Dorsal and Ventral Stimuli in Cell–Material Interactions: Effect on Cell Morphology
© The Author(s) 2012
Received: 22 April 2012
Accepted: 15 May 2012
Published: 2 June 2012
Cells behave differently between bidimensional (2D) and tridimensional (3D) environments. While most of the in vitro cultures are 2D, most of the in vivo extracellular matrices are 3D, which encourages the development of more relevant culture conditions, seeking to provide more physiological models for biomedicine (e.g., cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. In this study, cells were cultured between two protein coated surfaces (sandwich-like culture). Cells used both dorsal and ventral receptors to adhere and spread, undergoing morphological changes with respect to the 2D control. Combinations of fibronectin and bovine serum albumin on the dorsal and ventral sides led to different cell morphologies, which were quantified from bright field images by calculating the spreading area and circularity. Although the mechanism underlying these differences remains to be clarified, excitation of dorsal receptors by anchorage to extracellular proteins plays a key role on cell behavior. This approach—sandwich-like culture—becomes therefore a versatile method to study cell adhesion in well-defined conditions in a quasi 3D environment.
Cells in multicellular organisms live within tissues, where they are surrounded by the extracellular matrix (ECM), a complex fibrous matrix that provides mechanical support as well as specific biochemical and biophysical signals able to direct cell function [1–3]. Since the natural habitat of most living cells is a tridimensional (3D) mesh surrounding them, culturing cells on bidimensional (2D) surfaces imposes an unnatural environment totally different from the natural ECM. Changes in cell behavior as a way to self-adaptation to the situation occur [4, 5]. For instance, fibroblasts spread on 2D surfaces , whereas they adopt a bipolar shape in vivo; moreover, spindle-like shape similar to in vivo is recovered if cells are cultured in 3D collagen gels [7, 8] and tissue-derived matrices,  suggesting that morphological alterations are related to the dimensionality of the surroundings. Likewise, the more physiologically relevant 3D environment is increasingly preferred when doing research on cellular processes in vitro, including matrix secretion, cell differentiation, morphogenesis, cancer research and drug development [10–13].
We have developed a new sandwich-like methodology to investigate the role of dorsal and ventral stimuli on cell morphology, using different material substrates (Fig. 1a). Concretely, we have used as upper substrate thin poly(ethyl acrylate) film and different bottom substrates: (i) spincoated poly(ethyl acrylate) on which FN assembles spontaneously into fibrillar networks in a physiological way ; (ii) then, we have used FN adsorbed on glass, which allows cells to reorganize FN at the material interface [24, 25]; (iii) finally, we have investigated the role of topological cues making use of aligned fibers obtained via electrospinning. These substrates were coated with different proteins (fibronectin or albumin) and assembled in a sandwich-like configuration. As a widely studied model of cell adhesion and migration in 2D and 3D, NIH3T3 fibroblasts were used; such cells do not naturally display apical–basal polarity, thus allowing to observe specific effect of anchorage of cells on either one or both sides and the influence of its temporal course.
2 Materials and Methods
Polymer sheets of ethyl acrylate (EA) (Sigma-Aldrich, Steinheim, Germany) 0.4 mm of thickness were obtained by radical polymerization of a solution of EA using 0.2 wt% benzoin (98 % pure, Scharlau, Barcelona, Spain) as photoinitiator. The polymerization was carried out up to limiting conversion. After polymerization, low molecular-mass substances were extracted from the material by drying in vacuum to constant weight. Rounded samples were cut from the polymerized film to be used as the top substrates of the sandwich. PEA films were washed in an ultrasonic bath for 5 min and hydrated overnight in Dulbecco’s phosphate buffered saline (DPBS, Invitrogen) the day before cell culture.
2.2 Spin Coater
Thin films of poly(ethyl acrylate) (PEAspc) were prepared by making use of a spin-coater (Brewer Science, Rolla, USA). PEA was dissolved in toluene at a concentration of 2.5 wt%. Spin casting was performed on glass coverslips at 2,000 rpm for 30 s. Samples were dried under vacuum at 60 °C before use.
