A simple soft lithographic route to fabrication of poly(ethylene glycol) microstructures for protein and cell patterning

We present a simple, direct soft lithographic method to fabricate poly(ethylene glycol) (PEG) microstructures for protein and cell patterning. This lithographic method involves a molding process in which a uniform PEG film is molded with a patterned polydimethylsiloxane stamp by means of capillary force. The patterned surfaces created by this method provide excellent resistance towards non-specific protein and cell adsorption. The patterned substrates consist of two regions: the molded PEG surface that acts as a resistant layer and the exposed substrate surface that promotes protein or cell adsorption. A notable finding here is that the substrate surface can be directly exposed during the molding process due to the ability to control the wetting properties of the polymer on the stamp, which is a key factor to patterning proteins and cells.     

Selective Surface Modification in Silicon Microfluidic Channels for Micromanipulation of Biological Macromolecules

Interactions between biological macromolecules and micrometer- and sub-micrometer-scale surface structures are directly influenced by the surface wettability, chemical reactivity and surface charge. Understanding these interactions is crucial for developing integrated microsystems for biological and biomedical processing and analysis. We report development of selective surface modification techniques based on microcontact printing and polyelectrolyte adsorption. These techniques were applied to lithographically patterned silicon microfluidic channels and flat silicon substrates to create surface microstructures with contrasting wetting properties and surface charges. These controls enabled us to devise various techniques for controlled loading and processing of biomaterials in the channels. Solutions containing long chain biological macromolecules DNA and microtubules were directly loaded into the microchannels by using a micromanipulator/microinjector system. Structural arrangements of these linear macromolecules, which were probed by using fluorescence and laser scanning confocal microscopy, were found to be quite different from bulk solutions. As expected, the filamentous molecules were observed to align linearly along the channels, with the degree of alignment dependent on channel width as well as the length of the molecule. This molecular alignment, which is induced by both the surface confinement effect and capillary flow during sample loading, may be used to enhance processing of biological materials in silicon biomedical microdevices. It also opens up the possibility of carrying out direct combinatorial structural characterization of proteins in the microchannels utilizing X-ray diffraction, which so far has not been possible.     

Chemical and Topographical Surface Modification for Control of Central Nervous System Cell Adhesion

We describe methods of fine scale chemical and topographical patterning of silicon substrates and the selected attachment and growth of central nervous system cells in culture. We have used lithography and microcontact printing to pattern surfaces with self-assembled monolayers and proteins. Chemical patterns can be created that localize and guide the growth of cells on the surfaces. Self-assembled surface texturing with structures at the tens of nanometers scale and lithographic based methods at the micrometer scale have been used to produce a variety of surface topographical features. These experiments suggest that surface texture at the scale of tens of nanometers to micrometers can influence the attachment of these cells to a surface and can be used as a mechanism of isolating cells to a particular area on a silicon substrate.     

High-throughput screening of gene function in stem cells using clonal microarrays

We describe a microarray-based approach for the high-throughput screening of gene function in stem cells and demonstrate the potential of this method by growing and isolating clonal populations of both adult and embryonic neural stem cells. Clonal microarrays are constructed by seeding a population of cells at clonal density on micropatterned surfaces generated using soft lithographic microfabrication techniques. Clones of interest can be isolated after assaying in parallel for various cellular processes and functions, including proliferation, signal transduction, and differentiation. We demonstrate the compatibility of the technique with both gain- and loss-of-function studies using cell populations infected with cDNA libraries or DNA constructs that induce RNA interference. The infection of cells with a library prior to seeding and the compact but isolated growth of clonal cell populations will facilitate the screening of large libraries in a wide variety of mammalian cells, including those that are difficult to transfect by conventional methods.     

Functionalized hydrogel surfaces for the patterning of multiple biomolecules

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 2007.     

Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate

Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Compartmentalized Devices as Tools for Investigation of Human Brain Network Dynamics

Neuropsychiatric disorders have traditionally been difficult to study due to the complexity of the human brain and limited availability of human tissue. Induced pluripotent stem (iPS) cells provide a promising avenue to further our understanding of human disease mechanisms, but traditional 2D cell cultures can only provide a limited view of the neural circuits. To better model complex brain neurocircuitry, compartmentalized culturing systems and 3D organoids have been developed. Early compartmentalized devices demonstrated how neuronal cell bodies can be isolated both physically and chemically from neurites. Soft lithographic approaches have advanced this approach and offer the tools to construct novel model platforms, enabling circuit-level studies of disease, which can accelerate mechanistic studies and drug candidate screening. In this review, we describe some of the common technologies used to develop such systems and discuss how these lithographic techniques have been used to advance our understanding of neuropsychiatric disease. Finally, we address other in vitro model platforms such as 3D culture systems and organoids and compare these models with compartmentalized models. We ask important questions regarding how we can further harness iPS cells in these engineered culture systems for the development of improved in vitro models. Developmental Dynamics 248:65–77, 2019. © 2018 Wiley Periodicals, Inc.     

Substrate-mediated delivery from self-assembled monolayers: effect of surface ionization, hydrophilicity, and patterning

Gene transfer has many potential applications in basic and applied sciences. In vitro, DNA delivery can be enhanced by increasing the concentration of DNA in the cellular microenvironment through immobilization of DNA to a substrate that supports cell adhesion. Substrate-mediated delivery describes the immobilization of DNA, complexed with cationic lipids or polymers, to a biomaterial or substrate. As surface properties are critical to the efficiency of the surface delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to correlate surface chemistry of the substrate to binding, release, and transfection of non-specifically immobilized complexes. Surface hydrophobicity and ionization were found to mediate both DNA complex immobilization and transfection, but had no effect on complex release. Additionally, SAMs were used in conjunction with soft lithographic techniques to imprint substrates with specific patterns, resulting in patterned DNA complex deposition and transfection, with transfection efficiencies in the patterns nearing 40%. Controlling the interactions between complexes and substrates, with the potential for patterned delivery, can be used to locally enhance or regulate gene transfer, with applications to tissue engineering scaffolds and transfected cell arrays.     

Microenvironments engineered by inkjet bioprinting spatially direct adult stem cells toward muscle- and bone-like subpopulations

In vivo, growth factors exist both as soluble and as solid-phase molecules, immobilized to cell surfaces and within the extracellular matrix. We used this rationale to develop more biologically relevant approaches to study stem cell behaviors. We engineered stem cell microenvironments using inkjet bioprinting technology to create spatially defined patterns of immobilized growth factors. Using this approach, we engineered cell fate toward the osteogenic lineage in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary muscle-derived stem cells (MDSCs) isolated from adult mice. This patterning approach was conducive to patterning the MDSCs into subpopulations of osteogenic or myogenic cells simultaneously on the same chip. When cells were cultured under myogenic conditions on BMP-2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time-lapse microscopy was used to visualize the formation of multinucleated myotubes, and immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off BMP-2 pattern. This work provides proof-of-concept for engineering spatially controlled multilineage differentiation of stem cells using patterns of immobilized growth factors. This approach may be useful for understanding cell behaviors to immobilized biological patterns and could have potential applications for regenerative medicine.     

Microfluidic patterning of cells in extracellular matrix biopolymers: effects of channel size,cell type,and matrix composition on pattern integrity

The organization of cells within an extracellular matrix is critical to promote appropriate cellular interactions and tissue function in vivo. The ability to design and create biologically relevant cellular arrangements via microfluidic patterning on surfaces provides new capabilities for tissue engineering and biomimetics. The purpose of this article is to describe techniques using microfluidic patterning of three-dimensional biopolymer matrices to improve cellular pattern integrity and to provide microscale control over cellular microenvironments. Results demonstrated that the incorporation of extracellular matrix biopolymers in cell microfluidic patterning results in a more stable pattern of adherent human endothelial cells than patterning without matrix components after several days in vitro. This may be important for carrying out long-term biological experiments and tissue engineering in vitro. Moreover, chemical components in the patterned biopolymer matrices, such as collagen, chitosan, and fibronectin, influenced the ability of the matrices to control cell migration and pattern stability over time. Thus, microfluidic patterning of cells in extracellular matrix biopolymers was shown to be useful in patterning multiple cell types in well-defined three-dimensional geometries.     

