A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas

A route towards immune protection

This work describes a route towards an immune protective device for islet of Langerhans transplantation. We developed a protocol to use MIN6 β cells aggregates as pseudo-islets to overcome the donor shortage issue (chapter 3). In this thesis we explored two different immune protective strategies; a multibore hollow fiber and flat microwell membranes. In chapter 4 we investigated the possibility to use a commercial multibore hollow fiber. In this chapter we showed that we were capable to physically separate islets using agarose macrospheres. Photoacoustic imaging in chapter 5 confirmed these findings. The flat membrane microwell device was developed based on work of Buitinga et al. (1) who developed a poly(ethylene glycol terephthalate)-poly(butylene terephthalate) (PEOT/PBT) microwell scaffold, made by micro thermoforming of dense thin films. We developed a microwell device that consisted of a thin PES/PVP membrane for islet encapsulation and a 0.45μm pore sized PES lid (chapter 6). These membranes were sealed prior to seeding by a custom made seal device. Encapsulated human islets were still responsive to glucose stimulation after 7 days of culture. As a lack of proper vascularization after transplantation and thereby hypoxia is a major contributor to islet death, we studied solutions to overcome this issue. In chapter 7 we showed that micropatterned membranes in combination with MSCs could be used to enhance vascularization in vivo. Finally, we combined the oxygen generating materials of Pedraza et al. (2) with the microwell membrane device of chapter 6 in chapter 8. We showed, similar to Pedraza et al. that the oxygen generating disk had a positive effect on islet viability and function. In this thesis we took the first steps towards an immune protective device, although successful in many aspects additional work is needed to further optimize the devices developed in this work. Therefore in chapter 9 an outlook is presented of different options in order to reach a functional immune protective device for islet encapsulation

A Protocol to Enhance INS1E and MIN6 Functionality—The Use of Theophylline

In vitro research in the field of type I diabetes is frequently limited by the availability of a functional model for islets of Langerhans. This method shows that by the addition of theophylline to the glucose buffers, mouse insulinoma MIN6 and rat insulinoma INS1E pseudo-islets can serve as a model for islets of Langerhans for in vitro research. The effect of theophylline is dose- and cell line-dependent, resulting in a minimal stimulation index of five followed by a rapid return to baseline insulin secretion by reducing glucose concentrations after a first high glucose stimulation. This protocol solves issues concerning in vitro research for type I diabetes as donors and the availability of primary islets of Langerhans are limited. To avoid the limitations of using human donor material, cell lines represent a valid alternative. Many different β cell lines have been reported, but the lack of reproducible responsiveness to glucose stimulation remains a challenge

A Protocol to Enhance INS1E and MIN6 FunctionalityThe Use of Theophylline

In vitro research in the field of type I diabetes is frequently limited by the availability of a functional model for islets of Langerhans. This method shows that by the addition of theophylline to the glucose buffers, mouse insulinoma MIN6 and rat insulinoma INS1E pseudo-islets can serve as a model for islets of Langerhans for in vitro research. The effect of theophylline is dose- and cell line-dependent, resulting in a minimal stimulation index of five followed by a rapid return to baseline insulin secretion by reducing glucose concentrations after a first high glucose stimulation. This protocol solves issues concerning in vitro research for type I diabetes as donors and the availability of primary islets of Langerhans are limited. To avoid the limitations of using human donor material, cell lines represent a valid alternative. Many different cell lines have been reported, but the lack of reproducible responsiveness to glucose stimulation remains a challenge

An important step towards a prevascularized islet macroencapsulation device-effect of micropatterned membranes on development of endothelial cell network

The development of immune protective islet encapsulation devices could allow for islet transplantation in the absence of immunosuppression. However, the immune protective membrane / barrier introduced there could also impose limitations in transport of oxygen and nutrients to the encapsulated cells resulting to limited islet viability. In the last years, it is well understood that achieving prevascularization of the device in vitro could facilitate its connection to the host vasculature after implantation, and therefore could provide sufficient blood supply and oxygenation to the encapsulated islets. However, the microvascular networks created in vitro need to mimic well the highly organized vasculature of the native tissue. In earlier study, we developed a functional macroencapsulation device consisting of two polyethersulfone/polyvinylpyrrolidone (PES/PVP) membranes, where a bottom microwell membrane provides good separation of encapsulated islets and the top flat membrane acts as a lid. In this work, we investigate the possibility of creating early microvascular networks on the lid of this device by combining novel membrane microfabrication with co-culture of human umbilical vein endothelial cell (HUVEC) and fibroblasts. We create thin porous microstructured PES/PVP membranes with solid and intermittent line-patterns and investigate the effect of cell alignment and cell interconnectivity as a first step towards the development of a stable prevascularized layer in vitro. Our results show that, in contrast to non-patterned membranes where HUVECs form unorganized HUVEC branch-like structures, for the micropatterned membranes, we can achieve cell alignment and the co-culture of HUVECs on a monolayer of fibroblasts attached on the membranes with intermittent line-pattern allows for the creation of HUVEC branch-like structures over the membrane surface. This important step towards creating early microvascular networks was achieved without the addition of hydrogels, often used in angiogenesis assays, as gels could block the pores of the membrane and limit the transport properties of the islet encapsulation device. [Figure not available: see fulltext.]

