A CULTURE SUBSTRATUM APPROPRIATE FOR BRAIN-CELLS IS A CHONDROITIN SULFATE GLYCOSAMINOGLYCAN IN SERUM

When cells dissociated from the neonatal rat brains are plated on a po ly-lysine-coated surface in a serum-free medium, they display a strang e morphology: a dark and extended cell body. Preincubation of the surf ace with fetal bovine serum was found to inhibit the appearance of thi s strange contraction of the basal cell sheets in a dose-dependent man ner. This finding indicated the presence of a factor(s) in the serum, which might be an appropriate substratum for prolonged survival of bra in neurons. In the current study, this factor was highly purified thro ugh DEAE ion-exchange chromatography followed by gel filtration. The f actor was eluted from a Superose column at fractions corresponding to a molecular weight greater than 1000 kDa. By SDS-PAGE analysis, these fractions were found to contain a major band (greater than or equal to 1000 kDa) positive for alcian blue and few minor bands faintly staina ble with Coomassie blue. The activity of the purified sample, inducing the morphological change in cells, was diminished by incubation with chondroitinase ABC. Neither heparitinase II, hyaluronidase, nor trypsi n modified the activity. An authentic chondroitin sulfate (type B) mim icked the serum action on the morphology of brain cells in early stage s of culture. Taking these findings together, it is suggested that the factor in serum beneficial for the attachment of brain cells is compo sed of a chondroitin sulfate with a Mr greater than 1000 kDa. Cortical cells dissociated from the neonatal rat brain attached well to the pu rified factor-coated surface and displayed a healthy morphology: an op tically-reflective cell body with thick neurites for at least 3 days i n the absence of serum.