High Resolution Imaging of Primary Pancreatic Cell Attachment
and Expansion on Cellulose Scaffolds
Due to the non-transparent nature of the cellulose scaffold, we examined
cell attachment and extent of coverage on the scaffolds by SEM on days
1, 3, 5 and 10 after seeding (Figure 3). On day one, for all surface
modifications, cells were distributed throughout the scaffolds. By day
three, cells produced visible cytoplasmic extensions and were
distributed more widely on the scaffolds. We observed a clear preference
for non-coated unmodified (U) and amine-modified (A) and gelatin-coated,
NaOH-modified (NG) surfaces compared with other conditions (Supplemental
Figure 1). Significant cell expansion was observed by day five, along
with cell migration into the scaffold interior pockets. Cell aggregates
were also observed as well as extracellular matrix (ECM) on the
scaffolds as shown in the images. These results were consistent with our
MTT-assay quantification of viable cells over time under each condition.
Cell aggregates were seen on uncoated, unmodified (U) and amine-modified
(A) scaffolds more than on other surface conditions, which show a more
extensive cell distribution (Supplemental Figure 1).
hiPSC-Derived
NKX6-1+/PDX-1+ Pancreatic
Cells Bound to Both NaOH- and Amine-modified Scaffolds
hiPSC-derived pancreatic cells were generated and matured to beta cells
by combining two existing protocols (Kroon 2008, Millman 2016) and
validated by immunocytochemical analysis of pancreatic markers (Figure
4) similar to previous experiments with primary pancreatic cells. On day
12 of differentiation, cells were detached and seeded onto cellulose
scaffolds as single cells or as small aggregates. Based on the results
of the MTT assay and SEM analysis using primary pancreatic cells, two
conditions, uncoated amine-modified (A) scaffolds and gelatin-coated,
NaOH-modified (NG) scaffolds were evaluated. Similar to previous
experiments, 20,000 hiPSC-derived endocrine precursors were seeded on
scaffolds and differentiation was continued in stage 5 and 6
differentiation media (Millman 2016) for 10-12 additional days (10 days
for aggregates, 12 days for single cells), which should result in
immature beta cells expressing markers such as insulin, NKX6-1, PDX-1
and MAF BZIP transcription factor A (MafA). The viable cell density was
analyzed by MTT assay on days 1, 5 and 10/12, and cells successfully
attached and proliferated on both surfaces (Figure 5A and Figure 5B). In
comparison with single cells, the seeded aggregates had higher initial
viable cell density; however, the viable cell density declined over time
for cells on both surface conditions, with a very significant decrease
on the gelatin-coated, NaOH-modified (NG) scaffolds on day 5, which
reduced the number of viable cells to less than half of the initial
number (Figure 5B). In contrast, the viable cell density for single
cells on the uncoated amine-modified (A) scaffolds increased linearly
over time. Even though single cells on the gelatin-coated, NaOH-modified
(NG) scaffolds demonstrated slightly higher viable cell density than
single cells on the amine-modified scaffolds, the viable cell number
dropped suddenly on day 5, similarly to aggregates. To determine if cell
viability was being affected or if cells were detaching from the
scaffold surface, we used SEM to assess the attachment and morphology of
the differentiating hiPSC-derived pancreatic cells on the scaffolds on
days 1, 5 and 12. During differentiation, we observed a significant
amount of ECM produced on uncoated amine-modified scaffolds loaded with
single cells and aggregates (Figure 5E). In contrast, ECM deposition was
lower on gelatin-coated, NaOH-modified (NG) scaffolds seeded with
aggregates and single cells (Figure 5F). Images of aggregates on
scaffolds from day 12, entering stage 6 (immature beta cells), reveal an
almost empty scaffold, in agreement with MTT data showing a decrease in
number of viable cells (Figure 5A). In both single cells and aggregates
assays, uncoated amine-modified surfaces showed more uniform cell
attachment and growth compared to gelatin-coated, NaOH-modified
surfaces, and therefore, proved to be a better candidate for pancreatic
cell culture.
