Bioengineering’s Dan Huh and colleagues have developed a number of organ-on-a-chip devices to simulate how human cells grow and perform in their natural environments. Their latest is a muscle-on-a-chip, which carefully captures the directionality of muscle cells as they anchor themselves within the body. See the full infographic at the bottom of this story. (Illustration by Melissa Pappas).
Studying drug effects on human muscles just got easier thanks to a new “muscle-on-a-chip,” developed by a team of researchers from Penn’s School of Engineering and Applied Science and Inha University in Incheon, Korea.
Muscle tissue is essential to almost all of the body’s organs, however, diseases such as cancer and diabetes can cause muscle tissue degradation or “wasting,” severely decreasing organ function and quality of life. Traditional drug testing for treatment and prevention of muscle wasting is limited through animal studies, which do not capture the complexity of the human physiology, and human clinical trials, which are too time consuming to help current patients.
An “organ-on-a-chip” approach can solve these problems. By growing real human cells within microfabricated devices, an organ-on-a-chip provides a way for scientists to study replicas of human organs outside of the body.
Using their new muscle-on-a-chip, the researchers can safely run muscle injury experiments on human tissue, test targeted cancer drugs and supplements, and determine the best preventative treatment for muscle wasting.
Dan Huh, Ph.D.
This research was published in Science Advances and was led by Dan Huh, Associate Professor in the Department of Bioengineering, and Mark Mondrinos, then a postdoctoral researcher in Huh’s lab and currently an Assistant Professor of Biomedical Engineering at Tulane University. Their co-authors included Cassidy Blundell and Jeongyun Seo, former Ph.D. students in the Huh lab, Alex Yi and Matthew Osborn, then research technicians in the Huh lab, and Vivek Shenoy, Eduardo D. Glandt President’s Distinguished Professor in the Department of Materials Science and Engineering. Lab members Farid Alisafaei and Hossein Ahmadzadeh also contributed to the research. The team collaborated with Insu Lee and professors Sun Min Kim and Tae-Joon Jeon of Inha University.
In order to conduct meaningful drug testing with their devices, the research team needed to ensure that cultured structures within the muscle-on-a-chip were as close to the real human tissue as possible. Critically, they needed to capture muscle’s “anisotropic,” or directionally aligned, shape.
“In the human body, muscle cells adhere to specific anchor points due to their location next to ligament tissue, bones or other muscle tissue,” Huh says. “What’s interesting is that this physical constraint at the boundary of the tissue is what sculpts the shape of muscle. During embryonic development, muscle cells pull at these anchors and stretch in the spaces in between, similar to a tent being held up by its poles and anchored down by the stakes. As a result, the muscle tissue extends linearly and aligns between the anchoring points, acquiring its characteristic shape.”
The team mimicked this design using a microfabricated chip that enabled similar anchoring of human muscle cells, sculpting three-dimensional tissue constructs that resembled real human skeletal muscle.
The National Science Foundation’s CAREER Award is given to early-career researchers in order to kickstart their careers in innovative and pivotal research while giving back to the community in the form of outreach and education. Alex Hughes, Assistant Professor in Bioengineering and in Cell and Developmental Biology, is among the Penn Engineering faculty members who have received the CAREER Award this year.
Hughes plans to use the funds to develop a human kidney model to better understand how the development of cells and tissues influences congenital diseases of the kidney and urinary tract.
The model, known as an “organoid,” is a lab-grown piece of human kidney tissue on the scale of millimeters to centimeters, grown from cultured human cells.
“We want to create a human organoid structure that has nephrons, the filters of the kidney, that are properly ‘plumbed’ or connected to the ureteric epithelium, the tubules that direct urine towards the bladder,” says Hughes. “To achieve that, we have to first understand how to guide the formation of the ureteric tubule networks, and then stimulate early nephrons to fuse with those networks. In the end, the structures will look like ‘kidney subunits’ that could potentially be injected and fused to existing kidneys.”
The field of bioengineering has touched on questions similar to those posed by Hughes, focusing on drug testing and disease treatment. Some of these questions can be answered with the “organ-on-a-chip” approach, while others need an even more realistic model of the organ. The fundamentals of kidney development and questions such as “how does the development of nephrons affect congenital kidney and urinary tract anomalies?” require an organoid in an environment as similar to the human body as possible.
“We decided to start with the kidney for a few reasons,” says Hughes. “First, because its development is a beautiful process; the tubule growth is similar to that of a tree, splitting into branches. It’s a complex yet understudied organ that hosts incredibly common developmental defects.
“Second,” he says, “the question of how things form and develop in the kidney has major medical implications, and we cannot answer that with the ‘organ-on-a-chip’ approach. We need to create a model that mimics the chemical and mechanical properties of the kidney to watch these tissues develop.”
The fundamental development of the kidney can also answer other questions related to efficiency and the evolution of this biological filtration system.
“We have the tendency to believe that systems in the human body are the most evolved and thus the most efficient, but that is not necessarily true,” says Hughes. “If we can better understand the development of a system, such as the kidney, then we may be able to make the system better.”
Hughes’ kidney research will lay the foundation for broader goals within regenerative medicine and organ transplantation.
Andrei Georgescu (left) and Dan Huh are the co-founders of Vivodyne, a spin-off of Huh’s BIOLines lab.
Dan Huh, Associate Professor in the Department of Bioengineering, has been steadily growing a collection of organs-on-chips. These devices incorporate human cells into precisely engineered microfluidic channels that mimic an organ’s natural environment, providing a way to conduct experiments that would not otherwise be feasible.
Huh’s previous research has involved using a placenta-on-a-chip to study which drugs are able to reach a developing fetus; investigating microgravity’s effect on the immune system by sending one of his chips to the International Space Station; and testing treatments for dry eye disease using an eye-on-a-chip, complete with a mechanical blinking eyelid.
Now, he and his colleagues are taking this technology out of their lab and into industry with their company, Vivodyne.
Andrei Georgescu, Huh’s lab-member and co-founder of Vivodyne, recently spoke with Technical.ly Philly’s Paige Gross about the growth of their company.
Research into potential drugs is usually performed first on mice, and success is only found in a fraction of humans once implemented in clinical trials, Andrei Georgescu, cofounder and CEO of Vivodyne, told Technical.ly. The genetic makeup just isn’t similar enough. But technology that allows scientists to test therapies on lab-grown human organs called “organs on chip” is allowing for testing without human subjects.
The organs on chip allow for a drug to react to tissue in a more similar way to the body than it would in a petri dish, Georgescu said. Cells sense their environment very well, he added.
“We’re making the environment more complicated, making its spacial features complicated enough to match the native complexity of the organs,” he said. “When [cells] sense a softer environment, they start to behave more realistically. Their response to the drug is more realistic.”
Huh’s organ-on-a-chip devices contain human cells, allowing for experiments that could not otherwise be practically or ethically performed.
Chlorine gas is a commonly used industrial chemical. It is also highly toxic and potentially deadly; it was used as a chemical weapon in both World War I and the Syrian Civil War and has led to multiple deaths from industrial accidents. Mixing certain household cleaners can also produce the toxic gas, leading to lasting lung injuries for which there are currently no effective treatments.
Now, researchers at Penn Engineering and Penn’s Perelman School of Medicine are collaborating with BARDA, the U.S. Office of Health and Human Services’ Biomedical Advanced Research and Development Authority, to address this need using their lung-on-a-chip technology.
The laboratory of Dan Huh, associate professor in the Department of Bioengineering, has developed a series of organ-on-a-chip platforms. These devices incorporate human cells into precisely engineered microfluidic channels that mimic an organ’s natural environment, providing a way to conduct experiments that would not otherwise be feasible.
Dan Huh, PhD
Huh’s previous research has involved using a placenta-on-a-chip to study which drugs are able to reach a developing fetus; investigating microgravity’s effect on the immune system by sending one of his chips to the International Space Station; and testing treatments for dry eye disease using an eye-on-a-chip, complete with a mechanical blinking eyelid.
