Control of Cell Culture Microenvironment Using Programmable Systems

Control of cell culture microenvironment is a useful technique to close the experimental gap between in vitro and in vivo experimental models. The increasing popularity of microfluidic methods necessitates the study of PDMS’ tendency to absorb hydrophobic molecules into the polymer matrix and thus their apparent reduction in concentration can cause inconsistencies in assay results performed in PDMS systems. This absorptive behavior of PDMS can be studied using fluorescent dyes that act as model molecules to simulate drug/protein behavior and can be used to understand the effects of hydrophobicity of PDMS in microfluidic applications. Control of culture microenvironment also plays an important role when studying the effects of gasotransmitters in vitro. Conventional methods using H2S, and CO precursor molecules cause the generation of inconsistent and unreliable data due to the precursor-dependent release characteristics of gasotransmitter exposure. To alleviate this, and to establish a standardized experimental platform to stabilize exposures of short-lived gasotransmitters to more experimentally relevant timescales and concentrations, an Arduino-controlled was developed that made possible the pilot-scale experiments of multi-hour and multi-day exposures of in vitro models to gasotransmitters, as well as multi-gasotransmitter experiments into the same in vitro system. NaSH, when used as a precursor molecule results in short-lived exposure on the order of a few minutes, but that could be stabilized and extended to multiple hours and days when coupled with an active control system that regulated the concentration and temporal profiles of H2S and CO treatment. The tailored gasotransmitter-delivery also enabled the study of their concentration dependent physiological effects in vitro along with the novel ability to control multiple gasotransmitters. The active control system of gasotransmitter delivery also enables dose control with delivery profiles that are not currently possible. Exploratory in vitro studies are done with the active dosing system used for high throughput studies as well as dose-dependent ROS response studies. Cell viability of A549 cells in response to treatment with multiple gasotransmitters was quantified as downstream effects of exposure to ROS in a combinatorial scheme.