Scaffold-Supported hiPSC-Derived
NKX6-1+/PDX-1+ Pancreatic
Cells Secrete Insulin
Insulin release by pancreatic cells on uncoated amine-modified (A)
scaffolds and gelatin-coated, NaOH-modified (NG) scaffolds was
quantified by ELISA on days 1, 5 and 10/12. Single cells on
amine-modified (A) scaffolds produced the highest amount of insulin on
day 1 (Figure 5C); however, insulin production declined on days 5 and
12, similar to single cells on gelatin-coated, NaOH-modified (NG)
scaffolds (Figure 5D). The decrease in insulin production was consistent
with loss of cells from the scaffolds. It is unclear if the remaining
cells attached to the scaffolds exhibited any change in insulin
production. Cell aggregates on both scaffold modifications had a very
low insulin release at day 1. Even though insulin release increased by
day 5 on both surfaces, it was still approximately half of the insulin
released from single cells. On both surface modifications, insulin
concentration surpassed that produced by a similar number of pancreatic
cells cultured in a dish (data not shown). Based on ELISA results,
single cells seeded on scaffolds secreted higher amounts of insulin than
aggregates cultured on scaffolds, which in turn secreted more insulin
than cells cultured on tissue culture dishes, with the highest amount
produced by single cells on uncoated amine-modified (A) scaffolds.
Insulin Secretion by Scaffold-Attached hiPSC-Derived
NKX6-1+/PDX-1+ Pancreatic
Cells Increases with Integration into the Bioreactor
A miniature bioreactor was manufactured with the uncoated amine-modified
(A) wicking matrix scaffold as that scaffold modification demonstrated
the greatest potential for cell expansion and insulin production in our
previous experiments. The bioreactor scaffold was pre-wetted with medium
and then seeded with 5 x 106 or 1 x
107 hiPSC-derived pancreatic cells (Figure 6A).
Insulin release and metabolite profiles were determined from assays of
the culture medium. At the conclusion of experiments, MTT was added to
the bioreactor to visualize viable cells (Figure 6B), and SEM
visualization of cell attachment and distribution throughout the
scaffold from the bioreactor was performed (Figure 6C).
Insulin ELISA of bioreactor samples from 1 x 107seeded cells showed an insulin production greater than 0.28 µg on day 2
of the bioreactor run. Insulin production was again evaluated at day 4,
6, 8, 10 and 13. Insulin production on day 6 increased to 0.3 µg and
remained high through the last examined time point, day 13 (Figure 6D).
When fewer cells were seeded (5 x 106) onto the
bioreactor, an initial lag in insulin production was observed, but by
days 7 to 13 higher insulin output was achieved, although slightly
reduced versus seeding with 1 x 107 cells.
In addition to insulin production, we evaluated the metabolic profile of
cells seeded at the 1 x 107 cell density for glucose
and lactate production that are typically good indicators of process
robustness. The metabolic profile of cells was determined using a YSI
biochemical analyzer (Figure 6E). Glucose consumption in the bioreactor
seeded with 1 x 107 cells increased
~2-fold between day 2 and day 6, after which the glucose
consumption decreased ~15% between day 6 and 8 and
another 10% by the end of experiment. Lactate production was much lower
than glucose consumption and showed an opposite trend from glucose
consumption, with a substantial drop in lactate production between day 2
and 8, followed by an increase until the end of the culture. The
relatively high glucose consumption throughout the experiment is
indicative of sustained metabolic activity of the cells in the
bioreactor over 13 days. These results show cells in the bioreactor were
able to attach to and grow on an amine-modified scaffold, maintain a
steady metabolic activity, and produce high amounts of insulin for 13
days. A bioreactor inoculated with 5 x 106 cells was
disassembled after 14 days and cells were harvested from the
amine-modified scaffold by trypsinization and sonication; 50 x
106 cells were counted, demonstrating
~10-fold expansion of differentiating pancreatic cells
in bioreactor.