The Chan Zuckerberg Initiative (CZI) has announced $14 million in funding to support 29 interdisciplinary teams who are investigating the role of inflammation in disease. Among these recipients is Dan Huh, Associate Professor in Bioengineering, whose placenta-on-a-chip research will “explore how maternal and fetal cells respond to specific inflammatory signals and analyze the network of placental cells and immune cells that impact pregnancy outcomes in chronic inflammatory diseases.”
Kellie Ann Jurado, Presidential Assistant Professor in the Perelman School of Medicine’s Department of Microbiology, will lead the research team. She and Huh will collaborate with Monica Mainigi, William Shippen, Jr. Assistant Professor of Human Reproduction in Penn Medicine.
A version of the Huh Lab’s placenta-on-a-chip from 2018
Huh’s placenta-on-a-chip consists of a small block of silicone containing microfluidic channels separated by a membrane of human cells. Variations in designs and cell types allow researchers to study how different molecules cross that barrier, allowing for experiments that would be otherwise impossible or unethical. For example, Huh and his group previously used a placenta-on-a-chip designed to model the placental barrier to research the effect of maternally-administered medications on the fetal bloodstream.
In this new study, Huh, Jurando and Mainigi were motivated by even more fundamental questions of pregnancy.
“It has been known for quite some time that women with chronic inflammatory diseases are at increased risk of developing various complications during pregnancy,” Huh says. “Despite accumulating clinical evidence, we understand little about how inflammation contributes to adverse pregnancy outcomes.”
Rachel Young, a graduate student in Huh’s lab, holds up the new eye-on-a-chip device. The latest iteration of the lab’s eye-on-a-chip has a mechanical eyelid to simulate blinking, and was used to test an experimental drug for dry eye disease. By incorporating human cells into an engineered scaffolding, the eye-on-a-chip has many of the benefits of testing on living subjects, while minimizing risks and ethical concerns.
People who spend eight or more hours a day staring at a computer screen may notice their eyes becoming tired or dry, and, if those conditions are severe enough, they may eventually develop dry eye disease (DED). DED is a common disease with shockingly few FDA-approved drug options, partially because of the difficulties of modeling the complex pathophysiology in human eyes. Enter the blinking eye-on-a-chip: an artificial human eye replica constructed in the laboratory of Penn Engineering researchers.
This eye-on-a-chip, complete with a blinking eyelid, is helping scientists and drug developers to improve their understanding and treatment of DED, among other potential uses. The research, published in Nature Medicine, outlines the accuracy of the eye-on-a-chip as an organ stand-in and demonstrates its utility as a drug testing platform.
They collaborated with Vivian Lee, Vatinee Bunya and Mina Massaro-Giordano from the Department of Ophthalmology in Penn’s Perelman School of Medicine, as well as with Vivek Shenoy, Eduardo D. Glandt President’s Distinguished Professor in Penn Engineering’s Department of Materials Science and Engineering. Other collaborators included Woo Byun, Andrei Georgescu and Yoon-suk Yi, members of Huh’s lab, and Farid Alisafaei, a member of Shenoy’s lab.
Huh’s lab specializes in creating organs-on-a-chip that provide microengineered in vitro platforms to mimic their in vivo counterparts, including lung and bone marrow proxies launched into space this May to study astronaut illness. The lab has spent years fine-tuning its eye-on-a-chip, which earned them the 2018 Lush Prize for its promise in animal-free testing of drugs, chemicals, and cosmetics.
In this study, Huh and Seo focused on engineering an eye model that could imitate a healthy eye and an eye with DED, allowing them to test an experimental drug without risk of human harm.
The Huh lab’s eye-on-a-chip attached to a motorized, gelatin-based eyelid. Blinking spreads tears over the corneal surface, and so was a critical aspect to replicate in the researchers’ model of dry eye disease. cells. The cells of the cornea grow on the inner circle of scaffolding, dyed yellow, and the cells of the conjunctiva grow on the surrounding red circle. Artificial tears are supplied by a tear duct, dyed blue.
