Preparing Microfluidic Devices For Neuron Cell Culture
1) Clean Corning No.1 24 mm X 40 mm glass slides
<ul>
<li>Sonicate the glass slides in a water bath sonicator for 30 minutes</li>
<li>Rinse the glass slides with 70% EtOH</li>
<li>Wash slides three times with dH2O</li>
<li>Dry slides in a tissue culture hood for several hours, preferably overnight</li>
</ul>
Note: We have special metal trays that we load with the slides. The slides are separated individually and placed in slots in this metal rack. We can then place these metal loading trays in a glass dish that we fill with water, place in the water bath sonicator, and sonicate for 30 minutes. Subsequent washes are carried out in this dish.
2) Preparing the PDMS devices
<ul>
<li>Cut out the PDMS mold from the silicon wafer, punch holes in the PDSM cast and quarter it</li>
<li>Clean the PDMS devices by first blowing inert gas (Argon or Nitrogen) across them. Then use 3M Scotch Brand 471 tape to lift off any remaining debris</li>
</ul>
Note: It is important to keep the devices face up. Initially, when we cut the PDMS away from the silicon wafer, the side in contact with the wafer is the clean side: it contains the microfluidic channels. We try to be as careful as we can as to not damage the device, and keep the clean side face up until we are ready for plasma bonding.
<ul>
<li>Autoclave devices in plastic containers</li>
<li>After autoclaving, clean No. 1 Corning glass slides and plasma-treat devices using a Harrick brand plasma cleaner, <a href="http://www.harrickplasma.com/" target="_blank">www.harrickplasma.com</a> </li>
<li>Place the devices clean side down on the glass slide.</li>
</ul>
The device is now plasma bonded to the glass slide.
3) Coating The Devices With Poly L-Lysine (PLL)
<ul>
<li>PLL is added to a well on each side of the device and allowed to flow through into the next well</li>
</ul>
=&gt; 150 &micro;l of PLL for the large wells and 30 &micro;l PLL for the smaller wells. It does not have to be exact as long as the wells are full.
Note: If you look at a device you will see 4 wells: each two are connected. You want to put the PLL in one well so that it will flow through the device into the next well.
<ul>
<li>Allow the PLL to flow through the device for about 10 minutes and then add more PLL to the wells to fill them up</li>
<li>Place the devices containing PLL in an incubator at 37&deg;C for a minimum of 4 hours (overnight is preferable)</li>
<li>Vacuum out the excess PLL after treatment, but be careful not to suck all the liquid out of the device</li>
</ul>
Note: You do not want to introduce air bubbles to the channels or chamber. Just vacuum out the excess PLL in the wells.
<ul>
<li>Add autoclaved dH2O to each well on either side of the device, and allow to flow to the other well by capillary action</li>
</ul>
Figure 1 is a diagram of a microfluidic device. Notice how the Blue (Soma side) is connected and the Red (Axonal side) are connected. When we say put the PLL, or water or media, on one well on each side of the device, what we mean, for example, is: Place 150 ul of autoclaved dH2O in one Blue Well then place 150 &micro;l of autoclaved dH20 into one Red Well. The water (or PLL, or Media, or Cells) will flow through the device to the other corresponding well.
<img src="/files/ftp_upload/old/410_2008_8_27_3/410_Figure-01.png" alt="Figure 1" width="362" height="216" />
&nbsp; Figure 1

