Liver microsystems in vitro for drug response.

Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells.

Triggered immobilization of proteins in the plasma membrane of living cells into functional micropatterns is established by using an adaptor protein, which is comprised of an antiGFP nanobody fused to the HaloTag protein. Efficient in situ reorganization of the type I interferon receptor subunits as well as intact, fully functional signaling complexes in living cells are achieved by this method.

Facile and Rapid Microcontact Printing of Additive-Free Polydimethylsiloxane for Biological Patterning Diversity.

The development and application of micropatterning technology play a promising role in the manipulation of biological substances and the exploration of life sciences at the microscale. However, the universally adaptable micropatterning method with user-friendly properties for acceptance in routine laboratories remains scarce. Herein, a green, facile, and rapid microcontact printing method is reported for upgrading popularization and diversification of biological patterning. The three-step printing can achieve high simplicity and fidelity of additive-free polydimethylsiloxane (PDMS) micropatterning and chip fabrication within 8 min as well as keep their high stability and diversity. A detailed experimental report is provided to support the advanced microcontact printing method. Furthermore, the applications of easy-to-operate PDMS-patterned chips are extensively validated to complete microdroplet array assembly with spatial control, cell pattern formation with high efficiency and geometry customization, and microtissue assembly and biomimetic tumor construction on a large scale. This straightforward method promotes diverse micropatternings with minimal time, effort, and expertise and maximal biocompatibility, which might broaden its applications in interdisciplinary scientific communities. This work also offers an insight into the establishment of popularized and market-oriented microtools for biomedical purposes such as biosensing, organs on a chip, cancer research, and bioscreening.

Development of a Probability-Based In Vitro Eye Irritation Screening Platform.

Traditional eye irritation assessments, which rely on animal models or ex vivo tissues, face limitations due to ethical concerns, costs, and low throughput. Although numerous in vitro tests have been developed, none have successfully reconciled the need for high experimental throughput with the accurate prediction of irritation potential, attributable to the complexity of irritation mechanisms. Simple cell models, while suitable for high-throughput screening, offer limited mechanistic insights, contrasting with more physiologically relevant but less scalable complex organotypic corneal tissue constructs. This study presents a novel strategy to enhance the predictive accuracy of screening-compatible simple cell models in eye irritation testing. Our method combines the results of two in vitro assays-cell apoptosis and nociceptor (TRPV1) activation-using micropatterned chips to partition human corneal epithelial cells into numerous discrete small populations. Following exposure to test compounds, we measure apoptosis and nociceptor activation responses. The large datasets collected from the cell micropatterns facilitate binarization and statistical fitting to calculate a mathematical probability, which assesses the compound's potential to cause eye irritation. This method potentially enables the amalgamation of multiple mechanistic readouts into a singular index, providing a more accurate and reliable prediction of eye irritation potential in a format amenable to high-throughput screening.

Single-Cell Micropatterning by Non-fouling Hydrogels.

Micropatterned substrate is a unique method for studying cell biology at the single-cell level. Using photolithography to create binary patterns of cell-adherent peptide surrounding by non-fouling cell-repellent poly(ethylene glycol) (PEG) hydrogel, this patterning method allows for controlling cell attachment with desired sizes and shapes up to 19 days. Here we provide the detailed procedure of fabrication for such patterns. This method will allow monitoring of prolonged reaction of single cells such as cell differentiation upon induction or time-resolved apoptosis stimulated by drug molecules for cancer treatment.

Engineering spatial-organized cardiac organoids for developmental toxicity testing.

