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Establishment of a cell micropatterning method for the quantitative assessment of the organization of the keratin filament network

3.4 Normalized cell shape
To investigate the role of geometric features on the KIF network and to identify the appropriate size of patterned features, AK13-1 cells and HaCaT B10 cells spread on micropatterns with an average cell spreading area of 700 µm², 900 µm², 1100 µm² and 1600 µm² were analyzed. After cell attachment and spreading an increased number of malformed micropatterns could be observed (Fig. 10). These were mostly micropatterns occupied by one or multiple cells, suggesting that the printed proteins were peeled off by the cells, resulting in pattern degradation. A retreat of the micropattern boundaries is observed, which correlates with the cell boundaries (Fig. 10 A‘ and B‘). As the overall shape of the patterned cells was unchanged, these cases were also documented and the effects of pattern degradation on the distribution of KIFs assessed as negligible or insignificant.

Fig. 10 Pattern degradation on PDMS. After protein patterning and passivation of non-printed regions HaCaT B10 cells (A-A'') and AK13-1 cells (B-B'') were seeded on the micropatterns and allowed to spread for 24 h before fixation. Bar, 5 µm. FN micropatterns (magenta) display degradations (A and B) caused by cells peeling off the ECM coating as the correlation of cell boundaries and the boundaries of degraded micropatterns indicate (A' and B'). The keratin network (green) of AK13-1 cells and HaCaT B10 cells braids the nucleus of each cell type. Bar, 10 µm.

Fig. 10 Pattern degradation on PDMS. After protein patterning and passivation of non-printed regions HaCaT B10 cells (A-A“) and AK13-1 cells (B-B“) were seeded on the micropatterns and allowed to spread for 24 h before fixation. Bar, 5 µm. FN micropatterns (magenta) display degradations (A and B) caused by cells peeling off the ECM coating as the correlation of cell boundaries and the boundaries of degraded micropatterns indicate (A‘ and B‘). The keratin network (green) of AK13-1 cells and HaCaT B10 cells braids the nucleus of each cell type. Bar, 10 µm.

Fully spread single cells were found often on small micropatterns (Fig. 11 A-A“‘) whereas an increase in micropattern size led to an increase in the number of cells per micropattern (Fig. 11 B-B“‘) or to incomplete cell spreading of single cells (Fig. 11 C-C“‘). Hence, less or no fully spread single cells could be found for many large micropatterns.

Fig. 11 HaCaT B10 cells spread on Y-shaped micropatterns.

Fig. 11 HaCaT B10 cells spread on Y-shaped micropatterns. HaCat B10 cells were seeded on the CYTOOchip™ and allowed to spread for 24 h before fixation. Fully spread single cell found on a 700 µm² micropattern (A-A“‘). Larger micropatterns are either occupied multiple cells as seen on 1100 µm² micropatterns (B-B“‘) or by incompletely spread single cells (C-C“‘). The micropattern is detected by fluorescently labeled FN (A, B, C), nuclei by DAPI staining (superposed on contrast image [TL] in A‘, B‘, and C‘) and human keratin 5 through EGFP signal (A“‘, B“‘ and C“‘) in the immortalized human keratinocyte-derived cell line HaCaT B10. A merged image of fluorescently labeled FN, DAPI and human keratin 5-EYFP is shown in A“‘, B“‘ and C“‘. Bar, 10 µm.

First results showed that the human keratin 13 network of AK13-1 cells spread on micropatterns with an average cell spreading area of 700 µm² (Fig. 12 A-A‘) appeared extremely dense compared to fully spread cells on larger micropatterns (Fig. B-B‘) and on non-printed substrates (Fig. C-C‘). Same could be observed for the keratin 5 network of HaCaT B10 cells (no comparative data shown), which suggests the assumption that KIFs of cells spread on micropatterns seem in general compact compared to cells with unlimited cell spreading area on non-printed substrates.

Fig. 12 Human keratin 13 network density of AK13-1 cells

Fig. 12 Human keratin 13 network density of AK13-1 cells. A) HK5 network of an AK13-1 cell spread on a 700 µm² round micropattern. B) HK5 network of a AK13-1 cell spread on a 1100 µm² round micropattern. C) HK5 network of AK13-1 cells spread on FN coated PDMS. Bar, 10 µm. A‘, B‘ and C‘) Merged images of the human keratin 13-EGFP (green) and DAPI staining (blue).

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