The following protocol is based upon work carried out
with robust cell lines such as CHO and may require modification
for other cell lines. The CulturSil Cell Culture Bags
are supplied in a sterile, pre-wetted form and require
only rinsing with the culture medium of choice before
the addition of cell culture medium and inoculum.
The vessel should be handled in a similar fashion to
conventional cell culture vessels with all manipulations
being carried out in flow cabinets. Inoculum for the
growth of mammalian cells in CulturSilT vessels should
be prepared according to standard mammalian cell sub-culture
procedures.
We recommend that for initial studies, bags be filled
with 30-50mL of culture medium at an initial density
of 1 x105 cells/mL.
Note: Liquid handling is most easily
achieved for these vessels by employing a large capacity
(i.e. 50mL) syringe with a standard Luer fitting.
1. Add 15-25mL of complete culture media to the CulturSilT
bag via one of the Luer fittings on the inlet/outlet
ports. After the addition of growth medium the air space
in the vessel should be minimized by fitting a 50mL
syringe to the open Luer of the bag and hanging the
bag, allowing the air to rise to the open Luer end of
the bag, the syringe should then be drawn until the
culture medium starts to enter the filling tube. The
Luer cap should then be replaced and the vessel gently
agitated by rocking in the hand for 30 seconds. The
media should then be removed from the vessel by syringe
and discarded. This rinsing step should then be repeated.
2. Add 15-25mL of complete culture media to the CulturSilT
bag via one of the Luer fittings on the inlet/outlet
ports. The plug should then be replaced in the Luer
fitting and the CulturSilT vessel placed in a standard,
humidified cell culture incubator to equilibrate for
at least 1 hour.
3. Prepare the required inoculum (approx. 3-5 x 106
cells) in the log phase, at >90% viability, in 15-25mL
of pre-warmed, complete media and add this to the CulturSilT
bag.
4. After the addition of inoculum the CulturSilT bag
should be CulturSil Protocol rocked gently in the hand
to disperse the inoculum and the air space in the vessel
should again be minimized as described in section 1
above.
5. The CulturSilT growth vessel should then be returned
to a standard humidified CO2 cell culture incubator
with the vessel being gently 'flipped' every 15-30 minutes
during the first 4 hours to provide for effective seeding
of both faces of the vessel.
Note: 1.
Studies have indicated that the vessel should not require
additional maintenance for at least 72 hours. In order
to achieve maximum cell density we would recommend periodic
sampling of media and assay for glucose concentration,
with media removal and replacement as necessary (or
as required for the harvest of secreted product).
2. During the culture period the
cells can be observed through the viewing window on
the surface of the CulturSilT bag using a light microscope.
3. Cells can be harvested by use
of solutions such as PBS and/or Trypsin EDTA, The CulturSilT
bag is a robust system that can be agitated at harvest
to detach the cell population if required.
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