Sterilization of lung matrices by supercritical carbon dioxide

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO(2)) that can achieve a sterility assurance level 10(−6) in decellularized lung matrix. The effects of ScCO(2) treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO(2) did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO(2), indicating that ScCO(2) produces a matrix that is stable during storage. The current study's results indicate that ScCO(2) can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes

Method and system for aligning fibers during electrospinning

A method and system are provided for aligning fibers in an electrospinning process. A jet of a fiberizable material is directed towards an uncharged collector from a dispensing location that is spaced apart from the collector. While the fiberizable material is directed towards the collector, an elliptical electric field is generated via the electrically charged dispenser and an oppositely-charged control location. The field spans between the dispensing location and the control location that is within line-of-sight of the dispensing location, and impinges upon at least a portion of the collector. Various combinations of numbers and geometries of dispensers, collectors, and electrodes can be used

Influence of pH on Extracellular Matrix Preservation During Lung Decellularization

The creation of decellularized organs for use in regenerative medicine requires the preservation of the organ extracellular matrix (ECM) as a means to provide critical cues for differentiation and migration of cells that are seeded onto the organ scaffold. The purpose of this study was to assess the influence of varying pH levels on the preservation of key ECM components during the decellularization of rat lungs. Herein, we show that the pH of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-based decellularization solution influences ECM retention, cell removal, and also the potential for host response upon implantation of acellular lung tissue. The preservation of ECM components, including elastin, fibronectin, and laminin, were better retained in the lower pH conditions that were tested (pH ranges tested: 8, 10, 12); glycosaminoglycans were preserved to a higher extent in the lower pH groups as well. The DNA content following decellularization of the rat lung was inversely correlated with the pH of the decellularization solution. Despite detectible levels of cyotoskeletal proteins and significant residual DNA, tissues decellularized at pH 8 demonstrated the greatest tissue architecture maintenance and the least induction of host response of all acellular conditions. These results highlight the effect of pH on the results obtained by organ decellularization and suggest that altering the pH of the solutions used for decellularization may influence the ability of cells to properly differentiate and home to appropriate locations within the scaffold, based on the preservation of key ECM components and implantation results

Use of bioengineered human acellular vessels to treat traumatic injuries in the Ukraine–Russia conflict

On February 24th 2022, the Russian Federation invaded Ukraine. Consistent with other modern conflicts, blastsand shrapnel penetration have caused a large fraction ofthe traumatic injuries, creating contaminated woundsthat often compromise the vascular supply to limbs andorgans. Although autogenous veins are the preferredoption for repair, their limited availability leads to the useof synthetic grafts. However, usage of synthetic vasculargrafts carries a high risk of infection, meaning that thereis a lack of suitable conduit in some war-injured patients. Human Acellular Vessels (HAVs) are bioengineeredvascular conduits that are cultured from human donors’smooth muscle cells and subsequently decellularized.1 TheHAV is an investigational biological product in late-stageclinical development, with over 1,000 patient-years ofexposure. After clinical implantation, the HAV repopulateswith cells, producing a living vascular tissue that may behighly resistant to infection.2,3 In March 2022, surgeons inUkraine requested the HAV for vascular repair underHumanitarian conditions. In response, the manufacturerof the HAV (Humacyte Global Inc.) worked with the International Office of the US FDA and the Ukrainian Ministry of Health to provide HAVs to five hospitals in Ukraine.Surgeons in Ukraine were trained remotely, by videoconferencing, on the procedures applicable to the HAV.Since June 2022, 13 patients who lacked autologousvein for repair have been treated with the HAV to repair arange of arteries including superficial femoral, commonfemoral, popliteal, and brachial arteries. Of these, 11 sustained limb vascular injuries in the ongoing conflict, mostlycomprising blast and shrapnel wounds. One such patient isshown in Fig. 1, who underwent repair of the commonfemoral artery on day 1, had patency of the HAV confirmedat day 52, and who began ambulating on day 119.Three patients received HAVs after failure of eithersaphenous vein (n = 2) or synthetic graft (n = 1) to repair anarterial injury. As of January 29th 2023, all HAVs retainedprimary patency, and no infections nor amputations of theaffected limbs were reported. After follow-up times rangingfrom 1 to 7 months, there have been no reports of HAVconduit infection or mechanical failure. The Ukrainereal-world trauma experience demonstrates the potentialfor regenerative medicine technologies to improve patientoutcomes in resource-limited environments

Alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor

Traditional stem cell differentiation protocols make use of a variety of cytokines including growth factors (GFs) and inhibitors in an effort to provide appropriate signals for tissue specific differentiation. In this study, iPSC-derived type II pneumocytes (iPSC-ATII) as well as native isolated human type II pneumocytes (hATII) were differentiated toward a type I phenotype using a unique air–liquid interface (ALI) system that relies on a rotating apparatus that mimics in vivo respiratory conditions. A relatively homogenous population of alveolar type II-like cells from iPSC was first generated (iPSC-ATII cells), which had phenotypic properties similar to mature human alveolar type II cells. iPSC-ATII cells were then cultured in a specially designed rotating culture apparatus. The effectiveness of the ALI bioreactor was compared with the effectiveness of small molecule-based differentiation of type II pneumocytes toward type 1 pneumocytes. The dynamics of differentiation were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), flow cytometry and immunocytochemistry. iPSC-ATII and hATII cells cultured in the ALI bioreactor had higher levels of type I markers, including aquaporin-5(AQ5), caveolin-1, and T1α, at both the RNA and protein levels as compared with the flask-grown iPSC-ATII and hATII that had been treated with small molecules to induce differentiation. In summary, this study demonstrates that a rotating bioreactor culture system that provides an air–liquid interface is a potent inducer of type I epithelial differentiation for both iPS-ATII cells and hATII cells, and provides a method for large-scale production of alveolar epithelium for tissue engineering and drug discovery

Bioengineered lungs generated from human iPSCs‐derived epithelial cells on native extracellular matrix

The development of an alternative source for donor lungs would change the paradigm of lung transplantation. Recent studies have demonstrated the potential feasibility of using decellularized lungs as scaffolds for lung tissue regeneration and subsequent implantation. However, finding a reliable cell source and the ability to scale up for recellularization of the lung scaffold still remain significant challenges. To explore the possibility of regeneration of human lung tissue from stem cells in vitro, populations of lung progenitor cells were generated from human iPSCs. To explore the feasibility of producing engineered lungs from stem cells, we repopulated decellularized human lung and rat lungs with iPSC‐derived epithelial progenitor cells. The iPSCs‐derived epithelial progenitor cells lined the decellularized human lung and expressed most of the epithelial markers when were cultured in a lung bioreactor system. In decellularized rat lungs, these human‐derived cells attach and proliferate in a manner similar to what was observed in the decellularized human lung. Our results suggest that repopulation of lung matrix with iPSC‐derived lung epithelial cells may be a viable strategy for human lung regeneration and represents an important early step toward translation of this technology.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142929/1/term2589.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142929/2/term2589_am.pd

Fate of Distal Lung Epithelium Cultured in a Decellularized Lung Extracellular Matrix

Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-βR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-βR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix metalloprotease-2 (MMP-2) genes after 1 day in culture (p=0.0135 and 0.0128, respectively) and elevated collagen I expression at 7 days (p=0.0016). These data suggest that the original type II-enriched population underwent a transition to increased expression of mesenchymal markers, perhaps as part of a survival or wound-healing program. These results suggest that additional medium components and/or the application of physiologically appropriate stimuli such as ventilation may be required to promote lung-specific epithelial phenotypes

Human iPS cell–derived alveolar epithelium repopulates lung extracellular matrix

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII–like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung

Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary–venous ECs (COL15A1(neg)) localized to the lung parenchyma and systemic–venous ECs (COL15A1(pos)) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic–venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice