Cell culture basic techniques

1. Cell culture laboratory equipment
Necessary supplies:
• cell culture vessels (flasks, Petri dishes,
multi-well plates)
• pipettes and motorized pipette
controller
• syringe and needles
• plastic centrifuge tubes
• cryovials, cryo-boxes,
• filters for syringes and bottles,
• waste containers,
• deionized water,
• laboratory glass dishes
• disinfectants (isopropyl or 70 %
ethanol, Na-hypochlorite)

2. Work in aseptic conditions
Figure 8. Layout of work area. An example of proper arrangement of working area allowing laminar airflow. During
manipulation, in the middle of working surface, cell culture vessels are placed while pipets are set on the left and
motorized pipette controller is on the right.

3. Basic protocols for cell subculturing
Subculture of adherent cells
• Time between reseeding (passage)
varies with cell line and growth rate
Remove and discard the spent cell culture media
from the culture vessel
Detach cells by proteolytic enzymes (trypsine)
and/or mechanical (scraper) from the culture vessel
Trypsin neutralization
Gathering of a detached cells by fresh media
Continuation of a cell cultivation in a new vessel
until they reach confluence from 80 to 100%

4. Basic protocols for cell subculturing
Subculture of suspension cell lines
• Cells with intention to form agregates or
clumps must be disrupt to the single cell
suspension for further cultivation or for
the purpose of cell counting
View cells and assess condition
Add fresh media to obtaine
appropriate seeding density
Continuation of a cell cultivation
in a new vessel
Repeat every 2 to 3 days

5. Basic protocols for cell subculturing
Suspension cells Adherent cells
Easier to culture, can be diluted
without removing all old media
More steps needed for culture, require
complete media change
Do not require mechanical or chemical
dissociation
Require trypsinization to subculture, is
stressful for the cells
Not easy to determine confluency, require
daily cell count
Can be easily inspected under a microscope
to determine confluency
Growth limited by cell concentration Growth limited by surface area
Table 4. Comparison between subculturing of suspension and adherent cell cultures

6. Basic protocols for cell subculturing
Cell cryopreservation and towing workflow
•Remove medium by
centrifugation
•Collect cells into cold vials
• Add cold media and cryo-
protectant
Cells preparation
for freezing
• Slowly cells freezing at -1 o
C/min rate in an refrigerator
Freezing cells
•Transfer vials to -80 o C or/
and liquid nitrogen
Long-term
storage
Towing and
recovering cells
• Slightly heat up in hands
• Dilute with medium
• Centrifuge and discard medium
• Add fresh medium and transfer
to a cell culture dish

7. Cell culture applications
Cell culture
applications
Cancer
Research
Virology
Toxicity
Testing
Vaccine
Production
Genetically
Engineered
ProteinReplacement
Tissue or
Organ
Genetic
Engineering
Genetic
Therapy
Drug
Screening and
Development
Model System