Influence of Aerosolization on Endothelial Cells for Efficient Cell Deposition in Biohybrid and Regenerative Applications

Abstract

The endothelialization of gas exchange membranes can increase the hemocompatibility of extracorporeal membrane oxygenators and thus become a long-term lung replacement option. Cell seeding on large or uneven surfaces of oxygenator membranes is challenging, with cell aerosolization being a possible solution. In this study, we evaluated the endothelial cell aerosolization for biohybrid lung application. A Vivostat® system was used for the aerosolization of human umbilical vein endothelial cells with non-sprayed cells serving as a control. The general suitability was evaluated using various flow velocities, substrate distances and cell concentrations. Cells were analyzed for survival, apoptosis and necrosis levels. In addition, aerosolized and non-sprayed cells were cultured either static or under flow conditions in a dynamic microfluidic model. Evaluation included immunocytochemistry and gene expression via quantitative PCR. Cell survival for all tested parameters was higher than 90%. No increase in apoptosis and necrosis levels was seen 24 h after aerosolization. Spraying did not influence the ability of the endothelial cells to form a confluent cell layer and withstand shear stresses in a dynamic microfluidic model. Immunocytochemistry revealed typical expression of CD31 and von Willebrand factor with cobble-stone cell morphology. No change in shear stress-induced factors after aerosolization was reported by quantitative PCR analysis. With this study, we have shown the feasibility of endothelial cell aerosolization with no significant changes in cell behavior. Thus, this technique could be used for efficient the endothelialization of gas exchange membranes in biohybrid lung applications.

Towards a Biohybrid Lung? Impacted Endothelial Layer Integrity in Inflammatory Conditions

“…After dynamic culture, HUVECs were fixed with ice-cold methanol for 10 min at -20 °C and washed three times with DPBS. The membranes were carefully cut out of the microfluidic devices, and ICC was performed with antibodies against CD31 and von Willebrand factor (vWf) as previously described [30]. Briefly, blocking with 3% bovine serum albumin (BSA, Sigma Aldrich) in DPBS was performed to block the non-specific binding sites.…”

“…PDMS membranes were washed as described above, followed by gluing of the membranes to the microfluidic devices (sticky Slides 0.2 Luer, Ibidi) with 250 µm channel height as previously described [17] and coated with RGD. For endothelialisation, HUVECs were resuspended in EGM2 medium with 1% ABM at a concentration of 5 × 10 6 cells/mL and seeded in the microfluidic channels.…”

“…The endothelialised membranes were carefully cut from the microfluidic devices, and ICC was performed with antibodies against CD31 and von Willebrand factor (vWf) and DAPI (Carl Roth) to counterstain nuclei as previously described [19]. For microscopy, a drop of mounting medium (Ibidi) was placed in a microscopy-suited µ-Slide chambered coverslip (Ibidi), and the sample was placed into the chamber with the cell-side down.…”