This work was supported by the National Institute of Health grant # 1R01CA120825-01A1, the Collaborative Biomedical Research (CBR) Program at the University of Massachusetts Amherst, and the Isenberg Scholarship for Bhushan J. Toley. We gratefully acknowledge the valuable contribution of James Schafer, the videographer, narrator, and editor of this video.

1. Device Fabrication
Replication of microfluidic features in elastomeric materials was based on the method described by Duffy et al.1
<ol>
	<li>Mix elastomer (polydimethylsiloxane; PDMS) and curing agent from the Silicone Elastomer Kit (Dow Corning, Midland, MI) in a 9:1 weight ratio and pour over the master to form a 4 mm thick layer. Degas to remove air bubbles and cure the mixture at 60&deg;C for 5 hours. Peel cured PDMS from the mold to obtain a stamp of flow features on the elastomer.</li>
	<li>Punch holes for inlets and outlets using a 1.5 mm biopsy punch (Miltex, York, PA) mounted on a drill press. Pull out any debris.</li>
	<li>Subject the features side of the stamp and a clean glass slide to oxygen plasma for 8 minutes in an oxygen plasma etcher. Bring the treated surfaces in contact immediately to form a bond between them. Maintain the assembly at 60&deg;C on a slide warmer for at least 5 hours to strengthen the bond.</li>
	<li>Connect 0.032" ID PTFE tubing (Cole Parmer, Vernon Hills, IL) to the inlets and outlets using an interface of male luer lock connectors attached to barbed female luer lock connectors (Qosina, Edgewood, NY).</li>
	<li>Set up the flow assembly using shut off valves and a Y-connector (Upchurch Scientific, Oak Harbor, WA; Figure 1). Mount the device on to the microscope. Attach syringes to tubing using 20G 1.5" needles (BD Bioscience, Rockville, MD).</li>
</ol>
2. Formation of Uniform Spheroids
Spheroids were formed by the hanging drop method2,3.
<ol>
	<li>Trypsinize cells under a sterile cell culture hood.</li>
	<li>Centrifuge trypsinized cells at 2000 rpm for 5 minutes and resuspend in 6 ml fresh medium. Dilute the stock solution to a desired final concentration (Table 1).</li>
	<li>Remove the cover of a 48 well plate and place it upside down in the sterilized hood. Circular regions corresponding to each well are apparent on the cover. Fill each well of the plate with 1 ml of sterilized water to maintain humidity. Put a 20 &mu;l droplet of the dilute cell solution in each circular region using a micropipette. Carefully invert the cover and place it over the well plate, ensuring that the drops do not touch the edges of the wells. </li>
	<li>Incubate the well plate at 37&deg;C for a specified number of days (Table 1). A spheroid will form in each hanging drop.</li>
</ol>
3. Introduction of Spheroids into Device
Refer to Figure 1 and Table 2 for this section.
<ol>
	<li>Sterilize the device by flushing with 70% ethanol introduced through the flow inlet. Subsequently, flush with PBS followed by HEPES buffered cell culture medium. Leave the medium syringe SF attached to the tubing.</li>
	<li>Draw 2-3 spheroids into syringe SP, tap syringe to remove air bubbles, and attach to the packing inlet of the device.</li>
	<li>Open inlet valve VPin and close VFin. Open outlet valve VPout and close VFout. Hold the packing syringe vertical with the needle pointing downward; watch as spheroids settle to the bottom of the syringe into the luer lock connector of the needle. Push the plunger on the packing syringe and watch spheroids enter the tubing and flow into the device. Because only the packing outlet valve is open, a spheroid will enter the chamber of the device and be retained by posts at the back (Fig. 2).</li>
	<li>Close inlet valve VPin and outlet valve VPout. Mount the syringe SF on a syringe pump. Open valve VFin and valve VFout. Flow medium into the device at 3 &mu;l/min.</li>
</ol>
4. Introduction of Apoptosis Detecting Agent (CaspGLOW)
After packing chambers with spheroids, allow equilibration for up to 24 hours to establish nutrient gradients and microenvironments, before introducing apoptosis detecting or therapeutic agents. Because the tissues in chambers are in contact with impenetrable walls from top and bottom, there are no nutrient gradients along the thickness (0.15mm) of the tissue. After 24 hours of incubation, all cell layers along the thickness of the tissue are therefore equivalent and the only heterogeneity is in a direction away from the flow channel.
<ol>
	<li>Shut off VFin, stop the syringe pump, remove syringe SF from the pump and replace it with a syringe having medium containing 0.25 &mu;l/ml of CaspGLOW Red Active Caspase-3 marker (Red-DEVD-FMK; Biovision, Mountain View, CA). </li>
	<li>Manually flush 0.7 ml of this solution through the device to ensure displacement of older medium. Mount the syringe on the pump, restart flow, and open VFin. Over a period of 5-8 hours, the apoptosis detecting agent diffuses into the tissue and fluoresces brighter in apoptotic regions. Apoptosis detecting agent at this concentration is maintained in all subsequent flow solutions into the device.</li>
</ol>
Note:
<ol class="lroman">
	<li>Tissue heterogeneity can be confirmed by obtaining linear intensity profiles of fluorescence as described in Section 6. The profiles should show spatial gradients in apoptosis, confirming that the tissue is heterogeneous with respect to cell viability.</li>
	<li>CaspGLOW Green Active Caspase-3 marker (Fluorescein-DEVD-FMK; Biovision) can also be used to detect apoptosis. This marker contains the green fluorophore fluorescein instead of the red fluorophore rhodamine.</li>
</ol>
5. Introduction of Therapeutic Agent
<ol>
	<li>Follow the procedure in steps 4.1-4.2 to introduce 10 &mu;M doxorubicin hydrochloride (Dox; Sigma-Aldrich, St. Louis, MO) containing apoptosis detecting agent.</li>
