Expansion of Primary Pancreatic Cells on the Cellulose Scaffold is Affected by Scaffold Surface Modification
To determine optimal conditions for pancreatic cell growth on the porous cellulose scaffold, cellulose pieces (5 mm x 3mm x 2 mm) from wicking matrix material subjected to various treatments were evaluated in a multi-well platform (Figure 1C) for cell attachment/retention, viability, cell penetration into the scaffold, morphology, and functionality of cells on the scaffold. Purchased human primary pancreatic cells (Islets of Langerhans) were cultured and expanded in coated flasks to reach the desired number for seeding onto scaffold and validated by immunocytochemistry for relevant intracellular pancreatic biomarkers, including PDX-1, NeuroD1, NGN3 and NKX6-1 (Figure 2A). These pancreatic cells, referred to asNKX6-1+/PDX-1+ , were collected by trypsinization, and 20,000 cells were seeded onto cellulose scaffolds that were either unmodified, amine- or NaOH-modified, with or without gelatin coating. Cells were maintained for 10 days on these scaffolds and evaluated at days 1, 3, 5 and 10 by MTT assay (Figure 2B) and scanning electron microscopy (Figure 3). The time course of cell growth on the six different cellulose scaffolds was measured by MTT assay. On day 1, viable cells were observed to attach efficiently to all six scaffold modifications (Figure 2B, Figure 3). On day 3, amine-modified (A), amine-modified, gelatin-coated (AG), and NaOH-modified, gelatin-coated (NG) scaffolds showed significantly greater viable cell densities than the other scaffolds (one-way ANOVA, p < 0.001), while on day 5, the unmodified, non-coated scaffolds showed lower viable cell densities than any of the other scaffolds (one-way ANOVA, p < 0.001). Interestingly, by day 10, cells grown on the unmodified, uncoated scaffolds had recovered, and no statistically significant differences in cell density between any of the modifications were observed based on MTT assay, though qualitative differences were observed based on SEM imaging as described below. The MTT assay data were further used to calculate the expansion of pancreatic progenitors under the six modification conditions to find the optimum surface conditions for attachment and growth of pancreatic progenitors (Table 1). The MTT assay results indicate that theNKX6-1+/PDX-1+ pancreatic progenitor cells attached and expanded to some degree in all six conditions. The largest expansion achieved was 6.6-fold on the gelatin-coated, NaOH-modified (NG) scaffolds; whereas only 3.8-fold expansion was observed on similarly modified NaOH scaffolds lacking gelatin coating (N). Gelatin coating was not always required, since unmodified (U) and amine-modified (A) scaffolds without gelatin coating had the next highest expansion with 5.6- and 5.4-fold expansion respectively. Based on our observations, uncoated, amine-modified (A) scaffolds and gelatin-coated, NaOH-modified (NG) scaffolds provided better surface attachment and higher growth rates for primary pancreatic cells (Figure 2B) and were selected for further studies.