The authors acknowledge financial support from a variety of sources including the UK Engineering and Physical Sciences Research Council (grant refs. EP/K011685/1, EP/K024485/1) and a graduate studentship for JH; the Leverhulme Trust (RPG-2014-292); the Wellcome Trust Institutional Strategic Support Fund (105610/Z/14/Z); the British Council (216196834); and the University of Manchester for a University of Manchester Research Institute (UMRI pump priming fund) and a Presidential Doctoral Scholarship to SW. Technical assistance by Dr. Andreas Lieb (Nanosurf AG) is also gratefully acknowledged.

<ol>
	<li>Saavedra, H. M., 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=Hybrid+Strategies+in+Nanolithography.">Hybrid Strategies in Nanolithography.</a> Rep. Prog. Phys. 73, 036501 (2010).</li><li>Gates, B. D., 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=New+Approaches+to+Nanofabrication:+Molding,+Printing,+and+Other+Techniques.">New Approaches to Nanofabrication: Molding, Printing, and Other Techniques.</a> Chem. Rev. 105, 1171-1196 (2005).</li><li>Garcia, R., Knoll, A. W., Riedo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Scanning+Probe+Lithography.">Advanced Scanning Probe Lithography.</a> Nat. Nanotechnol. 9, 577-587 (2014).</li><li>Piner, R. D., Zhu, J., Xu, F., Hong, S., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Dip-Pen"+Nanolithography.">"Dip-Pen" Nanolithography.</a> Science. 283, 661-663 (1999).</li><li>Salaita, K., Wang, Y., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Dip-Pen+Nanolithography.">Applications of Dip-Pen Nanolithography.</a> Nat. Nanotechnol. 2, 145-155 (2007).</li><li>Lewis, A., 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=Fountain+Pen+Nanochemistry:+Atomic+Force+Control+of+Chrome+Etching.">Fountain Pen Nanochemistry: Atomic Force Control of Chrome Etching.</a> Appl. Phys. Lett. 75, 2689-2691 (1999).</li><li>Kim, K. -. H., Moldovan, N., Espinosa, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nanofountain+Probe+with+Sub-100+Molecular+Writing+Resolution.">A Nanofountain Probe with Sub-100 Molecular Writing Resolution.</a> Small. 1, 632-635 (2005).</li><li>Gruter, R. R., Voros, J., Zambelli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FluidFM+as+a+Lithography+Tool+in+Liquid:+Spatially+Controlled+Deposition+of+Fluorescent+Nanoparticles.">FluidFM as a Lithography Tool in Liquid: Spatially Controlled Deposition of Fluorescent Nanoparticles.</a> Nanoscale. 5, 1097-1104 (2013).</li><li>Salaita, K., 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=Massively+Parallel+Dip-Pen+Nanolithography+with+55+000-Pen+Two-Dimensional+Arrays.">Massively Parallel Dip-Pen Nanolithography with 55 000-Pen Two-Dimensional Arrays.</a> Angew. Chem. Int. Ed. 45, 7220-7223 (2006).</li><li>Huo, F., 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=Polymer+Pen+Lithography.">Polymer Pen Lithography.</a> Science. 321, 1658-1660 (2008).</li><li>Shim, W., 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=Hard-Tip,+Soft-Spring+Lithography.">Hard-Tip, Soft-Spring Lithography.</a> Nature. 469, 516-520 (2011).</li><li>Liao, X., Braunschweig, A. B., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Force-Feedback"+Leveling+of+Massively+Parallel+Arrays+in+Polymer+Pen+Lithography.">"Force-Feedback" Leveling of Massively Parallel Arrays in Polymer Pen Lithography.</a> Nano Lett. 10, 1335-1340 (2010).</li><li>Wang, S., Hosford, J., Heath, W. P., Wong, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Area+Scanning+Probe+Nanolithography+Facilitated+by+Automated+Alignment+of+Probe+Arrays.">Large-Area Scanning Probe Nanolithography Facilitated by Automated Alignment of Probe Arrays.</a> RSC Adv. 5, 61402-61409 (2015).</li><li>Noh, H., 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="Multipoint+Force+Feedback"+Leveling+of+Massively+Parallel+Tip+Arrays+in+Scanning+Probe+Lithography.">"Multipoint Force Feedback" Leveling of Massively Parallel Tip Arrays in Scanning Probe Lithography.</a> Small. 11, 4526-4531 (2015).