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Tissue Engineering

Tissue Engineering - Poster Session C

Poster K19 - Exploring the effects of serum on the performance and phenotype of tissue-engineered smooth muscle cell microvessels

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Vascular tissue engineering aims to create functional blood vessel models and grafts that can be used to study vascular diseases and develop regenerative medicine applications. A critical component of this field is the development of smooth muscle cell (SMC) microvessels, which consist of a monolayer of SMCs devoid of endothelium. This structure mimics the tunica media of physiological arterioles and serves as a foundation for endothelial cell (EC) seeding to replicate the tunica intima.

Sera, particularly fetal bovine serum (FBS), are widely utilized in cell and tissue culture due to their rich composition of growth factors and nutrients. However, the impact of serum on three-dimensional (3D) microvessels is not well characterized. This study aims to examine the effects of FBS on the performance of tissue-engineered SMC microvessels, focusing on critical parameters such as patency and SMC phenotype. A patent, occlusion-free SMC microvessel is essential for subsequent EC seeding to create functional arteriole models or vascular grafts. Moreover, controlled proliferation and a contractile phenotype of SMCs are crucial for accurately modeling healthy physiological arterioles. Conversely, to model vascular diseases such as intimal hyperplasia and atherosclerosis, SMCs need to exhibit a synthetic phenotype characterized by increased proliferation, motility, and extracellular matrix (ECM) synthesis.

Understanding the performance of SMC microvessels under varying serum conditions is vital for advancing tissue engineering and vascular biology. Insights from this study will contribute to developing more physiologically relevant and disease-specific vascular models, enhancing our ability to study vascular pathologies and develop therapeutic interventions.

Materials and

Methods:  A microfluidic device was assembled by adhering glass coverslips to a 3D-printed, biocompatible housing using double-sided adhesive. A 150 μm diameter nitinol wire was inserted as a templating needle. Collagen I, titrated to pH 9, was injected into the device and incubated at 37°C for 15 minutes. The templating needle was then removed, creating a cylindrical channel surrounded by a collagen I matrix, which was crosslinked by perfusing 20 mM genipin through the channel for 2 hours (Figure 1).

Primary human brain vascular SMCs were cultured in complete smooth muscle cell medium (SMCM) (Table 1) until 80% to 90% confluency. At this point, they were detached and seeded into the hydrogel channel to form a microvessel with an adherent, confluent monolayer of cells (Figure 1). Seeded devices were incubated in complete SMCM under static conditions for 2 to 4 hours, after which a 400 Pa hydrostatic pressure was applied to create medium flow. After the static incubation period (D0), all microvessels were matured in basal SMCM (Table 1) for 12 hours (D0.5). Subsequently, microvessels were maintained in chemically defined SMCM (CD-SMCM), either serum-free or supplemented with 10% FBS (Table 1), for a total duration of 7 days (D7).

On D1, D4, or D7, SMCs were detached from the microvessels and lysed. Purified RNA from isolated cells was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Results, Conclusions, and Discussions: Serum-free microvessels maintain a complete monolayer of SMCs with a constant minimum luminal cross-sectional area and negligible overproliferation (OP). By D4, SMCs exhibit an elongated morphology characteristic of the contractile phenotype (Figure 2A, C, D, E). In contrast, microvessels cultured in 10% FBS become occluded by D4 due to dense masses of SMCs, which persist through D7 (Figure 2A). While serum-free microvessels maintain a constant flow rate and patency (Figure 2B), microvessels in 10% FBS experience decreased flow from D1 to D4, indicating reduced patency as overproliferating SMCs occlude the microvessels at random loci and reduce the luminal cross-sectional area. Between D4 and D7, the flow rate dropped to zero, suggesting the formation of fully developed occlusion sites (Figure 2B, C, D, E).

ACTA2 and TAGLN are two genes highly expressed in contractile SMCs. In 10% FBS microvessels, TAGLN expression is significantly higher than that in serum-free microvessels on D4 (Figure 3). This could imply a more contractile SMC phenotype following FBS exposure, which would contradict the observations of SMC OP and morphology. To make a conclusive claim about the effect of FBS on SMC phenotype, additional supporting experiments are required, including qPCR for other SMC phenotype-related genes and immunohistochemistry for phenotypic marker proteins.

This study presents the complex, multifaceted effects of FBS on the performance and phenotype of tissue-engineered SMC microvessels, guiding future efforts to model healthy and diseased arterioles. Key innovations include the quantification of microvessel occlusion, providing a valuable metric for evaluating other tissue-engineered vascular constructs.

Acknowledgements (Optional):