Mechanical properties of human amniotic fluid stem cells using nanoindentation
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.
Section snippets
Materials
The hAFS cells (passage 21) were kindly provided by Wake Forest Institute for Regenerative Medicine (Winston-Salem, NC, USA). Chang media containing alpha minimum essential medium (α-MEM) (GIBCO), 18% Chang Medium B (Irvine Scientific), 2% Chang Medium C (Irvine Scientific), 15% Fetal Bovine Serum (FBS) (GIBCO), and 1% Pen Strep (GIBCO) were used to prepare cell culture medium. OB6 cell vials were received from Dr. Lecka Czernik at the University of Toledo. α-MEM supplemented with 10% FBS and
Cell imaging and characterization
Fig. 3 shows fluorescence microscopy images of hAFS and OB6 cells. Live and healthy cells were stained as green. Investigation of the hAFS cell morphology showed distinct regions. hAFS cells were well-elongated in one direction as expected (Kim et al., 2007, Kolambkar et al., 2010) and the length of cells were normally longer than 200 μm. hAFS cell area was significantly larger compared with OB6 cell. Microtubules and filamentous structures spread out over hundreds of micrometers prevented the
Conclusion
The goal of this article was to find the elastic modulus of hAFS cells as a novel source of stem cells and compare its mechanical properties and cell morphology with OB6 cells as one of the widely used cell lineages. In addition, we presented a new method for cell imaging and indentation in an aqueous environment. In the new method, we tried to eliminate the inherent concerns of the current method of cell imaging and indentation. Our results displayed that hAFS cells were well elongated cells
Conflict of interest statement
We declare that none of the authors involved in this research work have conflict of interest.
Acknowledgments
This work was supported by the National Science Foundation (NSF) Grant ♯0652024 and the National Institute of Health (NIH) Grant ♯DE019508.
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