</li><li>Dalby, M. J., 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=The+Control+of+Human+Mesenchymal+Cell+Differentiation+Using+Nanoscale+Symmetry+and+Disorder.">The Control of Human Mesenchymal Cell Differentiation Using Nanoscale Symmetry and Disorder.</a> Nat. Mater. 6, 997-1003 (2007).</li><li>Connell, C. D., Higgins, M. J., Moulton, S. E., Wallace, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-Bioelectronics+via+Dip-Pen+Nanolithography.">Nano-Bioelectronics via Dip-Pen Nanolithography.</a> J. Mater. Chem. C. 3, 6431-6444 (2015).</li><li>Giam, L. R., 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=Scanning+Probe-Enabled+Nanocombinatorics+Define+the+Relationship+Between+Fibronectin+Feature+Size+and+Stem+Cell+Fate.">Scanning Probe-Enabled Nanocombinatorics Define the Relationship Between Fibronectin Feature Size and Stem Cell Fate.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 4377-4382 (2012).</li><li>Eichelsdoerfer, D. J., 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=Large-Area+Molecular+Patterning+with+Polymer+Pen+Lithography.">Large-Area Molecular Patterning with Polymer Pen Lithography.</a> Nat. Protoc. 8, 2548-2560 (2013).</li><li>Aran, K., Sasso, L. A., Kamdar, N., Zahn, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible,+Direct+Bonding+of+Nanoporous+Polymer+Membranes+to+PDMS+or+Glass+Microdevices.">Irreversible, Direct Bonding of Nanoporous Polymer Membranes to PDMS or Glass Microdevices.</a> Lab Chip. 10, 548-552 (2010).</li><li>Liao, X., Braunschweig, A. B., Zheng, Z., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-+and+Time-Dependent+Feature+Size+and+Shape+Control+in+Molecular+Printing+via+Polymer-Pen+Lithography.">Force- and Time-Dependent Feature Size and Shape Control in Molecular Printing via Polymer-Pen Lithography.</a> Small. 6, 1082-1086 (2010).</li><li>Sheehan, P. E., Whitman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol+Diffusion+and+The+Role+of+Humidity+in+"Dip+Pen+Nanolithography".">Thiol Diffusion and The Role of Humidity in "Dip Pen Nanolithography".</a> Phys. Rev. Lett. 88, 156104 (2002).</li><li>Geissler, M., 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=Fabrication+of+Metal+Nanowires+Using+Microcontact+Printing.">Fabrication of Metal Nanowires Using Microcontact Printing.</a> Langmuir. 19, 6301-6311 (2003).</li><li>Gmeiner, B., Leibl, H., Zerlauth, G., Seelos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Binding+of+Distinct+Functional+Fibronectin+Domains+to+Immobilized+Metal+Chelates.">Affinity Binding of Distinct Functional Fibronectin Domains to Immobilized Metal Chelates.</a> Arch. Biochem. Biophys. 321, 40-42 (1995).</li><li>Curran, J. M., 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=Introducing+Dip+Pen+Nanolithography+as+a+Tool+for+Controlling+Stem+Cell+Behaviour:+Unlocking+the+Potential+of+the+Next+Generation+of+Smart+Materials+in+Regenerative+Medicine.">Introducing Dip Pen Nanolithography as a Tool for Controlling Stem Cell Behaviour: Unlocking the Potential of the Next Generation of Smart Materials in Regenerative Medicine.</a> Lab Chip. 10, 1662-1670 (2010).</li><li>Davey, M. R., Anthony, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Cell Culture: Essential Methods. , 153-172 (2010).</li><li>Brinkmann, F., 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=Interdigitated+Multicolored+Bioink+Micropatterns+by+Multiplexed+Polymer+Pen+Lithography.">Interdigitated Multicolored Bioink Micropatterns by Multiplexed Polymer Pen Lithography.</a> Small. 9, 3266-3275 (2013).</li><li>Senesi, A. J., Rozkiewicz, D. I., Reinhoudt, D. N., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose-Assisted+Dip-Pen+Nanolithography+of+Oligonucleotides+and+Proteins.">Agarose-Assisted Dip-Pen Nanolithography of Oligonucleotides and Proteins.</a> ACS Nano. 3, 2394-2402 (2009).</li><li>Carnally, S. A. M., Wong, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+Catalysis+to+Enhance+Scanning+Probe+Nanolithography.">Harnessing Catalysis to Enhance Scanning Probe Nanolithography.</a> Nanoscale. 6, 4998-5007 (2014).</li></ol>

The alignment algorithm and software were developed by, and are proprietary to, the University of Manchester. It is available for download at http://www.click2go.umip.com/.

