Cell Plating, Media Change, and Culture Maintenance Protocol

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About Cell Plating or Cell Passaging

Most types of cells stop or slow down their growth at a characteristic population density in any particular medium.

Cell to cell contact, localized or generalized depletion of nutrients or production of cell generated inhibitors leads to density dependent inhibition or sometimes called as contact inhibition of cell growth.

Cells at this stage are said to be confluent.

This is important to avoid Contact Inhibition of Proliferation (CIP), exhibited by most cancerous cell lines.

The phenomenon of a cell ceasing to proliferate after contact with other cells is called as CIP.

Adherent cells cover the entire growth surface available to them is referred to as confluence.

Hence they need to be subcultured, passaging, cell plating (indicated by passage number).

The passage number is the number of times that the culture has been subcultured.

Why Cell Plating or Cell Passaging

Cells are often passaged at semiconfluency when they are in log phase.

Failure to subculture or cell plating, the proliferating cells results in reduced mitotic index and eventually cell death.

The most important step in subculturing or cell plating of monolayers is to detach cells from the surface of the primary culture vessel by either enzymatic means such as trypsinization or mechanical means such as scraping of monolayer cells.

The resultant cell suspension is then reseeded, into fresh cultures.

Secondary cultures are checked for growth, fed periodically, and may be subsequently subcultured to produce tertiary cultures, etc.

The time between passaging cells depends on the growth rate and varies from cell line to cell line.

Detachment of cells by mechanical means is accomplished by scraping monolayer of cells with commercially available silicon rubber spatula known as a scrapper.

Whereas proteases such as trypsin, collagenase and pronase etc. are used for enzymatic dissociation.

Prolonged enzymatic dissociation may damage cell membrane.

Thus, it is wise to decrease the incubation period and physical injury when in enzyme solution.

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Cell Plating or Cell Passaging Overview. Images are created with BioRender

Cell Plating or Cell Passaging Requirements

1. Confluent culture of cells

2. Appropriate culture media

3. Antibiotics and antifungal agent

4. Sterile PBS (Phosphate buffer saline)

5. Trypsin/EDTA solution (0.25% Trypsin in 1mM EDTA in PBS)

6. Sterile pipettes, culture vessels, sterile beakers

7. Cotton swabs, 70% IPA,

8. 15 ml & 50 ml Centrifuge tubes

Cell Plating or Passaging of Adherent Cells

Check all the cell lines under the microscope for contamination, cell morphology, density and presence of clumps or detachment.

1. The protocol described here is for a typical confluent culture in a 25 cm2 flask

2. Remove medium with a sterile pipette without disturbing the cells.

3. Wash adhering cell layer with sterile PBS (Phosphate buffer saline) to remove traces of serum that can inhibit trypsin.

4. Add 2 ml trypsin/EDTA at 37ºC to cover the cell layer.

5. Incubate for 2 minutes at 37ºC in CO2 incubator. Tap occasionally to verify that the cells are releasing. Check in microscope to visualize detachment of cells.

6. Remove trypsin-EDTA. Add fresh 2 ml of medium and rinse cell layer two or three times to dissociate cells and to dislodge any remaining adherent cells.

7. Transfer the required number of cells to a new labelled flask containing pre-warmed medium. Maintain the split ratio as recommended.

8. Incubate cells in humidified incubator at 37ºC, 5% CO2.

Cell Plating or Passaging of Suspension Cells

In suspension culture cells are suspended in complete growth medium rather than attached to a culture flask surface, it is not necessary to disperse them enzymatically before passaging.

However, before passaging, cells must be maintained in culture by feeding every 2 to 3 days until they reach confluency (i.e. until the cells clump together in the suspension and medium appears turbid when the flask is swirled).

1. With a pipette, mix the cells thoroughly by pipetting up and down and rinsing the side of the flask.

2. Decant the cell suspension into the centrifuge tube, spin at 1300 rpm for 7 minutes at 4 °C temperature to pellet down all the cells.

3. Add 1 ml of fresh media.

4. Split into two flasks with 0.5 ml of cell suspension and add 6.5 ml of fresh media.

5. Label new passage number on both the flasks.

6. Incubate cells in humidified incubator at 37 C, 5% CO2.

Precautions During Cell Plating or Passaging

Cell degeneration may lead to change in morphology i.e. rounding up of cells and their detachment from the surface of the culture vessel.

The most frequent reasons for rapid cell degeneration include use of too high seeding density, use of poor quality or too high concentration of fetal calf serum when preparing cultures.

Whenever rapidly growing, continuous cell lines are maintained in a laboratory there is a risk of cell line cross-contamination.

The problem of cell degeneration can usually be overcome by appropriate adjustment of the cell count or foetal calf serum concentration.

Only one cell line should be used in at any one time.

After removal of the cell cultures from the cabinet, the cabinet should be swabbed down with a suitable disinfectant and the cabinet UV run for five minutes before introduction of another cell line.

Bottles or aliquots of medium should be dedicated for use with only one cell line.

Regularly return to frozen stocks — never grow a cell line for more than three months 15 passages from stock passage level, whichever is the shorter period.

All culture vessels must be carefully and correctly labelled. (including name of cell line, passage number and date of transfer)

Some cultures whilst growing as attached lines adhere only lightly to the flask called as fragile cells, thus it is important to ensure that the growth medium is retained and the flasks are handled with care to prevent any cells detachment prematurely.

Although majority of cells will detach in the presence of trypsin alone the EDTA is added to enhance the activity of the enzyme.

Presence of serum inactivate Trypsin activity. Therefore, it is essential to wash the monolayer of cells with PBS without calcium and magnesium in order to remove all traces of serum from the culture medium.

Cells should only be exposed to trypsin/EDTA long enough to detach cells.

Prolonged exposure could damage cell surface receptors.

In case of cells lost in trypsin removal, trypsin should be neutralised with serum prior to seeding cells into new flasks otherwise cells will not attach.

Trypsin may also be neutralised by the addition of soyabean 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 as above.

This is especially necessary for serum-free cell cultures.

If the cells harvested are at too low a cell density to re-seed at the appropriate cell density into fresh flasks it may be necessary to centrifuge the cells e.g. 5 mins at 1300 rpm, and resuspend in a smaller volume of medium.

The cells are then centrifuged, resuspended in fresh culture medium and counted as above.

This is especially necessary for serum-free cell cultures.

If the cells harvested are at too low a cell density to re-seed at the appropriate cell density into fresh flasks it may be necessary to centrifuge the cells e.g. 5 mins at 1300 rpm, and resuspend in a smaller volume of medium.

Precautions During Cell Plating or Passaging Ciations:


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