Name: Microfluidic Device for Recreating a Tumor Microenvironment in Vitro (Video) | JoVE Journal
Uploaded: 2011-11-20T14:00:00.000+00:00
Duration: 16 min 18 s
Description: Learn to fabricate a microfluidic device that recreates 3D tumor microenvironments in vitro for testing chemotherapeutic drugs like doxorubicin. This method enables accurate drug diffusion studies and improved cancer research.

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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°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°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 μl 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°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 μl/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 μl/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 μM 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 –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 μm x 300 μm), 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="Static equilibrium ΣFx=0 diagram, forces balance analysis, physics concept, vector representation." data-altupdatedat="1788235205000" data-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="Diffusion equation \( \tilde{c}(x,t) = \text{erfc}\left(\frac{x}{2\sqrt{D \cdot t}}\right) \) formula." data-altupdatedat="1788235205000" data-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/μL</td>
			<td>2-3 days</td>
		</tr>
		<tr>
			<td>T47D</td>
			<td>750 cells/μL</td>
			<td>3-4 days</td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>150 cells/μL</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="Culture chamber flow diagram, showing fluid dynamics setup with valves and tubing connectors for cell growth." data-altupdatedat="1788235205000" data-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="Spheroid chamber diagram with filter posts, indicating medium flow direction." data-altupdatedat="1788235205000" data-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="Microfluidic device showing Doxorubicin diffusion and fluorescence intensity graph." data-altupdatedat="1788235205000" data-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 μm. 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/pubmed?term=((Rapid+Prototyping+of+Microfluidic+Systems+in+Poly%28dimethylsiloxane%29%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">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/pubmed?term=((Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">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/pubmed?term=((Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method%5BTitle%5D)+AND+%22Methods.+Mol.+Med%22%5BJournal%5D)">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/pubmed?term=((Salmonella+typhimurium+specifically+chemotax+and+proliferate+in+heterogeneous+tumor+tissue+in+vitro%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">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/pubmed?term=((A+multipurpose+microfluidic+device+designed+to+mimic+microenvironment+gradients+and+develop+targeted+cancer+therapeutics%5BTitle%5D)+AND+%22Lab.+Chip%22%5BJournal%5D)">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/pubmed?term=((A+mathematical+model+of+drug+transport+in+human+breast+cancer%5BTitle%5D)+AND+%22Microvasc.+Res%22%5BJournal%5D)">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/pubmed?term=((Microvascular+architecture+in+a+mammary+carcinoma%3A+branching+patterns+and+vessel+dimensions%5BTitle%5D)+AND+%22Cancer.+Res%22%5BJournal%5D)">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/pubmed?term=((The+unique+physiology+of+solid+tumors%3A+opportunities+%28and+problems%29+for+cancer+therapy%5BTitle%5D)+AND+%22Cancer.+Res%22%5BJournal%5D)">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/pubmed?term=((The+histological+structure+of+some+human+lung+cancers+and+the+possible+implications+for+radiotherapy%5BTitle%5D)+AND+%22Br.+J.+Cancer%22%5BJournal%5D)">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/pubmed?term=((Drug+penetration+in+solid+tumours%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">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><tbody><tr><td>Silicone elastomer kit</td><td>Ellsworth Adhesives</td><td>184 Sil Elast Kit</td><td></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&quot; ID</td></tr><tr><td>Male luer lock connector</td><td>Qosina</td><td>65111</td><td></td></tr><tr><td>Barbed female luer lock connector</td><td>Qosina</td><td>11556</td><td></td></tr><tr><td>Shut-off valve</td><td>Idex Health and Science</td><td>P-721</td><td></td></tr><tr><td>Y-connector</td><td>Idex Health and Science</td><td>P-513</td><td></td></tr><tr><td>20G 1.5&quot; needles</td><td>BD Biosciences</td><td>305176</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td></td></tr><tr><td>CaspGLOW Fluorescein</td><td>Biovision Inc.</td><td>K183-25</td><td></td></tr><tr><td>CaspGLOW Red</td><td>Biovision Inc.</td><td>K193-25</td><td></td></tr><tr><td>Doxorubicin Hydrochloride</td><td>Sigma-Aldrich</td><td>44583</td><td></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

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.

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.

