This research was partially supported by National Science Foundation (NSF 1227359), WVU EPSCoR program funded by the National Science Foundation (EPS-1003907), WVU ADVANCE office sponsored by the National Science Foundation (1007978), and WVU PSCoR, respectively. The microfabrication work was done in WVU Shared Research Facilities (Cleanroom facilities) and Microfluidic Integrative Cellular Research on Chip Laboratory (MICRoChip Lab) at West Virginia University. The confocal imaging was done at WVU Microscope Imaging Facility.

1. Photolithography Fabrication of Photoresist Master Mold
The following protocol shows the process to fabricate the microchannels with diameters between 30-60 &#956;m. To get a microchannel with a smaller diameter (less than 30 &#956;m), a single spin-coating of photoresist is needed.
<ol>
	<li>Transfer the reflow photoresist from the refrigerator at 4 &#176;C to the cleanroom 24 hr prior to use and allow it to warm to room temperature.</li>
	<li>Clean a silicon wafer and bake it for one hr at 150 &#176;C to allow it to dehydrate. The dehydration will assist the photoresist adhesion to the silicon substrate.</li>
	<li>Spin-coat the first&#160;layer of photoresist using the following recipe:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Time (Seconds)</td>
			<td>Speed (rpm)</td>
			<td>Acceleration</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>300</td>
			<td>highest</td>
		</tr>
		<tr>
			<td>2</td>
			<td>10</td>
			<td>0</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>3</td>
			<td>300</td>
			<td>highest</td>
		</tr>
		<tr>
			<td>4</td>
			<td>60</td>
			<td>600</td>
			<td>highest</td>
		</tr>
		<tr>
			<td>5</td>
			<td>10</td>
			<td>500</td>
			<td>highest</td>
		</tr>
		<tr>
			<td>6</td>
			<td>10</td>
			<td>600</td>
			<td>highest</td>
		</tr>
	</tbody>
</table>
<ol start="4">
	<li>Then, bake the wafer on a hotplate with a temperature of 110 &#176;C for 90 sec. After the soft bake the thickness of the photoresist will be 20-30 &#956;m.</li>
	<li>Spin coat a second&#160;layer of photoresist following the same recipe used for the first&#160;layer.</li>
	<li>Soft bake the wafer again by placing it on a hotplate with a temperature of 110 &#176;C for 90 sec. After this soft bake the thickness of the photoresist will be 40-60 &#956;m.</li>
	<li>Generate positive master patterns by exposing the photoresist to UV light with an exposure dose of 14,500 mJ/cm2 through a film mask.</li>
	<li>Dilute the developer with deionized (DI) water (1:2 (v/v)). Rinse the wafer repeatedly in the solution until the pattern is fully developed. Then&#160;wash the wafer with DI water and dry it using nitrogen gas.</li>
	<li>Reflow: Place the wafer on a hotplate with a temperature of 120 &#176;C for 4 min and cover with a glass Petri dish to prevent solvent evaporation. Remove the wafer from the hotplate and allow it to cool to room temperature. The photoresist thickness after reflow will be 50-60 &#956;m.</li>
</ol>
2. Soft Lithography Fabrication of PDMS Microchannel Network
<ol>
	<li>Prepare polydimethylsiloxane (PDMS) solution at the weight ratio of 10:1 (base:curing agent) and mix it thoroughly using a planetary centrifugal mixer.</li>
	<li>Cast the PDMS solution onto the reflowed photoresist master mold. Place the casted PDMS in a desiccator for 15 min to degas. Use nitrogen gas to remove any remaining bubbles if necessary.</li>
	<li>Bake the PDMS in an oven at a temperature of 60 &#176;C for 3 hr to allow it to cure. Then remove the cured PDMS layer from the master mold.</li>
	<li>Use a sharpened puncher to create inlet/outlet holes by punching holes in the channel network. Clean the surface of the PDMS using nitrogen gas.</li>
	<li>Treat two PDMS layers with oxygen plasma for 30 sec inside a plasma cleaner at an operating pressure of 4.5 x 10-2 Torr and an oxygen flow rate of 3.5 ft3/min. Then, align the surfaces of the PDMS manually under an optical microscope. Use a drop of water if necessary for a better control of the alignment.</li>
	<li>Bake the device in an oven at 60 &#176;C for 30 min to achieve permanent bonding.</li>
</ol>
3. HUVEC Culture and Seeding in the Chip
<ol>
	<li>Culture the HUVECs with culture medium with L-glutamine supplemented with 10% fetal bovine serum (FBS). For the experiment passages between 2 and 5 were used.</li>
	<li>Once the HUVECs are confluent, count the cells and harvest them by first rinsing the cells with HEPES buffered saline solution (HEPES-BSS), and then treat the cells with trypsin/EDTA and incubate for 2-6 min at 37 &#176;C. After the trypsinization is complete, neutralize the trypsin/EDTA with trypsin neutralizing solution. Centrifuge and suspend the cells in culture medium with 8% dextran to collect them. Dextran was used to increase the medium viscosity to aid in better cell seeding and attachment.</li>
	<li>Treat the device with oxygen plasma for 5 min with an operating pressure of 4.5 x 10-2 Torr and an oxygen flow rate of 3.5 ft3/min. Then&#160;load the device with DI water and treat with UV light for 8 hr in a laminar biosafety hood for sterilization.</li>
	<li>One day before the cells are ready, wash the device with 1x phosphate buffered saline (1x PBS) then coat with fibronectin (100 &#956;g/ml, diluted with 1x PBS) and incubate in the refrigerator at 4 &#176;C overnight.</li>
	<li>After the fibronectin coating, wash the device with 1x PBS then load with culture medium. Incubate the device at a temperature of 37 &#176;C for 15 min.</li>
	<li>Load the HUVEC cells in 8% dextran culture medium with a cell concentration of 3-4 x 106 cells/ml. Place a 20 &#956;l droplet of cells at one inlet of the device and tilt it to introduce the cells into the microfluidic channel. After 15-20 min the cells will begin to attach to the side walls of the channels. Rotate the device every 15 min to create a more uniform distribution of cells. If necessary, additional loading can be performed.</li>
</ol>
4. Long-term Perfusion Setup
After 5-6 hr of static culture, the attached HUVECs will start to fully spread out. Set up perfusion using a remote controlled syringe pump system with a steady flow of 10 &#956;l/hr. Perfusion can be adjusted for a higher flow rate, and can last for a time period between 4 days to&#160;2 weeks.
5. Cell Staining and Microscope Characterization
<ol>
	<li>When the cells reach confluence inside the device, firstly, wash the device with 1x PBS to thoroughly remove the medium. Then load the device with red dye diluted with diluent (4 &#956;l-1 ml). Load the dye similar to the cell loading procedure. Incubate the device in the dark for 5 min at room temperature, and then wash the device with culture medium to stop the staining. Long incubation of the dye can cause cellular toxicity and disadhesion.</li>
	<li>Load the device with blue dye diluted with 1x PBS (2 droplets per ml). Incubate in the dark for 5 min at room temperature then thoroughly wash the device with 1x PBS.</li>
	<li>Examine the cell staining under an inverted optical microscope. If the staining was good, load the microchannels with fixing medium (3.5% paraformaldehyde diluted with 1x PBS), then submerge the device in fixing medium and completely cover it with aluminum foil. Store the device in the refrigerator at a temperature of 4 &#176;C to prevent the device from drying out and photobleaching. The fixed device is now ready for confocal imaging, which can be done by a laser scanning confocal microscope.</li>
</ol>

