We thank T. Hunt, M. Rosenbluth, and the Lam Lab for their advice and useful discussions. We acknowledge the support from G. Spinner and the Institute for Electronics and Nanotechnology at the Georgia Institute of Technology. Financial support for this work was provided by an NIH grant K08-HL093360, UCSF REAC award, an NIH Nanomedicine Development Center Award PN2EY018244, and funding from the Center for Endothelial Cell Biology of Children's Healthcare of Atlanta.

1. Fabrication of the Endothelial Microdevice
<ol>
 <li>Create a photomask by submitting a computer assisted design (CAD) drawing of the microfluidic device to an outside mask vendor. The mask used was composed of a chrome layer on soda lime glass. In this case the microfluidic channel width was 30 &mu;m.</li>
 <li>Clean a bare silicon wafer with piranha (10:1 ratio of sulfuric acid and hydrogen peroxide) for 15 minutes and dip in hydrofluoric acid for 30 seconds. Rinse with deionized (DI) water for approximately 10 seconds.</li>
 <li>Using a spin coater, spin Microchem SU-8 2025 photoresist onto the wafer to a height of 30 &mu;m. For SU-8 2025, a spin speed of 3000 rpm is recommended. Other viscosities of SU-8 are available which will achieve this height. Full instructions for SU-8 use are available at <a href="http://www.microchem.com" target="_blank">www.microchem.com</a></li>
 <li>Place the wafer with SU-8 on a hotplate at 95 &deg;C for 5 minutes to drive off excess solvent. 
 Note: An oven will dry the wafer differently than a hotplate and is not recommended.</li>
 <li>Place a mask with the desired feature shape over the wafer, and expose to UV light (160 mJ/cm2 measured at 365 nm) in a mask aligner (Karl Suss, MA-6). This cross links the photoresist.</li>
 <li>Place the wafer back on a hotplate at 95 &deg;C for an additional 5 minutes to further accelerate the polymerization of SU-8.</li>
 <li>Immerse the wafer in SU-8 Developer, composed primarily of PGMEA (propylene glycol methyl ether acetate) for 4 minutes to remove the non-cross-linked SU-8.</li>
 <li>Rinse the newly developed wafer with 100% isopropyl alcohol (IPA) for 10 s. The wafer may then be dried using pressurized nitrogen or by allowing the solvent to evaporate from the wafer in a clean fume hood for several minutes.</li>
 <li>Tape the edges of the dry wafer with patterned SU-8 into a petri dish to prevent movement.</li>
 <li>Apply 1 mL of Sigmacote to the wafer using a pipette, cover with the top of the petri dish, and swirl wafer to ensure complete coating of the wafer, remove cover, and allow wafer to dry for several minutes until all solvent has evaporated.</li>
</ol>
2. PDMS (Polydimethylsiloxane) Preparation
<ol>
 <li>Mix PDMS polymer and curing agent at a 10:1 ratio (w/w) and remove air bubbles using a vacuum desiccator. The length of time needed to degas the PDMS varies on the strength of the available vacuum system, but typically ranges from several minutes to one hour. For a six inch petri dish with no previously poured PDMS, a total volume of 60 mL of polymer is recommended.</li>
 <li>Pour the mixture onto the wafer, approximately 5 mm thick. Also pour onto a flat bottom dish to create a thin sheet of PDMS, approximately 1 mm thick. Cure at 60 &deg;C in an oven overnight.</li>
 <li>Using a knife or scalpel, cut out around the cured PDMS device and remove it from the wafer. Additionally, cut a thin sheet of PDMS slightly larger than the device.</li>
 <li>Create inlet and outlet holes in the PDMS device using a 1.0 mm hole punch. This may be accomplished using either a pin vice, Harris Uni-Core, or similar device.</li>
 <li>Clean the surfaces of the device and the sheet using scotch tape.</li>
 <li>Using a plasma cleaner, expose the surfaces of the PDMS device and PDMS sheet to oxygen plasma for 30 s. The oxygen plasma creates reactive species on the exposed surface of the PDMS which bond when brought into physical contact.</li>
 <li>Connect smaller tubing to a small length (several centimeters) of large tubing, which in turn is connected to a syringe with a blunt-point needle, filled with 50 &mu;g/ml fibronectin from human plasma in PBS. The tubing and needle are sized such that a friction fit is created between the tubing. Insert smaller tubing in the inlet of PDMS microdevice and apply pressure to the syringe to fill the channels completely with fibronectin solution and to create a small 100 &mu;L drop at the outlet port, to ensure that the channels stay wet. Incubate the device at 37 &deg;C for 40-60 min.</li>
 <li>Connect a fresh syringe filled with PBS to the blunt point needle. Apply pressure to the syringe to rinse the device with PBS.</li>
</ol>
3. Seeding the Microfluidic Device with Endothelial Cells
<ol>
 <li>Prepare 1,000,000 cells/mL of human umbilical vein endothelial cells (HUVECs) in endothelial growth media with 8% dextran. A range of 500,000 to 2,000,000 cells/mL has been successfully used. Addition of dextran to the cell loading medium increases the viscosity of the fluid, which decreases the velocity of endothelial cells as they enter the fibronectin-coated microfluidic system. This, in turn, increases the likelihood that the cells will adhere and culture successfully within the microdevice.</li>