Opening the ‘‘White Box’’ in Tissue Engineering: Visualization of Cell Aggregates in Optically Scattering Scaffolds

The noninvasive and longitudinal imaging of cells or cell aggregates in large optically scattering scaffolds is still a largely unresolved problem in tissue engineering. In this work, we investigated the potential of near-infrared (NIR) photoacoustic (PA) tomography imaging to address this issue. We used clinically relevant sizes of highly light scattering polyethersulfone multibore hollow fiber scaffolds seeded with cells. Since cells have little optical absorption at NIR wavelengths, we studied labeling of cells with absorbers. Four NIR labels were examined for their suitability based on absorption characteristics, resistance to bleaching, and influence on cell viability. On the basis of these criteria, carbon nanoparticles proved most suitable in a variety of cells. For PA imaging, we used a research setup, based on computed tomography geometry. As proof of principle, using this imager we monitored the distribution and clustering of labeled rat insulinoma beta cell aggregates in the scaffolds. This was performed for the duration of 1 week in a nondestructive manner. The results were validated using fluorescence imaging, histology, and light microscopy imaging. Based on our findings, we conclude that PA tomography is a powerful tool for the nondestructive imaging of cells in optically scattering tissue-engineered scaffolds

Pancreatic islet macroencapsulation using microwell porous membranes

Allogeneic islet transplantation into the liver in combination with immune suppressive drug therapy is widely regarded as a potential cure for type 1 diabetes. However, the intrahepatic system is suboptimal as the concentration of drugs and nutrients there is higher compared to pancreas, which negatively affects islet function. Islet encapsulation within semipermeable membranes is a promising strategy that allows for the islet transplantation outside the suboptimal liver portal system and provides environment, where islets can perform their endocrine function. In this study, we develop a macroencapsulation device based on thin microwell membranes. The islets are seeded in separate microwells to avoid aggregation, whereas the membrane porosity is tailored to achieve sufficient transport of nutrients, glucose and insulin. The non-degradable, microwell membranes are composed of poly (ether sulfone)/polyvinylpyrrolidone and manufactured via phase separation micro molding. Our results show that the device prevents aggregation and preserves the islet's native morphology. Moreover, the encapsulated islets maintain their glucose responsiveness and function after 7 days of culture (stimulation index above 2 for high glucose stimulation), demonstrating the potential of this novel device for islet transplantation

An important step towards a prevascularized islet microencapsulation device: in vivo prevascularization by combination of mesenchymal stem cells on micropatterned membranes

Extrahepatic transplantation of islets of Langerhans could aid in better survival of islets after transplantation. When islets are transfused into the liver 60-70% of them are lost immediately after transplantation. An important factor for a successful extrahepatic transplantation is a well-vascularized tissue surrounding the implant. There are many strategies known for enhancing vessel formation such as adding cells with endothelial potential, the combination with angiogenic factors and / or applying surface topography at the exposed surface of the device. Previously we developed porous, micropatterned membranes which can be applied as a lid for an islet encapsulation device and we showed that the surface topography induces human umbilical vein endothelial cell (HUVEC) alignment and interconnection. This was achieved without the addition of hydrogels, often used in angiogenesis assays. In this work, we went one step further towards clinical implementation of the device by combining this micropatterned lid with Mesenchymal Stem Cells (MSCs) to facilitate prevascularization in vivo. As for HUVECs, the micropatterned membranes induced MSC alignment and organization in vitro, an important contributor to vessel formation, whereas in vivo (subcutaneous rat model) they contributed to improved implant prevascularization. In fact, the combination of MSCs seeded on the micropatterned membrane induced the highest vessel formation score in 80% of the sections. [Figure not available: see fulltext.]