Integration of real time oxygen measurements with a 3D perfused tissue culture system

Abstract

In vitro models that capture the complexity of human tissue and organ behaviors in a scalable and easy-to- use format are of increasing interest for both technological applications in drug development and in basic biology research. Tissues and organs are perfused continuously with blood, which delivers nutrients, oxygen, and macromolecular regulatory molecules. In vitro culture models that incorporate local micro-perfusion in a format that allows accesses to cells and their microenvironment are desirable to a broad research community. This thesis describes a platform that features an array of bioreactors that foster three dimensional tissue organization under continuous perfusion. Each bioreactor contains a scaffold that supports formation of hundreds of 3D microscale tissue units. Perfusion through the tissue is achieved using integrated pneumatic diaphragm micropumps. Pumps continuously circulate cell culture medium within each of the fluidically isolated bioreactors in the array. Pulsatile flow from the pumps is filtered using integrated fluidic capacitors such that the flow rate through the scaffold is constant. The format of the device mimics the familiar multiwell tissue culture plate and is easily integrated into existing laboratory facilities. One desirable feature for both parsing metabolic function and assessing response to treatments is a real time read out of oxygen tension at key points in the bioreactor. Such added dimension of real time measurement significantly enhances the value of a cue-response experiment such as a liver drug toxicology study. The thesis describes optical oxygen sensors that measure the florescence decay time of a ruthenium complex, which varies predictably in different oxygen environments. The sensors excite a layer of ruthenium glued to the end of an optical fiber using a stochastic signal from a light emitting diode (LED). The response is then measured on a photodiode. System identification techniques are used to determine the relevant time constants which are subsequently converted to oxygen measurements. Application to real time monitoring of liver tissue function is used for illustration of the utility of the measurements.