7. It is important to use fresh cultures of bacteria at the late log
phase of growth for invasion. Prior analysis of strain-specific
growth profiles may be required to determine appropriate bacterial counts for the desired multiplicity of infection (MOI)
during invasion. When bacteria are harvested, they should be
kept at room temperature throughout harvesting and during
complexation with any exogenous reagents. Placing bacterial
cultures on ice or at 4
C will significantly inhibit the vectors
ability to bind to the cell membrane during invasion.
8. Observe the bacterial pellet carefully to ensure that it does not
dissociate while the supernatant is removed. The 50 mL tube
can be inverted on tissue briefly for leftover BHI media to be
absorbed.
9. Vials of cryopreserved cells should be kept on ice when they are
transferred between liquid nitrogen storage and a water bath.
Thawed cells that have been transferred into complete culture
media should be pelleted and resuspended in fresh complete
culture media to remove traces of cyropreservants like glycerol
or DMSO before incubation overnight. Omitting this step may
significantly reduce the viability of the thawed cells.
10. Trypsinization rate will vary slightly depending on the cell line
that is used. The flask can be tapped lightly to detach cells. A
5 mL serological pipette can be used to gently wash off cells
using the trypsin contained in the flask. Cells should be
observed under the microscope to ensure that proper detachment from the cell culture flask is achieved.
11. Exposing cells to trypsin for too long may negatively impact
the receptors present on the cell membrane. Trypsin should be
inhibited using complete media containing serum immediately
cells are sufficiently detached.
12. Ensure that the cell pellet is not disturbed while discarding the
spent media supernatant. If the pellet is disturbed, additional
centrifugation for 5 min at 225 Â g can be performed.
13. The density of cells seeded for invasions will vary according to
cell type. Cells should ideally be seeded at approximately 60%
confluency. Slow growing cells can be seeded at higher densities provided crowding does not occur. When cells are incubated following the invasion process, non-exponential growth
of the cells resulting from high confluency will limit the maximum achievable gene delivery efficiency. Cells should not be
seeded for invasion a day after thawing from cryopreservation
and should be allowed sufficient time to multiply until approximately 80% confluency.
14. The duration that cells are exposed to PBS or invasion media
which do not contain serum should be minimized. Cells will
begin to round or shrink the longer they are left in PBS or
24
Andrew N. Osahor and Kumaran Narayanan
phase of growth for invasion. Prior analysis of strain-specific
growth profiles may be required to determine appropriate bacterial counts for the desired multiplicity of infection (MOI)
during invasion. When bacteria are harvested, they should be
kept at room temperature throughout harvesting and during
complexation with any exogenous reagents. Placing bacterial
cultures on ice or at 4
C will significantly inhibit the vectors
ability to bind to the cell membrane during invasion.
8. Observe the bacterial pellet carefully to ensure that it does not
dissociate while the supernatant is removed. The 50 mL tube
can be inverted on tissue briefly for leftover BHI media to be
absorbed.
9. Vials of cryopreserved cells should be kept on ice when they are
transferred between liquid nitrogen storage and a water bath.
Thawed cells that have been transferred into complete culture
media should be pelleted and resuspended in fresh complete
culture media to remove traces of cyropreservants like glycerol
or DMSO before incubation overnight. Omitting this step may
significantly reduce the viability of the thawed cells.
10. Trypsinization rate will vary slightly depending on the cell line
that is used. The flask can be tapped lightly to detach cells. A
5 mL serological pipette can be used to gently wash off cells
using the trypsin contained in the flask. Cells should be
observed under the microscope to ensure that proper detachment from the cell culture flask is achieved.
11. Exposing cells to trypsin for too long may negatively impact
the receptors present on the cell membrane. Trypsin should be
inhibited using complete media containing serum immediately
cells are sufficiently detached.
12. Ensure that the cell pellet is not disturbed while discarding the
spent media supernatant. If the pellet is disturbed, additional
centrifugation for 5 min at 225 Â g can be performed.
13. The density of cells seeded for invasions will vary according to
cell type. Cells should ideally be seeded at approximately 60%
confluency. Slow growing cells can be seeded at higher densities provided crowding does not occur. When cells are incubated following the invasion process, non-exponential growth
of the cells resulting from high confluency will limit the maximum achievable gene delivery efficiency. Cells should not be
seeded for invasion a day after thawing from cryopreservation
and should be allowed sufficient time to multiply until approximately 80% confluency.
14. The duration that cells are exposed to PBS or invasion media
which do not contain serum should be minimized. Cells will
begin to round or shrink the longer they are left in PBS or
24
Andrew N. Osahor and Kumaran Narayanan