Cellular adhesion is a central element in tissue mechanics, biological cell-cell signaling, and cell motility. In this context, the cell-substrate distance has been investigated in the past by studying natural cells and biomimetic cell models adhering on solid substrates. The amount of water in the membrane substrate gap, however, is difficult to determine. Here, we present a neutron reflectivity (NR) structural study of confluent epithelial cell monolayers on silicon substrates. In order to ensure valid in vitro conditions, we developed a cell culture sample chamber allowing us to grow and cultivate cells under proper cell culture conditions while performing in vitro neutron reflectivity measurements. The cell chamber also enabled perfusion with cell medium and hence allowed for contrast variation in situ by sterile exchange of buffer with different H2O-to-D2O ratio. Contrast variation reduces the ambiguity of data modeling for determining the thickness and degree of hydration of the interfacial cleft between the adherent cells and the substrate. Our data suggest a three-layer interfacial organization. The first layer bound to the silicon surface interface is in agreement with a very dense protein film with a thickness of 9 ± 2 nm, followed by a highly hydrated 24 ± 4 nm thick layer, and a several tens of nanometers thick layer attributed to the composite membrane. Hence, the results provide clear evidence of a highly hydrated intermediate region between the composite cell membrane and the substrate, reminiscent of the basal lamina.