Emerging technologies in stem cell engineering have produced sophisticated organoid platforms by controlling stem cell fate via biomaterial instructive cues. By micropatterning and differentiating human induced pluripotent stem cells (hiPSCs), we have engineered spatially organized cardiac organoids with contracting cardiomyocytes in the center surrounded by stromal cells distributed along the pattern perimeter. We investigated how geometric confinement directed the structural morphology and contractile functions of the cardiac organoids and tailored the pattern geometry to optimize organoid production. Using modern data-mining techniques, we found that pattern sizes significantly affected contraction functions, particularly in the parameters related to contraction duration and diastolic functions. We applied cardiac organoids generated from 600 µm diameter circles as a developmental toxicity screening assay and quantified the embryotoxic potential of nine pharmaceutical compounds. These cardiac organoids have potential use as an in vitro platform for studying organoid structure-function relationships, developmental processes, and drug-induced cardiac developmental toxicity.

Self-Organization of Fibroblast-Laden 3D Collagen Microstructures from Inkjet-Printed Cell Patterns.

One of the major challenges encountered in engineering complex tissues in vitro is to increase levels of complexity at the micron scale in 3D structures. Here, a strategy to create self-organized 3D collagen microstructures by 2D micropatterning of fibroblasts is developed. Drop-on-demand inkjet printing is used to pattern fibroblast cells on a collagen substrate in pre-designed patterns and with controlled density. It is found that cell-to-ECM interaction promotes cellular self-organization of 3D microstructures on collagen hydrogel, whereas the formation of 3D microstructure is inhibited by disruption of actin polymerization. Using this phenomena, the controlled sizes and morphologies of the 3D collagen microstructures is demonstrated by manipulating the designs of cell patterns and the density of cells. Finally, this technique is applied to build a human skin model with papillary microstructures at the dermo-epidermal junction. This approach to create 3D cell-laden collagen microstructures by cell patterning provides a simple and powerful way to mimic the structures and functions of complex tissues and organs, and can make a contribution to reduce the gap between the human body and in vitro tissue models.

Photo-Cleavable Peptide-Poly(Ethylene Glycol) Conjugate Surfaces for Light-Guided Control of Cell Adhesion.

Photo-responsive cell attachment surfaces can simplify patterning and recovery of cells in microdevices for medicinal and pharmaceutical research. We developed a photo-responsive surface for controlling the attachment and release of adherent cells on a substrate under light-guidance. The surface comprises a poly(ethylene glycol) (PEG)-based photocleavable material that can conjugate with cell-adhesive peptides. Surface-bound peptides were released by photocleavage in the light-exposed region, where the cell attachment was subsequently suppressed by the exposed PEG. Simultaneously, cells selectively adhered to the peptide surface at the unexposed microscale region. After culture, the adhered and spread cells were released by exposure to a light with nontoxic dose level. Thus, the present surface can easily create both cell-adhesive and non-cell-adhesive regions on the substrate by single irradiation of the light pattern, and the adhered cells were selectively released from the light-exposed region on the cell micropattern without damage. This study shows that the photo-responsive surface can serve as a facile platform for the remote-control of patterning and recovery of adherent cells in microdevices.

Manipulation of living cells with 450 nm laser photobiomodulation.

Increasing studies demonstrated that photobiomodulation (PBM) influenced specific biological effects in cells, tissues and organs, and these effects rely on the production of light irradiation. In this study, we aimed to precisely manipulate the spatial arrangement of adhesion cells in a traditional culture condition with 450 nm low intensity laser. Through 450 nm laser PBM, the adhesion of the cultured cells was significantly improved and resisted the digestion of 0.1% trypsin. Combined with a computer aided design system (CAD) and computer numerical control (CNC) system, the designed laser irradiation pattern induced the specific cell micropattern in the culture dish. RNA sequencing and biochemical experiments confirmed that the 450 nm laser prompted low-density lipoprotein (LDL) bonding to the cell surface and induced lipid peroxidation, which crosslinked and modified the protein molecules on the irradiated cell surface. In this way, the peroxidation product-modified proteins resisted trypsin proteolysis, ultimately leading to a differential detachment between the irradiated and non-irradiated cells under trypsin treatment. This convenient method did not require special biomaterial processing, has no impact on cell viability and functions, and required no changes to the conventional cell culture conditions. The photo-induced cell capturing is a great complement to existing tools by providing spatial resolution.

