The Tissue Culture Follies

Image: Anne MacNamara/R. Ian Freshney Like most researchers, you probably hate tissue culture work. But, if such 'trivialities' as experimental quality and reproducibility are important to you, then you and your cells will need to come to an understanding. Perhaps a short trip through the looking glass--that is, culture flask--will be of value. What do you have to lose? It's free, and all the seats are front row center! Cast: (in order of appearance) First researcher Cells Narrator Second researcher First technician Second technician Setting: A laboratory where live-cell research is ongoing. The first researcher is in his office working on his computer. The cells are in flasks in a water-jacketed cell-culture incubator 50 feet away. ACT 1: DINNER TIME! in which the researcher--and his cells--are hungry First researcher: (checking his watch) It's five-thirty, already? Shoot, I'm late for dinner, and I forgot to pass my cells! Can they wait? What is it, Tuesday? Let's see (thinking for a moment), the split ratio was 1:20 ... done Thursday afternoon. They should be fine. I'll do the cells first thing tomorrow. Narrator: Meanwhile, in the incubator... Cells: Good grief, it's really crowded in here. Hey, move over, would you? This is my patch of polystyrene; you can sit over there. Oh, for pity's sake, do you have to divide right now? Get a room! Can't you wait 'til those idiots feed us or subculture us? The pH has dropped at least three-tenths of a point in the last 12 hours and there's nothing left to eat. I'm either going to have to kill myself, die of starvation, or nod off in G0--and so help me, if you don't stop, I'm taking you with me. Just watch: tomorrow, I bet they'll expect us to do something we do only in log phase. What do they think we are, magicians? Narrator: An all-too-familiar scene? Alas, yes, and one likely to be repeated ad infinitum. An organized rebellion is not in the cards for these poor souls--I mean cells--and, in fact, it's unnecessary. Payback comes in many forms, and in this case, the currency is data--faulty data. 2:00 p.m., the next day. The second researcher is tracking down the first one in the coffee room. The second researcher is desperate because her boss asked her for some results that she doesn't have yet. Second researcher: I'm really in a bind: I need to make RNA today or I'm toast. Do you still have those cells going? Maybe you've got an extra flask hanging around? First researcher: Let me take a look. I owe you one. (As he ambles off toward the cell culture room, he mutters under his breath) Crud! I forgot to pass my cells this morning. The second researcher is right on the heels of the first researcher, and anxious. The first researcher yanks the tissue culture flask out of the 37°C incubator, sloshing medium into the cap. Leaving the inside and outside incubator doors open, he walks to the inverted microscope and examines the cells in the flask. Cells: Whoa, what was that tidal wave? Must mean they're going to transfer us. It's about time: I lost 60 friends overnight. Maybe this time they could actually count us instead of just splitting us. I bet they don't even know our doubling time. Second researcher: Boy, the medium is pretty yellow. How do they look? First researcher: Well, they're a tad overgrown, but they're not falling off the flask or anything. Second researcher: I'll take them. How bad could they be? Narrator: That depends on whether the mRNA of interest will be expressed in adequate amounts, or at all, when the cells are starving and dying. One way to avoid this kind of problem is to designate one lab member as the tissue culture aficionado--someone whose sole job is to make sure the cell lines are healthy and ready when a researcher needs them. Some organizations establish cell-culture core facilities to give the cells the best possible care and free up the investigators' time. The key is finding someone who enjoys taking care of things and is willing to spend time getting to know each cell line's individual characteristics. Some culturists liken the process to gardening: observing and nursing seedlings along, knowing what nutrients they require, and putting them in the right environment to elicit the best response. For in vitro assays, this means understanding the endpoint to be measured and using your knowledge of the cell line to assist in creating the largest window of detection. The shrewd cell culturist will manipulate cell density, substrate, and type of medium, and will help run a time course to determine when the strongest signal occurs with the lowest background in the control. Meanwhile, in another incubator labeled "Hybridoma Running: Hands Off!!"... Cells: Man, am I pumped! The technician just flushed us and hooked up a fresh bottle of medium. I've got room to flex, chow down, and get into hyperdrive. That researcher thought he got lots of antibodies yesterday; just wait until he does his next harvest. This new technician who's feeding and changing us is awesome. She's always checking the glucose level and making sure we're not overgrown or underfed. Sometimes she even talks to us. I feel so special! First technician: (to the cells) Oh, please don't die! I just need another 100 milligrams of antibody. I know you've been working hard for two weeks but I promise to come in Saturday and Sunday to make sure you're flushed and fed, and that the pH is okay. Narrator: It's like taking care of a baby. For two to four weeks, a successful hybridoma requires constant, almost obsessive-compulsive attention. Sometimes, when they're really cooking, the color of the medium will go from red to light orange in just eight hours. A decision to go home, rather than flush and refeed, can completely crash the system overnight, with no chance of recovery. Since hybridomas run for such a long time, excellent aseptic technique and careful resterilization of the ports using 70% ethanol is critical. ACT 2: A NEW ARRIVAL Image: Anne MacNamara/R. Ian Freshney It's 7:00 a.m. A Styrofoam box labeled "Dry Ice: RUSH," sits on the floor of the lab from a late evening delivery the previous night. The first researcher enters the lab, sees the box, and