Tissue cultures of the nervous tissue have been widely used among neurocytologists, using mainly a roller-tube method. In the ordinary roller-tube method tissue fragments in approximately 1mm square are used as explants, which fact makes it hard to observe neurons, coexisting with numerous other cells in tissue fragments, in the early stage of tissue culture under the phase contrast microscope. In order to overcome this disadvantage, various enzymes have been used to dissociate neurons from other cellular components in tissues prior to cultivation.
Since the procedure of the dissociation method utilizing enzymes, however, is rather complicated and time-consuming, the cells are apt to die, and thus far the cultivation of neurons from central nervous tissues has been failed by this method. OKAMOTO introduced the mechanical dissociation method in lieu of the enzymatic dissociation method particulary for this purpose.
This is to report the results obtained from the cultures of cerebellar cortices of various animals cultivated by OKAMOTO's dissociation method.
Materials and methods.
Cerebellar cortices of various new-born animals (31 puppies, 23 kittens, 2 rabbits, 2 guinea-pigs, 1 rat, 1 goat, 1 ox and 8 chickens) as well as embryos (16 human and 30 chicken) were used in the present investigation.
Cerebellar tissues, obtained from young animals and embryos by decapitation, were cut in fragments as small as possible in GEY's balanced salt solution on the slide glass according to OKAMOTO's dissociation method.
One drop of the suspension obtained by this method was mounted in plasma clot on 12×50mm cover glass No. 1. The clot consisted of equal parts of heparinized rooster plasma and embryonic extract from 7-day chiken embryos. After clot had become firm, each cover glass was inserted respectively in one roller-tube. One hour after explantation, 2ml of medium, consisting of 50% human ascitic fluid, 45% GEY's balanced salt solution and 5% embryonic extract, was added to each tube. The glucose content of this medium was increased to somewhat 300mg-% and 1000 units of Penicillin were added per 1ml medium. The cultures were incubated at 37°C. The medium was changed once a week. Cultures were observed from time to time with low power microscopy, and photographs were taken with still and time-lapse cinematographic technique, using phase contrast microscope.
Several staining methods such as supravital staining with methylene blue, JACOBSON's method, BODIAN's copper protargol method and NISSL stain were applied to the cultures.
Results.
1. Although various animals were used in the present observation, in terms of cellular growth in vitro the satisfactory results were obtained only in puppy and kitten as it was in the case of an ordinary roller-tube method.
2. The suspension obtained by OKAMOTO's method contained mechanically dissociated cells as well as small fragmented explants measuring 0.1-0.5mm in diameter. Cellular growth was observed both from the dissociated cells and coexisting tissue fragments. Although the dissociated nonneuronal elements appeared to start growing later and have less reproducibility than the ones growing from coexisting small fragmented explants, by and large they did not seem to have any significant morphological differences among them.
3. Pomerat classified astrocytes grown in vitro into 2 types; fibrous and protoplasmic, according to the usual classical classification.
However, since the astrocyte changes its morphological features depending on the age of culture and its localization in the outgrowth, astrocytes cultured in vitro should rather be classified according to the morphological characteristics observed under phase contrast microscope in the living state as follows:
Type I. Fusiform astrocyte.
Type II. Astrophorous Astrocyte.
A. Oligodendro-astrocyte, B. Standard astrocyte, C. Multidendroastrocyte.
Type III. Membranous astrocyte.
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