Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

Abstract

Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among potential applications as culture platforms for drug screening.

The importance of being a lumen

“…Lee et al (28) found that stem cells patterned with rounded phenotypes differentiate into adipocyte‐like cells compared to cells with more spread phenotypes, which differentiate into neuron‐like cells. It has also been shown that ECM structure and the topography encountered by a cell can influence cell fate (29–32).…”

A Parameter Study for 3D-Printing Organized Nanofibrous Collagen Scaffolds Using Direct-Write Electrospinning

“…Various groups have developed methods to finely pattern electrospun nanofibers into organized structures. Fibers have been directed onto spinning mandrel collectors to create fiber spools [7], micropatterned via insulator-collector geometries to induce specific shapes [8] and positioned in precise locations by rapidly translating the collector with an X-Y stage [9][10][11][12]. The latter technique, sometimes termed direct-write electrospinning (DWES) is a relatively new additive manufacturing approach to electrospinning into highly organized confirmations.…”

Solution fibre spinning technique for the fabrication of tuneable decellularised matrix-laden fibres and fibrous micromembranes

“…First, the ability of EA.hy926 cells to deform the as-produced fibres were investigated; this was performed by comparing cell cultures maintained on three pure gelatin fibre configurations: fibres attached on glass, fibres suspended sparsely across a channel, and fibres forming a membrane with dense mesh. In the first test, the fibre pattern configuration was mostly fixed on the glass substrate like in our previous work [21]. Figure S4a shows that at day 1, cells form aligned patterns following those of the fibres, showing distinct orientations differing from the cells attached on the fibre-free space (i.e.…”

“…For the endothelial cell line (EA.hy926) culture, a protocol was adapted from [21] For the glomerular cell culture, conditionally immortalised human glomerular endothelial cell (GEnC) lines and podocytes cell lines were obtained and cultured according to [22,23]. A 3D ring-membrane device was immersed in DMEM for 1 hour before cell seeding, with the fibrous membrane facing the bottom of the 6-well plate.…”

“…We attribute this ability to incorporate high concentrations of ECM 'fillers' to the LEP configuration, which uses initiators to break up the pendant droplet for fibre drawing (see detailed mechanism in [18]). Without initiators, pure gelatin fibres can be processed using a near-field electrospinning (NFES) method at a higher applied voltage [21]. However, as ECM content was added beyond 10 wt%, no fibres can be produced with NFES even at high voltages (>1000 V).…”