Setting Up of Primary Cultures from Fetal Dog Brain: An Alternative Method in Line with the 3Rs Principles

In the last years, alternatives to animal testing were applied, aimed at reduce
the laboratory use of animals, in agreement with the 3Rs principles (Reduction,
Refinement and Replacement). In the approach to the study of neurological
disorders, several in vitro models and alternative animal species like mammals
with a more complex nervous system have been proposed as translational
model for human. Lately, canines have been considered an excellent model
for study on neurodegenerative disorders since they may develop central
nervous system disease close to those observed in humans. In the present
report, we describes a method to obtaining primary cultures from dog fetuses
brain collected accidentally and cryopreserved until the use. The brain tissues
were dissociated through a mild enzymatic papain system and cultured, and the
development, morphology and the amount of neuronal cells were assessed. We
appreciated the capability of cryopreserved brain tissues to provide a vital and
proliferating neuronal primary culture. Moreover, we noticed which explants from
early gestation days fetuses (25 days) yielded the higher number of neurons
in comparison with those obtained from older fetuses (45days). Our data may
suggest a good alternative experimental approach to obtain a primary neuronal
culture, without necessarily having to use experimental animals.