	<li>Continue flow of the therapeutic agent solution for a fixed period of time. Shut off valve VFin and turn off the syringe pump. Replace the treatment syringe with a fresh medium syringe containing apoptosis detecting agent. Allow flow of fresh medium for 24 &#x2013;36 hours while continuously monitoring the tissue under a microscope.</li>
</ol>
6. Time-lapse Microscopy and Estimation of Drug Diffusivity Coefficients
For obtaining cleanest fluorescence data, acquire all images by focusing the microscope on the bottom layers of tissues. Because there are no nutrient gradients in the vertical direction (see Section 4), the bottom layers of cells are good representatives of all other layers above.
<ol>
	<li>The entire acquisition process was automated using a customized script in IPLab (BD Bioscience, Rockville, MD). Acquire transmitted light 
	and fluorescence images of the packed spheroid at 10x magnification every 30 minutes (Fig. 3A). Acquire an image of the background 
	fluorescence prior to introduction of Dox. To accommodate for the large size of the chamber (1000 &mu;m x 300 &mu;m), acquire two adjoining 
	images and tile them together4.</li>
	<li>For mathematical estimation of drug diffusivity coefficients, the first step is to generate averaged linear intensity profiles of Dox fluorescence using ImageJ. Select a rectangular region of interest (ROI) encompassing the tissue in the chamber. Use the Plot Profile command to generate a profile of average intensities as a function of distance from the flow channel. Repeat for up to 3 different time points.</li>
	<li>Obtain a background fluorescence image before introducing Dox to measure autofluorescence from the tissue. Subtract the average background fluorescence intensity from the obtained intensity profiles and normalize each profile by the corresponding maximum intensity to obtain <img src="/files/ftp_upload/2425/2425eq0.jpg" alt="c" />(x,t)(Fig. 3B).</li>
	<li>The next step is to evaluate the effective diffusion coefficient D of Dox within tumor tissue. Dox diffusion can be represented by the following equation5 
	<img src="/files/ftp_upload/2425/2425eq1.jpg" alt="Equation 1"/> 
	where erfc is the complementary error function, x is distance into the tissue from the channel, and t is time after introduction of Dox (refer to Fig. 3). Use the following iterative scheme at each time point considered:
		<ul>
			<li>Guess a value for D</li>
			<li>Calculate right side of the equation at each location x</li>
			<li>Calculate the sum of squared errors (residual) between the two sides</li>
			<li>Modify D to minimize the residual</li>
		</ul>
	</li>
	<li>Average the optimum values of D obtained at each time point to estimate the average effective diffusion coefficient of Dox in the tissue.</li>
</ol>
7. Representative Results:
The microfluidic devices provided 1mm x 0.3mm x 0.15mm optically accessible culture chambers for growth of 
three-dimensional tumor tissue. Multicellular tumor spheroids were flown into these chambers and were retained by two filter posts at 
the back. The hanging drop method allowed quick formation of spheroids of consistent size and shape from several cell lines. Spheroids 
were successfully grown on the device for up to 3 days. Growth in the chambers was associated with a reproducible modification of 
microenvironments within spheroids. Apoptosis occurred less in cells in proximity of the flow channel and higher deeper into the tissue. 
The device was used to estimate the diffusion coefficient of doxorubicin in tumor tissue. The obtained value of 8.75 x 10-7 cm2s-1 agrees 
with the value of 9.1 x 10-7 cm2s-1 reported previously6 in human breast cancer.
<table border="1">
	<tbody>
		<tr>
			<td>Cell Line</td>
			<td>Required Cell Concentration</td>
			<td>Incubation Time</td>
		</tr>
		<tr>
			<td>LS174T</td>
			<td>300 cells/&mu;L</td>
			<td>2-3 days</td>
		</tr>
		<tr>
			<td>T47D</td>
			<td>750 cells/&mu;L</td>
			<td>3-4 days</td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>150 cells/&mu;L</td>
			<td>5-6 days</td>
		</tr>
	</tbody>
</table> 
Table 1. Parameters for Hanging Drop Spheroids
<table border="1">
	<tbody>
		<tr>
			<td>Part</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>SF</td>
			<td>Flow Syringe</td>
		</tr>
		<tr>
			<td>SP</td>
			<td>Packing Syringe</td>
		</tr>
		<tr>
			<td>VFin</td>
			<td>Flow Inlet Valve</td>
		</tr>
		<tr>
			<td>VPin</td>
			<td>Packing Inlet Valve</td>
		</tr>
		<tr>
			<td>VFout</td>
			<td>Flow Outlet Valve</td>
		</tr>
		<tr>
			<td>VPout</td>
			<td>Packing Outlet Valve</td>
		</tr>
	</tbody>
</table>
Table 2. Syringes and Valves in Flow Setup
<img src="/files/ftp_upload/2425/2425fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic of the flow setup VFin and VFout: Flow inlet and outlet 
valves; VPin and VPout: Packing inlet and outlet valves; SF and SP: Flow and packing syringes. The 
packing syringe and packing inlet and outlet valves are used when a spheroid is flown into the chamber. The flow syringe and flow inlet 
and outlet valves are used to flow medium subsequently.
<img src="/files/ftp_upload/2425/2425fig2.jpg" alt="Figure 2" /> 
Figure 2. Schematic of a spheroid trapped in a chamber on the device. A spheroid is flowed into the chamber on the device and is blocked by two filter posts at the back of the chamber.
<img src="/files/ftp_upload/2425/2425fig3.jpg" alt="Figure 3" /> 
Figure 3. Doxorubicin diffusion in tumor tissue on the device. A. Merged transmitted light and red fluorescent image of tissue, showing the location and concentration of doxorubicin (in red). Scale bar represents 250 &mu;m. B. Normalized linear intensity profile from doxorubicin fluorescence.

