No abstract

Various neural tissue engineering approaches that are under development for applications ranging from guidance conduits to cell-based therapies rely on the ability to encapsulate cells in three-dimensional (3D) scaffolds. Schwann cells play a key role in peripheral nerve regeneration by forming oriented paths for regrowing axons. We have engineered collagen and hyaluronic acid interpenetrating polymer network (IPN) hydrogels with and without laminin as a 3D culture system for Schwann cells in an attempt to devise novel neural regeneration therapies. Encapsulation of Schwann cells in 3D hydrogel constructs did not affect cell viability and cells were viable for 2 weeks in all hydrogel samples. Moreover, in hydrogels with high cell density, cells underwent spreading and proliferation, and the cell numbers increased by day 14 as assessed qualitatively using a Live/dead assay and scanning electron microscopy (SEM), and quantitatively using a CellTiter 96 AQueous non-radioactive cell proliferation assay. In some cases, the cells aligned parallel to each other and formed structures reminiscent of Bands of Büngner. Schwann cells in cell-hydrogel constructs with high cell density were not only viable but also actively secreting nerve growth factor and brain-derived neurotrophic factor. Of particular importance was the observation that addition of laminin in these hydrogels increased the overall production of nerve growth factor and brain-derived neurotrophic factor from the cells. Immunostaining revealed that S100 expression and cell spreading were differentially affected by cell density. Interestingly, in the co-culture of dissociated neurons with Schwann cells, neurons were able to extend neurites and some neurites were observed to follow Schwann cells. Therefore, we conclude that Schwann cells encapsulated in the 3D extracellular matrix-mimicking hydrogel may hold promise in nerve regeneration therapies and may form the basis for understanding the underlying mechanisms of Schwann cell interactions with neurons and various extracellular matrix components.

Polymer-based, injectable systems that can simultaneously deliver multiple bioactive agents in a controlled manner could significantly enhance the efficacy of next generation therapeutics. For immunotherapies to be effective, both prophylactically or therapeutically, it is not only critical to drive the antigen (Ag) specific immune response strongly towards either T helper type 1 (Th1) or Th2 phenotype, but also to promote recruitment of a high number of antigen-presenting cells (APCs) at the site of immunization. We have recently reported a microparticle-based system capable of simultaneously delivering siRNA and DNA to APCs. Here we present an in situ crosslinkable, injectable formulation containing dendritic cell (DC)-chemoattractants and dual-mode DNA-siRNA loaded microparticles to attract immature DCs and simultaneously deliver, to the migrated cells, immunomodulatory siRNA and plasmid DNA antigens. These low crosslink density hydrogels were designed to degrade within 2–7 days in-vitro and released chemokines in a sustained manner. Chemokine carrying gels attracted 4–6 folds more DCs over a sustained period in vitro, compared to an equivalent bolus dose. Interestingly, migrated DCs were able to infiltrate the hydrogels and efficiently phagocytose the siRNA/DNA carrying microparticles. Hydrogel embedded microparticles co-delivering Interleukin-10 siRNA and plasmid DNA antigens exhibited efficient Interleukin-10 gene knockdown in migrated primary DCs in-vitro.

The ability to efficiently isolate undifferentiated human induced pluripotent stem cells (UD-hiPSCs) as colonies from contaminating non-pluripotent cells is a crucial step in the stem cell field to maintain hiPSC survival, purity, and karyotype stability. Here we demonstrate significant differences in ‘adhesive signature’ among UD-hiPSCs, parental cells, partially reprogrammed cells, and differentiated progeny. The distinct adhesive signature of hiPSCs was exploited to rapidly (~10 min) and efficiently isolate fully reprogrammed bona fide hiPSCs as intact colonies from heterogeneous reprogramming cultures and differentiated progeny using microfluidics. hiPSCs were isolated in a label-free fashion and enriched to > 95–99% purity and survival without adversely affecting the transcriptional profile, differentiation potential or karyotype of the pluripotent cells. This rapid and label-free strategy is applicable to isolate UD-hPSCs (hiPSCs, hESCs) from heterogeneous cultures during reprogramming and routine cultures and can be expanded to purify stem cells of specific lineages, such as neurons and cardiomyocytes.

The field of tissue engineering and regenerative medicine will tremendously benefit from the development of three dimensional scaffolds with defined micro- and macro-architecture that replicate the geometry and chemical composition of native tissues. The current report describes a freeform fabrication technique that permits the development of nerve regeneration scaffolds with precisely engineered architecture that mimics that of native nerve, using the native extracellular matrix component hyaluronic acid (HA). To demonstrate the flexibility of the fabrication system, scaffolds exhibiting different geometries with varying pore shapes, sizes and controlled degradability were fabricated in a layer-by-layer fashion. To promote cell adhesion, scaffolds were covalently functionalized with laminin. This approach offers tremendous spatio-temporal flexibility to create architecturally complex structures such as scaffolds with branched tubes to mimic branched nerves at a plexus. We further demonstrate the ability to create bidirectional gradients within the microfabricated nerve conduits. We believe that combining the biological properties of HA with precise three dimensional micro-architecture could offer a useful platform for the development of a wide range of bioartificial organs.