An optimized method for obtaining adult rat spinal cord motor neurons to be used for tissue culture.
There is a paucity of detailed methods describing how to harvest and process motor neurons obtained from the adult rat spinal cord.



Removal of intra-cardiac perfusion step. The spinal cord is extruded intact from the rat in under 60s post-decapitation then processed without differentiation of ventral and dorsal regions. The temperature during processing was maintained at room temperature (22°C) except during the Papain processing step where the temperature was increased to 30°C.



Cell debris interfered with the counting of cells at the time of plating. Also, cell types could not be identified since they appear rounded structures with no projections. Cell viability counts reduced to 91% and 63% from day 7 to day 14 and days 7-28 respectively. Red blood cell counts in stepped density gradient layers 2 and 3 were low.



No requirement for intra-cardiac perfusion. No requirement to cool to 4°C post harvesting, No requirement for specialized substrates. Reduces processing time by at least 2h and reduces the potential for processing errors through a reduction in complexity. Procedures are also explained suitable for those new to the culture of primary adult motor neurons.



Cell viability counts indicate that removal of the perfusion step has a minimal effect on the viability of the cultured nerve cells, which may be due to the reduction in the spinal cord harvesting time and the inclusion of Hibernate based media during extrusion and processing.
Discipline of Anatomy and Pathology, University of Adelaide, Adelaide, Australia. Electronic address: malcolm.brinn@adelaide.edu.au.
Malcolm
M
Brinn
Discipline of Pharmacology, University of Adelaide, Adelaide, Australia.
Katie
K
O'Neill
Discipline of Pharmacology, University of Adelaide, Adelaide, Australia.
Ian
I
Musgrave
Department of Spinal Surgery Royal Adelaide Hospital, Australia; Discipline of Orthopaedics and Trauma, School of Medicine, University of Adelaide, Adelaide, Australia.
Brian J C
BJC
Freeman
Discipline of Anatomy and Pathology, University of Adelaide, Adelaide, Australia.
Maciej
M
Henneberg
Discipline of Anatomy and Pathology, University of Adelaide, Adelaide, Australia.
Jaliya
J
Kumaratilake
D000818:Animals; D018929:Cell Culture Techniques; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D005260:Female; D005998:Glycine / Q000494:pharmacology; D007382:Intermediate Filaments / Q000378:metabolism; D008297:Male; D009046:Motor Neurons / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology*; D013997:Time Factors; D046509:Tissue Culture Techniques
D002795:Choline O-Acetyltransferase; D005998:Glycine
Adult rat motor neurons; Neuroscience protocol; Primary cell culture; Serum free medium; Spinal cord; Tissue culture