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

The overall goal of this procedure is to demonstrate the fabrication and operation of a microfluidic device that recreates tumor microenvironments in vitro and can be used for testing chemotherapeutic drugs. This is the microfluidic device. This device can be useful in the following areas, easy testing for chemotherapeutic drugs in vitro.It provides consistent and accurate results for testing three dimensional tumor tissue, and this device provides a very accurate representation of in vivo tumor. Microenvironments tumors exist in 3D microenvironments and the ability to test in one is significant. The first step of the procedure is to assemble the device, which involves fabrication of microfluidic chip using soft lithography, punching holes at the inlets and outlets, bonding to a glass slide and connecting tubing for flow inlet and outlet.Next, the uniform multicellular tumor steroids are generated for use in the device using the hanging drop method. The spheroids are then introduced into the device, followed by apoptosis, detecting agents and chemotherapeutic drugs. Finally, time lapse microscopy is performed, followed by mathematical estimation of drug diffusivity coefficients to quantify the penetration ability of the chemotherapeutic agent.Ultimately, results can be obtained that show tissue growth in the device formation of heterogeneous microenvironments within the tissue and drug diffusion into the tissue through the use of time lapse fluorescence microscopy. Limited penetration of cancer drugs into solid tumors is an important cause of their failure. Most current cancer drug testing models do not mimic the three dimensional nature of solid tumors.We have developed a microfluidic device consisting of continuously perfused three dimensional tumor tissues. This model more accurately represents the delivery of drugs to solid tumors and their systemic clearance. Multicellular tumor steroids are a well-established model of three dimensional tumor tissues.In this procedure, the hanging drop method is used to create steroids for use in the microfluidic device. This method allows formation of consistent steroids in a short period of time. Testing chemotherapeutic drugs in vitro in three dimensional tissue can help screen for drugs in a more efficient manner compared to monolayers of cells.Mix the PDMS and curing agent from the silicon elastomer in a nine to one weight ratio. Then pour this mixture over the master to form a four millimeter thick glare Degas. To remove air bubbles cure at 60 degrees Celsius for five hours.Peel the cured PDMS from the mold to obtain a stamp of flow features on the elastomer. Then using a one And a half millimeter biopsy punch mounted on a drill, press punch holes for inlets and outlets. Pull out any debris.Subject the feature side of the stamp and a clean glass slide to oxygen plasma for eight minutes. In an oxygen plasma etcher, bring the treated surfaces in contact immediately to form a bond between them. Maintain the assembly at 60 degrees Celsius on a slide warmer for at least five hours.To strengthen the bond connect 0.032 inches ID PTFE tubing to the inlets and outlets of the microfluidic device. Using an interface of male lure lock connectors attached to barbed female lure lock connectors, these specific connectors are used to allow to easily pass through them without breaking. Finally, set up the flow assembly using shutoff valves and a Y connector, mount the device onto the microscope After mounting the device, connect tubing to appropriate inlets and outlets to complete the flow setup.As shown in this figure, this configuration allows for the ease and versatility of packing a spheroid into the device or create a flow of medium through the device to pack. Open the packing inlet valve VPN and the packing outlet valve VP out a tumor. OID can then pass through the inlet and flow into the culture chamber where the post in this chamber will hold the tumor in place to flow medium.Close the packing valves and open the flow inlet valve VF in and the flow outlet valve VF out to initiate the formation of uniform PHE. Using the hanging drop method trypsin ice cells under a culture hood. Take the resulting trypsin cells from the centrifuge.Dilute the stock solution to a desired concentration by referring to table one. Next, remove the cover of a 48 well plate and place it upside down. In a sterilized hood, fill each well of the plate with one milliliter of sterilized water.To maintain humidity, put a 20 microliter droplet of dilute cell solution in each circular region on the cover. Using a micro pipette, carefully invert the cover and place it over the plate, ensuring that drops Do not touch the edges of the wells. Incubate the plate at 37 degrees Celsius.Refer to table one for the duration of the incubation based on the particular cell used, a steroid will form in Each hanging drop. Start By sterilizing the device by flushing it with 70%ethanol through the flow inlet. Follow with a flush each of PBS, then H-E-P-E-S buffered cell culture medium.Leave the medium syringe, attach to the tubing. Next, draw two to three Steroids into the syringe tap syringe. To remove Air bubbles, Attach the syringe to the packing inlet of the device.Open the inlet valve VPN and close VFN open outlet valve VP out and close VF out. Hold the packing syringe vertical with the needle pointing downward. Watch as the steroid settle to the bottom of the syringe into the lure lock connector of the needle.Push the plunger on the packing syringe and watch PHE enter the tubing and flow into the device because only the packing outlet valve is open. A steroid will enter the chamber of the device and be retained by posts at the back. Once inside the chamber, the S spheroid conforms to the shape of the chamber and acquires a rectangular geometry.Close inlet valve VPN and out outlet valve VP out mount the flow