Fact Sheet: Subculturing and Maintenance of Monolayer Cell Culture
Culture Examination / Medium Preparation:
- Observe the contents of the culture vessel for signs of microbial contamination, checking that the culture is 90-95% confluent using an inverted microscope.
- Prepare fresh growth medium recommended for the cell line ensuring that any supplements are added, pre-warming the medium to the temperature that the cell line is growing at.
- Remove and discard the culture medium from the culture vessel, washing the cell sheet with a balanced salt solution such as Phosphate Buffered Saline; this is to remove traces of serum which may inhibit the reaction of the dissociating solution (such as Trypsin) that is added in the next stage.
- Incubate the culture vessel and observe the cells under the microscope at regular intervals for detachment, tapping the vessel gently to aid detachment.
- When the cells have detached, transfer them to a sterile container and add pre-warmed growth medium before centrifuging the cell suspension (time & speed dependent on the cell type) and pouring off the supernatant.
- Resuspend the cell pellet in an appropriate volume of growth medium and count the live cells in the suspension using a haemocytometer with the aid of vital stain such as trypan blue
- Finally, calculate the volume of suspension required and add an appropriate volume of growth medium to achieve the seeding density recommended for the cell line. Transfer the cell suspension to new culture vessels before incubating
- Instead of counting cells, in certain circumstances (such as maintenance of cell line where the requirement for exact seeding density is not important) the cell suspension can be split among several culture vessels; for example, a split ratio of 1:2, means transferring the cell suspension of one vessel to two new vessels of the same size
- If cells are difficult to remove from the culture vessel then you can use Trypsin with a higher enzyme concentration. Incubate (37°C for most mammalian cell lines) with pre-warmed Trypsin, rinse the cell sheet thoroughly prior to adding the dissociating solution and do not leave the culture to become over confluent before sub culturing
- To prevent clumping of cells, add growth medium as soon as most of the cells have detached from the vessel surface
- Use gentle centrifugation speed and do not centrifuge for too long
- Pipette gently when resuspending the pellet
In the Lab / at Wickham Laboratories Ltd
- L929 fibroblast cell line is used in cytotoxicity testing by the Extraction / Elution or agar Diffusion methods to ascertain the absence of extractable cytotoxic substances in medical devices or materials used in their manufacture
- Testing methods comply with ISO 10993-5 & USP <87>