RESUMO
The development of an endothelialized membrane oxygenator requires solution strategies combining the knowledge of oxygenators with endothelial cells' biology. Since it is well known that exposing cells towards pure oxygen causes oxidative stress, this aspect has to be taken into account in the development of a biohybrid oxygenator system. N-Acetylcysteine (NAC) is known for its antioxidant properties in cells. We tested its applicability for the development of an endothelialized oxygenator model. Cultivating human umbilical vein derived endothelial cells (HUVEC) up to 6 days with increasing concentrations of NAC from 1 to 30 mM revealed NAC toxicity at concentrations from 20 mM. Cell density clearly decreased after radical oxygen species exposure in non-NAC pretreated cells compared to 20 mM NAC precultured HUVEC after 3 and 6 days. Also the survival rate after ROS treatment could be restored by incubation with NAC from 15 to 25 mM for all time points. NAC treated cells changed their morphology from typical endothelial cells' cobblestone pattern to a fusiform, elongated configuration. Transformed cells were still positive for typical endothelial cell markers. Our present results show the potential of NAC for the protection of an endothelial cell layer in an endothelialized membrane oxygenator due to its antioxidative properties. Moreover, NAC induces a morphological change in HUVEC similar to dynamic cultivation procedures.
RESUMO
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.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Pulmão/metabolismo , Oxigênio/metabolismo , Troca Gasosa Pulmonar/fisiologia , Linhagem Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fenômenos Fisiológicos RespiratóriosRESUMO
OBJECTIVE: This study focusses on the development of a biomimetic oxygenator test device. Due to limited biocompatibility, current oxygenators do not allow mid- to long-term therapy. Tissue engineering uses autologous cell sources to overcome the immunogenic barriers of biomaterials. Surface coating with endothelial cells might improve hemocompatibility and thus prevent immunogenic reactions of the body. In this study this concept is applied to endothelialise a gas-permeable membrane to develop a biomimetic oxygenator test-device (ENDOXY). METHODS: ENDOXY-a multifunctional test-system was developed to endothelialise a gas-permeable membrane suitable for cell culture and to test the cell retention under shear stress and to measure gas transfer through it. RESULTS: Successful endothelialisation of the membrane was achieved and cells showed characteristic endothelial morphologies. They stained positive for endothelial markers. The number of cells aligned with shear stress and cell retention after blood perfusing experiments was high. Gas transfer is observed via uncoated and endothelialised membranes. CONCLUSION: The study showed promising results with regard to system design, endothelialisation, and cell retention under shear stress conditions. It strongly encourages further research into the system by testing different membrane materials to design a biomimetic membrane surface and pave way for a fully hemocompatible oxygenator.