Direct observation of CD4 T cell morphologies and their cross-sectional traction force derivation on quartz nanopillar substrates using focused ion beam technique
- Dong-Joo Kim†1,
- Gil-Sung Kim†1,
- Jung-Hwan Hyung1,
- Won-Yong Lee2,
- Chang-Hee Hong1 and
- Sang-Kwon Lee†2Email author
© Kim et al.; licensee Springer. 2013
Received: 31 May 2013
Accepted: 14 July 2013
Published: 23 July 2013
Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported. Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.
KeywordsCell traction force Cell adhesion CD4 T cell Cell migration Focused ion beam
Cell adhesion is the initial step upon interactions of substrate materials with loaded cells. In particular, it was shown that nanotopography influences diverse cell behaviors such as cell adhesion, cytoskeletal organization, apoptosis, macrophage activation, and gene expression [1, 2], which in turn leads to proliferation, differentiation, and migration on various nanostructures including nanofibers , nanopillars , and nanogrooves [5, 6]. As a result, cell behaviors are critically determined by the interaction between nanoscale cellular surface components such as microvilli, filopodia, extracellular matrix (ECM), and the underlying nanostructure topography . However, little is known of how the use of size and shape-matched diverse nanometer-scale topographies interact to not only the forthcoming cells but also the nanoscale cellular surface components of cells bound on the nanotopographic substrates in cell adhesion steps even at the very early stage of incubation (<20 min).
Cell traction force (CTF) is crucial to cell migration, proliferation, differentiation, cell shape maintenance, mechanical cell-signal generation, and other cellular functions just following adhesion step on the nanotopographic substrates. Once transmitted to the ECM through stress fibers via focal adhesions, which are assemblies of ECM proteins, transmembrane receptor, and cytoplasmic structural and signaling proteins (e.g., integrins), CTF directs many cellular functions . In addition, CTF plays an important role in many biological processes such as inflammation , wound healing , angiogenesis , and cancer metastasis . Thus, a complete knowledge of CTF regulation and the improvement of the ability to measure CTFs are currently critical in clear understating physiological and pathological events at both the tissue and organ levels. To date, various techniques have been developed and have refined over the years to measure CTFs of single cells or population of cells, including cell-populated collagen gel method , micromechanical cantilever beam-based force sensor array , cell traction force microscopy , and elastomeric micropost array [16, 17]. In 2009, Li et al. reported another favorable method to quantify the traction force of a single cell by aligned silicon nanowire (SiNW) arrays . They reported that the CTFs of the cells cultured on this SiNW arrays could be calculated from these underlying SiNW deflections. However, no further lateral CTF information (cross-sectional) inside the cell underlying on the nanotopographic substrates was provided.
In this letter, we first report on direct observations of the primary mouse CD4 T cell morphologies by culturing CD4 T cells on streptavidin (STR)-functionalized quartz nanopillar arrays (QNPA) using a scanning electron microscopy (SEM) method and then demonstrate a new alternative technique to measure cross-sectional cell traction force distribution of surface-bound CD4 T cells including those inside the cells on QNPA substrates by culturing the cells on the top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam (FIB)-assisted technique. It conducted both a high-performance etching and imaging scheme from FIB and finite element method (FEM)-based computer simulation tools with well-defined QNPA substrates. We suggest that the use of the FIB-based technique combined with QNPA and FEM simulation would be a powerful and fine process to evaluate cross-sectional CTFs of single cells.
Results and discussion
These results suggest that the microvilli (filopodia or lamellipodia) of CD4 T cells closely react with the QNPA substrates via high-affinity STR-biotin conjugation as we have proven previously  and extend filopodia of widths depending on the diameter of the QNPAs to identify the size of the structures underneath the cells using filopodia as illustrated in Figure 3c. This strong linear response in the filopodia extending from the T cells bound on the solid-state surfaces with the nanopillar diameters of the surface could be explained by a contact guidance phenomenon. This is usually used to explain the behavior of fibroblast filopodia on nanostructured substrates with long incubation [5, 26, 27]. According to the contact guidance phenomenon, the T cells extend the filopodia to recognize and sense the surface features of nanotopographic substrates when they are bound on the surface at the early state of the adhesion and then form themselves on the substrates with a similar size of the nanostructure underneath the cells (Figure 3c). Our observation corresponds well with previous results from Dalby et al.  even if we conducted it on T cells instead of epithelial cell line.
