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[Junhong Zhou](https://orcid.org/0000-0001-6327-6512), [Jun Nakanishi](https://orcid.org/0000-0003-4457-6581)

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[Impact of Interfacial Viscosity on the Robustness of Phospholipid‐Decorated Fluid Cell Scaffolds](https://mdr.nims.go.jp/datasets/37d80649-4f40-4141-ad74-33f7c11c52f9)

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Impact of Interfacial Viscosity on the Robustness of Phospholipid‐Decorated Fluid Cell ScaffoldsImpact of Interfacial Viscosity on the Robustness ofPhospholipid-Decorated Fluid Cell ScaffoldsJunhong Zhou and Jun Nakanishi*1. IntroductionCells in biological tissues reside within theextracellular matrix (ECM). The ECM, con-sisting of water, proteins, and polysacchar-ides, not only provides structural support tocells but also regulates cell behaviorthrough biochemical and biophysical cues,influencing normal development and path-ological processes. Recently, it has beenwidely recognized that the biophysicalproperties of ECM, particularly its mechan-ical properties, strongly affect cell behav-ior.[1] Mesenchymal stem cells adapt tothe elasticity of the model ECM and changetheir morphology on variable-compliantpolyacrylamide gels by rearranging theircytoskeletons to differentiate into specificlineages.[2] However, native ECM not onlyexhibits elastic properties but also has a vis-cous nature with time-dependent mechan-ical properties.[3]It has been found that the loss modulus(G 00) measured at 1Hz is �10% of the stor-age modulus (G 0) in soft tissues (brain) andstiffer skeletal tissues (bone).[3a] Recently, hydrogels with tunableviscoelastic properties have been developed by changing theirmolecular weight and through covalent or ionic crosslinking,revealing that viscosity also plays a significant role in influencingcell behavior, such as cell spreading.[3] Further to this bulkmechanic tuning, a simple model system for manipulating viscos-ity has been developed using supported lipid bilayers (SLBs). SLBs,which exhibit a long range of lipid lateral mobility within lipidmembranes assembled on a solid-supported surface, have revealedthe effect of the interfacial viscosity on cell behavior.[4–8] For exam-ple, Bennett et al. used SLB systems composed of either saturatedor unsaturated phospholipids with melting temperatures higheror lower than ambient temperature to investigate cell behaviorat the SLBs with two distinct states of viscosities. The viscosityof these SLBs drives the mobility of the ligands present on thesurface, resulting in an enhanced cell-spreading area and mecha-nosensitivity with increasing viscosity.[8] Furthermore, by chang-ing the underlying substrate from a rigid solid support to a softpolymer support, SLBs can mimic the physiological flexibilityof the cellular environment.[4] However, these SLBs, together withhydrogels, are limited in their ability to reach the super-soft regionof mechanics, which is needed to further expand the viscoelasticwindow of model ECMs.A fluid interface consisting of two liquid phases has beenemployed to expand the ultimate soft range of cell scaffolds inJ. Zhou, J. NakanishiResearch Center for Macromolecules and BiomaterialsNational Institute for Materials Science (NIMS)1-1 Namiki, Tsukuba, Ibaraki 305-0044, JapanE-mail: NAKANISHI.Jun@nims.go.jpJ. Zhou, J. NakanishiGraduate School of Advanced Science and EngineeringWaseda University3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, JapanJ. NakanishiGraduate School of Advanced EngineeringTokyo University of Science6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, JapanJ. NakanishiResearch Center for Autonomous Systems Materialogy (ASMat)Institute of Integrated Research (IIR), Institute of Science Tokyo (ScienceTokyo)4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, JapanThe ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/anbr.202500076.© 2025 The Author(s). Advanced NanoBiomed Research published byWiley-VCH GmbH. This is an open access article under the terms ofthe Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the originalwork is properly cited.DOI: 10.1002/anbr.202500076The mechanical properties of the cellular microenvironment contribute signifi-cantly to cell behavior. Thus, deformable phospholipid-decorated perfluoro-carbon interfaces have emerged for further expansion of material mechanics toan ultimate soft range as cell scaffolds. In addition, a highly deformable staterequires the material to be robust enough to adapt to dynamic cellular forces.However, the effect of interfacial viscosity on the cell adhesion behavior andmaterial robustness remains unknown on the super-soft substrate. To addressthese issues, an interfacial phospholipid membrane (IPLM) with tunable viscosityis constructed by varying the mixing ratio of saturated and unsaturated lipidlayers. By co-assembling a cell adhesive and fluorescent lipid into the IPLM, it isshown that higher viscosity interfaces with lower unsaturated lipid content arepreferred from the viewpoint of cell spreading. However, a viscosity that is toohigh for 0% unsaturated lipid alters the lipid layer to a brittle solid-like nature,making it less adaptive to cell traction-induced high deformation. Therefore, atleast a trace amount of unsaturated lipids is required to maintain the robustnessof fluid scaffolds. These findings are useful for the design of biomimetic materialsand the long-term investigation of cell-matrix mechanical interactions in highlyadaptive environments.RESEARCH ARTICLEwww.