ARTICLESPUBLISHED ONLINE: 3 AUGUST 2015 | DOI: 10.1038/NMAT4357

Unjamming and cell shape in the asthmaticairway epitheliumJin-Ah Park1*†, Jae Hun Kim1†, Dapeng Bi2, Jennifer A. Mitchel1, Nader Taheri Qazvini1,3,Kelan Tantisira4, Chan Young Park1, Maureen McGill1, Sae-Hoon Kim1, Bomi Gweon1, Jacob Notbohm1,Robert Steward Jr1, Stephanie Burger1, Scott H. Randell5, Alvin T. Kho6, Dhananjay T. Tambe1,7,Corey Hardin1, Stephanie A. Shore1, Elliot Israel4, David A. Weitz8, Daniel J. Tschumperlin9,Elizabeth P. Henske4, Scott T. Weiss4, M. Lisa Manning2, James P. Butler1,4, Je�rey M. Drazen1

and Je�rey J. Fredberg1

From co�ee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems—both inert and living—have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. Theunjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phasesremain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked tocell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scalingpredicts a counter-intuitive relationship between jamming, cell shape and cell–cell adhesive stresses that is borne out bydirect experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigationof bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelialdynamics play a prominent role.

One of the central unresolved mysteries of asthma is howthe asthmatic airway remodels itself1. This remodellingis often progressive and has long been thought to be

the end product of a cascade of cell-signalling events that areinitiated in the bronchial epithelium and driven by repetitiveinflammatory, allergic, or viral insults1. Tissue-remodelling eventsmore broadly, including those underlying morphogenesis, woundrepair and cancer invasion, have been linked to collective cellularmigration2–5, often in the context of the epithelial-to-mesenchymaltransition (EMT; ref. 6). But no clear physical picture has yetemerged that can capture these collective biological processes andtheir interconnections. Here we provide evidence that the physicalprocess of cell jamming and unjamming5,7–11, which has beenmissing from descriptions of tissue remodelling, might tie togetherat least some of them.

Primary human bronchial epithelial cells (HBECs) were derivedfrom non-asthmatic and asthmatic donors, plated on a porousTranswell insert, and established in air/liquid interface (ALI)culture12 (Methods). Initial culture in submerged conditions causesbasal cells to proliferate, and subsequent culture in ALI conditionsinhibits any further proliferation and triggers differentiation ofthose basal cells into a mature confluent pseudostratified bronchialepithelial layer comprising goblet cells and ciliated cells13. Here weshow that such a pseudostratified layer derived from non-asthmaticdonors is quiescent. Cellular motions are relatively small, cellular

rearrangements among neighbouring cells are rare, and each cellremains virtually caged by those immediate neighbours. Statisticalanalyses of these motions confirm that such a layer is solid-likeand jammed. However, application of an apical-to-basal mechanicalstress mimicking the compressive effect of bronchospasm12,14,15

(Supplementary Fig. 1) is sufficient to trigger large cellular motionsand cooperative cellular rearrangements. Cells move chaotically,but the motions exhibit cooperative packs and swirls; such a layeris fluid-like and unjammed. We then examine the more complexprocess of progressive layer maturation, where we find an innatetendency of the maturing layer to transition from an immature,fluid-like, unjammed phase in which cells readily rearrange andflow, into a mature, solid-like, jammed phase in which cells becomevirtually frozen in place8,9,16. As compared with non-asthmaticdonors, however, in the maturing layer derived from asthmaticdonors this jamming transition is delayed substantially or disruptedaltogether. In all these cases, the transition between unjammedand jammed phases is continuous; as the cell layer approaches thejamming transition, cellular motions become progressively slower,pack sizes become progressively larger, and pack lifetimes becomeprogressively longer.

One might have expected that cell jamming would be causedby increasing mutual cell–cell adhesive stresses such that cellsbecome stuck to immediate neighbours and, as a result, thecellular collective rigidifies, themutual cellular rearrangements stop,

1Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA. 2Syracuse University, Syracuse, New York 13244, USA. 3School ofChemistry, College of Science, University of Tehran, Tehran 14179, Iran. 4Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts02115, USA. 5The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514, USA. 6Children’s Hospital, Boston, Massachusetts 02215,USA. 7Department of Mechanical Engineering, University of South Alabama, Mobile, Alabama 36688, USA. 8Harvard University, Cambridge,Massachusetts 02138, USA. 9Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. †These authors contributed equally to this work.*e-mail: jpark@hsph.harvard.edu

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Figure 1 | In a confluent layer of well-di�erentiated HBECs, compressive stress mimicking bronchospasm, as in asthmatic bronchospasm, provokes thetransition from a solid-like jammed phase to a fluid-like unjammed phase. a, Speed maps (left panels) showed compressive stress at a magnitude of30 cm H2O induced hypermobility of HBECs on ALI day 16. Within any optical field the migration speed was spatially heterogeneous but increased stronglywith increasing P. Colour scale is shown at the bottom of the left panels. The size of vectors (right panels) increased with increasing P and showedlarge-scale dynamic heterogeneity. Vector scale is shown at the bottom of the right panels. b, As P was progressively increased to 30 cm H2O (red filledcircles), the mean square displacement, MSD, and the self-di�usion coe�cient Ds increased (inset; Ds= lim1t→∞MSD(1t)/(41t)), and the systembecame strongly super-di�usive. Error bars in the inset represent the standard deviation. c, When P was less than 20 cm H2O, the relative overlap of eachcell with its initial position was nearly perfect for time intervals (1t) of less than 144 min, as quantified by the ensemble average, 〈Q(1t)〉, close to 1. WhenP was 30 cm H2O (red filled circles), the overlap decreased to 0.17. d, The four-point susceptibility χ4(1t) is approximated by N[〈Q(1t)2

〉−〈Q(1t)〉2],where N is the number of cells. When movements are cooperative, χ4(1t) exhibits a peak whose position corresponds roughly to pack lifetime, and whosemagnitude corresponds roughly to pack size. When pressure was 30 cm H2O (red filled circles), χ4(1t) showed a peak indicative of cooperative packs offaster-moving cells with a pack lifetime of 45 min and a pack size of approximately 70 cells.

and the constituent cells cannot move17. Much to our surprise,direct measurements defied this expectation—in layers that becomejammed, the adhesive stresses between a cell and its neighbourswere attenuated, not augmented. To explain this paradox, weturned to the well-known vertex model, wherein a competitionbetween cell–cell adhesive stresses and cell cortical tension controlchanges of cell shape18–20. Novel analysis of this model, includinga critical scaling analysis, predicts that increased adhesion leads toincreased fluidity, and that cell jamming occurs as a well-definedindex of cell shape approaches a critical value. We show thatthe shape index acts as a simple structural order parameter thattakes on different values on either side of the jamming transition.

