Fig. 1. Schematic illustration of manipulation technique and ECM coatings. (a) Schematic illustration displays the fabrication of ECM coatings utilizing gelatin hydrogels. By this method, cell sheets can be reused. (b) Microscope images before and after removing HUVEC cell sheet; (c) SEM images of blank glass substrate (Control) and decellularized glass substrate (ECM); (d) Representative immunofluorescence images of collagen I in the ECM coatings generated by HUVECs on different kinds of substrate (ECM). Control represents the related blank substrates.

Fig. 2. ECM coating characterization. (a) Young's modulus distribution of different types of the hydrated ECM coatings as measured by AFM with different Peak Force Setpoint (0.5nN, 1Nn, 2nN). (b) Anhydrous thickness of ECM coatings on SiO2 surfaces as determined by ellipsometry (n = 6, one-way ANOVA). (c) Representative immunofluorescence images of collagen I in ECM coatings. Control group represents a blank glass substrate. (d) QCM frequency shift (Δf) of the desorption of ECM coatings from Au sensor surfaces. The blue curve stands for PBS rinsing. The red curve indicates trypsin washing, and the black curve demonstrates the SDS flushing. (e) Hydrated mass of the ECM coatings as calculated from the QCM results.

Fig. 3. Reuse of cell sheets. (a) Viability of the cells after being detached from the culturing glass surfaces and transferred to new glass surfaces. (b) ECM coatings generated by the fresh (above) and reused (below) L929 cells. (c) L929 cell fragment residues left in the ECM coatings as detected by cell membrane probe after decellularizing by the presented method (left) and the traditional cell lysis (right). (d) Area ratio of the cell membrane residual as detected by the membrane probe (n = 5, t-tests).

Fig. 4. Cell adhesion on ECM coatings. (a) Representative fluorescence images of F-actin and paxillin staining MC3T3 cells and the related area of the spreading cells and focal adhesions as well as the circularity of the cells on the ECM coatings generated by MC3T3 cells for 4 h. (b) Representative fluorescence images of F-actin and paxillin staining L929 cells and the related area of the spreading cells and focal adhesions on the ECM coatings generated by L929 cells for 24 h. (c) Representative fluorescence images of F-actin and paxillin staining HUVEC cells and the related area of the spreading cells and focal adhesions on the ECM coatings generated by HUVEC cells for 30 min. The control groups represented the cells adhered on the pristine glass surfaces. For all analysis n > 100, t-tests.

Fig. 5. Stem cell differentiation on the functional ECM coatings. (a) Representative immunofluorescence images of hMSCs stained with osterix on different types of ECM coatings after culturing for 2 days. (b) Representative ALP staining images of hMSCs on different types of ECM coatings after culturing for 7 days. (c) Representative immunofluorescence images of hMSCs stained with α-SMA on different types of ECM coatings after culturing for 7 days. (d) Representative immunofluorescence images of hMSCs stained with CD31 on different types of ECM coatings after culturing for 7 days. (e) Nuclear/cytoplasmic ratios of the average fluorescent intensity of osterix as shown in (a) (n > 50, one-way ANOVA). (f) Percentage of the ALP positive cells as shown in (b) (n = 3, one-way ANOVA). (g) Quantification of the average fluorescent intensity of α-SMA as shown in (c) (n > 50, one-way ANOVA). (h) Quantification of the average fluorescent intensity of CD31 as shown in (d) (n > 50, one-way ANOVA). The control groups represented the hMSCs on the pristine glass surfaces. M-ECM, L-ECM and H-ECM groups represented the hMSCs on the MC3T3, L929 and HUVEC generated ECM coatings, respectively.