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (1 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbHmailto:NAKANISHI.Jun@nims.go.jphttps://doi.org/10.1002/anbr.202500076http://creativecommons.org/licenses/by/4.0/http://www.advnanobiomedres.commechanobiology, as the fluid interface is intrinsic to a super-softnature derived from its original liquid phase. For example, ourgroup succeeded in using an interfacial phospholipid membrane(IPLM), which utilizes planar phospholipid membranes assem-bled at the water-perfluorocarbon (PFCL) interface. This IPLMhas identified a unique cell adhesion behavior, termed cellularadaptive wetting. This adaptive wetting involves a high out-of-plane deformation of the IPLM in response to cellular forces,enabling the readout of the cellular mechanical energy outputwith negligible energy dissipation by taking advantage of thesuper-soft nature of the fluid interface.[9] Nevertheless, howthe interfacial viscosity of IPLM affects cell adhesion remainspoorly understood. In addition, high-strain conditions can eithermake materials prone to cracks that propagate to a large scalewithin materials or exhibit resistance to cracks (robustness) inmaterials science in general. The high out-of-plane deformationcapability of the IPLM may also exhibit such characteristicsunder different viscosities. Such a correlation between IPLMrobustness and interfacial viscosity needs to be explored to opti-mize the material properties of cell scaffolds.To address these two aspects, this study utilized a facilemethod simply altering the mixing ratio of saturated and unsat-urated lipids for the systematic manipulation of IPLM viscosity toinvestigate the influence of systematically tunable interfacial vis-cosity on cell adhesion behavior and materials robustness. Here,we defined viscosity as the range of the mobility of lipid mole-cules within the IPLM, in a similar fashion to the viscosity ofreported SLBs.[8] Specifically, we used lipids with unsaturated(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and saturated(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC) tails as pri-mary components (Figure 1a). In this system, saturated DSPCexhibits a gel-like and poorly mobile state,[10,11] while mixing withunsaturated lipids like DOPC disrupts the intermolecular pack-ing between saturated lipid tails, increasing lipid mobility andsignificantly affecting overall membrane viscosity. Typical lateralmobility was expected to be manipulated by altering the mixingcontent of the unsaturated lipids (Figure 1b).[5,12] For the assem-bly process, the lipids were prepared as mixed lipid vesicles, andthe lipid membrane was formed spontaneously by incubatingthe vesicles and fluid interface under the proposed vesicle fusionat the water-PFCL interfaces (Figure 1c).[13] By introducingarginine-glycine-aspartic acid (RGD)-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and fluorophore-conjugated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine(DPPE), the IPLM allowed cell adhesion and visualization by fluo-rescence microscopy. In this study, Madin-Darby caninekidney (MDCK) cells stably expressing lifeact-green fluorescentprotein (GFP) were chosen because these cells undergomesenchymal-like phenotypic changes upon manipulation ofenvironmental factors, and fluorescent labeling of the actinFigure 1. Viscous tuning of phospholipid membranes at the fluid interface depending on the mixing ratio of saturated and unsaturated lipids. a) Chemicalstructure of two primary lipids, namely DOPC and DSPC. DOPC and DSPC are shown in cyan and black, respectively. b) Schematic drawing illustrating thevariation of IPLM viscosity as a function of unsaturated lipid content. The bar increments represent the content of DOPC changes. The bar decrementsrepresent the viscous changes of IPLM. c) Scheme of the proposed formation process of IPLM at the water–PFCL interface via vesicle fusion. The lipidmembranes also contain cell-adhesive RGD-DSPE (blue headgroup with saturated C-18 tail) and fluorescent Liss Rhod-DPPE (red headgroup with satu-rated C-16 tail).www.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (2 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.comcytoskeleton facilitates morphological observation.[14,15] Afterseeding the MDCK cells onto the IPLM systems, we hypothesizedthat cell spreading morphology and materials robustness inresponse to cellular forces would change depending on the viscos-ity of the IPLM.2. Results2.1. Impact of Unsaturated Lipid Contents on the PhaseBehavior in IPLMTo manipulate the viscosity of the substrate, we changed the mix-ing ratio of the saturated and unsaturated lipids, which allowedus to construct IPLM with different mobilities. Figure S1,Supporting Information, shows a scheme of IPLM coating ata planar interface. Briefly, a continuous phospholipid membranewas expected to form at the water-PFCL interface via adsorption,fusion, rupture, and merging.[13,16] In this IPLM, RGD-DSPE,and Liss Rhod-DPPE with saturated acyl chains were introducedas cell adhesion ligands and fluorescent probes, respectively, forthe membranes (Figure S1a, Supporting Information). The per-centages of these two lipids in the total lipid content were fixed at2% and 0.5%. Subsequently, varying the molar fractions (up to50%) of DOPC and saturated lipids (DSPCþ 2% RGD-DSPEþ 0.5% Liss Rhod-DPPE) were used to change the mobilityof the IPLM, in which DOPC and DSPC acted as primarycomponents.First, we studied the phase behavior at the liquid–liquid inter-face using fluorescence microscopy. Figure 2a shows represen-tative images of the distribution of Liss Rhod-DPPE fluorophoresin the six mixed lipid samples (0%, 2.5%, 5%, 10%, 20%, and50%) investigated at ambient temperature. At 0% DOPC, theimages showed apparent homogeneity based on the fluorescenceimages, as all lipids with saturated acyl chains were tightlypacked.[17] As the DOPC content increased to 2.5%, 5%, and10%, the fluorescence was evenly distributed over the entire sur-face, similar to that of the pure saturated lipid surface (0% case).Within this range (0%–10%), it was likely that the small amountsof unsaturated DOPC molecules were trapped as nanoscaledomains within the DSPC-rich homogeneous backgroundwithout affecting the overall fluorescence appearance, owingto the diffraction limit of optical imaging.