Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition.

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

The glycocalyx affects the mechanotransductive perception of the topographical microenvironment.

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.

3D Interfacial and Spatiotemporal Regulation of Human Neuroepithelial Organoids.

Neuroepithelial (NE) organoids with dorsal-ventral patterning provide a useful three-dimensional (3D) in vitro model to interrogate neural tube formation during early development of the central nervous system. Understanding the fundamental processes behind the cellular self-organization in NE organoids holds the key to the engineering of organoids with higher, more in vivo-like complexity. However, little is known about the cellular regulation driving the NE development, especially in the presence of interfacial cues from the microenvironment. Here a simple 3D culture system that allows generation and manipulation of NE organoids from human-induced pluripotent stem cells (hiPSCs), displaying developmental phases of hiPSC differentiation and self-aggregation, first into NE cysts with lumen structure and then toward NE organoids with floor-plate patterning, is established. Longitudinal inhibition reveals distinct and dynamic roles of actomyosin contractility and yes-associated protein (YAP) signaling in governing these phases. By growing NE organoids on culture chips containing anisotropic surfaces or confining microniches, it is further demonstrated that interfacial cues can sensitively exert dimension-dependent influence on luminal cyst and organoid morphology, successful floor-plate patterning, as well as cytoskeletal regulation and YAP activity. This study therefore sheds new light on how organoid and tissue architecture can be steered through intracellular and extracellular means.

Generation of Multicue Cellular Microenvironments by UV-photopatterning of Three-dimensional Cell Culture Substrates.

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems. This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Mechanical and Physical Regulation of Fibroblast-Myofibroblast Transition: From Cellular Mechanoresponse to Tissue Pathology.

Fibroblasts are cells present throughout the human body that are primarily responsible for the production and maintenance of the extracellular matrix (ECM) within the tissues. They have the capability to modify the mechanical properties of the ECM within the tissue and transition into myofibroblasts, a cell type that is associated with the development of fibrotic tissue through an acute increase of cell density and protein deposition. This transition from fibroblast to myofibroblast-a well-known cellular hallmark of the pathological state of tissues-and the environmental stimuli that can induce this transition have received a lot of attention, for example in the contexts of asthma and cardiac fibrosis. Recent efforts in understanding how cells sense their physical environment at the micro- and nano-scales have ushered in a new appreciation that the substrates on which the cells adhere provide not only passive influence, but also active stimulus that can affect fibroblast activation. These studies suggest that mechanical interactions at the cell-substrate interface play a key role in regulating this phenotype transition by changing the mechanical and morphological properties of the cells. Here, we briefly summarize the reported chemical and physical cues regulating fibroblast phenotype. We then argue that a better understanding of how cells mechanically interact with the substrate (mechanosensing) and how this influences cell behaviors (mechanotransduction) using well-defined platforms that decouple the physical stimuli from the chemical ones can provide a powerful tool to control the balance between physiological tissue regeneration and pathological fibrotic response.