Protocol – Subculturing Adherent Cells Growing in Serum-containing Growth Medium using Trypsin-EDTA


  • The Trypsin-EDTA method, also referred to as trypsinization, is the most commonly used method for passaging/subculturing of adherent cells.
  • Cells to be subcultured are first washed with Ca2+-free and Mg2+-free PBS and subsequently treated with Trypsin-EDTA solution. 
  • Using PBS free of Ca2+ and Mg2+ is necessary to minimize the amount of these divalent metal ions around the adherent cells as these metal ions strengthen the cell-cell and cell-substratum interactions.
  • Brief incubation with Trypsin-EDTA disrupts cell-cell and cell-substratum interactions, resulting in single-cell suspension.
  • Trypsin, a proteolytic enzyme, disrupts these interactions by proteolysis and EDTA by chelating divalent cations.
  • The treatment time and concentration of trypsin must be optimized for each cell line as under-treatment results in cell clump formation whereas over-treatment results in cell death due to excessive proteolysis. 
  • To stop the excessive proteolysis, trypsin must be either removed or inactivated. As serum is known to contain trypsin inhibitory activity, adding a serum-supplemented culture medium is sufficient to inhibit trypsin activity in cell lines grown in serum-supplemented medium. For those cell lines which are maintained in a serum-free culture medium, trypsin is removed after dilution of the reaction or inactivated by adding another trypsin inhibitor such as  soybean trypsin inhibitor.
  • Once a single cell suspension is ready, the cell number can be counted and fresh culture dishes can be seeded with a suitable number of cells.

Subculturing subconfluent adherent cell line growing in a serum-supplemented culture medium.

You must know all the important details of the cell line you want to subculture:

  • Cell culture medium
  • Split ratio (or seeding density)
  • Harvesting time
  • The temperature at which the cell line is maintained 

Take an example of the C2C12 cell line:
C2C12 is a mouse myoblast cell line which is maintained in DMEM supplemented with 10% FBS. Cells are maintained at 37°C in a humidified CO2 incubator. C2C12 cell line should not be allowed to reach 100 % confluency as it induces differentiation in this cell line. Therefore, cells must be harvested when the confluency is ≈80%. Recommended seeding density is 5.0 x 103 cells/cm2.


Equipment and disposables
♦ T25 flask/Tissue culture dishes

♦ Pipette (5 ml, 10 ml) (sterile glass or disposable plastic pipettes)
♦ Pipette aid
♦ Pasteur pipettes and vacuum source (for aspiration) (optional)
♦ Laminar flow hood (Certified Class II type)
♦ Beaker to discard the waste

Reagents and solutions
♦ 1 x Trypsin-EDTA solution
♦ Ca2+-free and Mg2+-free PBS
♦ Culture medium

1. Complete culture medium (basal medium + serum + supplements) and trypsin solution are usually stored at +4°C. You must warm up the culture medium, trypsin solution as well as PBS to the temperature at which cells are maintained e.g., 37°C for C2C12 cell line) in order to avoid unnecessary cold shock to cells.
2. Trypsin concentration is crucial for the successful subculturing of adherent cells. For most serum-grown cell lines, a 0.25% trypsin (w/v)/0.53mM EDTA solution can be used as recommended by ATCC for most of their serum-grown cell lines. However, depending on cell line sensitivity, either trypsin concentration can be changed or commercially available cell dissociation reagents such as Accutase can be used.

1. Subculture one cell line at a time. If you need to subculture two cell lines, you must clean the work surface of the laminar flow hood before operating the second cell line. This will reduce the risk of cross-contamination.
2. Do all operations aseptically. All reagents and equipment that come in direct contact with cells must be sterile.
3. All transfer of medium should be inside the laminar flow hood.
4. Wear a lab coat and disposable latex gloves during the procedure.
5. All the steps which involve changing the medium from culture must be done quickly to avoid any risk of drying of cells.
5. Don’t decant the medium from the culture flask/dish. This can increase the risk of contamination.

Starting material
Subconfluent (≈80 – 90% confluent) adherent cell culture growing in a T25 flask/60 mm culture dish

Prior to start
1. Examine the culture under an inverted phase-contrast microscope. Make sure cells are healthy and free of any contamination. Proceed further if cells are ready to subculture.
2. Make the laminar flow hood ready for cell culture work (wipe the work surface with 70% ethanol, turn the UV-light on, and after 20 min switch off the UV-light and turn on the laminar flow).
3. Warm-up reagents (culture medium, trypsin-EDTA solution, and PBS) to the temperature at which cells are maintained (for example place reagent bottles in a 37°C water bath for subculturing mammalian cell lines)
4. Place all reagents and other materials required for cell culture inside the laminar flow hood. Make sure they are sterile. You must wipe the surface of the reagent bottles with 70% ethanol before transferring them from the water bath to the laminar flow hood. 
5. Decide how many fresh culture dishes you want to prepare (you must know the split ratio or seeding density). Label them with the date of the subculture, passage number, cell name, and your name.


