Growing cells to log phase
Note: If your starting material is a biofluid from a liquid biopsy, you can skip this tip.
In most laboratories, growing cells to log phase is second nature. All that’s needed is a little extra time and a Vi-CELL BLU cell counter, so it’s not the growing of the cells to log phase that requires extra attention, it’s the plating of cells before growing to log phase.
Often, despite a scientist’s best efforts, the cell count obtained via hemocytometer can be skewed by several variables, including aggregation, an inhomogeneous mixture, and improper calculation. To keep errors in cell density from derailing your EV isolation from the very beginning, reliable cell counters and liquid-handling automation for cell seeding can provide the accuracy and standardization that is sometimes missing from day-to-day, hands-on lab work.
Under- or over-seeded cells require vigilant monitoring by spectrophotometer so as not to miss log phase growth, and plates seeded with inaccurately counted cells at the same time may not be ready for EV harvest at the same time.
Cells seeded at a predictable density should reach log phase at a predictable time, keeping your assay on schedule. When dealing with adherent mammalian cells, several concurrent cultures are typically grown, enabling the assessment of growth phase by spectrophotometer without disruption of the experimental cells.
Beyond the obvious benefits of standardizing your method of cell seeding, getting to know your cells’ typical growth attributes can help alert you to problems with your cells or culture conditions. If your cells typically achieve confluence in 18 hours, but your cells are lagging behind or racing ahead, it may be time to characterize a new aliquot of cells, or it may be signaling a change in media components or incubation conditions.
Why does log-phase growth matter when harvesting exosomes?
Because EVs are the desired endpoint, the conditions under which cells are grown–and under which EVs are released–can directly impact the quality of the EV and their cargoes. For example, cells grown under stressful conditions similar to those encountered in overcrowded, stationary-phase cultures exhibited a shift in EV cargoes to reflect the harsher environment.(1)
If your experimental goal is to recapitulate EV cargoes from normal conditions, ensuring your EVs are harvested at the right time matters.
- Villarroya-Beltri C, Baixauli F, Gutierrez-Vazquez C, Sanchez-Madrid F, Mittelbrunn M. Sorting it out: regulation of exosome loading. Semin Cancer Biol. 2014;28:3-13.