Using that shape index, we show not only that cell shape in thebronchial epithelial layer differs between cells derived from non-asthmatic versus asthmatic donors, but also that, regardless of cellorigin, cell shape at the jamming transition matches theoreticalpredictions, and thus resolves the paradox. This newphysical pictureraises questions about the relationship between the epithelial-to-mesenchymal transition and unjamming and, conversely, betweenthe mesenchymal-to-epithelial transition and jamming. Similarly,it suggests new, testable hypotheses concerning asthma aetiologyand asthma therapy. The more fundamental significance of thesefindings, however,may be to broaden notions of jammedmatter andgeneralize understanding of jamming mechanisms.

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Figure 2 | In HBECs over the course of ALI culture, a spontaneous phase transition occurs from a hypermobile, unjammed, fluid-like phase into aquiescent, jammed, solid-like phase, which was delayed in cells from asthmatic donors. a–c, Speed maps (left panels) and vector maps (right panels)showed that HBECs from a representative non-asthmatic donor were hypermobile on an early ALI day (a; day 3), but spontaneously became quiescent onlater ALI days (b, day 6; and c, day 8). Colour and vector scales are shown at the bottom of c. d–f, Speed maps (left panels) and vector maps (right panels)showed that HBECs from a representative asthmatic donor were hypermobile until later ALI days (d, day 6; and e, day 10) and became quiescent on ALIday 14 (f). Colour and vector scales are shown at the bottom of f. g, Four-point susceptibility χ4(1t) for HBECs from a non-asthmatic donor showed peaksindicative of cooperative packs of faster-moving cells with a lifetime of 81 min with a corresponding pack size of approximately 20 cells on ALI day 3 (bluetriangles), whereas peak was undetectable either on ALI day 6 (blue circles) or 8 (blue asterisks). Inset: MSD. h, Four-point susceptibility χ4(1t) for HBECsfrom an asthmatic donor showed peaks indicative of cooperative packs of faster-moving cells with lifetimes of 72 and 90 min with corresponding packsizes of approximately 26 and 12 cells on ALI day 10 (red circles) and 6 (red triangles), respectively, whereas peak was undetectable on ALI day 14 (redasterisks). Inset: MSD.

1042 NATUREMATERIALS | VOL 14 | OCTOBER 2015 | www.nature.com/naturematerials

NATURE MATERIALS DOI: 10.1038/NMAT4357 ARTICLESCompression unjams the jammed HBEC layerMechanical compression of the bronchial epithelium occurs duringsevere bronchospasm (Supplementary Fig. 1) and is sufficientto induce maladaptive airway remodelling even in the completeabsence of inflammatory events14,15. Although certain cell-signallingmodules have been identified, such as autocrine signalling throughthe epidermal-growth-factor-receptor family of ligands, the physicalmechanism remains unclear. To trigger these events we followedthe protocol of ref. 14; on day 16 of ALI culture we exposed theHBEC layer to a compressive apical-to-basal stress, P , spanningthe physiologic range (0, 3, 10, 20, or 30 cm H2O; Fig. 1 andSupplementary Fig. 1), where 3 cm H2O corresponds roughly to themaximal compressive stress expected to be exerted on HBECs byquiet tidal breathing, and 30 cm H2O corresponds to that expectedduring severe bronchospasm21,22.

The manner in which a cell moves provides clues aboutmechanisms that promote or impede its mobility (Fig. 1a).Therefore, we began by quantifying cellular motions using theirmean square displacement (MSD) over a given time interval, 1t ,averaged over many cells in several optical fields (Methods). Inmany physical systems, the MSD increases with time as a powerlaw, 1tα , where the exponent α is determined empirically. Whenthe exponent is unity (α=1), as in uncorrelated random Brownianmotion, particlemotions are diffusive.Whenα<1, particlemotionsare sub-diffusive, as when a particle might, in time, escape thecage comprising its immediate neighbours only to become quicklyrecaged by its new immediate neighbours; this uncaging–recagingprocess thereupon repeats in a stochastic fashion. When α > 1,particle motions are super-diffusive, and when α= 2, as occurs insimple linear translation at constant velocity, motions are ballistic.

In HBECs from non-asthmatic donors in ALI culture, cellularmotions were smallest when the compressive stress, P , was zeroand increased systematically as P was increased (Fig. 1b andSupplementary Movie 1). When 1t was small and P was zero,the MSD increased slowly with time in a sub-diffusive manner asif each cell were caged or jammed by its immediate neighbours.But as 1t exceeded 10min the MSD grew as 1t 1.3, indicatingthat motions at longer times were slightly super-diffusive, andsuggesting that each cell was eventually able to escape its cageand move in a persistent direction. But as P was increasedprogressively to 30 cm H2O, the MSD at the highest P increasedby about two orders of magnitude and varied as 1t 1.8. Cells in thiscompressed state were seen to become highly mobile and stronglysuper-diffusive (Fig. 1b).

