Composite lipid bilayers from cell membrane extracts and artificial

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Biological and Environmental Phenomena at the Interface

Composite lipid bilayers from cell membrane extracts and artificial mixes as a cell culture platform Anastasia Svetlova, Jana Ellieroth, Frano Milos, Vanessa Maybeck, and Andreas Offenhäusser Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00763 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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Composite lipid bilayers from cell membrane extracts and artificial mixes as a cell culture platform Anastasia Svetlova§, Jana Ellieroth§, Frano Milos§, Vanessa Maybeck§, and Andreas Offenhäusser*§ §

Institute of Bioelectronics (ICS-8), Forschungszentrum Jülich GmbH, Wilhelm-Johnen Straße,

52425 Jülich, Germany

ABSTRACT. Artificial lipid bilayer is the closest possible model for the cell membrane. Despite that, current methods of lipid bilayer assembly and functionalization do not provide a satisfactory mimic of cell-cell contact due to the inability to recreate an asymmetrical multicomponent system. In the current work, a method to produce an integrated solid-supported lipid bilayer combining natural extracts from cell membranes and artificially made lipid vesicles is proposed. This simple method allows delivery of transmembrane proteins and components of the extracellular matrix into the substrate. Biocompatibility of the composite natural/artificial lipid bilayers is evaluated by their interactions with the cardiomyocyte-like HL-1 cell line. Compared to fully artificial mixes, composite lipid bilayers allow cells to adhere and develop a morphologically more normal cytoskeleton.

1. Introduction

ACS Paragon Plus Environment

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Within a living tissue a plethora of biochemical and physical factors affect cell functions, such as proliferation, differentiation, growth, and survival1. To mimic a natural microenvironment that guides cellular responses in a controlled way is a challenging task. In the organism, cells interact with each other via docking of molecules on the outside part of the cell membrane, called the extracellular matrix (ECM)2 and with receptors, which are mostly integrins3–5. The majority of the common systems utilized in the laboratory rely on physically adhered proteins of the extracellular matrix on solid surfaces6,7. However, homogeneously distributed immobile proteins lack the structural complexity of the native ECM, or even change the conformation of proteins due to interactions with the surface8,9. As a cell culture platform, lipid bilayers are highly appealing due to their biomimetic properties – lipid molecules are the main structural component of cell membranes and insertion into the lipid bilayer is required for transmembrane proteins to perform their biologic function. Yet, the nature of the platform presents not only advantages but also a challenges. Although it was shown that some types of cells are able to survive on the unmodified surface of planar solid-supported lipid bilayer (SLB)10, the biologically inert nature of the lipid headgroups does not provide a starting point for adhesion complex formation. To facilitate cell adhesion, additional functionalization is required, for example, by covalent attachment of components of the ECM11,12 or by surfactant-assisted insertion of transmembrane proteins13. An alternative approach to cell membrane mimetics was utilized by Liu et al and Simonsson et al14,15. By delivering components of a cell membrane into an artificial lipid bilayer, it was possible to create cell membrane models for in vivo tests. Formation of extracellular vesicles (blebs) was facilitated by addition of diluted formaldehyde to a culture of adherent cells. It was further shown that these blebs, which are carrying the components of the cell membrane in their


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native form and orientation, are able to fuse with the artificially prepared lipid vesicles, resulting in the composite bleb-lipid bilayer (Bl-SLB) on the planar support. The most interesting feature of cell membrane blebs is their specific mechanism of parachuting fusion into bleb-SLBs induced by addition of small unilamellar vesicles (SUV). Unlike the SUV, blebs do not spontaneously rupture upon the contact with the substrate, but their parachuting occurs when SLB from ruptured SUV contacts a bleb sedimented on the surface. That way when fused into Bl-SLB, the outer part of the bleb – originally the extracellular side of the cell membrane – faces the solution, and the inside part of it faces the supporting substrate, resulting in an asymmetrical distribution of the components. After the Bl-SLB formation, the ECM components preserved on blebs are accessible for cells that are seeded on these substrates. Several types of mechanical cells of the organism have distinct stress fibers in the actin cytoskeleton, which are generated only when the cell-substrate traction forces are high enough to facilitate the focal adhesion complex formation16–18. Stretch of the initial adhesion protein complex by the actin fiber causes talin to change its conformation and unfold a vinculin binding site that facilitates further stabilization of actin fibers. In the absence of traction forces, cell shapes are altered16. The unique range of SLB fluidity allows one to modify the system for a range of mechanobiology applications19. Although high lateral fluidity of SLBs made from artificial lipid mixes prevents formation of focal adhesions16, additional adjustment of lateral fluidity helps with adhesion of cells: on the substrates with the less mobile ligands, cells spread more20. It is also possible to use physical barriers on the solid support that separate patches of SLB and prevent free diffusion of the components16,21. Overall, trying to adjust the substrate for the cell seeding by increasing the complexity of SLB requires a lot of modifications to the basic procedure.


