AVS 50th International Symposium
    Biomaterial Interfaces Tuesday Sessions
       Session BI-TuM

Paper BI-TuM3
The Use of XPS, SIMS, and Immunostaining to Examine the Behavior of Extracellular Matrix upon Cell Detachment from a Smart Polymer

Tuesday, November 4, 2003, 9:00 am, Room 307

Session: Cell/Surface Interactions
Presenter: H.E. Canavan, University of Washington
Authors: H.E. Canavan, University of Washington
X. Cheng, University of Washington
B.D. Ratner, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
The temperature-dependent behavior of poly(n-isopropylacrylamide) (NIPAM) is directly transmitted to cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS); after being cooled to room temperature, cells cultured on NIPAM spontaneously detach as contiguous sheets. In comparison, cells grown atop the TCPS surface remain attached for hours or days, requiring the use of enzymatic digestion or physical scraping to detach them. In addition, cell sheets detached from NIPAM surfaces appear to retain their function upon transfer to another growth surface, possibly due to the concurrent detachment of at least one protein of the Extracellular Matrix (ECM), fibronectin (FN). However, the extent to which ECM detaches from the NIPAM surface has remained unknown. We present a thorough examination of the cellular response to NIPAM using X-ray Photoelectron Spectroscopy (XPS), Secondary Ion Mass Spectrometry (SIMS), and immunostaining. XPS is used to make a quantitative comparison of the amount of protein atop NIPAM after cell removal. The primary proteins of the ECM (FN, laminin, and collagen) are examined using immunostaining to determine which of the ECM proteins studied lift off with the cellular layer. In addition, SIMS is used to identify the presence and identity of proteins left at the NIPAM surface after liftoff. Finally, the low-temperature liftoff technique is compared to other traditional cell removal protocols. Our SIMS, XPS, and immunoassay results suggest that low-temperature liftoff of the cell monolayer from the NIPAM surface is accompanied by the majority of the components of the ECM.