The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

Abstract

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffness-defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverse-transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretch-induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterial-associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.

Shaping the Cell and the Future: Recent Advancements in Biophysical Aspects Relevant to Regenerative Medicine

“…Another highlight at the TERMIS 2017 meeting was use of different methods for measuring cell morphology parameters. Our group showed how an automated, non-destructive, high-throughput quantification method, using images of calcein-stained cells, combined with binary thresholding, could be used to efficiently and accurately measure a panel of cell morphological parameters on a large number of cells (e.g., 60,000 cells) within a short time [18,19]. This automated high-throughput quantification method was significantly more efficient, precise and reproducible than manual methods.…”

“…Hence, studies have begun to increasingly and successfully use quantitative approaches to access morphological parameters and to correlate them to biological parameters and cell fate outcome. For example, our group has used a semi-automated high-throughput method for calculating a specific panel of shape descriptors [18,19,24], which was used to describe MSC shape in correlation to biophysical stimuli such as biomaterial type and nanoscale surface stiffness, as well as in response to externally applied biomechanical forces [19,24] (Figure 1). Therefore, quantitation of cellular morphology is useful for understanding the effects of different microenvironmental or biophysical stimuli on cells and for helping to define a morphological fingerprint for cells [20,25].…”

“…Cell behavior can be dictated by microenvironmental biophysical cues including (1) mechanical forces such as compression, tension, and shear forces, (2) substrate properties such as biomaterial type, ligand presentation, surface topography, viscoelasticity, and stiffness, or (3) biochemical cues such as autocrine and paracrine signals [19,24,31,[36][37][38][39]. Collectively, such cues can ultimately lead to a specific differentiation behavior of cells such as MSCs [38].…”

“…Recent developments in image processing technology and computer-aided analyses now allow for high-throughput calculation of quantitative shape descriptors for mathematically describing cellular morphology. Our lab has actively used algorithms in ImageJ, an open-source program for analyzing images [18,19]. In this software, shape descriptors can be calculated based on objective mathematical functions and produce numerical values.…”

“…We refer the biologically interested reader to [19] and the mathematically interested reader to the ImageJ homepage [50,51] and specifically to the "Analyze" menu section "set measurements", which details the mathematical calculations. In the context of quantifying MSC shape, we advocate using a defined panel of shape descriptors such as those introduced in our previous studies [18,19,24]. Other programs like MetaMorph [52] provide additional, albeit proprietary means for quantifying cell morphology and cell networks.…”