[18] At 20% DOPC,the interface was divided into two regions: island-like fluorescentmicrodomains surrounded by a dark background (Figure S2,Supporting Information). Nanoscale phase separation may alsooccur in both fluorescent and dark domains.[18] At 50% DOPC,domain separation became more apparent, with fluorescentdomain sizes reaching hundreds of microns, suggesting macro-scopic phase separation of the primary components (DSPC andDOPC). In addition, another IPLM system incorporating unsat-urated Liss Rhod-DOPE fluorophore and RGD-DOPE ligand—replacing Liss Rhod-DPPE and RGD-DSPE—was fabricated todetermine which lipid dominated the fluorescent regions.Similar to Figure 2, homogeneity was observed for 0%–10% sam-ples and macroscopic phase separation for 20%–50% samples(Figure S3, Supporting Information).To confirm that the homogeneous phases observed in0%–10% samples were not unstable, nonequilibrium states,we incubated the interface for 15 h. Homogeneous distributionswere maintained within the 0%–10% DOPC range (Figure 2b),indicating no optically detectable phase separation occurred evenover long timescales, proving stable lipid distribution during theexperiments. The segregation of fluorescent and dark domainswas also retained in the 20% and 50% IPLM samples, with ran-domly distributed fluorescent domains persisting in the darkbackground after 15 h of incubation. In addition, the contrastbetween bright and dark regions was slightly reduced owingto increased fluorescence in the dark regions, likely from minormigration of saturated Liss Rhod-DPPE fluorophore from fluo-rescent to dark domains (Figure 2b, 20% and 50%). Thisphenomenon did not appear with the unsaturated Liss Rhod-DOPE fluorophore (Figure S3, Supporting Information). Thephase separation-mediated domain formation at sizes compara-ble to cells led us to propose that excessively large phase domains(microns to hundreds of microns) may cause local heteroge-neous mobility.[19] Such domains could hinder investigation ofthe relationship between interfacial viscosity and cell adhesionowing to their nonuniformity. To determine whether thephase behavior was unique to fluid interfaces, we also con-structed solid-supported phospholipid membranes with thesame DOPC contents (0%, 2.5%, 5%, 10%, 20%, and 50%)(a)(b)Figure 2. Fluorescence microscopy images of lipid distribution by altering DOPC content in saturated lipids from 0% to 50%. The top and bottom panelscorrespond to each image of a) immediately after DOPC addition and b) after 15 h. The images shown in Figure 2 are the representative fluorescenceimages of the lipid distribution from more than 20 independent experiments.www.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (3 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.com(Figure S4, Supporting Information). As shown in Figure S5,Supporting Information, apparently homogeneous distributionswere observed in 0%–10% samples and sustained over one day,similar to the fluid interface. Notably, in 20%–50% samples,phase separation domains appeared but were smaller than thoseon the fluid interface, indicating that the effect of DOPC contenton phase behavior was not specific to the fluid interface, althoughhigher fluidity of the fluid interfaces accelerated domain coales-cence into larger phase-separated regions. Overall, at low DOPCcontent, IPLMs remained in a homogeneous phase withoutmacroscopic phase separation, while at high DOPC content,large-scale phase separation occurred, potentially complicatinganalyses of interfacial viscosity and cell adhesion owing tononuniformity.2.2. Evaluation of Interfacial Mobility Change by AlteringUnsaturated Lipid ContentsTo investigate the relationship between interfacial mobility andlipid composition in IPLM, we performed fluorescence recoveryafter photobleaching (FRAP) experiments (Figure S6, SupportingInformation, and Section 5.5). This also helped indicate whetherunsaturated lipids dominate the fluorescent region upon phaseseparation. Figure 3a shows fluorescence image changes duringFRAP experiments for varying DOPC contents. At low DOPCcontent (0%–2.5%), little to no recovery was observed, and thebleached dark spots remained unchanged during the observationperiod. As DOPC content increased to 5%–10%, fluorescenceintensity (F.I.) within the bleached spots increased, and their areadecreased by 30min. For the 20% sample, the phase-separateddomains were similar in size to the bleached regions, making itdifficult to selectively bleach either dark or fluorescent regions.Photobleaching the mixed domains resulted in complex recoverybehavior, presumably owing to differing recovery rates betweenregions. In the 50% sample, a clear difference in recoverybetween the red fluorescent and dark regions was observed, eventhough the dark regions contained small fluorescent islands(Figure 3a, 50% bright and 50% dark). The bright region showednear-complete recovery at 30min, while the dark mixed regionsshowed heterogeneous but detectable recovery, with bleachedspots visible immediately after irradiation (Figure S7,Supporting Information, 0 min). These differing recovery ratesfor the bright and dark regions indicated that unsaturated lipidsmay be partitioned into the fluorescent domains. To further con-firm whether the bright regions were dominated by unsaturatedDOPC, we also investigated the system using the unsaturatedLiss Rhod-DOPE fluorophore in FRAP experiments (FigureS8, Supporting Information). A similarly fast recovery wasobserved in the 50% sample’s bright regions. Thus, regardlessof the fluorophore conjugated lipid base, the bright regions weremobile phase-dominated. Considering Liss Rhod-DPPE behavedin a similar fashion to the unsaturated Liss Rhod-DOPE fluoro-phore, probably the bright regions were DOPC-rich regions.In addition, gradual drift of the entire membrane was observed(a)(b) (c)Figure 3. Evaluation of the mobility of the assembled IPLM by FRAP measurement. a) Fluorescence images of each sample taken before photobleaching,at 0 min, and at 30min after photobleaching. White circles indicate the position of the bleached spots. b) A typical schematic plot of time-dependentfluorescence recovery profiles. c) Quantitative analysis of the percentage of fluorescence recovery in the FRAP experiment shown in (a). F.I. values in thebleached regions were normalized to the value before photobleaching. Each condition was tested three times. Statistical differences were analyzed usingOne-way ANOVA: *p< 0.05, **p< 0.01, ***p< 0.001, N.S., not significant (p≥ 0.05).www.