(K-424) An Integrated Microfluidic Platform with Controlled Pressure Gradients for 3D Self-Assembled Microvasculature

Reliable methods to replicate vascular networks in vitro is of critical importance to the advancement of microphysiological systems (MPS) or organ-on-a-chip platforms as nearly every cell in the human body is located within 50-100μm of a blood vessel [1]. Currently, some of the most common approaches to generate 3D microvasculature utilize naturally-derived hydrogels (e.g. fibrin, collagen, or Matrigel) and devices made of polydimethylsiloxane (PDMS) [2,3]. Unfortunately, these hydrogels are prone to batch-to-batch variability and have poor tunability. Further, PDMS is not well-suited for mass production and has a tendency to absorb small molecules including some drugs and hormones.

Here, we introduce a microfluidic platform capable of reproducing highly-controlled microvasculature studies. Cells suspended in a synthetic polyethylene glycol (PEG) hydrogel are seeded into cyclic olefin copolymer (COC) thermoplastic devices, mitigating unwanted absorption of small molecules and unlocking the potential for large-scale manufacturing. The PEG-based hydrogel increases the tunability and reliability of the extracellular matrix, allowing the platform to be adapted for other MPS applications [4]. Each COC device interfaces with a pneumatic manifold that is used to control various on-chip functions. Other studies have utilized simple hydrostatic pressure gradients to promote perfusability in microvascular networks [5]. In our platform, diaphragm-based COC pumps, valves, and back-pressure regulators provide deterministic control over flow rates and pressure gradients across the hydrogel. With this platform, we demonstrate the successful generation of self-assembled, perfusable vascular networks and explore the effects of different tissue compartment geometries, cell densities, and pressure gradients on vessel formation.

Two types of COC devices were fabricated. A 5-layer device (Figure 1A) with integrated membrane pumps, valves, and back-pressure regulators serves as the primary configuration for the platform and interfaces with the pneumatic manifold and controller. 2-layer devices (Figure 1B) were also fabricated for easier prototyping and were connected to an external pump, pressure source, and reservoir via external tubing. Both devices and a supporting aluminum base plate (Figure 1C) were made via CNC machining. The pneumatic manifold was 3D printed and receives pressure and vacuum signals from electronically controlled solenoid valves outside of the incubator. The COC membranes and optical films were thermally bonded to the thicker fluidic and pneumatic layers prior to ethylene oxide sterilization.

GFP-labeled human umbilical vein endothelial cells (HUVECs) and normal human lung fibroblasts (NHLFs) were mixed in the PEG hydrogel solution and seeded into the tissue channel of the COC devices prior to polymerization. After allowing the gel in the device to form at 37 °C for 30 minutes, endothelial cells suspended in cell culture media were seeded into the side channels of each device to promote microvasculature network interaction with the media channels. Over 7 days, network formation was monitored using fluorescence microscopy. On Days 4-6, vessel perfusability is tested by mixing either fluorescent dextran or microbeads into the cell culture media in one side of the device and watching to see if the fluorescent molecules flow through the vessels to the opposite media channel (Figure 1D).

By optimizing the hydrogel properties, cell densities, ratio of endothelial to stromal cells, device geometry, and fluidic conditions, we are able to achieve self-organized vasculature in COC devices and with a synthetic PEG hydrogel. Establishing a pressure drop across the PEG hydrogel is critical to reliably produce perfusable vessel formation. A more controllable pressure gradient was established by introducing a tunable fluidic resistance between the side channels in the device or incorporating two independently controlled back-pressure regulators upstream of the tissue compartment channels. The back-pressure regulators are operated by an externally regulated air pressure; the fluid pressure must slightly exceed the regulator pressure for culture media to flow. Because the pneumatic pumps have a deterministic volumetric flowrate (typically 1 μL/s), the pressure difference across the hydrogel channel can be precisely controlled using either method.

We have observed optimal vessel formation under pressure gradients between 2-10 mmH₂O. Additionally, allowing the cells to establish networks for 1-3 days without pressure enhanced vessel perfusability; the application of the pressure gradient is beneficial for inducing patency in already-established vessels. The pressure gradient is also able to orient network formation horizontally, aligned with the induced interstitial flow through the hydrogel. Ongoing work involves image analysis to quantify vessel diameter, length, and perfusability in addition to measuring oxygen consumption along the hydrogel channel.

Our platform’s performance was compared to microvasculature generated in identical PDMS devices and/or in a fibrin hydrogel. Producing vascular networks of a similar quality to the current standard materials, our platform will equip researchers to conduct repeatable micovasculature studies at scale, investigate hydrophobic hormones and drugs more effectively, and eventually enable accurate modeling of microphysiological systems in vitro with vascularized organoids.

References

1.      Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Blood Vessels and Endothelial Cells. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26848/.

2.     Shirure VS, Hughes CCW, George SC. Engineering Vascularized Organoid-on-a-Chip Models. Annu Rev Biomed Eng. 2021 Jul 13;23:141-167. https://www.annualreviews.org/doi/10.1146/annurev-bioeng-090120-094330. Epub 2021 Mar 23. PMID: 33756087.

3.     Coughlin, M. F., Kamm, R. D., The Use of Microfluidic Platforms to Probe the Mechanism of Cancer Cell Extravasation. Adv. Healthcare Mater. 2020, 9, 1901410. https://doi.org/10.1002/adhm.201901410

4.     Gnecco, J.S., Brown, A.T., et al. Organoid co-culture model of the cycling human endometrium in a fully-defined synthetic extracellular matrix reveals epithelial-stromal crosstalk. Cell Med 2023 (accepted). https://www.biorxiv.org/content/10.1101/2021.09.30.462577v2.full (preprint).

5.     Yue, T., Zhao, D., Phan, D.T.T. et al. A modular microfluidic system based on a multilayered configuration to generate large-scale perfusable microvascular networks. Microsyst Nanoeng 7, 4 (2021). https://doi.org/10.1038/s41378-020-00229-8