Biofunctionalized Patterned Polymer Brushes via Thiol-Ene Coupling for the Control of Cell Adhesion and the Formation of Cell Arrays

Thiol–ene radical coupling is increasingly used for the biofunctionalization of biomaterials. Thiol–ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions with peptides or proteins and the patterning of cells, such as reactivity in physiological conditions and photoactivation. In this work, we synthesized alkene-functionalized (allyl and norbornene residues) antifouling polymer brushes (based on poly­(oligoethylene glycol methacrylate)) and studied thiol–ene coupling with a series of thiols including cell adhesive peptides RGD and REDV. The adhesion of umbilical vein endothelial cells (HUVECs) to these interfaces was studied and highlighted the absence of specific integrin engagement to REDV, in contrast to the high level of cell spreading observed on RGD-functionalized polymer brushes. This revealed that α₄β₁ integrins (binding to REDV sequences) are not sufficient on their own to sustain HUVEC spreading, in contrast to α_vβ₃ and α₅β₁ integrins. In addition, we photopatterned peptides at the surface of poly­(oligoethylene glycol methacrylate) (POEGMA) brushes and characterized the quality of the resulting arrays by epifluorescence microscopy and atomic force microscopy (AFM). This allowed the formation of cell patterns and demonstrated the potential of thiol–ene based photopatterning for the design of cell microarrays

Photoconfigurable, Cell-Remodelable Disulfide Cross-linked Hyaluronic Acid Hydrogels.

Dynamic photoresponsive synthetic hydrogels offer important advantages for biomaterials design, from the ability to cure hydrogels and encapsulate cells in situ to the light-mediated control of cell-spreading and tissue formation. We report the facile and effective photocuring and photoremodeling of disulfide-cross-linked hyaluronic acid hydrogels, based on photo-oxidation of corresponding thiol residues and their radical-mediated photodegradation. We find that the mechanical properties of disulfide hydrogels and the extent of their photoremodeling can be tuned by controlling the photo-oxidation and photodegradation reactions, respectively. This enables not only the photopatterning of the mechanical properties of hydrogels but also their self-healing and photomediated healing. Finally, we demonstrate the ability to encapsulate mesenchymal stromal cells within these materials and to regulate their protrusion and spreading in 3D matrices by controlling the mechanical properties of the disulfide networks. Therefore, synthetically accessible photoconfigurable disulfide hydrogels offer interesting opportunities for the design of soft biomaterials and the regulation of cell encapsulation and matrix remodeling for tissue engineering

Contractile myosin rings and cofilin-mediated actin disassembly orchestrate ECM nanotopography sensing

The nanotopography and nanoscale geometry of the extra-cellular matrix (ECM) are important regulators of cell adhesion, motility and fate decision. However, unlike the sensing of matrix mechanics and ECM density, the molecular processes regulating the direct sensing of the ECM nanotopography and nanoscale geometry are not well understood. Here, we use nanotopographical patterns generated via electrospun nanofibre lithography (ENL) to investigate the mechanisms of nanotopography sensing by cells. We observe the dysregulation of actin dynamics, resulting in the surprising formation of actin foci. This alteration of actin organisation is regulated by myosin contractility but independent of adapter proteins such as vinculin. This process is highly dependent on differential integrin expression as β3 integrin expressing cells, more sensitive to nanopattern dimensions than β1 integrin expressing cells, also display increased perturbation of actin assembly and actin foci formation. We propose that, in β3 integrin expressing cells, contractility results in the destabilisation of nanopatterned actin networks, collapsing into foci and sequestering regulators of actin dynamics such as cofilin that orchestrate disassembly. Therefore, in contrast to the sensing of substrate mechanics and ECM ligand density, which are directly orchestrated by focal adhesion assembly, we propose that nanotopography sensing is regulated by a long-range sensing mechanism, remote from focal adhesions and mediated by the actin architecture

Surface-Initiated Poly(oligo(2-alkyl-2-oxazoline)methacrylate) Brushes

Polymer brushes are particularly performant antifouling coatings, owing to their high grafting density that prevents unwanted biomacromolecules to diffuse through the coating and adhere to the underlying substrate. In addition to this structural feature, polymer brushes require a relatively high level of hydrophilicity and a globally neutral structure to display ultrahigh protein resistance. Poly­(2-alkyl-2-oxaolines) are attractive building blocks for such coatings as they can display relatively high hydrophilicity, owing to their amide repeat units, but can also be side-chain and end-chain functionalized relatively readily. However, poly­(2-alkyl-2-oxazolines) have not yet been introduced through a radical-mediated grafting from polymer brush structure that would confer the high level of grafting density that is the hallmark of highly protein resistant brushes. Here, we present the formation of a series of poly­(oligo­(2-alkyl-2-oxazoline)­methacrylate) brushes generated via a grafting from approach, via atom transfer radical polymerization. We characterize the chemical structure of the resulting coatings via ellipsometry, Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy. We show that allyl end groups can be introduced as a side chain of these brushes to allow functionalization via thiol-ene chemistry. We demonstrate the excellent protein resistance of these coatings in single protein solutions as well as serum solutions at concentration typically used for cell culture. Finally, we demonstrate the feasibility of using these brushes for the micropatterning of cells and the generation of cell-based assays

Vascularised cardiac spheroids-on-a-chip for testing the toxicity of therapeutics.

