A Cost‐Effective Culture System for the In Vitro Assembly, Maturation, and Stimulation of Advanced Multilayered Multiculture Tubular Tissue Models

Abstract

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus.

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells

“…HUASMCs were selected as cellular model. These cells are highly employed in TE studies since they can be easily extracted from a tissue (i.e., the umbilical cord) that is obtained by a non‐invasive procedure. In addition, thanks to the possibility to store umbilical cords by cell and tissue banking technologies, they represent a promising and attractive source of autologous cells for future clinical applications of vTE …”

“…As arteries and many soft biological tissues, collagen gels exhibit a viscoelastic behavior and consequently a time‐dependent response to applied stress . For this reason, stress‐relaxation tests consisting of a sustained tensile deformation for a certain period are commonly used to gain information about the viscoelastic mechanical properties of engineered vascular tissues . The stress response obtained by this test is well represented by the Maxwell–Wiechert model, composed of one spring in parallel with several spring‐dashpot (Maxwell) elements (a spring with modulus E i and a dashpot with viscosity η i in series).…”

“…The viscoelastic mechanical properties were evaluated by tensile stress relaxation tests in the circumferential direction performed on ring‐shaped samples (ring test). Samples of ≈5 mm‐long and 3.8 mm of diameter (the diameter of the mandrel, corresponding to small caliber arteries) were cut from the central part of the tubular constructs, whose terminal ends were discarded as they displayed a less homogeneous thickness due to boundary effects. The samples were mounted on ad hoc made L‐shaped grips and tested using an Instron E1000 (Instron Corporation, Norwood, MA, USA) equipped with a 5N load cell.…”

“…Since the introduction of collagen gels in vTE by Weinberg and Bell in 1986, several collagen‐based vascular models and maturation techniques of increasing complexity have been developed, including systems with co‐culture of vascular cells and physiological‐like mechanical stimulation . Cell density constitutes one of the most recurrent discrepancy of these models with respect to a native vessel, with cell seeding densities between 0.5 and 1 × 10 6 cells/mL .…”

Cell Seeding on UV‐C‐Treated 3D Polymeric Templates Allows for Cost‐Effective Production of Small‐Caliber Tissue‐Engineered Blood Vessels

“…Alignment analysis of cells in the adventitia and the media was performed as previously described . Briefly, complete vessels were cut open, fixed in 3.7% formaldehyde (VWR, Radnor, USA) and actin filaments were stained as described in Section 2.5.…”