To construct their eye-on-a-chip, Huh’s team starts with a porous scaffold engineered with 3D printing, about the size of a dime and the shape of a contact lens, on which they grow human eye cells. The cells of the cornea grow on the inner circle of scaffolding, dyed yellow, and the cells of the conjunctiva, the specialized tissue covering the white part of human eyes, grow on the surrounding red circle. A slab of gelatin acts as the eyelid, mechanically sliding over the eye at the same rate as human blinking. Fed by a tear duct, dyed blue, the eyelid spreads artificial tear secretions over the eye to form what is called a tear film.
“From an engineering standpoint, we found it interesting to think about the possibility of mimicking the dynamic environment of a blinking human eye. Blinking serves to spread tears and generate a thin film that keeps the ocular surface hydrated. It also helps form a smooth refractive surface for light transmission. This was a key feature of the ocular surface that we wanted to recapitulate in our device,” says Huh.
For people with DED, that tear film evaporates faster than it’s replenished, resulting in inflammation and irritation. A common cause of DED is the reduced blinking that occurs during excessive computer usage, but people can develop the disease for other reasons as well. DED affects about 14 percent of the world’s population but has been notably difficult to develop new treatments for, with 200 failed clinical drug trials since 2010 and only two currently available FDA-approved drugs for treatment.
Huh’s lab has been considering the drug-testing potential of organs-on-a-chip since their initial conceptualization, and, because of its surface-level area of impact, DED seemed the perfect place to start putting their eye model to the test. But before they started a drug trial, the team had to ensure their model could really imitate the conditions of DED.
“Initially, we thought modeling DED would be as simple as just keeping the culture environment dry. But as it turns out, it’s an incredibly complex multifactorial disease with a variety of sub-types,” Huh says. “Regardless of type, however, there are two core mechanisms that underlie the development and progression of DED. First, as water evaporates from the tear film, salt concentration increases dramatically, resulting in hyperosmolarity of tears. And second, with increased tear evaporation, the tear film becomes thinner more rapidly and often ruptures prematurely, which is referred to as tear film instability. The question was: Is our model capable of modeling these core mechanisms of dry eye?”
The answer, after much experimentation, was yes. The team evoked DED conditions in their eye-on-a-chip by cutting their device’s artificial blinking in half and carefully creating an enclosed environment that simulated the humidity of real-life conditions. When put to the test against real human eyes, both healthy and with DED, the corresponding eye-on-a-chip models proved their similarity to the actual organ on multiple clinical measures. The eyes-on-a-chip mimicked actual eyes’ performance in a Schirmer strip, which tests liquid production; in an osmolarity test, which looks at tear film salt content; and in a keratography test, which evaluates the time it takes for a tear film to break up.
Having confirmed their eye-on-a-chip’s ability to mirror the performance of a human eye in normal and DED-inducing settings, Huh’s team turned to the pharmaceutical industry to find a promising DED drug candidate to test-drive their model. They landed on an upcoming drug based on lubricin, a protein primarily found in the lubricating fluid that protects joints.
“When people think of DED, they normally treat it as a chronic disease driven by inflammation,” says Huh, “but there’s now increasing evidence suggesting that mechanical forces are important for understanding the pathophysiology of DED. As the tear film becomes thinner and more unstable, friction between the eyelids and the ocular surface increases, and this can damage the epithelial surface and also trigger adverse biological responses such as inflammation. Based on these observations, there is emerging interest in developing ophthalmic lubricants as a topical treatment for dry eye. In our study, we used an lubricin-based drug that is currently undergoing clinical trials. When we tested this drug in our device, we were able to demonstrate its friction-lowering effects, but, more importantly, using this model we discovered its previously unknown capacity to suppress inflammation of the ocular surface.”
By comparing the testing results of their models of a healthy eye, an eye with DED, and an eye with DED plus lubricin, Huh and Seo were able to further scientists’ understanding of how lubricin works and show the drug’s promise as a DED treatment.
