DISCUSSION
The need for innovative biomanufacturing platforms that sustain cell
viability and functionality are critical to broadly advancing cell
therapies. In this study, we demonstrated production of insulin from
metabolically competent pancreatic cells seeded onto a wicking matrix
cellulose scaffold. We tested multiple surface-modification conditions
for cell attachment and expansion in a multi-well test platform and
small-scale bioreactor. Cells seeded onto the wicking matrix cellulose
scaffold in the bioreactor exhibited greater expansion (up to 10-fold)
and increased insulin secretion, when compared with traditional 2D
cultures. Metabolic profiling indicates that the oxygenation and
physiological conditions in bioreactor were favorable. This
biomanufacturing platform provides an efficient 3D microenvironment for
pancreatic cells to support mass production of these insulin producing
cells for potential use in cell therapy to treat Type 1 diabetes
patients. The ability to use human induced pluripotent stem cell-derived
pancreatic cells also opens up the possibility of patient-specific
treatments.
Multiple surface modifications enabled attachment and growth of primary
pancreatic cells as evaluated by MTT metabolic assay and scanning
electron microscopy. Before seeding cells, a fraction of the culture was
validated in parallel for relevant biomarker expression by
immunocytochemistry, including PDX-1, NGN3, NKX6-1, NeuroD1 and insulin,
which are indicators of pancreatic lineage-committed cells (Takahashi
2016, Kieffer 2014, Benthuysen 2016). Pancreatic cells seeded at a
density of 20,000 cells/well on various modified scaffolds in a
multi-well platform were able to attach similarly to cellulose with all
six surface modifications; however, their expansion varied considerably.
Growth on uncoated, amine-modified cellulose (A) and gelatin-coated,
NaOH-modified cellulose (NG) scaffolds provided the most favorable
surface conditions for expansion of cells with 5 to 6-fold expansion. By
high resolution SEM imaging of cells on day 1 and day 3, we observed
spreading of cells on these scaffolds with visible lamellipodia
formation. As discussed by several authors (Banik 2015, Le 2013,
Krishnamurthy 2009), surface topography of various scaffolds contributes
to cell attachment and can impact long-term viability and
differentiation of cells. The cellulose scaffold supports cell spreading
and retention of phenotypic function. Seeded pancreatic cells displayed
an affinity towards all modified surfaces of the porous cellulose
scaffold that was apparent by formation of structures that resemble
lamellipodia and production of a film of extracellular matrix. In
addition, cells were able to form larger aggregates over 5-10 days,
consistent with survival and proliferation seen in other studies (Kim
2016, Salvatori 2014). Between day 1 and day 10, sustained culture on
the amine-modified scaffolds resulted in better expansion, based on a
greater distribution of cell clusters throughout the scaffold.
Our analysis revealed that the uncoated, amine-modified (A) and
gelatin-coated, NaOH-modified (NG) scaffolds were most favorable, and
these were chosen for additional analysis using hiPSC-derived pancreatic
cells. Viability, morphology and insulin secretion of hiPSC-derived
pancreatic cells on these two scaffold modifications were examined. The
human iPSC-derived endocrine cells were generated by multiple stages of
differentiation. In the first stage of pancreatic differentiation,
expression of definitive endoderm biomarkers, such as Sox 17 and
Brachyury increase while pluripotency markers decrease. Moving
developmentally toward posterior foregut, pancreatic biomarkers such as
PDX-1 appear. Following commitment to the pancreatic lineage, expression
of biomarkers such as NKX2-2, NKX6-1, Neurogenin-3 and insulin increase
(D’Amour 2006, Rezania 2012, Russ 2015). From committed pancreatic
cells, the hormone-releasing beta cells arise; however, these cells are
initially immature, and the amount of insulin released in response to
changes in glucose level is low. Further differentiation to mature, beta
islet cells is facilitated by addition of Triiodothyronine (T3) which is
a Thyroid receptor agonist, with observed upregulation of MafA in
cultures (Aguayo-Mazzucato 2013, Millman 2016). We tested hiPSC-derived
pancreatic progenitors,
NKX6-1+/PDX-1+ on our uncoated,
amine-modified (A) and gelatin-coated, NaOH-modified (NG) cellulose
scaffolds for 10 days as single cells and as lightly dissociated cell
aggregates. By MTT assay, we observed a predictable trend of decreased
cell proliferation as cells underwent differentiation on amine-modified
scaffolds from seeded aggregated cells. We observed that cell aggregates
had higher viable cell density on amine-modified (A) scaffolds compared
to gelatin-coated, NaOH-modified (NG) scaffolds on all days of testing.