 <li>Connect the new syringe and tubing as in Step 2.7. but with a longer tubing (approximately 1 meter in length). </li>
 <li>To optimize the performance of this system, it is critical at this point to prevent any leaks or bubbles in the entire perfusion system; any leakage of solution or presence of bubbles in the media will change the flow and prevent successful seeding of endothelial cells. Therefore, tight fitting of the tubing is essential to eliminate leakage between any connections. Bubbles in the syringe and/or tubing must be eliminated before cells are introduced in the microdevice and the entire perfusion system should be primed with the cell solution before attaching the tubing to the microfluidic to avoid the introduction of air bubbles.</li>
 <li>Using a syringe pump, infuse cell suspension into PDMS device at volumetric flow rate of 1.23 &mu;l/min for 2 hours at 37 &deg;C and 5% CO2. Optimal results were achieved when the syringe pump was at the same height as the PDMS device. For the inlet, the longer tubing, which can be coiled within the incubator, ensures that the cells and media are adequately warmed before coming in contact with the device. In our system, the volumetric flow rate of 1.23 &mu;l/min approximates a midstream velocity of ~1 mm/s in the smallest microchannels, corresponding to a wall shear stress of ~1 dyne/cm2 at those channels using whole blood.</li>
 <li>Using the same syringe pump and long tubing, perfuse fresh growth media for 2-8 days at ratio of 1.23 &mu;l/min. A successful device will have a monolayer of endothelial cells growing on the inside of the device within 24-48 hours. Previous experiments have shown that confluent monolayers appropriately express VE-cadherin at cell-cell junctions throughout the device14.</li>
 <li>Inject blood or cell suspension into system for experimentation.</li>
</ol>
4. Representative Results
Using this protocol, standard lithographic microfabrication techniques are used to create the mold needed to produce the microfluidic channels that physiologically mimic the sizescale of the microvasculature (Figure 1A). Using an optimized perfusion technique, endothelial cells then seed and confluently culture the entire inner surface of the microfluidic system within 24-48 hours of cell seeding (Figure 1B). As the microfluidic system is transparent, the entire microdevice can be placed on a brightfield/fluorescence microscope stage for imaging and data collection.
Our system can then be applied to study hematologic diseases that involve altered biophysical properties, such as sickle cell disease, in which the increased rigidity of sickled red cells and aberrant leukocyte and endothelial adhesion contribute to microvascular obstruction. A clinically approved medication, hydroxyurea, ameliorates symptoms but its direct effect on microvascular flow is unknown. Our assay takes into account both cell rigidity and adhesion, and demonstrates that hydroxyurea significantly ameliorates flow in sickle cell disease (Figure 2).
Sickle cell disease is only one example of an application for the microvasculature-on-a-chip, as this system is ideally suited to study any hematologic process in which blood cells interact with each other and endothelial cells in the microvasculature. Other clinically relevant applications include inflammatory disorders, sepsis/lung injury, thrombotic microangiopathies, malaria, and cancer metastasis while more basic applications include leukocyte biology and hematopoietic stem cell biology, among many others.
<img src="/files/ftp_upload/3958/3958fig1.jpg" alt="Figure 1"/> 
Figure 1. A) the initial PDMS microfluidic device before endothelialization. Here, the microdevice is injected with food coloring to illustrate sizescale and overall design of the system. B) Brightfield microscopy shows the microfluidic system is completely endothelialized within 48 hours of cell seeding using the protocol described here.
<img src="/files/ftp_upload/3958/3958fig2.jpg" alt="Figure 2"/> 
Figure 2. A) The microvasculature-on-a-chip with microchannels approximates the size of post-capillary venules (30 &mu;m), the site of most sickle cell microvascular obstructive events. Whole blood from sickle cell patients receiving hydroxyurea and from patients not receiving hydroxyurea is flowed through two different microvasculature-on-a-chip devices. B) Whole blood from sickle cell patients receiving hydroxyurea flows with relative ease within the endothelialized microchannels. C) Under the same hemodynamic conditions, whole blood from sickle cell patients not receiving hydroxyurea, however, exhibits much more sluggish flow with microchannel obstruction. The bottom channel is completely obstructed with no flow and the flow velocities in the other endothelialized microchannels are significantly lower than in the hydroxyurea condition. The scale bar in all three images is 30 &mu;m.

