This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. Photomask Fabrication
<ol>
	<li>Clean a 4&#8221; glass wafer with piranha solution (H2SO4/H2O2 in 2:1 ratio) for 20 min at 120 &#176;C by immersion in a quartz tank.</li>
	<li>Deposit a layer of Chromium/Gold (30 nm of Cr/1 &#181;m of Au) layer on the glass wafer using an e-beam evaporator7 (Figure 1A).
	<ol>
		<li>Place the wafers in an e-beam evaporator. Once the vacuum reaches 5 x 10-6 Torr, turn on the high voltage source (10 kV). Control the thickness by the monitor control panel.</li>
		<li>Pre-clean each material for 30 sec using the e-beam gun, keeping the shutter &#8220;OFF&#8221; (in order to avoid the deposition on the wafers).</li>
	</ol>
	</li>
	<li>Generate a Cr/Au photoresist masking layer for deep wet etching of the glass.
	<ol>
		<li>Apply a 2 &#181;m thick photoresist by spinning 5 ml of the solution for 30 sec at 3,000 rpm using a spin-spray coater system. Prebake the photoresist on a hotplate at 100 &#176;C for 1.5 min.</li>
		<li>Expose and hard bake the photoresist at 120 &#176;C for 30 min on a hot plate. It is critical to generate a hydrophobic surface and a strong adhesion of the photoresist to the metal layer. Pattern the Cr/Au layer using Cr and Au etchants through the photoresist mask8,9.</li>
	</ol>
	</li>
	<li>For protection of the un-patterned glass surface, temporarily bond the glass wafer to a dummy silicon wafer.9
	<ol>
		<li>Place the glass wafer on a hot plate at 110 &#176;C and melt the wax on the opposite side of the glass wafer (in such a way that the entire surface of the wafer is covered with wax).</li>
		<li>Place a dummy silicon wafer in contact with the glass wafer and press to remove the excess wax. In order to avoid spilling of the wax, place a clean room tissue paper on the hotplate.</li>
	</ol>
	</li>
	<li>Perform isotropic etching of the lens using optimized hydrofluoric acid (49% v/v) and hydrochloric acid (37% v/v) solution ( in a volumetric ratio of 10:1) with a magnetic stirrer for 8.5 min.10 Presence of the HCl is critical in achieving a good surface quality of generated lenses.
	<ol>
		<li>Ensure that the etching rate is 7 &#181;m/min; using a total volume of 200 ml of etching solution. Perform etching in a plastic container and take safety precaution for this processing step.</li>
		<li>Clean the wafer in the deionized (DI) water by rinsing and further drying at RT.</li>
	</ol>
	</li>
	<li>After the process completion, separate the glass wafer from the dummy silicon wafer and warm up the wax using a hot plate at 100 &#176;C for 15 sec. As the wax melts at this temperature, detach the glass wafer from the dummy silicon wafer.</li>
	<li>Remove remaining wax, the photoresist and the overhanging Cr/Au layers at the edges of the lenses using ultrasonication for 1 hr using N-methyl-2-pyrrolidone as the solvent at 80 &#176;C in an ultrasonic tank.</li>
	<li>Create a PDMS mold replica of the microlenses fabricated on the photomasks11.</li>
	<li>Characterize the photomask dimensions (length and width) and the microlenses PDMS mold (depth and diameter) replicas using a scanning electron microscope and stereomicroscope respectively.12-14</li>
</ol>
2. MN Shafts Fabrication
<ol>
	<li>Create a cavity of 2.5 cm &#215; 0.9 cm using the glass slides mounted on either sides of a glass. The number of the glass slides stacked on either side will determine the height of the cavity known as spacer thickness (Figure 1B).</li>
	<li>Secure each layer of the glass slide by applying a thin layer of the prepolymer solution containing Poly(ethylene glycol) diacrylate (PEGDA, MW = 258 Da) with 0.5% w/w 2-hydroxy-2-methyl-propiophenone (HMP) onto the glass slide followed by irradiation of the set up with the high intensity ultraviolet (UV) light for 2 sec.</li>
	<li>Position the photomask (previously fabricated) with the Cr/Au coated surfaces facing the interior of the cavity. Ensure that the sides of the cavity walls are not obscuring the lenses embedded in the photomask.</li>
	<li>Fill the cavity with the prepolymer solution until the Cr/Au coated surface is in contact with the solution without any visible bubbles.</li>
	<li>Irradiate the setup with high intensity UV light of a desired intensity for 1 sec at a distance of 3.5 cm from the UV source using the UV curing station with a UV filter range of 320-500 nm. Use a collimating adaptor with the UV light probe.</li>
	<li>Measure the intensity of UV light used using a radiometer.</li>
	<li>Following UV exposure, remove the photomask with the array of MNs. Pour the excess prepolymer solution that is not polymerized in the process back into its original container for reuse.</li>
	<li>Quantify the length and tip diameter of the MNs using a stereomicroscope according to manufacturer&#8217;s instructions.</li>
</ol>
3. MN Backing Layer Fabrication
<ol>
	<li>With forceps, place the MNs (previously fabricated) attached onto the photomask in a well of a 24-well plate as shown in the Figure 1C.</li>
	<li>Add a specified volume (300 - 400 &#181;l) of the prepolymer solution into the well until the needles are submerged to a desired height. This volume determines the thickness of the resultant backing layer.</li>
	<li>Irradiate the setup with high intensity UV light (15.1 W/cm2), 10.5 cm away from the UV source for a duration of 1 sec.</li>
	<li>Separate the backing layer on the MN array from the photomask using a sharp blade.</li>
	<li>Quantify the length, tip diameter and base diameter of the MNs with the backing layer using a stereomicroscope according to manufacturer&#8217;s instructions.</li>
</ol>

