Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics

Abstract

Recellularization of ex vivo‐derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four‐step culture approach were assessed. Using an ex vivo‐derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0–100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial‐like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein‐based scaffold and its positive remodeling toward arterial biomechanics were obtained.

A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture

“…Dynamic ow culture has been reported to improve the recellularization e ciency in various kinds of decellularized tissues 11,17,28 . In conventional methods of recellularization aiming for ex vivo tissue reconstruction before in vivo implantation, seeding cells by circulating a cell suspension (cells + culture medium) requires a relatively long duration of recellularization incubation, and results in a relatively low e ciency of recellularization 29,30 . We modi ed the protocol from previous reports to pre-seed the cells with a su ciently high cell density and conducted dynamic ow incubation with cell-free medium for 2 weeks, which resulted in improved endothelialization.…”

A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture

“…Dynamic flow culture has been reported to improve the recellularization efficiency in various kinds of decellularized tissues 11 , 17 , 28 . In conventional methods of recellularization aiming for ex vivo tissue reconstruction before in vivo implantation, seeding cells by circulating a cell suspension (cells + culture medium) requires a relatively long duration of recellularization incubation, and results in a relatively low efficiency of recellularization 29 , 30 . We modified the protocol from previous reports to pre-seed the cells with a sufficiently high cell density and conducted dynamic flow incubation with cell-free medium for 2 weeks, which resulted in improved endothelialization.…”

Flow with variable pulse frequencies accelerates vascular recellularization and remodeling of a human bioscaffold

“…As the vascular wall is mainly composed of SMC, its development first implies the presence of SMC throughout the entire wall thickness. By promoting a nutrient‐driven migration of SMC from the ablumen to the lumen of our human‐based scaffold (Tosun & McFetridge, 2013; Van de Walle, Moore, & McFetridge, 2020), such a repopulation was obtained for both the PMPD and CF grafts. This recellularization was however more advanced in the case of PMPD as depicted by an increased number of SMC as well as increased GAG content, reaching values close to those of native arteries where GAGs typically compose 2–5% of the dry weight (Wight, 1989).…”

“…In vitro methodologies have advanced to improve SMC migration and positive response of SMC and EC to ex vivo ‐derived ECM structures, such as the decellularized human umbilical vein (HUV) (Tosun & McFetridge, 2013; Uzarski et al, 2015; Uzarski, Van de Walle, & McFetridge, 2013; Van de Walle, Moore, & McFetridge, 2020). Among these methodologies, chemotactic gradients have been used to drive SMC migration throughout thick scaffolds such as obtaining a repopulated vascular wall (Tosun & McFetridge, 2013), and progressive flow as well as pressure conditioning have been exploited for the sequential adaptation of vascular cells to arterial biomechanics (Uzarski et al, 2015; Van de Walle et al, 2020). Herein, we proposed to apply these advanced methodologies for the in vitro development of a vascular graft composed of SMC as well as EC and based on the decellularized HUV.…”