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In the current work the first attempt to utilize Bl-SLB as a substrate for a cell growth is made. Figure 1A shows a scheme for the Bl-SLB formation process. Cell membranes are extracted in the form of blebs, then with the help of artificial lipids they are fused into a composite natural/artificial lipid bilayer. Performance of Bl-SLBs is evaluated by their interactions with a cardiomyocyte-like cell line, HL-122 and compared to the control substrate, fibronectin/gelatin mix. When cultured on the fluid bilayers in the absence of ECM proteins, HL-1 are unable to build a cytoskeleton in a proper manner and lose their specific shape. It is demonstrated that the composite Bl-SLBs are able to provide the environment required to build the morphologically normal cytoskeleton.

2. Materials and methods Cell cultures HL-1 cells were maintained in Claycomb’s media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich F2442), 100 U/mL penicillin-streptomycin (Gibco), 2 mM Lglutamine (Life technologies), and 0.1 mM nor-adrenaline (Sigma-Aldrich A0937) in a humidified 5% CO2 atmosphere at 37°C. HEK293 cells were maintained in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich D6429) supplemented with 10% fetal bovine serum (Life Technologies 10270106), 100 U/mL penicillin-streptomycin (Gibco) in a humidified 5% CO2 atmosphere at 37°C.

Cell membrane bleb preparation 70% confluent layers of HEK293 cells were washed twice with HEPES buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4). Then formation of cell membrane blebs was


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induced by changing the buffer for a blebbing buffer which was made from the same HEPES buffer with addition of 25 mM formaldehyde and 2 mM dithiothreitol. After incubation (6 hours unless specified in the text) at 37°C blebs were collected with the supernatant and stored at 4°C. For visualization on the microscope cell membranes were loaded with the fluorescent DiO dye prior to blebing by 30 min incubation in medium with 6 μM DiO Solution (Invitrogen).

Small unilamellar vesicle (SUV) preparation 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) was purchased from Avanti Polar Lipids,

Inc.

Texas

Red™

1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine

Triethylammonium Salt (TR-DHPE) was purchased from Invitrogen. In this work mixes of 100% POPC and 99% POPC with 1% TR-DHPE (TR-SUV) were utilized. Lipid solutions in chloroform were mixed at the proper ratio in a glass vial and the solvent was evaporated by the flow of nitrogen and further desiccation in vacuum chamber for 1 h. Lipid films were then rehydrated with HEPES buffer, sonicated in the bath sonicator to form large multilamellar vesicles that were extruded through a polycarbonate membrane with the pore size 100 nm. Resulting SUVs were stored at 4°C.

Dynamic light scattering The hydrodynamic diameters of the blebs were measured using a Zetasizer Nano ZS instrument (Malvern Instruments, Malver, UK). All measurements were performed at 21°C.

Bleb bilayer (Bl-SLB) formation


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Ibidi® 35 mm μ-Dishes with a high glass bottom (ibidi GmbH) were used as substrates for the characterization of a composite lipid bilayer formation. The dishes were cleaned with 100% isopropanol for 5 min, then extensively rinsed with MilliQ water ten times and dried in a stream of nitrogen gas. The cleaned glass bottoms were activated by oxygen plasma in a Pico plasma system (Diener Electronics) for 5 min at 0.8 mbar and 100 W generator power. For the experiments with cell cultures cleaned and activated dishes were sterilized by UV for 1h. Bleb dispersions collected from cells were deposited on prepared dishes, allowed to sediment at 37°C, then SUVs were added to a final concentration 0.3 mg/mL of the lipids. After 1 h incubation at 37°C the substrates were thoroughly rinsed with HEPES buffer to remove unfused blebs and SUVs. For the cell culture experiments the buffer was exchanged for the culture medium before seeding the cells. Exposure of the Bl-SLB to air should be avoided. Fibgel coatings for the control experiments were prepared by applying stock solution of 0.02% gelatin, 0.005 mg/mL fibronectin (Sigma-Aldrich G9391 and F1141, respectively) to pre-cleaned dishes for 1 h, followed by rinsing with Milli-Q water.

Quartz Crystal Microbalance with Dissipation Monitoring A Q-sense E4 instrument, equipped with open modules and QSOFT software (BiolinScientific/Q-Sense, Sweden), was used for the monitoring of the lipid bilayer formation. SiO2 coated AT-cut quartz crystals with a fundamental frequency of 5 MHz (BiolinScientific/QSense, Sweden) were cleaned with oxygen plasma (5 min, 0.8 mbar, 100 W generator power) and immersed in a 2% sodium dodecyl sulphate solution for 30 min at room temperature. After that the sensors were thoroughly rinsed with Milli-Q and dried with a nitrogen gas flow. These sensors were again activated with the oxygen plasma at the same conditions before experiments.


