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.