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Taking the brain apart to put it all together again

Fluidically linked Blood-Brain Barrier and Brain Organ Chips offer new method for studying the effects of drugs and disease on the brain and its blood vessels

By Lindsay Brownell

(BOSTON) — The human brain, with its 100 billion neurons that control every thought, word, and action, is the most complex and delicate organ in the body. Because it needs extra protection from toxins and other harmful substances, the blood vessels that supply the brain with oxygen and nutrients are highly selective about which molecules can cross from the blood into the brain and vice versa. These blood vessels and their unique network of supporting pericyte and astrocyte cells comprise the blood-brain barrier (BBB). When the BBB is disrupted, as happens with exposure to drugs such as methamphetamine (“meth”), the brain’s sensitive neurons become susceptible to harmful damage.

Beyond forming a physical barrier, the BBB is thought to directly interact with the brain and help regulate its function, but figuring out exactly how the cells of the BBB and the brain influence each other has been a challenge, as in vitro models (i.e., cells in a dish) are too simple and in vivo models (i.e., natural human brain tissue) too complex. Now, researchers at the Wyss Institute for Biologically Inspired Engineering have created a “just right” model of the BBB-brain interface using microfluidically linked Organ Chips that allows an unprecedented look into how the brain’s vasculature influences and regulates its metabolic function, and how meth and other drugs disrupt that interaction. The research is reported in Nature Biotechnology.

One Brain Chip (top) containing neurons and astrocytes is connected via microfluidic channels to two blood-brain barrier (BBB) chips containing endothelial cells and their supporting astrocytes and pericytes. The researchers were able to trace the flow of molecules from the vasculature across the BBB and into the brain, and found that substances produced by the endothelial cells help maintain neuronal function. Credit: Wyss Institute at Harvard University

“Most of today’s research on Organ Chips is focused on trying to pack more cell types onto each chip to approximate the complexity of whole organs, but the brain is already so complex that we decided to do the opposite and divide one organ onto multiple chips,” said first author Ben Maoz, Ph.D., a former Technology Development Fellow at the Wyss Institute who is currently an Assistant Professor at Tel Aviv University, Israel. “The beauty of this work is that it opens up another dimension for neurological research that no other method could by decoupling a very dense organ to unveil new interactions between the different structures within the brain.”

Joining Maoz as co-first authors of the paper are former Wyss Institute colleagues Anna Herland, Ph.D., who is now an Associate Professor at the Royal Institute of Technology and the Karolinska Institute in Stockholm, Sweden; and Edward FitzGerald, Ph.D., currently an Industrial Marie Skłodowska-Curie Fellow at Beactica AB and Uppsala University, Uppsala, Sweden.

The BBB-Brain Chip system consists of three polymer chips containing human cell-lined channels: one “influx” BBB Chip, a Brain Chip, and a second “efflux” BBB Chip. The BBB Chips have one microfluidic channel lined with endothelial cells through which flows culture medium that mimics blood, separated by a porous membrane from a parallel channel containing pericytes and astrocytes that is perfused with artificial cerebrospinal fluid (aCSF). The Brain Chip has a similar aCSF flow channel that is separated by another semipermeable membrane from a compartment containing human brain neurons and their supporting astrocytes to mimic brain tissue. The three chips’ aCSF channels are connected together in series, creating a fully linked system in which substances can diffuse from the vascular channel across the first BBB into the aCSF, enter the brain neuronal cell compartment, flow back into the aCSF, and ultimately diffuse out across the second BBB into the second vascular channel, as happens in vivo.

The team cultured human cells in the linked BBB-Brain Chips and exposed them to meth, which is known to disrupt the junctions between the cells of the BBB in vivo and cause it to “leak.” When meth was flowed through the blood vessel channel of the BBB Chip, it compromised the junctions of the BBB’s vascular endothelial cells and allowed the passage of molecules into the Brain Chip that normally wouldn’t be able to cross the BBB. This experiment confirmed that the model responds to meth the same way that the human brain does, making it a valuable tool for understanding how drugs and other substances work and developing new treatments.

Something else in the chips that were not exposed to meth also caught the scientists’ attention. They realized that the proteins expressed by the cells on fluidically linked BBB and Brain Chips were different from those expressed by cells on unlinked chips. For example, cells in all of the linked chips expressed higher levels of metabolism-associated proteins and lower levels of proteins involved in proliferation and migration than cells in unlinked chips, suggesting the cells in the Brain and BBB Chips were somehow influencing each other to maintain proper cell function.

