Workflow patterns effect pH variance in the microdroplet culture system

Abstract

Objective Determine if disruptive incubator work patterns have prolonged observable effects on pH value variability in culture microdroplets. Design This is a quantitative study evaluating real-time pH value variation during incubator disturbance conditions. Materials and methods Real time pH data was evaluated under two workflow patterns: low disturbance (incubator opened at beginning/end of the working day for 15 seconds) and high disturbance (incubator opened every hour for 15 seconds). A membrane impregnated with a fluorescent dye-protein conjugate that emits characteristic wavelength spectra at different pH levels was placed at the bottom of a cell culture dish. 50 µL of culture media was placed directly on the membrane under oil and allowed to equilibrate for 24 hours at 37°C and 5% CO2. pH values were collected from the microdroplet every 30 minutes using a continuous pH monitoring device (SAFE Sens® TrakStation® pH monitoring technology) inside a large format incubator. Results The average pH value in the microdroplet before testing was 7.32±0.002 (n = 32) for low disturbance testing and 7.33±0.005 (n = 32) for high disturbance testing. The pH coefficient of variance during high disturbance testing (0.066) was not significant compared to the pH coefficient of variance (0.060) of low disturbance testing. The average pH value collected during low disturbance testing was 7.32±0.003 (n = 32), and the average pH value collected during high disturbance testing was 7.35±0.013 (n = 32). The pH coefficient of variance during high disturbance testing was 0.182 and the coefficient of variance during low disturbance testing was 0.067. pH values collected 8 hours after disturbance averaged to 7.32±0.002 (n = 32) for low disturbance testing, and 7.34±0.013 (n = 32) for high disturbance testing. The pH coefficient of variance during high disturbance testing was 0.135 and the coefficient of variance was 0.076 for low disturbance. Conclusions Previous pH studies have used indirect measurement techniques that are not representative of the true pH of culture media, as pH can vary due to differences in media volume, oil overlay usage, and geometric shape. Using direct microdroplet pH testing, frequent disturbances in the incubator environment have an observable effect on microdrop culture dish pH variability. The act of opening and closing the incubator door also affects pH variability for at least 8 hours after ending incubator disturbances. Disclosures Author's company sells and markets SAFE Sens products. Funding None