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In order to provide an alternative treatment option to lung transplantation for patients with end-stage lung disease, we aim for the development of an implantable biohybrid lung (BHL), based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. Complete hemocompatibility of all blood contacting surfaces is crucial for long-lasting BHL durability and can be achieved by their endothelialization. Autologous endothelial cells (ECs) would be the ideal cell source, but their limited proliferation potential excludes them for this purpose. As induced pluripotent stem cell-derived ECs enable the generation of a large number of ECs, we assessed and compared their capacity to form a viable and confluent monolayer on HFM, while indicating physiologic EC-specific anti-thrombogenic and anti-inflammatory properties. ECs were generated from three different human iPSC lines, and seeded onto fibronectin-coated poly-4-methyl-1-pentene (PMP) HFM. Following phenotypical characterization, ECs were analyzed for their thrombogenic and inflammatory behavior with or without TNFα induction, using FACS and qRT-PCR. Complementary, leukocyte- and platelet adhesion assays were carried out. The capacity of the iPSC-ECs to reendothelialize cell-free monolayer areas was assessed in a scratch assay. ECs sourced from umbilical cord blood (hCBECs) were used as control. iPSC-derived ECs formed confluent monolayers on the HFM and showed the typical EC-phenotype by expression of VE-cadherin and collagen-IV. A low protein and gene expression level of E-selectin and tissue factor was detected for all iPSC-ECs and the hCBECs, while a strong upregulation of these markers was noted upon stimulation with TNFα. This was in line with the physiological and strong induction of leukocyte adhesion detected after treatment with TNFα, iPSC-EC and hCBEC monolayers were capable of reducing thrombocyte adhesion and repopulating scratched areas. iPSCs offer the possibility to provide patient-specific ECs in abundant numbers needed to cover all blood contacting surfaces of the BHL with a viable, non-thrombogenic and non-inflammatory monolayer. iPSC-EC clones can differ in terms of their reendothelialization rate, and pro-inflammatory response. However, a less profound inflammatory response may even be advantageous for BHL application. With the proven ability of the seeded iPSC-ECs to reduce thrombocyte adhesion, we expect that thrombotic events that could lead to BHL occlusion can be avoided, and thus, justifies further studies on enabling BHL long-term application.

© 2018 John Wiley & Sons, Ltd. Several key prerequisites need to be fulfilled for the development of a biohybrid lung, which can offer an actual alternative to lung transplantation. A major aspect is an optimized haemocompatibility of the device's artificial surfaces via endothelial cell seeding. In this study, four different types of polymeric gas exchange hollow fibre membranes (HFMs) were analysed utilizing four different seeding protocols in order to identify the ideal combination for sufficient long-term endothelialization. Human cord blood-derived endothelial cells (HCBECs) were used for the endothelialization of polypropylene HFMs with two different pore sizes and poly-4-methyl-1-pentene HFMs, both with and without heparin/albumin coating. The qualitative and quantitative impact of four different rotational seeding protocols regarding long-term HFM endothelialization and the impact of inflammatory stimulation on the seeded HCBECs were examined by fluorescence microscopy, cell counting, and analysis of relative expression levels of activation, shear stress, and thrombogenic state markers. Optimized endothelial cell seeding and long-term cultivation were only achieved using heparin/albumin-coated poly-4-methyl-1-pentene HFMs, applying 24 hr of rotational speed at 1 rpm followed by 120 hr of static culture. Neither cell-to-HFM contact nor the rotational cultivation procedure showed an impact on the physiological anti-thrombogenic and anti-inflammatory HCBEC activation status. Additionally, the cells maintained their physiological responsiveness towards inflammatory stimulation. Rotational seeding strategies and a seamless heparin/albumin coating of the HFMs are crucial requirements for a sufficient and long-lasting endothelialization and thus a key element in the future development and in vivo application of the biohybrid lung.

