Tubeless microfluidic angiogenesis assay with three-dimensional endothelial-lined microvessels - PubMed (original) (raw)

Tubeless microfluidic angiogenesis assay with three-dimensional endothelial-lined microvessels

Lauren L Bischel et al. Biomaterials. 2013 Feb.

Abstract

The study of angiogenesis is important to understanding a variety of human pathologies including cancer, cardiovascular and inflammatory diseases. In vivo angiogenesis assays can be costly and time-consuming, limiting their application in high-throughput studies. While traditional in vitro assays may overcome these limitations, they lack the ability to accurately recapitulate the main elements of the tissue microenvironment found in vivo, thereby limiting our ability to draw physiologically relevant biological conclusions. To bridge the gap between in vivo and in vitro angiogenesis assays, several microfluidic methods have been developed to generate in vitro assays that incorporate blood vessel models with physiologically relevant three-dimensional (3D) lumen structures. However, these models have not seen widespread adoption, which can be partially attributed to the difficulty in fabricating these structures. Here, we present a simple, accessible method that takes advantage of basic fluidic principles to create 3D lumens with circular cross-sectional geometries through ECM hydrogels that are lined with endothelial monolayers to mimic the structure of blood vessels in vitro. This technique can be used to pattern endothelial cell-lined lumens in different microchannel geometries, enabling increased flexibility for a variety of studies. We demonstrate the implementation and application of this technique to the study of angiogenesis in a physiologically relevant in vitro setting.

Copyright © 2012 Elsevier Ltd. All rights reserved.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Passive pumping-based microfluidic angiogenesis assay with 3D cylindrical lumens. (A) Illustration of a triple channel design with connecting microchannels. (B–D) Microchannel systems can be (B) single, (C) double, or (D) triple channel designs, and are arrayable. Devices are plasma-treated and bonded to glass-bottom Petri dishes. Scalebar ~ 10 mm.

Fig. 2

Fig. 2

(A) Viscous finger patterning is used to pattern the initial lumen structure through an ECM hydrogel. A microchannel is filled with a pre-polymerized ECM hydrogel solution. Passive pumping is used to flow media through the ECM solution, pushing out the center. Following incubation for 10 minutes at 37 deg C to complete polymerization, a lumen is patterned through the hydrogel. (B) Methods used to line lumens with endothelial cells to generate ELLs. (C) Methods used to pattern two ELLs lumens in a double microchannel. (D) Methods used to pattern one or three ELL in a triple microchannel. Note: When patterning an interconnected network in double channels, the diffusion channels are the same height as the main channels. The entire channel is filled at once and viscous finger patterning is performed prior to incubation.

Fig. 3

Fig. 3

ELLs were cultured for 48hours and stained for CD31 (green) and nuclei (blue). (A)–(C) Confocal slices of an ELL at the specified height. (D) Volume-rendered image of an ELL in a single microchannel. (E) Volume-rendered cross section of an ELL in a single microchannel. (F) Volume-rendered cross section of two ELLs in a double microchannel. (G) Volume-rendered cross section of three ELLs in a triple microchannel. Scale bar represents 100µm.

Fig. 4

Fig. 4

(A) Two interconnected ELLs were patterned in a double microchannel. (B) Volume-rendered cross-sectional view. Scale bar represents 500µm.

Fig. 5

Fig. 5

(A) Images of an ELL and an unlined lumen at the specified time point after the addition of FITC-labeled BSA. Scale bar represents 100µm. (B) Fluorescent images taken at the specified time point following the addition of 50ng/ml of 40kDa FITC-labeled Dextran to the left channel in a triple microchannel. Scale bar represents 500µm.

Fig. 6

Fig. 6

(A) Sprouting was induced on one side of an ELL patterned in the center channel of a triple microchannel when 50ng/ml of VEGF was added to the right side channel to generate a VEGF gradient. VEGF gradient was started 24 hours after HUVEC seeding and maintained for 48 hours. Scale bar represents 50µm. (B) Fluorescent images of the wall of an ELL (top) and an ELL exposed to a VEGF gradient (bottom) stained for Jagged-1 (red) and nuclei (blue). Scale bar represents 50µm. (C) Quantitative analysis of endothelial sprout number, (D) sprout length, and (E) sprout area when ELLs were exposed to a variety of conditions. Error bars represent standard deviation.

References

    1. Carmeliet P, Jain R. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–257. - PubMed
    1. Gerstner ER, Duda DG, di Tomaso E, Ryg PA, Loeffler JS, Sorensen AG, et al. VEGF inhibitors in the treatment of cerebral edema in patients with brain cancer. Nat Rev Clin Oncol. 2009;6:229–236. - PMC - PubMed
    1. Chi AS, Sorensen AG, Jain RK, Batchelor TT. Angiogenesis as a therapeutic target in malignant gliomas. Oncologist. 2009;14:621–636. - PMC - PubMed
    1. Jain R, Schlenger K, Hockel M, Yuan F. Quantitative angiogenesis assays: progress and problems. Nat Med. 1997;3:1203–1208. - PubMed
    1. Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis assays: A critical overview. Clin Chem. 2003;49:32–40. - PubMed

Publication types

MeSH terms

Grants and funding

LinkOut - more resources