New cytometer measures cell stiffness to improve disease diagnosis

Researchers from Brown University and their collaborators have developed a new way to measure the properties of cells - an important development, they say, because accurate measurements of changes in cell elasticity can be used to better understand diseases, diagnose patient symptoms and provide more accurate prognoses.

For example, cancer cells from tumors typically soften as they become more dangerous and likely to spread, while blood diseases like malaria and sickle cell can cause red blood cells to stiffen. Mechanical changes on a cellular level are also seen in neurodegenerative, cardiovascular and chronic inflammatory illnesses.

As detailed in a study in the journal Lab on a Chip, the researchers developed what they call a "mechanophenotyping cytometer" - a microfluidic device designed to measure a cell's physical size and squishiness, known as its mechanical phenotype.

Mechanophenotyping is an underused tool, said lead study author Graylen Chickering, a Ph.D. candidate in biomedical engineering in the lab of Brown University associate professor Eric Darling. This is mostly because measurement technologies lag behind other ways of analyzing cell properties.

The gold standard for measuring the squishiness or stiffness of a cell is atomic force microscopy, Chickering explained, which requires cells to be adhered on a surface and tested one by one with a tiny indenter.

This method essentially works by poking a cell. Imagine looking at a water balloon, and if you poke right on the edge of the balloon versus the center, it might feel different. Poking cells is also fairly slow, making it difficult to study large numbers of cells in a reasonable amount of time."

Graylen Chickering, a Ph.D. candidate in biomedical engineering, Brown University

Cell travel time: a key measurement

In developing the new technique, the scientists instead focused on a measurement called time-of-flight, which is the time it takes a cell to travel through tiny liquid-filled channels.

"The cell is essentially traveling from one checkpoint to another, and we take timestamps from each checkpoint to determine the time-of-flight," Chickering said.

The researchers used existing fluorescence signals from the cytometer, which is an apparatus for counting and measuring cells, to determine cell size and then used time-of-flight to determine cell stiffness. Softer cells move toward the center of the channel where fluid is moving fastest, while stiffer cells stay by the edges where fluid moves more slowly.

Chickering said that compared to atomic force microscopy, which allows experienced scientists to measure one cell every 30 seconds or so, she was able to use the new approach to look at 60 to 100 cells per second, with rates up to hundreds or even thousands of cells per second being possible.

"The proof of concept was when Graylen produced data showing that cell particles of different stiffnesses and different sizes had different correlational time of flights, which aligned with, theoretically, what we were expecting," said study author Darling, an associate professor of medical science, engineering and orthopaedics at Brown. "The method was so clean and reproducible compared to previous methods, which can result in different measurements depending on how they're used."

The findings are the result of a multi-year collaboration between researchers from Brown's Institute for Biology, Engineering and Medicine and a team from the National Institute of Standards and Technology (NIST) in Maryland. Brown contributed synthetic cell-like particles that were ideal for experimentation, Darling said, while NIST scientists created the foundational design for the cytometer device.

"We brought to the collaboration our polymer cell mimics, which served as calibration particles of specific sizes and stiffness, mapping out how these properties influence different recorded metrics from the device," Darling said. "The NIST cytometer has the unique feature of multiple measurement regions, providing error quantification for each particle flowing through it. That allowed us to show how much variability - both biological and technical - existed in our measurements."

Future work will use the mechanophenotyping cytometer to study the mechanical properties of cells from human blood and tissue samples provided by Brown's clinical partners.

"We expect to see differences between healthy individuals and those with certain types of disease, such as cancer," Darling said. "The ultimate hope is that a device of this sort could help with diagnosis or prognosis alongside existing methods."

Funding for the study was provided by the National Science Foundation (grant CMMI 2054193) and the National Institute of Standards and Technology.