How do we culture cells in the laboratory?

1. How do we
culture cells in
the laboratory?
LECTURE OF SUBJECT :
Dr. sharafaldin Al-musawi
College of Biotecholgy
LECTURE: 6
SUBJECT: Animal Tissue culture
LEVEL: 4

2. Revive frozen cell population
Isolate from tissue
Maintain in culture
(aseptic technique)
Sub-culture (passaging)
Cryopreservation
Count cells
Containment level 2
cell culture laboratory
Typical
cell culture flask
‘Mr Frosty’
Used to freeze cells

3. Why passage cells?
To maintain cells in culture
(i.e. don’t overgrow)
To increase cell number for
experiments/storage

4. Materials
Media– pre-warmed to 37ºC (refer to the ECACC Cell Line Data
Sheet for the correct medium)
70% ethanol in water
PBS without Ca2+/Mg2+
0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+
Trypsin
Soybean trypsin Inhibitor

5. Equipment
Personal protective equipment (sterile gloves, Laboratory coat, safety
visor)
Waterbath set to appropriate temperature
Microbiological safety cabinet at appropriate containment level
CO2 incubator
Pre-labeled flasks
Marker Pen
Pipettes

6. 1. View cultures using an inverted microscope to assess the
degree of confluency (70-90%) and confirm the absence
of bacterial and fungal contaminants.
2. Remove spent medium.
3. Wash the cell monolayer with PBS without Ca2+/Mg2+
using a volume equivalent to half the volume of culture
medium to remove dead cells and serum.
(Repeat this wash step if the cells are known to adhere strongly).
Procedure

7. 4. Pipette trypsin/EDTA onto the washed cell monolayer for
digests protein-surface interaction to release cells
(collagenase also useful) using 1ml per 25cm2 of surface
area.
• Rotate flask to cover the monolayer with trypsin.
Procedure

8. 5. Return flask to the incubator and leave for 2-10 minutes
(depending on cell type).
6. Examine the cells using an inverted microscope to ensure
that all the cells are detached and floating.
The side of the flasks may be gently tapped to release any
remaining attached cells.
Procedure

9. 7. Resuspend the cells in a small volume of fresh serum-
containing medium to inactivate the trypsin.
(Remove 100-200uL and perform a cell count).
8. Transfer the required number of cells to a new labeled
flask containing pre-warmed medium.
(refer to Cell Line Data Sheet for the required seeding density).
• Most cell lines will adhere in approx. 3-4 hours
Procedure

10. Why EDTA in trypsin?
EDTA enhances trypsin activity.
Actually trypsin/EDTA is a combined method for detaching cells.
Trypsin cuts the adhesion proteins in cell-cell and cell-matrix
interactions
EDTA is a calcium chelator, which integrins needs to interact with
other proteins for cell adhesion. no calcium = no cell adhesion.
EDTA: can decrease the clumping of cells.

11. • Some cultures whilst growing as attached lines adhere only lightly
to the flask, thus it is important to handled the flask with care to
prevent the cells detaching prematurely.
• Although most cells will detach in the presence of trypsin alone the
EDTA is added to enhance the activity of the enzyme.
Key Points

12. Key Points
• Trypsin is inactivated in the presence of serum. Therefore, it is
essential to remove all traces of serum from the culture medium by
washing the monolayer of cells with PBS without Ca2+/Mg2.
• Cells should only be exposed to trypsin/EDTA long enough to
detach cells. Prolonged exposure could damage surface receptors.
• Trypsin should be neutralized with serum prior to seeding cells
into new flasks otherwise cells will not attach.

13. • Trypsin may also be neutralized by the addition of soybean
trypsin inhibitor , where an equal volume of inhibitor at a
concentration of 1mg/ml is added to the trypsinised cells.
• The cells are then centrifuged, resuspended in fresh culture
medium and counted .
• This is especially necessary for serum-free cell culture.
Key Points

14. Confluency
• Confluency referring to the proportion of the surface
which is covered by cells.
• For example, 50 percent confluence means roughly half
of the surface is covered and there is still space for cells
to grow.
• 100 percent confluence means the surface is completely
covered by the cells, and no more space is left for the
cells to grow as a monolayer.

15. • Cells are typically passaged before becoming fully
confluent in order to maintain their
proliferative phenotype.
• Cells to be kept in healthy & in growing state have to be
sub-cultured or passaged , It’s the passage of cells when
they reach to 75-90% confluency in flask/dishes/plates.
Confluency

16. 30 % confluency10 % confluency 20 % confluency
40 % confluency 50 % confluency 60 % confluency
70 % confluency 85 % confluency 100 % confluency