A simple cell patterning method using magnetic particle-containing photosensitive poly (ethylene glycol) hydrogel blocks: a technical note

All human organs consist of multiple types of cells organized in a complex pattern to meet specific functional needs. One possible approach for reconstructing human organs in vitro is to generate cell sheets of a specific pattern and later stack them systematically by layer into a three-dimensional organoid. However, many commonly used cell patterning techniques suffer drawbacks such as dependence on sophisticated instruments and manipulation of cells under suboptimal growth conditions. Here, we describe a simple cell patterning method that may overcome these problems. This method is based on magnetic force and photoresponsive poly (ethylene glycol) diacrylate (PEG-DA) hydrogels. The PEG-DA hydrogel was magnetized by mixing with iron ferrous microparticles and then fabricated into blocks with a specific pattern by photolithography. The resolution of the hydrogel empty space pattern was approximately 150 μm and the generated hydrogel blocks can be remotely manipulated with a magnet. The magnetic PEG-DA blocks were used as a stencil to define the area for cell adhesion in the cell culture dish, and the second types of cells could be seeded after the magnetic block was removed to create heterotypic cell patterns. Cell viability assay has demonstrated that magnetic PEG-DA and the patterning process produced negligible effects on cell growth. Together, our results indicate that this magnetic hydrogel-based cell patterning method is simple to perform and is a useful tool for tissue surrogate assembly for disease mechanism study and drug screening.     

Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation

Most of the surface patterning methods currently applied are based on lithography techniques and microfabrication onto silicon or glass substrates. Here we report a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilising both electron beam (EB) polymerisation and local laser ablation techniques for microfabrication. Polyacrylamide was grafted onto tissue culture polystyrene (TCPS) dishes using EB irradiation. Water contact angles for these PAAm-grafted TCPS surfaces were less than 10 degrees (costheta = 0.99) with PAAm grafted amounts of 1.6 microg/cm(2) as determined by ATR/FT-IR. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces composed of hydrophilic PAAm and hydrophobic basal polystyrene layers. Bovine carotid artery endothelial cells adhered only to the ablated domains after pretreatment of the patterned surfaces with 15 microg/mL fibronectin at 37 degrees C. The ablated domain sizes significantly influenced the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without loss of pattern fidelity, indicating that more durable cell arrays can be obtained compared to those prepared by self-assembled monolayers of alkanethiols, as described in previous reports. The surface fabrication techniques presented here can be utilised for the preparation of cell-based biosensors as well as tissue engineering constructs.     

Lineage specification in neural crest cell pathfinding.

There are two principal models to explain neural crest patterning. One assumes that neural crest cells are multipotent precursors that migrate throughout the embryo and differentiate according to cues present in the local environment. A second proposes that the neural crest is a population of cells that becomes restricted to particular fates early in its existence and migrates along particular pathways dependent on unique cell-autonomous properties. Although it is now evident that the neural crest cell population, as a whole, is actually heterogenous (composed of both multipotent and restricted progenitors), evidence supporting the model of prespecification has increased over the past few years. This review will begin by telling the story of melanoblasts: a neural crest subpopulation that is biased toward a single fate and subsequently acquires intrinsic properties that guide cells of this lineage to their final destination. The remainder of this review will explore whether this model is exclusive to melanoblasts or if it can also be used to explain the patterning of other neural crest cells like those of the sensory, sympathoadrenal, and enteric lineages.     

