Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures

Abstract

Neuronal cell cultures offer a crucial tool to mechanistically analyse regeneration in the nervous system. Despite the increasing importance of zebrafish (Danio rerio) as an in vivo model in neurobiological and biomedical research, in vitro approaches to the nervous system are lagging far behind and no method is currently available for establishing enriched neuronal cell cultures. Here we show that magnetic-activated cell sorting (MACS) can be used for the large-scale generation of neuronal-restricted progenitor (NRP) cultures from embryonic zebrafish. Our findings provide a simple and semi-automated method that is likely to boost the use of neuronal cell cultures as a tool for the mechanistic dissection of key processes in neuronal regeneration and development.

Figure 1. Without further treatment zebrafish embryonic cell cultures contain only few neuronal cells.

(a) Embryonic zebrafish at 30 hours post fertilization (hpf) were dissociated into a single cell suspension by a semi-automated dissociation process (see Methods). The primary cells were cultured on laminin (50.000 cells/cm²) in a defined serum-free MACS Neuro Medium. Representative light microscope images of the in vitro cultures show cells with different morphologies, which started to form cell aggregates that became interconnected by neurites after a few days in vitro. (b) Immunofluorescent staining of 48 h and (c) 7 d primary cultures identified few neuronal (neurofilament, NF) and glial (glial fibrillary acid protein, GFAP) cell types located within these aggregates. Nuclei were stained with DAPI (blue). All scale bars, 50 μm.

Figure 2. Homogeneous and viable cell cultures of MACS-isolated PSA-NCAM positive cells from embryonic zebrafish.

(a) Schematic representation of magnetic-activated cells sorting (MACS) via magnetic anti-PSA-NCAM microbeads. The single cell suspension of dissociated embryonic zebrafish was incubated with anti-PSA-NCAM microbeads and loaded onto a MACS Column. The magnetically labelled cells were retained in the magnetic field of a MACS Separator, whereas the unlabelled cells flowed through the column (negative fraction). The labelled PSA-NCAM positive cells were then flushed out and collected (positive fraction). (b) Coronal and (c) transversal paraffin sections of embryonic zebrafish (30 hpf) showing PSA-NCAM expression (green) in the tail (grey arrow) as well as in several regions of the brain (white arrows) and the eyes (red arrow). Note the autofluorescence of the yolk. Nuclei were stained with DAPI (blue). tel, telencephalon; di, diencephalon. (d) Primary cultures of adherent cells from the positive fraction (50.000 cells/cm²) showed unipolar (white arrow), bipolar (black arrow) and multipolar (red arrow) morphologies and formed interconnected cell aggregates after 24 h. (e) Viability was assessed by using the Live/Dead assay showing live cells stained with calcein (green) and dead cells with EthD-1 (red). All scale bars, 25 μm. (f) Cells from each fraction showed high viability at 3 h in vitro (n = 3, mean ± SD). (g) Metabolic activity of cells from each fraction was detected by a resazurin-based assay at 3 h, 24 h and 48 h in vitro (n = 5, mean ± SD, P < 0.001). See Fig. 3 for further characterization of PSA-NCAM positive cells and their viability.

Figure 3. Enrichment of immature neuronal-restricted progenitors using PSA-NCAM mediated magnetic cell separation.

Cells from the original, the negative and the positive fraction were cultured on laminin (50.000 cells/cm²). After 3 h in vitro, the expression of (a) polysialilated-neural cell adhesion molecules (PSA-NCAM), (b) neurofilaments (NF), (c) glial fibrillary acid proteins (GFAP) and (d) A2B5 was analysed immunocytochemically in each cell fraction. Nuclei were stained with DAPI (blue). Scale bar, 25 μm. (e) The positive fraction showed a significantly higher content of PSA-NCAM- and NF-positive cells (n = 5, mean ± SD, P < 0.001). GFAP was detected in glial cells in all fractions (n = 5, mean ± SD, P = 0.41). A2B5 on the surface of glial-restricted progenitors (GRPs) is only detected in the positive fraction (n = 5, mean ± SD, P = 0.001).

Figure 4. Spontaneously formed aggregates predominantly contain neuronal cells.

(a) NRP cultures from embryonic zebrafish were cultured for 2 and 7 d, respectively. Representative images show the formation of cell aggregates that became interconnected by neurites. Both the size of the aggregates and the density of neurites extending from the aggregates increased until day 7. Immunocytochemical analyses demonstrated the expression of (b) polysialilated-neural cell adhesion molecule (PSA-NCAM) and (c) neurofilaments (NF) in most of the cells within the aggregates at 2 and 7 d in vitro. (d) Only a few cells showed the expression of glial fibrillary acid proteins (GFAP). Nuclei were stained with DAPI (blue). All scale bars, 50 μm.

Figure 5. Formation of neuronal networks induced by retinoic acid (RA).

(a) NRPs were cultured in a differentiation medium containing retinoic acid (1 μM in DMSO). After 7 d in vitro many cells left the neuronal aggregates, formed adherent monolayers and extended small processes. (b) The immunocytochemical staining of cells forming these monolayers revealed the expression of neurofilaments (NF). Nuclei were stained with DAPI (blue). (c) After two weeks in vitro further neuronal maturation and the formation of neuronal networks were observed. (d) The NF positive neurons seemed to be connected to adjacent neurons indicating the formation of synapses (white arrows). Nuclei were stained with DAPI (blue). All scale bars, 50 μm.