Titanium Phosphate Glass Microspheres as Microcarriers for In Vitro Bone Cell Tissue Engineering - Biointerfaces: Where Material Meets Biology - page 124

Serological pipettes - 10 and 25 mL (Fisher Scientific, UK)
SEM specimen stubs (Agar Scientific, UK)
Tissue culture flasks, 75 cm 2 (TPP; Helena Biosciences, UK)
Transwell s inserts (Corning Costar s , USA)

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5.4.3 Seeding of MG63 Cells on Microspheres
1. The medium used for cell culture is standard low-glucose DMEM
containing 10% FBS and 1% penicillin-streptomycin. To prepare this,
pre-warm all the components in a water bath at 37 1C. In a sterile
fume hood, remove 50 mL of medium from a 500 mL bottle of DMEM
and add 50 mL of FBS and 5 mL of penicillin-streptomycin.

NOTE: An increasing number of laboratories are using antibiotic-free
culture systems in order to avoid antibiotic resistance of contaminant
microbes and also because of possible effects on cell function. If cell cul-
ture is carried out without antibiotics, extra care must be taken to ensure
complete sterility of the cell culture environment and equipment used.

2. The MG63 cells are grown in a 75 cm 2 cell culture flask containing the
cell culture medium described in Step 1. The growth process is carried
out in a 37 1C/5% CO 2 incubator till the cells reach up to 90% con-
fluence on the flask surface.
3. At 1 day prior to cell seeding, weigh out the required quantity of
microspheres.

.

NOTE: The weight of microspheres used per well will depend on the
amount of microspheres required to cover the cell seeding surface with
a thin layer of microspheres, which in turn depends on the surface area
of the Transwell s insert mesh. This should be determined prior to the
experiment.

4. Add the microspheres to a glass vial (one vial per well) and seal the
open end of the vial with aluminium foil and autoclave tape. Place the
vials in a drying oven and sterilise the contents under dry heat at a
temperature of 180 1C for 3 h before leaving to cool overnight.

TIP: If a drying oven is not available, sterilisation can be carried out by
exposing the microspheres to UV light for 1 h. However, autoclaving of the
microspheres must be avoided since the microspheres are water-soluble
and will degrade under the effects of moisture and high temperature.

5. On the day of the experiment, remove the medium from the flask in a
sterile fume hood, wash the cells with 5 mL PBS and trypsinise them
using 3 mL trypsin-EDTA to detach the cells from the flask. Ensure