COMMENTARY

Toward in vitro models of brain structureand functionMichael L. Shulera,1 and James J. Hickmanb

aDepartment of Biomedical Engineering, Cornell University, Ithaca, NY 14853; andbNanoScience Technology Center, University of Central Florida, Orlando, FL 32826

The development of effective tissue-engi-neered models of the brain remains anelusive challenge because of its inherentcomplexity. Such models would be extremelyimportant to understanding brain develop-ment, and for exploring therapeutic optionsfor disorders of the CNS, including thetreatment of traumatic brain injury (TBI)and related damage to the brain. One million,seven hundred thousand TBIs occur in theUnited States annually (1). These in vitromodels would also be invaluable test bedsfor drug-discovery investigations and in tox-icology evaluations. In PNAS, Tang-Schomeret al. (2) describe a promising model of a cor-tical tissue mimic and demonstrate its appli-cations to a better understanding of responseto TBI.Several classes of in vitro models of the

brain have been described (3), includingacute preparations (or explants of CNS tis-sues), organotypic cultures or thin slices ofCNS maintained for greater than 7 d, cerebralorganoids (which can be formed from theself-organization of human pluripotent stemcells in 3D cultures) (3), and tissue-engi-neered constructs (2, 4, 5). Here we focuson the cell-based techniques for organoidsand tissue-engineered constructs.In organoids, the formation of cortex-like

structures that are reminiscent of the humandeveloping cerebral cortex have been ob-served (6). These structures promise to beuseful models for brain development andneurodevelopmental disorders. Using humanpatient-specific induced pluripotent stemcells (iPSC), Lancaster et al. (6) were able tomodel microcephaly through observationsof premature neuronal differentiation. Theauthors used a spinning bioreactor to groworganoids up to 4 mm in diameter that couldbe maintained for up to 10 mo. Although thistechnology is truly impressive, there are keylimitations to these models. Currently, adultneuronal behavior is difficult to mimic withiPSC technology and when—and if—this ispossible remains an open question. Further-more, the self-organization in the organoids

is incomplete, resulting in a model that lacksthe spatial organization observed in naturalhuman tissue. For example, the complex six-layered architecture of the cerebral cortex isnot emulated. Although the organoids cangrow to a 4-mm diameter, they lack an or-ganized “blood” supply and are starved fornutrients and oxygen in the center of theorganoid. In addition, the assembly processfor the organoids does not lend itself to readyinterrogation with assays or sensors to deter-mine cell health, cellular function, or a higherlevel of communication in the system. Fi-nally, the interest in combining multipleorgans in body-on-a-chip systems would pre-clude the utilization of organoid models be-cause of the inability to control the structuresand growth of the system. Although theseorganoid models represent exciting progress,the lack of human adult structure and diffi-culties of integration with sensors or otherplatforms will limit their use for many dis-ease and injury models.Tissue-engineered constructs can be

formed to address these issues by providingan architecture and environment that emu-lates key aspects of the organization in themature brain more completely. Models ofthe brain using tissue-engineering techni-ques and relatively mature cells from ani-mals have been constructed and shown tobe useful 3D neural cell models (e.g., ref. 4).Typically, such models are based on hydro-gels and often use rodent cells (Fig. 1). Inearly work, Xu et al. used cell printingof hippocampal neurons in hydrogels toform 3D constructs that demonstrated elec-trical synaptic activity using patch-clampelectrophysiology but lacked a defined ar-chitecture (7). Frampton et al. (4) used al-ginate with rat astroglioma cells, astrocytes,microglia, and neurons, but did not at-tempt to mimic the layered architecture ofthe cerebral cortical tissue or demonstrateelectrical connections that will be criticalto a more complete understanding of brainfunction. Functional culture has been shownwith neuromuscular junction systems for rat

(8) and human (9), but without the accom-panying 3D architecture.Recently Odawara et al. (5) reported on

the construction of a six-layered neuronalsystem using a reconstructed neuronal tis-sue mimic based on collagen fibers andpolydimethylsiloxane microchambers. Theseauthors used this system to produce alternat-ing layers with somata and neurite outgrowthdirected by the orientation of collagen fibrils.Interlayer synchronous firing in the artificialneuronal network was observed using multi-electrode arrays from the bottom layer of thestructure. Odawara et al. reconstructed theseneuronal networks using rat cells and humaniPSC-derived neurons. Thus, they were ableto mimic some aspects of the 3D layer struc-ture of the cerebral cortex and achieved a celldensity approximating the in vivo value, butlacked a true 3D functional characterizationof the system.The work of Tang-Schomer et al. (2)

extends beyond these studies to achieve a3D model of a specific brain structure bymimicking the cerebral cortex with increasedauthenticity. These constructs seek to providean effective model of the mechanical, chem-ical, and electrophysiological environment forthe cerebral cortex, along with correlative

Fig. 1. An example of the 3D culture of rat hippo-campal neurons in a collagen-based hydrogel immuno-stained with microtubule-associated protein-2. The cellsformed signature connections in 3D and had superiorsurvival to 2D-only cultures.

Author contributions: M.L.S. and J.J.H. wrote the paper.

The authors declare no conflict of interest.

See companion article on page 13811.

1To whom correspondence should be addressed. Email: mls50@cornell.edu.

