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.
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Method Article
Microfluidic Device for Recreating a Tumor Microenvironment in Vitro
* These authors contributed equally
In This Article
Summary
Abstract
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.
Protocol
1. Device Fabrication
Replication of microfluidic features in elastomeric materials was based on the method described by Duffy et al.1
- 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.
- Punch holes for inlets and outlets using a 1.5 mm biopsy punch (Miltex, York, PA) mounted on a drill press. Pull ou....
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Discussion
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 .......
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Disclosures
No conflicts of interest declared.
Acknowledgements
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.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 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 | |
| Trypsin-EDTA | Invitrogen | 25300-054 | |
| HEPES | Sigma-Aldrich | H-4034 | |
| CaspGLOW Fluorescein | Biovision Inc. | K183-25 | |
| CaspGLOW Red | Biovision Inc. | K193-25 | |
| Doxorubicin Hydrochloride | Sigma-Aldrich | 44583 | |
| 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 |
References
- Duffy, D. C., McDonald, J. C., Schueller, O. J., Whitesides, G. M. Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). Anal. Chem. 70, 4974-4984 (1998).
- Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K.
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