1. The protocol below provides a general procedure for subculturing adherent culture using Trypsin-EDTA. For specific details, we recommend you to go through the instruction manual provided with the cell line.
2. Remember to handle culture aseptically. Use one hand to operate the flask and the other hand to add or remove liquid. Usually, an experienced worker opens the lid of the culture dish and lifts it slightly with the help of two fingers of one hand, and uses the other hand to remove or add medium from the culture flask.

Step 1: Remove and discard culture medium from the culture flask/dish and wash cell monolayer gently with Ca2+-free and Mg2+-free PBS.
◊ Take out the culture vessel from the incubator and place it inside the laminar flow hood.
◊ Slightly tilt the culture dish. All the culture medium will be collected on the tilted side. Now remove and discard all the culture medium.
◊ Add 3 – 4 ml PBS. Swirl or tilt the culture dish in the opposite direction 2-3 times. Remove and discard PBS.

PBS washing will remove dead cells, cell debris, and the remaining culture medium.

1. A vacuum aspirator can be used to remove liquid from the culture vessel. Vacuum aspirator is a very quick and convenient way to remove liquid from the dish. If you don’t have a vacuum aspirator, use a pipette and pipette aid to remove liquids from the flask.
2. Always keep the flow of PBS on the sidewalls of the culture vessel to reduce the risk of cell detachment due to the flow of the liquid.

1. While pipetting PBS into the flask, take care that the flow of PBS should not disturb the cell monolayer.
2. Some cell lines are loosely adherent. In such cases, care must be taken to avoid any loss while removing the culture medium and washing with PBS.
3. Serum has trypsin inactivating activity. Traces of serum can impair the trypsin activity.

Step 2: Add Trypsin – EDTA solution and incubate 1 – 5 min at 37°C.
◊ Add a sufficient amount (0.5 ml – 1 ml for a T25 flask/60 mm dish) of Trypsin-EDTA solution and gently spread trypsin solution all over the monolayer by swirling and tilting the culture dish. Incubate at 37°C (in the incubator) until cells appear rounded and start detaching from the substratum (it can take 1-5 min for most of the cell lines).
◊ Gently tap the flask to remove all cells from the surface.

1. The amount of Trypsin-EDTA should be sufficient to cover all the cell surfaces of the culture vessel. A 0.5 – 1 ml Trypsin-EDTA solution is sufficient for a T25 flask/60 mm dish. Depending on the convenience, one can add more trypsin-EDTA solution. 
2. For each cell line, one should optimize the trypsin-EDTA treatment condition (incubation time and concentration). Some cell types are strongly adherent and require a higher concentration of the trypsin-EDTA solution as well as longer incubation time.
3. Trypsin has maximum activity at 37°C.

Tip: Examine the cell morphology under the microscope every minute if you don’t have any idea how long trypsin-EDTA treatment is required for your specific cell line.

1. Make sure trypsin – EDTA solution reaches all over the monolayer. Each and every cell should come in contact with a trypsin solution. Cells will not be detached or form clumps upon detachment if the trypsin treatment is insufficient.
2. The right trypsin-EDTA treatment condition is crucial for a successful trypsinization process. While overtreatment will cause cell death, undertreatment results in cell clump and undetached cells in the culture dish.

Step 3: Inactivate trypsin by adding fresh serum-containing medium
◊ Wash out all the cells from the surface by pipetting the fresh culture medium (4 ml) all over the surface.
◊ Disperse all cell clumps by pipetting 2 – 3 times.

Note: The serum has trypsin inactivating activity.

1. While transferring the medium in the vessel, keep the flow of the culture medium toward the surface where cells were attached.
2. If there are cell clumps, disperse them by several rounds of pipetting. More pipetting can cause cell death.

Step 4 (optional): Determine cell number
◊ Transfer all cell suspension to a centrifuge tube. Collect cells by centrifugation.
◊ Resuspend cells in an appropriate amount of complete medium (5 ml) and determine cell number using a haemocytometer or any other method.

1. Cell counting is not necessary for the regular maintenance of cell culture. Most researchers use a split ratio to seed cells in a fresh culture dish.
2. Split ratio is a rough estimation of how many culture dishes can be prepared from the existing cell culture. For e.g., you can prepare 5 to 10 culture dishes from the culture (90% confluent) if the split ratio is 1:5 to 1:10.

Step 5: Prepare a fresh culture dish from the cell suspension
◊ Transfer an aliquot of cells into a fresh culture flask/dish.
◊ Add fresh growth medium. Pipette cells to resuspend cells again.
◊ Discard the remaining cell suspension if you don’t need them.

Note: Use the recommended seeding density or split ratio to decide how many cells should be transferred to a fresh culture vessel. Generally, a 1 : 5 – 1 : 10 split ratio works well for fast-growing cell lines. For a slow-growing cell line, a 1: 3 split ratio is recommended.

Tip: If you need to make many culture dishes, prepare a master cell suspension before transferring cell suspension to the culture dishes.

Precaution: Make sure that important details (your name, cell line name, passage number, subculture date) are clearly written on the culture dish.

Step 6: Examine the culture dishes under an inverted phase-contrast microscope and place them back in the incubator.
◊ Open the lid of the flask slightly for air exchange if you are using sodium bicarbonate containing culture medium. Tighten the lid if you are using a vented cap flask.

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