Electrospun fibers of PEA were collected as described elsewhere . Briefly, PEA 1 % Benzoin was dissolved in hexafluoroisopropanol (HFIP, Sigma) at 20 mg/mL. Polymer solution was electrospun at a constant feed rate of 900 µL/h using a programmable syringe pump (New Era Pump Systems, Wantagh, NY, USA) with a voltage of 12.5 kV (Glassman High Voltage, High Bridge, NJ, USA) and a collector distance of 20 cm. In order to obtain aligned fibers (PEAa) the polymer solution was electrospun onto a rotating drum (rotating at 900 rpm, equivalent to a linear speed of 337.5 cm/s) where glass coverslips were stuck.
2.4 Scanning Electron Microscopy
The electrospun fibers were characterized by scanning electron microscope (SEM) (JEOL JSM 6300, JEOL Ltd., Tokyo, Japan) at 15 kV.
2.5 Protein Adsorption
Fibronectin (FN) from human plasma (Gibco) at 20 µg/mL in DPBS or heat-denatured Bovine Serum Albumin Fraction V (BSA) (Roche) at 10 mg/mL in water were adsorbed on the different substrates by immersing the sample in the protein solutions for 1 h. After adsorption, samples were rinsed in DPBS to eliminate the non-adsorbed protein
2.6 Atomic Force Microscopy
Atomic force microscopy (AFM) was performed in a NanoScope III from Digital Instruments (Santa Barbara, CA) operating in the tapping mode; the Nanoscope 5.30r2 software version was used for image processing and analysis. Si-cantilevers from Veeco (Manchester, UK) were used with force constant of 2.8 N/m and resonance frequency of 75 kHz. The phase signal was set to zero at a frequency 5–10 % lower than the resonance one. Drive amplitude was 200 mV and the amplitude setpoint (Asp) was 1.4 V. The ratio between the amplitude setpoint and the free amplitude (Asp/A0) was kept equal to 0.7.
2.7 Cell Culture
NIH3T3 fibroblasts (European Collection of Cell Cultures) were maintained in DMEM medium with 10 % Calf Serum (Thermo Scientific) and 1 % penicillin–streptomycin (Lonza). Prior to seeding on the substrates, samples (both the top and the bottom ones) were sterilized by UV exposure for 30 min and then coated with FN or BSA as explained. Then, 7,000 cells/cm2 were seeded in serum free conditions on the different bottom surfaces placed in a multi-well dish. Afterwards a film of PEA was gently laid over the bottom substrate either immediately (sandwich 0-y) or after 3 h of culture (sandwich 3-y). A highly concentrated cellular suspension was used in order to avoid cell loss after laying the upper substrate. Also, for the sandwich 3-y, excess of medium on the bottom surface was removed before laying the film of PEA. After assembling the sandwich a gentle pressure of approx. 103 Pa was applied for 3 min on the top surface to facilitate the initial stability of the system. Finally, pressure was released and the medium replenished. Sandwich-like cultures were then maintained at 37 °C in a humidified atmosphere under 5 % CO2. For longer cultures (24 h) medium was changed by serum-containing medium after 3 h of culture.
Finally, samples were fixed with 10 % formalin solution (Sigma) at 4 °C for 1 h, rinsed with DPBS and observed in a Leica DMI6000 inverted microscope.
2.8 Live/Dead Assay
Viability of cells was measured by live/dead assay (Invitrogen) and analyzed by fluorescence microscopy (Leica DMI 6000). Viability is given as the percentage of living cells.
2.9 Image Processing
All image processing and analysis was done using Adobe Photoshop CS5 and ImageJ. Briefly, brightness and contrast were modified in bright field images in order to define the cell shape with Adobe Photoshop CS5. Thereafter morphology was quantified by calculating cell area and circularity (4π × area/perimeter2), which corresponds to a value of 1 for a perfect circle using ImageJ software of at least 20 cells for each condition.
2.10 Statistical Analysis
Results are shown as average ± standard deviation. All experiments were performed in triplicate unless otherwise noted. Results were analyzed by one-way ANOVA. If treatment level differences were determined to be significant, pair-wise comparisons were performed (n ≥ 20). Statistically significant differences are depicted with the following signs. Let sandwich x–y stand for x-hours of ventral contact and y-hours of dorsal contact, then * is for the significance of y comparing among equal total culture times, † for the significance of y comparing for the same x. shows significance comparing with control for the same culture time and ¥ for conditions with same total culture time but different y (i.e. cells cultured in sandwich 3–3 and sandwich 0–6 adhere similarly on the ventral FN-coated substrate but not to the upper substrate).
