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.

The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis.

The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue) system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti–epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01). Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing.

Artificial tissues constructed from therapeutic cells offer a promising approach for improving the treatment of severe peripheral nerve injuries. In this study the effectiveness of using CTX0E03, a conditionally immortalised human neural stem cell line, as a source of allogeneic cells for constructing living artificial nerve repair tissue was tested. CTX0E03 cells were differentiated then combined with collagen to form engineered neural tissue (EngNT-CTX), stable aligned sheets of cellular hydrogel. EngNT-CTX sheets were delivered within collagen tubes to repair a 12 mm sciatic nerve injury model in athymic nude rats. Autologous nerve grafts (autografts) and empty tubes were used for comparison. After 8 weeks functional repair was assessed using electrophysiology. Further, detailed histological and electron microscopic analysis of the repaired nerves was performed. Results indicated that EngNT-CTX supported growth of neurites and vasculature through the injury site and facilitated reinnervation of the target muscle. These findings indicate for the first time that a clinically validated allogeneic neural stem cell line can be used to construct EngNT. This provides a potential ‘off the shelf’ tissue engineering solution for the treatment of nerve injury, overcoming the limitations associated with nerve autografts or the reliance on autologous cells for populating repair constructs.

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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.

The effects of biofouling on a wave measurement buoy are examined using concurrent data collected with two Datawell Waveriders at Ocean Station P: one heavily biofouled at the end of a 26-month deployment, the other newly deployed and clean. The effects are limited to the high-frequency response of the buoy and are correctly diagnosed with the spectral “check factors” that compare horizontal and vertical displacements. A simple prediction for the progressive change in frequency response during biofouling reproduces the check factors over time. The bulk statistical parameters of significant wave height, peak period, average period, and peak direction are only slightly affected by the biofouling because the contaminated frequencies have very low energy throughout the comparison dataset.

Neurodegenerative conditions remain difficult to treat, with the continuing failure to see therapeutic research successfully advance to clinical trials. One of the obstacles that must be overcome is to develop enhanced models of disease. Tissue engineering techniques enable us to create organised artificial central nervous system tissue that has the potential to improve the drug development process. This study presents a replicable model of neurodegenerative pathology through the use of engineered neural tissue co-cultures that can incorporate cells from various sources and allow degeneration and protection of neurons to be observed easily and measured, following exposure to neurotoxic compounds – okadaic acid and 1-methyl-4-phenylpyridinium. Furthermore, the technology has been miniaturised through development of a mould with 6 mm length that recreates the advantageous features of engineered neural tissue co-cultures at a scale suitable for commercial research and development. Integration of human-derived induced pluripotent stem cells aids more accurate modelling of human diseases, creating new possibilities for engineered neural tissue co-cultures and their use in drug screening.