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The experiments were performed at 37°C and the chambers were equilibrated at 37°C with HEPES buffer prior to recording a baseline. Collected blebs were added to the buffer in chambers. After 1 h of recording, SUV POPC solution was added to the chambers to get a final concentration of 0.3 mg/ml of lipids in the chambers. After equilibration, excess SUVs were rinsed with the gentle flow of the buffer. For clarity, all results displayed here are the representative result of the 11th overtone.

Determination of proliferation Bl-SLB or fibgel coatings were formed in Ibidi® dishes as described above. HL-1 cells were seeded at a density of 170 cells/mm2 for fibgel and 90 cells/mm2 for Bl-SLB. After the indicated period of time, cells in dishes were detached from the coatings by applying a 0.05% trypsin/EDTA mix, cells were collected and counted again in the Neubauer Chamber. After 5 days of culturing, the maximum cell number was selected as 100% confluency (indicating that the whole surface of the dish is covered with cells), and the cell numbers for other days are represented as a % confluency in relation to this number. On the figure averages of 3 dishes for fibgel and 2 dishes for Bl-SLB are presented.

Fluorescence imaging CalceinAM (Life technologies) 1mM stock in DMSO was diluted 1:3000 in the cell culture medium. Samples were incubated for 20 min and rinsed with HEPES buffer. Cells were visualized by the Zeiss Axio Imager Z1 from Carl Zeiss GmbH with the light souce Illuminator HXP120, 20x water objective and AxioCam MR R3 using the imaging software ZEN blue edition 2012.


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Immunostaining For the immunostaining a PBS buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) was used. All operations were done in a way that avoids a sample exposure to air. Cell culture medium was exchanged for PBS. Then the cells were fixed by 4% paraformaldehyde in PBS solution for 20 min. If not specified in the text, the samples then were permeabilized with 0.003% Tryton X-100 in PBS for 10 min. Unspecific binding was reduced by incubation in PBS buffer with 0.5% bovine serum albumin and 2% heat inactivated goat serum (blocking buffer) for 1 h. To visualize fibronectin a 1:200 dilution of anti-fibronectin antibody (rabbit polyclonal, Abcam ab2413) in the blocking buffer was incubated on the sample overnight at 4°C. Together with the primary antibody, DAPI (25 ng/mL, Millipore 90229) and TRITCphalloidin (0.3 ng/μL, Millipore 90228) were added to the sample. After the incubation, samples were rinsed 6 times with PBS and incubated in a 1:500 solution of a secondary antibody (goat anti-rabbit AlexaFluor488, Life technologies A11034) in blocking buffer. Samples were rinsed 6 times with PBS, mounted with coverslips while still immersed in liquid and visualized on the Axio Observer LSM880 equipped with AiryScan detector using the imaging software ZEN black edition 2012.

3. Results and discussion 3.1 The model Firstly, we tried to produce blebs from HL-1 cells by the PFA/DTT induction method23,24 for use in Bl-SLBs. However, our attempts were unsuccessful. Our results indicated that when using the PFA/DTT method mechanical properties of the cell membrane should be considered. Cardiac


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cells are one of the most stiff cells in the organism25 overall. How the crosstalk between organelles contributes to overall stiffness of the cell is a complicated issue26, however, some correlation between cell membrane stiffness and overall stiffness is to be expected. This suggests that releasing blebs from HL-1 cells may be impossible or require drastically different protocols. In contrast, HEK293 cells, as a kidney cell line, are much softer27 than the cardiac cells. We found that the membranes of these cells produce a large number of blebs when immersed in PFA/DTT solution (blebing buffer). HEK293 cells have the added advantage that they are an established system for expressing transgenes for future modification of their surfaces. Figure 1A illustrates the process of composite Bl-SLB assembly on the glass surface. Mechanism of small bleb fusion described in Liu et al.14 enables access from solution to proteins expressed on the extracellular side of HEK293 cells.

3.2 Characterization of bleb size distribution The process of bleb formation was monitored with fluorescence microscopy and dynamic light scattering. Membranes of HEK293 cells were labelled with the lipophilic tracer DiO and then the medium was exchanged for the blebing buffer. As clearly seen on photos, even after only 15 min of incubation, there are blebs of micrometer-range diameter formed on the surface of cells (Figure 1B). Blebs continue to be formed on the surface of cells while they are immersed in the blebing buffer (Figure 1C). After a gentle shaking of the sample, the blebs detach from the surface of cells and float in the buffer. Diameters of blebs that are visible in the microscope vary from 5 μm to 20 μm. To investigate the presence of blebs that can’t be resolved by an optical microscope, we incubated cells in a blebing buffer for different durations, collected the blebing buffer from the


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samples, allowed blebs to sediment for 1 day and measured the supernatant by dynamic light scattering. Presence of a nanometer-sized population of particles in the supernatant was confirmed (Figure 1D). The mean diameter of these particles increases with the increasing incubation time.

