Fig. 4.

Reliable anchorage of native cell-secreted ECM to a glass surface. (A) Schematic overview of the chemical layers needed to reliably anchor cell-secreted ECM to a glass coverslip. Fibronectin (FN) is immobilized by a covalent linkage with poly(octadecene-alt-maleic anhydride (POMA) on an aminosilanized glass coverslip. Immobilized FN allows stable binding of cell-secreted ECM proteins. (B) Workflow for anchoring smooth muscle cell (SMC) ECM to glass coverslips: 1) FN is covalently immobilized on a POMA coverslip. 2) SMCs are grown to confluency. 3) During this culture time, the SMCs will deposit their own ECM. 4) The SMCs are removed by a decellularization process with NH₄OH, leaving the secreted ECM attached to the POMA-FN coverslip. 5) Evaluation of renal cell function on captured ECM. (C) Quantification of collagen type IV area deposited by 50,000, 100,000 or 150,000 SMCs cultured for 3, 6 or 9 days on POMA-FN coverslips. Shown is mean ± SEM; **P < 0.01, ***P < 0.001. N ≥ 23 fluorescent Z-stacks derived from N = 3 samples. (D) Representative immunofluorescence Z-stacks (100× magnification) of anchored collagen type IV on POMA-FN coverslips, deposited by 50,000, 100,000 or 150,000 SMCs cultured for 3, 6 or 9 days. Scale bar represents 300 μm. (E) Quantification of collagen type IV area deposited by 150,000 SMCs cultured for 6 days on coverslips coated with POMA-FN, POMA, FN, gelatin or collagen or no coating. Shown is mean ± SEM; ***P < 0.001. N ≥ 19 fluorescent Z-stacks derived from N = 4 samples. (F) Representative immunofluorescence Z-stacks (100× magnification) of anchored collagen type IV deposited by 150,000 SMCs cultured for 6 days on coverslips coated with POMA-FN, POMA, FN, gelatin or collagen or no coating. Scale bar represents 300 μm.