Ultrathin transparent membranes for cellular barrier and co-culture models

Abstract

Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100–300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives.

Membrane Pore Spacing Can Modulate Endothelial Cell–Substrate and Cell–Cell Interactions

“…Both porous membranes had approximately 23% porosity and 77% contact area (Figure 1A and B). Fabrication details have been reported previously 28 and can be found in Supporting Information. Non-porous SiO 2 membranes were fabricated in the same manner and used as control substrates.…”

Micro‐Phase Separation within Epoxy Resin Yields Ultrathin Mesoporous Membranes with Increased Scalability by Conversion from Spin‐ to Dip‐Coating Process

“…Since PCGF is devoid of nitrogen, this strongly suggests that PEI accumulates at the bottom side during casting and corroborates the presence of a compositional gradient as a consequence of the proposed phase separation mechanism. Regardless of the underlying mechanism, these janus‐type membrane properties with the distinctly different undulating bottom surface might be of considerable interest for applications in adherent cell culture, cellular‐ and tissue barrier and coculture models given the thickness of under 100 nm.…”

Use of Nanosphere Self-Assembly to Pattern Nanoporous Membranes for the Study of Extracellular Vesicles

“…34,35 Such platform should provide ultrathin membranes with close control on pore size over the range of small EVs. Another reason for minimal thickness is to improve tissue barrier function in a direct co-culture format by allowing physical contact and fast exchange of soluble factors, [36][37][38] establishing a more physiologically relevant model. Several publications have reported on processes for the production of nanoscale pores but often produce thick membranes, do not successfully demonstrate membrane release, or are based on patterning approaches that do not offer control over the full range of EVs sizes.…”

“…We have also demonstrated the viability of utilizing those membranes for cell culture studies. 38,54 In the current work, we utilize a patterning approach with close control of pore size over the range of small EVs, together with a simple methodology for the release and integration of the ultrathin membrane into a silicone based device for the study of EVs. To the best of our knowledge, such a comprehensive process has not yet been demonstrated.…”

“…These membranes can be made of SiN for increased mechanical robustness or SiO 2 for optimum optical transparency and surface functionalization. 38,54 The pores have been patterned through the use of self-assembled nanospheres, similar in size and shape to small EVs. This patterning technique is a bottom-up approach known as nanosphere lithography (NSL) and offers pore size control over a large area at an affordable cost.…”