Figure 1. A. Schematic representation of the Bl-SLB assembly. Naturally ocurring phospholipids produced by the cell are shown in blue and yellow. Trans-membrane proteins in orange and extracellular matrix in dark green. SUVs of synthetic lipids are in red. The membrane


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separated from the cell in the form of the bleb recombines with SUV lipids on the surface. Extracellular and intracellular sides of the original cell membrane are labelled “E” and “I” respectively. Then Ccells can then be grown on the Bl-SLB, light greenare cultured on Bl-SLB. B-C. HEK293 cell membrane stained with DiO after 15 min (B) 3 hours (C) of incubation in the blebing inducing solution. Scale bar 20 μm. D. The fraction of collected blebs after removal of the giant blebs as characterized by the dynamic light scattering method. These results may be attributed to different mechanisms of bleb formation triggered by the chemically induced method. One mechanism of the blebing involves the exocytosis of vesicles14. Another mechanism involves the actin cytoskeleton detachment from the cell membrane and subsequent bulging of the membrane and the bleb formation24. It is worth mentioning that by the second mechanism both nanometer- and micrometer-sized blebs can be produced and both of these populations are carrying the transmembrane proteins located on the cytoplasmic membrane of the cells28,29. We decided to use all fractions mixed - the way they are collected from the cells, to achieve a maximum delivery of the cell membrane components to Bl-SLB.

3.3 Bleb-SUV fusion monitoring by QCM-D Interactions of blebs and SUVs with the SiO2 surface were monitored by QCM-D. A typical example of a recorded curve is presented in Figure 2A.


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Figure 2. A. Typical view of the QCM-D curves for the frequency (blue) and dissipation (red), with the numbers indicated: equillibration with the buffer (1), after addition of blebs (2), after addition of POPC SUV (3), after rinsing with the buffer (4). Bleb density is 11 μL/mm2. Abrupt changes indicated with the grey lines are artefacts caused by opening of the QCM chamber. B-C. Frequncy shift and dissipation shift (respectively) dependence on the amount of blebs per surface (N=3). D. DiO stained giant blebs (blue areas) spontaneously rupture on the glass surface in the absence of POPC SUV. E. Composite Bl-SLB (2.08 μL/mm2) made with TR-SUV 45 minutes after addition of the vesicles. Intensity of red color indicates coverage of the surface by the artificial component of Bl-SLB. F. Immunostaining of the composite Bl-SLB and HEK 293 cell (insert) with anti-fibronectin antibodies. Scale bars D-F and the insert 20 μm. Open chamber was chosen for the experiment because, unlike a microfluidic system, this setup recreates the environment in which Bl-SLB for the cell culture experiments was formed; in the absence of a liquid flow. Rapid shifts at the beginning of each segment are caused by opening the chamber and subsequent thermal equilibration. Upon addition of blebs (zone 2), sedimentation of


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particles is observed, as indicated by the decrease in the resonance frequency and increase in the dissipation. However, the dissipation value (maximum observed D is less than 6*10-6) does not reach the level for a layer of unruptured vesicles from the artificial mix (usually30 around 25*106).

After sedimentation of blebs, POPC vesicles were added to the chamber to facilitate bleb

fusion (zone 3). As POPC SUV tend to aggregate in solution, we found that the best results can be achieved if the vesicles are sonicated directly before addition. The phosphatidylcholine headgroup is a zwitter-ionic compound and these vesicles don’t fuse into the cell membranes31, but rupture on the SiO2 surface32. It was demonstrated in literature that patches of SLB formed on the surface that is not covered with blebs contact the edges of sedimented blebs and cause parachuting-like fusion of the blebs into the composite Bl-SLB. Thorough rinsing of the chambers after step 3 should remove all unruptured blebs and SUVs. The resulting change of frequency value (maximum observed |ΔF| is 60 Hz) is noticeably higher than the usual value for SLB made from artificial SUVs, but still not high enough for the layer of unruptured vesicles (for a pure POPC SLB obtained |ΔF| is 25.5 Hz, for a saturated layer of vesicles |ΔF| 220 Hz as observed by Cho at al30). Since blebs are supposed to carry more components than just the lipids, that can explain an increased |ΔF|. The Bl-SLB contains additional mass from proteins and is expected to be slightly thicker than a POPC SLB due to projection of transmembrane and bound extracellular components. However, Bl-SLB is flatter, and has less mass than water containing unruptured vesicles. In Richards at al23, the resulting |ΔF| and ΔD on the composite Bl-SLB are ~50 Hz and ~5 * 10-6, respectively. Vafaei et al report that20 dissipation changes dramatically upon the binding of proteins to the SLB and increases by ~12-35 *10-6 relative to the SLB value depending on the amount of covalently bound protein. We observed that the dissipation value


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decreases over time, which can be attributed to the restructuring of the proteins on the surface of Bl-SLB33, or slow parachuting of blebs into a planar layer. Figures 2B-C show the values 2 hours after the final step of washing away unfused POPC vesicles and blebs. We did not count the number of blebs from different preparations. We decided to keep the cell density and blebing conditions the same for all preparations and count the dilution by “μl of the dispersion of collected blebs per mm2 of the surface”.