Viable cell density from single-seeded cells on amine-modified scaffolds
(A) increased linearly throughout the experiment. However, single cells
seeded on gelatin-coated, NaOH-modified scaffolds (NG) showed a
significant decrease in viable cells on day 5. These findings suggest
that the differentiating seeded pancreatic cells were more compatible
with the amine-modified scaffolds. Images taken by SEM support this
observation. SEM images acquired on day 5 showed production of
significant amount of ECM on the amine-modified scaffolds seeded with
cell aggregates. The presence of ECM was also observed on day 5 and day
10 on amine-modified scaffolds seeded with single cells. As noted
previously (Hammer 2004, Stendhal 2009, Llacua 2018, Smink 2018), ECM
production is a necessary step for support of multiple cell activities
on a scaffold including survival and migration, and in some cases, ECM
components can integrate into the scaffold structure to enhance cell
attachment, differentiation and proliferation for tissue engineering.
A sustained ability to produce insulin is critical to biomanufacturing
efforts with pancreatic cells. Insulin secretion was measured for cells
grown on both the uncoated, amine-modified (A) scaffold and
gelatin-coated, NaOH-modified (NG) scaffold, and both demonstrated
greater insulin release than was observed from the same cells in 2D
culture (data not shown). This analysis demonstrates the enhanced
functional potential of chemically modified 3D cellulose scaffolds for
supporting hiPSC-derived pancreatic cells and differentiation into
mature, functional, insulin-secreting pancreatic cells. The amount of
insulin secreted from single cells seeded on both cellulose
surface-modified conditions decreased over time, in contrast to cell
aggregates seeded, which showed an increase in insulin secretion on day
5. We speculate that the higher insulin secretion and ECM production by
cell aggregates on amine-modified scaffolds is due to higher cell
density and increased cell-cell contact on the scaffolds. As a follow
up, we tested insulin release from single cells seeded on the scaffold
in the bioreactor at two seeding densities. The hiPSC-derived endocrine
precursors were seeded onto the amine-modified wicking matrix scaffold
in the bioreactor, and insulin release and metabolic activity of cells
were assessed for 13 days. We observed a significant increase in insulin
secretion at both cell seeding densities that remained high until the
conclusion of the experiment, demonstrating an improvement of cell
functionality in this 3D environment. The porous structure of cellulose
fibers along with adequate oxygenation and continuous accessibility of
all cells to media in the bioreactor appear to meet critical
microenvironment needs enabling ~2-fold increase in
insulin release in the bioreactor. Doubling the cell density enhanced
insulin production in the bioreactor, reaching the higher insulin
secretion by day 2 rather than day 7. Given that cell seeding density
affects differentiation and maturation of pancreatic cells in 2D
cultures (Gage 2013), we suspect that delayed insulin production at the
lower cell seeding density in the bioreactor is a result of an arrested
or slowed maturation process.
Metabolically, glucose consumption and lactate production in the
bioreactor were reflective of good cell viability and activity. Low
glucose consumption levels, which can be a sign of lowered cell
viability, were not observed. Higher inoculation densities (1 x
107) exhibited increased glucose consumption and
lactate production compared to lower cell densities (i.e., 5 x
106 cells, Supplemental Figure 2). The relatively low
levels of lactate produced in the bioreactor indicate sufficient
aeration, as would be expected in the wicking matrix bioreactor with the
thin film of liquid. In addition, the low lactate concentration
demonstrates a low rate of aerobic glycolysis. Elevated aerobic
glycolysis is a hallmark of tumor cells as well as dedifferentiation in
culture. In summary, the metabolic profile further supports the design
and use of the bioreactor for cell biomanufacturing.