<ol>
	<li>Zhou, C. P., Liu, Y. L., Wang, H. L., Zhang, P. X., Zhang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transdermal+delivery+of+insulin+using+microneedle+rollers+in+vivo.">Transdermal delivery of insulin using microneedle rollers in vivo.</a> International journal of pharmaceutics. 392, 127-133 (2010).</li><li>Lee, J. W., Choi, S. O., Felner, E. I., Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissolving+microneedle+patch+for+transdermal+delivery+of+human+growth+hormone.">Dissolving microneedle patch for transdermal delivery of human growth hormone.</a> Small. 7, 531-539 (2011).</li><li>Raphael, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=needle-free+vaccinations+in+skin+using+multi+layered,+densely-packed+dissolving+microprojection+arrays.">needle-free vaccinations in skin using multi layered, densely-packed dissolving microprojection arrays.</a> Small. 6, 1785-1793 (2010).</li><li>Lee, J. W., Han, M. R., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+microneedles+for+transdermal+drug+delivery.">Polymer microneedles for transdermal drug delivery.</a> Journal of drug targeting. 21, 211-223 (2012).</li><li>Kochhar, J. S., Goh, W. J., Chan, S. Y., Kang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+microneedle+array+fabrication+for+transdermal+drug+delivery.">A simple method of microneedle array fabrication for transdermal drug delivery.</a> Drug development and industrial pharmacy. 39, 299-309 (2013).</li><li>Kochhar, J. S., Zou, S., Chan, S. Y., Kang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+encapsulation+in+polymeric+microneedles+by+photolithography.">Protein encapsulation in polymeric microneedles by photolithography.</a> International journal of nanomedicine. 7, 3143-3154 (2012).</li><li>Tay, F. E. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preshaping+photoresist+for+refractive+microlens+fabrication.">Preshaping photoresist for refractive microlens fabrication.</a> P Soc Photo-Opt Ins. 1992, 275-282 (1993).</li><li>Friedman, G. B., Sandhu, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+Spherical+Aberration+of+a+Thin+Lens.">Longitudinal Spherical Aberration of a Thin Lens.</a> Am J Phys. 35, 628 (1967).</li><li>Xu, Q. A., Li, J., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collimated+the+laser+diode+beam+by+the+focus+lens.">Collimated the laser diode beam by the focus lens.</a> Semiconductor Lasers and Applications IV. 7844, (2010).</li><li>Lin, T. W., Chen, C. F., Yang, J. J., Liao, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual-directional+light-control+film+with+a+high-sag+and+high-asymmetrical-shape+microlens+array+fabricated+by+a+UV+imprinting+process.">A dual-directional light-control film with a high-sag and high-asymmetrical-shape microlens array fabricated by a UV imprinting process.</a> J Micromech Microeng. 18, (2008).</li><li>Dunne, S. M., Millar, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+distance+from+curing+light+tip+to+restoration+surface+on+depth+of+cure+of+composite+resin.">Effect of distance from curing light tip to restoration surface on depth of cure of composite resin.</a> Prim Dent Care. 15, 147-152 (2008).</li><li>Kochhar, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+integrated+transdermal+patch+for+fast+onset+and+sustained+delivery+of+lidocaine.">Microneedle integrated transdermal patch for fast onset and sustained delivery of lidocaine.</a> Molecular pharmaceutics. 10, 4272-4280 (2013).</li><li>Kochhar, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+microneedle+array+fabrication+off+a+photomask+to+deliver+collagen+through+skin.">Direct microneedle array fabrication off a photomask to deliver collagen through skin.</a> Pharmaceutical research. 31, 1724-1734 (2014).</li></ol>