Purpose Lung transplantation is the only curative therapy option for patients suffering from end stage lung diseases. However, it is associated with high risks and only available for selected patients. In order to provide an alternative treatment option, this project aims for the development of an implantable biohybrid lung (BL), based on hollow fibre membrane (HFM) technology used in extracorporeal membrane oxygenators. A crucial requirement to achieve long-lasting durability of the BL is the optimized bio- and haemocompatibility of all blood contacting surfaces, which can be achieved by endothelialisation. Methods Albumin/Heparin (A/H) and Fibronectin (FN) coated HFM were compared for their eligibility to mediate the establishment of a viable, confluent and non-thrombogenic endothelial cell (EC) monolayer. Activation status of the seeded ECs was analyzed and verified by leukocyte- and thrombocyte adhesion assays. The behavior of HFM-seeded ECs under workload conditions, i.e. fluid flow and oxidative stress exposure, was assessed in a customized flow chamber setup. To identify a clinically relevant EC source, comparative studies including human cord blood derived endothelial cells (hCBECs) induced pluripotent stem cell derived endothelial cells and immunotolerable MHC-silenced hCBECs were conducted. Results A physiologic, viable and confluent EC monolayer could be sustained under both, static and flow dynamic culture conditions. All tested EC types preserved their non-thrombogenic and non-inflammatory status on both tested HMF. Exposure to relevant levels of hyperoxia or hypoxia did not affect the physiological function of the ECs. However, FN coated HFMs demonstrated an improved EC-monolayer resistance towards flow conditions. Conclusion Endothelialisation of HFM improves their hemocompatibility. Moreover, the endothelial monolayer on HFM can resist clinically relevant settings, like flow exposure and hyperoxia/hypoxia. MHC-silenced ECs could be considered as suitable cell source, escaping the recipient's immune response. These results may bring BL as alternative of lung transplantation for patients with end-stage lung diseases closer to reality.

Purpose Left ventricular assist devices (LVADs) are established implantable devices in patients with end-stage heart failure. Due to their artificial materials, anticoagulative therapy is needed, though leading to partly lethal complications and limited clinical applications. Improved LVAD hemocompatibility of all blood contacting surfaces can be achieved by their endothelialisation. Additionally, the endothelial monolayer need to resist clinically relevant flow-conditions, for which we established a new approach using magnetic forces and intracellular superparamagnetic nanoparticles (NP). Methods Endothelial cells (EC) were incubated with nanoparticles of different concentrations and coatings (sodium citrate, polyvinylpyrrolidone) for various time periods. Nanoparticulate absorption behaviour was visualized by Prussian blue staining. Batches with high intracellular nanoparticle quantities were analysed for cell viability by immunofluorescence staining and proliferation assay, possible cytotoxicity by flow cytometry. Following, cellular alignment induced by different magnetic forces was examined and transferred to LVAD impeller endothelialisation. Results Concentration-, coating- and time-dependent changes in cell viability, proliferation and cytotoxicity could be shown, indicating best results for low concentrated sodium citrate nanoparticles, which were used for subsequent cell migration experiments. Magnetic field sufficiently affected endothelialisation as significantly more NP loaded ECs adhered to the impeller surface compared to the unloaded control group. Conclusion These results are promising steps towards the effective biologisation of the LVAD, as the LVAD itself may provide the needed magnetic field for sufficient and flow-resistant endothelialisation. This hypothesis needs to be analysed within the following experiments.

Working towards establishing a biohybrid lung with optimized hemocompatibility, this study analyzed the feasibility of establishing flow-resistant endothelium on heparin/albumin coated poly-4-methly-1-pentene hollow fiber gas exchange membranes (PMP-HFs). The seeding efficiency and proliferation of human cord blood derived endothelial cells (HCBEC) on PMP-HFs were analyzed under static conditions by WST-8 cell proliferation assay and fluorescence microscopy. The HCBEC monolayer integrity under different flow conditions was also assessed. Endothelial-specific phenotype verification, expression activation levels and thrombogenic state markers were quantified by real-time RT-PCR for cell-to-PMP-HF contact under static and dynamic conditions. The results demonstrated the feasibility of establishing a viable, confluent, and flow-resistant endothelial monolayer on the blood-contact surface of PMP-HFs, which maintained a physiological response to TNFα-stimulation and flow conditions. The endothelial phenotype, expression levels of adhesion molecules and thrombogenic state markers were unaffected by cell-to-PMP-HFs contact. These results represent a significant step towards establishing a biohybrid lung.