The

reproducibility shown by the error bars in Figure 2B-C indicates that the total material transferred from lot to lot using this method is consistent. The oscillation frequency change between the buffer and formed Bl-SLB states increases with the increasing bleb dispersion density at the low densities. At the density around 2 μl/mm2, the surface coverage of blebs reaches saturation, and the further increase in bleb density does not cause changes in the mass of the adsorbed layer.

3.4 Mobility of the components Blebs made from DiO stained HEK293 cells were collected and deposited on the glass surface, incubated there 1h at 37°C and then thoroughly rinsed with HEPES buffer. Due to their small size, fluorescence from the nanometer-sized population of blebs is not detectable. However, we observed spontaneously ruptured giant blebs that are shown on the Figure 2D. The puddle of ruptured giant blebs forms a shape typical for another standard study model – electrically formed giant unilamellar vesicles (GUVs) – when they rupture on the glass surfaces34. Although it was demonstrated that small blebs don’t rupture spontaneously due to the presence of proteins that mechanically stabilize their shape15,28, we suggest that the giant blebs will rupture spontaneously by the same mechanism as GUVs. Lipid bilayers are bendable when


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the curvature radius of the vesicle is high. The vesicle loses the round shape when it touches the support causing local changes in the radius of curvature and destabilizing the membrane34. However, as a result of the rupture, the membrane orientation, and consequently the orientation of transmembrane proteins, inverses. Nevertheless, if fused with the SUV layer, lipid components of the cell membrane still can be delivered into the Bl-SLB. To check the distribution of lipids originating from the cell membranes vs. those originating from the artificial mix in the composite Bl-SLB, uncolored blebs were fused by POPC SUVs containing a small fraction of the fluorescent lipid (TR-SUVs). The Figure 2E pictures the sample rinsed with the buffer 45 minutes after the addition of TR-SUVs. The uniform zone of bright fluorescence is where TR-SUVs were able to rupture and fuse small blebs unhindered. Dark zones are the areas that were covered by the puddles of the ruptured giant blebs. From the blurred edges between the two zones it is visible that diffusion of TR-DHPE into these areas already started. Membranes of cells have many components, such as cholesterol and gel-phase lipids that hinder the lateral diffusion of the molecules in the plane of the lipid bilayer35. These components would also be carried by the blebs and delivered into the Bl-SLB.

3.5 Delivery of fibronectin into Bl-SLB The delivery of the ECM component fibronectin into the Bl-SLB was investigated. Fibronectin is one of the major components that can bind to integrins and become a starting point for focal adhesion formation2. It is secreted by various cell types, including kidney cells36, and is bound by integrins to the surface of the cell that produced it37. The insert on Figure 2F demonstrates immunostaining of a HEK293 cell with rabbit anti-fibronectin and alexa564 anti-rabbit


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antibodies. As no permeabilization was done to the sample, conjugated fluorescent complex is located on the membrane surface of the cell only. The Bl-SLB made according to our standard protocol, incubated for 4 days at 37°C, and immunostained for fibronectin are shown in Figure 2F. We did not permeabilize the substrate before the incubation with the antibodies because only the fibronectin that is oriented to the solution and not to the dish is relevant for our application. There is a large-scale homogenous distribution of that protein with areas of low density of fibronectin that resemble zones of ruptured giant blebs, as seen in Figures 2D-E. Because the membranes of the giant blebs inverse as they rupture on the glass surface, the extracellular proteins are facing the dish and thus were not stained. The fibronectin from the tiny blebs that parachuted is facing the solution and accessible to the antibodies. Diffusion of the fibronectin can be hindered by its form on the cell surface as an insoluble structured matrix38. The immunostaining indicates that some proteins were able to diffuse into the inversed areas, although these zones are still depleted relative to the homogenous zones. The biggest of these low-density zones reach the size range of an HL-1 cell, and the possible effects of these inverted regions of membrane on the cell morphology when used as culture substrates are discussed further below.

3.6 Interactions with cells It was interesting to test Bl-SLB performance as a cell culture platform. We chose to investigate its stability by culturing HL-1 cells on Bl-SLB coated dishes. Cells of this mouse heart tumor-derived line are known to preserve several typical features of the cardiomyocyte such as spontaneous electrical action potentials and even mechanical contractions22. As mechanically active cells, they are expected to generate high tension forces when they develop


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stress fibers to maintain their shape as shown for motile cell lines39,40. Additionally, we aimed to observe an impact on a cell morphology of Bl-SLB with different ratios of natural to artificial components.

Figure 3. A-B. HL-1 cells 3 days in culture on (A) and 2.08 μL/mm2 and (B) 0.16 μL/mm2 BlSLB stained with CalceinAM. Scale bars 20 μm. C-D. Schemes of the cell-substrate interaction on a rigid (C) and fluid (D) substrate. When enough force can be generated, the cell forms stress fibers that help to hold its stretched form (C). When resistance to a pulling force at a focal adhesion complex is low, the cell only expresses cortical actin and takes a round shape (D). We discovered that Bl-SLB are able to support cell spreading on their surfaces and cell growth: HL-1 cells are spread and flattened on the surface, forming a monolayer after several cell divisions (Figure 3A). The behavior of cells is remarkably different from the case when cells that are seeded on the bilayer made from POPC only, where cells don’t adhere at all and are washed away during the medium change. On a diluted Bl-SLB (Figure 3B), cells don’t stretch and remain circular after seeding, in accordance with the low density of fibronectin in the diluted Bl-SLB and inert nature of POPC that composes most of the diluted Bl-SLB surface. In that case, when they are dividing cells don’t spread on the surface and instead grow in round clusters


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adhering mostly to each other. Surprisingly, some of these clusters are resistant to rinsing with buffer. It can be, that a cell on the bottom of the cluster is connected to low-density fibronectin on the diluted Bl-SLB, although overall high fluidity of the components results in inability to stretch via stress fibers. This observation corresponds with previous reports about the impact of the substrate softness on the cell shape17. POPC lipids exist in a liquid-like phase at 37°C, and the amount of the membrane components that lower the overall fluidity of the Bl-SLB in the diluted Bl-SLB is low, resulting in overall high fluidity of the substrate. A traction force of 5 pN is required to change the conformation of the initial adhesion protein complex into the state that allows further development of the focal adhesion18. In the absence of traction forces, the position of the integrin complexes is not defined by the position of the integrin ligands on the surface. Due to mobility of the ligands, the integrin complexes end up compactly gathered under the cell and actin stress fibers can’t be assembled (Figure3D). As a result of that, a cell takes a round shape, as previously described for fibroblasts cultured on a substrate with mobile RGD peptides (an integrin binding motif found in fibronectin)16. Lowered density of integrin ligands on the surface of diluted Bl-SLB may also play the role.

3.7 Stress fiber formation


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Figure 4. A-F: HL-1 cells after 3 days in culture on 0.05 μL/mm2 Bl-SLB (A), 0.52 μL/mm2 BlSLB (B), 2.08 μL/mm2 Bl-SLB (C, E), fibgel (D). Green color represents fibronectin-antibodies complex, red – TRITC-phalloidin, blue – DAPI. Arrows are indicating an elongated cells (C) and actin stress fibers (E). Scale bars 50 μm (A-D), 20 μm (E). F: Cell growth on Bl-SLB compared to the control. To investigate the proposed mechanism for Bl-SLB interaction with the cells (Figure 4C-D), we observed the state of the cytoskeleton on the fibgel substrate and on Bl-SLBs with different dilutions. As previously described, adhesion and spreading of cells improves with the increasing fraction of the cell membrane extract in the Bl-SLB (Figures 4A-C). On 2.08 μL/mm2 Bl-SLB some of the cells cells were elongated (pointed on the figure), although areas with this morphology appear only seldomly on the substrate. Good cell adhesion was also achieved on


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0.52 μL/mm2 Bl-SLB, but not on 0.05 μL/mm2 Bl-SLB, where cells were growing in floating clusters, occasionally anchored to Bl-SLB. We found that on Bl-SLBs with the bleb density 1 μL/mm2 or more, cell shapes are comparable to the shapes on a standard immobilized coating. This coating, commonly referred to as fibgel, consists of gelatin, which is a mix of hydrolyzed collagen, another ECM component, and fibronectin. The orientations of the molecules are distributed randomly on the surface. Moreover, fibronectin for the coating preparation is usually derived from the blood plasma, where it exists in the form of a soluble monomer, unlike the ECM38. However, HL-1 cells cultured on fibgel coating are able to maintain a differentiated cardiac phenotype. We observed that the surface density of the protein on this coating is higher than in 2.08 μl/mm2 Bl-SLB, although the distribution of fibronectin is inhomogenous (see areas in green on the Figure 4D). Interestingly, in some areas on Bl-SLB, HL-1 cells were even more stretched than on the standard control fibgel coating (Figure 4C indicated with arrow compared to Figure 4D). We suggest that this effect could be caused by several reasons. Firstly, the fact that ECM is structured on the surface of HEK293 plasma membrane38 creates a more physiological environment for the cells. Moreover, the process of rinsing can cause additional structuring of the protein into fibrills. Lastly, the effect may be caused by triggering additional biochemical pathways by other components of the blebs. HEK293 cells are known to express several components that can interact with HL-1 cell components, for example, Connexin43 protein41, which forms gap junction plaques in heart42. Unfortunately, it was not possible to determine the distribution of fibronectin on the substrate under the cells, since fibronectin on HL-1 membranes was also stained and it was not possible to distinguish between the fibronectin from the HL-1 cell membrane and from the substrate.


