Abstract
Methods are described for the generation and analysis of 3D co-culture models in which astrocyte and neuronal behavior can be studied. Cells may be obtained from a variety of sources, then cultured within collagen hydrogels to explore cellular responses and interactions in response to substances under test or under conditions that mimic physiological or pathological environments. Cell populations are labelled then either mixed within gels or arranged as separate adjacent populations, with further options including directing the self-alignment of cells to form anisotropic 3D cultures. Immunofluorescence staining and confocal microscopy can be used to capture image data from 3D structures and detailed protocols are provided for obtaining reliable results. Finally, 3D image analysis of confocal microscopy data is discussed, providing guidance on how astrocyte and neuronal features can be quantified.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang DD, Bordey A (2008) The astrocyte odyssey. Prog Neurobiol 86(4):342–367
Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5(2):146–156
Lee J, Cuddihy MJ, Kotov NA (2008) Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev 14(1):61–86
East E, Phillips JB (2008) Tissue engineered cell culture models for nervous system research. In: Greco GN (ed) Tissue engineering research trends. Nova Science, New York, pp 141–160
LaPlaca MC et al (2007) CNS injury biomechanics and experimental models. Prog Brain Res 161:13–26
Kofron CM, Fong VJ, Hoffman-Kim D (2009) Neurite outgrowth at the interface of 2D and 3D growth environments. J Neural Eng 6(1):016002
Li GN et al (2007) Genomic and morphological changes of neuroblastoma cells in response to three-dimensional matrices. Tissue Eng 13(5):1035–1047
East E, Golding JP, Phillips JB (2009) A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis. J Tissue Eng Regen Med 3(8):634–646
Puschmann TB et al (2013) Bioactive 3D cell culture system minimizes cellular stress and maintains the in vivo-like morphological complexity of astroglial cells. Glia 61(3):432–440
Bailey JL et al (2011) In vitro CNS tissue analogues formed by self-organisation of reaggregated post-natal brain tissue. J Neurochem 117(6):1020–1032
Dubois-Dauphin ML et al (2010) The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells. Biomaterials 31(27):7032–7042
Honegger P et al (2011) Preparation, maintenance, and use of serum-free aggregating brain cell cultures. Methods Mol Biol 758:81–97
Gil V, del Rio JA (2012) Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue. Nat Protoc 7(2):268–280
Ge D et al (2013) Culture and differentiation of rat neural stem/progenitor cells in a three-dimensional collagen scaffold. Appl Biochem Biotechnol 170(2):406–419
East E et al (2010) Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit. Tissue Eng Part A 16(10):3173–3184
Cullen DK, Simon CM, LaPlaca MC (2007) Strain rate-dependent induction of reactive astrogliosis and cell death in three-dimensional neuronal-astrocytic co-cultures. Brain Res 1158: 103–115
East E, Golding JP, Phillips JB (2012) Engineering an integrated cellular interface in three-dimensional hydrogel cultures permits monitoring of reciprocal astrocyte and neuronal responses. Tissue Eng Part C Methods 18(7):526–536
Cullen DK, Vernekar VN, LaPlaca MC (2011) Trauma-induced plasmalemma disruptions in three-dimensional neural cultures are dependent on strain modality and rate. J Neurotrauma 28(11):2219–2233
Phillips JB, Brown R (2011) Micro-structured materials and mechanical cues in 3D collagen gels. Methods Mol Biol 695:183–196
East E, Johns N, Georgiou M, Golding JP, Loughlin AJ, Kingham P, Phillips JB (2013) A 3D in vitro model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury. Regen Med 8(6):739–746
Gromnicova R, Davies HA, Sreekanthreddy P, Romero IA, Lund T, Roitt IM, Phillips JB, Male DK (2013) Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro. PLoS One 8(12):e81043
Grinnell F, Petroll WM (2010) Cell motility and mechanics in three-dimensional collagen matrices. Annu Rev Cell Dev Biol 26: 335–361
Sundararaghavan HG et al (2009) Neurite growth in 3D collagen gels with gradients of mechanical properties. Biotechnol Bioeng 102(2):632–643
Georgiou M et al (2013) Engineered neural tissue for peripheral nerve repair. Biomaterials 34(30):7335–7343
Brown RA et al (2005) Ultrarapid engineering of biomimetic materials and tissues: fabrication of nano- and microstructures by plastic compression. Adv Funct Mater 15: 1762–1770
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Phillips, J.B. (2014). Monitoring Neuron and Astrocyte Interactions with a 3D Cell Culture System. In: Murray, A. (eds) Axon Growth and Regeneration. Methods in Molecular Biology, vol 1162. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0777-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0777-9_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0776-2
Online ISBN: 978-1-4939-0777-9
eBook Packages: Springer Protocols