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NEWS FEATURECorrection for “News Feature: Building benchtop humanmodels,” by Amber Dance, which appeared in issue 22, June2, 2015, of Proc Natl Acad Sci USA (112:6773–6775; 10.1073/pnas.1508841112).

The editors note that image credits for Fig. 1 and Fig. 2 appearedincorrectly. Fig. 1, on page 6773, was courtesy of Mandy BridgetteEsch and Michael Louis Shuler (Cornell University, Ithaca, NY).Fig. 2, on page 6774, was courtesy of Harvard’s Wyss Institute. Thefigures and their corrected legends appear below.

www.pnas.org/cgi/doi/10.1073/pnas.1511286112

In hopes of improving and accelerating drug testing, researchers in the laboratory of Michael Shuler at Cornell University designed interconnected chambersto culture cells from 10 different tissue types as part of a microphysiological system. Image courtesy of Mandy Brigitte Esch and Michael Louis Shuler (CornellUniversity, Ithaca, NY).

A chip engineered to imitate lung function has multiple components (Inset). It’s part of a planned multiorgan microphysiological system. Image courtesy ofHarvard’s Wyss Institute.

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News Feature: Building benchtophuman modelsResearch teams are linking mini model organs to simulate whole-bodyresponses to medicines and potential terror agents.

Amber DanceScience Writer

The scientists inspect their “patient”; then,quite deliberately, they administer an over-dose of the painkiller Tylenol. They watchover the next 24 h as the medicine attacksthe liver. Levels of the amino acid tryptophanrise, indicating the organ is not workingproperly. Then production of bile acid drops,a sign of liver failure. This much Tylenol islike hitting the organ with a hammer, saysRashi Iyer, a toxicologist at the Los AlamosNational Laboratory in New Mexico. “Theliver falls apart.”But no one was harmed in this case. The

patient was but a snippet of engineered livertissue, part of a system meant to model thecomplex responses of human organs, which

Iyer and her collaborators are building. Andas the first of four planned “organoids” toundergo validation before being linked to-gether by a yet-to-be devised artificial circu-latory system, the little liver had just passedan important test. It responded to the Tylenolthe way a real person’s liver would. Iyer’steam affectionately refers to their modelas “Homo minutus,” but its official nameis the Advanced Tissue-engineered HumanEctypal Network Analyzer, or ATHENA forshort. They hope that like the namesake god-dess, it will bring some much-needed wis-dom to the development of new medicines.They’re not alone. The Los Alamos group

is one of several building multiorgan models,

called microphysiological systems, which rep-licate the physiology of a person on a simpler,smaller scale. These models come in a varietyof shapes and sizes, but commonly featurecultured human cells, arranged so they caninteract like they do in actual people andconnected by shared media or a series oftubes standing in for blood vessels. The de-velopment of these kinds of benchtop humanmodels has accelerated recently, spurred onby technological advances and a $200 millionboost in funding from the US government.Longstanding pitfalls of drug developmentare also driving commercial interest.Inventing and validating a new drug reg-

ularly takes a decade or longer and can costhundreds of millions of dollars. However, themajority of medicines that start human trialsfail to pass muster, often because of un-expected side effects or toxicity (1). Part ofthe problem is that scientists must test newmedicines in isolated human cells, in animals,or in silico. None of these does a great job offoretelling what a drug will really do inside aperson. Desktop human models aspire to al-low for a faster and more realistic way toidentify toxic medicines early on, so re-searchers can abandon dangerous medicinesand speed the path of useful ones.But scientists have only just begun ex-

ploring and testing these approaches. Lots ofresearch, testing, and validation remainsbefore it’s clear whether these systems willlive up to their promise and prove to be asuperior alternative. After all, animal mod-els, despite their flaws, have the distinct ad-vantage of being complete; only a wholeorganism can be exercised, for example, orhas the capacity to demonstrate how drugsaffect behavior.

Seeking New SolutionsThe current push to make microphysiologicalsystems was inspired, in part, by a De-partment of Defense (DOD)-sponsoredNational Academy of Sciences panel onmodel systems for evaluating counter-measures against biological or chemical

In hopes of improving and accelerating drug testing, researchers in the laboratory ofMichael Shuler at Cornell University designed interconnected chambers to culture cells from10 different tissue types as part of a microphysiological system. Image courtesy of Harvard’sWyss Institute.