13682–13683 | PNAS | September 23, 2014 | vol. 111 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1414484111

functional responses to various inputs, suchas diffuse axonal injury in brain trauma.Tang-Schomer et al.’s (2) approach is

based on a modular design that emulatesthe mechanical structure of the brain usingsilk protein-based scaffolds of high porositycombined with a collagen gel. A unit moduleconsists of neuron-rich regions to emulate“grey matter” and axon-only regions thatmimic “white matter”; the composite structurefacilitates the formation of 3D axon connec-tions. The neurons were anchored to the silksurface and extended axons into the collagengel. The silk, coated with polylysine, enabledcortical neuron adhesion, and the stiffness ofthe material promoted axon outgrowth. Thiscomposite, during a 9-wk-long experimentalperiod, promoted in 3D the formation ofinterconnected mini-networks, increased axonlength, and improved viability compared withcollagen gel-only controls. Additionally, invivo-like electrophysiology was monitored in3D and was sustained better in the compositesystem than a collagen-only system.The system was then tested for response

(i.e., surrogates for cellular damage, electro-physiological response, and neurochemicalchanges) to simulated mechanical injury toemulate TBI. In response to simulated injury,there was a rapid increase in both localelectrical field potential measurements andglutamate release, which mimic the observedresponse in animal studies. The compositemodel demonstrated an increase in neuronalclustering as well as improved cell viabilitywhen compared with gels. Tang-Schomeret al. (2) suggest that these composites im-prove the transport of oxygen and nutrientscompared with hydrogels alone, as the hydro-gel structure may collapse because of gel deg-radation. The ability to sustain cellular activitywill be important to the study of many diseasestates and potential response to treatment infuture studies.Tang-Schomer et al. (2) recognize possibil-

ities for further improvement in such models.The current work was mostly focused on

reproducing the brain’s in vivo mechanicalproperties and contains only neurons, whereasthe addition of astrocytes, oligodendro-cytes, and microglial cells would certainlyincrease authenticity and allow it to becompared directly to earlier work. As mod-els composed of hydrogels with up to fourcell types have been constructed (4), the

Tang-Schomer et al.describe a promisingmodel of a cortical tis-sue mimic and demon-strate its applications toa better understandingof response to TBI.addition of other cellular components to thiscomposite model should be straightforward.The authors also suggest the use of humaniPSCs, as done in Lancaster et al. (6) andOdawara et al. (5), to transition from a rat tohuman system. One difficulty with the hu-man iPSC approach is whether a more ma-ture phenotype can be achieved; here theextended period of culture in Tang-Schomeret al. (2) should aid in that goal. The use ofhuman CNS cells would also make it moreapplicable to creating an in vitro model toverify work being done on the Brain Initiativenow being investigated by the NationalInstitutes of Health, the Defense Advanced

Research Projects Agency, the National Sci-ence Foundation, and the European Union.In addition, the modular approach to con-struction and development of this systemmake it an ideal candidate for further in-tegration into body-on-a-chip or organ-on-a-chip systems currently being developed toreduce and eventually eliminate the use ofanimals in drug discovery and toxicologicaltesting (10).Overall, during the last few years we have

seen a significant improvement in the de-velopment of in vitro models of the brain.However, tissue-engineered models of thebrain are still in their infancy. A tissue-engineered model demonstrating complexCNS function is a distant goal! What Tang-Schomer et al. (2) have done is to make a su-perior model emulating response to mechan-ical insults. This approach is clever, makeseffective use of the basic tools of tissue engi-neering, and has direct applicability to animportant human health issue (TBI). How-ever, more complex tissue-engineered modelswill be required to address the full range ofCNS response to exposure to chemicals,pharmaceuticals, and disease. The establish-ment of an effective tissue-engineered model[such as found in Tang-Schomer et al. (2)]provides a basis upon which more completemodels can be constructed, and these modelswill allow preliminary evaluation of possibletherapeutic treatments.

1 Faul M, Xu L, Wald MM, Coronado VG (2010) Traumatic BrainInjury in the United States: Emergency Department Visits,Hospitalizations and Deaths 2002–2006 (National Center for InjuryPrevention and Control, Centers for Disease Control and Prevention,Atlanta).2 Tang-Schomer MD, et al. (2014) Bioengineered functionalbrain-like cortical tissue. Proc Natl Acad Sci USA 111:13811–13816.3 Morrison B III, Elkin BS, Dollé JP, Yarmush ML (2011) In vitromodels of traumatic brain injury. Annu Rev Biomed Eng13:91–126.4 Frampton JP, Hynd MR, Shuler ML, Shain W (2011) Fabrication andoptimization of alginate hydrogel constructs for use in 3D neural cellculture. Biomed Mater 6(1):015002.5 Odawara A, Gotch M, Suzuki I (2013) A three-dimensionalneuronal culture technique that controls the direction of neurite-

elongation and the portion of soma to mimic the layered structure ofthe brain. RSC Adv 3(45):23620–23630.6 Lancaster MA, et al. (2013) Cerebral organoids modelhuman brain development and microcephaly. Nature 501(7467):373–379.7 Xu T, et al. (2009) Electrophysiological characterization ofembryonic hippocampal neurons cultured in 3D collagen hydrogel.Biomaterials 30(26):4377–4383.8 Das M, et al. (2007) Embryonic motor neuron-skeletal muscle co-culture in a defined system. Neuroscience 146:481–488.9 Guo X, Gonzalez M, Stancescu M, Vandenburgh H, Hickman JJ(2011) Neuromuscular junction formation between human stemcell-derived motoneurons and human skeletal muscle in a definedsystem. Biomaterials 32(36):9602–9611.10 Esch MB, et al. (2014) How multi-organ microdevices can helpfoster drug development. Adv Drug Deliv Rev 69-70:158–169.

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