How many passages can I expect to get out of my cells in long-term culture and on what days do you recommend splitting them? Also do you have any suggestions on how to increase the rate of expansion of cells? (2024)

FAQs

How many passages can a cell line be cultured for? ›

Continuous immortalized cell lines are comprised of a single cell type that can be serially propagated in culture either for a limited number of cell divisions (approximately thirty) or otherwise indefinitely. Cell lines of a finite life are usually diploid and maintain some degree of differentiation.

In comparison, a continuous cell line, e.g. derived from a human cancer, can be passaged an infinite number of times. After prolonged passaging the difference in phenotype over 10 passages is likely to be much less than the difference between the first 10 passages of primary cells.

Moreover, multiple passages can increase susceptibility of the primary culture to microbial contamination. Generally for primary cultures, passaging primary cells in between 2 to 5 are suitable for maintaining the genetic and phenotypic properties.

As a general guide, from a confluent flask of cells: 1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days. 1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days. 1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days.

Passaging is the procedure of harvesting cells from a culture, transferring the cells to one or more culture vessels with fresh growth medium, and using those cells to start new cultures. It is also referred to as subculturing.

Practically speaking, cell passages should be limited to prevent population and genetic drift, and ideally, experiments performed with similar passage numbers. There is no defined maximum number of passages you should perform for a cell line, but it is highly recommended to keep passage numbers low.

The concept of the Hayflick limit was advanced by American anatomist Leonard Hayflick in 1961, at the Wistar Institute in Philadelphia, Pennsylvania. Hayflick demonstrated that a normal human fetal cell population will divide between 40 and 60 times in cell culture before entering a senescence phase.

A cell can divide upto 60 to 80 times in its lifetime before attaining the apoptosis that is cell death.

A549 cells were passaged when ~80% confluent and used at passages of 4–18 for all tissue culture experiments.

Why is cell passaging important? ›

One of the first cell culture techniques taught is cell passaging. Passaging is important for many reasons, the most obvious being to create a critical number of cells. The immortal cell line is vital to obtain enough cells to perform experiments and studies and to provide consistency.

5) Sub-culturing

Also referred to as cell splitting and cell passaging. Split ratios or seeding densities can be used to ensure cells are ready for an experiment on a particular day or maintain cell cultures for future use or as a backup.

A confluency of 80% means that 80% of the culture vessel surface is covered with cells.

It's best to split cells before the plates are more than 80-85% confluent because cells like their space. If they reach confluence, they stop growing (contact inhibition) and it takes them time to recover after they are passaged.

Immortalized cell lines are cells that continue to grow and divide indefinitely in vitro under optimal culture conditions [44].

In some cases, researchers may achieve substantial T cell expansion in an average time frame of 10-14 days. However, for long-term research applications, T cells may need to be cultured for several weeks.

Cell lines obtained from in vitro transformed cell lines or cancerous cells are indefinite cell lines and can be grown in monolayer or suspension form. These cells divide rapidly with a generation time of 12–14 hours and have a potential to be subcultured indefinitely.