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Tissue Engineering

Forming a Functional Medial Layer of Smooth Muscle Cells within Engineered Microvasculature

(H-320) Forming a Functional Medial Layer of Smooth Muscle Cells within Engineered Microvasculature

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row H - Poster # 320

Presenting Author(s)

VV

Victoria Vest, BS

Graduate Student
Vanderbilt University
Nashville, Tennessee, United States

Co-Author(s)

IH

Isabella Holtz

undergraduate student
Vanderbilt University, United States
EC

Eliam Chang

undergraduate researcher
Vanderbilt University, United States
FT

Fiona Thompson

undergraduate researcher
Vanderbilt University, United States

Primary Investigator(s)

LB

Leon Bellan (he/him/his)

Associate Professor of Mechanical Engineering; Associate Professor of Biomedical Engineering
Vanderbilt University, United States

Introduction:: Many methods have been developed to form perfusable microvasculature to support, via the delivery of oxygen and nutrients and the removal of waste products, cells in the interior of thick, hydrogels. Existing work, however, has generally demonstrated vessels with mismatched size and vessel wall architecture, researchers have largely fabricated vessels on the scale of arterioles or small resistance arteries, but with a simple endothelial cell (EC) layer most similar to the wall architecture of capillaries. These engineered microvessels notably lack the medial layer of smooth muscle cells (SMCs) seen in similarly sized vessels in natural tissue, a feature which enables the regulation of local vascular resistance. As regulation of vascular resistance is one of the primary functions of such vessels in vivo, we seek to fabricate small resistance artery and arteriole-scale vessels with coaxial layers of ECs and SMCs, leveraging appropriate matrix characteristics along with cyclic stretch to drive SMCs to a contractile phenotype and to induce circumferential alignment of the SMC layer. Herein, we demonstrate a method for seeding SMCs onto the surface of a microchannel and show that matrix characteristics affect SMC organization. We also show that we can seed a layer of ECs on top of the SMCs to create a multilayer vessel wall. We have previously shown that we can induce physiological levels of cyclic stretch in these channels with appropriate pulsatile flow.

Materials and Methods::

Straight channels with a diameter of 300µm were formed in a hydrogel using nylon monofilament as a removable template. We employed a 10% gelatin hydrogel, formed by crosslinking with 2% w/v microbial transglutaminase at 37° for 15 minutes. Various solutions of ECM proteins were incubated inside the channels for several hours to promote cell adherence. RFP-expressing human aortic SMCs were then introduced into the channel at a seeding density of 5x10⁴ cells/cm² and allowed to adhere for several hours. Smooth Muscle Differentiation Media (SMDM) was introduced into the channels 8 hours after seeding. After 5 days of culture in SMDM, a suspension of human umbilical vein endothelial cells at 10⁶ cells/mL was introduced into the channels. Whole channels were regularly imaged using a confocal microscope to monitor cell viability, adherence, and confluence. Cell morphology and orientation were analyzed in MATLAB. To determine SMC phenotype, 𝛼-SMA levels were characterized via immunohistochemistry.

Results, Conclusions, and Discussions::

SMCs seeded in channels with collagen IV + fibronectin + vitronectin orient obliquely around the channel circumference, while those seeded on only collagen IV, or in channels with no ECM coating, align randomly or parallel to the channel’s longitudinal axis, indicating that ECM coating composition affects SMC organization in this system. ECs seeded interior to the SMC layer were able to adhere and create a monolayer.

We have cultured SMCs on the walls of microchannels formed within a gelatin hydrogel, achieving helical organization around the channel circumference. We have also added an intimal EC layer. Ongoing work will determine how cyclic stretch, induced by pulsatile flow, can be employed to further control architecture and phenotype of the SMC layer. Production of a circumferentially aligned SMC layer capable of contraction or relaxation in response to vasoactive stimuli would be a transformative step in engineered microvasculature, enabling local regulation of vascular resistance in artificial tissue constructs and serving as a model for study of arterioles in isolation.

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