Biomedical Engineering Reference
In-Depth Information
3.5. Method V: Astroglia-Enriched Cultures
Enriched astroglia are prepared by a repeated subculturing of the mixed
glia cultures after the removal of the majority of microglia by shaking. After
fi ve consecutive passages in vitro, a near pure preparation of astroglia can be
obtained (
see
Note 8
).
1. After the removal of microglia by shaking, rinse the fl ask twice with 30 mL of
prewarmed (37°C) sterile PBS in a cell culture hood.
2. Add 10 mL of the trypsin-EDTA solution (37°C) to each fl ask and incubate for
5 min in a 37°C incubator. The cells should be detached. If needed, gently tap the
side of the fl ask to ensure a complete dislodging of the cells.
3. Transfer the cells to a 50-mL conical tube. Pellet the cells by centrifugation for
8 min at 200
g
at room temperature.
4. Reseed the cells at a ratio of one 150-cm
2
fl ask to two 75-cm
2
fl asks. Maintain
a total of 15 mL of microglia/astroglia maintenance medium for each 75-cm
2
fl ask. The cultures usually reach confl uence in 5-7 d.
5. Subculture by first rinsing the flasks with sterile PBS, detaching the cells
with trypsin-EDTA and then reseeding the cells to 75-cm fl asks at a ratio of
1 to 1.5.
6. When the cells grow to confl uence again (in 5-7 d), subculture at a ratio of 1 to
1.5 and repeat the process two more times.
7. After a total of fi ve consecutive subculturings, the enriched astroglia are seeded
ready for use.
8. The purity of the cultures can be determined by immunostaining with astroglia-
and mciroglia-specifi c markers. Usually a >98% pure astrocyte-enriched culture
can be obtained (
see
Note 8
).
4. Notes
1. We have been routinely using the E13/14 embryos of Fisher 344 rats supplied
by Charles River for preparing mixed neuron-glia cultures. Differences in the
assignment of exact embryonic age by different vendors and in the fetuses
among different strains of rats may call for pilot studies to fi nd the most suitable
age to use.
2. Use of anesthetic agents should be avoided, and rapid asphyxiation with CO
2
gas
appears to have a minimal impact on the embryonic brain cells.
3. Detailed illustrations are provided in the
ref.
11
to help locate the ventral
mesencephalic region for microdissection.
4. The number of passages of the tissue suspension through the various pipet and
pipet tips is empirical. The actual number of passages may depend on the balance
between achieving a single cell suspension and not damaging too many cells,
which will be obvious when estimating the density of cell suspension with trypan
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