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Cytogenetic Analysis:
pseudodiploid, pseudodiploid [48963]
Age:
8 days
Comments:
Clonal permanent cell lines with astrocytic or microglial properties have been established from explant cultures of 8-day postnatal mouse cerebella after in vitro spontaneous transformation. [48963] [49701]
Sister flasks of lethally irradiated astrocytes, which are known to synthesize the macrophage-microglia growth factor, M-CSF, were used as a substrate for cloning the cells. [48963] [49701]
The cell lines were derived in a multistage process. Slowly proliferating foci with several morphologies appeared 4 months after initiation of the cultures and became progressively enriched by cells with a homogeneous appearance. [48963]
Some of these cloned cell lines bound anti-glial fibrillary acidic protein (GFAP) antibodies and therefore appeared to be astrocytic. No other glial neuronal or microglial markers have been detected in these clones. [48963]
According to their morphology, 3 separate types of these GFAP-positive clones could be distinguished. Type I and II cells had small somata. [48963]
Type I had several short processes, while type II had two processes, one of which was very thin and long (greater than 200 microns). Type III cells had large flat somata and no processes. [48963]
The astrocyte type II cloned cell line named C8-S is available as ATCC CRL-2535 and the astrocyte type III cloned cell line named C8-D30 is available as ATCC CRL-2534.
One clone with microglial properties named C8-B4 is available as ATCC CRL-2540.
The C8-D1A cell line has the morphology of fibrous astrocytes.
These astrocytic clones might be the in vitro counterparts of fibrous (type I), or velamentous (type III) astrocytes and of Golgi epithelial cells (type II). [48963]
Propagation:
ATCC complete growth medium: The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%. Temperature: 37.0°C Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Subculturing:
Protocol:
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended Medium Renewal: Every 2 to 3 days