This protocol serves to provide users with a convenient methodology to rapidly carry out nanolithographic patterning with high uniformity and controllable feature size over large (cm2) areas. Substrates bearing these large area nanopatterns can then be further elaborated for a variety of applications. One major application of this technology is in the generation of nanofabricated surfaces for cell-surface interaction studies. This report shows some illustrative examples of cell culture on these materials, demonstrating control of hMSC morphology by nanofabricated substrates.
The key enabler of this protocol is the automation of the alignment procedure (step 4) that allows highly uniform and high-throughput production of features on surfaces, down to nanoscale resolution, which enables the rapid turnover of cell culture experiments. The polymer pen lithography carried out using this alignment algorithm is able to generate nanoscale features within approximately 30 min. The reproducibility and accuracy of automated alignment, and thus the uniformity of the patterned features, is however critically dependent on the quality of the probe arrays that are produced (step 1 and 2). Any flaws in their preparation that result in blunt, broken or missing probes; such as trapped air bubbles (step 1.5) or improper separation of the probes from the master (step 1.8) can result in inaccurate alignment and poor quality lithography.
This reported method shares a limitation in common with other alignment methods that rely on force feedback. The accurate determination of when the probes are in contact with the surface is constrained by the need to account for background vibrations caused by the ambient environment and the movement of the sample stage. In general, the sensors have a force sensitivity in the &#181;N regime (2 &#181;N in this case), but the alignment algorithm is designed to only register a force of at least 490 &#181;N as definitive contact between the probes and the surface, in order to avoid any &#39;false positives&#39; resulting from background noise.13 Thus, this method tends to produce large features (1-2 &#181;m) since the probes must extended a large distance on the z-axis (with a consequent higher force) in order to register contact. In order to compensate, smaller features can be generated by reducing the z-axis distance travelled during the lithography step (e.g., entering the &#39;Black&#39; setting in step 5.2.3.2 as 3 &#181;m instead of 5 &#181;m).
Nevertheless, even with this limitation, the automation algorithm is able to address a critical aspect in the application of parallelized scanning probe lithography methods, as alignment was previously the most time demanding and imprecise step in the implementation of these techniques. This automation now shifts the rate-limiting step of the fabrication process from the alignment to the lithographic writing itself. While this protocol demonstrates the application of this alignment procedure to PPL, the framework could be applied to a number of SPL techniques such as lipid-DPN26 and matrix-assisted lithography27 as well as potential future catalytic probe systems.28

Progress in nanotechnology is dependent on the development of techniques capable of efficiently and reliably fabricating nanoscale features on surfaces.1,2 However, generating such features over large areas (multiple cm2) reliably and at relatively low cost is a non-trivial endeavor. Most existing techniques, derived from the semiconductor industry, rely on ablative photolithography to fabricate &#39;hard&#39; materials. More recently, lithographic techniques derived from scanning probe microscopy (SPM) have emerged as a convenient and versatile approach for the rapid prototyping of nanoscale designs.3 SPM-based techniques are able to conveniently and rapidly &#39;write&#39; any user-defined pattern. The most well-known of these is dip-pen nanolithography (DPN), pioneered by Mirkin et al.,4 where a scanning probe is used as a &#39;pen&#39; to transfer a molecular &#39;ink&#39; to the surface producing features in a fashion analogous to writing. Under ambient conditions, as a probe is scanned across a surface the &#39;ink&#39; molecules are transferred to the surface via a water meniscus that forms between the probe and the surface (Figure 1). DPN thus allows the nanolithographic deposition of a wide range of materials, including &#39;soft&#39; materials such as polymers and biomolecules.5 Related techniques using probes engineered with channels for fluid delivery, variously referred to as &#39;nanopipettes&#39; and &#39;nano-fountain pens&#39;, have also been reported.6,7,8
The main obstacle to the wider application of SPM-derived lithography is throughput, as it requires an excessively long time to pattern centimeter-scale areas with a single probe. Early efforts to address this issue focused on the parallelization of cantilever-based DPN, with both &#39;one- dimensional&#39; and &#39;two-dimensional&#39; (2D) probe arrays being reported for the lithography of centimeter-sized areas.5,9 However, these cantilever arrays are produced through relatively complex multistep fabrication methods and are relatively fragile. The invention of polymer pen lithography (PPL) addressed this issue by replacing the standard SPM cantilevers with a 2D array of soft siloxane elastomer probes bonded to a glass slide.10 This simple probe setup significantly decreases the cost and complexity of patterning large areas, opening up nanolithography to a wider range of applications. This cantilever-free architecture has also been expanded to hard-tip soft-spring lithography,11 which provides a hybrid of soft elastomeric backing with hard silicon tips giving improved resolution in comparison to patterns produced using soft elastomer tips.
