Cell patterning using magnetite nanoparticles and magnetic force

Technologies for fabricating functional tissue architectures by patterning cells precisely are highly desirable for tissue engineering. Although several cell patterning methods such as microcontact printing and lithography have been developed, these methods require specialized surfaces to be used as substrates, the fabrication of which is time consuming. In the present study, we demonstrated a simple and rapid cell patterning technique, using magnetite nanoparticles and magnetic force, which enables us to allocate cells on arbitrary surfaces. Magnetite cationic liposomes (MCLs) developed in our previous study were used to magnetically label the target cells. When steel plates placed on a magnet were positioned under a cell culture surface, the magnetically labeled cells lined on the surface where the steel plate was positioned. Patterned lines of single cells were achieved by adjusting the number of cells seeded, and complex cell patterns (curved, parallel, or crossing patterns) were successfully fabricated. Since cell patterning using magnetic force may not limit the property of culture surfaces, human umbilical vein endothelial cells (HUVECs) were patterned on Matrigel, thereby forming patterned capillaries. These results suggest that the novel cell patterning methodology, which uses MCLs, is a promising approach for tissue engineering and studying cell-cell interactions in vitro.     

Photolithographic patterning of C2C12 myotubes using vitronectin as growth substrate in serum-free medium

The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In our serum-free defined culture system, differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate-adsorbed vitronectin or laminin. On the basis of this substrate requirement of myotube formation, we developed a photolithography-based method to pattern C2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We have determined that the optimal line width to form single myotubes is approximately 30 mum. To illustrate a possible application of this method, we patterned myotubes on the top of commercial substrate-embedded microelectrodes. In contrast to previous experiments where cell patterning was achieved by selective attachment of the cells to patterned surfaces in a medium that contained all of the factors necessary for differentiation, this study illustrates that surface patterning of a signaling molecule, which is essential for skeletal muscle differentiation in a defined system, can result in the formation of aligned myotubes on the patterns. This technique is being developed for applications in cell biology, tissue engineering, and robotics.     

Topographical and physicochemical modification of material surface to enable patterning of living cells.

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Stepwise assembly of micropatterned co‐cultures using photoresponsive culture surfaces and its application to hepatic tissue arrays

Cell micropatterning, a method to place cells at arbitrary regions, is becoming an essential tool to conduct cell biology and tissue engineering. Conventional cell patterning techniques usually allow only single patterning with single cell type on the same culture surface. However, biomedical research today requires even sophisticated fabrication methods that require spatiotemporal control of multiple cell arrangements. Here we introduce in situ cell micropatterning system which enables stepwise cell patterning using a photoresponsive cell culture surface (PRCS) whose cell adhesiveness could be altered by the UV irradiation. To demonstrate an application to tissue engineering, a liver‐mimic tissue array was fabricated and liver‐specific gene expressions were quantified with real time PCR. Patterned co‐culture systems composed of HepG2 spheroids with Balb/3T3 were fabricated, and the optimum spheroid diameter, which yielded the highest cellular functions, was determined to be 150 µm. After 20 days of patterned co‐culture of HepG2 spheroids and Balb/3T3, CYP3A4 expression increased 50‐fold higher than conventionally cultured HepG2; CYP3A4 expression was 20% higher than randomly co‐cultured HepG2 and Balb/3T3. Thus the combination of PRCS and the photomask‐free irradiation apparatus showed the versatility of experimental setups and proved to be a powerful tool for biomedical studies. Biotechnol. Bioeng. 2009;103: 552–561. © 2009 Wiley Periodicals, Inc.     

Dielectrophoretic registration of living cells to a microelectrode array

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 micromdiameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20000 cells onto a 1cm(2) grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70+/-1% fidelity versus 17+/-1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.     

A method for micrometer resolution patterning of primary culture neurons for SPM analysis

In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near-field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra-fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-L-lysine was selectively deposited using a silicone-based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8-day-old chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule-associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.     

