Elsevier

Biomaterials

Volume 35, Issue 10, March 2014, Pages 3281-3297
Biomaterials

Comparative decellularization and recellularization of normal versus emphysematous human lungs

https://doi.org/10.1016/j.biomaterials.2013.12.103Get rights and content

Abstract

Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.

Introduction

Devastating lung diseases, such as pulmonary fibrosis and chronic obstructive pulmonary diseases (COPD), are increasing in prevalence and remain without a cure except for lung transplantation. However, there are not enough donor lungs to match the current clinical need and transplantation efficacy is further inhibited by acute and chronic rejection and by complications from immunosuppressive drugs. Alternative options need to be explored to increase the potential supply of donor lungs and the subsequent efficacy of transplantation. A rapidly growing body of literature suggests that decellularized (acellular) whole lung scaffolds recellularized with autologous stem or progenitor cells obtained from the intended transplant recipient may provide a potential means of utilizing failed donor or even cadaveric lungs in clinical transplantation approaches [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. However, lungs available for producing acellular lung scaffolds may come from older individuals with a history of pre-existing pulmonary disorders, such as emphysema or pulmonary fibrosis. It is currently unknown whether these lungs could be used to produce suitable acellular scaffolds for ex vivo tissue regeneration. In previous studies, we found that murine alveolar epithelial cells (C10) had limited survival when inoculated into acellular mouse lung scaffolds obtained from mice with experimentally-induced emphysema compared to normal mice [13]. To assess whether these results translated to repopulation of acellular emphysematous human lung tissue, acellular scaffolds were generated from cadaveric lungs obtained from emphysematous and healthy individuals. Lung architecture and residual protein content were assessed by histology, immunohistochemistry, and mass spectrometry as well as the ability of the acellular scaffolds to support short and long term recellularization with a variety of human cell types including human bronchial epithelial cells (HBEs), endothelial progenitor cells (CBFs), lung fibroblasts (HLFs), and bone marrow-derived MSCs (hMSCs). Potential scaffold toxicity and the ability of matrix components to support cell growth was also assessed on tissue culture plates coated with solubilized extracellular matrix from decellularized normal and emphysematous lungs.

Section snippets

Human lungs

Human lungs were obtained from autopsy services at Fletcher Allen Hospital in Burlington, Vermont. Classification of normal versus COPD (emphysematous) was based on review of available clinical records including known previous COPD or other lung diseases, smoking history, chest radiographs, pulmonary function testing, and use of respiratory medications. Subsequent confirmation of emphysematous changes on histologic sections were utilized to corroborate clinical data. A total of 18 lungs (7

Histologic and proteomic characterization of acellular emphysematous and normal lungs

Whole cadaveric lungs or isolated pulmonary lobes were decellularized using a protocol we have developed for lungs from larger animals and humans (Fig. 1A) [22]. The lungs were predominantly from an aged population (mean age at death 65.7 years, range 38–89 years) with no statistically significant differences (p < 0.015) between time from death until autopsy, cause of death, or additional findings at autopsy. Relevant clinical characteristics are detailed in Table 1.

Histological assessment

Discussion

Ex vivo lung bioengineering using decellularized whole lungs as scaffolds represents one potential new strategy for lung transplantation and in particular addresses the shortage of available donor lungs [1]. The acellular donor or cadaveric lungs could be inoculated with autologous stem or progenitor cells obtained from the transplant recipient and then clinically implanted after a suitable ex vivo regeneration strategy. Our group and others have demonstrated in a range of animal and human lung

Conclusions

Our data suggests that emphysematous lungs support initial engraftment and proliferation of a variety of human cell lines, but for reasons presently unknown, do not support prolonged viability. However, COPD, including emphysema, is a heterogenous collection of disorders and more extensive exploration of these findings needs to be performed in a wider range of diseased human lungs. Further, other acute pulmonary pathologies at the time of lung harvest from autopsy may also conceivably impact

Acknowledgments

The authors would like to thank the autopsy staff at Fletcher Allen Hospital, particularly Dr. Nick Hardin, Dr. Barbara Waters from the UVM Department of Pathology for technical assistance in methodologic development for identifying and securing small airways and blood vessels for cell inoculations, Dr. Rachael Oldinski from UVM College of Engineering for assistance with the calcium alginate coating, Dr. Mervin Yoder from Indiana University – Purdue University Indianapolis for supplying the

References (27)

  • J. Cortiella et al.

    Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation

    Tissue Eng Part A

    (2010)
  • T.H. Petersen et al.

    Tissue-engineered lungs for in vivo implantation

    Science

    (2010)
  • H.C. Ott et al.

    Regeneration and orthotopic transplantation of a bioartificial lung

    Nat Med

    (2010)
  • Cited by (139)

    • Regional and disease specific human lung extracellular matrix composition

      2023, Biomaterials
      Citation Excerpt :

      With regards to the lung, decellularized lungs from animal or cadaveric human lungs can be assessed by mass spectrometry to provide semi-quantitative characterization of the lung ECM [17–21]. Current studies, including our own, have previously demonstrated that decellularized lungs retain much of the native (i.e. non-decellularized) ECM matrix composition, including larger structural ECM proteins, such as Col1 and type-III collagen (COL3) as well as smaller bioactive ECM proteins, such as basement membrane associated laminins, proteoglycans, and glycoproteins [17,19,20,22]. As such, decellularized lungs from patients with no history of lung disease (ND) and patients with end-stage lung diseases (i.e. COPD and IPF) have been directly compared using semi-quantitative mass spectrometry.

    View all citing articles on Scopus
    1

    Dr. Wagner and Mr. Bonenfant are co-first authors.

    2

    Tel.: +1 802 656 8110; fax: +1 802 656 8926.

    3

    Tel.: +1 802 656 9722; fax: +1 802 656 2914.

    4

    Tel.: +1 802 656 2526; fax: +1 802 656 3632.

    5

    Tel.: +39 70 675 8638; fax: +39 70 666 062.

    View full text