A crucial factor in the execution of these 2D array technologies is that the probe array must be exactly parallel to the surface substrate so that when lithography is utilized, all the probes come into contact with the surface simultaneously. Even a small misalignment can cause a large difference in feature size from one side of the array to the other, since some probes will come into contact with the surface earlier during the descent of the array, while others will come into contact later or not at all.12 Exact alignment is especially important with PPL due to the deformability of the soft elastomer probes, where the probes contacting the surface earlier will be compressed, leaving a larger footprint on the surface.
The early work on PPL employed purely visual inspection to guide the alignment process, using a camera mounted above the array to observe the deformation of the pyramidal probes as they were brought into contact with the surface.10 Alignment was judged by observing which side of the probes came into contact with the surface first, then adjusting the angle and repeating the procedure in an iterative manner until the difference in contact on each side of the probe was indistinguishable to the eye. As this alignment procedure relies on subjective visual inspection by the operator, reproducibility is low.
Subsequently, a more objective approach has been developed, consisting of a force sensor mounted beneath the substrate to measure the force applied upon contact of the probes on the surface.12 Alignment was thus achieved by adjusting the tilt angles to maximize the force exerted, which indicated that all the probes were simultaneously in contact. This method showed that alignment to within 0.004&#176; of the surface parallel was possible. This &#39;force feedback levelling&#39; has now been implemented into fully automated systems in two independent reports.13,14 Both use a triad of force sensors mounted either beneath the substrate or above the array and measure the amount of force exerted upon contact between the probe arrays and surface. These systems give high precision, reporting misalignments of &#8804;0.001&#176; over a 1 cm length scale,14 or &#8804; 0.0003&#176;over 1.4 cm.13 These automated alignment systems also provide major savings in operator time and overall time taken to complete the lithography process.
One major application of high-throughput surface fabrication enabled by this technology is the generation of cell culture substrates. It is now well established that cell phenotype can be manipulated by controlling the initial interaction between cells and surface features, and that this can be enhanced at the nanoscale.15 Specifically, scanning probe lithography methods have been shown to be a facile method to produce a variety of nanofabricated surfaces for such cell culture experiments.16 For example, surfaces presenting nanoscale patterns of self-assembled monolayers and extracellular matrix proteins templated by PPL and DPN have been used to study the potential of nano-modified materials in material induced differentiation of stem cells.17
This protocol describes the utilization of a modified atomic force microscope (AFM) system that enables large-area PPL. We detail the detection of force using multiple force sensors as the means of determining probe-surface contact, together with an algorithm that automates the iterative alignment process. Subsequent functionalization of these patterns with the extracellular matrix protein fibronectin and the culture of human mesenchymal stem cells (hMSC) are described, as a demonstration of PPL-fabricated surfaces applied for cell culture.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlexAFM mounted on a motorised 5-axis (XYZ&#920;&#934;) translation and goniometer stage</td>
			<td>NanoSurf</td>
			<td>P40008</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM control software</td>
			<td>NanoSurf</td>
			<td>C3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Engraving pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z225568</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick plasma</td>
			<td>PDC-32G-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PlasmaFlo</td>
			<td>Harrick plasma</td>
			<td>PDC-FMG-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Economy Dry Oxygen Service Pump</td>
			<td>Harrick plasma</td>
			<td>PDC-OPE-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rotator</td>
			<td>Stuart</td>
			<td>SB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Thermo Fisher Scientific</td>
			<td>5311-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Water Purification System</td>
			<td>Merck Millipore</td>
			<td>ZRXQ015WW</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Humidity Generator</td>
			<td>proUmid</td>
			<td>MHG32</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline Plus Pipette 100-1000 &#181;L</td>
			<td>Sartorius</td>
			<td>728070</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon masters</td>
			<td>NIL Technology</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright snapshot fluorescence microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope objectives</td>
			<td>Olympus</td>
			<td>10x and 60x UPlan FLN &#8734;/-/FN 26.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright bright field microscope</td>
			<td>Leica</td>
			<td>DM 2500M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>Ultrawave Ltd.</td>
			<td>U95</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet for recording and intepreting automated alignment results</td>
			<td>Microsoft</td>
			<td>Excel</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>34863</td>
			<td>FLAMMABLE</td>
		</tr>
		<tr>
			<td>Microscope Sildes, Clear, Ground</td>
			<td>Thermo Fisher Scientific</td>
			<td>451000</td>
			<td></td>
		</tr>
