A practical method for patterning lumens through ECM hydrogels via viscous finger patterning

Abstract

Extracellular matrix (ECM) hydrogels with patterned lumens have been used as a framework to generate more physiologically relevant models of tissues, such as vessels and mammary ducts, for biological investigations. However, these models have not found widespread use in research labs or in high-throughput screening applications in large part because the basic methods for generating the lumen structures are generally cumbersome and slow. Here we present viscous finger patterning, a technique to generate lumens through ECM hydrogels in microchannels that can be accomplished using manual or automated pipetting. Passive pumping is used to flow culture media through an unpolymerized hydrogel, creating a lumen through the hydrogel that is subsequently polymerized. Viscous finger patterning takes advantage of viscous fingering, the fluid dynamics phenomenon where a less viscous fluid will flow through and displace a more viscous fluid. We have characterized the technique and used it to create a variety of channel geometries and ECM hydrogel compositions, as well as for the generation of lumens surrounded by multiple hydrogel layers. Because viscous finger patterning can be performed with automated liquid handling systems, high-throughput generation of ECM hydrogels with patterned lumen is enabled. The ability to rapidly and cost-effectively create large numbers of lumens in natural polymers overcomes a critical barrier to the use of more physiologically relevant tissue models in a variety of biological studies and drug screening applications.

Figure 1

Process used for patterning lumens through an extracellular matrix (ECM) hydrogel via viscous finger patterning. First a microchannel is filled with an ECM hydrogel solution (A). The microchannels are incubated at 37 °C for a period of time depending on desired lumen dimensions, channel geometry, and ECM composition (B). Then passive pumping is performed by placing a droplet of culture media on the inlet (C), flushing out the ECM solution in the center of the microchannel (D), and leaving a continuous lumen through the ECM hydrogel (E).

Figure 2

Images of a lumen through a collagen I hydrogel. (A) Volume-rendered cross section. (B) Top view. For this image, a concentration of 4.5 mg/mL collagen I was used for the hydrogel. The microchannel was incubated for 2 min at 37 °C before viscous finger patterning. The microchannel was 500 μm tall, 500 μm wide, and 5 mm long, with inlet and outlet diameters of 1 mm and 1.75 mm, respectively. A z-stack of images was taken using multiphoton laser-scanning microscopy (MPLSM) imaging and volume-rendered using Osirix. Figure 3B represents a slice taken at a height of approximately 250 μm in the microchannel. Scale bars represent 100 μm.

Figure 3

Lumen widths can be controlled by varying channel geometry, incubation time, and hydrogel composition. (A) Lumens were generated through 4.5-mg/mL collagen I hydrogels after a 4-min incubation in straight microchannels of varying widths (no constrictions). (B) Lumens were generated through 4.5-mg/mL collagen I hydrogels after a 4-min incubation in microchannels with constrictions of varying widths adjacent to the inlet and outlet ports. (C) Lumens were generated through 4.5-mg/mL collagen I hydrogels after a 4-min incubation in microchannels with varying inlet port diameters. (D) Lumens were generated through 4.5-mg/mL collagen I hydrogels with varying incubation times. (E) Lumens were generated through collagen I hydrogels with varying concentrations after a 4-min incubation.

Figure 4

Image of dye flowed through lumens formed through a 4.5-mg/mL collagen I hydrogel in branching (A) or curving (B) microchannels. Microchannels were 300 μm in height with inlet and outlet diameters of 1 mm and 1.75 mm, respectively. The collagen I hydrogel was incubated for 2 min at 37 °C before lumen formation. (C) Dye flowed through two lumens formed through a 4.125-mg/mL collagen I hydrogel after incubation for 2 min. Two 400-μm tall, 500-μm wide chambers with inlet and outlet port diameters of 1 mm were separated by a 200-μm tall, 500-μm wide center chamber. Scale bars represent 500 μm.

Figure 5

Volume-rendered image (A) and an image taken at the widest portion (B) of a lumen surrounded by multiple collagen I hydrogel layers. First, a lumen was formed through a 4.0-mg/mL collagen I hydrogel after a 2-min incubation. After polymerization was complete, a 4.0-mg/mL collagen I solution with FITC-conjugated microbeads was pumped into the lumen, and the channels were incubated for 2 min. Passive pumping was then used to form a lumen through this second layer. Microchannels were 500 μm tall, 500 μm wide, and 5 mm long, with inlet and outlet diameters of 1 mm and 1.75 mm, respectively. Images were taken using multiphoton laser-scanning microscopy (MPLSM). Scale bar represents 100 μm.