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	<li>Mezzano, D., Quiroga, T., Pereira, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Level+of+Laboratory+Testing+Required+for+Diagnosis+or+Exclusion+of+a+Platelet+Function+Disorder+Using+Platelet+Aggregation+and+Secretion+Assays.">The Level of Laboratory Testing Required for Diagnosis or Exclusion of a Platelet Function Disorder Using Platelet Aggregation and Secretion Assays.</a> Semin. Thromb. Hemost. 35, 242-254 (2009).</li><li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Young, E. W. K., Simmons, C. 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No conflicts of interest declared.

Our endothelialized microdevice system is best suited when used in conjunction with in vivo experiments, and its reductionist approach may help elucidate the biophysical mechanisms of hematologic processes that are observed in humans and animal models. Furthermore, our system is not without limitations. For instance, our microfluidic channels are square in cross-section. Although technically circular microchannels can be fabricated10,11, we opted to use a more simplified and standard fabricatio...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>blunt point needle</td>
 <td>OK International</td>
 <td>920050-TE</td>
 <td>Precision TE needle 20 Gauge x 1/2", pink</td>
 </tr>
 <tr>
 <td>dextran</td>
 <td>Sigma-Aldrich</td>
 <td>31392</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>Sigma-Aldrich</td>
 <td>F0895</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hole puncher (pin vise)</td>
 <td>Technical Innovations</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human umbilical cord endothelial cells (HUVECs)</td>
 <td>Lonza</td>
 <td>CC-2519</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plasma cleaner</td>
 <td>Plasma</td>
 <td>PDC-326</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polydimethylsiloxane (PDMS)</td>
 <td>Fisher Scientific</td>
 <td>NC9285739</td>
 <td>Sylgard 184 Silicone Elastomer KIT</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma-Aldrich</td>
 <td>SL2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SU-8 2025</td>
 <td>Microchem</td>
 <td>Y111069</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SU-8 Developer</td>
 <td>Microchem</td>
 <td>Y020100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Harvard Apparatus</td>
 <td>70-3008</td>
 <td>PHD-ULTRA</td>
 </tr>
 <tr>
 <td>tubing(larger)</td>
 <td>Cole-Parmer Instrument Company</td>
 <td>06418-02</td>
 <td>Tygonreg microbore tubing, 0.020" ID x 0.060" OD</td>
 </tr>
 <tr>
 <td>tubing(smaller)</td>
 <td>Cole-Parmer Instrument Company</td>
 <td>06417-11</td>
 <td>PTFE microbore tubing, 0.012" ID x 0.030" OD</td>
 </tr>
 </tbody>
</table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

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15.8K Views.  Emory University School of Medicine. A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (

Video: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...