In conclusion, we have studied the behaviors (e.g., cell adhesion and spreading) of CD4 T cells captured on STR-functionalized QNPA substrates at the very early stage of incubation (less than 20 min). For this study, we prepared four different sizes of QNPA substrates using a modified self-assembly method. On the basis of our results, we found that the distribution of extended filopodial width of the captured CD4 T cells was highly related to the diameter of QNPA (200 to 450 nm), indicating that extended filopodia of CD4 T cells increased in width with the increasing diameter of QNPA from 200 to 450 nm. Furthermore, we demonstrated cross-sectional CTF distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on the tip of the QNPA and further analysis in the deflection of underlying QNPA via FIB technique. We promise that this technique can be powerful tools for evaluation of the CTF distribution on the nanopatterned substrates.
This study was supported by the Priority Research Centers Program and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010–0019694). This study was also supported by a grant from the Global Excellent Technology Innovation R&D Program funded by the Ministry of Knowledge Economy, Republic of Korea (10038702-2010-01).
- Arnold M, Cavalcanti-Adam EA, Glass R, Blummel J, Eck W, Kantlehner M, Kessler H, Spatz JP: Activation of integrin function by nanopatterned adhesive interfaces. Chem Phys Chem 2004, 5: 383–388.
- Zamir E, Geiger B: Components of cell-matrix adhesions. J Cell Sci 2001, 114: 3577–3579.
- Zhang NA, Deng YL, Tai QD, Cheng BR, Zhao LB, Shen QL, He RX, Hong LY, Liu W, Guo SS, Liu K, Tseng HR, Xiong B, Zhao XZ: Electrospun TiO2 nanofiber-based cell capture assay for detecting circulating tumor cells from colorectal and gastric cancer patients. Adv Mater 2012, 24: 2756–2760.View Article
- Koh LB, Rodriguez I, Venkatraman SS: The effect of topography of polymer surfaces on platelet adhesion. Biomaterials 2010, 31: 1533–1545.View Article
- Dalby MJ, Gadegaard N, Riehle MO, Wilkinson CDW, Curtis ASG: Investigating filopodia sensing using arrays of defined nano-pits down to 35 nm diameter in size. Int J Biochem Cell 2004, B36: 2005–2015.View Article
- Dalby MJ, Riehle MO, Johnstone HJH, Affrossman S, Curtis ASG: Nonadhesive nanotopography: fibroblast response to poly(n-butyl methacrylate)-poly(styrene) demixed surface features. J Biomed Mater Res A 2003, 67: 1025–1032.View Article
- Hart A, Gadegaard N, Wilkinson CDW, Oreffo ROC, Dalby MJ: Osteoprogenitor response to low-adhesion nanotopographies originally fabricated by electron beam lithography. J Mater Sci-Mater Med 2007, 18: 1211–1218.View Article
- Wang JHC, Lin JS, Yang ZC: Cell traction force microscopy. In Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials: Techniques and Applications. Edited by: Qin L, Genant HK, Griffith JF, Leung KS. Heidelberg: Springer; 2007:227–235.View Article
- Li B, Xie LK, Starr ZC, Yang ZC, Lin JS, Wang JHC: Development of micropost force sensor array with culture experiments for determination of cell traction forces. Cell Motil Cytoskel 2007, 64: 509–518.View Article
- Bromberek BA, Enever PAJ, Shreiber DI, Caldwell MD, Tranquillo RT: Macrophages influence a competition of contact guidance and chemotaxis for fibroblast alignment in a fibrin gel coculture assay. Exp Cell Res 2002, 275: 230–242.View Article
- Tranqui L, Tracqui P: Mechanical signalling and angiogenesis: the integration of cell-extracellular matrix couplings. Cr Acad Sci Iii-Vie 2000, 323: 31–47.View Article