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (4 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.comin the 20% and 50% samples. This drift was likely owing to theincreased presence of unsaturated lipids forming a continuousphase, where the highly mobile unsaturated lipids were no lon-ger constrained by saturated lipid-rich domains.[20]By considering the general fluorescence profile during FRAP(Figure 3b and S6), we quantified the mobility of IPLM in termsof the percentage of fluorescence recovery from the F.I. of thebleached region before and after 30min of photobleaching (SeeExperimental for details). As shown in Figure 3c, we observeda gradual increase in the recovery of the apparently homogeneousmembrane with a DOPC content of 0%–10%. Similar trends wereobserved for systems using unsaturated Liss Rhod-DOPE fluoro-phores (Figure S8, Supporting Information) and solid-supportedlipid membranes in the 0%–10% range (Figure S9, SupportingInformation). Therefore, the presence of trace amounts (up to10%) of unsaturated lipids enabled control of the overall mobilityof the IPLM samples, depending on the DOPC content. Moreover,there was no significant difference inmembranemobility betweenthose on the solid support and the fluid interface for the 0% and2.5% samples (Figure S10, Supporting Information), which maybe attributed to the roles of intermolecular interactions of eachlipid (Van der Waals force) in the overall membrane mobilityrather than the mobility and/or friction of lipid molecules againstthe subphase. Slower mobility was observed for the 5% and 10%samples on the solid-supported surface than for the same sampleson the fluid interface, suggesting that the type of sub-phase regu-lated the mobility of the coating in this range. Further increasingthe DOPC content to 20% and 50% abolished the dependence ofthe mobility on the subphase, owing to the high mobility of thelipid membranes. Therefore, the subphase type affected lipidmobility within a limited range of DOPC content.However, at 20% DOPC content, with comparable sizes of thephase separation domains and bleached spots, the heteroge-neous recovery in the mixed domains resulted in a large variationin the average recovery rate. When the DOPC content was furtherincreased to 50%, with an enlarged phase separation of the redfluorescent and dark domains, the recovery rate within the redfluorescent domains was significantly higher than that in thedark region, where the recovery rate was somewhat overesti-mated owing to the presence of mixed small fluorescent islands.Simultaneously, a 50% DOPC-containing sample using theunsaturated Liss Rhod -DOPE fluorophore exhibited a similarrate of recovery (Figure S11, Supporting Information), furthersupporting that the red fluorescent domain corresponded tothe unsaturated lipid-rich phase. To quantify the viscosity changeof the respective sample, the diffusion coefficient (D) and therelationship between the diffusion coefficient and the viscosity(η) based on the Saffman–Delbruck model were roughly mea-sured from FRAP recovery profiles using unsaturated LissRhod-DOPE fluorophore for 10%–50% samples (Figure S11,Supporting Information).[4a,8,21] The general increase in the dif-fusion coefficient (Figure S12a, Supporting Information) anddecrease in viscosity (Figure S12b, Supporting Information) wereindicative of a much higher mobility of the IPLM by increasingthe DOPC content. Overall, the DOPC-dependent increase influorescence recovery of the IPLMs across the 0%–50% rangedemonstrated that the viscous nature of IPLM was finely tunedby altering DOPC content.2.3. The Impact of IPLM Viscosity on Cell Spreading BehaviorAfter establishing the viscosity-tunable platform, we culturedLifeact-GFP MDCK cells on the IPLMs for 3 h and 6 h to observehow tunable interfacial mobility influences cell adhesion, partic-ularly during early cell–IPLM interactions. As shown inFigure 4a, spreading cells were observable on the 0%–5%DOPC samples, with a general decreased tendency in spreadingarea among these samples at the 3 h time point (Figure 4b, inset).In contrast, for DOPC contents above 10%, almost no cell spread-ing was observed, indicating that cells were unable to form a(a) (b)Figure 4. Representative images of cellular adhesion behavior at the IPLM with different DOPC content. a) Cell spreading morphology at the IPLMs afterseeding cells for 3 h and 6 h, respectively. Green: Lifeact-GFP. b) Quantitative analysis of the relationship between cell spreading area and unsaturatedDOPC content. Inset: magnified data from the 3 h condition. The number of cells was 50 for each condition. Statistical differences were analyzed usingOne-way ANOVA: *p< 0.05, **p< 0.01, and ***p< 0.001, N.S., not significant (p≥ 0.05).www.