Methods in Molecular Biology (2022) 2436: 157–165

DOI 10.1007/7651_2021_398

© Springer Science+Business Media, LLC 2021

Published online: 06 May 2021

Tracheal In Vitro Reconstruction Using a Decellularized

Bio-Scaffold in Combination with a Rotating Bioreactor

Georgia Pennarossa, Matteo Ghiringhelli, Fulvio Gandolfi,

and Tiziana A. L. Brevini

Abstract

Long-segment airway stenosis as well as their neoplastic transformation is life-threatening and still currently

represent unsolved clinical problems. Indeed, despite several attempts, definitive surgical procedures are not

presently available, and a suitable tracheal reconstruction or replacement remains an urgent clinical need. A

possible innovative strategic solution to restore upper airway function may be represented by the creation of

a bioprosthetic trachea, obtained through the combination of tissue engineering and regenerative medicine.

Here we describe a two-step protocol for the ex vivo generation of tracheal segments. The first step

involves the application of a decellularization technique that allows for the production of a naturally derived

extracellular matrix (ECM)-based bio-scaffold, that maintains the macro- and micro-architecture as well as

9 the matrix-related signals distinctive of the original tissue. In the second step chondrocytes are seeded

onto decellularized trachea, using a rotating bioreactor to ensure a correct scaffold repopulation.

This multi-step approach represents a powerful tool for in vitro reconstruction of a bioengineered trachea

that may constitute a promising solution to restore upper airway function. In addition, the procedures here

described allow for the creation of a suitable 3D platform that may find useful applications, both for

toxicological studies as well as organ transplantation strategies.

Key words Bioprosthetic trachea, Chondrocytes, Decellularization, ECM-based bio-scaffold, Rotat-

ing bioreactor, Tissue engineering

1

Introduction

Upper airway dysfunctions and diseases, such as stenosis and neo-

plastic transformation, represent life-threatening conditions and

seriously affect the duration and quality of life due to altered

breathing, speech, and swallowing [1]. Several studies have pro-

posed different techniques to help cartilage repairing, however

definitive surgical procedures are not presently available and a

suitable tracheal reconstruction or replacement remains an unmet

clinical need [2]. In this context, tissue engineering represents one

of the most promising approach to generate bioprosthetic tracheas,

suitable for organ transplantation. During the past 50 years, several

attempts have been made to cope with the challenging problem of

reconstructing

long

segmental

tracheal

defects

and

diverse

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