To assess relative metabolic activity and proliferation of c

To assess relative metabolic activity and proliferation of TG101209 attached to various substrates we used the MTT assay [29], [30]. In order to compare the metabolic activity of cells under the various conditions irrespective of the number or viability of the cells, we performed a PicoGreen DNA assay to normalize the metabolic activity to DNA content of the cultures. Thus, the relative metabolic activity (rUMTT) divided by DNA amount (ng DNA) allowed us to assess the normalized metabolic activity of the cells (rUMTT/ng DNA), which takes into account the possible different proliferation rates and viability of the cells during the time period of assessment. Our results demonstrate that both the relative (Fig. 3(A)) as well as the normalized (Fig. 3(C)) metabolic activity of the cells grown on Col-1 and PO was slightly higher than that of the cells grown on SF for 48h. Since the relative metabolic activity can be indicative of cell proliferation, we observe a corresponding increase in cell number at 96h compared to 48h on Col-1 and PO. Such an increase in cell number was not observed on SF at the 96h time point compared to the 48h time point; therefore, the normalized metabolic activity of the cells increased significantly at this later time point. The normalized metabolic activity further revealed the dramatic increase in metabolism of cells grown on SF at 96h compared to 48h and compared to the other substrates at the 96-h time point. This indicates that there is a slight delay in cells grown on SF to reach their maximal metabolic activity and we would expect the number of cells on SF to increase accordingly after the 96h time point. Despite an initial delay in cell proliferation of cultures grown on SF, cells on all substrates differentiated into astrocytes after growth factor withdrawal for 5 days (Fig. 4), as indicated by the positive stain for GFAP, a marker of astrocytes, and the phenotypic change indicative of astrocytic differentiation. This suggests that both Col-1 and SF supported the capacity of the glial precursor cells to differentiate into astrocytes to the same extent as that observed on PO-coated tissue culture plastic surfaces. Since our SF membranes are mainly in beta-sheet conformation and do not contain any cell-binding domains, such as RGD domains, cell differentiation processes were not expected to be influenced by the film itself, a prediction that was confirmed here. This is an important finding since the differentiation of ESC-derived glial progenitor cells into astrocytes needs to be controlled by exogenous cues [7], [31]. As determined previously for terminally differentiated cells [23], the inclusion of cell-binding domains or of Pronectin-F or Pronectin-L into the silk membrane may help to augment the release of bioengineered products. To assess whether SF supports the release of adenosine in the absence of such cell-binding domains, adenosine release was determined from Adk−/− glial precursor cells grown on SF and PO. Adk−/− cells cultured on SF for 48 and 96h and after 5 days of differentiation released the same amount of adenosine as the respective Adk−/− cells cultured on PO. Thus, the inclusion of cell-binding domains is not necessary to promote cell-mediated adenosine release. In contrast to our previous study, here we observed that differentiated cells do not release significantly more adenosine than proliferating cells [7]. One difference between the two experiments is the time period the cells were in culture before sampling the medium. In the previous study adenosine release was measured 24h after plating of glial precursor cells on PO, and glial differentiation was initiated 24h after plating. Therefore, the delayed sampling of adenosine performed in the present study resulted in higher levels of adenosine release from the proliferating cells when compared to the differentiated cells. Since in the present study the actual number of cells growing on SF could not be determined, cell counts were replaced by DNA quantification with the PicoGreen assay. Therefore, it is difficult to compare the current release data with that of our previous study, where the release was normalized according to cell numbers.