These swirling cellular motions and their changes were visuallystriking (Supplementary Movie 1). To provide further quantitativecharacterization of these motions, we measured the fractionalchange of cellular position in a given time increment 1t . Much asis done conventionally in studies of cooperative particle motionsin jammed or glassy inert systems23, here we chose 15% of averageparticle (that is, cellular) diameter as the reference length scale;if a region moved less than 15% of the average cellular diameterover the time interval 1t , we considered this as 100% overlap withthe initial position, whereas if a region moved more than 15% ofa cellular diameter, we considered this as 0% overlap (Methods).The average value of this overlap function over the entire opticalfield was defined as Q(1t) (ref. 23) and the ensemble average overall sequence images, 〈Q(1t)〉, was computed. Uncompressed cellsand those compressed with P less than 20 cm H2O showed nearlyperfect overlap (〈Q(1t)〉 = 1) for as much as 144min, as if cellswere immobile and jammed. However, for cells compressed with Pof 30 cm H2O, 〈Q(1t)〉 fell to 0.17 at 144min (Fig. 1c), suggestingappreciable mobility and, potentially, unjamming.

The MSD and the overlap function Q are useful metrics ofcellular motions. However, these metrics by themselves fail todistinguish uncorrelated cellular motions from the cooperative

motions that comprise the strings, clusters, swirls or eddies that arethe hallmark of the structurally heterogeneous dynamics and typifyjammed matter close to a jamming transition16,24. In such systems,as the jamming transition is approached, the cooperativity of thesemotions increases and, as a result, the length scale and the timescaleof these motions tend to grow sharply23,25,26. Therefore, from theoverlap function Q(1t) we computed the four-point susceptibility,χ4(1t), which exhibits a peak whose position corresponds roughlyto pack or swirl lifetime and whose magnitude corresponds roughlyto pack or swirl size (Methods)4,24,27. Accordingly, after severalpack lifetimes a sufficient degree of structural rearrangementswill have occurred such that any individual cell will find itselfsurrounded by a different set of immediate neighbours, and theoriginal pack will have become shuffled with its neighbours to theextent that it becomes unrecognizable. As in the well-establishedglass transition28, there is no objective cutoff that defines a jammingtransition; rather, the transition is continuous, and jamming issaid to occur when pack lifetime grows to exceed the laboratorymeasurement window, which we take here as 144min.

In HBEC layers derived from non-asthmatic donors, χ4(1t)revealed no peak for P less than 20 cm H2O and time intervals lessthan 144min (Fig. 1d); if a peak exists at all in these cases, it must befor substantially longer times. However, for P=30 cm H2O, a well-defined peak in χ4(1t) emerged, corresponding to faster-movingcooperative packs comprising roughly seven cells with an averagepack lifetime of 42min (Fig. 1d). Compressive stress of 30 cm H2Opressure was therefore sufficient to unjam the layer comprised ofHBECs from non-asthmatic donors, whereas at lower compressivestresses the induced motions were far smaller, and structurallyheterogenetic dynamics was not evident, as if the layer were frozen.

In cells from asthmatic donors, jamming is delayedIn submerged conditions, primary HBECs grow until confluenceand, subsequently, in ALI culture they differentiate without furtherproliferation. These events recapitulate repair processes that areknown to occur in vivo following epithelial injury or sloughing13.Roughly by day 3 in ALI culture the early phase of rapidproliferation was completed and the maturation and differentiationof the layer then continued; cell densities varied from well-to-well and from donor-to-donor, ranging roughly from 4,700 to5,300 cellsmm−2 and averaging 5,116 ± 233 cellsmm−2, but didnot increase systematically with ALI day (p = 0.37). The celllayer increasingly exhibited a well-differentiated pseudostratifiedphenotype that included goblet cells and ciliated cells, apico-basal polarization, tight junctions, and an increasingly tight barrierfunction, as reflected in progressive increases of the trans-epithelialelectrical resistance (TEER; ref. 29). As shown previously in HBECs(refs 29,30), we also found on the same ALI day lower TEERin cells from asthmatic donors compared with non-asthmaticdonors (Supplementary Fig. 2a), suggesting defective barrierfunction in those asthmatic cells. In uncompressed layers, wethen examined how these differences in barrier function mightcorrespond with changes in cellular jamming. Whether from non-asthmatic or asthmatic donors, the MSD and χ4(1t) showed thatcells were highly motile and unjammed on early days in ALIculture (Fig. 2a,d and Supplementary Fig. 2b and SupplementaryMovie 2). For example, on day 3 (Fig. 2g, triangles), HBECsfrom a non-asthmatic donor showed peaks in χ4(1t) indicative offaster-moving cooperative packs comprising roughly 20 cells withan average lifetime of 81min, but by days 6 (Fig. 2g, circles) and 8(Fig. 2g, asterisks), χ4(1t) showed no peak, thus indicating that thelayer had jammed.

Importantly, in cells from asthmatic donors compared withthose from non-asthmatic donors this transition to the jammedphase in ALI culture was substantially delayed. For example, cellsfrom a representative non-asthmatic donor jammed by day 6

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ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT4357

(Fig. 2a–c,g and Supplementary Movie 2), whereas cells from arepresentative asthmatic donor became jammed only by day 14(Fig. 2d–f,h and Supplementary Movie 3). On days 10 (Fig. 2h,circles) and 6 (Fig. 2h, triangles), HBECs from an asthmatic donorshowed peaks in χ4(1t) indicative of faster-moving cooperativepacks comprising approximately 26 and 12 cells with lifetimesof 72 and 90min, respectively, but by day 14 (Fig. 2h, asterisks)showed no peaks in χ4(1t), thus indicating that the layerhad jammed. This delayed jamming transition correlated wellwith delayed increases of TEER in cells from asthmatic donorscompared with cells from non-asthmatic donors. For example, incells from a representative non-asthmatic donor (SupplementaryFig. 2 and Supplementary Movie 2), TEER progressively increasedto 370� cm2 by day 6 as cells jammed, and continued toincrease to 700� cm2 until day 14, indicating that the earlyjamming transition corresponded to a rapid increase of barrierintegrity. However, in cells from a representative asthmatic donor,TEER reached only 421� cm2 even on day 21 (SupplementaryFig. 2 and Supplementary Movie 3). Moreover, this delay in thespontaneous jamming transition was not donor specific, but wasconsistently observed in cells obtained from multiple asthmaticdonors compared with those from non-asthmatic donors acquiredfrom the same source (The University of North Carolina, Methods;Supplementary Fig. 3).Whereas cells from all non-asthmatic donors(n= 5) became jammed between day 4 and 10 (SupplementaryFig. 3, blue circles), cells from asthmatic donors (n= 4) mostlyremained unjammed as late as day 14 (Supplementary Fig. 3,red circles)