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (5 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.comstable bond with the ligands on these highly mobile membranes.These results suggested that less mobile IPLMs (0%–10%DOPC) were necessary to support initial cell adhesion withina 3 h timeframe.After extending the incubation time to about 6 h (Figure 4a,b,6 h case), the cells enhanced spreading with expanding lamelli-podia, which was the appearance for early-stage cell spreading.No significant difference in the cell spreading area was observedat 0–2.5%, indicating that the effect of IPLM viscosity on thecell spreading behavior reached a saturation level in the rangeof 0–2.5% DOPC content. Subsequently, a slight decrease inthe spreading area was observed at 2.5%–5%. Thereafter, a sharpdecrease was found to be significantly affected by 5%–10%DOPC content. As the mobility increased from 10% to 20%IPLM, no significant difference was observed between thesetwo cases, while cells started to spread compared to their 3 h con-dition. For the 50% sample, no further spreading was observedcompared to the 3 h, 50% sample. In addition, some cells tendedto form aggregates in the 50% sample (Figure 4a, 50%, 6 h). Thisbehavior was consistent with that reported in the literature,[14]where MDCK cells either spread or coalesced to form aggregatesdepending on the interfacial relaxation time of the polymericscaffolds. Pulling all 6 h data together confirmed that cell spread-ing was influenced by IPLM mobility. In the 2.5%–10% DOPCrange, increasing mobility correlated with reduced cell spread-ing. Additionally, a mobility threshold appeared to be requiredto support significant cell spreading on less-mobile IPLMs, spe-cifically below 10%. In contrast, cell spreading was poor, with around morphology observed on more mobile IPLMs (above10%). Thus, the key difference in the initial cell adhesion onthe IPLMs was the rate of spreading, which was strongly modu-lated by interfacial mobility.In contrast, cells on the solid surface showed an enhancedspreading morphology compared to those on the fluid counter-part (Figure S13, Supporting Information). This is not surprisingconsidering that cell adhesion is not only regulated by interfacialviscosity but also by bulk mechanics. It should be noted thatinstead of limited spreading, the fluid interface allows adaptivewetting with out-of-plane deformation[9] where another criterion,mechanical robustness, plays a critical role in maintaining theintegrity of the cell scaffold; this will be discussed in the nextsection.2.4. Impact of Viscosity on the Robustness of IPLMThe observed similar dependence of fluorescent lipid mobility(Figure 3c) and cell spreading (Figure 4b) on DOPC content indi-cated successful control of the interfacial viscosity of the entirelipid membrane. In addition, we observed the condensation ofthe fluorescent lipids in terms of increased F.I. under the cells(Figure 5a, magnified area of the dashed rectangle for the 0%sample and the corresponding line scan). Furthermore, this con-densation was observed regardless of whether the fluorophorewas conjugated to a saturated or an unsaturated backbone(Figure S14, Supporting Information). Considering that cellsapplied forces to the IPLM by dragging the RGD ligands ratherthan the fluorescent lipid, these observations indicated that otherlipid components also moved along the cellular traction forces.(a) (b)(c) (d)Figure 5. Evaluation of the robustness of IPLMwith FC-40 subphase. a) Representative fluorescencemicrograph of adherent cells inducing defect regionsin the 0% sample and b) slight fluorescence depletion in the 2.5% sample at the FC-40–water interface. The area of the dashed rectangle is magnified onthe right. The locations of the edge of the defect or depletion and cells are indicated as a, b, c, and d. The corresponding line scan of F.I. and the markedlocations a, b, c, and d in the magnified image are shown below. Green: Lifeact-GFP. Red: Liss Rhod-labeled lipid. c) In situ fluorescence image of IPLM inits pristine (left) and manually fractured states (right). The area of the dashed rectangle is magnified in the inset (top right). d) The process of crackformation at the FC-40–water interface over time at the cell periphery. The area of the dashed rectangle is magnified in the upper image. The location ofthe edge of the crack is marked as a and b. The corresponding line scan of F.I. and marked locations a and b are shown at the lower right.www.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (6 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.comAt the same time, a dark zone with depleted fluorescence wasobserved around the cells after 3 h for the 0% sample.According to the reported findings,[22] a dilatational storage mod-ulus of phospholipid monolayer at the oil–water interface ishigher than the loss modulus. Especially, lipids with phosphati-dylethanolamine (PE) headgroup at high-strain state (higher than20%) could exhibit wrinkle formation, indicating jamming(which is solid-like elastic transition) at the oil–water interface,whose behavior could be linked to the formation of highly elasticinterfaces. Thus, the observed condensation of Liss Rhod-DPPEand RGD-DSPE, leading to dark zones (namely defect regions),suggested a limited range of lateral movement of the entire lipidsin the timescale of experimental 3 h against cellular force drag-ging, that overall lipid mobility became limited under prolongedcellular force dragging, exhibiting a solid-like elastic behavior.Furthermore, trace levels of fluorescence within the defectregions (Figure 5a, line scan of the 0% sample) suggested coex-istence of lipids and proteins from the culture medium, likelyowing to a relatively diluted lipid layer exposed to a critical pro-tein concentration.[23] To confirm the composition of the cellforce-induced defect regions, fluorescein isothiocyanatelabeled-bovine serum albumin (FITC-BSA) was introduced intothe culture medium. Both medium-derived protein labeled ingreen and red fluorescent lipids were observed in a coexistencefashion (Figure S15, Supporting Information). In contrast, IPLMwith a small amount of DOPC content (2.5% ratio) showed localaccumulation but less macroscopic depletion (consumption offluorophore) around the cells (Figure 5b, 2.5% sample). Sucha reduction in lipid depletion in the dark region could be causedby enhanced lipid reorganization and/or insufficient ligand drag-ging due to the increased mobility of the lipid layers in the pres-ence of 2.5% unsaturated DOPC. In addition, considering thathigh out-of-plane deformation was involved in the adaptive wet-ting behavior, as reported in our previous work,[9] tailoring theinterfacial tension was required to induce a high deformationstate with cellular force efficiently transferred to the IPLM.This approach could be utilized to exclude the factor of insuffi-cient ligand dragging. When the cell force induced high out-of-plane deformation by changing the oil phase from FC-40 toFC-70 (lower interfacial tension), the dark region of the 0% sam-ple became more significant in the top and side views (FigureS16, Supporting Information, 0% sample). The low interfacialtension rendered the interface much more deformable, and thus,the cells invaded most of their body below the initial level of thewater-PFCL interface.