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weapons in 2011, says Thomas Hartung,director of the Center for Alternatives toAnimal Testing at The Johns HopkinsUniversity in Baltimore. The NationalAcademy of Sciences panel’s charge: howto test treatments for toxins or pathogens,without any human victims to study orenroll in clinical trials? Their consensuswas that neither animals nor computer orcell-culture models could do the job. Some-thing new was needed (2).Recent events have only reinforced what’s

at stake. The 2014 outbreak of Ebola—a viruslong on the list of potential bioweapons thathad no vaccine or treatment—underscoredthe need for quick, accurate drug testing.Among the bottlenecks to quickly deployinga treatment, Iyer points out, were the testingand approval that had yet to take place.A handful of past experiments with two-

or three-organ systems hint at why the DODis betting the multiorgan models could revealunknown or unexpected toxicities better thancurrent systems. For example, the pesticidenaphthalene, found in mothballs, can causeheadaches, nausea, or anemia, although it’snot clear exactly how naphthalene sickenspeople. Michael Shuler, a biomedical engi-neer at Cornell University in Ithaca, NewYork, used a model with cells from both theliver and lung to find that the liver cells turnnaphthalene into naphthoquinone, and thiscompound in turn damages lung cells (3). Amodel with only liver or only lung tissuecould never have illustrated this process.

Millions for Micro-ModelsThe DOD initiated two grant programs formicrophysiological systems in 2012, onethrough the Defense Advanced Research Pro-jects Agency (DARPA) and the other via theDefense Threat Reduction Agency (DTRA).Under DARPA’s aegis, researchers at HarvardUniversity’s Wyss Institute for BiologicallyInspired Engineering in Boston and the Mas-sachusetts Institute of Technology (MIT) inCambridge are building machinery that canlink individual organs modeling 10 body sys-tems: circulatory, endocrine, gastrointestinal,immune, integumentary, musculoskeletal,nervous, reproductive, respiratory, and urinary.Meanwhile, DTRA selected two research

teams, one led by Iyer and the other by sci-entists at the Wake Forest Institute forRegenerative Medicine in Winston-Salem,North Carolina. Unlike DARPA’s plan to testcountermeasures, DTRA wants to quicklyidentify novel agents that might be a threatto people, says Hartung, who is part of theWake Forest project. For that purpose,DTRA asked for four-organ models.The NIH has also doled out $76 million

for scientists to grow individual tissues as wellas to connect them. NIH and DARPA re-searchers meet regularly to share progressand brainstorm solutions to common prob-lems. After two years, the NIH grantees havedeveloped several stand-alone organ models,including several kinds of organs and eventumors and are poised to start connectingthem via some sort of artificial circulatory

system, says Danilo Tagle, associate directorfor special initiatives at the NIH’s NationalCenter for Advancing Translational Sciencesin Bethesda, Maryland, who is coordinatingthe NIH-funded efforts.The DOD teams, midway through a five-

year grant program, have also just started tojoin their organs together. In contrast to theUnited States efforts, the European Commis-sion has devoted only a few million euros tosuch projects, but companies, such as Berlin-based TissUse and InSphero AG of Schlieren,Switzerland, are working onmultiorganmodels.

Mix and Match PartsThe teams pursuing microphysiological sys-tems seek not to replicate human physiologyexactly, but to create a stripped-down versionthat behaves like the real McCoy. The orga-noids have to mimic the body’s drug re-sponse, but can do that without matching thesize or shape of real organs. A surgeon mightnot even recognize the cultures as organs;indeed, the Wyss Institute’s models lookmore like computer chips than they do lungsor livers. However, the models match the realthing at the microscopic level. For example,researchers can copy kidney function with afew tubules—the organ’s subunits—ratherthan building a to-scale kidney replica.While Wyss and MIT are modeling all

