Mouse fibroblast cell adhesion studied by neutron reflectometry

Abstract

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be approximately 180 A. The membrane region ( approximately 80 A thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.

Figure 1

Schematic of the solid-liquid interface cell used in NR measurements. The quartz substrate with adherent cells is clamped against a Macor disk with a 0.2–0.3 mm thick gap created by an o-ring. The subphase, in this case dPBS, is injected into the gap. The neutron beam penetrates the lateral face of the quartz substrate to reach the solid-liquid interface where the cells reside.

Figure 2

NR data and SLD profile for a media-coated quartz substrate. (a) The NR profile for the media sample with no cells present. The model-dependent fit based on the simplest, physically relevant box model is in black, and the data are shown with open circles and error bars indicating 1 standard deviation (SD). (b) The corresponding SLD profile (black line) is depicted on top of an interpretation of the media distribution at the interface. Large regions in grayscale represent varying concentrations of the proteins, salts, and sugars that reside in the media. Concentration is highest close to the quartz and gradually diffuses into the subphase with increasing distance from the substrate. Small D₂O molecules represent the increasing volume fraction of water as a function of distance from the quartz substrate.

Figure 3

NR data and SLD profile for a high cell surface density sample. (a) NR data are shown with open circles and error bars that indicate 1 SD. The model-dependent fit is shown in black and the model-independent fit is shown in gray. (b) The corresponding SLD profiles for each fitting method. The model-independent method produces a family of SLD profiles (gray ribbon) where no curve differs from the lowest found χ² value by more than χ. The shading between 300 and 400 Å (b) represents the transition from hydrogen-rich material adjacent to the membrane to bulk dPBS in the cells' interior.

Figure 4

SLD profile of the highest surface concentration of cells depicted on top of a cartoon representation of how the cell behaves in the adherence region. Immediately adjacent to the quartz substrate is a layer of adherence proteins (∼120 Å thick), on top of which sits the membrane region (∼80 Å thick), followed by a diffuse profile representing the interior of the cell. Because of instrument limitations, the more distant cell membrane is not visible. Small D₂O molecules represent the water content as a function of distance from the quartz substrate.

Figure 5

NR profiles (a) and corresponding SLD profiles (b) for high (black) and low (gray) cell surface densities. NR data are shown by open circles, and error bars indicate 1 SD. The lower surface cell density is evident from the decreased scattering intensity (a) and the increased SLD in the membrane region (120–200 Å) and interior of the cell (200–320 Å) (b).

Figure 6

NR data (a) and corresponding SLD profiles (b) show changes to the cell sample due to the introduction of distilled water. Data obtained before (gray) and after (black) exposure to distilled water are shown by open circles and error bars indicating 1 SD. After modeling, the changes in the NR data indicate that the introduction of distilled water effectively removed almost all material from the substrate. The grayscale gradient (300–400 Å) only corresponds to the SLD profile before the introduction of distilled water.

Figure 7

NR and SLD profiles before and after introduction of trypsin. NR data (a) and corresponding SLD profiles (b) before (gray curve) and after (black curve) exposure. NR data are shown with open circles and error bars indicating 1 SD. The gray curves show typical low cell surface density profiles. After the introduction of trypsin, the changes in the NR data suggest that trypsin has digested most of the adherence proteins, leaving only trace amounts of protein on the quartz surface. The grayscale gradient (300–400 Å) only corresponds to the SLD profile before the introduction of trypsin.