[9] Such a high degree of deformation pro-vided a more severe mechanical stimulus to the IPLM. Withinthis high-deformation state, the 0% sample exhibited an enlargeddefect area toward the distal position from the cell periphery.This indicated that cellular force-induced fracture occurred inlipid membranes with a solid-like nature. The viscous 2.5%IPLM behaved similarly to that observed at the FC-40 interface,although a similar level of high out-of-plane deformation of themembranes was observed (Figure S16, Supporting Information,side view of the 2.5% sample).To directly demonstrate the impact of interfacial viscosity onthe robustness of the IPLM, the mechanical compliance of theIPLM with 0% and 2.5% DOPC content was tested by physicallyscratching the interface under an external force. As shown inFigure 5c, after manual scratching, distinct edges of completedefect were observed for 0% IPLM, which was typical for brittlematerials with little tendency to deform before fracture.[24] Themoderate mobility of the 2.5% sample made it difficult to formcracks by scratching (Figure S17, Supporting Information).Even when periodic scratching caused the appearance of cracks,diffuse fluorescent fragments remained attached to the fractureedges (Figure 5c, bottom panel). This observation suggestedthat the purely saturated one (0%) was brittle and easily frac-tured by a large force owing to the short range of lipid reorga-nization, whereas the one with moderate mobility (2.5%) wasrobust and capable of lateral reorganization against large defor-mations.[25,26] These material characterizations provided a rea-sonable expectation that cell-applied forces also caused thebrittle fracture of the 0% IPLM, as shown in Figure 5d. Thecrack formation process in the 0% sample was detected froma line scan of the F.I. around the cell periphery. The profileshowed an expansion of the sharp drop in intensity over time(line scan of the marked location between locations a and b foreach time point). Overall, the robustness of IPLM was con-trolled by altering the viscosity of the lipid membrane.Furthermore, rather than a solid-like brittle property, theIPLM required a certain degree of mobility to be robust againstcellular dynamic force exertion.3. DiscussionIn this study, we aimed to investigate how the viscosity of IPLMaffected cell adhesion behavior by mixing saturated and unsatu-rated lipids at the water–PFCL interface. DSPC was used as theprimary component to construct phospholipid membranes withvarying mobility by increasing the DOPC content. This furtherexpanded the viscoelastic window into an ultra-soft range. Thiscontrasted with previous studies based on hydrogels and solidSLBs, where viscosity tuning relies on cholesterol content,[7] lipidsaturation,[8] substrate hydrophilicity,[27] and other factors. At thisfluid interface, the omniphobic nature of the PFC enabled a con-tinuous lipid monolayer to coat the interface through van derWaals interactions between hydrophobic acyl chains.[28] This sug-gested that the degree of acyl chain saturation may alter mem-brane mobility through altering Van der Waals interactionsand, consequently, overall viscosity.Thereafter, the viscosity tunability of each IPLM sample wasinvestigated using FRAP by measuring the percentage of fluores-cence recovery in a bleached spot. IPLM exhibited high viscositywith macroscopic homogeneity at low DOPC content (0%–10%),while less viscous, phase-separated domains emerged at ≥20%DOPC. The apparent homogeneity of low-DOPC samples wasmaintained over a one-day time scale. A similar dependenceof interfacial mobility and cell adhesion behavior on DOPC con-tent was observed on phospholipid-decorated perfluorinatedsolid surfaces, indicating that the observed effects were not spe-cific to fluid interfaces.Furthermore, the apparently consistent lipid movement of theoverall lipid in response to cell traction forces was observed. Celladhesion to IPLM (0%) led to the occurrence of defect regions,whereas IPLM (2.5%) remained relatively intact at the cell periph-ery. This characteristic of IPLM robustness was found to bestrongly influenced by IPLM viscosity (Figure S18, Supportingwww.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (7 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.comInformation). Specifically, when cells were cultured at FC-40,local cracks driven by cell forces were observed in the 0% sample.To assess whether the saturated IPLM (0%) showed solid-like,brittle properties, high deformation driven by cell forces at theFC-70–water interface was investigated. The continuous mem-brane structure was stochastically disrupted, leading to macro-scale lipid defects. Manual scratching also resulted in a brittlefracture. Therefore, when cellular traction forces exceeded a crit-ical threshold, stochastic cracks and progressive fluorescentdefects appeared in the 0% sample. In contrast, the robustIPLM (2.5%) consumed some fluorophores and showed lessmacroscopic depletion. As this IPLM could dissipate mechanicalload and maintain structural integrity through lipid reorganiza-tion (Figure S18, Supporting Information), even when the entirecell body was wrapped within the PFCL phase after switchingfrom FC-40 to FC-70 (lower interfacial tension). However, theobserved relationship between IPLM mobility and robustnessproperty contrasted with previous findings on collagen I- or fibro-nectin-functionalized SLBs mixed with cholesterol, where highECM protein translocation led to local depletion.