Many biotechnology studies require isolating cells from living tissues. This is often done by mixing tissue samples with enzyme proteins that digest the matrix holding cells together, producing cultures of living cells.
Those digestive enzymes are often mixed with other tissue processing reagents (chemicals) in pre-formulated, “ready-to-use” solutions. However, while he was refining an advanced neuron isolation protocol, Department of Civil and Environmental Engineering (CEE) Research Associate Professor Larry Millet noticed something was off.
“As you add advanced components to enhance tissue dissociation, that cumulative addition of reagents can change the final solution pH (a measure of acidity),” Millet said. “But these protocols—and the papers that use them—don’t usually report pH explicitly.”
Cells can only operate within a narrow pH range. Neurons are sensitive and work best around pH 7.4, a bit less acidic than pure water. Low brain pH (acidity) is associated with multiple neurodegenerative diseases, including Alzheimer’s disease.
In spite of these tangible consequences, Millet’s team found that a common neuronal cell digestion formulation, as published, has a final solution pH of 6.6. Only three of the 50 most-cited neuronal digestion studies from the last 15 years report their final solution pH.
“pH is important in chemistry, biology, and tissue engineering, but any parameter can be overlooked in routine processes,” explained Millet. “With so many off-the-shelf products available, pH can become a background assumption rather than an active variable that experimenters need to verify.”
Since Millet’s interdisciplinary research program integrates the physical, chemical, and life sciences, his lab is ideally placed to set rigorous pH standards. With funding from the National Institutes of Health, Millet recruited two Engineering Vols from the Department of Biomedical Engineering (BME), Joshua Hilner and Allison Turner (both BS ‘25), to refine a reliable, easy method to monitor pH before or during cell digestion protocols.
“With persistence, repeated experimentation, extensive literature review, and guidance from Dr. Millet, we were able to optimize the protocol and achieve reliable, reproducible results,” said Turner, who is applying to medical school while working as an operating room attendant at the University of Tennessee Medical Center.
The team’s study was published in the open access journal Biosensors in November, 2025.
“I am honored to have led a publication in a journal that offers strong visibility and broad impact,” said Hilner, now a medical student at the Edward Via College of Osteopathic Medicine–Carolinas Campus. “I hope this study highlights the research potential of undergraduate students and underscores the importance of intentional faculty mentorship.”
Seniors are Ready for Research
Reproducibility—the idea that performing the same study multiple times will give the same result—is fundamental to scientific research. Failing to account for a fundamental variable like pH means that a single lab could conduct a published tissue digestion protocol with exactly the same materials multiple times but achieve different results.
“Enhancing reproducibility requires working under carefully controlled conditions and continuously identifying, reviewing, and reassessing potential sources of error,” Millet said.
In the spring of 2024, Millet overheard Hilner and Turner’s insightful conversation about material from their BME classes and immediately recognized their aptitude for this pH validation project.
“Undergraduate seniors are equipped to contribute meaningfully to research—the transition from a senior undergraduate to a new graduate student is marked by just one summer,” Millet said. “Joshua and Allison were driven, thoughtful, and used to collaborating as classmates. They were well prepared to drive this study to completion.”
Patience and Phenol Red
Over the next year, Hilner, Turner, and a visiting undergraduate from Texas State University worked to standardize the qualitative (descriptive) readings of phenol red, a widely used and inexpensive pH indicator that shows acidic solutions as yellow and basic (alkaline) solutions as red or purple.
The undergraduates used a spectrophotometer, a machine that measures light wavelengths, to analyze the color profile of a solution containing phenol red as they altered the solution’s pH. They compared those values with readings from a specialty, small volume glass pH probe, creating a set of references that translated the colors into quantitative (numerical) data.
The refined protocol allowed them to isolate and culture healthy cells that would otherwise have been poisoned by acidic enzyme media.
“Research like this plays a vital role in advancing medical knowledge and improving patient care,” said Turner, an aspiring surgeon. “This project helped me develop a structured, analytical mindset grounded in curiosity, which I believe will be invaluable as I prepare to make high-stakes decisions in patient care.”
Hilner agreed, “I am deeply grateful to Dr. Millet for fostering an environment that encourages students to reach their full potential.”
Now that the students’ detailed method is freely available, other labs can automate pH readings for cell dissolution protocols—not to mention other biochemical and medical applications.
“Tissue engineering and organ-on-a-chip platforms are at the forefront of multidisciplinary research,” Millet said. “Publishing in Biosensors makes this work freely available to readers worldwide; highlights the University of Tennessee’s strengths in applied, high-impact science with real-world societal relevance; and shows undergraduates that they, too, can conduct meaningful research with faculty mentorship.”
Contact
Izzie Gall ([email protected])