Substrate mechanics controls adipogenesis through YAP phosphorylation by dictating cell spreading.

The mechanoregulated proteins YAP/TAZ are involved in the adipogenic/osteogenic switch of mesenchymal stem cells (MSCs). MSC fate decision can be unbalanced by controlling substrate mechanics, in turn altering the transmission of tension through cell cytoskeleton. MSCs have been proposed for orthopedic and reconstructive surgery applications. Thus, a tight control of their adipogenic potential is required in order to avoid their drifting towards fat tissue. Substrate mechanics has been shown to drive MSC commitment and to regulate YAP/TAZ protein shuttling and turnover. The mechanism by which YAP/TAZ co-transcriptional activity is mechanically regulated during MSC fate acquisition is still debated. Here, we design few bioengineering tools suited to disentangle the contribution of mechanical from biological stimuli to MSC adipogenesis. We demonstrate that the mechanical repression of YAP happens through its phosphorylation, is purely mediated by cell spreading downstream of substrate mechanics as dictated by dimensionality. YAP repression is sufficient to prompt MSC adipogenesis, regardless of a permissive biological environment, TEAD nuclear presence or focal adhesion stabilization. Finally, by harnessing the potential of YAP mechanical regulation, we propose a practical example of the exploitation of adipogenic transdifferentiation in tumors.

A Micropatterning Strategy to Study Nuclear Mechanotransduction in Cells.

Micropatterning techniques have been widely used in biology, particularly in studies involving cell adhesion and proliferation on different substrates. Cell micropatterning approaches are also increasingly employed as in vitro tools to investigate intracellular mechanotransduction processes. In this report, we examined how modulating cellular shapes on two-dimensional rectangular fibronectin micropatterns of different widths influences nuclear mechanotransduction mediated by emerin, a nuclear envelope protein implicated in Emery-Dreifuss muscular dystrophy (EDMD). Fibronectin microcontact printing was tested onto glass coverslips functionalized with three different silane reagents (hexamethyldisilazane (HMDS), (3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) using a vapor-phase deposition method. We observed that HMDS provides the most reliable printing surface for cell micropatterning, notably because it forms a hydrophobic organosilane monolayer that favors the retainment of surface antifouling agents on the coverslips. We showed that, under specific mechanical cues, emerin-null human skin fibroblasts display a significantly more deformed nucleus than skin fibroblasts expressing wild type emerin, indicating that emerin plays a crucial role in nuclear adaptability to mechanical stresses. We further showed that proper nuclear responses to forces involve a significant relocation of emerin from the inner nuclear envelope towards the outer nuclear envelope and the endoplasmic reticulum membrane network. Cell micropatterning by fibronectin microcontact printing directly on HMDS-treated glass represents a simple approach to apply steady-state biophysical cues to cells and study their specific mechanobiology responses in vitro.

Photocleavable Peptide-Poly(2-hydroxyethyl methacrylate) Hybrid Graft Copolymer via Postpolymerization Modification by Click Chemistry To Modulate the Cell Affinities of 2D and 3D Materials.

Controlling the surface properties of engineered materials to enhance or reduce their cellular affinities remains a significant challenge in the field of biomaterials. We describe a universal technique for modulating the cytocompatibilities of two-dimensional (2D) and three-dimensional (3D) materials using a novel photocleavable peptide-grafted poly(2-hydroxyethyl methacrylate) (PHEMA) hybrid. The reversible addition-fragmentation chain transfer copolymerization of HEMA and propargyl acrylate was successfully controlled. The resultant alkyne-containing PHEMA was then used to modify the azide-terminated oligopeptides [Arg-Gly-Asp-Ser (RGDS)] with a photolabile 3-amino-3-(2-nitrophenyl)propanoic acid moiety via the copper-catalyzed alkyne-azide click chemistry. This strategy was readily used to decorate the surfaces of both hydrophilic and hydrophobic materials with RGDS peptides due to the high film-forming abilities of the PHEMA unit. The resultant thin film acted as an effective scaffold for improving cell adhesion and growth of NIH/3T3 fibroblasts and MC3T3-E1 osteoblast-like cells in vitro. In addition, UV irradiation of the surface led to the detachment of cells from the material surface accompanied by the photocleavage of RGDS grafts and enabled the 2D-patterning of cells and cell sheet engineering. The applicability of this system to 3D materials was investigated, and the cell adhesion was remarkably enhanced on a 3D-printed poly(lactic acid) object. This facile, biocompatible, and photoprocessable peptide-vinyl polymer hybrid system is valuable for its ability to advance the fields of tissue engineering, cell chips, and regenerative medicine.

Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS.

Microcontact printing (µCPr) is one of the most popular techniques used for cell micropatterning. In conventional µCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique--microcontact peeling (µCPe)--that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS. This technique employs microfeatured materials with a relatively high surface energy such as copper, instead of using a microfeatured PDMS stamp, to peel off a cell-adhesive layer present on the surface of substrates. Consequently, cell-nonadhesive substrates are exposed at the specific surface that undergoes the physical contact with the microfeatured material. Thus, although µCPe and µCPr are apparently similar, the former does not comprise a process of transferring biomolecules through hydrophobic PDMS. © 2017 by John Wiley & Sons, Inc.

Cell-Shaping Micropatterns for Quantitative Super-Resolution Microscopy Imaging of Membrane Mechanosensing Proteins.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Stencil Micropatterning for Spatial Control of Human Pluripotent Stem Cell Fate Heterogeneity.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns, although this requires the presentation of spatial environmental cues, i.e., biochemical and mechanical gradients. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Here, we describe stencil micropatterning as a simple and robust method to generate hPSC micropatterns for controlling hPSC differentiation patterns. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where the underlying extracellular matrix and hPSCs can access and attach to the substrate. This confers the unique capability of stencil micropatterning to work with a variety of culture substrates and extracellular matrices for optimal hPSC culture. We present the detailed steps of stencil micropatterning to successfully generate hPSC micropatterns, which can be used to investigate how spatial polarization of cell adhesion results in cell fate heterogeneity.

Facile endothelial cell micropatterning induced by reactive oxygen species on polydimethylsiloxane substrates.

Sophisticated cell pattern provides unique cellular assay platform for studying cell to cell interaction, cellular differentiation and signaling, high-throughput cell response to chemicals. In this study, we demonstrated reactive oxygen species (ROS) mediated endothelial cell micropatterning on a polydimethylsiloxane (PDMS) substrate. The exposure of UV/O radiation led to the formation of ROS on the surface of PDMS, which could selectively prevent adhesion of endothelial cells. The degree of ROS amount was monitored according to the UV/O irradiation time, and at least 36 µM of ROS resulted in the precise cellular micropattern on the PDMS. The presence of ROS affected not only cellular detachment from the substrate, but also endothelial cell morphology such as cell spreading area, confluence, nuclear area and nuclear inverse aspect ratio. In addition, we could observe that the actin cytoskeleton of cells was also constricted due to ROS, thereby minimizing the focal adhesion area of vinculin. Compared with previously reported methods which use chemical treatment or nano/microstructure on the substrate, the proposed methodology is quite simple, accurate, and harmless to the patterned endothelial cells.

Large area micropatterning of cells on polydimethylsiloxane surfaces.

