Name: expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior
Uploaded: 2016-12-08T14:00:00
Description: Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

1. Replication of PDMS Molds from an EBL Mold
<ol>
	<li>Fabricate silicon mold29
	<ol>
		<li>Spin coat 200 &#956;l polymethyl methacrylate (PMMA) solution on a 1&#160;&#215; 1 cm silicon (Si) substrate at 2,500 rpm for 1 min to form a thin film.</li>
		<li>Bake the PMMA film on the Si substrate at 180 &#176;C for 2 min.</li>
		<li>Write the designed nanopattern on the PMMA film by using a focused electron beam at an area dose of 300 &#181;C/cm2.</li>
		<li>Develop the PMMA nanopattern in developer...

<ol>
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We present a simple, affordable, yet versatile method to generate a large area of nanopatterned substrate. To faithfully expand highly defined nanopatterns, great attention should be paid to several critical steps. The first one is to trim the multiple PDMS molds. Unpatterned areas of the PDMS molds need to be removed. Additionally, the sidewalls of the molds should be cut vertically as perfect as possible to minimize the gaps between the molds. Collectively, the portion of unpatterned areas in the final stitch mold can ...

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>JEOL field emission SEM</td>
			<td>JEOL</td>
			<td>JSM-7600F</td>
			<td>EBL</td>
		</tr>
		<tr>
			<td>E-beam evaporator</td>
			<td>Kurt J. Lesker</td>
			<td>Model: LAB 18 e-beam evaporator</td>
			<td>nickel deposition</td>
		</tr>
		<tr>
			<td>Trion Minilock III ICP/RIE</td>
			<td>Trion technology</td>
			<td>Model: Minilock-phantom III</td>
			<td></td>
		</tr>
		<tr>
			<td>Press machine</td>
			<td>PHI Hydraulic Press</td>
			<td>Molde: SQ-230H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>Modle: WS-400A-6NPP-LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 critical dryer</td>
			<td>Tousimis</td>
			<td>Modle: Autosamdri-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>1080</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum plates</td>
			<td>McMaster-carr</td>
			<td>9057K123</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon sheets</td>
			<td>McMaster-carr</td>
			<td>8711K92</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dish</td>
			<td>FALCON</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable weighing boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td>Fisher Scientific</td>
			<td>02-913-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic desiccator</td>
			<td>Bel-Art Products</td>
			<td>F42025-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Fisher Scientific</td>
			<td>1110049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Ted Pella, inc.</td>
			<td>5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal blocks</td>
			<td>McMaster-carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Brettuns Village Leather Craft Supplies</td>
			<td>Arch punch</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methyl methacrylate)</td>
			<td>MicroChem</td>
			<td>495 PMMA A4</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184 kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Dow Chemical</td>
			<td>Styron 685D</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td>
			<td>Oakwood Chemical</td>
			<td>7142</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem</td>
			<td>MIBK/IPA at 1: 3 ratio</td>
			<td></td>
		</tr>
		<tr>
			<td>Remover</td>
			<td>MicroChem</td>
			<td>Remover PG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T324-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microsopy Science</td>
			<td>15712-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde&#160;</td>
			<td>Fisher Chemical</td>
			<td>G151-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-185</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

The stitch technique can generate a large area of nanopatterned substrates with high fidelity. Figure 1a and 1b display the large area of nanopatterns transferred from the stitched PDMS mold to PS plate and PS thin film on a Si substrate, respectively. The comparison between the original EBL-written mold (Figure 1c) and the final PDMS working substrate (Figure 1d) confirms that the EBL-written nanopatterns can be faithful...

