Designing a retrievable and scalable cell encapsulation device for potential treatment of type 1 diabetes.

Cell encapsulation has been shown to hold promise for effective, long-term treatment of type 1 diabetes (T1D). However, challenges remain for its clinical applications. For example, there is an unmet need for an encapsulation system that is capable of delivering sufficient cell mass while still allowing convenient retrieval or replacement. Here, we report a simple cell encapsulation design that is readily scalable and conveniently retrievable. The key to this design was to engineer a highly wettable, Ca2+-releasing nanoporous polymer thread that promoted uniform in situ cross-linking and strong adhesion of a thin layer of alginate hydrogel around the thread. The device provided immunoprotection of rat islets in immunocompetent C57BL/6 mice in a short-term (1-mo) study, similar to neat alginate fibers. However, the mechanical property of the device, critical for handling and retrieval, was much more robust than the neat alginate fibers due to the reinforcement of the central thread. It also had facile mass transfer due to the short diffusion distance. We demonstrated the therapeutic potential of the device through the correction of chemically induced diabetes in C57BL/6 mice using rat islets for 3 mo as well as in immunodeficient SCID-Beige mice using human islets for 4 mo. We further showed, as a proof of concept, the scalability and retrievability in dogs. After 1 mo of implantation in dogs, the device could be rapidly retrieved through a minimally invasive laparoscopic procedure. This encapsulation device may contribute to a cellular therapy for T1D because of its retrievability and scale-up potential.

Dynamic self-organization of microwell-aggregated cellular mixtures.

Cells with different cohesive properties self-assemble in a spatiotemporal and context-dependent manner. Previous studies on cell self-organization mainly focused on the spontaneous structural development within a short period of time during which the cell numbers remained constant. However the effect of cell proliferation over time on the self-organization of cells is largely unexplored. Here, we studied the spatiotemporal dynamics of self-organization of a co-culture of MDA-MB-231 and MCF10A cells seeded in a well defined space (i.e. non-adherent microfabricated wells). When cell-growth was chemically inhibited, high cohesive MCF10A cells formed a core surrounded by low cohesive MDA-MB-231 cells on the periphery, consistent with the differential adhesion hypothesis (DAH). Interestingly, this aggregate morphology was completely inverted when the cells were free to grow. At an initial seeding ratio of 1 : 1 (MDA-MB-231 : MCF10A), the fast growing MCF10A cells segregated in the periphery while the slow growing MDA-MB-231 cells stayed in the core. Another morphology developed at an inequal seeding ratio (4 : 1), that is, the cell mixtures developed a side-by-side aggregate morphology. We conclude that the cell self-organization depends not only on the cell cohesive properties but also on the cell seeding ratio and proliferation. Furthermore, by taking advantage of the cell self-organization, we purified human embryonic stem cells-derived pancreatic progenitors (hESCs-PPs) from co-cultured feeder cells without using any additional tools or labels.

Microgel encapsulated nanoparticles for glucose-responsive insulin delivery.

An insulin delivery system that self-regulates blood glucose levels has the potential to limit hypoglycemic events and improve glycemic control. Glucose-responsive insulin delivery systems have been developed by coupling glucose oxidase with a stimuli-responsive biomaterial. However, the challenge of achieving desirable release kinetics (i.e., insulin release within minutes after glucose elevation and duration of release on the order of weeks) still remains. Here, we develop a glucose-responsive delivery system using encapsulated glucose-responsive, acetalated-dextran nanoparticles in porous alginate microgels. The nanoparticles respond rapidly to changes in glucose concentrations while the microgels provide them with protection and stability, allowing for extended glucose-responsive insulin release. This system reduces blood sugar in a diabetic mouse model at a rate similar to naked insulin and responds to a glucose challenge 3 days after administration similarly to a healthy animal. With 2 doses of microgels containing 60 IU/kg insulin each, we are able to achieve extended glycemic control in diabetic mice for 22 days.

Amyloid fibrillation of hen egg-white lysozyme is inhibited by TCEP.

This work examines the inhibitory effect of TCEP on the in vitro fibrillation of hen lysozyme at pH 2. We demonstrate that the inhibition of hen lysozyme fibrillation by TCEP follows a dose-dependent manner. Our data show that the addition of TCEP prevents alpha-to-beta transition and promoted unfolding of lysozyme. Moreover, our findings suggested that the TCEP-induced attenuated fibrillation is associated with disulfide disruption and structural unfolding of HEWL.

An Atmosphere-Breathing Refillable Biphasic Device for Cell Replacement Therapy.

Cell replacement therapy is emerging as a promising treatment platform for many endocrine disorders and hormone deficiency diseases. The survival of cells within delivery devices is, however, often limited due to low oxygen levels in common transplantation sites. Additionally, replacing implanted devices at the end of the graft lifetime is often unfeasible and, where possible, generally requires invasive surgical procedures. Here, the design and testing of a modular transcutaneous biphasic (BP) cell delivery device that provides enhanced and unlimited oxygen supply by direct contact with the atmosphere is presented. Critically, the cell delivery unit is demountable from the fixed components of the device, allowing for surgery-free refilling of the therapeutic cells. Mass transfer studies show significantly improved performance of the BP device in comparison to subcutaneous controls. The device is also tested for islet encapsulation in an immunocompetent diabetes rodent model. Robust cell survival and diabetes correction is observed following a rat-to-mouse xenograft. Lastly, nonsurgical cell refilling is demonstrated in dogs. These studies show the feasibility of this novel device for cell replacement therapies.

Physical confinement induces malignant transformation in mammary epithelial cells.

