Effect of Osmotic Pressure on Neurogenesis in Cultures of Chick Embryo Spinal Cords

The osmotic pressure of the medium in stoppered, roller tube cultures increased by an average of 17 ± 6 mOsM per kg of water during 3 days of incubation at 37°C irrespective of the initial osmolality (280 to 340 mOsM) of the medium. The increase was apparently due to evaporation of water from the medium into the gas phase of the roller tube. This observation led us to study the effect of osmotic pressure on neuronal differentiation in cultures of chick embryo spinal cords. Spinal cords were excised from stage 16 to 19 (2.5 to 3 days of incubation) or stage 36 (10 days) chick embryos and cultured as fragments on collagen-coated cover slips in roller tubes at 37°C for 21 days. The medium was adjusted to 283 ± 3, 300 ± 3, 323 ± 3, or 342 ± 3 mOsM per kg with saturated choline chloride solution or distilled water. The results indicate that the nature of the neuronal differentiation in vitro was not altered by the osmolality of the medium. The proportion of cultures containing neurons was influenced by osmolality. In the 300 ± 3 mOsM medium, 75% of all the stage 36 cultures initiated contained neurons, and 52% of all the stage 16 to 19 cultures initiated contained neurons. In the other media the proportion of neuron-containing cultures was lower. Two conclusions were drawn. Neurogenesis in cultures of embryonic chick spinal cord fragments is sensitive to an increase in the initial osmotic pressure of the medium as small as 20 mOsM above the optimal 300 mOsM. As a result of the 17 mOsM increase which always occurred in the culture medium between feedings, the optimum osmolality for neuronal development is in fact a range, from 300 to 317 mOsM.