires proportionality. 10

Problems and Solutions P1: Matrigel migrated from the well of bioreactor to the surface of PDMS layer. S1: Let the second layer of Matrigel with cells sit for 1 hour instead of 20 minutes to polymerize. P2: Some cells migrated to the surface of PDMS layer and some even to the cell culturing dish. S2: The final bioreactor has a filter that prevents the cells from migrating out of the well. Current Progress Cells in the chamber slides grew for more than 20 days to form into acinar morphology Cells in bioreactors are staying inside of the well and forming spheriods, and they are 3 weeks old and still alive 11

System Diagram + - IA Acidified Iridium Oxide pH-Sensing Electrode DAQ Device - Fresh Media Acidified Bioreactor Iridium Oxide pH-Sensing Electrode Quasi-Reference Cavro® XLP 6000 Syringe Pump pH 8 Calibration Solution pH 6 Pt Wire for Bias Current Return Path

Illustration of Stop-Flow Operation 2. Stop and allow media acidification 3. Flow 4. Stop and Measure

Stop-flow Over Continuous Flow More equal nutrient/metabolite exchange Easier to conceptualize/model exchange Less delay in acquiring current pH measurement If response time of sensor is long, allows measurement of endpoint pH rather than moving average of pH signal (calculation of acidification rate better with former)

Integration of Electrodes and Wires into Fluidic Lines Iridium Oxide Electrode Epoxy Resin Tygon Tubing: 500 μm inner diam. Electrode: 125 μm diam. Flow Tygon Tubing Tygon Tubing 23-Gauge Syringe Needle

Automated Sensor Characterization pH-sensing electrode + IA DAQ Device - Automatically exchanges solution immersing the pH-sensing electrode and acquires measurements. Can be used to create a low pH bolus within a higher pH stream to test the system’s ability to detect an acidified bolus.