Note: Please note that, from now on, when I say "place media, cells, water or PLL in the TOP wells", I am referring to the diagram above where the Soma would be on the left and the Axons will grow to the right.
<ul>
<li>Aspirate off the water (again being careful not to fully remove all the liquid from the device), then add another 150 &micro;l of autoclaved dH2O to one of each connected well and allow it to flow through to the corresponding well.</li>
<li>Place the device containing dH2O in an incubator at 37&deg;C for 1 hour, then repeat the wash step again. Wash the devices three times with dH2O for one hour each, which is the total.</li>
</ul>
Note: Due to the possibility that borate from the PLL can absorb into the PDMS, some in the Jeon Lab recommend incubating the devices overnight with autoclaved dH2O after a couple of initial quick rinses. This will ensure&nbsp;that&nbsp;all free borate will leach out of the PDMS prior to the loading of cells. If this is not done, there may be a release of borate into the media over time, which could lead to cytotoxicity.
<ul>
<li>Add Neural Basal Media (NBM) with the necessary factors (glutamax, B27, PenStrep) to the top wells </li>
</ul>
Note: The devices can be incubated overnight and plated with cells the next day, or incubated a minimum of 3 hours with NMB + factors before plating the cells.
Preparing The Neurons For Loading Into The Devices
We use 18 day old fetal rat cortex that we either buy from a company called Brain Bits or that is prepared here on campus for us. We prefer to get the tissue fresh.
<ol>
<li>Obtain 1 fetal brain cortex (2 pieces of tissue) stored in Hibernate E</li>
<li>Take 3 long glass pipettes and fire them each into successively smaller sizes using an alcohol lamp</li>
<li>remove tissues from Hibernate E with a glass pipette and place in 2 ml of NMB + Factors in a sterile 15 ml tube.</li>
<li>Trituration: Using a bulb and glass pipette the cortex is passed up and down about 5 to 10 times. Then, the next smaller pipette is used to pipette up and down the tissue. Finally, the smallest pipette is used to pipette up and down the tissue. We like to make sure that there are no visible chunks. Note: Recently, the Jeon Lab began comparing cells from tissue stored in Hibernate E to cells that were prepared from Tissue initially treated with 0.125% Trypsin in 50% Ca++ free and Mg++ free dissection buffer. The consensus is that tissue prepared using trypsin yield more viable cells than tissue placed initially in Hibernate E. If you are NOT going to use the tissue right away, then you need to store the tissue in Hibernate E. If you are going to use the tissue right away and would like increased viability, then treat the tissue with Trypsin prior to mechanical trituration as following: 1) dilute 1 ml 0.25% Trypsin (Gibco, Invitrogen) with 1 ml of Ca++ free Mg++ free dissection buffer (final Trypsin = 0.125%) into a 15 ml tube (keep ice cold); 2) place tissue in buffer and incubate immediately at 37&deg;C for 8 minutes; 3) add 10 ml DMEM/10% FBS to stop the Trypsin reaction and continue protocol below. </li>
<li>Centrifuge cells at 1100 rpm for 1 minute</li>
<li>Aspirated media carefully off the cell pellet</li>
<li>Add 1 ml of NMB + factors to the pellet and re-suspended it by pipetting up and down</li>
<li>Pass the re-suspended cells by gravity flow through a filter to remove any clumps (BD Falcon cell strainer 40 um nylon)</li>
<li>Count and plate cells: 
<ul>
<li>Into a microfuge tube, add 60 &micro;l NMB + factors, 20 &micro;l of cell suspension, and 20 &micro;l of trypan blue then mix</li>
<li>Add about 10 &micro;l of this solution to a hemocytometer and count the live cells (not blue)</li>
<li>Adjust concentration to 2.5 - 4.5 x106 cells/ml Note: We generally obtain a concentration of about 2.5 - 4.5 x 106 cells/ml. depending on the volume the cells are resuspended in (normally 1 ml NBM + B27/Glutamax/PenStrep percortex). If the tissue was prepared with trypsin, the yield will likely be increased.</li>
</ul>
</li>
</ol>
Plating The Devices
<ol>
<li>Load cells (primary neurons) into one well of the device </li>
<li>Plate 5 &micro;l per small device and 20 &micro;l per big device Note: You can load 10 &micro;l of cells on the top of the device and 10 &micro;l on the bottom of the device (half total volume), or directly all 20 &micro;l of cells in the top somal well (5 &micro;l in the top somal well for the small devices). </li>
<li>Incubate for 10 minutes in a 37&deg;C incubator so cells can start to attach</li>
<li>Add approximately 150/30 &micro;l of NMB + factors to each well depending on device size Note: Volumes may vary depending on the thickness of the PDMS. All that matters is that the wells are full.</li>
</ol>
Supplemental On Using The Devices Without Plasma Bonding
Without plasma treating, Christina Tu, a technician, who does it successfully every week&nbsp;without plasma, says to just load the cells right away.
Remember to remove the media from both wells on the soma side (axon side won't matter if you leave the media in). It's the fluid flow that allows the cells to enter the main channel. If you have media in the wells, you won't get fluid flow.

<ol>
	<li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 1 (4), 2128-2136 (2006).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2 (8), 599-605 (2005).</li></ol>

The authors have nothing to disclose.

Here we demonstrated how to use the neuron microfluidic device without needing a plasma cleaner. We refer to this as non-plasma bonding.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PDMS</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>Sylgard 184 with curing agent Please consult Dow Corning to find a vendor near you</td>
</tr>
<tr>
<td>Cornig No. 1 Cover Glass</td>
<td>Cover Glass</td>
<td>Corning</td>
<td>Corning No. X2935 244</td>
<td>Available through Fisher Scientific, Fish Catalog number 12-531D</td>
</tr>
<tr>
<td>B27</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>Glutamax</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.</td>
</tr>
<tr>
<td>60 mm Petri Dish</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>08-757-13A</td>
<td>60 mm polystyrene sterile petri dishes are used to house the device bound to glass.</td>
</tr>
<tr>
<td>Poly-L-Lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-1274</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon 50 ml Tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352098</td>
<td>Available through Fisher Scientific catalog number 14-959-49A</td>
</tr>
<tr>
<td>BD Falcon 15 ml tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352097</td>
<td>Available through Fisher Scientific catalog number 14-959-70C</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

non-plasma bonding of pdms for inexpensive fabrication of microfluidic devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

Watch this Scientific Journal Video about Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices at JoVE.com

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

non-plasma-bonding-pdms-for-inexpensive-fabrication-microfluidic

Research

JoVE Journal

Biology

34.3K ビュー.  University of California, Irvine (UCI). このビデオでは、プラズマの結合なしニューロンマイクロ流体デバイスを使用する方法を示します。

ビデオ: Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

PDMS Device Fabrication and Surface Modification

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...