[7] Consideringthe difference of the subphase between IPLM and SLBs, this oppo-site trend may be attributed to the presence of a hydration layerbetween lipid membranes and the substrate in SLBs, whereas Vander Waals interactions mediated lipid assembly on tethered orliquid PFCLs. Therefore, the contrasting observations arised fromdifferences in the adaptive nature of lipid layers under cellulartraction forces. Furthermore, cells adhering to fluid interfacesexperienced varying degrees of deformation, especially at highout-of-plane deformation, necessitating IPLM robustness towithstand dynamic traction forces. These findings highlightedthe importance of IPLM robustness in supporting cell adhesionbehavior.4. ConclusionIn summary, we demonstrated that the mobility-tunable IPLMbased on different mixing ratios of saturated and unsaturated lip-ids affected the cellular adhesion behavior and robustness of cellscaffolds. In the lower DOPC content range, the viscosity ofIPLM evidently affected the cell adhesion behavior. MDCK cellson IPLM with high viscosity showed good spreading, while thoseon IPLM with low viscosity showed limited spreading, indicatingthe existence of a critical threshold to determine significant cellspreading. In addition, cells adhering to IPLM, composed of puresaturated lipids with a solid-like nature, caused a lipid defect onthe platform. A small amount of unsaturated lipids in the IPLMmay enable lateral reorganization to dissipate the loading forcesand maintain an intact structure at the macroscale. Overall, theviscous nature not only affected the cellular adhesion ability butalso made the IPLM robust enough to adapt to the cellulardynamic traction force. Given the role of viscosity and crackoccurrence in cell adhesion, further detailed studies on the rela-tionship between systematic mobility tuning and cellular adhe-sion behavior are of great interest. In addition, our study willbe useful for designing biomimetic materials with varying viscos-ities and for technical applications requiring cell adhesion tosuper-soft fluid surfaces.5. Experimental SectionLipid Vesicle Preparation: DSPC, DOPC, RGD-DSPE, and Liss Rhod-DPPEwere purchased from Avanti Polar Lipids (Alabaster, AL). The powders weredissolved in chloroform at concentrations of 10, 10, 1, and 1mM. Stock lipidvesicle solutions were prepared in phosphate buffered saline (PBS) at a con-centration of 1mM to the desired unsaturated DOPC: saturated (DSPC:RGD-DSPE:Liss Rhod-DPPE) molar ratios at x:[(97.5%–x):2%:0.5%] afterevaporating chloroform for 3 h under vacuum conditions. For changingthe saturated Liss Rhod-DPPE to unsaturated Liss Rhod-DOPE, the mixingratio was kept the same as saturated Liss Rhod-DPPE.Fluorination of the Bottom of the Culture Chamber: The custom-designedcell culture chamber (inner wall, 21mm; outer wall, 25mm) was fluorinatedas described previously.[9] In brief, the chambers were first treated with10 wt% potassium hydroxide at 60 °C for 1 h, followed by rinsing withdeionized water, and dried under an air stream. The bottom glass was thenfluorinated with a 0.5 vol% ethanol solution of trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) silane, followed by heating at 70 °C for 30 min.Finally, the glass was rinsed 2–3 times with ethanol and water for cleaningand sterilized via autoclaving.Construction of the IPLM: Fluorination allowed FC-40 (250 μL) to wet thelower layer of the well, followed by 2mL phosphate buffered saline (PBS).To assemble the IPLM at the water-PFCL interface, the stock lipid vesiclesolution was preheated at 70 °C for 30-min, then sonicated to clarity atRoom Temperature using a probe-type ultrasonic disruptor (UD-100;Tomy, Tokyo, Japan) with 10 cycles of intermittent operation (45 s ofhigh-power sonication separated by 30 s intervals). The unilamellarvesicles were then added to the upper PBS phase to achieve a final phos-pholipid concentration of 0.2mM. The IPLM self-assembly process wasperformed at Room Temperature for 2 h and then slowly washed by repeat-edly replacing the upper PBS phase with fresh PBS. FC-40 was used as thePFCL in all systems unless otherwise stated, in which case FC-70 was used.Construction of the Phospholipid Membrane at the Solid-SupportedSurface: Fluorination enabled phospholipids to coat the surface throughhydrophobic intermolecular interactions. To achieve this, the unilamellarvesicle solution (0.2 mM in 2.5 mL PBS solution) was added directly to thechamber. The self-assembly process was performed at RT for 2 h and thenslowly washed by repeatedly replacing with fresh PBS.FRAP: FRAP measurements exposed a fluorophore-labeled sample tostrong light, bleaching the fluorophore and creating a bleached region. Ifthe fluorescent molecules are highly diffusible, the bleached region recov-ers via diffusion from the surroundings. However, if the mobility is low, ableached region persists. Fluorescence images were obtained using aninverted microscope (IX-81; Olympus, Shinjuku, Japan) equipped with adisc-scan confocal unit CSU10 (Yokogawa, Tokyo, Japan), an Andor lasercombiner (Oxford Instruments, Oxfordshire, UK), an MD-695 CMOS cam-era, and a 60� water immersion objective (LUMPlanFL N; Olympus) toobserve the IPLM. Micropoint laser, ablation equipped within the abovemicroscope, was used to photobleach a spot in the desired region with561 nm light. Fluorescence imaging was performed after specified timeintervals. For the 2.5%, 5%, 10%, 20% and 50% samples, to accountfor potential rapid recovery immediately after bleaching, 30% of the pre-bleach intensity was used as the intensity at 0 min. This specific value wasdetermined using the 0% sample, as the bleached intensity from theinstrument should be consistent across all samples. The 0% sampleshowed almost no recovery during the 30min FRAP experiment, makingit a standard for evaluating the bleached intensity.To quantify the varied viscosity of the respective sample, diffusioncoefficient (D) and the relationship between the diffusion coefficientand the viscosity (ηm) based on Saffman–Delbruck equation were deter-mined from FRAP recovery profiles for 10%–50% samples.