Corneal transplantation constitutes one of the leading treatments for severe cases of loss of corneal function. Due to its limitations, a concerted effort has been made by tissue engineers to produce functional, synthetic corneal prostheses as an alternative recourse. However, successful translation of these therapies into the clinic has not yet been accomplished. 3D bioprinting is an emerging technology that can be harnessed for the fabrication of biological tissue for clinical applications. We applied this to the area of corneal tissue engineering in order to fabricate corneal structures that resembled the structure of the native human corneal stroma using an existing 3D digital human corneal model and a suitable support structure. These were 3D bioprinted from an in-house collagen-based bio-ink containing encapsulated corneal keratocytes. Keratocytes exhibited high cell viability both at day 1 post-printing (>90%) and at day 7 (83%). We established 3D bio-printing to be a feasible method by which artificial corneal structures can be engineered.

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C-23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperaturedependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 3 10 6 cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xenofree, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:339-349 SIGNIFICANCEDespite considerable advancement in the clinical application of cell-based therapies, major logistical challenges exist throughout the cell therapy supply chain associated with the storage and distribution of cells between the sites of manufacture and the clinic. A simple, low-cost system capable of preserving the viability and functionality of human adipose-derived stem cells (a cell with substantial clinical interest) at hypothermic temperatures (0°C-32°C) is presented. Such a system has considerable potential for extending the shelf life of cell therapy products at multiple stages throughout the cell therapy supply chain.

Human limbus-derived stromal/mesenchymal stem cells (hLMSC) can be one of the alternatives for the treatment of corneal scars. However, reliable methods of storing and transporting hLMSC remains a serious translational bottleneck. This study aimed to address these limitations by encapsulating hLMSC in alginate beads. Encapsulated hLMSC were kept in transit in a temperature-conditioned container at room temperature (RT) or stored at 4 °C for 3–5 days, which is the likely duration for transporting cells from bench-to-bedside. Non-encapsulated cells were used as controls. Post-storage, hLMSC were released from encapsulation, and viability-assessed cells were plated. After 48 and 96-hours in culture the survival, gene-expression and phenotypic characteristics of hLMSC were assessed. During transit, the container maintained an average temperature of 18.6 ± 1.8 °C, while the average ambient temperature was 31.4 ± 1.2 °C (p = 0.001). Encapsulated hLMSC under transit at RT were recovered with a higher viability (82.5 ± 0.9% and 76.9 ± 1.9%) after 3 (p = 0.0008) and 5-day storage (p = 0.0104) respectively as compared to 4 °C (65.2 ± 1.2% and 64.5 ± 0.8% respectively). Cells at RT also showed a trend towards greater survival-rates when cultured (74.3 ± 2.9% and 67.7 ± 9.8%) than cells stored at 4 °C (54.8 ± 9.04% and 52.4 ± 8.1%) after 3 and 5-days storage (p > 0.2). Non-encapsulated cells had negligible viability at RT and 4 °C. Encapsulated hLMSC (RT and 4 °C) maintained their characteristic phenotype (ABCG2, Pax6, CD90, p63-α, CD45, CD73, CD105, Vimentin and Collagen III). The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in transit at RT, therefore, potentially expanding the scope of cell-based therapy for corneal blindness.

Macroscopic capsules, with tunable properties based on hierarchical self-assembly on multiple lengthscales, are prepared from the co-operative self-assembly of polysaccharide and peptide amphiphiles. Different formulations can be used to create flexible membrane sacs in solution, soft capsules or rigid free-standing capsules. Samples are prepared by injecting a solution containing sodium alginate, with or without graphene oxide (GO), into a matrix consisting of a solution containing the peptide amphiphile PA C 16 -KKFF (K: lysine, F: phenylalanine), with or without CaCl 2 . Graphene oxide is added to the hybrid materials to modulate the mechanical properties of the capsules. Injection of sodium alginate solution into a pure PA matrix provides a flexible membrane sac in solution, while injection of NaAlg/GO solution into a PA matrix gives a soft capsule. Alternatively, a rigid free-standing capsule is made by injecting a NaAlg/GO solution into a PA + CaCl 2 matrix solution. A comprehensive insight into the hierarchical order within the capsules is provided through analysis of X-ray scattering data. A novel "Langmuir-Blodgett" mechanism is proposed to account for the formation of the sacs and capsules as the alginate solution is injected at the interface of the PA solution. The capsules show a unique antibacterial effect specific for the Gram positive bacterium Listeria monocytogenes, which is an important human pathogen. The hybrid nanostructured capsules thus have remarkable bioactivity and due to their tunable structural and functional properties are likely to have a diversity of other future applications.