Methods in Molecular Biology (2022) 2436: 167–182

DOI 10.1007/7651_2021_431

© Springer Science+Business Media, LLC 2021

Published online: 10 September 2021

Bioreactor-Based De-epithelialization of Long-Segment

Tracheal Grafts

Alba E. Marin-Araujo, Siba Haykal, and Golnaz Karoubi

Abstract

Tissue engineering techniques to generate a graft ex vivo is an exciting field of research. In particular, the

use of biological scaffolds has shown to be promising in a clinical setting. In this approach, decellularized

donor scaffolds are obtained following detergent-based enzymatic treatment to remove donor cells and

subsequently repopulated with recipient specific cells. Herein, we describe our bioreactor-based partial

decellularization approach to generate hybrid tracheal grafts. Using a short detergent-based treatment with

sodium dodecyl sulfate (SDS), we remove the epithelium and maintain the structural integrity of the donor

grafts by keeping the cartilage alive. The following will be a step-by-step description of the bioreactor

system setup and partial decellularization protocol to obtain a de-epithelialized tracheal graft.

Key words Biological scaffolds, Double-chamber perfusion bioreactor, Partial decellularization,

Tissue engineering, Tracheal de-epithelialization

1

Introduction

Tracheal transplantation using a tissue engineered graft is a

promising alternative for treatment of extensive tracheal injury.

The ultimate goal of tracheal tissue engineering is the generation

of a functional construct to replace damaged upper airways. While

synthetic grafts can provide structural integrity, recapitulation of

the ECM components and architecture is challenging. An ideal

approach allows for preservation of the extracellular matrix

(ECM) which provides important biochemical cues required to

support cellular function and maintains adequate biomechanical

support [1, 2]. Thus, decellularization and recellularization of

biological tracheal scaffolds is an exciting and promising area of

research. While the specific details of each protocol are dependent

on the tissue or organ of interest, the vast majority of decellulariza-

tion protocols require a combination of physical, enzymatic,

and/or chemical treatments [1]. In the trachea, fully decellularized

scaffolds have come across challenges with stenosis due to

Siba Haykal and Golnaz Karoubi are co-senior authors.

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