10 main body systems, other research teamshave selected a few key organs. The liver is afavorite because of its role in metabolizingmedications. And the heart is important forsafety testing because many would-be phar-maceuticals turn out to be cardiotoxic.Creating individual organs requires some

tissue-engineering choices. Bioengineers haveto decide on a source of human cells. Optionsinclude the standard cell lines that havestocked freezers and laboratory dishes for years,primary cells from surgical excisions, or stemcell-derived tissues. Ideally, scientists would liketo use the latter. In principle, stem cells can bemade from any living person and would allowresearchers to build a microphysiologicalmodel matching a specific patient, or repre-senting different genders, races, and ages.But today’s stem cell protocols typically

create immature cell types that don’t functionthe way fully differentiated cells would, Iyersays. One exception is heart muscle; scientistscan make operational cardiomyocytes fromstem cells, and both the ATHENA and Wyssteams are using them for their model hearts.To house those cultured cells, teams are

taking a variety of approaches. Wake Forest,for example, will use a bioprinter to “print”cells into a hydrogel matrix, creating 3Dorganoids (4). The ATHENA team is doingsomething similar, layering cells onto 3D

A chip engineered to imitate lung function has multiple components (Inset). It’s part of aplanned multiorgan microphysiological system. Image courtesy of Mandy Brigitte Esch andMichael Louis Shuler (Cornell University, Ithaca, NY).

6774 | www.pnas.org/cgi/doi/10.1073/pnas.1508841112 Dance

scaffolds. Shuler, who is participating in theNIH program, grows cells on silicon and usescomputer simulations to guide constructionof the models and interpret his results (5).Across the Atlantic, InSphero is making asimpler model with cellular spheroids floatingin wells connected by microchannels (6, 7).The Wyss Institute, pioneer of “organs-on-

chips” (8), houses its cells inside clear car-tridges about the size of a USB stick. Thelung chip, for example, mimics a single airsac with two sections separated by a porousmembrane. On the top of the membrane,lung cells live in the presence of air. On thebottom is a layer of endothelial cells forminga “blood vessel” wall, bathed in liquid me-dium. On either side are vacuum channels,which expand and contract, stretching andrelaxing the cells to simulate breathing (9).

Homo minutusUniting all those organoids into a physio-logical “system” presents another set ofchallenges, both biological and mechanical.To physically join its organs, the Wyss In-stitute has developed a device called the In-terrogator. In July 2014, the institute spun offa company, Emulate, Inc., of Boston, Mas-sachusetts, to perfect and commercialize themachine. Institute Director Don Ingbercompares the Interrogator to a compact diskchanger, although it’s about four feet wide.The miniorgans will exist in equally sizedchips that users can plug into the machine’sslots: 12 for now but eventually 36, Ingbersays. Within the Interrogator, users couldrun any organ combination they choose,such as 36 individual livers, 18 paired or-gans, or a few 10-organ setups. Users couldalso watch what their organs-on-chips areup to via an integrated microscope.When each chip’s blood vessel compart-

ment is linked to the others, the organoidswill be connected by a minicirculatory systemthat mimics how drugs would move in andout of organs and through the bloodstream.This is an important advantage over othermodels under development, Ingber notes.One problem that has yet to be solved,

however, is what scientists will use as a bloodsubstitute. Every tissue has a favorite me-dium, with specific buffers and growth fac-tors, and none of the teams has come up witha universal version. Thus far, Ingber andcolleagues have simply mixed the two mediafor the organs they’ve linked. He doubts thatapproach will work for 10 organs at once.“I think it’s more likely that we use [hu-man] plasma or blood,” he says.The Wyss scientists have already con-

nected their lung and liver chips. In a test, amedication they placed in the lung’s airspace

was “inhaled,” absorbed, and traveled to theliver, which responded by making enzymesthat prepare drugs for excretion. The nextmilestone on the DARPA checklist is to at-tach four different organs together by March2015. Ingber says the group has the lung,liver, gut, heart, and kidney to choose fromfor that quartet. By 2017, the researchers planto connect all 10 organ systems.ATHENA’s dime-sized liver, the furthest

along of the team’s organs, sits in housingabout as big as a credit card. The researchershave already run it through stringent tests,such as the Tylenol overdose. Now they’revalidating the heart, Iyer says. The team plansto link the heart and liver first, with a lungand then kidney to follow. The whole systemshould fit on a desktop.This Homo minutus’s claim to fame will

not be a particular organoid but an accessory:a mass spectrometer hooked up to the systemthat will allow the researchers to measurehundreds of compounds the model organs

“The hardest part is a goodmimic of the full immunesystem.”

