Objective: In this paper we have investigated the attachment and spreading kinetics of human embryonal carcinoma stem cells (TERA2.sp12) onto planar Si (Ti) O2 waveguides equipped with grating couples, and covered with poly-L-lysine (PLL), mucin acting as substrata for the cells. Analysis of the incoupling peak centre and width allows us to separate surface-molecular and bulk-cell effects of the three substrata investigated. The most extensive spreading is observed on PLL, coating with mucin completely inhibits cell spreading. We have also demonstrated the utility of OWLS for the quantitative label-free investigation of stem cell attachment and spreading.Materials and Methods: Cell culture & biochemicals Human EC cell lines, TERA2, were generously provided by S. Przyborski, Durham University, UK. Pharmaceutical grade porcine gastric mucin (PGM) was purchased from A/S Orthana Kemisk Fabrik, Kastrup, Denmark. The commercial mucin preparation with a mean molecular weight estimated as 565 kDa was dialysed to remove all salts and low molecular weight additives and lyophilized for storage. Substratum modification Waveguides were cleaned at room temperature with chromic acid (Fisher Scientific, U.K.) or under sonication in Colas Integra (Roche) cleaning solution or SDS/Colas Integra solution for 10 minutes, extensively rinsed using Elga ultrapure water (resistance 18.2 Mohm cm, filtered through 200 nm pores) and O2-plasma treated (20mW for 2 min). The PLL (0.01% solution; Sigma) and mucin (0.1% w/w) stock solution were made up by dissolving weighted dry material in ultra pure water this is sufficient to coat the waveguides and pre-equilibrated overnight. Solutions were applied to the waveguides for 20 minutes, washed twice with ultrapure water and incubated for at least 40 minutes in cell culture media (DMEM; high glucose (4500mg/L); pyridoxine HCl; NaHCO3; without Lglutamine) (Sigma) at 37 oC, 5% CO2 environment. Cell attachment Cells were detached from the culture flask using 0.05% trypsin/EDTA and collected using centrifugation (1500 rpm for 3 minutes). Cells were counted by eye using a hemocytometer, with the number of cells and surface coverage (60-70%) confirmed using phase contrast microscopy inspection of the waveguide after the experiments. Optical waveguide light mode spectroscopy Waveguides were made from amorphous silicatitania at a ratio of approximately 2:1 and incorporated a shallow (5-10 nm) grating coupler (type 2400, grating constant equal to 416.667 nm). The incoupling resonance peaks for the TM0 mode of the waveguides were measured every 40 seconds and saved for subsequent analysis.Results: The peak position is influenced by the presence of the cells in close contact with the substrata, their shape, and any protein exudates from the cells, which are deposited on the substratum. The two effects can be separated by considering simultaneously the surface coverage of the cells influencing the shape of the peaks. As explained above the appearance of a maximum in the plot of the overall width against cell development magnifies 50% cell coverage. The actual magnitude of the peak width depends on the Optical contrast between the uncovered and cell covered areas. The larger the cells, or the more tightly they are bound, the larger the contrast. We can exclude the first possibility since the cell numbers were identical. Therefore we infer that the cell bind about 4 times more strongly to the PLL than to the silica-titania. Finally we turn to mucin. Although there is a modest peak position shift, there is a total absence of peak broadening – implying no spreading (corroborated by the Optical micrographs. Therefore these type of stem cells most probably just exclude.Conclusion: Simultaneous measurement of incoupling peak position and width allowed us to get more detailed information about the surface behavior of living stem cells then previously demonstrated using only peak position data. We found that mucin totally inhibits the spreading of human embryonal carcinoma (TERA2) stem cells when compared with uncoated silica-titania, but the cells still register some physiological activity, while on the PLL surface cell spreading dominated the Optical signals.