Together, these systematic changes in cooperative cellularmotions establish that HBEC layers in ALI culture express spatiallyheterogeneous dynamics of the kind that in inert systems hasbeen taken as being the signature of matter that is close to ajamming transition23,26,27. As shown below, all the epithelial systemsstudied here existed in the vicinity of a jamming transition28.It is already known that asthmatic HBECs exhibit an aberrantinjury response and compromised differentiation31, and here wedemonstrate, further, that maturation of the airway epithelial layercoincides in time with the transition from an unjammed to ajammed condition, and that this transition is appreciably delayedin layers derived from asthmatic compared with non-asthmaticdonors (Fig. 2).

Jamming and the adhesion paradoxIn these collective cellular systems, what mechanical factors mightpromote cell jamming? Results from inert particulate matter32,33suggest that plausible mechanisms would include increased mutualcrowding and increased mutual adhesion4,9,16,17. As regards mutualcellular crowding34, we found no corresponding differences in thecase of the marked hypermobility in cells from asthmatic versusnon-asthmatic donors (Figs 1 and 2 and Supplementary Movies 1,2 and 3). As regards mutual adhesion, we hypothesized that justas increased particle–particle adhesion promotes jamming in close-packed inert collective systems7, so too increased cell–cell adhesionwould promote jamming in the confluent living cellular layer17.Superficially at least, such a notion would help to explain whymaturing HBEC layers derived from non-asthmatic donors tendto jam more rapidly than do those from asthmatic donors (Fig. 2and Supplementary Fig. 3), and would be consistent with theobservations that HBEC layers from asthmatic compared with non-asthmatic donors exhibit decreased TEER, increased permeability30and modestly diminished expression of E-cadherin35. We rejectedthis hypothesis, however, based on the experimental evidencedescribed below.

In the confluent intact epithelial layer, tugging (tensile) stresstransmitted across the cell–cell junction overwhelmingly dominatescompressive stress4,36. As shown below, each cell tugs on immediate

neighbours, but rarely pushes. Tugging stress exerted across thecell–cell junction can exist only to the extent that it is supportedby cell–cell adhesive stress, there being no other mechanismto transmit tensile stress across a cell–cell junction. As such,we can logically equate tugging stress transmitted across thecell–cell junction to adhesive stress. To measure this adhesivestress within the intact cellular layer, we first used MonolayerTraction Microscopy to map local tractions that each cell exertson its substrate, and then used Monolayer Stress Microscopy inthe same layer to map intercellular stresses that each cell exertson its immediate neighbours4,8,36 (Methods). We plated primaryHBECs obtained from non-asthmatic donors or from asthmaticdonors (Fig. 3) on a polyacrylamide gel (Young’s modulus= 1.2 kPa,thickness= 100 µm); an important difference here compared withALI culture conditions in Transwells described above is that platingHBECs on a gel precludes establishing an ALI culture. Hence, weresort to polyacrylamide only as a matter of necessity. Nonetheless,these HBECs showed cooperative fluctuations comparable tothose described above for cells in ALI culture (SupplementaryFig. 4a,b). On day 3 after seeding, HBECs from three asthmaticdonors showed peaks in χ4(1t) indicative of cooperative packscomprising approximately 80, 20 and 70 cells with lifetimes of20, 30 and 40min, respectively, but HBECs from non-asthmaticdonors showed no peaks in χ4(1t), thus indicating that thelayers had jammed (Supplementary Fig. 4c,d). In a representativelayer derived from a non-asthmatic donor, tractions exerted byHBECs on the substrate fluctuated markedly in time and in space(Fig. 3a) on a scale comparable to but somewhat larger thanthe size of a cell. The intercellular stress was heterogeneous andpredominantly tensile (Fig. 3c); in all experimental repetitions,stress averaged over the entire cell field was variable but tensile(average tension: 315±283 Pa; n=5). In the HBEC layers derivedfrom asthmatic versus non-asthmatic donors, r.m.s. tractionsexerted on the substrate were not statistically different, buttended to be larger (Fig. 3b,g; 114 ± 88 Pa versus 24 ± 7 Pa;p=0.22). However, intercellular tensions exerted by each cell on itsneighbours were larger by a factor 1.5 to 5 (Fig. 3d,h; 792±171 Paversus 257±82 Pa; p=0.02). In both non-asthmatic and asthmaticHBECs, cells moved in packs, with regions of high tension(Fig. 3c,d) that spanned many cells (Fig. 3e,f), indicating thatfluctuations in tension were cooperative over distances comparableto many cell diameters. To quantify the spatial extent of this stresscooperativity, we measured the spatial autocorrelation functionof tugging stress (tension), C(R), as a function of cell separationdistance, R (Methods). In every case the tension correlationC(R) decayed over several hundred micrometres, but with fasterspatial decay in cells derived from asthmatic versus non-asthmaticsubjects (Fig. 3i; the correlation values C(R) at 14 µm were0.29±0.02 and 0.48±0.03 in asthmatic and non-asthmatic HBEClayers, respectively; p = 0.003). HBECs derived from asthmaticversus non-asthmatic donors bear higher but more localizedintercellular tension.