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Nevertheless, on the areas of the substrate that are unoccupied by the cells it is possible to see fibronectin-conjugated fluorescent complexes in the fibgel and on the Bl-SLB 0.52 μL/mm2 and 2.08 μL/mm2 (Figures 4B-D and insert). There were very few of visible fibronectin points on 0.05 μL/mm2 Bl-SLB (Figure 4A). Figure 4E and the insert demonstrate stained actin fibers of cells. Most of the cells exhibit cortical actin filaments, but also distinct stress fibers are visible. That shows that the fluidity of the composite Bl-SLB allows a generation of forces high enough to enable focal adhesion formation. As fibronectin is regarded the model protein for study focal adhesions, we have tested its involvement in HL-1 adhesion to Bl-SLB. It was not possible to determine co-localization of the actin fiber starting point and any possible clusters of fibronectin for these samples (Figure 4E insert). That may be due to the more physiological density of fibronectin in Bl-SLB compared to commonly used layers of deposited proteins on solid supports5 making it not possible to visualize. Or it may beAnother possibility is that the reactivity between mouse integrin and human fibronectin is different from the bovine plasma fibronectin that is usually utilized. On the other hand, it is an interesting result. Plasma fibronectin exists in a soluble monomer form. However, in the tissue cells interact both with fibronectin in a form of a soluble monomer that is secreted by the cells, and in a polymerized form of pre-formed ECM. In most of the published experiments, adhesion of the cell and polymerization of pre-coated protein happen at the same time. Structured fibronectin in its in vivo form is rarely utilized due to its insolubility and the lack of protocols for the correct transfer of it for in vitro use. This shows the importance of Bl-SLB as a more biomimetic substrate for cell adhesion studies.

3.8 Cell growth on Bl-SLB and Bl-SLB stability


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We tested the ability of Bl-SLBs to support the cells while they are dividing. The growth rate is comparable to the growth on a fibgel coating as indicated on Figure 4F. After reaching confluency – which means, occupying the whole surface of the dish, on the control coating the cell sheet was stable for at least two more days, with cell numbers staying in the same range. However, after the full coverage is reached on the Bl-SLB, the next day the cell number radically drops. We suggest that happens due to the contractions of the HL-1 cells. These cells can develop spontaneous periodical contractions, like beating of the cardiac tissue22. This happens after the growing HL-1 cells form patches with tight contacts between the neighbor cells. After that they develop mature gap junctions that allow a cell patch to synchronously contract when a pacemaker cell sends an electrical signal. Since supported lipid bilayer is just a thin, unbound layer separated from the glass surface by 1-2 nm of water43, contraction of the cells linked to BlSLB will cause rupture of the layer. The patches of cells with an underlying patch of Bl-SLB will then float from the surface to the medium and be removed when the medium is changed, resulting in a decreased number of cells on the surface. Since it was demonstrated that contraction of cardiac myocytes is strong enough to cause wrinkling of soft polymer materials44, this mechanism of cell loss is the most likely one. Nevertheless, it was quite surprising that the mechanical properties of Bl-SLB enable them to hold the load from such mechanically strong cells up to the start of beating. Our results did not show Bl-SLB fibronectin – cell interactions. However, fibronectin, as well as a number of other macromolecular components of the cell membrane45, may play a role in mechanical stabilization of a lipid bilayer, so that can sustain cell growth. On the other side, easier detachment of a cell sheet can be an advantage for several applications, such as growth of substitute heart patches46. Indeed, we noticed that during the digestion with trypsin, cell patches in Bl-SLB coated dishes detach from the surface easier than


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in fibgel-coated dishes. EDTA that is a component of the digestion enzymatic mix may also remove calcium ions from the volume in between the lipid layer and the glass, making the interaction of two negatively charged surfaces no longer attractive. This process may be performed in the absence of enzymes, allowing cell patches to detach from the surface, but leaving cell-cell adhesions intact. It may be advantageous in a situation when a pre-formed cell layer should be transferred from a growth dish to another surface.

