In 2015, the National Institute of Neurological Disorders and Stroke released a report stating that more than 600 neurological conditions—including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and motor neuron disease, among others—affect an estimated 50 million Americans, a number that is growing each year. Many of these diseases share a common feature in the degradation of the blood-brain barrier (BBB), the cloak of endothelial cells that disposes of the brain’s waste while also providing necessary nutrients.
To better understand these diseases and to develop new ways to treat them, scientists rely on increasingly sophisticated cellular models that attempt to mimic the full complexity of the BBB. The advent of hydrogels, microfluidics, and so-called organs on a chip all rely on stable cell lines to build a useful proxy, but new research suggests that all cells may not respond equally to experimentation.
The Scientist spoke with Alisa Morss Clyne and Callie Weber, two bioengineers at the University of Maryland whose recent review, published March 16 in APL Bioengineering, makes the case for the inclusion of sex as a biological variable in cell-based experiments. Men and women, a growing body of evidence shows, respond differently to brain diseases in ways that can profoundly influence a study’s findings. Men, for example, are 1.5 times more likely to be diagnosed with Parkinson’s disease and often experience the condition more severely, perhaps because the higher levels of estrogen in premenopausal women shield the BBB from damage. When purchasing cells, Clyne says, scientists are rarely aware of the sex of the original cell donor, but it may ultimately have important consequences for the study of diseases, neurological or otherwise.
The Scientist: What is the relationship between the health of the blood-brain barrier and the onset of neurological disease?
Callie Weber: A lot of it is not really known. It’s a ‘chicken or the egg’ question of whether neurological diseases cause dysregulation of the blood-brain barrier, or if the blood-brain barrier breaks down and that leads to a lot of neurodegenerative diseases. But it is known that for pretty much all neurodegenerative diseases, there is a correlation with breakdown of the blood-brain barrier. You lose tight junctions between the cells, and then things from your bloodstream start leaching into your brain, which can cause further neurodegeneration.
TS: How does sex—being male or female—play into that?
CW: There are so many things that are unknown about what the differences are between male and female cells, and those differences are probably one of the keys to understanding a lot of these neurological diseases.
Women have more estrogen [and estrogen derivatives], and estrogen has been shown to be protective in the vasculature all around the body. It helps produce nitric oxide, which helps your blood vessels dilate and keeps them healthy. But then, in the brain specifically, scientists are more unsure about what estrogen can do. It probably helps keep the blood-brain barrier together. People think that it probably leads to improved tight junction formation between the cells that form the blood-brain barrier. And then, same with your blood vessels, estrogen in the brain probably also helps produce nitric oxide, but there’s a lot less research on that side of things.
TS: Why should scientists be thinking about where their cell lines come from as they design experiments?
Alisa Morss Clyne: In many neurological diseases, there are sex-based differences either in their incidence, in their progression, or sometimes in their treatment and how people respond to different drugs. But there’s hardly anything known about what’s going on at a cellular level. Is a male cell from the blood-brain barrier different from a female cell in the blood-brain barrier? Are the interactions among cells from a person of one sex versus the other sex different? The goal of our paper really was for people who do in vitro work with cells to think about where their cells are coming from.
Female animals, for example, are always thought to be more difficult to study because their hormone levels are so variable, and this goes with human clinical trials as well. Some of our collaborators and colleagues make it very clear that they always take their samples at one phase of the menstrual cycle. But it’s not like men’s hormones are exactly the same every day, either. Hormonal differences are just a fact of life.
TS: What first tipped you off to the sex-based differences [between cells] in your own work?
AMC: We were doing a study a few years ago on pulmonary hypertension, and we got our cells from the Pulmonary Hypertension Breakthrough Initiative. When we got the cells . . . we had 12 sets—six female and six male. Initially, we just threw them all together and studied all of them with the disease and without the disease. But when we were comparing the six with the disease to the six without the disease, we couldn’t see any differences.
We decided to look at all 12 individually, and right away you could see . . . that there are these differences across the sexes in our cells that were consistent. To me, that was really surprising, because I had never thought about the sex of my cells before. I just always assumed that when I take my cells and put them in culture, the sex of the cells didn’t matter, because the sex of a person is so based on your biochemical differences. If I’m culturing them all in the same way, they should all be the same. And I was really surprised to find out that that wasn’t true.
TS: Has the realization that there are these sex-based differences between cells led to any changes in how you structure your own experiments?
AMC: In some ways, yes. Those pulmonary endothelial cells we got were from adult patients, but now we’re looking at cells from the umbilical cord, so they’re from babies just before they’re born. There really shouldn’t be significant hormonal differences, and they should be more consistent in terms of what they’ve been exposed to.
We’re also continuing to look at male versus female cell differences, and also how they respond differently to things that cause some of these cardiovascular diseases or neurodegenerative diseases. We’ve started working more with [collaborators] who have human samples where we can look at what’s different in the blood from men and women. There are more complexities than just taking your cells and giving them estrogen. We want to expose our cells to serum from men and women in various states and see how the cells respond.
In other cases, we’re still just using endothelial cells and ignoring the sex entirely. But I think now that our eyes have been opened to the fact that this is important, it’s going to come through in more of our work.
C.M. Weber, A.M. Clyne, “Sex differences in the blood-brain barrier and neurodegenerative diseases,” APL Bioeng, doi:10.1063/5.0035610, 2021.
Editor’s note: The interview was edited for brevity.