On the basis of these observations we cannot determine if build-up of intercellular adhesive stresses might promote unjamming or,conversely, if unjamming might promote build-up of intercellularadhesive stresses. We can say, however, that unjamming coexistswith the amplification of local adhesive intercellular stresses,not with their attenuation (Fig. 3). This finding is counter-intuitive, but is in concert nevertheless with comprehensive recentfindings of others establishing by direct measurements that cell–celladhesive stress is not attenuated by reducing the expressionof E-cadherin37.

Cell jamming, mutual adhesion and perimeterTo shed light on this paradox we turned to the vertex model ofthe cellular layer18–20 (Supplementary Equations 1 and 2 in the

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Figure 3 | In HBECs derived from asthmatic donors compared with those from non-asthmatic donors, tractions and intercellular stresses are greater butthe spatial correlation of tension decays faster. a,b, Colour maps of tractions exerted by HBECs derived from a non-asthmatic donor (a; N2 in g–i) and anasthmatic donor (b; A2 in g–i) on their substrates. Colour scale is shown to the right of b. c,d, Colour maps of intercellular stresses exerted across cell–celljunctions for donors N2 (c) and A2 (d) show packs of high tension that span many cell diameters. Colour scale is shown to the right of d.e,f, Phase-contrast maps of HBEC layers on polyacrylamide gels for donors N2 (e) and A2 (f). g, In cells derived from asthmatic donors (red: A1, A2, A3)versus non-asthmatic donors (blue: N1, N2), root mean square (r.m.s.) tractions were not statistically di�erent, but tended to be larger (r.m.s. traction:114±88 Pa versus 24±7 Pa; variance: 7,821 versus 53; p=0.22). h, Intercellular tensions were larger by a factor of 1.5 to 5 in cells derived from asthmatic(red) versus non-asthmatic donors (blue; tension: 792± 171 Pa versus 257±82 Pa; variance: 29,426 versus 6,787; p=0.02). i, Spatial autocorrelationfunction, C(R), of tension as a function of cell separation distance, R, shows that the tension correlation decayed over several hundred micrometres in allcases, but extended to shorter distances in cells derived from asthmatic (red symbols) versus non-asthmatic donors (blue symbols), thus confirming thatintercellular stresses were larger in magnitude but more highly localized in the HBECs derived from asthmatic donors compared with non-asthmatic donors(C(R) at 140 µm in asthmatic versus non-asthmatic HBEC layers: 0.29±0.02 versus 0.48±0.03; variance: 0.0004 versus 0.0009; p=0.003). The r.m.s.tractions, intercellular tensions and spatial autocorrelation in g–i were averaged across three to five experimental repeats for each donor. Error bars in g,hrepresent the standard error.

Supplementary Information), which represents the projection ofeach cell in two-dimensions by an irregular curved polygon witha shape index, p= P/

√A, where P and A are the cell perimeter

and projected area, respectively (Supplementary Fig. 7). The modelproposes that the mechanical energy associated with each cell is afunction of three contributions: an energy associated with cell areaand attributable to area compressibility; an energy associated withcell perimeter attributable to the stiffness and contractility of theapical actomyosin ring; and an energy associated with interfacesof cell–cell contact, including the effects of adhesion moleculessuch as cadherins and associated actomyosin interactions, which,together, are expressible as a net line tension14,20,38,39. Whereas thefirst two contributions are always positive, the line tension decreaseswith increasing cell–cell adhesion or decreasing actin–myosincontractility, and therefore can be positive or negative18,39. Thesephysical effects compete to generate a preferred cell shape,characterized by its preferred perimeter, p0. In the vertex modelcells can exchange places with immediate neighbours, but any

such structural rearrangement requires changes in cell shapes.The change in energy required to complete such a structuralrearrangement defines an energy barrier (Supplementary Fig. 7).Analysing this model, we find a scaling collapse in which theaverage energy barrier to structural rearrangements, 1ε, exhibitstwo critical branches, f+ and f−, as a function of the parameter p0,with a crossover between them19 at a critical value of p0 denotedby p∗0 ,

1ε=|p0−p∗0|β f±(r |p0−p∗0|

−1)

Here r is the reciprocal of perimeter stiffness (SupplementaryInformation), β and 1 are critical scaling exponents. Importantly,p∗0∼=3.81 is a critical shape index derived from the analysis of criticalscaling behaviour; it is a pure number that rests on no adjustablemodel parameters or curve fitting to data19 (SupplementaryInformation). As the argument of f± becomes small, one branchapproaches a finite energy barrier height, thus implying solid-like

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ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT4357

Unjam

ming

Jam

min

g

Jamming

threshold

Day

Day

Day

Cell

shap

e (p

)

a b

3 6 8 6 10 14

p

2 6 10 14

Non-asthma Asthma 5.0

4.8

4.6

4.4

4.2

4.0

3.8

3.6

4.2

4.0

4.1

3.9

3.8

3.7

Figure 4 | With increasing maturation of HBECs in ALI culture, cell perimeter, as expressed by the non-dimensional parameter p̄, decreasessystematically towards the critical value p∗

0 (3.81) predicted to occur at jamming by the vertex model together with the theory of critical scalingexponents. a, Over the course of maturation in ALI culture, HBECs from a representative non-asthmatic donor (Fig. 2 and Supplementary Movie 2)approached the jammed state, and the median ratio of perimeter to the square root area of cells systematically approached the jamming threshold p∗0. InHBECs from a representative asthmatic donor (Fig. 2 and Supplementary Movie 3), however, the approach of p̄ to p∗0 was considerably delayed. Over time,and in both cases, p̄ systematically approached the jamming threshold of 3.81. Inset: p̄ for representative non-asthmatic and asthmatic donors plotted withthe same axis of ALI days to allow comparison of the jamming transition timing. Boxplot shows median and quartiles. Whiskers are maximum andminimum data points. b, Simulated tissues with input parameters of target cell-shape index p0=4.2, corresponding to a fluidized state (top panel), andp0=3.813, corresponding to a jammed tissue (bottom panel).

behaviour with a finite shear modulus of the cellular collective.However, the other branch approaches a vanishing energy-barrierheight and a vanishing shear modulus, thus implying no barrierto rearrangements and, therefore, unjammed fluid-like behaviourof the cellular collective. The former branch is predicted to prevailwhen p0<p∗0 and the latter when p0>p∗0 .

