Cells

You know those little guys that we all learned in high school biology that are the building blocks of life? Yea, they're important. I do a lot of work with cells, and I want to talk about some of those procedures.

To begin with, the type of cells that I work with are Human Embryonic Kidney 293T cells. Known as HEKs or 293Ts. A cell line is a group of immortalised cells. You can order them from a company, and you know what you are getting. HEK 293s were originally from a "healthy aborted fetus" in the 1970s. The number 293 comes from Frank Graham (the guy who immortalised this cell line), who numbered each of his experiments. Thus, HEK 293s came from his 293rd experiment.

HEK 293s are not just plain human cells. A bit of adenovirus 5 DNA was added and incorporated into the human chromosome. This is what gives them their immortality.

Ours are not just plain 293s, but 293Ts. This means that they are able to be transfected with plasmids easily. More on that later.

The way we work with these cells is very cyclical, so I really could start anywhere in describing the process.

We'll start with a flask filled with cells. The cells are in media (read food). We use DMEM as our media. DMEM is composed of amino acids, salts, glucose, vitamins, iron, and phenol red. Phenol red is very important. It is a dye that changes color based on how acidic it is. Mixed into our DMEM is fetal bovine serum (FBS) and a penicillin/streptomycin mixture. The FBS is what is left of the liquid portion of fetal cow blood after it is allowed to clot. It does not have blood cells in it, is low in antibodies, and has a good amount of growth factors. The pen/strep mixture is antibiotics so that the cells don't get infected.

You pull your flask out of the incubator (37 degrees C and 5% CO2), and you notice that the liquid (media) is a dull orange. Time to change the media! Why? Well, remember that phenol red? It starts out a reddish-pinkish color. When it gets too acidic (cell excrement makes it that way), the cells will die. Imagine living in your own poop. Even if you get nutrients from the air, after a while there is just too much poop, and you suffocate from it. Same sort of thing. The change in color signifies that it is becoming too acidic. These particular cells are called adherents. This means that they stick to a surface. In this case, they stick to a side of the flask (whichever end was down). So you flip the flask over, and all the liquid is on the opposite side from the cells. Now you can use an aspirating pipette (also known as a vacuum) to pull out the spent media. If you just want your cells to grow more, you will add more media. This is known as changing, swapping, or replacing the media.

Let's say that before you swapped the media, you looked at your flask under a microscope. You found that there was some overcrowding going on. This is another leading cause of cell death. If cells do not have space to grow in, they die. Sometimes something peculiar happens and the cells on the edge will start growing up the walls. Sometimes, they don't have good anti-gravity skills and the off-the-wallers become curlers. A film of cells will curl up off the wall and back into the cells. It may look cool, but it definitely means your cells are unhealthy (I know from experience).

How do you deal with cell-overcrowding? Decimation. You suck off the old media, and put in a little bit of trypsin-EDTA. The trypsin will cleave proteins (it's actually one of the digestive enzymes that digests proteins in humans), and the EDTA prevents the cells from clumping. You leave that in the incubator for five minutes, and all your cells will come off the walls/floor of the flask. You then spray them down with media so any clingers are forced into the solution. You pipette up and down to make sure everything is mixed nicely, and usually you pull off 9/10 of the liquid (and thus the cells) in the pipette. Then you vaccuum them. You are left with 1/10 your original cell count (-ish) and you add more media to get back to your original volume. I have found that doing this generally gives you three or four days before your cells are confluent (overcrowding) again.

We have cells. We know how to maintain them. What's the point? In our case, the point is usually transfection. Transfection is introducing foreign DNA for the cell to incorporate in its own genome. You put your cells in solution, and instead of vaccuuming a large portion of them, you then add that portion to a different flask. You then add an appropriate amount of media, and do some heavy mixing. Then you add about 10 ml of this new solution to your cell culture plates. You may know them as petri dishes. Your cells will grow and flourish (hopefully), and reach confluency.

After a couple of days, when it is time to change the media, you can transfect them. You put a bit of the DNA that you hope to express in a mix with media and a special reagent that will make a hole in the cell membrane for the DNA to go through. You add a few drops of this mix to each plate, and the new foreign DNA gets pulled into the cell. If everything worked well, the cell will think, 'Huh! There is DNA outside of the nucleus. Better put that back where it belongs!' The cell adds the new DNA to its own, and then acts as though nothing is different. In our case, the foreign DNA will code for a particular protein.

After another couple of days, the cells had enough time to produce that protein, and you can harvest them. This is done by basically scraping each of the plates, and sucking up the cells that were on there. There are a number of things that can be done after you have harvested the cells, but that is outside the scope of this post.

Now, you are an expert on cell culture! (-ish)