engineering human renal epithelial cells for transplantation in regenerative medicine

Abstract Cellular transplantation may treat several human diseases by replacing damaged cells and/or providing a local source of trophic factors promoting regeneration. We utilized human renal epithelial cells (hRECs) isolated from cadaveric donors as a cell model. For efficacious implementation of hRECs for treatment of kidney diseases, we evaluated a novel encapsulation strategy for immunoisolation of hRECs and lentiviral transduction of the Green Fluorescent Protein (GFP) as model gene for genetic engineering of hRECs to secrete desired trophic factors. In specific, we determined whether encapsulation through conformal coating and/or GFP transduction of hRECs allowed preservation of cell viability and of their trophic factor secretion. To that end, we optimized cultures of hRECs and showed that aggregation in three-dimensional spheroids significantly preserved cell viability, proliferation, and trophic factor secretion. We also showed that both wild type and GFP-engineered hRECs could be efficiently encapsulated within conformal hydrogel coatings through our fluid dynamic platform and that this resulted in further improvement of cell viability and trophic factors secretion. Our findings may lay the groundwork for future therapeutics based on transplantation of genetically engineered human primary cells for treatment of diseases affecting kidneys and potentially other tissues

Freeze Drying of Polymer Nanoparticles and Liposomes Exploiting Different Saccharide-Based Approaches

Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide-co-glycolide) (PLGA) nanoparticles and liposomes was evaluated. In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting

Smoothed Particle Hydrodynamics multiphase modelling of an experimental microfluidic device for conformal coating of pancreatic islets

•SPH model of the flow in a device for pancreatic islets conformal coating.•Encapsulation process suitably reproduced while assuring phase conservation.•Simulation of jet fragmentation and surface tension effects on islet coating process.•Evaluation of influence of process parameters on the shape of encapsulated islets. The paper discusses a Smoothed Particle Hydrodynamics (SPH) model for the analysis of the multiphase flow occurring in an experimental microfluidic device for conformal coating of pancreatic islets with a biocompatible and permeable polymer. The proposed numerical model, based on a weakly-compressible SPH approach, accurately mimics the encapsulation process while assuring phase conservation, thus overcoming potential limitations of grid-based models. The proposed SPH model is a triphasic multi-phase model that allows one: (i) to reproduce the physics of islet conformal coating, including the effects of surface tension at the interface of the involved fluids and of the islet diameter; and (ii) to evaluate how modulation of process parameters influences the fluid dynamics within the microfluidic device and the resulting coating characteristics. This model can represent a valuable, time- and cost-effective tool for the definition of optimized encapsulation conditions through in silico screening of novel combinations of conformal coating parameters, including polymeric coating blends, size range of insulin-secreting cell clusters, utilized chemical reagents, device geometry and scale

Multiphase modelling of an experimental device for conformal coating of pancreatic islets

This paper illustrates the development of an SPH model for the analysis of a relatively complex multiphase flow occurring in an experimental microfluidic device for encapsulation of islets of Langerhans (i.e. pancreatic cells aggregates) through a bio-compatible and permeable polymeric coating. Cell encapsulation guarantees optimal immunoisolation while allowing glucose and insulin diffusion, thus enhancing the possibility of transplantation without immunosuppression in Type-1 diabetic patients to restore correct glucose metabolism. A WCSPH numerical modelling, based on the formulation by Adami et al. (2010), of the multiphase flow in the experimental device proved to be phase conservative with respect to finite volume / finite element models early setups, and is able to accurately mimic the encapsulation process. The SPH model allows evaluating: (i) the feasibility of reproducing the physical phenomenon leading to islet encapsulation, such as surface tension at the interface and physical viscosity of both fluids (as shown experimentally by Suryo & Basaran, 2006); (ii) the influence of modulating different process parameters on the fluid dynamics and coating characteristics; and (iii) to overcome the limits of the early numerical model while increasing the resolution. The model represents a valuable, time-and cost-effective tool for further refinement of the experimental process

Device design and materials optimization of conformal coating for islets of Langerhans

