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

Frontiers in Bioengineering and Biotechnology, vol. 14, pp. 1772891

Abstract

Background 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. Methods 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). Results 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. Conclusion 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.

Authors 12

1. Eike B. Preuß

   Medizinische Hochschule Hannover · German Center for Lung Research

   Affiliation as printed

   Biomedical Research in Endstage and Obstructive Lung Disease Hanover (BREATH), German Center for Lung Research (DZL)

   Institute of Pathology, Lung Research Group, Hannover Medical School

2. Lara‐Jasmin Schröder

   Medizinische Hochschule Hannover · German Center for Lung Research

   Affiliation as printed

   Biomedical Research in Endstage and Obstructive Lung Disease Hanover (BREATH), German Center for Lung Research (DZL)

   Institute of Pathology, Lung Research Group, Hannover Medical School

3. Charlotte Hähner

   Medizinische Hochschule Hannover

   Affiliation as printed

   Institute of Pathology, Lung Research Group, Hannover Medical School

4. Jérôme A. von Bandemer

   Medizinische Hochschule Hannover

   Affiliation as printed

   Institute of Pathology, Lung Research Group, Hannover Medical School

5. Christopher Werlein

   Medizinische Hochschule Hannover

   Affiliation as printed

   Institute of Pathology, Lung Research Group, Hannover Medical School

6. Edith Katharina Plucinski

   Medizinische Hochschule Hannover · German Center for Lung Research

   Affiliation as printed

   Biomedical Research in Endstage and Obstructive Lung Disease Hanover (BREATH), German Center for Lung Research (DZL)

   Institute of Pathology, Lung Research Group, Hannover Medical School

7. Stephanie Schubert

   Medizinische Hochschule Hannover

   Affiliation as printed

   Institute of Pathology, Lung Research Group, Hannover Medical School

8. Holger Schlüter

   Boehringer Ingelheim (Germany)

   Affiliation as printed

   Boehringer Ingelheim

9. Matthew J. Thomas

   Boehringer Ingelheim (Germany)

   Affiliation as printed

   Boehringer Ingelheim

10. Danny David Jonigk Aachen Institute of Pathology

    Universitätsklinikum Aachen · German Center for Lung Research · RWTH Aachen University

    Affiliation as printed

    Biomedical Research in Endstage and Obstructive Lung Disease Hanover (BREATH), German Center for Lung Research (DZL)

    Institute of Pathology, RWTH Aachen University/UKA

11. Jan C. Kamp

    Medizinische Hochschule Hannover · German Center for Lung Research

    Affiliation as printed

    Biomedical Research in Endstage and Obstructive Lung Disease Hanover (BREATH), German Center for Lung Research (DZL)

    Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School

12. Mark Kühnel Aachen Institute of Pathology

    Universitätsklinikum Aachen · German Center for Lung Research · RWTH Aachen University

    Affiliation as printed

    Biomedical Research in Endstage and Obstructive Lung Disease Hanover (BREATH), German Center for Lung Research (DZL)

    Institute of Pathology, RWTH Aachen University/UKA