Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS

Sho Yokoyama1, Tsubasa S. Matsui2, Shinji Deguchi2

1 Current: Micro/Nano Technology Center, Tokai University, Hiratsuka, 2 Current: Division of Bioengineering, Graduate School of Engineering Science, Osaka University, Osaka
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.21
DOI:  10.1002/cpcb.22
Online Posting Date:  June, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique––microcontact peeling (μCPe)––that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS. This technique employs microfeatured materials with a relatively high surface energy such as copper, instead of using a microfeatured PDMS stamp, to peel off a cell‐adhesive layer present on the surface of substrates. Consequently, cell‐nonadhesive substrates are exposed at the specific surface that undergoes the physical contact with the microfeatured material. Thus, although μCPe and μCPr are apparently similar, the former does not comprise a process of transferring biomolecules through hydrophobic PDMS. © 2017 by John Wiley & Sons, Inc.

Keywords: cell micropatterning; protein micropatterning; microcontact printing; microcontact peeling

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Microcontact Peeling (μCPe)
  • Support Protocol 1: Cell Culture
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Microcontact Peeling (μCPe)

  • Sylgard 184 Silicone Elastomer Kit (e.g., Dow Corning) containing:
    • Base polymer
    • Cross linker
  • Pluronic F‐127 (e.g., Invitrogen)
  • Phosphate‐buffered saline (PBS; see appendix 2A)
  • Gelatin (e.g., Sigma‐Aldrich)
  • Cells of interest (see protocol 2Support Protocol)
  • Vacuum chamber
  • 35‐mm glass‐bottom or polystyrene cell culture dish
  • Spin coater (e.g., Kyowa Riken K‐359S1)
  • 65°C oven
  • Glow discharge plasma generator (e.g., Meiwafosis SEDE‐P or SEDE‐GE)
  • Copper electron microscopy grids (e.g., EM Japan, cat. no. G203)
  • Vacuum tweezer (e.g., Virtual Industries Tweezer‐Vac)
  • Fine tweezers
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Anderson, A. B., & Robertson, C. R. (1995). Absorption spectra indicate conformational alteration of myoglobin adsorbed on polydimethylsiloxane. Biophysical Journal, 68, 2091–2097. doi: 10.1016/S0006‐3495(95)80388‐7.
  Biasco, A., Pisignano, D., Krebs, B., Pompa, P. P., Persano, L., Cingolani, R., & Rinaldi, R. (2005). Conformation of microcontact‐printed proteins by atomic force microscopy molecular sizing. Langmuir, 21, 5154–5158. doi: 10.1021/la050010j.
  Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., & Ingber, D. E. (1997). Geometric control of cell life and death. Science, 276, 1425–1428. doi: 10.1126/science.276.5317.1425.
  Coyer, S. R., Delamarche, E., & García, A. J. (2011). Protein tethering into multiscale geometries by covalent subtractive printing. Advanced Materials, 23, 1550–1553. doi: 10.1002/adma.201003744.
  Coyer, S. R., García, A. J., & Delamarche, E. (2007). Facile preparation of complex protein architectures with sub‐100‐nm resolution on surfaces. Angewandte Chemie (International ed. in English), 46, 6837–6840. doi: 10.1002/anie.200700989.
  Deguchi, S., Matsui, T. S., & Iio, K. (2011). The position and size of individual focal adhesions are determined by intracellular stress‐dependent positive regulation. Cytoskeleton, 68, 639–651. doi: 10.1002/cm.20541.
  Deguchi, S., Nagasawa, Y., Saito, A. C., Matsui, T. S., Yokoyama, S., & Sato, M. (2014). Development of motorized plasma lithography for cell patterning. Biotechnology Letters, 36(3), 507–513. doi: 10.1007/s10529‐013‐1391‐3.
  Desai, R. A., Khan, M. K., Gopal, S. B., & Chen, C. S. (2011). Subcellular spatial segregation of integrin subtypes by patterned multicomponent surfaces. Integrative Biology, 3, 560–567. doi: 10.1039/c0ib00129e.
  McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., & Chen, C. S. (2004). Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Developmental Cell, 6, 483–495. doi: 10.1016/S1534‐5807(04)00075‐9.
  Ohishi, T., Noda, H., Matsui, T. S., Jile, H., & Deguchi, S. (2016). Tensile strength of oxygen plasma‐created surface layer of PDMS. Journal of Micromechanics and Microengineering, 27, 015015. doi: 10.1088/0960‐1317/27/1/015015.
  Pitaval, A., Tseng, Q., Bornens, M., & Théry, M. (2010). Cell shape and contractility regulate ciliogenesis in cell cycle‐arrested cells. The Journal of Cell Biology, 191, 303–312. doi: 10.1083/jcb.201004003.
  Rodriguez, N. M., Desai, R. A., Trappmann, B., Baker, B. M., & Chen, C. S. (2014). Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization. Langmuir, 30, 1327–1335. doi: 10.1021/la404037s.
  Ruiz, S. A., & Chen, C. S. (2007). Microcontact printing: A tool to pattern. Soft Matter, 3, 168–177. doi: 10.1039/B613349E.
  Théry, M. (2010). Micropatterning as a tool to decipher cell morphogenesis and functions. Journal of Cell Science, 123, 4201–4213. doi: 10.1242/jcs.075150.
  Théry, M., Jiménez‐Dalmaroni, A., Racine, V., Bornens, M., & Jülicher, F. (2007). Experimental and theoretical study of mitotic spindle orientation. Nature, 447, 493–496. doi: 10.1038/nature05786.
  Théry, M., Pépin, A., Dressaire, E., Chen, Y., & Bornens, M. (2006). Cell distribution of stress fibres in response to the geometry of the adhesive environment. Cell Motility and the Cytoskeleton, 63, 341–355. doi: 10.1002/cm.20126.
  Tseng, Q., Wang, I., Duchemin‐Pelletier, E., Azioune, A., Carpi, N., Gao, J., … Balland, M. (2011). A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels. Lab on a Chip, 11, 2231. doi: 10.1039/c0lc00641f.
  Yokoyama, S., Matsui, T.S., & Deguchi, S., (2014). Microcontact peeling as a new method for cell micropatterning. PLoS One, 9, e102735. doi: 10.1371/journal.pone.0102735.
  Zelisko, P. M., & Brook, M. A. (2002). Stabilization of α‐chymotrypsin and lysozyme entrapped in water‐in‐silicone oil emulsions. Langmuir, 18, 8982–8987. doi: 10.1021/la025867k.
PDF or HTML at Wiley Online Library