		<tr>
			<td>(7&#8211;8% vinylmethylsiloxane)-dimethylsiloxane copolymer, trimethylsiloxy-terminated</td>
			<td>Gelest</td>
			<td>VDT-731</td>
			<td></td>
		</tr>
		<tr>
			<td>1,3,5,7-tetramethyl-1,3,5,7-tetravinylcyclotetrasiloxane</td>
			<td>Gelest</td>
			<td>SIT7900.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution</td>
			<td>Sigma-Aldrich</td>
			<td>479527</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>(25&#8211;35% methylhydrosiloxane)-dimethylsiloxane copolymer, trimethylsiloxane-terminated</td>
			<td>Gelest</td>
			<td>HMS-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Boat 100 mL</td>
			<td>Scientific Laboratory Supplies</td>
			<td>BALI828</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Appleton Woods</td>
			<td>KS230</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SARSTEDT</td>
			<td>82.1473</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Thermo Fisher Scientific</td>
			<td>ST10-031T</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Carbon Tape</td>
			<td>Agar scientific</td>
			<td>AGG3939</td>
			<td></td>
		</tr>
		<tr>
			<td>16-Mercaptohexadecanoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>448303-1G</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>34852</td>
			<td>FLAMMABLE</td>
		</tr>
		<tr>
			<td>Gold coated microscope slide</td>
			<td>Sigma-Aldrich</td>
			<td>643203</td>
			<td>Once opened gold will remain reactive to thiols for at least 1 month</td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>T8656</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Iron(III) nitrate nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>529303</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>84415</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>(11-Mercaptoundecyl)hexa(ethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>675105</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt(II) nitrate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>203106</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCGM Mesenchymal Stem Cell Growth Medium</td>
			<td>Lonza UK</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells</td>
			<td>Lonza UK</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Multifuge X1 Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004210</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Centrifuge Tubes</td>
			<td>Greiner Bio-One</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>P/0840/53</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>&#34;Detergent&#34; in manuscript</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-fibronectin antibody</td>
			<td>Abcam</td>
			<td>ab2413</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37117</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3912</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>10253041</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 tissue culture flask</td>
			<td>Thermo Fisher Scientific</td>
			<td>10790113</td>
			<td></td>
		</tr>
		<tr>
			<td>cantilever</td>
			<td>BudgetSensor</td>
			<td>ContAl-G</td>
			<td></td>
		</tr>
	</tbody>
</table>

large-area scanning probe nanolithography facilitated by automated alignment and its application to substrate fabrication for cell culture studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

To check whether the automated alignment had been successful, the graphs plotted from the alignment data (in the spreadsheet from step 4.8) were examined. Where the alignment process had been successful the two plots, corresponding to the angle by which the sample stage has been tilted along the &#952; and &#966; axes, showed a series of rising and descending data points. In each of the plots, two linear fits of the data points showed a well-defined intersect &#34;peak&#34; indicating the maximum z-extension and the corresponding angle at which alignment was achieved (Figure 4A and 4B). This process is repeated four times (i.e., twice for each axis) and plotted as a set of four coordinates. The intersection of each pair of coordinates thus shows the overall optimum angles (Figure 4C).13 In cases where the alignment was not successful, it can be observed that their corresponding &#952; and &#966; angle plots do not give good quality linear fits, or do not intersect (Figure 5). Such failed alignments are typically as a result of the arrays being improperly trimmed or mounted to the probe holder (steps 1.7, 1.8, and 2.2). In these cases, the arrays were discarded and a new one prepared and mounted (steps 1 and 2), and the alignment process repeated (step 4).
Upon successful alignment and lithography with MHA by PPL, patterned gold substrates were then imaged using lateral force microscopy to examine whether deposition had taken place. A larger area examination of the printed surfaces was also conducted by optical microscopy of the substrates after etching of the gold not protected by the deposited thiol (Figure 6 and Figure 7). However, the etched patterns cannot be used for further functionalization and should only be used to confirm patterning on representative samples of a batch of printed surface substrates. If the etched patterns show blank areas corresponding to individual pens (Figure 8), this result indicates that the production of probe arrays has not been done successfully, and that some probes are damaged or missing. This inhomogeneity of the probes may be due to the use of an old master where the perfluorinated coating has worn away (step 1.3), resulting in some probes being torn away when the array is separated from the master. In these cases, a new master should be used. The result may also be due to the presence of air bubbles trapped between the glass backing and the master (step 1.5), or if the probe array was not cleanly separated from the master after curing (step 1.8).
