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.