Am J Physiol Cell Physiol 300: C657CC670, 2011 [PMC free article] [PubMed] [Google Scholar] 33. for variation in cell density throughout the seeded scaffold. We then applied this method to 9-Methoxycamptothecin the study of mouse lung scaffolds and found that decellularization of presliced mouse lungs produced matrices with preserved alveolar architecture and proteinaceous components including fibronectin, collagens I and IV, laminin, and elastin. Treatment with a 1-integrin-neutralizing antibody significantly reduced the repopulation index after 24 h of culture. Treatment with focal adhesion kinase (FAK) inhibitor and extracellular signal-regulated kinase (ERK) inhibitor further reduced initial repopulation 9-Methoxycamptothecin scores while treatment with AKT inhibitor increased initial scores. Rho-associated kinase inhibitor had no discernible effect. These data indicate that initial adhesion and survival of mouse fibroblasts in the decellularized mouse lung occur Rabbit polyclonal to Caspase 1 in a 1-integrin-dependent, FAK/ERK-dependent manner that is opposed by AKT. and and 0.01) and the 105 seeded sample ( 0.05) but that no difference was detected between the 104 and 105 values. At the 72-h time point, the 105 sample no longer exceeds the 104 sample, but the 106 value still exceeds the 104 value ( 0.05). At the 5-day time point, engraftment in the 104 seeded scaffold approximates the 105 and 106 samples and is increased from the baseline 104 9-Methoxycamptothecin value ( 0.05). From 7 days onward, the 104, 105, and 106 samples are equivalent and the 104 sample continues to be increased from 24 h ( 0.05). At and 0.05). Due to statistical corrections for multiple comparisons, no other comparisons reached significance. Graphical representation of these data is shown in Fig. 3 0.01) and the 105 seeded sample ( 0.05). No difference is seen in the 104 and 105 values. At the 72-h time point, the 105 sample no longer exceeds the 104 sample, but the 106 value still exceeds the 104 value ( 0.05). At the 5-day time point, the 104 seeded scaffold is usually increased from baseline ( 0.05) and now approaches the 105 and 106 samples. From on, the 104, 105, and 106 samples are equivalent, and the 104 sample continues to be increased from 24 h ( 0.05). At and 0.05). Due to statistical corrections for multiple comparisons, no other comparisons are significant. Decellularized rat lung slices support survival, proliferation, and collagen production of seeded fibroblasts. Based on these data, it seemed that 105 cells appeared to be the optimum dose for cell seeding in terms of two-dimensional initial engraftment. Thus further functional assessment of seeded cells was performed at this dosage. The finding that fibroblast quantities increased over time suggested that fibroblasts could both survive and proliferate in decellularized rat lung slices. To ascertain whether this assumption was valid, we assessed fibroblast cell death 9-Methoxycamptothecin by TUNEL staining and fibroblast proliferation by Ki67 immunostaining at serial time points during seeding and culture. Here we found little to no detectable TUNEL signal after 24 h of culture and only very few cells exhibiting TUNEL signal after 7 and 14 days (Fig. 4and ?andand data not shown), indicating that, in addition to engrafting in a two-dimensional fashion, cells seeded in this manner can migrate down into the matrix. One crucial function of fibroblasts is the synthesis and deposition of ECM proteins that maintain lung architecture and stability. To determine whether A9 fibroblasts displayed this function in the decellularized rat lung slices, collagen production and deposition were evaluated using immunofluorescence detection of procollagen I1 and Masson Trichrome staining. As shown in Fig. 4, ?,and ?andand ?and4and 0.0005). and ?and 0.05) or 106 ( 0.01) cells, and there is no difference in the index between the scaffolds seeded with 105 or 106 cells. At the 72-h.