| Clumping/aggregation
of cells |
Switch
baffled Erlenmeyer flasks and change media completely.
Monitor cell growth/clumping for 3–4 days. If not
improved, abandon cells and defrost new cells. |
| Formation
of a ring of cells inside the baffled
Erlenmeyer flask |
Transfer
cells/media to new baffled Erlenmeyer flask. However,
this ring has not shown to be detrimental in production
quality, so you may choose to continue culturing those
cells if you desire. |
| Slow
cell growth |
Monitor
cell growth every day and change media if necessary. If
the cells do not recover, abandon cells and defrost new
cells. |
| Cells
grow too fast |
Dilute
cells often with c-BalanCD HEK293 media. Always maintain
the cell density between 3 × 105 live
cells/mL and 3 × 106 live cells/mL. |
| Contamination
of cells in baffled Erlenmeyer flask (indicated by
excessive turbidity of cells and/or unnaturally fast
cell growth) |
Abandon
cells and defrost new ones. Make sure to also
appropriately discard any old media suspected of
contamination and use completely new baffled Erlenmeyer
flasks. You may consider adding 1×
antibiotic/antimycotic to the flask but be advised that
this may inhibit cell growth; proper aseptic technique
should suffice in preventing contamination. |
| Empty
capsid contamination |
The
protocol is optimized for maximizing the titer of full
capsids. If the presence of empty capsids is deemed
unacceptable for your specific application, a
conventional plasmid ratio (helper plasmid: Rep/Cap
plasmid: AAV shuttle vector = 2:1:1) can be used to
mitigate this concern. |
