A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture

Mohammadhossein Dabaghi,Alexander Noble,Spencer D Revill,Vibudha Nanduri,Neda Saraei,Jeremy Alexander Hirota,Boyang Zhang,Jose Moran-Mirabal,Abiram Chandiramohan,Martin Kolb,Julia Ungureanu,Mouhanad Babi,Mabel Barreiro Carpio,Yaron Shargall,Kjetil Ask

doi:10.3390/cells10061538

Abstract

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems

Highlights

We showed that primary human lung fibroblast cells could survive in decellularized ECM (dECM)-based hydrogels and the dECM hydrogels with various concentrations underwent different contraction
Histological analysis was performed to visualize the removal of cellular components, especially nucleic acid material, and investigate the disruptive impact of the detergents used in the decellularization process on extracellular matrix (ECM) proteins (Figure 1a)
The Double-stranded DNA (dsDNA) concentration was quantified using the PicoGreen assay, indicating that dsDNA was decreased from 2700 ± 600 to 40 ± 20, which is an acceptable reduction suggested in the literature [15,41] (Figure 1b)

Summary

Introduction

In vitro organ-on-chip devices have attempted to more accurately model the in situ environment by integrating mechanical forces, including airflow and stretch [12,13,14]. Most of these models still lack the use of primary cells and tissue-specific ECM components required for cell differentiation, as well as 3D geometries that are important for lung cell differentiation [9,15]. Pulmonary diseases including IPF and COPD frequently cause significant changes and remodeling in the ECM, which is rarely an integrated feature in in vitro models [16,17,18]. Ex vivo models have not been employed extensively and are limited by access to fresh human lung tissue and face complications to be scaled up to a high-throughput system [19]

Objectives

Methods

Results

Discussion

Conclusion