intends to open it and put the contents in the freezer, but becomes distracted by a phone call. The second technician trips over the box and shoves it into a kneehole under a benchtop, where it lies forgotten. Clock on the wall fast-forwards to 3:00 p.m. First technician: (Hollering across the lab) I'm expecting a shipment of cells. Has anyone seen a dry ice delivery? Second technician: I kicked a box this morning when I was using the pH meter. I stuck it under the bench over there. First technician: Thanks. I'll check it out. The first technician opens the box and notices that all the dry ice has evaporated. She removes a small vial of semi-frozen cells and stashes it in the -20°C freezer. She finds some documentation in the box and reads it over quickly. First technician: (again hollering, as she rummages through a refrigerator) Do we have any fetal calf serum thawed? What about RPMI and insulin? The cells will have to wait, or I could thaw them in whatever medium we have in the fridge. What are we doing with these cells anyway? Maybe I should put them back into liquid nitrogen. Narrator: Meanwhile, back in the -20°C freezer... Cells: Is it getting warm in here, or is it just me? I'm seriously considering metabolizing a little bit. What on earth is that garlicky smell? Oh yeah, they used DMSO when they froze us. This could mean they'll be letting us out soon. I wonder if they know we need insulin, and that nearly half of us won't survive the thawing process. I hope I'm one of the lucky ones: I've got 64 kids to think about. First technician: (talking to herself) Thank goodness I could get some fresh RPMI basal medium and insulin from down the hall. It wouldn't be good to leave those cells in the 'minus 20,' and maybe we need them right away. If not, I can still expand the culture so I can freeze back 10 or 20 vials of early-passage cells. That way I'll be sure we'll have enough to last for the whole project. Narrator: Though the technician was initially drawn to taking the easy way out, she went the extra mile to find the recommended medium for the new cell line and placed the already-thawing cells into culture. First technician: Let's see ... the spec sheet says that only 60% of them will survive the thaw. I'll add a little extra fetal bovine serum and dilute them 1 to 30 in the medium to lessen any residual DMSO effects. Cells: Aaah, paradise! I can feel the bottom of the flask. Pretty soon I'll be able to spread out a little, relax, and think about where I am in the cell cycle. Narrator: Each cell line is as individual--genotypically speaking--as the person or animal from which it was isolated. Purveyors of cell lines provide specifications that must be considered if the cells are to perform as closely as possible to the phenotype for which they were chosen. ACT 3: MOLD! in which the technician--and her cells--receive unwelcome guests First thing, the next morning. First technician: (to herself) I wonder how the cells I thawed are doing? I'll warm up their medium in the water bath and take a look. The first technician cleans the hood and her hands with 70% ethanol. Then she opens the outer door of the incubator and looks to see where the flask is located. Only then does she open the inner door. In one smooth motion, she tightens the cap and draws the flask out of the incubator, keeping the medium out of the neck and cap area. She scans the incubator shelves and walls for signs of mold and makes sure there's water in the humidity pan. Cells: I wonder if the technician noticed that dish touching the back wall. I definitely heard shouts from back there that mold spores were sneaking under the lid. I'm glad I'm in a flask and not a dish. It's gonna be curtains for them. First technician: Hmmm... The spec sheet was about right. There are plenty of dark, floating, dead, or dying cells, but the ones that are attached look bright and pretty happy. I'll wash them once with warm medium and then replace it with fresh medium to get rid of the residual DMSO. The first technician logs in her notebook the time and date of thawing and the relative viability for the next day. After replacing the medium, she carefully returns the flask to the incubator, again scanning for mold. She notices a dish toward the back of the chamber whose medium is pinker than normal, and removes the dish carefully. The tiny spots of mold are immediately apparent to her trained eye. Using the microscope she confirms the presence of tentacle-like hyphae extending beyond the moldy dots. Technician: Sorry guys, no hope for you: Mold is just too hard to get rid of. I'll have to wrap and autoclave you, but first I better check for other infected plates. I might need to clean the incubator, too. Narrator: Cleaning the incubator is never a bad idea, but just as when you clean your house, the incubator will be dirty again within hours of finishing. Cleaning the trays frequently with 70% ethanol, even buying new shiny ones--and keeping them shiny--is a good idea. Mold seems to love spilled medium and rough surfaces. Yet, the incubator is not the cells' biggest threat. More often than not the phrase 'We have met the enemy and he is us' rings true in the lab. Following aseptic technique is critical to preventing contamination. Learning from someone with a history of success, asking lots of questions, and having a book on cell culture on hand is a great way to start. There are probably as many ways to culture cells aseptically as there are people who culture cells. A tissue culture professional cannot become a cell and live in a tissue culture flask, of course, but viewing the world from that perspective can be an asset. As much art as science, tissue culture represents the starting point for so much of the work researchers do, and its quality can make or break an experiment. Painstaking attention to detail is not only a requirement for success, but often allows the culturist to contribute to both experimental design and analysis. Just remember the cell culture mantra: "Observe, observe, observe." And then, observe again. Susan Jenkins can be contacted at sjenkins@the-scientist.com.

The Tissue Culture Follies

Interested in reading more?

Become a Member of