Switchable Water‐Adhesive, Superhydrophobic Palladium‐Layered Silicon Nanowires Potentiate the Angiogenic Efficacy of Human Stem Cell Spheroids

Background

Three-dimensional (3D) stem cell spheroids are of interest for cell therapy, but spheroids made by conventional hanging-drop, spinner-flask or microwell methods need better viability, uniformity and function. Superhydrophobic surfaces that switch reversibly between water-repellent and water-adhesive states could hold culture droplets while keeping them compact, yet earlier stimulus-responsive surfaces (temperature, magnetic field, ultraviolet light, pH) took longer than a few minutes to switch and often formed hydrophilic domains that let droplets lose their spherical shape.

Design and approach

The surface is made by aqueous electroless etching of Si into vertically aligned nanowires, sputter deposition of a gas-sensitive Pd layer and coating with dodecylalkyltrichlorosilane (DTS), following the group's earlier Pd/Si NW work. When hydrogen dissolves in Pd to form Pd hydride, the lattice constant expands from 3.90 to 4.04 Å; the authors attribute the switch in water adhesion to the resulting morphological change of the Pd/Si NWs, which reverses in air. A culture-medium droplet placed on the hydrogen-exposed surface stays pinned, while the surrounding area that the droplet does not touch becomes water-repellent again within a minute, so the droplet keeps a nearly spherical shape during hanging-drop culture.

How it was evaluated

Wetting was assessed by static water contact angle (WCA), droplet roll-off on tilted surfaces in air and 8% hydrogen (H2), and the rebound of impacting droplets used to estimate adhesion energy. hADSC spheroids were grown for 4 days at seeding densities of 1.25, 2.5 and 5.0 × 10⁵ cells/mL in 10, 15 and 20 μL droplets and compared with spheroids from spinner-flask suspension culture and hanging drops on petri dishes by Live/Dead staining, size distribution and VEGF enzyme-linked immunosorbent assay (ELISA); immunostaining, MTT metabolic assay and quantitative real-time polymerase chain reaction (qRT-PCR) compared Pd/Si NW spheroids only with petri-dish spheroids, because spinner-flask spheroids showed low viability and low VEGF secretion. Angiogenic function was tested in vitro by culturing human umbilical vein endothelial cells (HUVECs) in endothelial basal medium (EBM) mixed 7:3 with spheroid-conditioned medium under normoxia (20% O2) and simulated ischemia (1% O2), with EGM-2 and EBM as positive and negative controls.

Key results

The Pd/Si NWs remained superhydrophobic in both ambients, with WCA decreasing slightly from 158.4 ± 3.37° in air to 153 ± 3.58° in H2. An 8 μL water droplet rolled off the surface in air, whereas after exposure to 8% H2 the surface became water-adhesive; the transition occurred within 5 seconds and could be repeated for several cycles. From the difference in the energy lost by impacting droplets in air and in H2, the authors calculated an adhesion energy of 161 nJ. A 10 μL droplet of culture medium stayed pinned when the substrate was tilted vertically or turned upside down, and droplets from 5 to 20 μL were held, while larger droplets rolled off.

On the Pd/Si NWs, spheroid viability was maintained over 4 days, spheroid size increased with seeding density and medium volume, and VEGF secretion normalized to DNA content depended on spheroid size. Compared with spinner-flask and petri-dish methods, the Pd/Si NWs produced more viable, compact spheroids with a narrower size distribution (n = 10) and much greater VEGF secretion (n = 3, p < 0.01 versus both groups).

Relative to petri-dish spheroids, spheroids on the Pd/Si NWs showed higher E-cadherin and fibronectin staining, higher mitochondrial metabolic activity (n = 5, p < 0.05), downregulated pro-apoptotic BAX and upregulated anti-apoptotic BCL2L1, and upregulated HIF-1α and VEGF gene expression (n = 3, p < 0.05). The authors interpret these changes as stronger cell-cell and cell-matrix interaction in compact spheroids together with hypoxic adaptation.

Conditioned medium from Pd/Si NW spheroids significantly enhanced HUVEC proliferation under both normoxic and hypoxic conditions compared with medium from spinner-flask and petri-dish spheroids. Under 1% O2, it increased the number of capillaries formed 5 hours after plating on Matrigel (n = 4, p < 0.01 versus both groups) to a level comparable to growth factor- and serum-supplemented EGM-2. The authors relate the improved spheroid quality to weaker horizontal hydrodynamic flow inside droplets on the Pd/Si NWs than on petri dishes.

Open questions

All functional tests were performed in vitro. The authors state that the efficacy of the spheroids for therapeutic angiogenesis in vivo remains to be examined, and that spheroid culture on Pd/Si NW surfaces needs to be scaled up and optimized for mass production.

How it connects to other research

The related study by Bartosh and colleagues, cited in this paper, prepared human mesenchymal stromal cell spheroids by a conventional hanging-drop protocol and examined their anti-inflammatory properties; this study keeps the hanging-drop format but replaces the culture substrate with a hydrogen-switchable superhydrophobic surface and measures angiogenic paracrine function in vitro.