Grande-Allen and her team designed their fluorescent particles to report on oxygen levels inside gels. Their work appears in the American Chemical Society journal ACS Biomaterials Science and Engineering.
“We’ve been collaborating with investigators in intestinal mechanobiology and wanted a straightforward way to tell what level of oxygen we had throughout our 3D tissue cultures,” Grande-Allen said. “Where we intend a specific level of oxygen, we want to be sure that’s what the cells are getting.
“There are multiple ways of doing this,” she said. “We can have computational models, but we’d have to make several assumptions about the way oxygen permeates the culture medium and 3D scaffold material. A better way is to measure it directly, so that was our goal.”
Lead author Reid Wilson, an M.D./Ph.D. student at Rice and Baylor College of Medicine, built on the work of Rice alumnus Matthew Sapp and Rice graduate student Sergio Barrios to develop soft microparticles that that incorporate an oxygen-triggered fluorescent molecule based on palladium and a reference fluorophore.
Wilson went through several iterations of dye combinations and concentrations to develop those microparticles. “The problem with using oxygen-responsive fluorophores in three-dimensional cultures is their signal isn’t bright enough to measure reliably,” he said. “So we loaded the microparticles with high concentrations of dye, which allowed more reproducible measurements of the oxygen concentration.”
The particles can be suspended in hydrogel along with living cells, and tests showed they are not toxic to those cells. Signals from the fluorescent components can be read at their individual wavelengths, but their power lies in combining the response from both, which gives clinicians the ability to measure oxygen content as far as 2 millimeters into tissues.
“That’s small, but oxygen diffusion limits are usually tiny,” Grande-Allen said. “Some cells are quite close to a blood supply, with a high oxygen level brought in by blood cells with hemoglobin. But some bacteria in the microbiome are normally anaerobic and survive better without oxygen.”
Grande-Allen said the particles aren’t susceptible to photobleaching (fading) when illuminated at the proper wavelength, nor did they sink out of the hydrogel, as larger fluorescent particles were prone to do, even after a year in storage.
She noted that tissues like cartilage and certain types of diseased heart valves don’t have vascular networks, yet their cells thrive. “I’ve always wondered how these cells get nourishment and what they need to survive,” she said. “With oxygen-sensing microparticles and other techniques we use in my lab to stretch living and engineered materials, we can start to work toward answering these questions.”
Rice research scientist Jennifer Connell is co-author of the paper. Grande-Allen is a professor of bioengineering and chair of the Rice Department of Bioengineering.
The National Institute of Allergy and Infectious Disease and the National Institute of Diabetes and Digestive and Kidney Diseases supported the research.