To test further the prediction that cells in late ALI days wereapproaching a jammed state, we measured the shape index pfor cells in HBEC layers as a function of ALI day in non-asthmatic and asthmatic HBECs (Methods); the median of pis denoted p̄. Data indicate highly significant differences in p̄between ALI days, between cells derived from healthy versusasthmatic donors, and often between the observed value of p̄ andthe critical value 3.81 for jamming (Fig. 4 and SupplementaryFig. 5). However, as ALI day progressed, the structural parameterp̄ progressively approached p∗0 (Fig. 4a and Supplementary Fig. 5).Note that because p̄ is a measure of cellular structure, notcellular dynamics or intercellular stresses, it is independent ofmeasurements of MSD, χ4 and cellular stresses. As such, thebehaviour in Fig. 4 independently validates the critical behaviouras jamming is approached, and implies that cells in the jammedstate are unable to surmount the mechanical energy barriers thatincrease as cell–cell adhesion decreases (Supplementary Fig. 7).Importantly, in asthmatic compared with non-asthmatic HBECs,the approach of p̄ to p∗0 is appreciably delayed. Moreover, afterapplication ofmechanical compressive stresses similar inmagnitudeto those expected during bronchospasm, the HBECs layer becomesunjammed and, as predicted from the vertex model, p̄ substantiallyexceeds p∗0 (Supplementary Fig. 6).

Even for cells within the same epithelial layer, an importantfeature of the observed values of p for individual cells is theirstriking variability (Fig. 4 and Supplementary Fig. 5), which is

much larger than measurement errors attributable to identificationof cell boundaries derived from phase-contrast images. Geometryimposes some constraints on this variability; p, and therefore p̄,have no upper limit, but can never be smaller than the shapeindex for a circle, roughly 3.54. In the limit of vanishing activeforce fluctuations, as described here, the vertex model predicts thevariability to be highly skewed19 and to increase away from thecritical point. Interestingly, the former of these predictions is borneout by observations, but the latter is not (Supplementary Fig. 5). Assuch, we suspected that active force fluctuations may contribute tothe observed variability.

Therefore, we next incorporated small but non-negligibleactive-force fluctuations into the vertex model (SupplementaryInformation). Not surprisingly, increased force fluctuations are seento work in concert with increased adhesion to unjam the cellularcollective, providing another path to unjamming (SupplementaryMovie 4). Cells in jammedmodel tissues fluctuate but do not changeplaces with immediate neighbours, whereas structural rearrange-ments occur frequently in the unjammed tissue. Interestingly, suchrearrangements cease and the model tissue becomes jammed whenthe shape index p̄ approaches the same critical value of 3.81, regard-less of the magnitude of the active fluctuations. This finding wouldexplain why active HBECs still jam at p̄=3.81, and suggests that theobserved variability in p near the critical point might be attributableto active-force fluctuations. If true, then it may be possible in thefuture to estimate the properties of these active forces by compar-ing shape fluctuations predicted by active-vertex behaviour versusexperimental observations. Another open question is the extent towhich p̄ can be used as a structural order parameter in other tissuetypes; finding that p̄ is experimentally close to 3.81 is sufficient for aninterpretation of a jammed state in our HBECs, which are isotropicin the plane. We expect that this analysis and this shape index may

1046 NATUREMATERIALS | VOL 14 | OCTOBER 2015 | www.nature.com/naturematerials

NATURE MATERIALS DOI: 10.1038/NMAT4357 ARTICLESbe useful for other isotropic tissues, but that it may fail in interestingand potentially predictable ways for anisotropic tissues, such as inthe endothelial layer under shear flow40.

The vertex model is therefore seen to capture essential features ofcollective cellular behaviour, especially the existence and propertiesof a jamming transition. Specifically, in some conditions each cellbecomes frozen in place relative to immediate neighbours, as ifcaged, whereas in other conditions each cell readily exchangesplaces with immediate neighbours and, as a result, the integratedtissue continuously remodels. Concerning rates of cellular motionand rearrangement, across the transition there exist qualitative aswell as quantitative differences in rates of cellular motion andrearrangement. The vertex model also predicts precisely how cellrearrangements across this transition—as independently assessed bydynamic metrics of jamming—are marked by changes in a specificindex of cell shape (Fig. 4). Nevertheless, interesting theoreticalquestions remain unanswered. In the vertex model, for example,propulsive stresses are not taken explicitly into account. Theoriesbased directly on self-propelled particles, by contrast, show thatpropulsive forces help to unjam particulate systems3,41,42, but insuch systems jamming is driven by mutual crowding rather thanby changes in mutual adhesion or line tension. As such, they donot account for the changes of cell shape reported here, and theypredict a transition that is therefore of a fundamentally differentkind (Supplementary Information)43. For that reason, addition ofpropulsive stresses to the vertex model would help to resolve thequestions of the extent to which propulsive stresses might alterthe nature of the transition or the centrality of the critical shapeparameter, p∗0 (refs 20,39,44–46).

The emerging physical pictureIt has been argued recently that a wide variety of living collectivesystems evolve spontaneously towards the neighbourhood of acritical phase transition47–49. In the particular case of the airwayepithelial cellular layer, cellular motions (Figs 1 and 2), cellularforces (Fig. 3) and cellular shapes (Fig. 4) all provide strong evidencethat a critical jamming transition occurs in these tissues. Ourfindings thereby suggest a new physical picture of airway epithelialremodelling, maturation and repair, in which epithelial cellsmigrate collectively in conditions close to a jamming transition17.When effective energy barriers are small or non-existent, thetissue is readily able to explore new configurations as the systemremodels into new states, some of which might be adaptive andothers maladaptive9,16,17. But, as energy barriers increase, the tissuebecomes locked into a jammed state.

