Dissolved oxygen determination in two-dimensional tissue cultures using optical sensors

Oxygen concentration determination is of importance across many different industries, biomedical research among them. In biological systems, in vivo oxygen levels vary across a wide spectrum and strongly influence function of cells and tissues. Direct measurements of oxygen tension and its progression in real-time are necessary in order to understand and control cell survival, proliferation and differentation. In this thesis, special attention is payed to skin tissue. An in-depth understanding of these aspects will allow to provide better diagnostic and therapeutic tools for wound care. The general objective of this thesis is to analyze dissolved oxygen concentration in a two-dimensional tissue culture of a keratinocyte cell line (HaCaT) via the development and application to this purpose of an optical sensor. The sensor is based on a metalloporphyrin membrane (PtOEP) that emits a fluorescence signal that is quenched by oxygen in the environment. Images of the membrane are captured using time-lapse microscopy with a filter at the desired wavelength. Average pixel value is then analyzed, as it is directly related to O2 concentration. The porphyrin-based optical sensor developed in this work is proved to be compatible with cell culture. Using this sensor, changes in oxygen concentration in a physiological range can be detected. Dissolved O2 concentration in culture medium is found to decrease at high cell densities as a result of O2 consumption. It is also possible monitor discontinuities in a monolayer of cells by detection of local O2 concentration using this sensor. Ingeniería Biomédica