AVS 50th International Symposium
    Biomaterial Interfaces Wednesday Sessions
       Session BI+SS-WeM

Paper BI+SS-WeM7
Analyzing Lymphocyte Adhesion, Membrane Receptors and Cytoskeletal Rearrangement on Micron Scale Mitogen Patterns

Wednesday, November 5, 2003, 10:20 am, Room 307

Session: Cell Interactions with Patterned Surfaces
Presenter: R.N. Orth, Cornell University
Authors: R.N. Orth, Cornell University
M.J.B. Flaminio, Cornell University
J. Kameoka, Cornell University
T.G. Clark, Cornell University
H.G. Craighead, Cornell University
Correspondent: Click to Email
In this study, we investigated an in vitro immune reaction on a planar surface between T cells, B cells, and micron scale patterned mitogens as a model system for analyzing cell surface ligand responses. To form functionalized biomaterial microdomains, a polymer-coated substrate was patterned using photolithography and reactive ion etching. The samples were incubated in antibody and mitogen solutions prior to polymer removal and cell application. Uniform mitogen patterns ranging from 76 µm to <1 µm were created to target cell surface receptors, upregulate intracellular signaling cascades and cell activity, and stimulate proliferation. Several methods were used to analyze the patterned mitogens' effects on the lymphocytes. Carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-stained lymphocytes harvested from the substrate demonstrated a proliferative response when assayed by flow cytometry. Mouse monoclonal antibodies against equine membrane cell receptors (anti-major histocompatibility (MHC) class II, anti-CD4, anti-CD3, and anti-leukocyte function associated-antigen (LFA)-1) provided a view of stimulated cells' surface receptor distribution. Secondary anti-mouse antibodies with a conjugated 1.5 nm gold sphere were bound to the primary antibodies. The samples were incubated in a silver solution to form 10-100 nm spheres as the silver nucleated off the gold particles. Scanning electron microscopy (SEM) imaging provided high resolution images of the cell surface ligands' spatial distribution as marked by the silver spheres. This patterning technique provided a precise and reproducible means to structure biomaterial surfaces at subcellular resolutions.