The bronchial epithelium is subject to repeated mechanicalperturbations through the action of spontaneous breathing, and issubject to injury through exposure to harmful environmentalpollutants, viruses, allergens, reactive oxygen species, orinflammatory mediators. Here we put forward the hypothesisthat in non-asthmatic subjects these external factors can cause thecellular collective to unjam, explore various possible configurations,and then resettle into an adaptive quiescent, jammed, solid-likestate; by so doing they effect self-repair in that remodelled state.Importantly, we have not yet determined themolecularmechanismsthat impact the jamming transition, and we expect that there couldbe many. Nevertheless, the observations reported here establisha simple and easily measured structural index that quantifies theproximity of the layer to a jamming transition, and suggests thatperhaps molecular mechanisms can be investigated and categorizedby how they affect jamming.

Bronchial epithelial cells and their mechano-sensitivity areincreasingly understood to play a major role in airway remodellingin asthma, but just how these events are linked to asthmapathobiology has remained unclear1,12,14,15,21,34,50–52. In cells fromasthmatic compared with non-asthmatic donors in ALI culture, we

show that the transition from a hypermobile, unjammed, fluid-like phase to a quiescent, jammed, solid-like phase is delayedsubstantially or disrupted altogether. Whether this delay arisesfrom layer injury, immaturity, or dysmaturity, the prolonged orsustained hypermobile fluid-like phase defines an unanticipatedmaladaptive phenotype which, to our knowledge, comprisesthe first known instance—in any disease—where pathobiologyis linked to the recent discovery of cellular jamming andunjamming4,17,53,54. It remains to be determined if these processesand their downstream effects might be favourably impacted bybronchodilators, corticosteroids, or other therapeutic interventions.

MethodsMethods and any associated references are available in the onlineversion of the paper.

Received 13 February 2015; accepted 18 June 2015;published online 3 August 2015

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AcknowledgementsAuthors thank the staff of the UNC CF Center, Tissue Procurement and Cell CultureCore at the University of North Carolina, Chapel Hill. This research was supported by theFrancis Family Foundation, the Alfred P. Sloan Foundation, the American HeartAssociation (13SDG14320004), the National Research Foundation of Korea(NRF-2013S1A2A2035518), the National Science Foundation (BMMB-1334611,DMR-1352184) and the National Institutes of Health (K25HL091124, P30DK065988,P30ES000002, HL007118, R01HL102373, R01HL107561, P01HL120839).

Author contributionsJ.-A.P. designed experiments, carried out time-lapse imaging of HBECs in ALI cultureand interpreted data. J.H.K. designed measurements of physical forces within HBECs andanalysed data. J.H.K. and M.M. carried out force-measurement experiments. J.H.K.,N.T.Q., C.Y.P. and C.H. analysed the dynamics of cellular motions. D.B. and J.A.M.analysed cell-shape parameters. D.B. modelled cell-shape parameters and cell motility.J.A.M. and S.-H.K. carried out time-lapse imaging of HBECs in ALI culture. D.T.T., B.G.,J.N., R.S. and S.B. contributed to preparation of physical-force measurements. S.H.R.contributed to the design of experiments and provided primary HBECs. D.A.W., D.J.T.,S.T.W., M.L.M., J.P.B., J.M.D. and J.J.F. guided data interpretation and analysis of cellularmigration and the jamming transition. S.H.R., A.T.K., S.A.S., E.I., S.T.W., E.P.H., K.T. andJ.M.D. guided data interpretation on the biological relevance of cellular migration. J.-A.P.,J.H.K., D.B., M.L.M. and J.J.F., wrote the manuscript. J.-A.P. and J.J.F. oversaw the project.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints.Correspondence and requests for materials should be addressed to J.-A.P.

Competing financial interestsThe authors declare no competing financial interests.

1048 NATUREMATERIALS | VOL 14 | OCTOBER 2015 | www.nature.com/naturematerials

NATURE MATERIALS DOI: 10.1038/NMAT4357 ARTICLESMethodsCulture of primary human bronchial epithelial cells.We used primary humanbronchial epithelial cells (HBECs) derived from ten non-asthmatic and nineasthmatic donors from two distinct sources. Primary HBECs at passage 1 werepurchased from Lonza or provided by S. Randell at the Marsico LungInstitute/Cystic Fibrosis Research Center at the University of North Carolina,Chapel Hill. Human lungs unsuitable for transplantation, including three cases offatal asthma and one with asthma in the medical social history, were obtainedunder protocol #03-1396 approved by the University of North Carolina at ChapelHill Biomedical Institutional Review Board. Informed consent was obtained fromauthorized representatives of all organ donors. All non-asthmatic lungs were fromnon-smokers with no history of chronic lung disease. HBECs from all donors werecultured as previously described12. Passage 2 HBECs were plated at a density of125,000 cells cm−2 on 12-Transwell plates with polyester membranes with 0.4 µmpores (Corning) coated with 50 ngml−1 of type 1 rat tail collagen (BD Biosciences).Cells were cultured for five to seven days under submerged conditions untilconfluence. Once cells reached confluence, submerged condition was switched tothe ALI condition by removing medium from the apical surface. The ALI culturewas maintained for another 16–18 days, unless described otherwise. By 10–11 daysin ALI culture, cells from all donors produced a substantial amount of mucus at theapical surface.

Exposure of HBECs to compressive mechanical stress. On day 16 of ALI culture,when HBECs had matured into a well-differentiated phenotype of confluentbronchial epithelium similar to that existing in vivo13, we exposed cells toapical-to-basal compressive stress spanning the physiologic ranges (0, 3, 10, 20, or30 cm H2O) for three hours as previously described14,21,52, along with appropriatetime-matched controls. For the comparison of cells from non-asthmatic versusasthmatic donors, we used cells on different ALI day, as indicated in Figs 2 and 4,during the course of ALI culture.

