How do we culture cells in the laboratory?

Revive frozen cell populationIsolate 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

Why passage cells?

To maintain cells in culture (i.e. don’t overgrow)

To increase cell number for experiments/storage

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

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

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

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

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

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

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.

• 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

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.

• 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

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.

• 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

30 % confluency10 % confluency 20 % confluency

40 % confluency 50 % confluency 60 % confluency

70 % confluency 85 % confluency 100 % confluency