We thank D. D. Zhang for insightful discussion and generously providing reagents. M. J. is supported by the NIH Cardiovascular Training Grant, the Arizona Technology Research Initiative Fund, and Achievement Rewards for College Scientists. This work is supported by the NIH Director's New Innovator Award (1DP2OD007161-01), the National Science Foundation (0855890), and the James S. McDonnell Foundation.

1. Creation of molds used for patterning
<ol>
<li>Conceptual design of patterns. Patterns are created which serve to form cell networks, control cell contact, isolate cells, mimic natural structures, or otherwise guide cell adhesion and placement.</li>
<li>Computer-aided design or CAD based creation of pattern and creation of photomask. The desired pattern is designed in CAD software, such as AutoCAD, and is then sent to an outside company for producing a photomask.</li>
<li>Photolithography to produce master pattern. Glass slides are patterned with either a thin positive resist or a thick negative resist depending upon the size of the structure being created. Glass slides are used for their benefits of being low cost, stronger than silicon wafers and not producing as much dust when broken. Alternatively, other convenient structures such as diffraction gratings can be used as the master pattern.</li>
<li> The patterned slide is attached to a stack of slides which have not been patterned. All steps are performed inside a clean flow hood to minimize dust.</li>
<li>Creation of master molds. A container of tinfoil slightly larger than the stack of slides is created to hold the 30 g of Polydimethylsiloxane (PDMS) which is then poured over the photolithographically patterned surface to create an initial mold. The PDMS is degassed and allowed to cure for one to two days.</li>
<li>Once cured the PDMS is peeled off of the master pattern and forms a mold for the next step.</li>
<li> 6 g of 2 part epoxy ( Devcon #14310(6 g) is mixed for 1 minute and then poured into the master mold, degassed and allowed to cure. Degassing the epoxy must be done quickly (&lt;8 min) using many bubble breaking cycles and using only a thin layer before it the epoxy sets and bubble removal becomes impossible to remove the bubbles. The 6 g used produces a thin layer which facilitates quick bubble removal.</li>
<li> After one hour, the 2 part epoxy will be partially cured and more epoxy can be added on top without degassing to produce a thicker structure which is easer to handle in later steps. The epoxy is then allowed to cure for one to two days.</li>
<li>Once cured, the epoxy is removed from the PDMS master mold and tape is wrapped around the epoxy mold to form a container. A working mold is then created by pouring PDMS onto the epoxy mold, degassing the PDMS, and curing for one to two days.</li>
<li> Once cured, the working mold is peeled off of the epoxy and stored to maintain cleanliness.</li>
</ol>
2. Patterning of surfaces with molds for cell guidance
<ol>
<li>Small sections of the working mold are cut out and placed onto a polystyrene Petri dish. The locations of these mold sections are labeled.</li>
<li>A tripod is formed from the small sections and weighted to ensure conformal contact between the working mold and the Petri dish surface. Good placement can be observed by looking at the bottom of the dish.</li>
<li>The assembly is placed into the plasma chamber. Plasma treatment is initiated and continued at 150 Pa at 29.6 W (max power) for 10 minutes using air plasma.</li>
<li> After plasma treatment, the mold and weight arraignment are removed.</li>
<li> The Petri dish is placed under UV light for 10 minutes of sterilization inside the biosafety cabinet and stored there until seeded with cells.</li>
</ol>
3. Seeding surfaces with cells
<ol>
<li>SH-SY5Y CRL-2266 Human Neuroblastoma or other cells are cultured using standard protocols for the cell type.</li>
<li>The confluence of the SH-SY5Y CRL-2266 Human Neuroblastoma cells is measured visually before seeding onto the patterned surface.</li>
<li>Standard cell splitting is carried out to remove the cells from the surface of their Petri dish.</li>
<li>The cells, once free floating, are seeded onto the surface of the patterned Petri dish after being diluted to achieve the desired confluence when placed on the patterned surface.</li>
<li>The cells are allowed to settle and attach to the surface.</li>
<li>The patterned Petri dish is incubated for several hours to several days while the cells assemble onto the pattern created by the plasma.</li>
<li>When culturing the neuroblastoma cells, retinoic acid (10 &mu;M) is added daily in order to induce the cells to differentiate into a neuron-like state. The addition of the retinoic acid is done in the dark.</li>
<li>Retinoic acid is added daily for several days, after which differentiation will be observable.</li>
<li>Media is replaced every 3-4 days.</li>
</ol>
4. Observation and Analysis
<ol>
<li>Periodic images of the live cells or videos are taken in order to observe the cell behavior over time. For live images, the cells are placed in a microscope stage top incubator which keeps the cells at 37 &deg;C, 100% humidity, and 5% CO2. Cells can also be fixed and stained for observation under fluorescence</li>
<li> The images or videos of the cells that have been captured are imported to an image analysis system and measurements are carried out including growth rate, cell size, migration speed, differentiation, alignment, location of specific proteins, and others.</li>
</ol>

