Cellular Contact Guidance on Liquid Crystalline Networks with Anisotropic Roughness

Cell contact guidance is widely employed to manipulate cell alignment and differentiation in vitro. The use of nano- or micro-patterned substrates allows efficient control of cell organization, thus opening up to biological models that cannot be reproduced spontaneously on standard culture dishes. In this paper, we explore the concept of cell contact guidance by Liquid Crystalline Networks (LCNs) presenting different surface topographies obtained by self-assembly of the monomeric mixture. The materials are prepared by photopolymerization of a low amount of diacrylate monomer dissolved in a liquid crystalline solvent, not participating in the reaction. The alignment of the liquid crystals, obtained before polymerization, determines the scaffold morphology, characterized by a nanometric structure. Such materials are able to drive the organization of different cell lines, e.g., fibroblasts and myoblasts, allowing for the alignment of single cells or high-density cell cultures. These results demonstrate the capabilities of rough surfaces prepared from the spontaneous assembly of liquid crystals to control biological models without the need of lithographic patterning or complex fabrication procedures. Interestingly, during myoblast differentiation, also myotube structuring in linear arrays is observed along the LCN fiber orientation. The implementation of this technology will open up to the formation of muscular tissue with well-aligned fibers in vitro mimicking the structure of native tissues.


Experimental methods for myotube differentiation and analysis
Murine C2C12 myoblasts were grown in standard cell culture conditions (37 ºC in 5% CO2 humidified atmosphere) on Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). When the cells are in sub-confluency, in order to differentiate them, the medium was changed to DMEM supplemented with 2% Horse Serum (HOS).
Confocal analysis. C2C12 myoblasts were grown on glass coverslips as a control and on the nematic and isotropic coating until sub-confluency and then differentiated for 96 hours. The samples were washed with PBS and fixed in 3% paraformaldehyde for 20 min at 4 °C. Subsequently, the fixed samples were permeabilized with 3 washes on TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) and blocked with 5.5% HOS in TBST for 1 hour at room temperature. Samples were immunostained with anti-MHC primary antibody (1:100 in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl)) overnight at 4 °C. Then washed once with TBST, once with TBST with 0.1% BSA following the incubation with the secondary antibody conjugated with Alexa Fluor 488 (1:100 in TBS with 3% BSA). For the nuclei staining 4′,6-diamidino-2-phenylindole (DAPI) was used at a final concentration of 10 µM in TBST for 5 minutes at room temperature. Several washes with TBST were performed before mounting the samples with a glycerol mounting medium. Leica TCS SP8 confocal fluorescence microscope was used to analyze the samples.
Immunoblot analysis. Cells were lysed in 500 µl of complete radio-immunoprecipitation assay (RIPA) buffer on ice for 20 minutes. Lysates were clarified by centrifugation, and total protein contents were obtained using Bradford assay (Bio-Rad Laboratories). SDS-PAGE was used to separate total proteins for each sample (20 µg) and transfer onto PVDF membranes. These membranes were incubated in a 2% milk solution, incubated with primary antibodies, then with secondary antibodies conjugated with horseradish peroxidase. Figure S1. Profilometer analysis of a nematic coating. a) 2D height map at the border in between the glass (blue area) and the polymer coating (green area) measured by optical profilometer; b) example of height profile at the border in between the glass (corresponding to 0 nm in the y axis) and the polymer coating.  Nematic coating observed at the border with tilted stub; scale bar: 5 μm, red arrows show the nematic director; c) Nematic coating at high magnification, scale bar: 2 μm; d) Isotropic coating at high magnification, scale bar: 2 μm. ef) SEM images of different samples of nematic coatings. Image J was used to estimate the fiber diameter report here as a medium from 3 samples. In particular, at higher magnification (f) we can observe mainly small fibers (some are highlighted in green) with diameter around 81 ± 4 nm and bigger fibers (some are highlighted in blue) with a medium diameter of 178 ± 6 nm.   Figure S6. Other images of cells cultured on LCN coating. a) Images of HDFs on nematic coating at different magnification; b) Images of C2C12 myoblasts on nematic coating at different magnification. All images are acquired on different days (to reach different confluency levels) and tests.

Further information on cell culture
Basic information on chosen cell lines. Human dermal fibroblast (HDF) is a primary cell culture from human origin. A primary cell culture is an ex vivo culture of cells obtained from a biopsy of the tissue of the multicellular organism (in this case the epidermis of a human). This type of culture is more representative of an in vivo tissue than an immortalized cell line and the cells behave in a similar way than in the living organisms. This aspect also means that, after several divisions, the cells acquire a senescent phenotype leading to a cessation of the cell division. On the other hand, C2C12 is an immortalized mouse myoblast cell line. An immortalized cell line also comes from a multicellular organism (that normally do not proliferate indefinitely) but, in this case, thanks to a mutation, the acquisition of that senescent phenotype has bypassed and the cell cycle and division keep going until some space is present on the scaffold. Those 2 different cell types were chosen because they come from different organisms (human and mouse), are widely used in research and representative of different tissues (muscular and dermal ones).
Estimation of cell density on different scaffolds. Cell density was estimated after the same number of days of culture on the different materials to compare their viability. Cells were seeded with the same density and let grow for 3 days and then stained. An area of 1 mm 2 was selected and cells were counted from 6 different images (from different experiments) on each coating and for each cell type. Then, mean and standard deviation of cell density were calculated for each scaffold (control, nematic and isotropic coating) and reported in Table S1.