Lab on a Chip Supplemental Information

On-chip CO2 control for microfluidic cell culture: Supplemental Information Samuel P. Forry and Laurie E. Locascio

Protocol for pH Determination Solution pH was measured using a ratiometric pH sensitive dye.1 Fluorescence images of SNARF solutions in microfluidic devices were acquired on an inverted microscope with 353 nm – 388 nm excitation and emission at 573 nm – 608 nm or 670 nm – 710 nm. Images were first flat field corrected, using a Schott glass image2, and then background subtracted (to remove PDMS autofluorescence) using a non-emissive area in the field of view. The pixels for each channel were identified manually and averaged for each image. The ratio of intensities from each emission band gave a dimensionless number that scaled with solution pH (Fig. SI 1). The system response was calibrated using buffer solutions of known pH (measured off-chip: Orion 3-Star pH Meter, Thermo, Waltham, MA). As indicated by the dye manufacturer, pH and dye fluorescence were related by:

!" = !"#!"#$% − !"#!!!!!!!!

∗ !!(!!)!!(!!)

Equation SI-1

where pKaSNARF=7.5, ! = !!!/!!!, F indicates fluorescence intensity, λ1 and λ2 refer to the two emission bands, and A and B refer to limiting values at acidic and basic endpoints (Fig. SI 2). Calibration was repeated in multiple devices over many days. From these, an average calibration curve and 95% confidence interval was determined and used for all pH measurements.

B!A !

C! D!

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Fig. SI 1. Fluorescence ratio pH measurement. Fluorescence emission was captured at two wavelengths (A and B) for calibration pH series. The ratio of intensity values (C) was quantified (D) to give an average value for each channel that scaled with pH according to Equation SI-1.

Electronic Supplementary Material (ESI) for Lab on a ChipThis journal is © The Royal Society of Chemistry 2011

2

References:

1. S. Bassnett, L. Reinisch, and D. C. Beebe, American Journal of Physiology - Cell Physiology, 1990, 258, C171 -C178. 2. P. C. Thomas, M. Halter, A. Tona, S. R. Raghavan, A. L. Plant, and S. P. Forry, Analytical Chemistry, 2009, 81, 9239-9246.

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Fig. SI 2. pH calibration curve. Data from individual channels (e.g. black squares) was fitted to Equation SI-1 to provide a calibration curve. The average calibration curve from seven curve fits (black line) and 95% confidence interval (grey band) were determined.

Electronic Supplementary Material (ESI) for Lab on a ChipThis journal is © The Royal Society of Chemistry 2011

Lab on a Chip Supplemental Information

1

On-chip CO2 control for microfluidic cell culture: Supplemental Information Samuel P. Forry and Laurie E. Locascio

Descriptions of Movies in Supporting Information Movie S1. Pervaporative Chamber Collapse

The device was loaded with water and heated to 37 C. Inlet and outlet channels leading to a central chamber were closed (t=0 min), and phase contrast images were acquired every 5 minutes while humidified gas was pumped through adjacent control channels. Multiple images (5x magnification) were stitched together digitally to display the whole field of view.

Movie S2. Microfluidic Culture with PCO2 Control at 0.05 atm

Suspended mouse fibroblasts were loaded into a microfluidic device prepared for cell culture. Flow was stopped for 2 hr to allow cells to attach. Subsequently, one volume-change (~50 nL) was pumped into the chamber over 90 s at the start of each hour. Water equilibrated at 5% CO2 was pumped through adjacent control lines throughout the experiment for PCO2 control. Phase contrast images of the culture were acquired at 20 min intervals.

Movie S3. Microfluidic Culture with PCO2 Control at 0.0 atm

Suspended mouse fibroblasts were loaded into a microfluidic device prepared for cell culture. Flow was stopped for 2 hr to allow cells to attach. Subsequently, one volume-change (~50 nL) was pumped into the chamber over 90 s at the start of each hour. Air-equilibrated water (0.0% CO2) was pumped through adjacent control lines throughout the experiment for PCO2 control. Phase contrast images of the culture were acquired at 15 min intervals.

Movie S4. Long-term Microfluidic Cell Culture

The culture depicted in Movie S2 was observed over one week in microfluidic culture. Conditions are described in the figure legend for Movie S2.

Electronic Supplementary Material (ESI) for Lab on a ChipThis journal is © The Royal Society of Chemistry 2011