Rite of passage

by Lorien E. Menhennett

Lung fibroblasts (pictured above) produce the “food” our lung epithelium cells need to survive, as I learned this week.

A few days ago, Olga told me she’d thrown out a flask of fibroblast cells. What?! I couldn’t believe my ears. Didn’t we need those cells for … um … something?

Every day, I learn new techniques. But sometimes I get caught up in the “doing” and miss the bigger picture. Monday, though, I got some clarity. The “doing” and the “bigger picture” came together.

Olga and I are taking care of two types of mouse lung cells: fibroblasts and epithelium. Up until Monday afternoon, I couldn’t keep them straight.

Monday morning — before the clarity — Olga had me work with 10 plates (round plastic dishes) of fibroblasts. For five of the plates, Olga instructed me to aspirate (remove) the high-calcium media (liquid they grow in) and pipette in low-calcium media. Simple enough. Aspiration, pipetting, easy stuff (which feels really good to say!). I wasn’t quite sure why I was replacing high-calcium media with low-calcium media, but Olga was busy setting up another experiment so I went ahead with her instructions.

Once I was done with that, Olga told me that we were going to “passage,” or split, the other five plates into 10 plates to give the cells more room to proliferate. Basically, we wanted them to grow more, and they needed space to do that. That’s all well and good, but I wasn’t quite sure why we needed so many fibroblasts … we already had a lot, and had more in the freezer; why keep so many in the incubator? A question for later, I decided.

The first step in passaging the cells was aspirating their media. Then I washed the plates out with PBS, a sterile buffered saline solution, to remove any traces of serum from the plates (serum is one of the ingredients in the media). After aspirating the PBS, I added a solution of trypsin and EDTA to the plates, one by one, to detach the cells from the bottom of the plastic plates. Trypsin is an enzyme found in the digestive tract, and it literally “digests” the proteins that help the cells stick to the bottom of the plates. Olga and I then waited a few short minutes for the cells to detach, then pipetted in more media to inhibit the trypsin (to keep it from completely breaking down the cells).

I then sucked up the cells with a pipette, put them in a centrifuge tube, and ran the centrifuge machine for a few minutes. The result was a tube with a “supernatant” – layer of light pink liquid on the top — and a “pellet” — solid collection of cells on the bottom. I aspirated the supernatant, leaving the pellet at the bottom (this is a bit tricky – you have to be REALLY careful not to suck up the pellet, because then you’ve lost all your cells and you’re screwed). Next step was to “resuspend” the cells in new media and divide them into 10 plates (remember, we started with five, so we’ve doubled the number of plates). Because they have more space, the cells will spread out and grow more until they reach “confluence” – saturation.

OK, I get all that. But why did we do all of that to the fibroblasts? And what about the epithelium?

Olga explained: we want to grow as many fibroblasts as possible (hence “passaging” the fibroblasts from five into 10 plates so that they will proliferate more) because fibroblasts produce the “food” that epithelium need to survive. And the epithelium are the cells that we are interested in looking at, from an experimental point of view, but we can’t cultivate them without also growing fibroblasts.

Basically, when you put fibroblasts into low-calcium media (as I did with five of those plates Monday), the fibroblasts produce what are called “growth factors” that epithelium need to live. Olga threw out those flasks of fibroblasts a few days ago because she had gotten what she wanted from them – the growth-factor-filled media to give to the epithelium – and the cells were no longer any good (i.e., capable of producing more growth factors).

Ah-ha. That’s why you ask questions. To discover the “why.” To give your activity context. To provide you with a goal.