# A novel agarose-free, standardized generation and versatile ECM characterization of decellularized scaffolds from normal and fibrotic human lung tissue

## TL;DR

This paper introduces a new method to create lung tissue scaffolds that preserve key structural components and support cell studies in lung diseases.

## Contribution

A novel agarose-free, apoptosis-assisted method for generating standardized decellularized lung scaffolds from fibrotic and normal human tissue.

## Key findings

- The protocol removed over 99.8% of nuclei while preserving collagen and elastin in both fibrotic and non-fibrotic lung tissue.
- Fibroblasts successfully colonized the scaffolds, with non-fibrotic scaffolds showing more contraction than fibrotic ones.
- A semi-quantitative histological analysis method was developed to assess multiple ECM components from a single sample.

## Abstract

The extracellular matrix (ECM) is a key regulator of tissue homeostasis and remodeling in interstitial lung diseases (ILD) such as idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (FHP). Decellularized lung scaffolds provide a physiologically relevant platform for studying cell-matrix interactions but generating structurally intact scaffolds from fibrotic human lungs remains challenging. Many existing protocols struggle with either insufficient removal of the cells or significant disruption of certain ECM components. In addition, all of them rely on agarose-embedding for the production of thin lung slices.

Tissue samples were decellularized using apoptosis inducers (Camptothecin, Raptinal) and a mild detergent (SB-10). Standardized decellularized lung scaffolds (SDLS) were generated using a gelatin-based temporary cutting support structure, which is completely removable prior to recellularization. Decellularization and preservation of the ECM was validated by DAPI nuclear counts and semi-quantitative image analysis of collagen (picro-sirius red under polarization) and elastin (elastic Verhoeff-Van Gieson (EVG)). For functional testing, SDLS were repopulated with primary human lung fibroblasts (CD90+CD31−CD45−) and assessed for colonization, viability (WST-1 assay), and cytotoxicity (LDH assay).

The protocol removed all nuclei (>99.8%) in both fibrotic and non-fibrotic lung tissue while preserving structural integrity and the major ECM components (collagen and elastin). The novel semi-quantitative analysis proved to be accurate and versatile for the analysis of multiple ECM components within one sample. Overall, both Collagen (fresh 15.5% ± 4.3 vs. decellularized 16.7% ± 4.4) and elastin (fresh 10.0% ± 3.7 vs. decellularized 9.8% ± 3.4) remained stable post-decellularization. Fibroblasts successfully colonized SDLS, with non-fibrotic scaffolds showing pronounced contraction, whereas fibrotic scaffolds retained their dimensions. Even though fibroblasts seeded on SDLS showed significantly lower viability than those seeded on plastic plates, cytotoxicity 1 day after repopulation was not increased and remained acceptable.

We present a reproducible, agarose-free, apoptosis-assisted method for the generation of residue-free and standardized decellularized lung scaffolds from normal and fibrotic human tissue. The novel semi-quantitative histological analysis allows the investigation of multiple ECM components from a single sample. The SDLS platform preserves key ECM components and supports controlled recellularization enabling physiologically relevant studies of matrix-cell interactions and therapeutic screening in ILD models.

Diagram illustrating an agarose-free, apoptosis-assisted lung decellularization workflow, starting with healthy and fibrotic lung tissue, followed by cell death induction, gelatin embedding, generation of standardized decellularized lung scaffolds, co-culture with primary human fibroblasts, and depiction of extracellular matrix changes from fresh to recellularized states.