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In this study, we improved a perfusion-based bioreactor (PBB) system to recellularize acellular lung scaffolds. A method was used for the online monitoring of the recellularization process in order to find more data during a re-seeding procedure. Lungs were harvested from young adult (2-month-old) male rats. After preparation and pre-decellularization, the lungs were decellularized with liquid detergent in order to obtain an acellular scaffold. Acelluar tissues were stained by hematoxylin and eosin to verify efficient removal of cell components and debris. Furthermore, the maintenance of the extracellular matrix architecture was evaluated using scanning electron microscopy. After rinsing for detergent removal, human umbilical cord vein endothelial cells were cultured on acellular tissue using a PBB. Data analysis of the dissolved oxygen through culturing indicated a specific growth rate of 0.0199 h−1 (doubling time, td = 34 h). The average oxygen uptake rate per cell was measured as 14.3 nmol/min per 106 cells. This study demonstrates that recellularization on acellular tissue can be monitored online to provide insight into the growth behavior of cells. The results show the high performance design of PBBs for tissue-engineered lungs.

Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.

Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.

Background: Lung transplantation is the only curative approach for end stage lung disease, but tissue shortage remains a problem. Generating lung tissue ex vivo could overcome tissue shortages. Both synthetic and biologic scaffolds have been explored, yet both have limitations. Biologic scaffolds have low reproducibility while synthetic scaffolds lack biologically inductive properties. Hybrid materials, comprised of both, have been used for tissue engineering, but have been limited to simple shapes. Recent advances in 3D printing may permit for complex shapes, but potential bioinks are unknown. We hypothesized that a hybrid hydrogel generated by combining a synthetic polymer with lung derived extracellular matrix (ECM) could be a suitable bioink for 3D bioprinting lung tissue. Methods: We generated hybrid hydrogels from a synthetic polymer, alginate, and ECM derived from decellularised mouse lungs to make hybrid hydrogels. Viability and proliferation of distal lung epithelial cell lines (MLE12 and A549) encapsulated in the hydrogel were measured via immunofluorescence (Ki67+) and flow cytometry (EdU+) after 7 days. Results: Both MLE12 and A549 cells remained viable and were distributed homogenously within manufactured hydrogels of various shapes. MLE12s in ECM-alginate hybrid hydrogels were significantly more proliferative (30% increase) than MLE12 cells seeded in an alginate hydrogel as assessed by flow cytometry. Conclusions: A hybrid hydrogel generated from alginate and decellularised lung derived ECM supports proliferation of lung epithelial cell lines and can be manufactured in a variety of shapes. The hybrid alginate-ECM solution is thus a promising candidate as a bioink for 3D bioprinting.