—Michael Shuler

are using or producing. Iyer likens ATHENAto an infant. It cannot say what is wrong, butby sampling the “blood” and—once theyhook up the kidney— “urine,” scientists candeduce what’s happening inside.

Clinical Trials on a BenchtopDARPA and the NIH plan for their micro-physiological systems to be commerciallyavailable from spin-offs like Emulate or othercompany partners. As for ATHENA, DTRAwill decide how it is distributed, Iyer says.For now, it’s hard to predict how muchan ATHENA or a human-on-a-chip mightcost. But these models have the potentialto save companies big-time dollars, saysDouglas Keller, global head of standardsand innovation in preclinical safety at SanofiUS, a pharmaceutical company in Bridgewater,New Jersey. For example, human studies

of how two drugs interact can swallow abouthalf a million dollars each, he says, so abenchtop human that could answer thatquestion could mean serious savings.Despite the tremendous potential, Keller,

who is not involved in any of the projects,counsels caution, having seen major tech-nology advances fall short of their promise.He cites toxicogenomics: the once-vaunted useof chips to analyze gene expression hasn’t yetenabled toxicologists to fully grasp how dif-ferent drugs are toxic. Microphysiological sys-tems will surely have limitations, he predicts.Would the DOD’s microphysiological sys-

tems have helped early in the Ebola outbreak?“Maybe,” says Shuler. Scientists might havebeen able to use microphysiological systems tonarrow down the best candidate treatmentsquickly. They could, for example, “infect” amicrophysiological system with the virus, thentreat it with different combinations of antivi-rals and look for a reduction in viral load, hespeculates. They’d still need to run humantrials of their favorite candidates, though. AndShuler is not certain that any of the systemsunder development now could do the job.“The hardest part is a good mimic of the fullimmune system,” he says. “You can modelaspects of the immune system, but a completemodel is difficult.”The Wyss model can circulate immune

cells, but other models do not. Ingber ac-knowledges other limits among the micro-physiological systems in the works. Theycannot, for example, model orthopedic dis-eases that result from pressure on joints. Mostlack fat, which can absorb drugs, although theWyss Institute is working on a fat chip.Tagle takes the long view, however. He

foresees a time when pharmaceutical com-panies could access thousands of differentbenchtop humans, each made with stem cellsfrom a different individual, to run early-stageclinical trials on chips. It may sound far-fetched, but then, Ingber thought the samewhen he first submitted his DARPA grantapplication. “It felt like it was impossible,” herecalls. “I’m amazed at how far we’ve gotten.”

1 Hartung T (2013) Look back in anger—What clinicalstudies tell us about preclinical work. ALTEX 30(3):275–291.2 Institute for Laboratory Animal Research (2011) Animal Models forAssessing Countermeasures to Bioterrorism Agents (NationalAcademies Press, Washington, DC).3 Sweeney LM, Shuler ML, Babish JG, Ghanem A (1995) A cellculture analogue of rodent physiology: Application tonaphthalene toxicology. Toxicol In Vitro 9(3):307–316.4 Xu T, et al. (2013) Complex heterogeneous tissue constructscontaining multiple cell types prepared by inkjet printing technology.Biomaterials 34(1):130–139.5 Sin A, et al. (2004) The design and fabrication of three-chamber microscale cell culture analog devices with

integrated dissolved oxygen sensors. Biotechnol Prog 20(1):338–345.6 Kim JY, et al. (2015) 3D spherical microtissues and microfluidictechnology for multi-tissue experiments and analysis. J BiotechnolS0168-1656(15):00012-7, 10.1016/j.jbiotec.2015.01.003.7 Kim J, Fluri DA, Kelm JM, Hierlemann A, Frey O (2014) 96-wellformat-based microfluidic platform for parallel interconnection ofmultiple multicellular spheroids. J Lab Autom 20(3):274-282, doi:10.1177/2211068214564056.8 Huh D, Torisawa YS, Hamilton GA, Kim HJ, Ingber DE (2012)Microengineered physiological biomimicry: Organs-on-chips. LabChip 12(12):2156–2164.9 Huh D, et al. (2010) Reconstituting organ-level lung functions ona chip. Science 328(5986):1662–1668.

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