[4a,8,21]According to the Equation (1), the diffusion coefficient D can be calculatedfor the 10% DOPC sample as an example.D ¼ rn24τ1=2(1)rn is roughly estimated radius of the bleach area (in this studyrn = 6.06 μm). τ1=2= 1288 s. Thus,D = 0.00716 μm2 s�1. The relationshipwww.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (8 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.combetween the diffusion coefficient D and the viscosity ηm can useEquation (2) according to the literature.[4a,8,21]D ¼ kBT4πηmhlnηmhηwR� �� 0.5772� �(2)kB is the Boltzmann constant, kB = 1.381� 10�23 m2 kg s�2 K�1,T = 310 K (37 °C), ηw = 0.691� 10�3 Pa·s (the viscosity of water),h = 3 nm (the thickness of a lipid), R = 0.5 nm (the radius of a singlelipid). Then the viscosi ty ηm of each sample can be calculated afterthe iteration calculation by Microsoft Excel.Cell Culture: The culture medium for MDCK cells (RCB0995, RIKEN CellBank) expressing Lifeact-GFP was prepared by diluting with 10� minimalessential medium (MEM, Thermo Fisher Scientific, Waltham,Massachusetts, U.S.). It was supplemented with 10% heat-inactivatedfetal bovine serum (EU origin, Biowest, Naullie, France),100 units mL�1 penicillin, 100 μgmL�1 streptomycin (Nacalai, Kyoto,Japan), 1% MEM nonessential amino acids (Nacalai), 1% sodium pyru-vate (Nacalai), 1% L-glutamate (Nacalai), and 2.2 g L�1 sodium hydrogencarbonate (Wako). The cells were cultured in a Petri dish at 37 °C in a 5%CO2 atmosphere. Prior to seeding the cells onto the IPLM, the upper aque-ous phase (PBS) of the IPLM platform was replaced with the prepared cellculture medium. For seeding onto the IPLM platform, the MDCK cellsexpressing Lifeact-GFP were detached from the culture dish using tryp-sin-ethylenediaminetetraacetic acid (Wako), plated at a density of 2� 104cells cm�2, and incubated at 37 °C with 5% CO2 for the desired time beforeobservation. Images were obtained using a confocal laser-scanning micro-scope equipped with a 60� water objective.Evaluation of the Robustness: First, a fluorescence microscopy image ofthe IPLM was obtained. The IPLM system was then manually scratched atthe fluid interface using a needle. The resulting cracks were observed insitu via fluorescence microscopy. The durability of the IPLM was charac-terized by the presence of fragment diffusion near the edge of the crack.Statistical Analysis: All data were not normalized prior to collation ofindependent experiments. Results are presented as mean� standard devi-ation. The sample size for each experiment is indicated in the correspond-ing figure legends. Statistical analyses were performed using one-wayANOVA followed by Tukey’s post-hoc test for pairwise comparisons usingan online statistical tool (One-way ANOVA with post-hoc Tukey HSD TestCalculator). P values below 0.05 were considered significant.Supporting InformationSupporting Information is available from the Wiley Online Library or fromthe author.AcknowledgementsThis study was supported in part by the Japan Society for the Promotion ofScience KAKENHI (grant nos. 22H00596 and 23K17481 to J.N.).Conflict of InterestThe authors declare no conflict of interest.Author ContributionsJun Nakanishi: conceptualization (lead); formal analysis: supporting; fund-ing acquisition (lead); methodology (equal); supervision (lead); validation(lead); writing—review & editing (equal). Junhong Zhou: conceptualiza-tion (equal); data curation (lead); formal analysis (lead); investigation(lead); methodology (equal); validation (equal); writing—original draft(lead); writing—review & editing (equal).Data Availability StatementThe data that support the findings of this study are available from thecorresponding author upon reasonable request.Keywordscell adhesion, extracellular matrix, fluid interface, phase separation,robustness, viscosityReceived: March 17, 2025Revised: July 4, 2025Published online: August 22, 2025[1] D. E. Discher, P. Janmey, Y. Wang, Science 2005, 310, 1139.[2] a) A. J. Engler, S. Sen, H. L. Sweeney, D. E. Discher, Cell 2006, 126, 677.H. Cao, Q. Zhou, C. Liu, Y. Zhang, M. Xie, W. Qiao, N. Dong, ActaBiomater. 2022, 143, 115. b) Y. Hou, L. Yu, W. Xie, L. C. Camacho,M. Zhang, Z. Chu, Q. Wei, R. Haag, Nano Lett. 2019, 20, 748.[3] a) O. Chaudhuri, J. Cooper-White, P. A. Janmey, D. J. Mooney,V. B. Shenoy, Nature 2020, 584, 535. b) C. Huerta-López,A. 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Bosch-Fortea, A. Chrysanthou, C. V. M. Alexis,C. Matellan, A. Zarbakhsh, G. Mastroianni, A. R. Hernandez,J. E. Gautrot, Biomaterials 2022, 284, 121494.[27] K. J. Seu, A. P. Pandey, F. Haque, E. A. Proctor, A. E. Ribbe, J. S. Hovis,Biophys. J. 2007, 92, 2445.[28] K. Ullmann, L. Poggemann, H. Nirschl, G. Leneweit, Colloid Polym.Sci. 2020, 298, 407.www.advancedsciencenews.com www.advnanobiomedres.comAdv. NanoBiomed Res. 2025, 5, 2500076 2500076 (10 of 10) © 2025 The Author(s). Advanced NanoBiomed Research published by Wiley-VCH GmbH 26999307, 2025, 11, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/anbr.202500076 by Jun Nakanishi - National Institute For , Wiley Online Library on [08/12/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advnanobiomedres.com Impact of Interfacial Viscosity on the Robustness of Phospholipid-Decorated Fluid Cell Scaffolds 1. Introduction 2. Results 2.1. Impact of Unsaturated Lipid Contents on the Phase Behavior in IPLM 2.2. Evaluation of Interfacial Mobility Change by Altering Unsaturated Lipid Contents 2.3. The Impact of IPLM Viscosity on Cell Spreading Behavior 2.4. Impact of Viscosity on the Robustness of IPLM 3. Discussion 4. Conclusion 5. Experimental Section