An important step towards a prevascularized islet macroencapsulation device-effect of micropatterned membranes on development of endothelial cell network

Abstract

The development of immune protective islet encapsulation devices could allow for islet transplantation in the absence of immunosuppression. However, the immune protective membrane / barrier introduced there could also impose limitations in transport of oxygen and nutrients to the encapsulated cells resulting to limited islet viability. In the last years, it is well understood that achieving prevascularization of the device in vitro could facilitate its connection to the host vasculature after implantation, and therefore could provide sufficient blood supply and oxygenation to the encapsulated islets. However, the microvascular networks created in vitro need to mimic well the highly organized vasculature of the native tissue. In earlier study, we developed a functional macroencapsulation device consisting of two polyethersulfone/polyvinylpyrrolidone (PES/PVP) membranes, where a bottom microwell membrane provides good separation of encapsulated islets and the top flat membrane acts as a lid. In this work, we investigate the possibility of creating early microvascular networks on the lid of this device by combining novel membrane microfabrication with co-culture of human umbilical vein endothelial cell (HUVEC) and fibroblasts. We create thin porous microstructured PES/PVP membranes with solid and intermittent line-patterns and investigate the effect of cell alignment and cell interconnectivity as a first step towards the development of a stable prevascularized layer in vitro. Our results show that, in contrast to non-patterned membranes where HUVECs form unorganized HUVEC branch-like structures, for the micropatterned membranes, we can achieve cell alignment and the co-culture of HUVECs on a monolayer of fibroblasts attached on the membranes with intermittent line-pattern allows for the creation of HUVEC branch-like structures over the membrane surface. This important step towards creating early microvascular networks was achieved without the addition of hydrogels, often used in angiogenesis assays, as gels could block the pores of the membrane and limit the transport properties of the islet encapsulation device.

Fig. 1

NHDF and HUVEC culture on PES/PVP membranes a Average number of NHDF and HUVEC attached per mm² on PES/PVP membranes coated with various fibronectin concentrations. Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001, n = 3. b Images of methylene blue stained NHDFs and HUVECs cultured for 4 days on coverslip-positive control, non-coated PES/PVP membranes and membranes coated with 200 µg/ml and 1 mg/ml fibronectin

Fig. 2

Membrane characteristics a Scanning electron microscopy images of micropatterned membranes with intermittent lines and combination of intermittent and solid lines; b Schematic representation of patterned on the silicon wafer used for micropatterned membrane fabrication: a = 100, b = 20, c = 40 d = 100, e = 540 µm; c Water transport through the patterned PES/PVP membranes; d Representative stress-strain curve obtained for PES/PVP membranes

Fig. 3

NHDF and HUVEC culture on micropatterned membranes a Images of methylene blue stained NHDF and HUVEC after 1 day of culture on micropatterned PES/PVP membranes. Nucleus alignment in relation to the surface topography for b NHDFs and c HUVECs

Fig. 4

Co-culture of NHDFs and HUVECs resulting in HUVEC network formation. In green the immunostaining for CD31 of HUVEC cells on a non-patterned membranes, b membrane with intermittent lines, c membrane with intermittent and solid lines, d example of elongated HUVEC branch-like structure of the network. The dotted line is drawn to guide the eye of the reader

Fig. 5

Quantification of HUVEC branch-like structure alignment relative to the x-axis of the immunostaining images (n = 3). Membrane patterns were aligned parallel to the x-axis