Development of a rotational cell‐seeding system for tubularized extracellular matrix (ECM) scaffolds in vascular surgery

Abstract

Tubularized porcine extracellular matrices (ECMs) are under investigation as adjuvant scaffolds for endovascular aneurismal repair (EVAR). Limitations with tubularized ECMs in this setting include difficulties in achieving a confluent endothelium on the scaffold's luminal surface prior to in vivo implantation. In this in vitro study a rotational "cell-seeding rig" (RCR) was constructed to assess the potential for endothelialization of tubular ECM constructs. Human aortic endothelial cells (HAECs) were cultured onto the luminal surfaces of tubular porcine urinary bladder matrix (UBM) scaffolds and rotated in the RCR at experimental rotational speeds. Results showed that endothelial attachment occurred at a rotation speed of six revolutions per hour. HAECs continued to proliferate after the initial attachment period of 24 h and formed a confluent endothelial monolayer after 14 days of growth. Our results demonstrate that RCRs facilitate attachment of HAECs in vitro at a speed of six revolutions per hour. The endothelialization technique presented in the current study may be important for advancing tissue-engineering approaches to address some of the current limitations in endovascular treatments of abdominal aortic aneurysms.

Hybrid cardiovascular sourced extracellular matrix scaffolds as possible platforms for vascular tissue engineering

“…Human umbilical vein endothelial cells (HUVECs) from an infant male Caucasian donor were obtained cryopreserved at passage 1 (Pro‐moCell GmbH) and expanded to passage 7 in a 5% CO 2 /37°C atmosphere. HUVECs were expanded using MCBD 131 medium (Life Technologies) supplemented with 5% v/v fetal bovine serum (FBS; ThermoFisher Scientific); 1% v/v L‐glutamine; 1% v/v penicillin/streptomycin (Life Technologies); 1 mg/L hydrocortisone; 50 mg/L ascorbic acid (Sigma); 2 μg/L fibroblast growth factor (PeproTech); 10 μg/L epidermal growth factor (PeproTech); 2 μg/L insulin‐like growth factor (PeproTech); and 1 μg/L vascular endothelial growth factor (PeproTech) (Callanan, Davis, McGloughlin, Walsh, ; Carroll, McGloughlin, O'Keeffe, et al, ; Davis et al, ).…”

“…Real‐time polymerase chain reaction was performed using a LightCycler® 480 Instrument II (Roche Life Science) and Sensifast™ SYBR® High‐ROX system (Bioline). Forward and reverse sequences were designed with Sigma‐Aldrich and are displayed in Table (Callanan et al, ; Fischer et al, ). Relative quantification of Reverse transcription polymerase chain reaction (RT‐PCR) results was carried out using the 2 −ΔΔ ct method (Livak & Schmittgen, ).…”

“…ThermoFisher Scientific); 1% v/v L-glutamine; 1% v/v penicillin/streptomycin (Life Technologies); 1 mg/L hydrocortisone; 50 mg/L ascorbic acid (Sigma); 2 μg/L fibroblast growth factor (PeproTech); 10 μg/L epidermal growth factor (PeproTech); 2 μg/L insulin-like growth factor (PeproTech); and 1 μg/L vascular endothelial growth factor (PeproTech) (Callanan, Davis, McGloughlin, Walsh, 2014b;Carroll, McGloughlin, O'Keeffe, et al, 2009;Davis et al, 2014).…”

Secreted Endothelial Cell Factors Immobilized on Collagen Scaffolds Enhance the Recipient Endothelial Cell Environment

“…Human umbilical vein endothelial cells (HUVECs) from an infant male Caucasian donor were obtained cryopreserved (500,000 cells) at passage 1 (PromoCell GmbH) and cultured and expanded to passage 5 (P5) in a humidified atmosphere of 5% CO 2 /37°C in T-75 vented flasks (Corning ® ) and grown to 80% confluency. HUVECs were cultured according to a previously used endothelial cell culture protocol, 37–39 and in brief, MCDB 131 medium (Life Technologies™) was supplemented with 2% fetal bovine serum (FBS; ThermoFisher Scientific); 1% l -glutamine; 1% penicillin/streptomycin (Life Technologies); 1 mg/L hydrocortisone; 50 mg/L of ascorbic acid (Sigma); 2 mg/L fibroblast growth factor; 10 mg/L epidermal growth factor; 2 mg/L insulin-like growth factor; and 1 mg/L VEGF (PeproTech).…”

Investigation of the Biomechanical Integrity of Decellularized Rat Abdominal Aorta

“…The native aortas showed a 3-layered structure (Fig 2C and Fig 2F), comprising an outer layer of cells (endothelium) generally called tunica intima (Fig 2AeFig 2F) [23], the tunica media consisting of elastic fibers (Fig 2C and Fig 2F) intercalated with smooth muscle fibers and collagen fibers (Fig 2B and Fig 2E), and the adventitia consisting mainly of elastic lamellae (Fig 2C and Fig 2F), collagen fibers, and fibroblasts ( Fig 2B and Fig 2E) [24]. The elastic fibers were organized in a concentric fashion in the tunica media (Fig 2C and Fig 2F), whereas they were distributed along random directions, together with the collagen fibers, in the adventitial layer ( Fig 2C and Fig 2F, and Fig 2B and Fig 2E, respectively).…”