Florescent microscopy images of the fibronectin functionalized surfaces incubated hMSCs were also collected (Figure 9). In general, all substrates were stable within the in vitro culture environment and the cells adhered and adapted their morphology to the printed patterns in case of smaller isolated 20 x 20 array of features.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56967/56967fig1.jpg" /> 
Figure 1. Schematic representation of polymer pen lithography showing molecular ink transport via a water meniscus on probe tip. (A) Side view and (B) top view of the polymer pen array indicate that when the probe array and surface substrate are fully aligned, all the probes come into contact with the surface simultaneously, resulting in parallelized lithography. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56967/56967fig2.jpg" /> 
Figure 2. Schematic diagram of polymer pen lithography set up. (A) Expanded side view of experimental set up where the prepared probe array is attached to probe holder and mounted to AFM scanner. The substrate is placed on the stage, below which are located the three force sensors. (B) A representation of the assembled instrumentation, showing the AFM scan head relative to the sample stage. (C) Bottom view showing force sensor location. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56967/56967fig3.jpg" /> 
Figure 3. Schematic representation depicting the spectroscopy program for the alignment procedure. The AFM scanner is set to move the probes toward sample by a distance of 10 &#181;m within 100 ms, held at position for 250 ms, followed by a retraction of 10 &#181;m within 100 ms, and then held for 250 ms at the retracted position. The motion is then repeated throughout the alignment process. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56967/56967fig4.jpg" /> 
Figure 4. Graphs illustrating a successful alignment. Graphs of z position against the tilt angles (A) &#952; and (B) &#966; for a successful alignment, where &#9679; indicates the actual values measured and + indicates the best fit with the least-squares method. (C) Graph of &#966; against &#952; fitted angles with the four points where the maximum z-position was reached. The intersection point marked is the final optimum tilt angle across both axes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56967/56967fig5.jpg" /> 
Figure 5. Graphs illustrating an unsuccessful alignment. Graphs of z position against the tilt angles (A) &#952; and (B) &#966; for an unsuccessful alignment, where &#9679; indicates the actual values measured and + indicates the best fit with the least-squares method. (C) Graph of &#966; against &#952; fitted angles with the four points where the maximum z-position was reached. No clear optima or intersection point are observed and therefore the optimum alignment angles are not resolved. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56967/56967fig6.jpg" /> 
Figure 6. Illustrative optical microscopy and atomic force microscopy images of gold substrates that were patterned with MHA by the aligned PPL arrays and then etched. (A) and (B) are sequentially magnified optical microscopy images of the etched patterns; (C) is an AFM topography image of a single grid of patterns. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56967/56967fig7.jpg" /> 
Figure 7. Illustrative optical microscopy images of gold substrates that were patterned with MHA by the aligned PPL arrays and then etched. (A) and (B) are sequentially magnified optical microscopy images of etched patterns and (C) is a lower magnification image that shows large area homogeneous patterns. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56967/56967fig8.jpg" /> 
Figure 8. Illustrative optical microscopy image of a gold substrate that was unevenly patterned with MHA and then etched. The intended patterns (shown in the inset) were repeating grids of 20 dot lines arranged in 20 lines, with every two lines produced by increasing the z-axis extension by 1 &#181;m (ranging from 5 to -5 &#181;m). It can be seen that in some areas no patterns are generated, due to missing probes in those locations. In the areas where only two lines of dots are produced, this result indicates that a probe is present but it is not of the same height as the fully functioning probes, so only deposit features when the array is extended to the full z-axis distance. In this image, the contrast has been deliberately altered to enable observation of the partially printed areas. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56967/56967fig9.jpg" /> 
Figure 9. Epifluorescence microscopy images of hMSCs cultured on the fibronectin arrays templated by PPL. (A) and (B) are high magnification images showing individual cells. (C) Shows an example pattern of the fibronectin array without an adherent cell and (D) is a wide field image of the cells cultured in a grid arrangement (a schematic of the printed pattern is also shown in the inlay). The cells are stained to show fibronectin (red), F-actin (green) and cell nuclei (blue). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies at JoVE.com

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

large-area-scanning-probe-nanolithography-facilitated-automated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K Views.  University of Manchester. Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Video: Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

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.

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 ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...