1. 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

2. 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.

3. 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.

4. 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.

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

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

7. 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: 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

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

9. 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

10. 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

11. 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

12. 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

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

14. 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)

15. 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

16. 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.