Researchers at the RIKEN Center for Biosystems Dynamics Research in Japan have developed a new system for keeping tissue viable for long-term study once transferred from an animal to a culture medium. The new system uses a microfluidic device made of polydimethylsiloxane (PDMS) with a porous membrane that can keep tissue from both drying out and from drowning in fluid. This study was published in the journal Analytical Sciences.
The team tested the device using tissue from the mouse suprachiasmatic nucleus, a complex part of the brain that governs circadian rhythms. By measuring the level of bioluminescence coming from the brain tissue, they were able to see that tissue kept alive by their system stayed active and functional for over 25 days with nice circadian activity. In contrast, neural activity in tissue kept in a conventional culture decreased by 6% after only 10 hours.
This new method will be useful in observing development and testing how tissues respond to drugs. Experiments with tissues are much more complex and provide important information such as cell to cell interaction, unlike seeded cells where such observation is difficult.
Researchers from MIT have developed “physiome on chip” technology that could be used to evaluate new drugs and detect side effects before the drugs are tested in human clinical trials. This could be used an alternative to animal testing for pharmacological testing before human clinical trials.
Body on a chip. Credit: Felice Frankel
They used a microfluidic platform that connects engineered tissues from ten different organs,. By doing so, the researchers were able to accurately replicate human organ interactions considerable periods at a time, allowing them to measure the effects of medication on different organs. This system is also well designed to test immunotherapy as the antibodies are unique to humans and could not be reliably tested in animals. This research is published in the journal Scientific Reports.
“Some of these effects are really hard to predict from animal models because the situations that lead to them are idiosyncratic,” says Linda Griffith, one of the senior authors of the study. “With our chip, you can distribute a drug and then look for the effects on other tissues and measure the exposure and how it is metabolized.”
Griffith believes that the immediate applications of this technology involve modeling on fewer organ systems. Griffith’s lab is now developing a model system to investigate the role of the gut microbiome in Parkinson’s disease by creating body on chip that contains brain, liver, and gastrointestinal tissue.
“An advantage of our platform is that we can scale it up or down and accommodate a lot of different configurations,” Griffith says. “I think the field is going to go through a transition where we start to get more information out of a three-organ or four-organ system, and it will start to become cost-competitive because the information you’re getting is so much more valuable.”
Citation: Edington, Collin D., Wen Li Kelly Chen, Emily Geishecker, Timothy Kassis, Luis R. Soenksen, Brij M. Bhushan, Duncan Freake, Jared Kirschner, Christian Maass, Nikolaos Tsamandouras, Jorge Valdez, Christi D. Cook, Tom Parent, Stephen Snyder, Jiajie Yu, Emily Suter, Michael Shockley, Jason Velazquez, Jeremy J. Velazquez, Linda Stockdale, Julia P. Papps, Iris Lee, Nicholas Vann, Mario Gamboa, Matthew E. Labarge, Zhe Zhong, Xin Wang, Laurie A. Boyer, Douglas A. Lauffenburger, Rebecca L. Carrier, Catherine Communal, Steven R. Tannenbaum, Cynthia L. Stokes, David J. Hughes, Gaurav Rohatgi, David L. Trumper, Murat Cirit, and Linda G. Griffith. “Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies.” Scientific Reports 8, no. 1 (2018). doi:10.1038/s41598-018-22749-0.
Research funding: U.S. Army Research Office and DARPA.
Scientists at Vanderbilt University have created a 3D organ-on-a-chip that can mimic the biomechanical properties of the heart. The device and the results of initial experiments are reported in the journal Acta Biomaterialia (for links see below).
View of the cardiac fiber in the I-Wire device at two levels of magnification. Credit: VIIBRE Vanderbilt University
The unique aspect of the new device, which represents about two-millionths of a human heart, is that it controls the mechanical force applied to cardiac cells. This allows the researchers to reproduce the mechanical conditions of the living heart in addition to its electrical and biochemical environment.