3 Results and Discussion
We have checked cell viability within the sandwich-like system at the longest time (1 day). By doing so, we intend to rule out any diffusion problem of the culture medium through the system. As expected, most cells remain viable during the experiment (viability higher than 80 % after 1 day of culture) since a permeable material, PEA, is used as the top surface of the sandwich construct (the diffusion coefficient of water in PEA is D ~ 3.4 × 107 cm2/s) .
To address the effect of the initial ventral material interaction before dorsal stimulation, sandwich-like cultures were established either immediately after cell seeding—to prevent any preferential role of ventral receptors—or after 3 h of 2D culture, to permit initial cell adhesion on material surfaces using ventral receptors. Cell culture within sandwiches was maintained up to 24 h to study the time evolution of cell morphology. Hereafter, a two variable nomenclature will be used to easily identify each culture condition: sandwich x–y; where x stands for the time (hours) of ventral stimulation and y for the time (hours) in full sandwich-like culture.
3.1 Dorsal and Ventral Stimulation Using Material-Driven FN Fibrils
We have used FN-coated poly(ethyl acrylate), PEA, for both dorsal and ventral stimulation as FN organizes into interconnected physiological-like fibrils upon adsorption on this material. The fibrillar organization of FN upon passive adsorption on PEA was named material-driven fibrillogenesis, since the assembled FN fibrils on PEA share some similarities with cell-assembled FN matrices . In addition, the resulting fibrillar FN structure on PEA recapitulates the native structure of FN matrices and displays enhanced biological activity [23, 26]. Figure 1c shows the FN network assembled upon adsorption on PEA observed with AFM in comparison with globular organization of FN on glass. FN organization on electrospun PEA fibers is also shown.
3.2 Effect of Topological Cues at the Ventral Material Interface
Cells tend to align and spread under the strong geometrical input coming from the fibers (2D). After 3 h, cells are already aligned and circularity does not change anymore as a function of time (2D). Our results show that upon dorsal stimulation, cells do not continue the interaction with the underlying fibers as in the 2D situation (compare e.g. sandwich 3–3 and sandwich 3–21, Fig. 3), which is somehow frozen in. In addition, this experiment suggests that signaling coming from the dorsal stimuli diminishes the strength of the inputs coming from the ventral topological cues: cells are not able to align on the fibers if the sandwich is assembled from the very beginning. By contrast, cells remain in a rounded-like morphology, with high circularity regardless the underlying ventral topological cues (sandwich 0–6 and sandwich 0–24, Fig. 3). Moreover, even when ventral adhesion on the electrospun fibers is allowed for 3 h before dorsal stimulation, more rounded cells are equally obtained (sandwich 3–3 and sandwich 3–21, Fig. 3). The time evolution of the morphological parameters has been included for this configuration in Figure S2 for easy reading.
3.3 Effect of FN Reorganization at the Ventral Material Interface
3.4 Effect of Non-Adhesive Dorsal Contact
3.5 Relevance and Limitations of the Model
Nutrient, oxygen and waste diffusion are important factors to take into account in cell culture, being more important therefore in 3D scaffolds, hydrogels, multi-layer and sandwich cultures. That is the reason why we use thin poly ethyl acrylate (PEA) films with a water diffusion coefficient of D ~ 3.4 × 107 cm2/s as upper substrates in our sandwich-like model. Cell viability was above 80 % during the experiment, showing that oxygen and nutrient diffusion is not a limiting factor in this system. This issue could become relevant at longer times or to transport molecules (such as growth factors) with higher molecular weight, that do not diffuse throughout the polymer. Considering the temporal framework of our experiments, as well as the absence of growth factors in the medium, we assume that our results observed in the sandwich-like cultures must be ascribed to the dorsal interaction, disregarding any diffusion problems that deprive cells from nutrients or other important chemical cues. Nevertheless, it must be taken into account that permeability of the upper substrate is a critical issue in the design of the sandwich-like system, and using less permeable materials as upper substrates could lead to a gradient of cell death, from the centre to the periphery, as happens in tumors. In fact, sandwich-like cultures with limited diffusion has been studied as an approach to supplement multicellular spheroids as tumor analogues [34–36].