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

Scalable Production and Cryostorage of Organoids Using Core-Shell Decoupled Hydrogel Capsules.

Organoids, organ-mimicking multicellular structures derived from pluripotent stem cells or organ progenitors, have recently emerged as an important system for both studies of stem cell biology and development of potential therapeutics; however, a large-scale culture of organoids and cryopreservation for whole organoids, a prerequisite for their industrial and clinical applications, has remained a challenge. Current organoid culture systems relying on embedding the stem or progenitor cells in bulk extracellular matrix (ECM) hydrogels (e.g., Matrigel™) have limited surface area for mass transfer and are not suitable for large-scale productions. Here, we demonstrate a capsule-based, scalable organoid production and cryopreservation platform. The capsules have a core-shell structure where the core consists of Matrigel™ that supports the growth of organoids, and the alginate shell form robust spherical capsules, enabling suspension culture in stirred bioreactors. Compared with conventional, bulk ECM hydrogels, the capsules, which could be produced continuously by a two-fluidic electrostatic co-spraying method, provided better mass transfer through both diffusion and convection. The core-shell structure of the capsules also leads to better cell recovery after cryopreservation of organoids probably through prevention of intracellular ice formation.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies.

Designing compartmentalized hydrogel microparticles for cell encapsulation and scalable 3D cell culture.

We describe here designs of compartmentalized hydrogel microparticles with a tunable extracellular matrix (ECM) support for cell encapsulation and scalable 3D cell culture. The microparticles, rapidly formed by a one-step, multi-fluidic electrostatic spraying technique (>10 000 min-1), have a uniform spherical shape, a nearly monodisperse size distribution and controlled compartmentalization. They not only have a high surface area for mass transfer but also offer defined space and essential ECM support for various scalable and efficient 3D cell culture, co-culture and microtissue production applications.

Developing robust, hydrogel-based, nanofiber-enabled encapsulation devices (NEEDs) for cell therapies.

Cell encapsulation holds enormous potential to treat a number of hormone deficient diseases and endocrine disorders. We report a simple and universal approach to fabricate robust, hydrogel-based, nanofiber-enabled encapsulation devices (NEEDs) with macroscopic dimensions. In this design, we take advantage of the well-known capillary action that holds wetting liquid in porous media. By impregnating the highly porous electrospun nanofiber membranes of pre-made tubular or planar devices with hydrogel precursor solutions and subsequent crosslinking, we obtained various nanofiber-enabled hydrogel devices. This approach is broadly applicable and does not alter the water content or the intrinsic chemistry of the hydrogels. The devices retained the properties of both the hydrogel (e.g. the biocompatibility) and the nanofibers (e.g. the mechanical robustness). The facile mass transfer was confirmed by encapsulation and culture of different types of cells. Additional compartmentalization of the devices enabled paracrine cell co-cultures in single implantable devices. Lastly, we provided a proof-of-concept study on potential therapeutic applications of the devices by encapsulating and delivering rat pancreatic islets into chemically-induced diabetic mice. The diabetes was corrected for the duration of the experiment (8 weeks) before the implants were retrieved. The retrieved devices showed minimal fibrosis and as expected, live and functional islets were observed within the devices. This study suggests that the design concept of NEEDs may potentially help to overcome some of the challenges in the cell encapsulation field and therefore contribute to the development of cell therapies in future.

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

INTRODUCTION: The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. METHODS: MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. RESULTS: Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. CONCLUSIONS: Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.

Nanofibrous microposts and microwells of controlled shapes and their hybridization with hydrogels for cell encapsulation.

A simple, robust, and cost-effective method is developed to fabricate nanofibrous micropatterns particularly microposts and microwells of controlled shapes. The key to this method is the use of an easily micropatternable and intrinsically conductive metal alloy as a template to collect electrospun fibers. The micropatterned alloy allows conformal fiber deposition with high fidelity on its topographical features and in situ formation of diverse, free-standing micropatterned nanofibrous membranes. Interestingly, these membranes can serve as structural frames to form robust hydrogel micropatterns that may otherwise be fragile on their own. These hybrid micropatterns represent a new platform for cell encapsulation where the nanofiber frames enhance the mechanical integrity of hydrogel and the micropatterns provide additional surface area for mass transfer and cell loading.

Role of mass transfer in overall substrate removal rate in a sequential aerobic sludge blanket reactor treating a non-inhibitory substrate.

A laboratory study was undertaken to explore the role of mass transfer in overall substrate removal rate and the subsequent kinetic behavior in a glucose-fed sequential aerobic sludge blanket (SASB) reactor. At the organic loading rates (OLRs) of 2-8 kg chemical oxygen demand (COD)/m3-d, the SASB reactor removed over 98% of COD from wastewater. With an increase in OLR, the average granule diameter (dp=1.1-1.9 mm) and the specific oxygen utilization rate increased; whereas biomass density of granules and solids retention time decreased (13-32 d). The intrinsic and apparent kinetic parameters were evaluated using break-up and intact granules, respectively. The calculated COD removal efficiencies using the kinetic model (incorporating intrinsic kinetics) and empirical model (incorporating apparent kinetics) agreed well with the experimental results, implying that both models can properly describe the overall substrate removal rate in the SASB reactor. By applying the validated kinetic model, the calculated mass transfer parameter values and the simulated substrate concentration profiles in the granule showed that the overall substrate removal rate is intra-granular diffusion controlled. By varying different dp within a range of 0.1-3.5 mm, the simulated COD removal efficiencies disclosed that the optimal granular size could be no greater than 2.5 mm.