“We created the I-Wire Heart-on-a-Chip so that we can understand why cardiac cells behave the way they do by asking the cells questions, instead of just watching them,” said Gordon A. Cain University Professor John Wikswo, who heads up the project. “We believe it could prove invaluable in studying cardiac diseases, drug screening and drug development, and, in the future, in personalized medicine by identifying the cells taken from patients that can be used to patch damaged hearts effectively.”
The I-Wire device consists of a thin thread of human cardiac cells 0.014 inches thick (about the size of 20-pound monofilament fishing line) stretched between two perpendicular wire anchors. The amount of tension on the fiber can be varied by moving the anchors in and out, and the tension is measured with a flexible probe that pushes against the side of the fiber. The fiber is supported by wires and a frame in an optically clear well that is filled with a liquid medium like that which surrounds cardiac cells in the body. The apparatus is mounted on the stage of a powerful optical microscope that records the fiber’s physical changes. The microscope also acts as a spectroscope that can provide information about the chemical changes taking place in the fiber. A floating microelectrode also measures the cells’ electrical activity.
According to the researchers, the I-Wire system can be used to characterize how cardiac cells respond to electrical stimulation and mechanical loads and can be implemented at low cost, small size and low fluid volumes, which make it suitable for screening drugs and toxins. Because of its potential applications, Vanderbilt University has patented the device. Unlike other heart-on-a-chip designs, I-Wire allows the researchers to grow cardiac cells under controlled, time-varying tension similar to what they experience in living hearts.
To demonstrate the I-Wire’s value in determining the effects that different drugs have on the heart, the scientists tested its response with two drugs known to affect heart function in humans: isoproterenol and blebbistatin. Isoproterenol is a medication used to treat bradycardia (slow heart rate) and heart block (obstruction of the heart’s natural pacemaker). Blebbistatin inhibits contractions in all types of muscle tissue, including the heart.
According to Veniamin Sidorov, the research assistant professor at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) who led its development, the device faithfully reproduces the response of cardiac cells in a living heart.
Citations 1. Sidorov, Veniamin Y., Philip C. Samson, Tatiana N. Sidorova, Jeffrey M. Davidson, Chee C. Lim, and John P. Wikswo. “I-Wire Heart-on-a-Chip I: Three-dimensional cardiac tissue constructs for physiology and pharmacology.” Acta Biomaterialia 48 (2017): 68-78. doi:10.1016/j.actbio.2016.11.009. 2. Schroer, Alison K., Matthew S. Shotwell, Veniamin Y. Sidorov, John P. Wikswo, and W. David Merryman. “I-Wire Heart-on-a-Chip II: Biomechanical analysis of contractile, three-dimensional cardiomyocyte tissue constructs.” Acta Biomaterialia 48 (2017): 79-87. doi:10.1016/j.actbio.2016.11.010.
Research funding: National Institutes of Health, National Science Foundation, Defense Threat Reduction Agency, American Heart Association, Department of Veterans Affairs. Adapted from press release by Vanderbilt University.
Harvard University researchers have developed a multiregional brain-on-a-chip that models the connectivity between three distinct regions of the brain. The in vitro model was used to extensively characterize the differences between neurons from different regions of the brain and to mimic the system’s connectivity. The research was published in the Journal of Neurophysiology.
Image of the in vitro model showing three distinct regions of the brain connected by axons. Credit: Disease Biophysics Group/Harvard University
“The brain is so much more than individual neurons,” said Ben Maoz, co-first author of the paper and postdoctoral fellow in the Disease Biophysics Group in the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS). “It’s about the different types of cells and the connectivity between different regions of the brain. When modeling the brain, you need to be able to recapitulate that connectivity because there are many different diseases that attack those connections.”
Researchers from the Disease Biophysics Group at SEAS and the Wyss Institute modeled three regions of the brain most affected by schizophrenia the amygdala, hippocampus and prefrontal cortex. They began by characterizing the cell composition, protein expression, metabolism, and electrical activity of neurons from each region in vitro.