Microfabricated organ-on-a-chips are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but recreating microvascularised tissue models remains difficult in many cases. This is particularly relevant to mimic the systemic delivery of therapeutics, to capture the complex multi-step processes associated with trans-endothelial transport or diffusion, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac spheroids embedded in microfluidic chips. Different protocols used for embedding spheroids within vascularised multi-compartment microfluidic chips were investigated first to identify the importance of the spheroid processing, and co-culture with pericytes on the integration of the spheroid within the microvascular networks formed. The architecture of the resulting models, the expression of cardiac and endothelial markers and the perfusion of the system was then investigated. This confirmed the excellent stability of the vascular networks formed, as well as the persistent expression of cardiomyocyte markers such as cTNT and the assembly of striated F-actin, myosin and α-actinin cytoskeletal networks typically associated with contractility and beating. The ability to retain beating over prolonged periods of time was quantified, over 25 days, demonstrating not only perfusability but also functional performance of the tissue model. Finally, as a proof-of-concept of therapeutic testing, the toxicity of one therapeutic associated with cardiac disfunction was evaluated, identifying differences between direct in vitro testing on suspended spheroids and vascularised models

The impact of pericytes on the stability of microvascular networks in response to nanoparticles.

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in-vitro models and organ-on-chip designs. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. The use of such co-culture for the testing of therapeutics and nanoparticle safety is increasingly considered for the validation of therapeutic strategies. This report presents the use of a microfluidic model for such applications. Interactions between endothelial cells and pericytes are first explored. We identify basal conditions required to form stable and reproducible endothelial networks. We then investigate interactions between endothelial cells and pericytes via direct co-culture. In our system, pericytes prevented vessel hyperplasia and maintained vessel length in prolonged culture (> 10 days). In addition, these vessels displayed barrier function and expression of junction markers associated with vessel maturation, including VE-cadherin, β-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and prevented vessel regression, in contrast to the striking dissociation of networks in endothelial monocultures. This response was also observed when endothelial/pericyte co-cultures were exposed to high concentrations of moderately toxic cationic nanoparticles used for gene delivery. This study highlights the importance of pericytes in protecting vascular networks from stress and external agents and their importance to the design of advanced in-vitro models, including for the testing of nanotoxicity, to better recapitulate physiological response and avoid false positives

Development of an in vitro microfluidic model to study the role of microenvironmental cells in oral cancer metastasis.

Metastasis occurs when cancer cells leave the primary tumour and travel to a secondary site to form a new lesion. The tumour microenvironment (TME) is recognised to greatly influence this process, with for instance the vascular system enabling the dissemination of the cells into other tissues. However, understanding the exact role of these microenvironmental cells during metastasis has proven challenging. Indeed, in vitro models often appear too simplistic, and the study of the interactions between different cell types in a 3D space is limited. On the other hand, even though in vivo models incorporate the TME, observing cells in real-time to understand their exact role is difficult. Horizontal compartmentalised microfluidic models are a promising new platform for metastasis studies. These devices, composed of adjacent microchannels, can incorporate multiple cell types within a 3D space. Furthermore, the transparency and thickness of these models also enables high quality real-time imaging to be performed. This paper demonstrates how these devices can be successfully used for oral squamous cell carcinoma (OSCC) metastasis studies, focusing on the role of the vascular system in this process. Conditions for co-culture of OSCC cells and endothelial cells have been determined and staining protocols optimised. Furthermore, several imaging analysis techniques for these models are described, enabling precise segmentation of the different cell types on the images as well as accurate assessment of their phenotype. These methods can be applied to any study aiming to understand the role of microenvironmental cell types in cancer metastatic dissemination, and overcome several challenges encountered with current in vitro and in vivo models. Hence, this new in vitro model capable of recapitulating important aspects of the cellular complexity of human metastatic dissemination can ultimately contribute to replacing animal studies in this field

Stem Cell Expansion and Fate Decision on Liquid Substrates Are Regulated by Self-Assembled Nanosheets

S.D.C. thanks the Institute of Bioengineering for a studentship. L.P. thanks the China Scholarship Council for a studentship (201708060335). J.E.G. and D.K. thank the Leverhulme Trust Foundation for financial support (RPG-2017-229, Grant 69241)

Differential integrin expression regulates cell sensing of the matrix nanoscale geometry

Funding from Institute of Bioengineering (Queen Mary, University of London) and from the Engineering and Physical Sciences Research Council (EP/J501360/1) is gratefully acknowledged