INTRODUCTION
Diabetes mellitus is one of the leading causes of death, and according
to the International Diabetes Federation (IDF), more than 420 million
individuals have been diagnosed with diabetes worldwide. Diabetes, or
elevated blood sugar, can lead to long-term complications including
cardiovascular diseases and damage to the retina, kidney and nerves
(Okere 2016, Kieffer 2018). There are two types of diabetes mellitus. In
type 1 diabetes, an autoimmune disorder, insulin-producing beta cells
are attacked by the immune system, while in type 2 diabetes,
insufficient insulin is produced by the beta cells and/or other cells
exhibit insulin resistance (Wang 2015). Current treatments are insulin
injection and islet transplantation. Insulin injection provides a
temporary solution, with risks of inappropriate pre-determined dosing
and dependence on external insulin. Islet transplantation remains the
only permanent treatment; however, it is complicated by the shortage of
donors, required immunosuppression, and risk of tissue rejection. Due to
these limitations, a new source of beta cells for type 1 diabetes
patients is needed (Vieira 2016, Jiang 2017), which will require
advances in human stem cell technologies and biomanufacturing platforms.
Stem cells offer opportunities for patient-specific cell therapies, and
induced pluripotent stem cells (iPSC) are considered an attractive
replacement for organ transplantation. However, generation and scale-up
of patient-specific cells using current protocols is time consuming and
costly (Wilmut 2015, Herberts 2011), and whether allogenic cell
transplantation provides sufficient immune match compared with
autologous cell transplantation is not yet resolved (Millman 2017).
Induced pluripotent stem cells, embryonic stem cells, and multipotent
stem cells, including bone-marrow mesenchymal stem cells have been
successfully differentiated to insulin-producing beta cells in culture
(Kieffer, 2016, Jacobson 2017). In order to prevent immune responses to
transplanted pancreatic cells, macro- or micro-encapsulation devices
have been studied (Shultz 2015, Vegas 2016, Bruin 2013). A Phase I
clinical trial of a macro-carrier by Viacyte was successfully completed,
and the macro-carrier is currently in Phase II clinical trials
(https://clinicaltrials.gov identifier: NCT02239354).
Strategies for moving from bench to clinic require mass production of
cells either in 2D cell factory cultures as currently performed by
Viacyte (Schultz 2015), or in 3D bioreactors, which offer different
limitations and advantages. Limited surface area in 2D cultures as well
as limited recapitulation of the native organ 3D environment make 2D
cultures impractical for scale-up (Kropp 2017). The advantage of 3D
culture systems is that cells tend to aggregate into three-dimensional
tissue structures that exhibit more natural functional behavior than
conventional 2D culture (Modulevsky 2014). Both static and dynamic 3D
platforms have been investigated, with the idea that a dynamic system is
better able to handle transport of metabolites in high density cell
systems than the static one (Kempf 2016). However, dynamic systems, such
as stirred and rotating bioreactors, create additional complications of
induced shear stress, loss of cells during media changes,
non-adaptability of some cell types to suspension culture, cell
retrieval and insufficient oxygenation, especially in the case of larger
cell aggregates. These limitations can result in cell death and lower
the cell yield in these system (Kempf 2016, Kropp 2017). Therefore, an
alternative, static, wicking-matrix bioreactor which provides a thin
film of medium dripped onto cells on the scaffold offers advantages for
3D culture such as improved oxygen transfer without the detrimental
properties of dynamic systems.
Differentiation of human stem cells to insulin-producing pancreatic
cells has been performed on synthetic 3D scaffolds including Activin
A-grafted gelatin-poly(lactide-co-glycolide) nanoparticle scaffolds,
poly(lactide-co-glycolide) microporous scaffolds layered with Exendin-4,
polyvinyl alcohol scaffolds, and polyether sulfone nanofibrous scaffolds
(Kuo 2017, Kasputis 2018, Enderami 2018, Nassiri-Mansour 2018).
Synthetic scaffolds offer control over mechanical properties and
reproducibility; however, expensive manufacturing techniques and
challenges in manipulating surface chemistry to enhance biocompatibility
and cell adhesion reduce desirability (Dhandayuthapani 2011, Gervaso
2013).
Cellulose is a naturally abundant, FDA-approved polymer used frequently
in biomedical applications including wound dressings and bone tissue
engineering (de Oliveira Barud 2016). Cellulose is economical,
biocompatible and is readily modifiable to match the desired mechanical
and chemical properties (Courtenay 2018). To optimize cell-scaffold
functional interactions, we examined six surface modifications of the
cellulose scaffold in a multi-well plate assay before scale-up (Figure
1A and Figure 1C). We chose two chemical surface modification
approaches. One is the commonly used amine-modification to provide
positively charged functional groups for cell binding (Richbourg 2019,
Courtenay 2017). The other is a simple NaOH-treatment, which has been
shown to enhance surface roughness, hydrophilicity and cell attachment
(Chen 2007, Park 2007, Park 2014, Bosworth 2019). We further evaluated
whether gelatin-coating enhanced cell attachment cellulose matrices
since gelatin (denatured collagen) generally can support cell attachment
and growth (Davidenko, 2016). Commercial and in-house hiPSC-derived
pancreatic cells expressing NK6 Homeobox 1
(NKX6-1)+/pancreatic and duodenal homeobox 1
(PDX-1)+ were seeded onto scaffolds and evaluated.
Upon determining an optimal scaffold chemistry, we further demonstrated
use of a cost-effective, wicking-matrix bioreactor, fabricated by
Sepragen Corporation for scale-up biomanufacturing of hiPSC-derived
pancreatic cells. The bioreactor consists of a porous amine-modified
cellulose scaffold with 20 to 50-µm wide fibers (Figure 1B) in a sterile
chamber with independent air and media inlets and a waste removal outlet
(Figure 1D). This system provides a thin film of fresh media on the
cell-seeded scaffold and a continuous oxygen environment and liquid
waste removal. Our comprehensive analysis includes insulin production,
viability, morphology and functionality of cells on scaffolds in culture
dishes and bioreactors. Within the scaled-up wicking matrix-bioreactor,
we achieved 10-fold expansion and insulin production on amine-modified
cellulose scaffolds. Our findings indicate the potential of
amine-modified cellulose for future biomanufacturing processes for
cell-based diabetic therapies.