BACKGROUND: Precise spatial control and patterning of cells is an important area of research with numerous applications in tissue engineering, as well as advancing an understanding of fundamental cellular processes. Poly (dimethyl siloxane) (PDMS) has long been used as a flexible, biocompatible substrate for cell culture with tunable mechanical characteristics. However, fabrication of suitable physico-chemical barriers for cells on PDMS substrates over large areas is still a challenge. RESULTS: Here, we present an improved technique which integrates photolithography and cell culture on PDMS substrates wherein the barriers to cell adhesion are formed using the photo-activated graft polymerization of polyethylene glycol diacrylate (PEG-DA). PDMS substrates with varying stiffness were prepared by varying the base to crosslinker ratio from 5:1 to 20:1. All substrates show controlled cell attachment confined to fibronectin coated PDMS microchannels with a resistance to non-specific adhesion provided by the covalently immobilized, hydrophilic PEG-DA. CONCLUSIONS: Using photolithography, it is possible to form patterns of high resolution stable at 37°C over 2 weeks, and microstructural complexity over large areas of a few cm(2). As a robust and scalable patterning method, this technique showing homogenous and stable cell adhesion and growth over macroscales can bring microfabrication a step closer to mass production for biomedical applications.

Dynamic photochemical lipid micropatterning for manipulation of nonadherent mammalian cells.

Cell micropatterning methods with stimuli-responsive dynamic surfaces are getting a lot of attention in a wide variety of research fields, ranging from cell engineering to fundamental studies in cell biology. The surface of a slide coated with photo-cleavable poly(ethylene glycol) (PEG)-lipid can be used to spatiotemporally control cell immobilization and release by light irradiation. On the basis of this surface, it is easy to design simple methods for making a fine micropattern of any kind of cell. Furthermore, target cells can be selectively and rapidly released from this surface by light irradiation. In this review, we first describe how to obtain the photo-cleavable PEG-lipid from commercially available compounds through a facile four-step synthesis. Next, as a cell-patterning method, the protocols of coating substrates with the PEG-lipid, irradiating a pattern of light onto the coated substrate, and loading cells onto the irradiated surface are described. These protocols require no expensive equipment and potentially apply to any substrates that can adsorb serum albumin or chemically expose amine moieties on their surfaces. Finally, as an advanced method, cell release from the PEG-lipid surface in microfluidic devices is introduced. We also discuss the advantages and the possible applications of the present dynamic cell-patterning method.

Development of micropatterned cell-sensing surfaces.

Microfabricated surfaces have been widely utilized for defining adhesion of single cells or groups of cells of various kinds. Beyond simple control of cell attachment, it is often important to monitor the molecules released by cells. Co-immobilizing miniature sensors alongside cells enables more sensitive detection of secreted factors and may allow for such detection to happen within the context of local microenvironment. Methods for interfacing cells and sensors are central to the notion of local in situ detection of cell function. This chapter describes the use of hydrogel photolithography for integrating cells and sensing elements on culture surfaces. Two types of micropatterned sensing surfaces are described: (1) arrays of microwells for single cell capture that contain antibodies against secreted proteins and (2) entrapment of enzymes inside hydrogel microstructures for local detection of cell metabolism. In both cases, poly(ethylene glycol) hydrogel lithography was employed to control cell attachment, in the second approach hydrogel structures also carried enzymes and functioned as sensors. The development of robust cell/sensor interfaces has implications for diagnostics, tissue engineering, and drug screening.

Directing GPCR-transfected cells and neuronal projections with nano-scale resolution.

Surface modification technology has made significant advances in recent years towards the miniaturization and organization of traditional cell culture systems. However, the capability of directing transfected cells and neuronal connections to probe small structures such as spines is still under development. In the current work, interactions of different micropatterned substrates with HEK 293, CF10 cell lines, and primary neuronal cultures are evaluated. Using conventional and confocal fluorescence microscopies, several morphological and behavioral aspects of all three cell types were investigated. The immortalized cell lines were able to attach to the substrate and interact with neighboring cells. Similarly, cortical neurons formed connections guided by the micropatterns. Transfection of HEK 293 or CF10 cell lines with specific members of the G protein-coupled receptor family did not alter the behavior of these cells in the micropatterns. On the other hand, neuronal projections were efficiently isolated by the patterns, simplifying the localization of spines with nano-scale resolution probed by atomic force microscopy. This work presents a valuable approach to isolate cells or to constrain important cell structures to grow along a desired pattern, thus facilitating advanced biological studies.