The authors declare that they have no competing financial interests.

1. Master mold fabrication
One of the designing and guiding principles for vascular morphometry is known as Murray&#39;s law16, which states that the distribution of vessel diameters throughout the network is governed by minimum energy consideration. It also states that the cube of the diameters of a parent vessel at a bifurcation equals the sum of the cubes of the diameters of the daughter vessels (<img alt="Equation 1" fo:content-width="0.9in" fo:src="/files/ftp_upload/50771/50771eq1...

Microvessels, as a part of the circulation system, mediate the interactions between blood and tissues, support metabolic activities, define tissue microenvironment, and play a critical role in many health and pathological conditions. Recapitulation of functional microvessels in vitro could provide a platform for the study of complex vascular phenomena. However, conventional in vitro microvessel assays, such as endothelial cell migration assays, endothelial tube formation assays, and rat and mouse aortic ring assays, are unable to recreate the in vivo microvessels with respect to three-dimensional (3D) geometry and continuous flow control1-8. Studies of microvessels using animal models and in vivo assays, such as corneal angiogenesis assay, chick chorioallantoic membrane angiogenesis assay, and Matrigel plug assay, are more time-consuming, high in cost, challenging with respect to observation and quantifications, and raise ethical issues1, 9-13.
Advances in micromanufacturing and microfluidic chip technologies have enabled a variety of insights into biomedical sciences while curtailing the high experimental costs and complexities associated with animals and in vivo studies14, such as easily and tightly controlled biological conditions and dynamic fluidic environments, which would not have been possible with conventional macroscale techniques.
Here, we present an approach to construct an endothelialized microchannels-on-a-chip which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow by using the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflow Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZP4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 400K</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Culture Medium</td>
			<td>Invitrogen</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr>
			<td>NacBlue Nuclei Staining</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH Red Stain</td>
			<td>Sigma</td>
			<td>MINI26 and PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Curing Agent</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Invitrogen, Molecular Probes</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>95771</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>BelArt Products</td>
			<td>999320237</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump System</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Fisher Scientific</td>
			<td>13-246-516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Invertoskop 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Barnstead/Thermolyne Cimarec</td>
			<td>SP131635</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Our approach to fabricate the multi-depth microchannel network mimics the complex 3D geometries of in vivo microvessels, in which the microchannels have rounded cross sections15. Additionally, the diameters of parent branching channels and the daughter channels approximately obey Murray&#39;s law for maintaining the fluid flow at a required level so that the overall channel resistance is low and flow velocities are more uniform throughout the network16-18. The processes and results for the ...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

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

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, ...

procedure-for-development-multi-depth-circular-cross-sectional

Research

JoVE Journal

Bioengineering

13.8K Views.  West Virginia University. 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 and can be applied for microvascular research.

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

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.

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 ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...