The development and characterisation of complex ovine neuron cultures from fresh and frozen foetal neurons

Cultures of ovine cerebral and cerebellar neurons from mid-term sheep foetal brains, 9-15 weeks old, have been established for the first time. These foetal brains are relatively mature, being at similar stages of development as peri and post-natal rodent brains. Cultures were routinely maintained for 3-4 weeks, and longer. Nearly all the cells from the younger foetuses adhered as neurons. The proportion of glial cells increased with age, as did the risk of cultures being overtaken by glial cells. Cultured neurons were bipolar, tripolar and multipolar, similar to the morphologies of neurons in vivo. Older foetuses also yield more complex neurons, notably giant cells. Other properties of the cultured neurons also mimic in vivo observations, including neurite beading, complexity in neurotransmitter class (GABAergic and glutamatergic) and calcium binding protein (calbindin and calretinin) content. Single cell divisions of neurons were observed in younger cultures by time-lapse photography and the occurrence of telophase nuclei. The advantage of the high yield of genetically identical cells obtained from a single sheep foetus, 150 million, was extended by cryopreservation of neurons after snap freezing, and later culture. These cultures showed the same characteristics as cultures from the freshly plated cells.