An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA.
BACKGROUND
Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-μm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells.


METHODS
Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required.


RESULTS
Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells.


METHODS
The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration.


CONCLUSIONS
The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications.
Department of Pathology and Experimental Therapeutics, Medical School (Bellvitge Campus), University of Barcelona-UB, Barcelona, Spain; Institute for Bioengineering of Catalonia-IBEC, Barcelona, Spain.
Zaida
Z
Álvarez
Department of Pathology and Experimental Therapeutics, Medical School (Bellvitge Campus), University of Barcelona-UB, Barcelona, Spain.
Elena
E
Sena
Department of Pathology and Experimental Therapeutics, Medical School (Bellvitge Campus), University of Barcelona-UB, Barcelona, Spain; Speech and Hearing Research Laboratory, School of Medicine, University of Split, Croatia.
Marta
M
Mattotti
Institute for Bioengineering of Catalonia-IBEC, Barcelona, Spain; Department Material Science and Metallurgical Engineering, Technical University of Catalonia-UPC, Barcelona, Spain; Centro de Investigaciòn Médica en Red, Biomecanica, Biomateriales y Nanotecnologìa-CiberBBN, Barcelona, Spain.
Elisabeth
E
Engel
Department of Pathology and Experimental Therapeutics, Medical School (Bellvitge Campus), University of Barcelona-UB, Barcelona, Spain. Electronic address: salcantara@ub.edu.
Soledad
S
Alcántara
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology*; D002540:Cerebral Cortex / Q000166:cytology*; D018920:Coculture Techniques; D005786:Gene Expression Regulation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects* / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D019904:Polymethyl Methacrylate / Q000378:metabolism / Q000494:pharmacology*; D014746:Vimentin / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; D014746:Vimentin; D019904:Polymethyl Methacrylate
Astrocytes; Bergmann glia; Micro-patterning; Poly-methyl methacrylate (PMMA); Progenitors; Radial glia; Surface topography