Isolation of a mouse motoneuron enriched fraction from spinal cord on a density barrier

The growth and differentiation of spinal motoneurons are dependent on various genetic and epigenetic factors, which influence both functional and morphological characteristics [1].

 Involvement of a number of trophic molecules is known to be an important part of these processes [1]. Consequently there is a considerable amount of research carried out on cultured motoneurons, which can be derived from spinal cord. The general procedure can be summarized as follows: Spinal cords are dissected from embryos and following a combined enzymic and mechanical disruption of the tissue, debris is removed by pelleting the cells through a cushion of bovine serum albumin. 

A motoneuron-rich fraction is then isolated from the cell pellet prior to cell culture. Bataille et al [1] layered the crude cell pellet from rat embryo spinal cord over two layers of Nycodenz of density,  = 1.047 and 1.065 g/ml and analyzed the cells that banded at the top of each Nycodenz layer. 

The majority of the cells banded at the lower interface and the diameter of these cells (approx 4.3 m) was much lower than the minor population of cells (motoneurons) around the upper interface (approx 6.7 m). 

These large low-density cells contained very high levels of acetylcholine, which was virtually absent from the smaller denser cells at the lower interface. It seems clear from this earlier method that the motoneurons, as a result of their larger size are less dense than the other cells. Martinou [2] reported that the denser layer contained cholinergic cells other than motoneurons.

 In some cases the density of the two layers was changed, e.g. 1.035 and 1.092 g/ml [3] and for mouse embryo spinal cord 1.042 and 1.065 g/ml [4]. The gradient format was later simplified to a single low-density Nycodenz cushion usually of density 1.055 g/ml [5,6], although densities as low as 1.035 g/ml have also been used [7].