Conclusion Overall, a simple protocol for the assembly of composite lipid bilayers was shown. A homogenous distribution of lipids in the bilayer can be achieved, although at least one of the protein components remains inhomogeneously distributed on the resulting surface. In general, a biocompatibility of the components from two different cell lines was observed. It was possible to culture HL-1 cells on a composite lipid bilayers made from the extracts from HEK293 cells. Use of the HEK293 cells opens possibilities for further studies, as this cell line is known for one of the highest transfection efficiency47. It is possible to make the cells overexpress and insert transmembrane proteins of interest in the cytoplasmic membrane. A protocol for BlSLB fusion provides a simple method to reconstitute these proteins in a biomimetic coating for other applications. The presented method allows to control the cell adhesion behavior by adjusting a natural/artificial ratio in the mix. Impact of the individual components of the Bl-SLB on the cell adhesion is still to be studied. Further studies may investigate the impact of the individual components of the Bl-SLB on the cell adhesion, as well as the exact components responsible for HL-1 cell adhesion.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We would like to thank Dr. Agnes Csiszar from the Institute of Biomechanics (ICS-7) for the use of Zetasizer Nano ZS. ABBREVIATIONS Bl-SLB, bleb-supported lipid bilayer; DiO, benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl2(3H)-benzoxazolylidene)-1-propenyl]-, perchlorate; DLS, dynamic light scattering; DTT, dithiothreitol; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; GUV, giant unilamellar vesicles; HEK293, human embryonic kidney 293 cells; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HL-1, HL-1 cardiomyocyte cells; PFA, paraformaldehyde; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; SLB, supported lipid bilayer; SUV, small unilamellar vesicles; TR, Texas Red; TR-SUV, Texas Red-small unilamellar vesicles; QCM-D, quartz crystal microbalance with dissipation REFERENCES (1)

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For table of contents only 82x44mm (300 x 300 DPI)


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Figure 1. A. Schematic representation of the Bl-SLB assembly. Naturally ocurring phospholipids produced by the cell are shown in blue and yellow. Trans-membrane proteins in orange and extracellular matrix in dark green. SUVs of synthetic lipids are in red. The membrane separated from the cell in the form of the bleb recombines with SUV lipids on the surface. Extracellular and intracellular sides of the original cell membrane are labelled “E” and “I” respectively. Then cells are cultured on Bl-SLB. B-C. HEK293 cell membrane stained with DiO after 15 min (B) 3 hours (C) of incubation in the blebing inducing solution. Scale bar 20 μm. D. The fraction of collected blebs after removal of the giant blebs as characterized by the dynamic light scattering method. 131x63mm (300 x 300 DPI)


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Figure 2. A. Typical view of the QCM-D curves for the frequency (blue) and dissipation (red), with the numbers indicated: equillibration with the buffer (1), after addition of blebs (2), after addition of POPC SUV (3), after rinsing with the buffer (4). Bleb density is 11 μL/mm2. Abrupt changes indicated with the grey lines are artefacts caused by opening of the QCM chamber. B-C. Frequncy shift and dissipation shift (respectively) dependence on the amount of blebs per surface (N=3). D. DiO stained giant blebs (blue areas) spontaneously rupture on the glass surface in the absence of POPC SUV. E. Composite Bl-SLB (2.08 μL/mm2) made with TR-SUV 45 minutes after addition of the vesicles. Intensity of red color indicates coverage of the surface by the artificial component of Bl-SLB. F. Immunostaining of the composite Bl-SLB and HEK 293 cell (insert) with anti-fibronectin antibodies. Scale bars D-F and the insert 20 μm. 169x89mm (300 x 300 DPI)


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Figure 3. A-B. HL-1 cells 3 days in culture on (A) and 2.08 μL/mm2 and (B) 0.16 μL/mm2 Bl-SLB stained with CalceinAM. Scale bars 20 μm. C-D. Schemes of the cell-substrate interaction on a rigid (C) and fluid (D) substrate. When enough force can be generated, the cell forms stress fibers that help to hold its stretched form (C). When resistance to a pulling force at a focal adhesion complex is low, the cell only expresses cortical actin and takes a round shape (D). 84x57mm (300 x 300 DPI)


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Figure 4. A-F: HL-1 cells after 3 days in culture on 0.05 μL/mm2 Bl-SLB (A), 0.52 μL/mm2 Bl-SLB (B), 2.08 μL/mm2 Bl-SLB (C, E), fibgel (D). Green color represents fibronectin-antibodies complex, red – TRITCphalloidin, blue – DAPI. Arrows are indicating an elongated cells (C) and actin stress fibers (E). Scale bars 50 μm (A-D), 20 μm (E). F: Cell growth on Bl-SLB compared to the control. 171x111mm (300 x 300 DPI)


Composite lipid bilayers from cell membrane extracts and artificial

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