This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A1A2057527 and NRF-2016R1D1A1B03934418).

1. Preparation of through-hole array aluminum plate and magnet array
<ol>
	<li>Prepare two 50 mm x 50 mm (or larger) aluminum plates. The thickness of each plate was 300 &#181;m that is half of the bead diameter.</li>
	<li>Form a 30 x 30 through-hole array on one of the aluminum plates by using a CNC rotary engraver with a &#934;550-&#181;m micro drill bit with 30 mm/s of plunge rate and 8000 RPM of spindle speed. The distance between each hole (center to center) was 1 mm (Figure 1a and Figure 2a, i).</li>
	<li>Form a 30 x 30 array of &#934;750-&#181;m through-holes on the other aluminum plate, using the same procedure as that described in 1.2 (Figure 1a and Figure 2a, ii).</li>
	<li>Attach the two plates each other by using a sticky tape and form &#934;3 mm alignment holes at each of the four corners of both aluminum plates.</li>
	<li>Soak the aluminum plates in 15% sulfuric acid for 12 h to clean their surfaces. Since the thin layer of aluminum oxide on the surface of the aluminum make it corrosion resistant, the hole diameter and thickness of the plate are not changed by this acid treatment.</li>
	<li>Form a 30 &#215; 30 array of 1 x 1 x 1 mm neodymium magnets (with a magnetic strength of 0.363 N). Ensure that each magnet is the opposite polarity to its neighbor. To prevent the breaking or scattering of the magnet array, attach a 30 x 30 mm aluminum plate to the bottom of the magnet array using double-sided tape (Figure 2a,&#160;iii and inset in Figure 2).</li>
</ol>
2. Bead trapping process
<ol>
	<li>Align and stack the two aluminum plates (top plate: 750-&#181;m-hole plate, bottom plate: 550-&#181;m-hole plate) using the prepared alignment holes at the four corners of each plate (Figure 1b).</li>
	<li>Lock the two plates together by inserting M3 bolts into the alignment holes, and then secure the bolts with nuts (Figure 1b).</li>
	<li>Stack the aluminum plate assembly on the prepared magnet array (Figure&#160;1b, 2b, and 2c). Align the array of magnets and the array of through holes in the aluminum plate during the stacking process. Then use a sticky tape to fix the position of the magnet array.</li>
	<li>Place a sufficient number of &#934;600 mm SUJ2 steel beads on the plate assembly and manipulate the beads using an acryl (or non-metallic) plate such that a bead becomes trapped in each hole (Figure 1c, 1d, and 1e) while simultaneously removing the excess beads which have not lodged in the holes.</li>
	<li>Carefully remove the top plate to avoid unwanted scattering and dislocation of the trapped beads (Figure 1f).</li>
</ol>
3. Concave microwell fabrication
<ol>
	<li>Move the concave microwell mold, produced in steps 2.1 to 2.5, above, to a Petri dish.</li>
	<li>Mix polydimethylsiloxane (PDMS) monomer and curing agent according to the manufacturer&#39;s instructions19 with a PDMS monomer:curing agent ratio of 10:1.</li>
	<li>De-gas the PDMS mixture by using a desiccator and vacuum pump to remove any bubbles trapped in the PDMS mixture.</li>
	<li>Pour the PDMS mixture into the concave microwell mold and de-gas again using the same procedure as that described in 3.3 (Figure 1f).</li>
	<li>Bake the PDMS mixture on a hotplate at 80 &#176;C for 2 h to form a bead-embedded PDMS substrate (Figure 1g).</li>
	<li>Remove the cured PDMS substrate from the mold (Figure 1g). In the removing process, spray methanol using washing bottle to detach PDMS substrate from the mold.</li>
	<li>Using a &#934;15 mm x 2 mm magnet, remove the trapped steel beads from the PDMS substrate (Figure 1h). For this process, any magnet that is sufficiently strong to extract the beads from the PDMS substrate can be used.</li>
</ol>
4. Spheroid culture
<ol>
	<li>Cut the concave microwell-patterned PDMS substrate by using a &#934;14 mm biopsy punch to be fitted in 24-well plate in this study.</li>
	<li>Sterilize the resulting &#934;14 mm PDMS substrate in an autoclave sterilizer at 121 &#176;C and 15 psi.</li>
	<li>Place the sterilized PDMS substrate into a 24 well plate.</li>
	<li>Coat the entire PDMS substrate with 4% (w/v) pluronic F-127 solution overnight to prevent cell attachment to the microwell surface. During the coating process, remove any air bubbles entrapped in the concave microwells by pipetting or by using an ultrasonic cleaner.</li>
	<li>Flush the F-127 solution three times by using phosphate-buffered saline (PBS).</li>
	<li>Seed 1 mL of cell-medium (Dulbecco&#39;s Modified Eagle Medium) solution (which contains 2 x 106 cells) on the PDMS substrate. Note that the seeding density can be changed according to the target spheroid size and/or target cell type. Here, adipose-derived stem cells (ASC) were used.</li>
	<li>Aspirate the 1 mL of medium using a 1000 &#181;L pipette to remove any excess cells that were not trapped in the microwells (Figure 3).</li>
	<li>Incubate the cells at 36.5 &#176;C, humidity of &#62;95%, and 5% CO2 condition. In the case of the ASCs used in our study, the cells aggregate to a spheroid in 48 h.</li>
</ol>

