Monday, June 14, 2010

Method Monday: Cell Culture

I have my training in genetics, and more specifically cancer genetics.  So a great deal of my work has been with little cells growing rapidly on plastic, in pink-colored media, in a warm, humid incubator.  This is the world of cell culture, also called tissue culture. It's also the iconic image of a laboratory worker: someone in a white coat, gloves and goggles, laboring away with their arms in a plastic chamber and transferring reddish liquid from one flask to another.

Hint: most of those pictures are totally staged.  I never wore goggles.

Here's what cell culture is all about.

Cells are harvested from a source, usually a hunk of tissue, (we'll come back to that) and are gently teased apart both mechanically via scissors and chemically with enzymes, typically trypsin, collagenase, or dispase, that chew up the connections between the cells.  Then, the cells are soaked in a liquid media, and placed in a vessel, either a flask or a petri dish.  These are usually plastic treated with poly-L-Lysine, creating a positively charged surface to make the cells stick better.  Then the cells sit in an incubator set to body temperature (37C, or 98.6F) that is infused with carbon dioxide.  This keeps the cells at a happy pH. Then let the experiments begin!

What is in the media?  Well, it's got some salts, some sugar, some minerals.  Think of it as a really expensive form of Gatoraide.  Usually there's antibiotics too, because good cells and bad bacteria don't mix.  That can be it, and that is called minimal media - just enough to keep the cells alive.  But most cells need more than that to grow.  So extras are added, such as amino acids, the building blocks of proteins, and often serum.  Harvested from calves or fetal calves (don't ask, you don't want to know), serum a blood product chock-full of growth factors and goodies that help cells grow.  Oh, and the pink color?  It's phenol red, a dye that indicates the pH.  Cherry red is good, purple is bad.

So where do these cells come from? Pretty much any hunk of tissue will do, from human, mouse, dog, Chinese hamster, you name it.  For a lot of purposes, cell lines have already made, and can be purchased.  Very convenient, because it allows multiple researchers to use the same cells in their experiments and it's easier to compare data.  For example, one frequently used cell line is a human cervical cancer cell line called HeLa.  The name comes from "Henrietta Lacks."  The cells were made in 1951, and nearly every cancer lab since has used those cells. Hows that for immortality? Other times researchers have to make their own cell lines.

Some main categories of cells people use are primary, immortal, and stem cell lines.  Primary means they're straight from a living being, and typically have a short time they can be used due to senescence - a process of aging that limits the number of times a cell can divide. In one lab, I routinely made primary cell culture from mouse hippocampal neurons. Not cells that can easily live and divide for many generations.  Immortal cell lines have supposedly limitless replicative potential are those that have been pushed through senescence, through crisis, and live on. Or they have been induced to live on and on by introducing viral genes that allow them to keep on growing.  Both methods rely on changing the expression of genes that normally keep cells from dividing out of control.  And stem cell culture takes cells from the inner cell mass of an embryo which are then grown on a feeder layer of cells, with careful media conditions.  These cells are undifferentiated and supposedly have limitless replicative potential.

So, there's books and books written on cell culture, and really neat things researchers use it for.  I remember in one lab working on muscle differentiation, and I could take muscle stem cells, add a couple growth factors into it and a few days later, watch the differentiated muscle cells twitch in the dish.  Freaky. But other than playing around, cell culture gives us a way to poke and prod into the biology of cells in a way not otherwise possible in a living organism. Yes, there's drawbacks and limitations.  Cells in a dish do not behave like cells living in a body.  But for a vast amount of basic biology and early drug testing, cell culture is a key way to gain insights and knowledge.


  1. shoot - I missed commenting on this one before you posted the next one! I used EBV to create our immortalized B cell lines (LCL). I'm in an office environment now but I sure do miss those cells sometimes. Easier than office politics :)

  2. True, true. When cells talk back to you... oh wait! They can't!

    Cool beans! One of my old lab mates should have talked to you - she had a pain of a time getting some B cells immortalized. I made lots of MEF cell lines. It was maybe as easy as it gets - I just let them sit and sit until something grew out. Then karyotype to see that they had near about 20 chromosomes. Then genotyped and called it a day. Well, it really took about 4 months.... I don't miss the waiting.