The authors declare no conflict of Interest.

The protocol described above for fabrication of the MNs array has been presented to fabricate the MNs array of ~1 cm2. The arrays can be scaled up by creating a large size cavity and by using a larger photomask. The increased cavity size can be created by increasing the width between the spacers on either side. Though each step to fabricate the MN arrays in the protocol was important, the most crucial steps were: the photomask positioning, the filling of prepolymer solution, and irradiation of the setup. Posit...

Transdermal drug delivery offers an attractive alternative approach for drug administration, especially for biomolecules, which are almost exclusively administered by hypodermic injections. However, skin, especially the top layer (the stratum corneum), is a formidable barrier preventing exogenous molecules from entering the human body. Recently, MN devices have emerged as enabling tools to deliver drugs through skin. The MN devices create temporary pores inside the stratum corneum to allow the passage of drug molecules to achieve the desired physiological activity with improved patient compliance and convenience1-3.
Various fabrication methods have been adopted to fabricate polymeric MNs4. However, they usually involve complicated and multiple step processes requiring long times and/or high temperatures to fabricate MNs arrays.4 To simplify the fabrication process, a single step mold-free process using a photomask was developed recently5,6. However, with this method, fabricated MNs had blunt needle tips, as no mechanism was in place to modify the ultraviolet (UV) light path involved in photolithography.
In this study, embedded microlenses in the photomask have been proposed to define the geometry of the MNs. The protocol to fabricate photomasks consisting of embedded microlenses and subsequently MN fabrication with sharp tips using the photomask are reported.

<table><tbody><tr><td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td><td>SIGMA&amp;nbsp;</td><td>475629-500ML</td><td></td></tr><tr><td>2-hydroxy-2-methyl-propiophenone (HMP)</td><td>SIGMA&amp;nbsp;</td><td>405655-50ML</td><td></td></tr><tr><td>Bovine collagen type 1, FITC conjugate&amp;nbsp;</td><td>SIGMA&amp;nbsp;</td><td>C4361</td><td></td></tr><tr><td>UV curing station&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>OmniCure S2000-XL</td><td></td></tr><tr><td>Collimating Adaptor&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>EXFO 810-00042</td><td></td></tr><tr><td>24-well plate</td><td>Thermo Fisher Scientific, USA</td><td></td><td></td></tr><tr><td>Nikon SMZ 1500 stereomicroscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Dillon GL-500 digital force gauge&amp;nbsp;</td><td>Dillon, USA</td><td></td><td></td></tr><tr><td>A-1R confocal microscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr></tbody></table>

polymeric microneedle array fabrication by photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

The geometry of the MNs can be significantly affected by the photomask characteristics and embedded microlens. The degree of refraction affects the transmission path of the UV rays, which influenced the MN geometry (Figure 2A). Each microlens was found to have a 350 &#181;m diameter, a 130 &#181;m flattened convex surface, and a 62.3 &#181;m depth (Figure 2B-D). Using the Pythagoras theorem, the radius of curvature of the first surface was found to be 272.89 &#181;m. The focal length was...

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Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

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12.0K Views.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Video: Polymeric Microneedle Array Fabrication by Photolithography

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

Engineering

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Developmental Biology

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Neuroscience

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...