3D Electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes

Abstract

Astrocytes are a target for regenerative neurobiology because in brain injury their phenotype arbitrates brain integrity, neuronal death and subsequent repair and reconstruction. We explored the ability of 3D scaffolds to direct astrocytes into phenotypes with the potential to support neuronal survival. Poly-e-caprolactone scaffolds were electrospun with random and aligned fibre orientations on which murine astrocytes were sub-cultured and analysed at 4 and 12 DIV. Astrocytes survived, proliferated and migrated into scaffolds adopting 3D morphologies, mimicking in vivo stellated phenotypes. Cells on random poly-e-caprolactone scaffolds grew as circular colonies extending processes deep within sub-micron fibres, whereas astrocytes on aligned scaffolds exhibited rectangular colonies with processes following not only the direction of fibre alignment but also penetrating the scaffold. Cell viability was maintained over 12 DIV, and cytochemistry for F-/G-actin showed fewer stress fibres on bioscaffolds relative to 2D astrocytes. Reduced cytoskeletal stress was confirmed by the decreased expression of glial fibrillary acidic protein. PCR demonstrated up-regulation of genes (excitatory amino acid transporter 2, brain-derived neurotrophic factor and anti-oxidant) reflecting healthy biologies of mature astrocytes in our extended culture protocol. This study illustrates the therapeutic potential of bioengineering strategies using 3D electrospun scaffolds which direct astrocytes into phenotypes supporting brain repair.

Astrocytes Grown in Alvetex® Three Dimensional Scaffolds Retain a Non-reactive Phenotype

“…Such cultures take longer to become confluent and whilst they too express high levels of GLAST; GLT-1 levels decrease over time, with the functional burden of glutamate transport assumed by GLAST [48,50]. Indeed, research has shown that GLAST mediates the majority of L-glutamate uptake in postnatal cortical astrocyte cultures (28)(29)(30)(31)(32)(33)(34)(35), correlating with increased cell surface expression of GLAST [22].…”

“…These results suggest the intriguing notion that reduced astrocyte reactivity is associated with lower levels of GLT-1. In disease or animal disease models, under conditions where astrocytes up-regulate GFAP there is often down-regulation of GLT-1 eg in Motor Neuron Disease [22,30,51]. Loss of GLT-1 during astrocyte reactivity is often associated with a loss of protective functions in astrocytes.…”

“…Several groups have shown that culture of cells on 3D scaffolds can enhance astrocyte phenotype, indicative of a less 'reactive' expression profile [26,30]. For first time we tested a commercially available material, Alvetex as a 3D scaffold.…”

“…Scaffolds have been used to recreate aspects of the 3 dimensional arrangement of astrocytes in vitro, for example using collagen [26,27], hydrogels [12,28], tripalmitin [29] and poly-ε-caprolactone electrospun scaffolds [28,30,31]. In this study, we report the use of Alvetex, a commercially available porous scaffold formed of polystyrene that is supplied as cell culture inserts of 200µm thickness [32].…”

Prospects for polymer therapeutics in Parkinson's disease and other neurodegenerative disorders

“…12), nanogels may be designed for therapeutic delivery [151,152]. Macroscopic hydrogels/scaffolds may be designed for neuron guidance applications, cell delivery systems or growth factor/drug depots for sustained release [153][154][155][156]. Network formation may be induced via a variety of methods.…”