Cell Passaging (Subculturing)
Passaging is essential for maintaining
healthy, proliferating adherent cells. As cells reach confluence (covering the
flask surface), they require fresh space and nutrients. Passaging involves:
-
Detaching cells (typically with
trypsin),
-
Diluting them in fresh medium,
and
-
Replating into new culture
vessels.
Routine subculturing keeps cells in log-phase
growth, ensuring optimal health for experiments like transfection or
protein expression.
Reagents
Required
-
Complete growth medium (e.g.,
DMEM + 10% FBS)
-
1× PBS (phosphate-buffered
saline) without Ca²⁺/Mg²⁺
-
0.05% or 0.25% Trypsin-EDTA
-
70% Ethanol (for sterilization)
Equipment
-
T25 flasks (sterile)
-
Biosafety cabinet (Class II)
-
CO₂ incubator (37°C, 5% CO₂)
-
Inverted microscope
Steps:
1. Preparation
-
Warm all reagents (medium, PBS,
trypsin) to 37°C in a water bath or incubator.
-
Label new T25 flask with cell
line name, passage number, and date.
2. Examine
Cells
-
Observe cells under microscope:
-
Check confluency (>80% is
typical for passage).
-
Look for contamination or
abnormal morphology.
3. Wash with
PBS
-
Aspirate old medium completely.
-
Add 2–3 mL of 1× PBS to wash
and remove serum (which inhibits trypsin).
-
Gently swirl and aspirate.
4.
Trypsinization
-
Add 0.5–1 mL of trypsin-EDTA
(enough to cover the cell layer).
-
Gently rock the flask to spread
evenly.
-
Incubate at 37°C for 2–5
minutes.
-
Monitor under microscope until
cells round up and detach.
5. Neutralize
Trypsin
-
Add 2–4 mL of complete growth
medium to stop trypsin activity.
-
Gently pipette up and down to
disperse cells.
6. Split and
Seed
-
Transfer 1/4 to 1/3 of the cell
suspension to a new T25 flask.
-
Add fresh 4–5 mL of complete
medium to the new flask.
-
Discard the old flask
-
Return nee flask to 37°C
incubator with 5% CO₂.
7. Post-Passage
-
Observe new flask after ~24
hours for attachment and morphology.
-
Change medium after 2–3 days if
needed.
Notes
-
Work inside a biosafety cabinet
using aseptic technique.
-
Discard all waste in biohazard
containers.
-
Keep trypsin exposure time
short to avoid damaging cells.