Publications

While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations.

JTD Keywords: Hydrogel scaffold, Confocal microscopy, Substrate curvature, Cell morphology, Cell orientation, Histological section, Small intestine, Villus

Mesenchymal stem cells (MSCs) are a promising cell source for cell-based therapies because of their self-renewal and multi-lineage differentiation potential. Unlike embryonic stem cells adult stem cells are subject of aging processes and the concomitant decline in their function. Age-related changes in MSCs have to be well understood in order to develop clinical techniques and therapeutics based on these cells. In this work we have studied the effect of aging on adhesive behaviour of bone marrow-derived MSC and MG- 63 osteoblastic cells onto three extracellular matrix proteins: fibronectin (FN), vitronectin (VN) and collagen I (Coll I). The results revealed substantial differences in adhesive behaviour of both cell types during 21 days in culture. Bone-marrow derived MSCs decreased significantly their adhesive affinity to all studied proteins after 7th day in culture with further incubation. In contrast, MG-63 cells, demonstrated a stable cell adhesive phenotype with high affinity to FN and Coll I and low affinity to vitronectin over the whole culture period. These data suggest that adhesive behaviour of MSCs to matrix proteins is affected by aging processes unlike MG-63 cells and the age-related changes have to be considered when expanding adult stem cells for clinical applications.

JTD Keywords: Cell morphology, Cell attachment and spreading, Fibronectin, Vitronectin, Collagen I

Chemical gradient surfaces are described as surfaces with a gradually varying composition along their length. Continuous chemical gradients have recently been proposed as alternative to discrete microarrays for the high throughput screening of the effects of ligand concentration in cells. Here we review some of the most recent examples in which gradients have been used to evaluate the effect of a varying ligand concentration in cell adhesion, morphology, growth and differentiation of cells, including some of our recent findings. They show the importance of the organization of ligands at the nanoscale, which is highlighted by abrupt changes in cell behavior at critical concentration thresholds.

JTD Keywords: Cell Adhesion, Cell Differentiation, Cell growth, Cell morphology, Molecular gradient

It is known that cells respond strongly to microtopography. However, cellular mechanisms of response are unclear. Here, we study wild-type fibroblasts responding to 25 μm2 posts and compare their response to that of FAK-/- fibroblasts and fibroblasts with PMA treatment to stimulate protein kinase C (PKC) and the small g-protein Rac. FAK knockout cells modulated adhesion number and size in a similar way to cells on topography; that is, they used more, smaller adhesions, but migration was almost completely stalled demonstrating the importance of FAK signaling in contact guidance and adhesion turnover. Little similarity, however, was observed to PKC stimulated cells and cells on the topography. Interestingly, with PKC stimulation the cell nuclei became highly deformable bringing focus on these surfaces to the study of metastasis. Surfaces that aid the study of cellular migration are important in developing understanding of mechanisms of wound healing and repair in aligned tissues such as ligament and tendon.

JTD Keywords: Adhesion, Cell migration, Cell morphology, Focal adhesion kinase, Microstructures

Substrate topography, independently of substrate chemistry, has been reported to have significant effects on cell behaviour. Based on the use of fabrication techniques developed by the silicon microtechnology industry, numerous studies can now be found in the literature analyzing cell behaviour as to various micro- and nanofeatures such as lines, wells, holes and more. Most of these works have been found to relate the micro- and nano-sized topographical features with cell. orientation, migration, morphology and proliferation. In recent papers, even the influence of substrate nanotopography on cell gene expression and differentiation has been pointed out. However, despite the large number of papers published on this topic, significant general trends in cell behaviour are difficult to establish due to differences in cell type, substrate material, feature aspect-ratio, feature geometry and parameters measured. This paper intends to compile and review the relevant existing information on the behaviour of cells on micro- and nano-structured artificial substrates and analyze possible general behavioural trends.

JTD Keywords: Microstructure, Topography, Cell behaviour, Cell morphology, Cell orientation

Topographic micro and nanostructures can play an interesting role in cell behaviour when cells are cultured on these kinds of patterned substrates. It is especially relevant to investigate the influence of the nanometric dimensions topographic features on cell morphology, proliferation, migration and differentiation. To this end, some of the most recent fabrication technologies, developed for the microelectronics industry, can be used to produce well-defined micro and nanopatterns on biocompatible polymer substrates. In this work, osteoblast-like cells are grown on poly(methyl methacrylate) substrates patterned by nanoimprint lithography techniques. Examination of the cell-substrate interface can reveal important details about the cell morphology and the distribution of the focal contacts on the substrate surface. For this purpose, a combination of focused ion beam milling and scanning electron microscopy techniques has been used to image the cell-substrate interface. This technique, if applied to samples prepared by freeze-drying methods, allows high-resolution imaging of cross-sections through the cell and the substrate, where the interactions between the nanopatterned substrate, the cell and the extracellular matrix, which are normally hidden by the bulk of the cell, can be studied.

JTD Keywords: Electron microscopy, Interface, Nanotopography, Osteoblast, Adhesion molecule, Cell morphology