Mechanical properties of human amniotic fluid stem cells using nanoindentation

Abstract

The aim of this study was to obtain nanomechanical properties of living cells focusing on human amniotic fluid stem (hAFS) cell using nanoindentation techniques. We modified the conventional method of atomic force microscopy (AFM) in aqueous environment for cell imaging and indentation to avoid inherent difficulties. Moreover, we determined the elastic modulus of murine osteoblast (OB6) cells and hAFS cells at the nucleus and cytoskeleton using force–displacement curves and Hertz theory. Since OB6 cell line has been widely used, it was selected to validate and compare the obtained results with the previous research studies. As a result, we were able to capture high resolution images through utilization of the tapping mode without adding protein or using fixation methods. The maximum depth of indentation was kept below 15% of the cell thickness to minimize the effect of substrate hardness. Nanostructural details on the surface of cells were visualized by AFM and fluorescence microscopy. The cytoskeletal fibers presented remarkable increase in elastic modulus as compared with the nucleus. Furthermore, our results showed that the elastic modulus of hAFS cell edge (31.6 kPa) was lower than that of OB6 cell edge (42.2 kPa). In addition, the elastic modulus of nucleus was 13.9 kPa for hAFS cell and 26.9 kPa for OB6 cells. Differences in cell elastic modulus possibly resulted from the type and number of actin cytoskeleton organization in these two cell types.

Introduction

Finding mechanical properties of biological samples, especially living cells, has been of great interest to researchers. Analysis of cellular mechanical properties can lead us to discover new methods of identifying various forms of cancers (Brandão et al., 2003, Cross et al., 2007, Lekka, 2012) and other diseases (Suresh, 2006). In tissue engineering, probing the elasticity and adhesion of live cells can provide physical insight into the mechanical and chemical properties of biomaterials and scaffold materials (Simon et al., 2004). Many methods including magnetic twisting cytometry, traction force microscopy, micro-pipette aspiration, optical trap, optical stretcher, micro- and nano-needle insertion and atomic force microscopy (AFM) have been used to find the mechanical properties of cells (Bao and Suresh, 2003, Lim et al., 2006). Compared to the other methods, AFM indentation has become the principal technique in measuring the cell mechanical properties of surface layers and especially cells with high spatial precision (Casuso et al., 2011, Quist and Lal, 2012). Although there are some limitations in using AFM for the measurement of cell mechanics such as lateral drag of the cell by the tip, the calibration (Kirmizis and Logothetidis, 2010) and not ideally sharp probes (Wu et al., 2012), this method is the most widely used.

The elastic modulus of a living cell is usually calculated by producing force–displacement curves with data from indentation tests. The common analysis models for cell indentation are based on the Hertz theory (Hertz, 1882, Sneddon, 1965). This model is based on perfectly elastic behavior of cells during indentation (Weisenhorn et al., 1993). In the application to the AFM measurements the generally considered indenter's geometries are the conical and spherical ones. Although nanoindentation is widely used to investigate the stem cell mechanical properties (Darling et al., 2008), these properties for some new types of stem cells have not yet been fully discovered.

In last few decades, main advancement has been facilitated by the discovery of stem cells, capable of converting to various cell lineages. Stem cells have been isolated from embryonic, fetal, and adult tissues and more recently, also from umbilical cord, placenta and amniotic fluid. Among the source of stem cells that have been studied, human Amniotic Fluid Stem (hAFS) cells have arisen as an attractive source of stem cells since 2007 (Siegel et al., 2008), as its procurement does not raise the ethical concerns associated with the use of human embryonic stem cells (Rodrigues et al., 2012a, Rodrigues et al., 2012b, Siegel et al., 2007). In addition, hAFS cells have the advantage of being primitive cells with little known antigenicity and great expansion capabilities. These cells can be induced to differentiate into cells that represent each germ layer, such as adipogenic, osteogenic, myogenic, endothelial, neuronal, hepatic, and chondrogenic lineages (Cananzi et al., 2009, Joo et al., 2012). hAFS cells are becoming an important source of cells for regenerative medicine and tissue engineering. In this way, it is necessary to characterize its mechanical properties. In other words, although hAFS cells have many properties that support their clinical usefulness (Skardal et al., 2012), little is known about the mechanical properties.

In this study, the aim was to determine the mechanical properties of hAFS cells and compare them to murine osteoblast (OB6) cells as a reference using AFM imaging and indentation with the Hertz model. Specifically, we chose OB6 cells as a reference cell source since our laboratory focuses on OBs for the bone regeneration studies. The OB6 cells are well known cells with the previously known mechanical properties (Charras and Horton, 2002, Darling et al., 2008) to verify the result of current technique and discuss the possible source of differences with other papers. Both nucleus and cytoskeleton play an important role in cell deformation and mechanical properties. Thus, indentation was done at nucleus and cytoskeleton regions of the cells. The tapping mode was used for cell imaging in physiological aqueous environment without using fluid cell or any other specific equipments. In addition, this physiological aqueous environment provided the advantage of tapping mode without requiring adhesive proteins to attach cells on the substrate. In addition, images obtained from AFM were compared with fluorescence microscopy images to verify the viability of cells used for AFM studies. Generally, there were two aims in this paper. First, a new and simple technique of imaging and indenting for living cells in tapping mode was introduced. Secondly, the elastic modulus for hAFS cells, as an attractive new stem cell source, was determined using the above technique and validated using OB6 cells.