<ol>
	<li>Duffy, D. C., McDonald, J. C., Schueller, O. J., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Prototyping+of+Microfluidic+Systems+in+Poly(dimethylsiloxane).">Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).</a> Anal. Chem. 70, 4974-4984 (1998).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnol. Bioeng. 83, 173-180 (2003).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods. Mol. Med. 140, 141-151 (2007).</li><li>Kasinskas, R. W., Forbes, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+specifically+chemotax+and+proliferate+in+heterogeneous+tumor+tissue+in+vitro.">Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro.</a> Biotechnol. Bioeng. 94, 710-721 (2006).</li><li>Walsh, C. 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+multipurpose+microfluidic+device+designed+to+mimic+microenvironment+gradients+and+develop+targeted+cancer+therapeutics.">A multipurpose microfluidic device designed to mimic microenvironment gradients and develop targeted cancer therapeutics.</a> Lab. Chip. 9, 545-554 (2009).</li><li>Lankelma, J., Fernandez Luque, R., Dekker, H., Schinkel, W., Pinedo, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mathematical+model+of+drug+transport+in+human+breast+cancer.">A mathematical model of drug transport in human breast cancer.</a> Microvasc. Res. 59, 149-161 (2000).</li><li>Less, J. R., Skalak, T. C., Sevick, E. M., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+architecture+in+a+mammary+carcinoma:+branching+patterns+and+vessel+dimensions.">Microvascular architecture in a mammary carcinoma: branching patterns and vessel dimensions.</a> Cancer. Res. 51, 265-273 (1991).</li><li>Brown, J. M., Giaccia, A. 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+unique+physiology+of+solid+tumors:+opportunities+(and+problems)+for+cancer+therapy.">The unique physiology of solid tumors: opportunities (and problems) for cancer therapy.</a> Cancer. Res. 58, 1408-1416 (1998).</li><li>Thomlinson, R. H., Gray, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histological+structure+of+some+human+lung+cancers+and+the+possible+implications+for+radiotherapy.">The histological structure of some human lung cancers and the possible implications for radiotherapy.</a> Br. J. Cancer. 9, 539-549 (1955).</li><li>Minchinton, A. I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nat. Rev. Cancer. 6, 583-592 (2006).</li></ol>