“It’s no surprise that neurons in distinct regions of the brain are different but it is surprising just how different they are,” said Stephanie Dauth, co-first author of the paper and former postdoctoral fellow in the Disease Biophysics Group. “We found that the cell-type ratio, the metabolism, the protein expression and the electrical activity all differ between regions in vitro. This shows that it does make a difference which brain region’s neurons you’re working with.”
Next, the team looked at how these neurons change when they’re communicating with one another. To do that, they cultured cells from each region independently and then let the cells establish connections via guided pathways embedded in the chip.
The researchers then measured cell composition and electrical activity again and found that the cells dramatically changed when they were in contact with neurons from different regions.
“When the cells are communicating with other regions, the cellular composition of the culture changes, the electrophysiology changes, all these inherent properties of the neurons change,” said Maoz. “This shows how important it is to implement different brain regions into in vitro models, especially when studying how neurological diseases impact connected regions of the brain.”
To demonstrate the chip’s efficacy in modeling disease, the team doped different regions of the brain with the drug Phencyclidine hydrochloride commonly known as PCP which simulates schizophrenia. The brain-on-a-chip allowed the researchers for the first time to look at both the drug’s impact on the individual regions as well as its downstream effect on the interconnected regions in vitro.
The brain-on-a-chip could be useful for studying any number of neurological and psychiatric diseases, including drug addiction, post-traumatic stress disorder, and traumatic brain injury.
“To date, the Connectome project has not recognized all of the networks in the brain,” said Parker. “In our studies, we are showing that the extracellular matrix network is an important part of distinguishing different brain regions and that, subsequently, physiological and pathophysiological processes in these brain regions are unique. This advance will not only enable the development of therapeutics, but fundamental insights as to how we think, feel, and survive.”
Citation: Dauth, Stephanie, Ben M. Maoz, Sean P. Sheehy, Matthew A. Hemphill, Tara Murty, Mary Kate Macedonia, Angie M. Greer, Bogdan Budnik, and Kevin Kit Parker. “Neurons derived from different brain regions are inherently different in vitro: A novel multiregional brain-on-a-chip.” Journal of Neurophysiology (2016): jn-00575. DOI: 10.1152/jn.00575.2016 Research funding: Defense Advanced Research Projects Agency. Adapted from press release by Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).
The blood-brain barrier is a network of specialized cells that surrounds the arteries and veins within the brain. It forms a unique gateway that both provides brain cells with the nutrients they require and protects them from potentially harmful compounds.
This is an illustration of the neurovascular unit on a chip. Credit: Dominic Doyle, Vanderbilt University
An interdisciplinary team of researchers from the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) headed by Gordon A. Cain University Professor John Wikswo report that they have developed a microfluidic device that overcomes the limitations of previous models of this key system and have used it to study brain inflammation, dubbed the “silent killer” because it doesn’t cause pain but contributes to neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. Recent research also suggests that it may underlie a wider range of problems from impaired cognition to depression and even schizophrenia.
The project is part of a $70 million “Tissue Chip for Drug Testing Program” funded by the National Institutes of Health’s National Center for Advancing Translational Sciences. Its purpose is to develop human organ-on-a-chip technology in order to assess the safety and efficacy of new drugs in a faster, cheaper, more effective and more reliable fashion.
The importance of understanding how the blood-brain barrier works have increased in recent years as medical researchers have found that this critical structure is implicated in a widening range of brain disorders, extending from stroke to Alzheimer’s and Parkinson’s disease to blunt force trauma and brain inflammation.
Despite its importance, scientists have had considerable difficulty creating faithful laboratory models of the complex biological system that protects the brain. Previous models have either been static and so have not reproduced critical blood flow effects or they have not supported all the cell types found in human blood-brain barriers.