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

The overall goal of this procedure is to produce a large area of nanopatterned substrate from small nanopatterned molds by using a simple and affordable yet versatile stitch technique. This method can help study substrate nanotopography modulation of cell phenotype and function at cellular and molecular levels, such as expression of focal adhesion proteins. The main advantage of this technique is that it is simple and cost effective.To begin, prepare the silanized silicon mold in the PDMS prepolymer, as described in the text protocol. Put the silanized silicon mold in a 60 millimeter Petri dish. Pour the PDMS prepolymer on the silicon mold in the Petri dish.Place the Petri dish in a plastic desiccator and degas for about 10 minutes, until all bubbles disappear. Transfer the Petri dish to a hot plate and cure the PDMS prepolymer at 70 degrees Celsius for four hours. Carefully peel off the PDMS mold from the silicon mold by using tweezers.Determine the orientation of anisotropic PDMS nanopatterns, such as nanogratings, under an optical microscope, and mark it on the backside of the PDMS molds with a marker pen. Clean the silicon substrate with ethanol in a fume hood, and then dry it with compressed air. Trim off the unpatterned areas of each PDMS mold with a blade.The PDMS molds should be aligned as close as possible, but not touching the surrounding mold, to minimize the unpatterned area and to avoid touching to the nanopattern deformation when the compressor force is applied. Place the trimmed PDMS mold with the nanopattern face-down on the mirror side of the silicon substrate, and then align other molds close to but not touching the surrounding molds. It is critical to try to cure the PDMS at the basal layer because uncured PDMS may flow through the gap between the multiple molds and damage the nanopatterns.However, the completely cured PDMS cannot bond the multiple molds together. To prepare a PDMS adhesive layer, cast one gram of degassed PDMS prepolymer on a clean glass slide to form a 0.5 millimeter thick layer. Bake the PDMS layer at 100 degrees Celsius on a hot plate for three to five minutes.Use a needle to touch the layer and ensure that the layer is partially but not completely cured. Place the PDMS layer on the backside of the aligned PDMS molds, quickly invert this assembly, and transfer it to the hot plate. Apply a compressive force using a metal block on top of the assembly to ensure good contact between the PDMS adhesive layer and the backside of the PDMS molds.Cure the PDMS adhesive layer at 100 degrees Celsius for one hour. Turn off the hot plate, remove the metal block, and peel off the single-stiched PDMS mold from the silicon substrate. To nanoimprint the stitched PDMS mold into the PS plate, place the PS plate in an aluminum spacer set on a three inch silicon wafer.Heat the PS plate on a hot plate at 250 degrees Celsius for 30 minutes. Then, place the stitched PDMS mold, with nanopatterns face-down, on the molten PS plate. Next, place an aluminum plate on the glass slide of the stitched PDMS mold.Apply a compressive pressure by using metal blocks on the aluminum plate, and wait for three minutes. Lift and replace the metal block from the aluminum plate, and increase the compressive pressure to 25 kilopascals. Then, repeat this step with the pressure increased to 50 kilopascals.Maintain the temperature of the hot plate at between 240 and 250 degrees Celsius under consistent pressure of 50 kilopascals for 15 minutes. Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature is below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS plate.To nanoimprint the PDMS mold on a PS thin film, place the stitched PDMS mold with nanotopography face-down on the PS thin film, which is set on a hot plate. Apply compressive pressure of 12 kilopascals on the PDMS mold by using metal blocks on the glass slide of the PDMS mold. Increase the temperature of the hot plate to 180 degrees Celsius, and maintain it for 15 minutes.Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature drops below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS film. Using a hollow steel arch punch, cut the nanopatterned PDMS substrates into discs to fit the configuration of a specific multi-well plate.Then, use tweezers to place the PDMS discs into the wells of the multi-well plate. Sterilize the PDMS substrates by using 70%ethanol for 30 minutes. Afterwards, aspirate the ethanol, and follow this by UV exposure for 30 minutes.Wash the PDMS substrates with 1X sterile phosphate-buffered saline three times. Next, coat the PDMS substrates with extracellular matrix protein for 30 minutes at room temperature. Rinse the PDMS substrates three times with sterile PBS, each for five minutes.Suspend human A549 lung cancer cells in Dulbecco's Modified Eagle Medium with 10%fetal bovine serum, and count the cells using a hemocytometer. Plate the cells at a seeding density of 2, 000 cells per square centimeter on the PDMS substrates. Culture the cells at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide for one day, and observe them afterwards.The generated nanopattern on the PS plate and the thin film are shown here. The arrows indicate the polymer raise in the interstices of the stitched PDMS molds. These SEM images demonstrate that PDMS-working substrates are faithfully transferred from the electron beam lithography-written nanopattern by applying the stitch technique.Representative SEM images of A549 cells grown on nanogratings and nanopillars are shown here. The cells show aligned cell morphology on the nanogratings, while the cells spread randomly on the nanopillars. After watching this video, you should have a good understanding of how to generate a large area of nanopatterned substrate by stitching multiple, small defined nanopatterned molds.Don't forget that working with toluene and paraformaldehyde can be extremely hazardous, and always wear appropriate personal protection equipment while performing the procedures.

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Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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 ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...