No conflicts of interest declared.

The vasculature in tumors is sparse and ill developed7,8. There are regions located far (>100 &mu;m) from the 
blood vessels that are inaccessible to nutrients and drugs supplied though the vasculature9. The resulting heterogeneous microenvironment contributes to the limited efficacy of many chemotherapeutics10. The microfluidic device developed here recreates a heterogeneous tumor microenvironment characterized by proliferating, quiescent and apoptotic or necrotic cells, in vitro. Cells ...

<table border="1">
	<tbody>
		<tr>
			<th>Name of reagent</th>
			<th>Company</th>
			<th>Catalog number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Silicone elastomer kit</td>
			<td>Ellsworth Adhesives</td>
			<td>184 Sil Elast Kit</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Miltex biopsy punch</td>
			<td>MedexSupply</td>
			<td>MTX-33-31AA</td>
			<td>1.5 mm</td>
		</tr>
		<tr>
			<td>PTFE tubing</td>
			<td>Cole Parmer</td>
			<td>EW-06417-31</td>
			<td>0.032" ID</td>
		</tr>
		<tr>
			<td>Male luer lock connector</td>
			<td>Qosina</td>
			<td>65111</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Barbed female luer lock connector</td>
			<td>Qosina</td>
			<td>11556</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shut-off valve</td>
			<td>Idex Health and Science</td>
			<td>P-721</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Y-connector</td>
			<td>Idex Health and Science</td>
			<td>P-513</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20G 1.5" needles</td>
			<td>BD Bioscience</td>
			<td>305176</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-4034</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaspGLOW Fluorescein</td>
			<td>Biovision</td>
			<td>K183-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaspGLOW Red</td>
			<td>Biovision</td>
			<td>K193-25 </td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Doxorubicin Hydrochloride</td>
			<td>Sigma</td>
			<td>44583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LS174T</td>
			<td>ATCC</td>
			<td>CCL-188</td>
			<td>Human Colon Carcinoma cell line</td>
		</tr>
		<tr>
			<td>T47D</td>
			<td>ATCC</td>
			<td>HTB-133</td>
			<td>Human Ductal Carcinoma cell line</td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td>Human Mammary Adenocarcinoma cell line</td>
		</tr>
	</tbody>
</table>

microfluidic device for recreating a tumor microenvironment in vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Watch this Scientific Journal Video about Microfluidic Device for Recreating a Tumor Microenvironment in Vitro at JoVE.com

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

microfluidic-device-for-recreating-a-tumor-microenvironment-in-vitro

Research

JoVE Journal

Bioengineering

11.5K 조회수.  University Of Massachusetts Amherst. 우리는 이기종 종양 microenvironments를 재현 마이크로 유체 장치의 제조 및 운영을위한 절차를 제시 체외에서. 종양 조직에서 apoptosis의 다양성이 평가 된 형광 얼룩과 종양 조직에 chemotherapeutic 약물 doxorubicin의 효과적인 확산 계수를 사용하여 정량되었다.

비디오: Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...