The new device, which the researchers call a NeuroVascular Unit (NVU) on a chip, overcomes these problems. It consists of a small cavity that is one-fifth of an inch long, one-tenth of an inch wide and three-hundredths of an inch thick – giving it a total volume of about one-millionth of a human brain. The cavity is divided by a thin, porous membrane into an upper chamber that acts as the brain side of the barrier and a lower chamber that acts as the blood or vascular side. Both chambers are connected to separate microchannels hooked to micropumps that allow them to be independently perfused and sampled.
To create an artificial blood-brain barrier, the researchers first flip the device over so the vascular chamber is on top and inject specialized human endothelial cells. They found that if they maintain a steady fluid flow through the chamber during this period, the endothelial cells, which left to themselves form shapeless blobs, consistently orient themselves parallel to the direction of flow. This orientation, which is a characteristic of the endothelial cells in human blood-brain barrier, has been lacking in many previous models.
After a day or two, when the endothelial cells have attached themselves to the membrane, the researchers flip the device and inject the two other human cell types that form the barrier — star-shaped astrocytes and pericytes that wrap around endothelial cells — as well as excitatory neurons that may regulate the barrier. These all go into the brain chamber that is now on top. The porous membrane allows the new cells to make physical and chemical contact with the endothelial cells just as they do in the brain.
The researchers were able to purchase the human endothelial cells, astrocytes and pericytes that they need from commercial sources. For the excitatory neurons required, they turned to Vanderbilt University Medical Center collaborators M. Diana Neely, research associate professor of pediatrics, and Aaron Bowman, associate professor of pediatrics, neurology and biochemistry. Starting with human induced pluripotent stem cells that are generated directly from adult cells they were able to produce the specialized neurons that the project needed.
“This is one of the most exciting projects I’m involved with,” said Neely. “Although it’s still in its infancy, it has tremendous potential.” According to Bowman, one potential application is to develop tissue chips that contain cells from individual patients, making it possible to predict their personal reactions to different drugs.
“Once we had successfully created the artificial barrier, we subjected it to a series of basic tests and it passed them all with flying colors. This gives us the confidence to state that we have developed a fully functional model of the human blood-brain barrier,” said VIIBRE staff scientist Jacquelyn Brown, who is first author of the paper “Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor” that described this achievement in the journal Biomicrofluidics.
“The NeuroVascular Unit (NVU) on a chip has reached the point where we can begin using it to test different drugs and compounds,” observed team member Donna Webb, associate professor of biological sciences who is interested in studying how different substances affect synapses — the junctions between neurons. “There is an urgent need for us to understand how various substances affect cognitive processes. When we do, we will be in for a number of surprises!
Already, the VIBRE team has used the NeuroVascular Unit (NVU) on a chip to overcome a basic limitation of existing studies of brain inflammation, which have only produced snapshots of the process at various stages. Because the NeuroVascular Unit (NVU) on a chip can be continuously monitored, it has provided the first dynamic view of how the brain and blood-brain barrier respond to systemic inflammation.
These results are summarized in a paper titled “Metabolic consequences of inflammatory disruption of the blood-brain barrier in an organ-on-chip model of the human neurovascular unit” accepted for publication in the Journal of Neuroinflammation.
The scientists exposed the NeuroVascular Unit (NVU) on a chip to two different compounds known to induce brain inflammation: a large molecule found on the surface of certain bacteria called lipopolysaccharide and a “cocktail” of small proteins called cytokines that play an important role in immune response to inflammation.
“One of our biggest surprises was the discovery that a critical component in the blood-brain barrier’s response to these compounds was to begin increasing protein synthesis,” said Brown. “Next will be to find out which proteins it is making and what they do.”
The researchers also found that the blood vessels in the barrier respond to inflammation by pumping up their metabolic rate while the metabolism of the brain cells slows down. According to Brown, “It might be that the vasculature is trying to respond while the brain is trying to protect itself.”
Citation: “Metabolic consequences of inflammatory disruption of the blood-brain barrier in an organ-on-chip model of the human neurovascular unit” accepted in Journal of Neuroinflammation. Research funding: NIH