Every Saturday I plan to write about a technique I’m using/have used in my project. I’ll explain what the purpose of it is, why I want the data it produces, and how to perform it. Even, and especially, if you don’t work in a science lab, I hope you still won’t just dismiss the entry as nerd-speak, and instead, read through. I promise to keep the entry concise and not assume you know anything more than high-school science.
Yesterday I “passaged my cells”. Passaging cells is the first thing any person starting in a cell culture lab learns. Cell culture is the process of growing cells in the lab. The cells can be from any source: animal, vegetable, or mineral – just kidding, minerals don’t have cells. I’m lucky in the almost all the cells I’ve worked with are adherent. This means that if you take a solution of cells and place it in a plastic dish, the cells will attach and grow on the bottom, instead of floating around. They key word here is “grow” because cells divide and multiply over time. Everyone, for example comes from a fertilized egg, a single cell, which divided in two, and those two divided, and those four divided, etc. until finally there’s you (very simplified and some major assumptions taken with that analogy, but the concept applies). Cells growing in a dish are no different. They multiply. Exponentially. This means that they’ll run out of empty space in the dish since they’re only growing in 2D and not on top of each other. When they completely run out of space we call this “confluent” because they look like a single layer. When cells get confluent they often change their behavior, because they touch each other a lot more, so we generally want to avoid this. We also want to avoid confluency for the sheer reason that if cells are growing in 2D and run out of space, they will stop growing, and generally, as scientist, we always want more cells, so we need to keep them growing. We prevent cells from becoming confluent by taking them off the dish they are currently in, and placing them in a dish with bigger surface area, so their density is decreased and they can expand again. This process is known as “passaging”.
1) Remove cell dishes from the incubator. We keep our cells at 37°C (human body temperature).
2) Remove the media (growing solution) from the dish and rinse with salt solution.
3) Add trypsin to the dishes to cause them to detach from the plastic (a few minutes at 37°C).
4) Remove the cell solution and add normal growth media to stop the trypsin reaction.
5) Count the cells (optional – but helps decide how big of a dish to replate them in).
6) Centrifuge the cells to make a cell pellet
7) Remove the liquid from the top, this ensures all trypsin is removed
8) Add in new growth solution to cells and place in the new, larger dish, or use as desired.