Optimising contraction and alignment of cellular collagen hydrogels to achieve reliable and consistent engineered anisotropic tissue.

Engineered anisotropic tissue constructs containing aligned cell and extracellular matrix structures are useful as in vitro models and for regenerative medicine. They are of particular interest for nervous system modelling and regeneration, where tracts of aligned neurons and glia are required. The self-alignment of cells and matrix due to tension within tethered collagen gels is a useful tool for generating anisotropic tissues, but requires an optimal balance between cell density, matrix concentration and time to be achieved for each specific cell type. The aim of this study was to develop an assay system based on contraction of free-floating cellular gels in 96-well plates that could be used to investigate cell-matrix interactions and to establish optimal parameters for subsequent self-alignment of cells in tethered gels. Using C6 glioma cells, the relationship between contraction and alignment was established, with 60-80% contraction in the 96-well plate assay corresponding to alignment throughout tethered gels made using the same parameters. The assay system was used to investigate the effect of C6 cell density, collagen concentration and time. It was also used to show that blocking α1 integrin reduced the contraction and self-alignment of these cells, whereas blocking α2 integrin had little effect. The approach was validated by using primary astrocytes in the assay system under culture conditions that modified their ability to contract collagen gels. This detailed investigation describes a robust assay for optimising cellular self-alignment and provides a useful reference framework for future development of self-aligned artificial tissue.

Challenges in the development of a reference standard and potency assay for the clinical production of RAFT tissue equivalents for the cornea.

AIM: To develop a reference standard and potency assay for Real Architecture For 3D Tissues (RAFT) tissue equivalents intended for use in limbal epithelial stem cell (LESC) therapy for the cornea. METHODS: RAFT, a cell-seeded plastic compressed collagen construct with LESCs cultured on the surface, was manufactured with the goal of achieving GMP compliance. RAFTs were tested for reproducibility of manufacture (reference standard) and subsequently wounded and monitored for re-epithelialization (potency assay). RESULTS: RAFT tissue equivalents produced with cells from different biological donors were capable of supporting multilayered epithelium in culture. The potency assay demonstrated re-epithelialization following wounding, indicating the potential efficacy of RAFT constructs. CONCLUSION: We have presented our attempts at creating a reference standard and potency assay for the clinical manufacture of RAFT for the treatment of LESC deficiency. However, it remains challenging for adult stem cell therapies (including LESC therapy) to fully meet regulatory requirements when dealing with a limited source of autologous cells with inherent biological variation between donors.

Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture.

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.

Plastic compressed collagen constructs for ocular cell culture and transplantation: a new and improved technique of confined fluid loss.

PURPOSE: Cultured limbal epithelial cell transplantation is a commonly used clinical treatment for ocular surface repair. We have previously shown that plastic compressed (PC) type I collagen constructs are a suitable substrate for human limbal epithelial cell (HLEC) culture for transplantation. For this process to achieve compliance with Good Manufacturing Practice, and therefore be suitable for therapeutic cell therapy manufacture, the original method required substantial modification. METHODS: The compression method was changed from unconfined (highly variable reproducibility) to confined compression (CC) (highly reproducible manufacture) and we assessed whether this altered the physical characteristics of the substrate. We have measured transparency, assessed scanning electron microscope images of the surface and performed live/dead cell viability assays of cells within the constructs. HLECs were then cultured on the surface of both types of construct and the resulting cell phenotype characterized. RESULTS: We have determined that the change in process does not alter the physical characteristics of the substrate. Furthermore, there is no change to the substrate's ability to support HLEC culture and maintenance of a mixed population of stem and differentiated cells. Additionally, cells were able to form a confluent sheet and multilayer to produce an intact epithelium. CONCLUSIONS: This modification allows scaling up of the process in a well-plate format, which is essential for creation of multiple corneal epithelial models for in vitro testing. This improvement to the original plastic compression method also allows the process to be employed in custom-made cassettes, the design of which takes into consideration the manufacturing and regulatory requirements for delivery of a cell therapy.

Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.