Methamphetamine (“meth,” left) is known to disrupt the tight junctions – shown here in green – between the cells of the blood-brain barrier (BBB), letting toxic substances enter the brain. When the researchers added meth to healthy BBB cells (middle), it caused the junctions to become leaky (right), confirming that the BBB-Brain Chip system can be used to study the effects of drugs on the human brain. Credit: Wyss Institute at Harvard University

“Blood vessels are frequently thought to just be a barrier or a transporter of chemicals. But when we looked at the linked BBB-Brain Chips, we noticed that there seemed to be some crosstalk between the endothelial cells and the neurons,” said Herland. “We also know from studies of long-term meth abusers that this drug affects the brain’s metabolism, so we started to dig deeper to see if we could characterize the metabolic link between the BBB and the brain.”

The modular nature of the BBB-Brain Chip system allowed the researchers to analyze all of the molecules secreted by the individual cell types alone, and then connect the chips to trace where those substances traveled. The chemicals secreted by the cells on the uncoupled BBB Chip were largely related to neuron maintenance and protection, demonstrating that the molecules produced by the BBB travel into the brain and provide chemical cues to neurons. The scientists then administered radioactive carbon-labeled glucose, pyruvate, and lactate as energy supplies to unlinked Brain Chips so they could trace the carbon molecules as they were metabolized. They found that the production of both glutamine and the neurotransmitter GABA from the energy-supplying molecules was lower in unlinked Brain Chips than in Brain Chips linked to the BBB.

By demonstrating that products of blood vessel cell metabolism are used to produce neurotransmitters that mediate information processing by the brain’s neurons, these results suggest that the health of our blood vessels could have a direct impact on how our minds function.

“The big breakthrough here is that not only have we created a new model for studying the effects of drugs on the human brain, along the way we teased out the communication networks between cells in a way that never could have been done with traditional brain research techniques,” said corresponding author Kit Parker, Ph.D., a Core Faculty member of the Wyss Institute and the Tarr Family Professor of Bioengineering and Applied Physics at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS). “We are seeing here an unanticipated level of complexity that raises the bar in terms of what it will mean to successfully map the brain’s connectome.”

“What’s really incredible is that we were able to do a highly multiplexed, massively parallel metabolomic analysis of many different chemicals produced by different cell types, all on these tiny chips,” said co-corresponding author and Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at SEAS. “We’re excited to push the limits of how complicated and sophisticated Organ Chips can be, and potentially use this decoupling approach to analyze how vascular endothelial cells contribute to the specialized functions of other organs as well.”

How do you study something as complex as the human brain? Take it apart. Wyss researchers have created Organ Chips that mimic the blood-brain barrier and the brain and, by linking them together, discovered how our blood vessels and our neurons influence each other. Credit: Wyss Institute at Harvard University

This work was supported by the Wyss Institute for Biologically Inspired Engineering, DARPA, the Sweden-America Foundation, the Carl Trygger Foundation, and the Erik and Edith Fernström Foundation.

Additional authors of the study include Edward FitzGerald, Ph.D., a former Graduate Fellow at the Wyss Institute who is now a Ph.D. student at Uppsala University; Thomas Grevesse, Ph.D., a former Postdoctoral Fellow at the Wyss Institute and SEAS and current Intern Fellow at Stellenbosch University; Charles Vidoudez, Ph.D., a Mass Spectrometrist at Harvard University; Alan Pacheco, a graduate student at the Wyss Institute and Boston University; Sean Sheehy, Ph.D., a former Postdoctoral Fellow at the Wyss Institute and SEAS who is currently a Vertex Fellow at Vertex Pharmaceuticals; Tae-Eun Park, Ph.D., a former Postdoctoral Fellow at the Wyss Institute who is now an Assistant Professor at ULSAN National Institute of Science and Technology in Korea; Stephanie Dauth, Ph.D., a former Postdoctoral Fellow at the Wyss Institute and SEAS who is currently a Scientific Project Manager at Fraunhofer IME Frankfurt; Robert Mannix, a Research Technologist at Boston Children’s Hospital and the Wyss Institute; Nikita Budnik, a student at McGill University; Kevin Shores, a Graduate Research Assistant at Harvard SEAS; Alexander Cho, a Bioengineering Fellow at the Wyss Institute and SEAS; Janna Nawroth, Ph.D., a former Technology Development Fellow at the Wyss Institute and SEAS who is now a Principal Investigator at Emulate, Inc.; Daniel Segrè, Ph.D., Professor of Biology, Bioinformatics, and Biomedical Engineering at Boston University; and Bogdan Budnik, Ph.D., Director of Proteomics at Harvard University.

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