Important differences with 3D environment include the lack of isotropy, limitation of cell mobility to the x–y plane, mechanical properties of the substrates and absence of a physiological nanofibrillar environment (although this is somehow mimicked by the fibrillogenesis of fibronectin on PEA). As a result, the round morphology observed in sandwiches 0-y is different from the spindle-like morphology observed in fibronectin-coated acrylamide sandwiches  or even in natural ECM . These differences might be sought in the mechanical modulus of the substrates (acrylamide substrates have lower stiffness), although protein composition also seems to be important as cells cultured in 3D collagen gels do not always display the characteristic spindle-like morphology . So, even if our sandwich-like system is not a truly representation of a 3D situation, it is a useful model beyond 2D systems, and it allows a controlled and versatile tuning of the substrates and composition, more difficult to tailor in standard 3D environments such as Matrigel or collagen gels.
The own nature of this system enables a wide range of possibilities. Such model enables to study the effect of external pressure on cell behavior, by simply changing the weight applied; substrates with varying chemistries and mechanical properties can be used. It could also be possible to study the role of cell–cell interaction by seeding both sides of the sandwich system with cells. Furthermore, several conditions can be studied at the same time such as the influence of the pressure on cell–cell interaction. Therefore sandwich-like cultures could become an important system for deciphering cell response under well-defined conditions and later, to translate this knowledge to multi-layer approaches used nowadays on tissue engineering and to 3D environments if a good agreement is achieved (between sandwich and 3D). Representative contrast phase images of cells in several conditions have been included as supplementary material (Figure S6).
The use of sandwich-like cultures has shown to be a robust tool to investigate the role of dimensionality in cell-materials interactions. It allows the combination of different adhesive proteins and geometrical inputs in both dorsal and ventral sides.
Overall, initial dorsal and ventral stimuli inhibit cell spreading and give rise to rounded-like cell morphology. However, if dorsal stimuli are applied once cells have already started (ventral) spreading on a material surface, cell stop spreading and somehow freeze into the attained morphology. By contrast, cell retraction into a rounded morphology is not observed as time goes by. Moreover, stimulation of dorsal receptors is strong enough to inhibit the geometrical inputs coming from the ventral side (e.g. alignment of cells along electrospun fibers). Strikingly, cell behavior in 3D environments might not be only a consequence of integrin-mediated adhesion to the surrounding matrix, as BSA-covered top-substrates elicit the same response as FN-coated one. That is to say, not only protein–protein interaction should be considered to explain cell behavior in 3D environments, but also the role of a pure mechanical-contact interaction must be considered.
Further studies are needed to get more insights into the role of dimensionality in cell behavior using this sandwich-like approach, where the mechanism for cell adhesion must be elucidated. The origin of cell behavior due to mere mechanical dorsal contact might be sought in the need for cell to adhere and build up ventral focal adhesions, in dependence on how protected cells feel their membrane surface. However, this would mean the existence of additional cellular mechanotransduction mechanisms to explore the environment other than integrin mediated ones.
The support of the project MAT2009-14440-C02-01 and FPU program AP2009-3626 is acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.
