Poster U10 - Strategies for Improved Endothelial Cell Adhesion in Microphysiological Vascular Model System

Introduction: Human tissue-engineered blood vessels (TEBVs) are increasingly utilized for in vitro research, such as studying the pathology of atherosclerosis and Hutchinson-Gilford Progeria Syndrome. These engineered constructs serve as crucial tools not only for disease modeling but also for drug screening and the development of regenerative therapies. Central to the efficacy of TEBVs is the successful seeding and maintenance of endothelial cells, which form the inner lining of blood vessels and are critical for vascular function. Despite advancements in this field, consistently establishing a stable endothelial layer in the vessel lumen remains challenging, primarily due to the complex interplay of cellular dynamics, material selection, and biomechanical forces. Furthermore, the lack of a standardized workflow in the field complicates the replication of results and the comparison of endothelialization strategies across different studies. Addressing these challenges, our study presents a systematic approach to optimize the fabrication and endothelialization process of TEBVs.

Materials and

Methods: We explored chamber design, material selection, and procedural methodologies to fabricate and perfuse TEBVs that exhibited natural vascular structures. We incorporated human neonatal dermal fibroblasts (hNDFs) in a tubular collagen scaffold and seeded human umbilical vein endothelial cells (HUVECs) to replicate the native blood vessel architecture. To systematically investigate the factors contributing to robust coverage of endothelial cells within the lumen, we used red fluorescent protein expressing HUVECs (RFP-HUVECs) to assess parameters such as endothelial cell seeding density, rotation time, and flow application. Based on prior work, we tested EC seeding densities ranging from 0.5 to 1 million cells for each TEBV with rotation duration of 3h (short), 6h (medium), or 12h (long). Additionally, we explored the impact of perfusion application by either connecting the vessels to a pump set at maximum speed or gradually increasing perfusion speed over a 3-hour period. To facilitate in situ observation of the vessels, we also designed a TEBV viewing chamber. The optimal conditions identified were subsequently validated through experiments using TEBVs constructed with HUVECs. We also exposed TEBVs to TNF-α treatment allowing for exploration of endothelial activation in the TEBV system.

Results, Conclusions, and Discussions: Our comprehensive workflow compromised five continuous steps: beginning with mixing high-concentration rat tail Type I collagen and hNDFs to form a vascular wall-like layer. After gelation, we employed plastic compression and dehydration to strengthen the TEBVs. Endothelial cells were then introduced into the lumen, with a 12-hour rotation ensuring their even distribution. The vessels were maintained with daily media changes and perfused under physiological shear stress (0.4 Pa) for a week to mature. The implemented protocol yields TEBVs with a robust and functional endothelial layer that exhibits stability over time. We demonstrated that the inclusion of a 12-hour rotation period and a gradual application of flow were pivotal factors in enhancing EC attachment and resistance to shear stress. After 7 days of perfusion, microscopic analysis showed a notable increase in the percentage of circumferential endothelial coverage in TEBVs subjected to 3-hour ramped-up perfusion (65.6±15.3%, n = 4 TEBVs) compared to TEBVs that underwent rapid perfusion initiation (3.3±2.9%, n = 3 TEBVs), as confirmed by positive von Willebrand Factor (vWF) staining. The endothelial cell coverage in the lumen showed no significant difference at 24 h, 48 h, 72 h and 7 days, revealing that our protocol supports the development of a stable endothelium under perfusion conditions. En face staining elucidated the morphological characteristics of ECs within the TEBV lumen after perfusion with localization of VE-cadherin at cell-cell junctions. To further evaluate the functionality of TEBVs, we exposed them to TNF-α to induce inflammatory responses in ECs. The treatment led to a marked increase in THP-1 monocyte accumulation within the lumens of the TEBVs. The upregulation of Intercellular Adhesion Molecule-1 (ICAM-1), a mediator of monocyte-EC interaction, further supported the responsive nature of the endothelial cells to inflammatory stimuli in the TEBV system. While this study showcases notable achievements, certain limitations remain unaddressed, such as prolonged perfusion viability assessment. Future studies may explore the use of primary cells and cells derived from induced pluripotent stem cells (iPSCs). Despite these limitations, the developed TEBVs serve as valuable models for screening tests and offer insights into optimizing tissue-engineered vascular models.

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