Stabilisation of hydrogels containing neural cells to form CNS tissue models for monitoring neuronal and glial cell behaviour <i>in vitro</i>

Recreating the 3D environment of the CNS using hydrogel matrices allows neurons and glial cells in vitro to behave similarly to their counterparts in vivo, providing a relevant tool for neurobiological studies. The overall aim is to develop engineered neural tissue models to resemble functional CNS tissue, with anisotropic tracts of neurons and glia arranged within a hydrogel, at a scale suitable for drug screening. Stability of cellular hydrogels is important for the generation of engineered tissue models and our approach employs RAFT™ technology (TAP Biosystems) that removes some interstitial fluid, increasing matrix density to stabilise the architecture of the hydrogels. In order to develop a robust CNS tissue model using this method, it was necessary to assess the sensitivity of neural cells to the stabilisation process. Initial experiments used C6 glioma cells seeded within collagen gels which were then subjected to RAFT for 15 min. Cell viability was assessed by propidium iodide staining in control constructs with no treatment and in RAFT-treated constructs at time points of 24 and 72 hours. Results demonstrated that the RAFT process did not reduce cell viability when compared to controls. After 24 hours, 0.58 ± 0.14 % cell death was seen in the treated constructs and 1.38 ± 0.26 % was viewed in controls. After 72 hours, a similar pattern was observed with cell death of 0.33 ± 0.06 % in treated and 1.80 ± 0.27 % in controls. Following initial tests with C6 glioma cells, subsequent experiments used the same approach to assess the viability of a range of primary rat CNS neural cells and other relevant cell lines including astrocytes, oligodendrocytes, microglia and neurons. CNS co-culture systems will incorporate various combinations of cells that can be tailored to suit specific neuroscience research requirements and assessing the viability of cells is a critical step in the development of culture models. Stabilisation of the cellular hydrogels through increasing the cell and matrix density in this way will be used to establish versatile engineered neural tissues. A consistent model system will provide experimental platforms to monitor neural cell morphology, proliferation, differentiation and sensitivity, as well as neuron-glial interactions to generate rapid, reliable and reproducible results