AVS 51st International Symposium
    Biomaterial Interfaces Friday Sessions
       Session BI-FrM

Paper BI-FrM1
The Effect of Cell Detachment Method on the Identity and Quantity of Residual ECM Proteins Retained at Surfaces

Friday, November 19, 2004, 8:20 am, Room 210D

Session: "Active" - Dynamic Biointerfaces
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
Treatment of tissue culture polystyrene (TCPS) with poly(n-isopropylacrylamide) (pNIPAM) has been developed as a technique for the harvest of intact cell monolayers. Although low-temperature liftoff from pNIPAM is known to be less damaging to cells than traditional cell removal methods, little is known about the effects these techniques have on the underlying Extracellular Matrix (ECM). Recently, we demonstrated that although immunoassay of ECM components show that low-temperature liftoff removes the majority of the ECM concurrently with the cells, XPS and SIMS results reveal that some protein does remain at the pNIPAM surface. In this work, we further examine the effect that low-temperature liftoff from pNIPAM and traditional cell removal methods have on the ECM. Using XPS, we compare the relative amount of ECM remaining at culture surfaces after cell removal by the different methods. Using SIMS, MALDI, and immunostaining, we identify the individual proteins left behind. Finally, LDH assay is used to ascertain the viability of the residual ECM left behind by each cell removal method. We find that in addition to its dramatic effects on cell viability and morphology, trypsin removes much of the underlying ECM and often adsorbs to the surface itself, drastically reducing the adhesion of new cells. Although mechanical dissociation of the cell layer is less damaging to the underlying ECM, harvest via this method results in incomplete cell layers with partially damaged appearance. Of these techniques, only low-temperature liftoff from the pNIPAM surface harvests a complete cell monolayer while leaving behind ECM proteins capable of promoting new cell adhesion.