- Folkman J, Moscona A (1978) Nature 273:345–349View ArticleGoogle Scholar
- Watt FM, Jordan PW, O’Neill CH (1988) Proc Natl Acad Sci USA 85:5576–5580View ArticleGoogle Scholar
- Weiss P (1959) Rev Mod Phys 31:449–454View ArticleGoogle Scholar
- Lewis WH, Lewis MR (1926) In: Cowdry EV (ed) General cytology. University of Chicago Press, ChicagoGoogle Scholar
- Weiss P (1959) Rev Mod Phys 31:11–20View ArticleGoogle Scholar
- Bard JB, Hay ED (1975) J Cell Biol 67:400–418View ArticleGoogle Scholar
- Elsdale T, Bard J (1972) J Cell Biol 54:626–637View ArticleGoogle Scholar
- Grinnell F (2003) Trends Cell Biol 13:264–269View ArticleGoogle Scholar
- Cukierman E, Pankov R, Stevens DR, Yamada KM (2001) Science 294:1708–1712View ArticleGoogle Scholar
- Hutmacher DW (2010) Nat Mat 9:90–93View ArticleGoogle Scholar
- Lutolf MP, Hubbell JA (2005) Nat Biotech 23:47–55View ArticleGoogle Scholar
- House DJM, Elstad K, Socrate S, Kaplan DL (2012) Tissue Eng Part A 5–6:499–507View ArticleGoogle Scholar
- Rimann M, Graf-Hausner U (2012) Curr Opin Biotech 23:1–7View ArticleGoogle Scholar
- Beningo KA, Dembo M, Wang YL (2004) PNAS 101:18024–18029View ArticleGoogle Scholar
- Dunn JC, Yarmush ML, Koebe HG, Tompkins RG (1989) FASEB J 3:174–177Google Scholar
- Ryan CM, Carter EA, Jenkins RL, Sterling LM, Yarmush ML, Malt RA, Tompkins RG (1993) Surgery 113:48–54Google Scholar
- Knop E, Bader A, Boker K, Pichlmayr R, Sewing KF (1995) Anat Rec 242:337–349View ArticleGoogle Scholar
- Guaccio A, Guarino V, Alvarez-Pérez MA, Cirillo V, Netti PA, Ambrosio L (2011) Biotechnol Bioeng 108:1965–1976View ArticleGoogle Scholar
- Gong YY, Xue JX, Zhang WJ, Zhou GD, Liu W, Cao Y (2011) Biomaterials 32:2265–2273View ArticleGoogle Scholar
- Fraley SI, Feng Y, Krishnamurthy R, Kim DH, Celedon A, Longmore GD, Wirtz D (2010) Nat Cell Biol 12:598–604View ArticleGoogle Scholar
- Kubow KE, Horwitz AR (2011) Nat Cell Biol 13:3–5View ArticleGoogle Scholar
- Fraley SI, Feng Y, Wirtz D, Longmore GD (2011) Nat Cell Biol 13:5–7View ArticleGoogle Scholar
- Salmerón-Sánchez M, Rico P, Moratal D, Lee T, Schwarzbauer J, García AJ (2011) Biomaterials 32:2099–2105View ArticleGoogle Scholar
- Altankov G, Groth T (1994) J Mat Sci Mat Med 5:732–737View ArticleGoogle Scholar
- Altankov G, Grinnell F, Groth T (1996) J Biomed Mat Res 30:385–391View ArticleGoogle Scholar
- Ballester-Beltrán J, Cantini M, Lebourg M, Rico P, Moratal D, García AJ, Salmerón-Sánchez MJ (2012) Mat Sci Mat Med 1:195–204View ArticleGoogle Scholar
- Gallego Ferrer G, Monleón Pradas M, Gómez Ribelles JL, Pissis P (1998) J Non-Cryst Soli 235–237:692–696. http://www.sciencedirect.com/science/article/pii/S0022309398005730
- Chaudhuri O, Parekh SH, Lam WA, Fletcher DA (2009) Nat Methods 6:383–387View ArticleGoogle Scholar
- Gugutkov D, González-García C, Rodríguez Hernández JC, Altankov G, Salmerón-Sánchez M (2009) Langmuir 25:10893–10900View ArticleGoogle Scholar
- Altankov G, Groth T, Krasteva N, Albrecht W, Paul D (1997) J Biomat Sci Polym E 8:721–740View ArticleGoogle Scholar
- Curtis ASG, Forrester JV (1984) J Cell Sci 71:17–35Google Scholar
- Zelzer M, Albutt D, Alexander MR, Russell NA (2012) Plasma Process Poly 9:149–156View ArticleGoogle Scholar
- Tamada Y, Ikada Y (1993) J Colloid Interf Sci 155:334–339View ArticleGoogle Scholar
- Hlatky L, Alpen EL (1985) Cell Tissue Kinet 6:597–611Google Scholar
- Hlatky L, Alpen EL, Yee MK (1986) Radiat Res 1:62–73View ArticleGoogle Scholar
- Hlatky L, Sachs RK (1988) Alpen EL 2:167–178Google Scholar
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