Control of cultured human cells with femtosecond laser ablated patterns on steel and plastic surfaces

Abstract

The purpose of the present study is to explore topographical patterns produced with femtosecond laser pulses as a means of controlling the behaviour of living human cells (U2OS) on stainless steel surfaces and on negative plastic imprints (polycarbonate). The results show that the patterns on both types of material strongly affect cell behaviour and are particularly powerful in controlling cell spreading/elongation, localization and orientation. Analysis by fluorescence and scanning electron microscopy shows that on periodic 1D grating structures, cells and cell nuclei are highly elongated and aligned, whereas on periodic 2D grid structures, cell spreading and shape is affected. The results also show that the density and morphology of the cells can be affected. This was observed particularly on pseudo-periodic, coral-like structures which clearly inhibited cell growth. The results suggest that these patterns could be used in a variety of applications among the fields of clinical research and implant design, as well as in diagnosis and in cell and drug research. Furthermore, this article highlights the noteworthy aspects and the unique strengths of the technique and proposes directions for further research.

Fig. 1

Laser ablated structures on steel (a-c) and hot embossed structures on polycarbonate (d-f). 2D periodic grating (a) and pseudo-periodic, coral-like (b) structures have been hot embossed to polycarbonate (d and e, respectively). It can be seen that despite the high aspect ratio, the replication process has been carried out without losing any submicron details. c A typical 1D grating structure employed in this study. f A polycarbonate replica in a position where two 1D gratings cross and form a 2D grating. Bar in a and d is 10 μm, b and e 30 μm, c 4 μm and f 3 μm

Fig. 2

A typical fluorescence microscope image of cultured U2OS human cells on a steel substrate after 2 days. Structures on the steel are visualized in an epifluorecence microscope without applying filters—the image is merged with blue and red emission channels representing nuclei and β-tubulin networks, respectively. The typical cell morphologies of U2OS cells on a smooth steel surface can be seen in area C. On this substrate, other areas consist of ‘horizontal’ lines (a) and ‘vertical’ lines (d) crossing to form a 2D grid structure (b). LIPPS nanogrooves can be either parallel to micron grooves (d) or perpendicular to micron grooves (a). Hence, all nanogrooves are orientated vertically in this picture—and also in the grid structure (b). The height of the grooves in A and D is 4 μm, and hence the height of the structures on the area B varies up to 8 μm. The orientation of LIPPS may in part explain why the cells are more elongated in area D than in A and prefer ‘vertical’ orientation on the grid B. The cell nuclei are stained blue with Hoechst and the red represents the cell skeletons (β-tubulin antibody). Bar is 100 μm

Fig. 3

Orientation of the cells and nuclei on 1D periodic structures after a 4 day culture. The fluorescent staining of the nuclei with Hoechst (a) and of the entire cells with SYBR orange protein stain (b) shows that the nuclei are also orientated. While the quantification is more easily done from the pictures with nuclei staining, c shows the degree of orientation of the cell nuclei on 12.5 μm (black) and 25 μm (dark gray) and on smooth steel (light gray). The percentage of the cell nuclei (Y-axis), whose orientation diverged less than 2, 2.5, 3, 3.5, 5, 10, 20 and 45 ° was calculated using the CellProfiler program. For random orientation, 50 % is reached at 45. Bars in a and b 100 μm

Fig. 4

Cell morphologies on a grid structure after a 2 day culture. a Fluorescence microscope image of the cells when the focal plane is on the top of the structure. b Focal plane is ten microns below the one in A. c Scanning electron microscope image of cells growing downwards. Bars a 25 μm, b 25 μm and c 4 μm

Fig. 5

Cell cultures on plastic replicas. a In the upper-left corner of the image (area F), confluent cells can be seen on the smooth surface after a 4 day culture, while on the upper and left margins there are “vertical” (area V1D) and “horizontal” 1D (area H1D) grating structures, respectively, which cross elsewhere in the picture (2D). b Horizontal structures (H1D in A) and 2D grid (2D in A). c, d Details of the grid structure can be seen down left in A (area 2D in A). e, f Cells on smooth and on coral-like structures. Bars a 1 mm, b 100 μm, c 4 μm, d 10 μm, e 100 μm and f 20 μm