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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=Concave+microwell+based+size-controllable+hepatosphere+as+a+three-dimensional+liver+tissue+model.">Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model.</a> Biomaterials. 32 (32), 8087-8096 (2011).</li><li>Yeon, S. E., 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=Application+of+concave+microwells+to+pancreatic+tumor+spheroids+enabling+anticancer+drug+evaluation+in+a+clinically+relevant+drug+resistance+model.">Application of concave microwells to pancreatic tumor spheroids enabling anticancer drug evaluation in a clinically relevant drug resistance model.</a> PloS one. 8 (9), (2013).</li><li>Park, J. Y., Hwang, C. M., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-lithographic+fabrication+of+concave+microwells+and+a+microfluidic+network.">Ice-lithographic fabrication of concave microwells and a microfluidic network.</a> Biomed Microdevices. 11 (1), 129-133 (2009).</li><li>Corning, 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> Sylgard 184 Silicone Elastomer. Technical Data Sheet. , (2008).</li><li>Giang, U. B. T., Lee, D., King, M. R., DeLouise, L. 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The authors have no conflicts of interest to disclose.

The major challenge facing this fabrication method was the secure fixing of the beads in the through-hole array in the aluminum plate. To solve this challenge, magnetic force in the form of a 30 x 30 magnet array was used to fix the beads securely, as shown in Figures 6 and 7. The magnetic flux density of the magnet array, which has the opposite polarity, is strongest at the center of each magnet surface. Because the strength of the magnetic force depends on the flux density, the beads w...