- Franck C, Maskarinec SA, Tirrell DA, Ravichandran G: Three-dimensional traction force microscopy: a new tool for quantifying cell-matrix interactions. PLoS One 2011, 6(3):e17833.View Article
- Campbell BH, Clark WW, Wang JHC: A multi-station culture force monitor system to study cellular contractility. J Biomech 2003, 36: 137–140.View Article
- Galbraith CG, Sheetz MP: A micromachined device provides a new bend on fibroblast traction forces. P Natl Acad Sci USA 1997, 94: 9114–9118.View Article
- Butler JP, Tolic-Norrelykke IM, Fabry B, Fredberg JJ: Traction fields, moments, and strain energy that cells exert on their surroundings. Am J Physiol-Cell Ph 2002, 282: C595-C605.View Article
- Fu JP, Wang YK, Yang MT, Desai RA, Yu XA, Liu ZJ, Chen CS: Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat Methods 2011, 8: 184.View Article
- Yang MT, Sniadecki NJ, Chen CS: Geometric considerations of micro- to nanoscale elastomeric post arrays to study cellular traction forces. Adv Mater 2007, 19: 3119–3123.View Article
- Li Z, Song JH, Mantini G, Lu MY, Fang H, Falconi C, Chen LJ, Wang ZL: Quantifying the traction force of a single cell by aligned silicon nanowire. Array Nano Lett 2009, 9: 3575–3580.View Article
- Kim DJ, Lee G, Kim GS, Lee SK: Statistical analysis of immuno-functionalized tumor-cell behaviors on nanopatterned substrates. Nanoscale Res Lett 2012, 7: 1–8.View Article
- Kim DJ, Seol JK, Wu Y, Ji S, Kim GS, Hyung JH, Lee SY, Lim H, Fan R, Lee SK: A quartz nanopillar hemocytometer for high-yield separation and counting of CD4(+) T lymphocytes. Nanoscale 2012, 4: 2500–2507.View Article
- Lee SK, Kim GS, Wu Y, Kim DJ, Lu Y, Kwak M, Han L, Hyung JH, Seol JK, Sander C, Gonzalez A, Li J, Fan R: Nanowire substrate-based laser scanning cytometry for quantitation of circulating tumor. Cells Nano Lett 2012, 12: 2697–2704.View Article
- Kim ST, Kim DJ, Kim TJ, Seo DW, Kim TH, Lee SY, Kim K, Lee KM, Lee SK: Novel streptavidin-functionalized silicon nanowire arrays for CD4(+) T lymphocyte separation. Nano Lett 2010, 10: 2877–2883.View Article
- Kim DJ, Seol JK, Lee G, Kim GS, Lee SK: Cell adhesion and migration on nanopatterned substrates and their effects on cell-capture yield. Nanotechnology 2012, 23: 395102.View Article
- Jakob M, Dimitrios G, Riehle MO, Nikolaj G, Peter S: Fixation and drying protocols for the preparation of cell samples for time-of-flight secondary ion mass spectrometry analysis. Anal Chem 2009, 81: 7197–7205.View Article
- Kaab MJ, Richards RG, Walther P, Ap Gwynn I, Notzli HP: A comparison of four preparation methods for the morphological study of articular cartilage for scanning electron microscopy. Scanning Microsc 1999, 13: 61–69.
- Borenstein JT, Langer R, Bettinger CJ: Engineering substrate topography at the micro- and nanoscale to control cell function. Angew Chem Int Edit 2009, 48: 5406–5415.View Article
- Dalby MJ, Hart A, Yarwood SJ: The effect of the RACK1 signalling protein on the regulation of cell adhesion and cell contact guidance on nanometric grooves. Biomaterials 2008, 29: 282–289.View Article
- Dalby MJ, Riehle MO, Johnstone HJH, Affrossman S, Curtis ASG: Polymer-demixed nanotopography: control of fibroblast spreading and proliferation. Tissue Eng 2002, 8: 1099–1108.View Article
- Fu JP, Wang YK, Yang MT, Desai RA, Yu XA, Liu ZJ, Chen CS: Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat Methods 2010, 7: 733–736.View Article
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