OVERVIEW

AGS cells grow as an adherent monolayer. Therefore, detaching cells from the substrate using specific means is required for subculturing (also called passaging) cells. The trypsin-EDTA solution is the most common method to detach cells from the culture flask for routine subculture. Subculturing required a brief treatment of cell monolayer with Trypsin-EDTA that causes cell detachment followed by resuspending the cells in culture medium and distribution of cells into fresh tissue culture flasks. The detailed procedure is given below.

REQUIREMENTS

Equipment and disposables
♦ T25 flask pr 60 mm dish
♦ 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 (0.25% trypsin (w/v), 0.53 mM EDTA solution) *
♦ Ca2+-free and Mg2+-free PBS *
♦ Culture medium *

* Culture medium (Serum-supplemented complete culture medium) and trypsin solution are usually stored at 4°C. Warm culture medium, trypsin solution, and PBS to 37°C in order to avoid unnecessary cold shock to cells. 

General precautions:

1. Do all operations aseptically. Remember to handle culture aseptically. Use one hand to operate the flask and the other hand to add or remove liquid using a pipette aid or a micropipette. 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.
2. All reagents and equipment that come in direct contact with cells must be sterile.
3. All transfers of the liquid 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.
6. Don’t decant the medium from the culture flask/dish. This can increase the risk of contamination.
   

Starting material
Subconfluent (≈90% confluent) AGS cell culture in a T25 flask or 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. 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. 
4. 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.

PROCEDURE

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 culture medium will be collected on one side. Now remove and discard all the culture medium.
◊ Add 5 ml PBS. Swirl and tilt the culture dish in the opposite direction 2-3 times gently. Remove and discard PBS.

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

Tips
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 5 ml pipette and pipette aid to remove liquid 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.

Precautions
1. While pipetting PBS into the flask, take care that the flow of PBS should not disturb the cell monolayer.
2. The serum has trypsin inactivating activity. Traces of serum can impair trypsin activity.

Step 2: Add Trypsin – EDTA solution and incubate for 4 – 8 min at 37°C.
◊ Add 2 ml Trypsin-EDTA solution and gently spread 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.
◊ Gently tap the flask to remove all cells from the surface.

Tips:
1. Examine the flask under an inverted microscope after 4 min, if all the cells appear rounded and loosely attached, tap the flask and detach all cells. Incubate for more time if cells still adhere to the flask.
2. Repeated warming of the Trypsin-EDTA solution can lead to reduced activity of the Trypsin-EDTA solution. So if you are using Trypsin-EDTA solution that has been warmed several times, cell detachment from the tissue culture flask may take longer time than expected.
3. Over-confluent culture can also take a longer incubation time in Trypsin-EDTA solution to detach cells from the flask.

Precautions:
1. Make sure that trypsin – EDTA solution reaches all over the monolayer. Each and every cell should come in contact with a trypsin solution. Cells will not detach 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 can cause cell death, undertreatment results in cell clumps 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 (8 ml) all over the surface.
◊ Disperse all cell clumps by pipetting 2 – 3 times.

Note:
The serum has trypsin inactivating activity.

Tips
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. Remember that more pipetting can also cause cell death.

Step 4 (optional): Determine cell number
◊ Transfer all cell suspension to a falcon tube and resuspend the cells homogeneously.
◊ Determine the cell number using hemocytometer or any other appropriate method available in your lab.

Tips
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. The split ratio is a rough estimation of how many culture dishes can be prepared from the existing cell culture. For the AGS cell line,  you can prepare 4 to 6 culture dishes from the 90% confluent culture.

Step 5: Prepare a fresh culture dish from the cell suspension
◊ Transfer right amount of cells into a fresh culture flask/dish. For example, If you are using a split ratio of 1: 6, transfer ⅙ volume of the cell suspension to a fresh T-25 flask/ 60 mm dish.
◊ Add fresh growth medium to a final volume of 5 ml in a T-25 flask/ 60 mm dish. Resuspend the cells homogeneously by a few rounds of gentle pipetting.
◊ Discard the remaining cell suspension if you don’t need them.

Note:
Use the recommended seeding density or split ratio or specific to decide how many cells should be transferred to a fresh culture vessel. Sometimes, depending on the experimental requirement, you need to prepare the culture dish with cells.

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

Preccaution: 
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. Tighten the lid if you are using a vented cap flask.