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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.  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. RefAlliot F, et al. A spontaneously immortalized mouse microglial cell line expressing CD4. Brain Res. Dev. Brain Res. 95: 140-143, 1996. PubMed: 8873987 RefAlliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

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. 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. RefAlliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

According to their morphology, 3 separate types of these GFAP-positive clones could be distinguished. Type I and II cells had small somata. RefAlliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977  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. RefAlliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

The astrocyte type I cloned cell line named C8-D1A is available as ATCC CRL-2541 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-S cell line has the morphology of Golgi Bergman radial cells. 

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). RefAlliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-053mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:4 to 1:6
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Alliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

Alliot F, et al. A spontaneously immortalized mouse microglial cell line expressing CD4. Brain Res. Dev. Brain Res. 95: 140-143, 1996. PubMed: 8873987

Alliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977