Cells grown into a spheroid form are more similar to real tissue in the body than a two-dimensional planar culture1.&#160;Given this advantage, the use of spheroids has been adopted to improve the study of cell to cell interaction2,3, immune-activation4, drug screening5, and differentiation6. In addition, spheroids incorporating multiple cell types have recently been applied to organoids (near-physiological three-dimensional (3D) tissue), which are very useful for studying human development and disease7.&#160;Several methods have been used to produce spheroids. The simplest method involves the utilization of a non-adhesive surface, such that the cells aggregate with each other and form spheroids. A Petri dish can be treated with bovine serum albumin, pluronic F-127, or a hydrophobic polymer (e.g. poly 2-hydroxyethl methacrylate) to make its surface non-adhesive8,9. The spinner-flask method is another well-known means of producing large amounts of spheroids10,11. In this method, cells are held in suspension by stirring to prevent them from becoming attached to the substrate. Instead, the floating cells aggregate to form spheroids. Both the non-adhesive surface method and spinner flask method can produce large amounts of spheroids. However, they are subject to limitations including difficulties in controlling the spheroid size, as well as the tracking and monitoring of each spheroid. As a remedy for such problems, another spheroid production method, namely, the hanging drop method can be employed12. This involves depositing cell suspension drops onto the underside of the lid of a culture dish. These drops are usually 15 to 30 &#181;L in size and contain approximately 300 to 3000 cells13. When the lid is inverted, the drops are held in place by surface tension. The microgravity environment in each drop concentrates the cells, which then form single spheroids at the free liquid-air interface. The benefits of the hanging drop method are that it offers a well-controlled size distribution, while it is easy to trace and monitor each spheroid, relative to the non-adhesive surface and spinner flask methods. However, this method incurs one disadvantage in that the massive production of spheroids and the production process itself is excessively labor intensive.
A microwell array is a flat plate with many micro-size wells, each having a diameter ranging from 100 to 1000 &#181;m. The spheroid production principle when using microwells is similar to that of the non-adhesive surface method. Benefits include the fact that microwells provide spaces between the microwells for separating the cells or spheroids, such that it is easy to control the spheroid size, while also making it easy to monitor each single spheroid. With a large number of microwells, high-throughput spheroid production is also possible. Another advantage of microwells is the option to form wells of different shapes (hexahedral, cylindrical, trigonal prismatic) depending on the users&#39; unique experimental purposes. Generally, however, a three-dimensional (3D) concave (or hemispherical) shape is regarded as being the most suitable for producing uniform-sized single spheroids. Therefore, the usefulness of concave microwells has been reported for many cell biology studies such as those examining the cardiomyocyte differentiation of embryonic stem cells14, the insulin secretion of islet cell clusters15, the enzymatic activity of hepatocytes16, and the drug resistance of tumor spheroids17.
Unfortunately, the fabrication of microwells often requires specialized micropatterning facilities; conventional photolithography-based methods require exposure and developing facilities while reactive ion-etching-based methods need plasma and ion-beam equipment. Such equipment is costly which, together with the complicated fabrication process, presents a high barrier to entry for biologists who do not have access to microtechnology. To overcome these problems, other cost-effective methods such as ice lithography18 (using frozen water droplets) and the flexible membrane method14 (using a membrane, through-hole substrate, and a vacuum) have been suggested. However, these methods also incur serious drawbacks such as it being difficult to control the pattern sizes, the attainment of high aspect ratios, and the production of larger-area microwells.
To overcome the above issues, we are proposing a novel concave microwell fabrication method utilizing a through-hole substrate, steel beads, and a magnet array. Using this method, hundreds of concave spherical microwells can be fabricated by taking advantage of the mechanism of magnetic-force-assisted self-locking metallic beads (Figure 1). The fabrication process involves the use of very few expensive and complicated facilities and does not demand many advanced skills. As such, even unskilled persons can easily undertake this fabrication method. To demonstrate the proposed method, human-adipose-derived stem cells were cultured in the concave microwells to produce spheroids.

<table><tbody><tr><td>CNC rotary engraver</td><td>Roland DGA</td><td>EGX-350</td><td></td></tr><tr><td>Micro drill bit</td><td>HAM Pr&amp;auml;zision</td><td>30-1301 TA</td><td>&amp;Phi; 0.55 and 0.75 mm</td></tr><tr><td>Sulfuric acid 98%</td><td>Daejung</td><td>7683-4100</td><td>For cleaning aluminum plate.&lt;br/&gt; Dilute with distilled water with 15% solution</td></tr><tr><td>Neodymium magnet</td><td>Supermagnete</td><td>W-01-N</td><td>1 x 1 x 1 mm</td></tr><tr><td>Bearing ball</td><td>Agami Modeling</td><td>SUJ2</td><td>&amp;Phi; 600 &amp;mu;m steel bead</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dowcorning</td><td>Sylgard 184</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma Aldrich</td><td>p2443</td><td>Dilute with phosphate buffered saline to 4% (w/v) solution</td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (D-PBS)</td><td>ATCC</td><td>30-2200</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Adipose-derived mesenchymal stem cells</td><td>ATCC</td><td>ATCC PCS-500-011</td><td></td></tr></tbody></table>

a paired bead and magnet array for molding microwells with variable concave geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

A convex mold and microwell pattern were successfully fabricated by following steps 2.1 to 3.7. (Figure 4). The commercial steel beads were trapped in the 30 x 30 through-hole array. The beads were held tightly without any gaps between the beads and the corresponding through-holes (Figure 4a). The shape of fabricated concave microwell is concave hemispherical, with a diameter of 600 &#181;m, which is the same as that of the steel...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

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8.6K Views.  Chung-Ang University. This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

Video: A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Chemistry

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive.
Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

We show a simple and rapid method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development.

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

Biology

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Engineering

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

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 ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Biochemistry

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

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

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

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