syringe SF on the syringe pump. This pump is run by a computer using syringe pump pro open inlet valve VFN and outlet valve VF out. Begin the flow of medium into the device at Three microliters a minute.Incubation Of spheroid for 24 hours is followed by introduction of apoptosis detecting agents. Before introducing apoptosis, detecting, or therapeutic agents, allow equilibrium of spheroid for up to 24 hours to establish nutrient gradients and microenvironments. Then shut off valve VFN.Stop the syringe pump. Remove syringe from pump and replace it with the syringe containing medium with 0.25 microliters per mil of CASP glow red active Caspase three marker. Wash the device with the solution by manually flushing 0.7 milliliters of it through the device.Then mount the syringe on the pump. Restart the flow and open valve VFN. Over a period Of five to eight hours, cask glove red active cph phase three will diffuse into the tissue and fluoresce brighter in apoptotic regions apoptosis detecting agent at this concentration is maintained in all subsequent Flow solutions.Doxorubicin Is introduced five to eight hours after introduction of apoptosis detecting agents. This treatment is cleared away from the system by flushing. To add chemotherapeutic agents, follow the procedure for apoptosis detection to introduce 10 micromolar doxorubicin hydrochloride.Then replace the treatment syringe with the syringe with 0.7 milliliters of medium. Continue the flow of the therapeutic agent for a fixed period of time. Then shut off valve VFN and turn off the syringe pump.Remove treatment syringe from the syringe pump and replace with the syringe with 0.7 milliliters of medium. Allow the flow of fresh medium for 24 to 36 hours while continuously monitoring the tissue under a microscope. The Entire acquisition process for time lapse microscopy was automated using a customized script in IP lab, acquire transmitted light and fluorescence images of the PAX Foid at 10 x magnification every 30 minutes For mathematical estimation of drug diffusivity coefficients, the first step is to generate average linear intensity profiles of docs fluorescence using image J.Select a rectangular region of interests, or 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 three different time points. Next, subtract the average background fluorescence intensity from the obtained intensity profiles and normalize each profile using the corresponding maximum intensity to obtain C as a function of X and T.Where C is the normalized concentration of doxorubicin, the value of C varies between zero to one.The next step is to evaluate the effective diffusion coefficient of docs within tumor tissue docs. Diffusion can be represented by the following equation where ERFC is the complementary error function. X is the distance into the tissue from the channel, and T is the time after introduction of doxorubicin.Use the following iterative scheme at each time point considered. Guess a value for D.Calculate right side of equation at each location. X.Calculate the sum of squared errors between the two sides, modify D to minimize the residual average.The optimum values of D obtained at each time point to estimate the average effective diffusion coefficient of doxorubicin in the tissue. The microfluidic devices provided a one millimeter by 0.3 millimeter by 0.15 millimeter optically accessible culture chambers for growth of three dimensional tumor tissue. The hanging drop method allowed quick formation of tumor PHE of consistent size and shape from several cell lines.PHE were successfully grown on the device for up to three days. Growth in the chambers was associated with the reproducible modification of microenvironments. Within phe, apoptosis occurred less in cells in close proximity to the flow channel and higher and deeper into the tissue.The device was used to estimate the diffusion coefficient of doxorubicin and tumor tissue. The experimentally tained value agreed with the value reported previously in human breast cancer While attempting this procedure. It's important to be extra cautious of maintaining sterility when flowing PHE into the device because it has to be performed outside of cell culture.Hood spray syringes and ends of tubing with ethanol generously to ensure sterility. After watching this video, you should have an understanding of the role of limited penetration of drugs into tumors. We have demonstrated a technique to mimic the delivery and systemic clearance of drugs on three dimensional tumor tissues and quantify their penetration.Eliminating poorly penetrating drugs will significantly streamline the process of cancer drug development.

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Research

JoVE Journal

Bioengineering

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

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

In this video, we generate microtumors from patient-derived tumor cells using 3D human biogel. These microtumors closely resemble the in vivo tumor microenvironment and serve as potential platforms for drug testing.

Generation of Microtumors: An In Vitro Culture Technique to Culture 3D Microtumors to Analyze Response to Drug Treatment

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering of a Human 3D in vitro Tumor Test System

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

We present a microfluidic cancer-on-chip model, the "Evolution Accelerator" technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Microfluidic Model to Mimic Initial Event of Neovascularization

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Learn to fabricate a microfluidic device that recreates 3D tumor microenvironments in vitro for testing chemotherapeutic drugs like doxorubicin. This method enables accurate drug diffusion studies and improved cancer research.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

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