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Refereed/Peer-reviewed The ability to create complex three-dimensional cellular models that can effectively replicate the structure and function of human organs and tissues in vitro has the potential to revolutionize medicine. Such models could facilitate the interrogation of developmental and disease processes underpinning fundamental discovery science, vastly accelerate drug development and screening, or even be used to create tissues for implantation into the body. Realization of this potential, however, requires the recreation of complex biochemical, biophysical, and cellular patterns of 3D tissues and remains a key challenge in the field. Recent advances are being driven by improved knowledge of tissue morphogenesis and architecture and technological developments in bioengineering and materials science that can create the multidimensional and dynamic systems required to produce complex tissue microenvironments. In this article, we discuss challenges for in vitro models of tissues and organs and summarize the current state-of-the art in biomaterials and bioengineered systems that aim to address these challenges. This includes both top-down technologies, such as 3D photopatterning, magnetism, acoustic forces, and cell origami, as well as bottom-up patterning using 3D bioprinting, microfluidics, cell sheet technology, or composite scaffolds. We illustrate the varying ways that these can be applied to suit the needs of different tissues and applications by focussing on specific examples of patterning the bone-tendon interface, kidney organoids, and brain cancer models. Finally, we discuss the challenges and future prospects in applying materials science and bioengineering to develop high-quality 3D tissue structures for in vitro studies. Published under an exclusive license by AIP Publishing.

Cells sense and respond to a variety of physical cues from their surrounding microenvironment, and these are interpreted through mechanotransductive processes to inform their behavior. These mechanisms have particular relevance to stem cells, where control of stem cell proliferation, potency, and differentiation is key to their successful application in regenerative medicine. It is increasingly recognized that surface micro- and nanotopographies influence stem cell behavior and may represent a powerful tool with which to direct the morphology and fate of stem cells. Current progress toward this goal has been driven by combined advances in fabrication technologies and cell biology. Here, the capacity to generate precisely defined micro- and nanoscale topographies has facilitated the studies that provide knowledge of the mechanotransducive processes that govern the cellular response as well as knowledge of the specific features that can drive cells toward a defined differentiation outcome. However, the path forward is not fully defined, and the "bumpy road" that lays ahead must be crossed before the full potential of these approaches can be fully exploited. This review focuses on the challenges and opportunities in applying micro- and nanotopographies to dictate stem cell fate for regenerative medicine. Here, key techniques used to produce topographic features are reviewed, such as photolithography, block copolymer lithography, electron beam lithography, nanoimprint lithography, soft lithography, scanning probe lithography, colloidal lithography, electrospinning, and surface roughening, alongside their advantages and disadvantages. The biological impacts of surface topographies are then discussed, including the current understanding of the mechanotransductive mechanisms by which these cues are interpreted by the cells, as well as the specific effects of surface topographies on cell differentiation and fate. Finally, considerations in translating these technologies and their future prospects are evaluated.

Cells are able to perceive complex mechanical cues across both the micro- and nanoscale which can influence their development. Whilst causative effects between surface topography and cellular function can be demonstrated, the variability in materials used in this screening process makes it difficult to discern whether the observed phenotypic changes are indeed a result of topographical cues alone, or the inherent difference in material properties. A novel approach to directly imprint both micro- and nanoscaled topographical features into the base of conventional cell cultureware is thus developed, facilitating its compatibility with standard biological techniques and methods of analysis. The utility of this technology is demonstrated by performing high-throughput screening across five distinct cell types to interrogate the effects of 12 surface topographies, exemplifying unique cell specific responses to both behavior and cell morphological characteristics. The ability of this technology to underpin new insights into how surface topographies can regulate key image descriptors to drive cell fate determination is further demonstrated. These findings will inform the future development of advanced micro- and nanostructured cell culture substrates that can regulate cell behavior and fate determination across the life sciences, including fundamental cell biology, drug screening, and cell therapy.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.

Cues from the extracellular environment, including physical stimuli, are well known to affect mesenchymal stem cell (MSC) properties in terms of proliferation and differentiation. Many therapeutic strategies are now targeting this knowledge to increase the efficacy of cell therapies, typically employed to repair tissue functions in the event of injury, either by direct engraftment into the target tissue or differentiation into mature tissues. However, it is now envisioned that harnessing the repertoire of factors secreted by MSCs (termed the secretome) may provide an alternate to these cell therapies. Of current interest are both direct protein secretions and two major subpopulations of bioactive extracellular vesicles (EVs), namely exosomes and microvesicles. EVs released by MSCs are reflective of their cells of origin, able to impact upon the activities of other cells in the local microenvironment, making the rational design of MSC paracrine activities an encouraging strategy to reproducibly modulate cell therapies. The precise mechanisms by which the secretome is modulated by the microenvironment, however, remain elusive. Controlling MSC growth conditions with oxygen tension, growth factor composition, and mechanical properties may serve to directly influence paracrine activity. Our growing understanding implicates components of the mechanotransduction machinery in translating both mechanical and chemical cues from the environment into alterations in gene regulation and varied paracrine activity. As technologies are developed to manufacture MSCs, advances in bioengineering and novel insight of how the extracellular environment affects MSC paracrine activity will play a pivotal role in the generation of widespread, successful, clinical MSC therapies.

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.