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EndOxy: Dynamic Long-Term Evaluation of Endothelialized Gas Exchange Membranes for a Biohybrid Lung
In the concept of a biohybrid lung, endothelial cells seeded on gas exchange membranes form a non-thrombogenic an anti-inflammatory surface to overcome the lacking hemocompatibility of today’s oxygenators during extracorporeal membrane oxygenation. To evaluate this concept, the long-term stability and gas exchange performance of endothelialized RGD-conjugated polydimethylsiloxane (RGD-PDMS) membranes was evaluated. Human umbilical vein endothelial cells (ECs) were cultured on RGD-PDMS in a model system under physiological wall shear stress (WSS) of 0.5 Pa for up to 33 days. Gas exchange performance was tested with three biological replicates under elevated WSS of 2.5 Pa using porcine blood adjusted to venous values following ISO 7199 and blood gas analysis. EC morphology was assessed by immunocytochemistry (n = 3). RGD-PDMS promoted endothelialization and stability of endothelialized membranes was shown for at least 33 days and for a maximal WSS of 2.5 Pa. Short-term exposure to porcine blood did not affect EC integrity. The gas transfer tests provided evidence for the oxygenation and decarboxylation of the blood across endothelialized membranes with a decrease of transfer rates over time that needs to be addressed in further studies with larger sample sizes. Our results demonstrate the general suitability of RGD-PDMS for biohybrid lung applications, which might enable long-term support of patients with chronic lung failure in the future. © 2019, The Author(s).
Porous PVDF Monoliths with Templated Geometry
Additive manufacturing of complex porous polymer geometries is a new field of advanced materials processing. Such new geometries can be used to fabricate porous polymer monoliths serving as a support for other material functions. Here, a novel fabrication technology to manufacture tailored 3D porous monoliths via additive manufacturing and templating is presented. The method is based on replicating a 3D-printed mold with a polymer solution of polyvinylidenfluorid-triethyl phosphate (PVDF-TEP) and induce phase separation of the polymer solution subsequently. In a second step, the mold is removed without affecting the porous PVDF phase. As a result, porous monoliths with a templated 3D architecture are successfully fabricated. The manufacturing process is successfully applied to complex structures and can be applied to any conceivable geometry. Coating the porous 3D monoliths with another PVDF solution allows applying a skin layer yielding an asymmetric membrane monolith. As a showcase, a polydimethylsiloxane coating even leads to a smooth and dense layer of micrometer size. The methodology enables a new generation of complex porous polymer monoliths with tailored surface coatings. For the combination of poly(dimethylsiloxane) on a porous support, gas/liquid mass transfer is used in blood oxygenation with reduced diffusion limitation is within reach.
EndOxy: Mid-term stability and shear stress resistance of endothelial cells on PDMS gas exchange membranes
Endothelialized oxygenator devices (EndOxy) with a physiological, nonthrombogenic, and anti-inflammatory surface offer the potential to overcome current shortcomings of conventional extracorporeal membrane oxygenation such as complications like thromboembolism and bleeding that deteriorate adequate long-term hemocompatibility. The approach of endothelialization of gas exchange membranes, and thus the formation of a nonthrombogenic and anti-inflammatory surface, is promising. In this study, we investigated the mid-term shear stress resistance as well as gas transfer rates and cell densities of endothelial cells seeded on RGD-conjugated polydimethylsiloxane (RGD-PDMS) gas exchange membranes under dynamic conditions. Human umbilical vein endothelial cells were seeded on RGD-PDMS and exposed to defined shear stresses in a microfluidic bioreactor. Endothelial cell morphology was assessed by bright field microscopy and immunocytochemistry. Furthermore, gas transfer measurement of blank, RGD-conjugated, and endothelialized PDMS oxygenator membranes was performed. RGD-PDMS gas exchange membranes proved suitable for the dynamic culture of endothelial cells for up to 21 days at a wall shear stress of 2.9 dyn/cm2. Furthermore, the cells resisted increased wall shear stresses up to 8.6 dyn/cm2 after a previous dynamic preculture of each one hour at 2.9 dyn/cm2 and 5.7 dyn/cm2. Also, after a longer dynamic preculture of three days at 2.9 dyn/cm2 and one hour at 5.7 dyn/cm2, increased wall shear stresses of 8.6 dyn/cm2 were tolerated by the cells and cell integrity could be remained. Gas transfer (GT) tests revealed that neither RGD conjugation nor endothelialization of RGD-PDMS significantly decrease the gas transfer rates of the membranes during short-term trials. Gas transfer rates are stable for at least 72 hours of dynamic cultivation of endothelial cells. Immunocytochemistry showed that the cell layer stained positive for typical endothelial cell markers CD31 and von Willebrand factor (VWF) after all trials. Cell density of EC on RGD-PDMS increased between 3 and 21 days of dynamic culture. In this study, we show the suitability of RGD-PDMS membranes for flow resistant endothelialization of gas-permeable membranes, demonstrating the feasibility of this approach for a biohybrid lung. © 2020 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC
Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes
In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.
Trypsin-Free Cultivation of 3D Mini-Tissues in an Adaptive Membrane Bioreactor
The production of large scaffold-free tissues is a key challenge in regenerative medicine. Nowadays, temperature-responsive polymers allow intact tissue harvesting without needing proteolytic enzymes. This method is limited to tissue culture plastic with limited upscaling capacity and plain process control. Here, a thermoresponsive hollow fiber membrane bioreactor is presented to produce large scaffold-free tissues. Intact tissues, rich in cell-to-cell connections and ECM, are harvested from a poly(N-vinylcaprolactam) microgel functionalized poly(ether sulfone)/poly(vinylpyrrolidone) hollow fiber membrane by a temperature shift. The harvested 3D tissues adhere in successive cultivation and exhibit high vitality for several days. The facile adsorptive coating waives the need for extensive surface treatment. The research is anticipated to be a starting point for upscaling the production of interconnected tissues enabling new opportunities in regenerative medicine, large-scale drug screening on physiological relevant tissues, and potentially opening new chances in cell-based therapies. © 2020 The Authors. Advanced Biosystems published by Wiley-VCH GmbH