Dielectrophoretic registration of living cells to a microelectrode array

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 Ipm diameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device.In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20,000 cells onto aI cm2 grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70 +/- 1% fidelity versus 17 +/- 1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Corrugation Enabled Asymmetrically Ultrastretchable (95%) Monocrystalline Silicon Solar Cells with High Efficiency (19%)

Stretchable solar cells are of growing interest due their key role in realizing many applications such as wearables and biomedical devices. Ultrastretchability, high energy‐efficiency, biocompatibility, and mechanical resilience are essential characteristics of such energy harvesting devices. Here, the development of wafer‐scale monocrystalline silicon solar cells with world‐record ultrastretchability (95%) and efficiency (19%) is demonstrated using a laser‐patterning based corrugation technique. The demonstrated approach transforms interdigitated back contacts (IBC) based rigid solar cells into mechanically reliable but ultrastretchable cells with negligible degradation in the electric performance in terms of current density, open‐circuit voltage, and fill factor. The corrugation method is based on the creation of alternating grooves resulting in silicon islands with different shapes. The stretchability is achieved by orthogonally aligning the active silicon islands to the applied tensile stress and using a biocompatible elastomer (Ecoflex) as a stretchable substrate. The resulting mechanics ensure that the brittle silicon areas do not experience significant mechanical stresses upon asymmetrical stretching. Different patterns are studied including linear, diamond, and triangular patterns, each of which results in a different stretchability and loss of active silicon area. Finally, finite element method based simulation is conducted to study the generated deformation in the different patterned solar cells.     

Design and Implementation of a Microelectrode Assembly for Use on Noncontact In Situ Electroporation of Adherent Cells

In situ electroporation of adherent cells provides significant advantages with respect to electroporation systems for suspension cells, such as causing minimal stress to cultured cells and simplifying and saving several steps within the process. In this study, a new electrode assembly design is shown and applied to in situ electroporate adherent cell lines growing in standard multiwell plates. We designed an interdigitated array of electrodes patterned on copper with printed circuit board technology and covered with nickel/gold. Small interelectrode distances were used to achieve effective electroporation with low voltages. Epoxy-based microseparators were constructed to avoid direct contact with the cells and to create more uniform electric fields. The device was successful in the electropermeabilization of two different adherent cell lines, C2C12 and HEK 293, as assessed by the intracellular delivery of the fluorescent dextran FD20S. Additionally, as a collateral effect, we observed cell electrofusion in HEK 293 cells, thus making this device also useful for performing cell fusion. In summary, we show the effectiveness of this minimally invasive device for electroporation of adherent cells cultured in standard multiwell plates. The cheap technologies used in the fabrication process of the electrode assembly indicate potential use as a low-cost, disposable device.     

Topographical and Physicochemical Modification of Material Surface to Enable Patterning of Living Cells

ABSTRACT:?

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes

Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron‐scale control of cell position can be achieved over centimeter‐length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro‐stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel?) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH‐3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. Biotechnol. Bioeng. 2012; 109: 2630–2641. © 2012 Wiley Periodicals, Inc.     

Generation of hierarchical nano- and microwrinkle structure for smooth muscle cell alignment

On the contrary to the complicated and high-cost photolithography based topographical patterning, the non-lithographical strain responsive wrinkling method can generate a variety of wrinkle structures with ease, high uniformity, and cost-effectiveness. The strain responsive wrinkling approach relied on the modulus difference between a thin and stiff film and a soft substrate, resulting in a periodic out-of-plane buckling deformation upon the release of the compressive stress. While the previous reports were dedicated to the micropattern generation, we, in this study, investigated the effect of the UV/O exposure time (10, 20, 30, 40, 50, and 60 min) and the stretching rate (10, 20, 30, and 40%) of the PDMS substrate on the wavelength and the amplitude of the wrinkle patterns in details. Sole nanowrinkle as well as hierarchical nano/microwrinkle patterns could be fabricated through the fine control of those factors. Furthermore, we examined the human aortic smooth muscle cell (HASMCs) alignment on the topographical patterned surface, and found that more than 80% of the HASMCs were cultured and aligned well along with the hierarchical nano/microwrinkle rather than the nanowrinkle pattern.     

Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three‐dimensional magnetic cell culture array

A three‐dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force‐based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array‐like multicellular patterns of melanoma were developed using a pin‐holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel‐coated surface as array‐like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct‐interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect‐interaction model) of melanoma spheroids, and (iii) fibroblast‐sheets coexisting under melanoma spheroids (fibroblast‐sheet model). The fibroblast‐sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast‐sheet model, the expression of IL‐8 and MMP‐2 increased by 24‐fold and 2‐fold, respectively, in real time RT‐PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Alignment of skeletal muscle myoblasts and myotubes using linear micropatterned surfaces ground with abrasives

Alignment of cells plays a significant key role in skeletal muscle tissue engineering because skeletal muscle tissue in vivo has a highly organized structure consisting of long parallel multinucleated myotubes formed through differentiation and fusion of myoblasts. In the present study, we developed an easy, simple, and low‐cost method for aligning skeletal muscle cells by using surfaces with linear microscale features fabricated by grinding. Iron blocks were ground in one direction with three kinds of abrasives (9 µm diamond suspension, #400 sandpaper, and #150 sandpaper) and then used as molds to make micropatterned polydimethylsiloxane (PDMS) substrates (type I, type II, and type III). Observation of the surface topography revealed that the PDMS substrates exhibited different degree of mean roughness (Ra), 0.03 µm for type I, 0.16 µm for type II, and 0.56 µm for type III, respectively. Murine skeletal muscle cell line C2C12 myoblasts were cultured and differentiated on the patterned PDMS substrates, and it was examined whether the alignment of C2C12 myoblasts and myotubes was possible. Although the cell growth and differentiation on the three types of patterned substrates were similar to those on the flat PDMS substrate as a control, the alignment of both C2C12 myoblasts and myotubes was obviously observed on types II and III, but not on type I or the control substrate. These results indicate that surfaces ground with abrasives will be useful for fabricating aligned skeletal muscle tissues. Biotechnol. Bioeng. 2009;103: 631–638. © 2009 Wiley Periodicals, Inc.     

A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells

Uniform patterning of cells is highly desirable for most cellular studies involving cell-cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6x6 PIRE array show that cells patterned within minutes with good uniformity (48+/-6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1+/-0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.     

Propagation of viruses on micropatterned host cells

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.     

Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.     

Electroporation: parameters affecting transfer of DNA into mammalian cells

Electroporation, the reversible breakdown of cell membranes caused by a high-voltage discharge, is a rapid, simple, and efficient method for introducing DNA into mammalian cells. An instrument for electroporation which permits the high-voltage discharge waveform to be varied with respect to rise time, peak voltage, and fall time is described. The uptake and expression of SV40 DNA following electroporation of two cell types, a human carcinoma-derived cell line, HEp-2, and a human lymphoblastoid cell line, 721, depended on the peak voltage and the fall time of the voltage discharge. The electronic parameters which produced optimum DNA transfer, however, differed for the two cell types. DNA as large as 150 kb was introduced into cells by electroporation. Cells can be electroporated in either phosphate-buffered saline or culture medium containing fetal bovine serum, and the efficiency of DNA transfer does not vary with cell densities from 10(6) to 2 X 10(7)/0.5 ml. Exposing the cells to multiple voltage discharges did not improve DNA transfer. DNA has been introduced by electroporation into all cell types tested, including human carcinoma-derived cell lines, human lymphoblastoid cell lines, human fibroblast strains, and primary human lymphocytes. To obtain maximal DNA transfer by this method, however, one must optimize the peak voltage and fall time of the discharge waveform for each cell type.     

An extracellular microelectrode array for monitoring electrogenic cells in culture

This paper describes a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes have been tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. It was found that extracellular stimulation of the cells was possible via the same electrodes used for recording.