Encapsulation of islets of Langerhans may represent a way to transplant islets in the absence of immunosuppression. Traditional methods for encapsulation lead to diffusional limitations imposed by the size of the capsules (600-1,000 m in diameter), which results in core hypoxia and delayed insulin secretion in response to glucose. Moreover, the large volume of encapsulated cells does not allow implantation in sites that might be more favorable to islet cell engraftment. To address these issues, we have developed an encapsulation method that allows conformal coating of islets through microfluidics and minimizes capsule size and graft volume. In this method, capsule thickness, rather than capsule diameter, is constant and tightly defined by the microdevice geometry and the rheological properties of the immiscible fluids used for encapsulation within the microfluidic system. We have optimized the method both computationally and experimentally, and found that conformal coating allows for complete encapsulation of islets with a thin (a few tens of micrometers) continuous layer of hydrogel. Both in vitro and in vivo in syngeneic murine models of islet transplantation, the function of conformally coated islets was not compromised by encapsulation and was comparable to that of unencapsulated islets. We have further demonstrated that the structural support conferred by the coating materials protected islets from the loss of function experienced by uncoated islets during ex vivo culture

Conformal Coating of Stem Cell-Derived Islets for β Cell Replacement in Type 1 Diabetes

The scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Transplantation of encapsulated stem cell-derived islets (SC islets) might extend the applicability of islet transplantation to a larger cohort of patients. Transplantation of conformal-coated islets into a confined well-vascularized site allows long-term diabetes reversal in fully MHC-mismatched diabetic mice without immunosuppression. Here, we demonstrated that human SC islets reaggregated from cryopreserved cells display glucose-stimulated insulin secretion in vitro. Importantly, we showed that conformally coated SC islets displayed comparable in vitro function with unencapsulated SC islets, with conformal coating permitting physiological insulin secretion. Transplantation of SC islets into the gonadal fat pad of diabetic NOD-scid mice revealed that both unencapsulated and conformal-coated SC islets could reverse diabetes and maintain human-level euglycemia for more than 80 days. Overall, these results provide support for further evaluation of safety and efficacy of conformal-coated SC islets in larger species. •Reaggregated human SC islets display glucose-stimulated insulin secretion in vitro•Conformal-coated human SC islets displayed physiological insulin secretion•Conformal-coated human SC islets in mice reversed diabetes to human euglycemic levels Scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Islet encapsulation may eliminate the need for chronic immunosuppression. Conformal coating seeks to overcome limitations of traditional microencapsulation. Transplantation of conformal-coated stem cell-derived islets might extend the applicability of islet transplantation to a larger cohort of patients

Fibrin gels engineered with pro-angiogenic growth factors promote engraftment of pancreatic islets in extrahepatic sites in mice

With a view toward reduction of graft loss, we explored pancreatic islet transplantation within fibrin matrices rendered pro-angiogenic by incorporation of minimal doses of vascular endothelial growth factor-A165 and platelet-derived growth factor-BB presented complexed to a fibrin-bound integrin-binding fibronectin domain. Engineered matrices allowed for extended release of pro-angiogenic factors and for their synergistic signaling with extracellular matrix-binding domains in the post-transplant period. Aprotinin addition delayed matrix degradation and prolonged pro-angiogenic factor availability within the graft. Both subcutaneous (SC) and epididymal fat pad (EFP) sites were evaluated. We show that in the SC site, diabetes reversal in mice transplanted with 1,000 IEQ of syngeneic islets was not observed for islets transplanted alone, while engineered matrices resulted in a diabetes median reversal time (MDRT) of 38 days. In the EFP site, the MDRT with 250 IEQ of syngeneic islets within the engineered matrices was 24 days, versus 86 days for islets transplanted alone. Improved function of engineered grafts was associated with enhanced and earlier (by day 7) angiogenesis. Our findings show that by engineering the transplant site to promote prompt re-vascularization, engraftment and long-term function of islet grafts can be improved in relevant extrahepatic sites

Prospective multicentre clinical performance evaluation of second and third generation zotarolimus-eluting stents to treat patients with bifurcated coronary lesions

To assess the "real world" clinical outcome of patients with bifurcated lesions undergoing percutaneous coronary intervention with implantation of second and third generations of zotarolimus-eluting stent