Measurement of cellular velocities in ALI culture. At 18 h after application ofcompressive stress, time-lapse phase-contrast images of HBECs in ALI culture wereacquired on an inverted optical microscope (Leica, DMI 6000B) equipped with aheated (37 ◦C), CO2-controlled (5%), humidified chamber. For each condition andeach donor, two to three wells were imaged at six different positions in each well at3-min intervals for 150min. In each region comprising roughly 1,200–1,500well-differentiated cells, we mapped cellular motions within the pseudostratifiedlayer in time and in space. Using phase-contrast images obtained in an apical focalplane, instantaneous cellular velocities were mapped by particle imagingvelocimetry (PIV; refs 10,16) in which the cross-correlation window size was64×64 pixels (pixel size of 0.44 µm). For each data set, the spatial distribution ofcellular migration velocities was mapped based on a running boxcar average ofinstantaneous velocities throughout 150-min periods. Cellular trajectories werecomputed by integration of the instantaneous velocity field obtained from the PIV,with interpolation of each displacement point to the nearest grid point. Thesetrajectories closely approximate cellular motions4,8,10,16,55; to achieve accuracycomparable to individual cell tracking, we chose a grid spacing (3.5 µm)substantially smaller than the size of a single cell (15–20 µm).

Quantifying spatially heterogeneous dynamics of cellular motions. As a metricof cellular motions, mean square displacements (MSD) were computed as afunction of time interval,1t :

MSD(1t)=〈|ri(t+1t)−ri(t)|2〉

where ri(t) indicates the position of cell i at time t and 〈. . .〉 denotes the averageover all time t , and all cells. The cellular self-diffusion coefficient defined asDs= lim1t→∞MSD(1t)/(41t) was then computed. To quantify the dynamics ofcellular motions, we computed the self-overlap order parameter27:

Q(1t)=N−1N∑i=1

wi

where N is the number of cells and w=1 if |ri(t+1t)−ri(t)|<0.15dc (where dc isaverage cell diameter) and w=0 otherwise. To quantify the size and lifetime ofthese cooperative fluctuations as proposed in ref. 24 proposed, spatially

heterogeneous cellular dynamics in each sample were quantified by the four-pointsusceptibility χ4, approximated by

χ4(1t)=N [〈Q(1t)2〉−〈D(1t)〉2]

where N is the number of cells and 〈. . .〉 denotes the ensemble average taken overall sequences of images and all time, t . This measure approximates the four-pointsusceptibility, χ4(1t), and possesses several useful attributes4,27, such asquantitative measures of the size of cooperatively moving cell packs and theirassociated rearrangement times.

Measurement of mechanical stresses within the HBEC layer. To measuremechanical stresses within the layer plane, we seeded HBECs on a polyacrylamidegel (Young’s modulus= 1.2 kPa, thickness= 100 µm). Gel preparation and seedingprotocols were similar to published protocols4,10,54. Briefly, we deposited apolydimethyl siloxane (PDMS) membrane with a rectangular opening (8×8mm)on the gel. After coating the gel with type 1 collagen (BD Biosciences), we seededHBECs derived from non-asthmatic and asthmatic subjects. We then removed thePDMS membrane and allowed HBECs to grow to confluence for three days.Fluorescence and phase-contrast images were acquired at 10-min intervals for 2 h.Local gel displacements were quantified from an image of embedded fluorescentmarkers at each experimental time point and a reference image obtained aftertrypsinization. Local migration velocities and gel displacements were obtained bythe PIV method10,16 using a cross-correlation window size of 32×32 pixels (pixelsize of 0.88 µm). To obtain substrate tractions, we used the numerical procedurefrom Fourier-Transform Traction Microscopy10,56. To obtain layer stresses, we usedthe numerical procedure fromMonolayer Stress Microscopy4,36,54. Briefly, wecomputed a map of the tractions exerted by the cells on their substrate using geldisplacements. From these tractions, we obtained the distribution of intercellularstresses within the cellular sheet. At each point in the cellular sheet, we computedthe two principal stress components σmax and σmin and their corresponding,mutually perpendicular, principal orientations. We then computed the local tensionwithin the cellular sheet, defined as σ̄ =(σmax+σmin)/2. The boundary edge of thepatterned cellular sheet was taken to be zero stress4,36. After recovery of a stressmap, an area near the edge (∼1mm) was cropped from the map (Fig. 3a–d).

Calculation of the spatial autocorrelation function of tension. To quantify thespatial extent of stress cooperativity, we computed the spatial autocorrelationfunction of intercellular tension4:

C(R)=1

Nvar(σ̄ )

N∑i,j=1

∑|Ri−Rj |=R

δσ̄iδσ̄i

where δσ̄i is the local departure of the tension at position Ri from its spatial mean〈σ̄ 〉, var(σ̄ ) is the variance of those departures, and the |Ri−Rj|=R denotesequality within a uniform bin width of 20 µm, within which there are N points.

Quantification of cell shapes in ALI culture. Time-lapse phase-contrast images ofHBECs in ALI culture were acquired as described above. For each condition anddonor, 130 cells were manually traced using ImageJ software (National Institutes ofHealth) from four different fields of view, and the shape index p=P/

√A, where P

and A are the cell perimeter and projected area, was computed for each traced cell.The median of this shape parameter p̄ was computed. With a ‘bootstrap’ method57,the data were uniformly randomly sampled with replacement 105 times, fromwhich the distribution of the resulting medians was used to determine confidenceintervals and p-values.

References55. Angelini, T. E., Hannezo, E., Trepat, X., Fredberg, J. J. & Weitz, D. A. Cell

migration driven by cooperative substrate deformation patterns. Phys. Rev.Lett. 104, 168104 (2010).

56. Butler, J. P., Tolic-Norrelykke, I. M., Fabry, B. & Fredberg, J. J. Traction fields,moments, and strain energy that cells exert on their surroundings. Am. J.Physiol. Cell Physiol. 282, C595–C605 (2002).

57. Efron, B. & Tibshirani, R. An Introduction to the Bootstrap(Chapman & Hall, 1993).

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