Purpose Lung transplantation remains the only treatment option for patients with end-stage lung disease. Although surgical techniques and organ preservation significantly improved over the last decades, ischemia and reperfusion injuries, which inter alia manifest in edema formation, still represent major limitations affecting clinical outcome. Here we investigated the hypothesis that different lung preservation solutions contribute to solution-specific degrees of transient leaks within the endothelial cell (EC) monolayer, therefore allowing liquid influx into the interstitial space leading to edema formation. Methods Intercellular connectivity within confluent EC monolayers was assessed after 1h, respectively 5.5h storage in the respective preservation solutions OCS™, Steen™, Perfadex™, PerfadexPlus™ or Celsior™ ± 10µM or 100µM 17β-Estradiol (17s), which was used to deliberate disintegration of the monolayer. Furthermore, 5.5h and 24h of regeneration in the preservation solution, respectively EC medium without 17s was analyzed. Monolayer permeability was evaluated using FITC-Dextran transwell-assays, cell viability (Calcein AM) and monolayer integrity (VE-Cadherin) were examined by fluorescence microscopy. Realtime qRT-PCR analyses were carried out to investigate responses on gene expression levels. Results FITC-Dextran assay indicated solution-specific monolayer permeability, after 1h incubation highest was identified for Celsior™, followed by Perfadex™ and OCS™, Steen™ and PerfadexPlus™ showed up with the lowest permeability. While addition of 10µM 17s did not affect solution-specific permeability, all groups treated with 100µM 17s become durable leaky. Comparable results were seen after 5.5h and 24h of regeneration, while previous incubation with 100µM 17s induced irreversible effects. These results could be confirmed by immunofluorescence staining. By contrast, independent of the treatment qRT-PCR analyses indicated negligible effects on gene expressions of activation and thrombogenic state markers. Conclusion The application of different preservation solutions results in solution-specific, partly irreversible endothelial leakage, which may partly explain edema and weight increase during organ preservation. Subsequent analysis will correlate these results with clinical parameters.

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality.

Utilization of living cells for therapies in regenerative medicine requires a fundamental understanding of the interactions between different cells and their environment. Moreover, common models based on adherent two-dimensional cultures are not appropriate to simulate the complex interactions that occur in a three-dimensional (3D) cell-microenvironment in vivo. In this study, we present a computer-aided method for the printing of multiple cell types in a 3D array using laser-assisted bioprinting. By printing spots of human adipose-derived stem cells (ASCs) and endothelial colony-forming cells (ECFCs), we demonstrate that (i) these cell spots can be arranged layer-by-layer in a 3D array; (ii) any cell-cell ratio, cell quantity, cell-type combination, and spot spacing can be realized within this array; and (iii) the height of the 3D array is freely scalable. As a proof of concept, we printed separate spots of ASCs and ECFCs within a 3D array and observed cell-cell interactions in vascular endothelial growth factor-free medium. It has been demonstrated that direct cell-cell contacts trigger the development of stable vascular-like networks. This method can be applied to study complex and dynamic relationships between cells and their local environment.

Objectives To evaluate bioartificial haemodialysis access grafts in a sheep model with respect to patency and morphology. Material and methods Bovine internal thoracic arteries (n = 28) were decellularised. Fourteen grafts (DC grafts) were directly implanted as cervical AV shunts, the remaining were re-seeded with endothelial cells (ECs) derived from blood samples of the later ovine recipient (EC grafts) first. Following simulated punctures and duplex ultrasound scans to determine patency, grafts were explanted for immunohistochemical characterisation after 3 and 6 months, respectively. DC grafts underwent biomechanical testing for compliance (C), suture retention strength (SRT), and burst pressure (BP) before (n = 6) and after (n = 6) implantation. Results Following 3 and 6 months, the majority of EC (n = 6/6; n = 6/7) and DC grafts (n = 5/6; n = 5/7) were patent and not relevantly stenosed (peak systolic velocity: EC grafts = 76 cm s−1 ± 4; DC grafts = 77 cm s−1 ± 5). Simulated haemodialysis punctures revealed significantly shorter bleeding times in all bioartificial grafts than in native jugular veins (P > 0.001). Comparing native carotid arteries with DC grafts prior to and post-implantation, the latter differed significantly with respect to C (P > 0.001; P = 0.005), whereas only pre-implant DC grafts differed regarding BP (P = 0.002); no differences were observed for SRT. Histology revealed complete endothelial surface coverage of EC, but not DC grafts. Furthermore, DC grafts exhibited areas of pronounced tissue calcification. Conclusion The preclinical development of a bioartificial haemodialysis access graft with promising mechanical and morphological properties in a sheep model is feasible.

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent of the Fpg-sensitive modifications were repaired within 2 h. Under conditions that induced 10 Fpg-sensitive modifications per 10(6) bp (six 8-oxoG residues per 10(6) bp), approximately 60 mutations per 10(6) cells were induced in the gpt locus of the AS52 cells. A rather similar mutation frequency was observed when a plasmid carrying the gpt gene was exposed to Ro19-8022 plus light under cell-free conditions and subsequently replicated in bacteria. Sequence analysis revealed that GC-->TA and GC-->CG transversions accounted for 90% of the base substitutions. A significant generation of micronuclei was detectable in AS52 cells exposed to the photosensitizer plus light as well.

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. The in vitro growth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein-Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity toward cis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity toward trans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations of o-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations of o-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.