Similarly, the process of building a blinking eye-on-a-chip pushed forward scientists’ understanding of the eye itself, providing insights into the role of mechanics in biology. Collaborating with Shenoy, director of the Center for Engineering MechanoBiology, the team’s attention was drawn to how the physical blinking action was affecting the cells they cultivated to engineer an artificial eye on top of their scaffolding.
“Initially, the corneal cells start off as a single layer, but they become stratified and form multiple layers as a result of differentiation, which happens when these cells are cultured at the air-liquid interface. They also form tight cell-cell junctions and express a set of markers during differentiation,” Huh says. “Interestingly, we found out that mechanical forces due to blinking actually help the cells differentiate more rapidly and more efficiently. When the corneal cells were cultured under air in the presence of blinking, the rate and extent of differentiation increased significantly in comparison to static models without blinking. Based on this result, we speculate that blink-induced physiological forces may contribute to differentiation and maintenance of the cornea.”
In other words, human cornea cells growing on the scientists’ scaffold more quickly became specialized and efficient at their particular jobs when the artificial eyelid was blinking on top of them, suggesting that mechanical forces like blinking contribute significantly to how cells function. These types of conceptual advances, coupled with drug discovery applications, highlight the multifaceted value that engineered organs-on-a-chip can contribute to science.
Huh and Seo’s eye-on-a-chip is still just dipping its toes into the field of drug testing, but this first step is a victory that represents years of work refining their artificial eye to reach this level of accuracy and utility.
“Although we have just demonstrated proof-of-concept,” says Seo, “I hope our eye-on-a-chip platform is further advanced and used for a variety of applications besides drug screening, such as testing of contact lenses and eye surgeries in the future.”
“We are particularly proud of the fact that our work offers a great and rare example of interdisciplinary efforts encompassing a broad spectrum of research activities from design and fabrication of novel bioengineering systems to in vitro modeling of complex human disease to drug testing,” says Huh. “I think this is what makes our study unique and representative of innovation that can be brought about by organ-on-a-chip technology.”
This work was supported by the National Institutes of Health through grants 1DP2HL127720–0, R01EY026972 and K08EY025742–01, the National Science Foundation through grants CMMI:15–48571, and Research to Prevent Blindness.
Dan Huh, the Wilf Family Term Assistant Professor in the Department of Bioengineering, focuses his research on creating organs-on-chips: specially manufactured micro-devices with human cells that mimic the natural cellular processes of organs. Huh’s lab has engineered chips that approximate the functioning of placentas and lung disease, some of which were launched into space in May. Most recently, Huh published a review of organ-on-a-chip technology in the journal Science with graduate students Sunghee Estelle Park and Andrei Georgescu.
The June 2019 issue of Science is a special issue centered around the science of growing human organ models in the laboratory. Such in vitro organs are known as organoids; they grow and develop much like organs do in the body, as opposed to Huh’s organs-on-chips, in which cells from the relevant organs are grown within a fabricated device that imitates some of the organ’s functions and natural environment.
In a video accompanying the review article, Huh explains how organoid and organ-on-a-chip technologies differ and the advantages that accompany each approach:
Unlike Organ-on-a-Chip, which are heavily engineered man-made systems, organoids allow us to mimic the complex of the human body in a more natural way. So organoids represent a more realistic model, but they have problems because they develop in a highly variable fashion and it’s not very easy to control their environment. So we think that Organ-on-a-Chip Technology is a promising solution to many of these problems.
Read Huh, Park, and Georgescu’s review article at Science.
SpaceX launched its 17th resupply mission to the International Space Station on May 4, with bioengineering professor Dan Huh’s organ-on-a-chip experiments in tow.
Dan Huh, the Wilf Family Term Assistant Professor in the Department of Bioengineering, researches human organs and the diseases that infect them by engineering devices made of living cells that act as stand-ins for organs. Huh’s lab has developed imitations of many organs, including the placenta and the eye, but it’s his lung-on-a-chip and his bone-marrow-on-a-chip that are reaching unprecedented heights as part of a new experiment taking place at the International Space Station (ISS).
On May 4, SpaceX launched a ISS-bound cargo capsule carrying Huh’s organ-on-a-chip experiments, which will remain in space for a month. Once back on Earth, the chips that spent time in space will be compared to control chips from Huh’s lab that are being monitored in parallel. Huh’s team is looking to see how being in space affects bacterial infections in lungs and white blood cell behavior in bone marrow. The researchers’ hope is that their studies will reveal important information about how human organs function both in space and on Earth.
Daniel Oberhaus of WIRED wrote an article describing the multiple organs-on-a-chip experiments being conducted at the ISS, including the two experiments headed by Huh:
Dan Huh is a bioengineer at the University of Pennsylvania and the lead researcher on the lung tissue chip headed to the ISS. This lung chip models a human airway and will be infected with Pseudomonas aeruginosa, a species of bacteria that had previously been found on the ISS. On Earth this bacteria is usually associated with respiratory infections, which are one of the leading types of illness on long-duration missions to the ISS.
Huh says scientists still know very little about why astronauts’ immune response seems to become suppressed in orbit, and the tissue chips are aimed at building a better understanding of the phenomenon.
Graduate student Andrei Georgescu and Assistant Professor Dan Huh in Huh’s lab. Adapting the organ-on-a-chip technology for a trip to the International Space Station presented Huh’s team with a number of engineering challenges. (Photo: Kevin Monko)
Throughout the 60-year history of the U.S. space program—from the Mercury capsules of the 1960s to today’s International Space Station—astronauts have been getting sick. Researchers know being in orbit seems to suppress their immune systems, creating a more fertile ground for infections to grow. But nobody really understands why.
Early on the morning of April 26, a SpaceX Falcon 9 rocket will launch a cargo mission to the ISS from Cape Canaveral Air Force Station. Along with fresh water, food, and other necessities for the crew, the craft will be carrying two experiments designed by Penn scientists that could help shed light on why bugs have bedeviled space travelers.
For more than a decade, Dan Huh, the Wilf Family Term Assistant Professor of Bioengineering in the School of Engineering and Applied Science, has been developing super-small devices that use living cells to stand in for larger organs. These organs-on-a-chip hold great promise for all kinds of research, from diagnosing disease to curing them. They’re also a way to test things, including drugs and cosmetics, in a way that mimics real life without relying on animal subjects.
The BE Seminar Series continues this week. We hope to see you there!
Shuichi Takayama, Ph.D.
Speaker: Shuichi Takayama, Ph.D.
Professor, GRA Eminent Scholar, Price Gilbert, Jr. Chair in Regenerative Engineering and Medicine
Wallace H. Coulter Department of Biomedical Engineering
Georgia Institute of Technology and Emory University
Date: Thursday, March 14th, 2019
Time: 12:00 pm
Location: Room 337, Towne Building
“Microfluidics and Immuno-Materials for Organs-on-a-Chip”
This presentation will describe microfluidic technologies to conveniently produce life-like pulsatile flows along with applications to study of lung injury, enhancement of in vitro fertilization, and analysis of frequency-dependent cellular responses. The microfluidic technologies range from adaptation of piezo-electric actuator arrays from Braille displays to design of microfluidic circuits that can be designed to switch fluid flow on and off periodically on their own. The presentation will also describe engineered materials to mimic an aspect of the innate immune system to combat bacterial infection. More specifically, reconstituted chromatin microwebs inspired by neutrophil extracellular traps. Using a defined composition reconstituted chromatin microweb, we reveal impact of microweb DNA-histone ratio on bacteria capture. Additionally, we found that E. coli, including clinical isolates and resistant strains, are killed more efficiently by the last-resort antibiotic, colistin, when bound to microwebs. Recent efforts towards incorporation of these materials into human cell systems will also be described. Time permitting, topics on organoids, fibrosis, liquid-liquid phase separation, and scaling may be incorporated.