5. Representative Results:
A typical result of implementing the plasma lithography technique is the formation of a pattern of cells which resembles some arbitrary or natural structure. This is seen in Figure 1a-b where lines and networks of neurons have been created. Other cell types can be used as well as seen in Figure 1c-d, which shows Human umbilical vein endothelial cells (HUVEC) and C2C12 skeletal muscle cells forming grids. Materials such as poly-l-lysine can also be patterned, Figure 1e to facilitate attachment of certain cell types and for other uses. In the case of the neurons shown, what was created are networks of cells which have connections between them. This replicates what occurs naturally in the brain where neurons have discrete connections between neighboring cells which influences the operation of the brain. With the creation of such a structure in the lab, the number, position, frequency and other factors of the connections can be systematically controlled. The result of these arraignments can be measured visually and additional inputs such as chemical or electrical stimulation can be implemented to probe the network behavior.
A negative result would be an overgrown or incomplete pattern or a contaminated sample. An incomplete or overgrown pattern would result from seeding the substrate with either too few or too many cells which would not supply the pattern with an ideal amount of cells. Additionally, if the pattern is not designed correctly (e.g., lines too narrow) the cells will not be able to attach and grow successfully on it. In the case of a contaminated sample, proper cleanliness would not have been maintained during one of the steps though this is rare when following proper cell culture protocol due to the fact that the plasma patterning also sterilizes the substrate.
<img src="/files/ftp_upload/3115/3115fig1.jpg" alt="Figure 1"/> 
Figure 1. Patterning of cells and protein.

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaporation-induced+assembly+of+biomimetic+polypeptides.">Evaporation-induced assembly of biomimetic polypeptides.</a> Appl Phys Lett. 93, 023120-023 (2008).</li><li>Langowski, B. A., Uhrich, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+Plasma-Initiated+Patterning+(&#956;PIP).">Microscale Plasma-Initiated Patterning (&#956;PIP).</a> Langmuir. 21, 10509-10514 (2005).</li><li>Tourovskaia, A., Barber, T., Wickes, B. T., Hirdes, D., Grin, B., Castner, D. 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No conflicts of interest declared.

The cell investigation method presented here enables creation of complex patterns of multicellular networks which mimic biological structures as well as provides a method to produce stimuli that are subcellular in nature which then facilitates investigations of both grouped cell behavior and single cell responses to environmental factors. The use of this method is simple yet robust as it can be quickly performed with low cost equipment in the same lab as the cell culture. It is also strongly cell sensitive, allows easy observation of the resultant behavior and is by nature stable for long time periods, which allows investigation of long term cell behavior. It additionally allows for a diverse range of experiments to be performed as it is compatible with many cell types and can create arbitrary patterns. The long term stability of the technique derives from the fact that the surface functionalization imparted by the plasma is part of the surface and is not a coating or other layer which can be removed or degraded. If kept under liquid, this type of modification can retain its cell guiding ability for months.
The most critical part of the experiment necessary to ensure meaningful results is that of creating the master pattern which will ultimately be used for cell guidance. If this pattern is not properly designed, cells will not appropriately respond to the pattern and may not produce useful behavior. Parameters such as line width, pattern spacing, and others can greatly influence the cell compatibility with a particular pattern, and typically a range of such parameters can be created on the initial photomask to screen for the most appropriate design. 
Other important parameters related to carrying out patterning include mold creation, maintaining a dust free environment, cell seeding and general sterility of cell culture. Mold creation may be effected by the various transfer steps which are undertaken to allow for repeated PDMS casting off an epoxy mold. The epoxy mold is created because it will not degrade in the same way as a resist mold with small, high aspect ratio structures under repeated casting. If the transfer molding is done correctly, the dimensions and yield will not be affected but if done incorrectly such as by poorly degassing, incomplete curing, or excessive heating, bubbles, roughness and deformation of a pattern can occur which affects the final outcome. In relation to maintaining a dust free environment, the molds must be kept as clean as possible as any dust can interfere with the proper contact between the working PDMS mold and the surface and thus proper plasma shielding and chemical patterning. The seeding density of the cells also must be optimized to ensure that neither too few or too many cells inhabit the pattern area and sterility must be maintained to avoid bacterial and other contamination of the cells being used.
The technique can also be incorporated with other elements such as microfluidics, microelectrodes, and mechanical probes. This provides additional stimuli to the cells in order to better replicate various physiological conditions during experimentation and future work is focused on studying the effects of these combinations

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Plasma Cleaner And Vacuum Chamber</td>
 <td>Harrick Plasma</td>
 <td>PDC-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inverted fluorescence and phase contrast microscope</td>
 <td>Nikon</td>
 <td>TE2000-U</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope stage top incubator</td>
 <td>AmScope</td>
 <td>Model TCS-100</td>
 <td>Modified to include an enclosure that supplies an appropriate atmosphere</td>
 </tr>
 <tr>
 <td>Polystyrene Petri dish</td>
 <td>VWR</td>
 <td>25384-090</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polydimethylsiloxane (PDMS)</td>
 <td>Dow Corning</td>
 <td>Sylgard 184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Epoxy</td>
 <td>Devcon</td>
 <td>2 ton clear epoxy #14310</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture media</td>
 <td>Various</td>
 <td>&nbsp;</td>
 <td>Media follows standard formulations for cell type</td>
 </tr>
 <tr>
 <td>Retinoic acid</td>
 <td>Sigma</td>
 <td>R2625</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

plasma lithography surface patterning for creation of cell networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

Watch this Scientific Journal Video about Plasma Lithography Surface Patterning for Creation of Cell Networks at JoVE.com

Plasma Lithography Surface Patterning for Creation of Cell Networks

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

plasma-lithography-surface-patterning-for-creation-of-cell-networks

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JoVE Journal

Bioengineering

12.6K Views.  University of Arizona. A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Video: Plasma Lithography Surface Patterning for Creation of Cell Networks

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Capillary Force Lithography for Cardiac Tissue Engineering

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

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

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

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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 ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...