Endothelial Cell Micropatterning: Methods,Effects, and Applications

The effects of flow on endothelial cells (ECs) have been widely examined for the ability of fluid shear stress to alter cell morphology and function; however, the effects of EC morphology without flow have only recently been observed. An increase in lithographic techniques in cell culture spurred a corresponding increase in research aiming to confine cell morphology. These studies lead to a better understanding of how morphology and cytoskeletal configuration affect the structure and function of the cells. This review examines EC micropatterning research by exploring both the many alternative methods used to alter EC morphology and the resulting changes in cellular shape and phenotype. Micropatterning induced changes in EC proliferation, apoptosis, cytoskeletal organization, mechanical properties, and cell functionality. Finally, the ways these cellular manipulation techniques have been applied to biomedical engineering research, including angiogenesis, cell migration, and tissue engineering, are discussed.     

Rapid Biocompatible Micro Device Fabrication by Micro Electro-Discharge Machining

Fabrication of a biocompatible micro device is predominantly done by silicon micromachining techniques. The lithographic and etching techniques require preparation and the use of masks which are time consuming and costly. Since bio research involves highly complex mechanisms, the modeling and simulation is difficult and experimental study is inevitable. To incorporate frequent design changes and to realize the hardware quickly, fabrication processes, complementary to the silicon micromachining techniques are required. In the present work the feasibility of using micro electro-discharge machining (EDM) for the fabrication of biocompatible microdevice has been studied. Micro channels with feature size as small as 25 microm are realized. The process is further improved by the introduction of ultrasonic vibration of the workpiece and the total time taken for the hardware realization is about 4 hours. The effects of ultrasonic vibration on the roughness of the spark eroded surface has been studied and reported. The potential of using micro EDM for making biocompatible devices for bio experiments is demonstrated and the surface finish achieved is well within the recommended Rz and Ra values of 3.4 and 0.4 microm respectively for biological studies like implant abutment.     

Methods for Fabrication of Nanoscale Topography for Tissue Engineering Scaffolds

Observations of how controlling the microenvironment of cell cultures can lead to changes in a variety of parameters has lead investigators to begin studying how the nanoenvironment of a culture can affects cells. Cells have many structures at the nanoscale such as filipodia and cytoskeletal and membrane proteins that interact with the environment surrounding them. By using techniques that can control the nanoenvironment presented to a cell, investigators are beginning to be able to mimic the nanoscale topographical features presented to cells by extracellular matrix proteins such as collagen, which has precise and repeating nanotopography. The belief is that these nanoscale surface features are important to creating more natural cell growth and function. A number of techniques are currently being used to create nanoscale topographies for cell scaffolding. These techniques fall into two main categories: techniques that create ordered topographies and those that create unordered topographies. Electron Beam lithography and photolithograpghy are two standard techniques for creating ordered features. Polymer demixing, phase separation, colloidal lithography and chemical etching are most typically used for creating unordered surface patterns. This review will give an overview of these techniques and cite observations from experiments carried out using them.     

Tissue engineering: the biophysical background

Tissue engineering is the construction, repair or replacement of damaged or missing tissue in humans and other animals. This engineering may take place within the animal body or as tissue constructs to be made in a bioreactor for later grafting into the animal. The minimal set of materials for this are the appropriate types of cell. Usually, however, non-living substrata are used as well. These substrata may be nothing more than materials that bulk up any voids in the damaged tissue and provide the mechanical strength that has been lost when the tissue is damaged or removed. They may serve a similar pair of functions in the bioreactor. They can do much more in terms of pattern formation. The orientations and morphology of the cells, the arrangement of intercellular material as it is laid down and the relationships between different cell types in the repairing or construct tissue are all of importance, for these should resemble the correct normal tissue as closely as possible. Most of these requirements are ones involving pattern formation. This review discusses the various ways in which tissue pattern can be engineered chiefly from a biophysical standpoint. Unpatterned cells are effectively not tissue. This engineering includes the use of topography on the substrata, chemical patterning of adhesive and other cues for the cells, mechanical force application to cause cell orientation and appropriate synthetic responses and electrical fields. The review also discusses the methods used to impart the appropriate cues to and through the materials which are often biodegradable polymers. The article gives particular attention to regions of research and practice where the involvement of the physicist or biophysicist is of importance.     

Cell patterning chip for controlling the stem cell microenvironment

Cell-cell signaling is an important component of the stem cell microenvironment, affecting both differentiation and self-renewal. However, traditional cell-culture techniques do not provide precise control over cell-cell interactions, while existing cell-patterning technologies are limited when used with proliferating or motile cells. To address these limitations, we created the Bio Flip Chip (BFC), a microfabricated polymer chip containing thousands of microwells, each sized to trap down to a single stem cell. We have demonstrated the functionality of the BFC by patterning a 50 x 50 grid of murine embryonic stem cells (mESCs), with patterning efficiencies >75%, onto a variety of substrates--a cell-culture dish patterned with gelatin, a 3-D substrate, and even another layer of cells. We also used the BFC to pattern small groups of cells, with and without cell-cell contact, allowing incremental and independent control of contact-mediated signaling. We present quantitative evidence that cell-cell contact plays an important role in depressing mESC colony formation, and show that E-cadherin is involved in this negative regulatory pathway. Thus, by allowing exquisite control of the cellular microenvironment, we provide a technology that enables new applications in tissue engineering and regenerative medicine.     

Fabrication of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) microstructures using soft lithography for scaffold applications

This paper reports two soft lithographic methods, micromolding and hot embossing, to produce biodegradable poly (3-hydroxybutyrate-co-3-ftydroxyhexanoate) (PHBHHx) arrays of microstructures for hosting and culturing cells in a local microenvironment by controlled shape. Silicon masters with high-aspect-ratio microfeatures were fabricated using KOH and DRIE anisotropic etching. These silicon masters were used as molds to construct PHBHHx microstructures using micromolding and hot embossing. Using silicon rather than conventional PDMS as molds allowed microstructures with feature size of 20 microm and height of 100 microm to be realized. PHBHHx microstructures with different configurations including circles, rectangles, and octagons were fabricated to investigate the effects of topography on cell culture. Mouse fibroblast cell lines L929 were cultured on PHBHHx microstructures in vitro to investigate the biocompatibility. This study demonstrates the feasibility of microfabrication of PHBHHx structures with micro-scale feature size using soft lithography, and the results show that PHBHHx microstructures can be created to mimic cellular microenvironment for cell culture, providing a convenient means to investigate relationships of microstructures and cell functions.     

Microstamp patterns of biomolecules for high-resolution neuronal networks

A microstamping technique has been developed for high-resolution patterning of proteins on glass substrates for the localisation of neurons and their axons and dendrites. The patterning process uses a microfabricated polydimethylsiloxane stamp with micrometer length features to transfer multiple types of biomolecules to silanederivatised substrates, using glutaraldehyde as a homobifunctional linker. To test the efficacy of the procedure, substrates are compared in which poly-d-lysine (PDL) was physisorbed and patterned by photoresist with those stamped with PDL. Fluorescein isothiocyanate labelled poly-I-lysine was used to verify the presence and uniformity of the patterns on the glass substrates. As a biological assay, B104 neuroblastoma cells were plated on stamped and physisorbed glass coverslips. Pattern compliance was determined as the percentage of cells on the pattern 8h after plating. Results indicate that the stamping and photoresist patterning procedure are equivalent. Substrates stamped with PDL had an average pattern compliance of 52.6±4.4%, compared to 54.6±8.1% for physisorbed substrates. Measures of background avoidance were also equivalent. As the procedure permits successive stamping of multiple proteins, each with its own micropattern, it should be very useful for defining complex substrates to assist in cell patterning and other cell guidance studies.