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1Department of Chemical Engineering, University Of Massachusetts Amherst
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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.
Toley, B. J., Ganz, D. E., Walsh, C. L., Forbes, N. S. Microfluidic Device for Recreating a Tumor Microenvironment in Vitro. J. Vis. Exp. (57), e2425, doi:10.3791/2425 (2011).
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.
1. Device Fabrication
Replication of microfluidic features in elastomeric materials was based on the method described by Duffy et al.1
2. Formation of Uniform Spheroids
Spheroids were formed by the hanging drop method2,3.
3. Introduction of Spheroids into Device
Refer to Figure 1 and Table 2 for this section.
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.
5. Introduction of Therapeutic Agent
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.
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.
|Cell Line||Required Cell Concentration||Incubation Time|
|LS174T||300 cells/μL||2-3 days|
|T47D||750 cells/μL||3-4 days|
|MDA-MB-231||150 cells/μL||5-6 days|
Table 1. Parameters for Hanging Drop Spheroids
|VFin||Flow Inlet Valve|
|VPin||Packing Inlet Valve|
|VFout||Flow Outlet Valve|
|VPout||Packing Outlet Valve|
Table 2. Syringes and Valves in Flow Setup
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.
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.
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.
The vasculature in tumors is sparse and ill developed7,8. There are regions located far (>100 μm) 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 in proximity of the channel are viable, but diffusion limitations give rise to nutritionally deficient apoptotic regions far from the channel. The flow channel represents a blood vessel and the spheroid represents a small region within a tumor adjacent to a blood vessel.
While introducing spheroids into the device, ensure that the punched inlet hole is clean and free of debris, because any obstruction to flow of tissue will result in failure to fill the chamber, or a broken spheroid. The packing syringe is operated manually during the process of introducing spheroids. The flow rate must be maintained as low as possible to avoid pushing the spheroid through the posts at the back of the chamber. As with all microfluidic devices, certain measures must be taken to avoid bubbles in the system, especially when switching syringes. Cutting a small piece of tubing each time a syringe is switched will help avoid bubbles.
Modifications to the device to allow use of multiple chambers and multiple treatments in a single experiment are ongoing. We believe that these devices can be used to understand the behavior of current cancer therapeutics in solid tumors. They can also be used as easy to construct drug-screening platforms to develop new cancer therapies.
No conflicts of interest declared.
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.
|Silicone elastomer kit||Ellsworth Adhesives||184 Sil Elast Kit|
|Miltex biopsy punch||MedexSupply||MTX-33-31AA||1.5 mm|
|PTFE tubing||Cole-Parmer||EW-06417-31||0.032" ID|
|Male luer lock connector||Qosina||65111|
|Barbed female luer lock connector||Qosina||11556|
|Shut-off valve||Idex Health and Science||P-721|
|Y-connector||Idex Health and Science||P-513|
|20G 1.5" needles||BD Biosciences||305176|
|CaspGLOW Fluorescein||Biovision Inc.||K183-25|
|CaspGLOW Red||Biovision Inc.||K193-25|
|LS174T||ATCC||CCL-188||Human Colon Carcinoma cell line|
|T47D||ATCC||HTB-133||Human Ductal Carcinoma cell line|
|MDA-MB-231||ATCC||HTB-26||Human Mammary Adenocarcinoma cell line|
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