Erica P. Johnson
Your stem cells have just arrived. Now, how do you convert those frozen vials into a thriving, human embryonic stem cell (hESC) research program? Last month I traveled to Wisconsin to get a primer from Daisy Manning, head instructor of a training course at the WiCell Research Institute in Madison, called "Introduction to Human Embryonic Stem Cell Culture Methods."
In a special student lab overlooking a nondescript research park through bulletproof glass, Manning took me through a gratis crash course on hESCs – a one-day version of the typically three-day class. Two hundred twenty students have rotated through this lab since WiCell began offering the class in 2003, paying $900 each for the opportunity. The class covers every aspect of basic stem cell culture, from preparation of the murine embryonic fibroblast (MEF) feeder layer to freezing down hESCs – everything but directed differentiation.
Manning encourages all...
1. HURRY UP AND WAIT
Don't expect to jump right into hESCs research, says Manning. "We want people to understand that even if your lab is ready to go, it is still going to take you about 16 weeks minimum, to go from deriving your MEFs to actually growing up a lot of human embryonic stem cells and being able to freeze them down. So even with a complete lab, you're looking at 4 to 6 months before you can even experiment with these cells."
2. DON'T BUY COMMERCIAL MEFs
Though it is possible to buy MEFs, WiCell recommends making them in-house. "It only costs $26 for one pregnant mouse, which can yield 12 to 15 vials of MEFs. It costs about $50 to get 2 to 4 vials of MEFs from a commercial vendor, so you're paying twice the cost for one-sixth the MEFs. ... In addition, you never know what you're going to get from a commercial vendor. We only use our MEFs to passage 5, and after that, they really start dying off. A lot of commercial vendors will sell MEFs at passage 3 or 4, which makes them useless for embryonic stem cell culture."
3. CHECK THE SOURCE
MEFs must be irradiated to halt their growth. WiCell's protocol calls for a dose of 8,000 rads, but this number is variable, says Manning. "You would think 8,000 rads is 8,000 rads, but we found it does vary with the source." With a cesium irradiator, 8,000 rads seems about right, she says, but with an x-ray machine the required dose is more like 12,000 rads. You can also inactivate chemically with mitomycin C, but Manning recommends irradiation if possible. "Embryonic stem cells are extremely sensitive to chemicals," she says. "When you're chemically inactivating your MEFs, you need to ensure there are no traces of the chemical on your cells before you add the embryonic stem cells."
4. THAW FAST!
Thaw cells as quickly as you can possibly do it without cutting corners, says Manning. "I've found a few cases where I've thawed a vial and a new person at WiCell thawed out a duplicate, and she got about 50% of the colonies I got even though they were the exact same cells... A difference of 30 to 45 seconds makes a big difference in the amount of cells you recover." Manning recommends practicing the protocol several times before actually thawing your hESCs, to minimize delays.
5. A FORTNIGHT TO RECOVER
Don't expect a completely dense culture right after a thaw, says Manning; only 0.1% to 1% of cells survive the procedure. "We always tell people, feed every single day because something might pop up in 3 days, 5 days, maybe even 7 or 8 days, all of a sudden a little colony will pop up." A thaw isn't deemed a failure until 14 days have passed. And don't neglect the plate's edges, she adds. "Frequently you find a lot of colonies around these edges." (Visualize these by placing your finger along the rim of the plate to block the diffraction of light.)
6. FEED YOUR CELLS DAILY
hESCs are much less forgiving than other cells, so feed and assess them every day. "You really have to nurture these cells to make them happy," says Manning. And never let them overgrow, she adds: They generally need to be split about once a week. hESC form tight, homogeneous, flat colonies with sharp borders, while differentiating colonies have ragged, uneven edges or transparent centers. Under low magnification, healthy colonies appear white, while colonies that have "balled up" in solution and then settled appear yellow; these latter colonies mimic a developing embryo and will likely differentiate.
7. WORRY, BE HAPPY
An experienced hESC veteran will worry about his or her cells, says Manning. "If you worry, you'll look at them under the microscope a lot, get a real sense of what they look like, what they look like before they differentiate. You learn to anticipate when the cells are going to do something tomorrow." And that's important, Manning adds, because hESC work really is more art than science. There's much the class cannot teach, "but we teach you how to make the correct judgement."
8. DIFFERENTIATION HAPPENS
Though it makes culture maintenance difficult, ESC differentiation is a good thing, because it indicates a healthy culture. A trouble-free culture should raise flags. "If it looks too good to be true, it probably is," says Manning. "When your embryonic stem cells are all of a sudden growing really well, and really fast, and they always look great, and they are never differentiating, that's when you should be suspicious that something is wrong." At that point, karyotype.
9. DECIDE ON A COURSE OF ACTION
"We have an informal rule for deciding what we want to do with our plate of human embryonic stem cells," says Manning. Under 10% differentiation, pass the cells as-is. Between 10% and 50% differentiation, remove those cells by a process called "pick-to-remove." Above 50% differentiation, mechanically remove the undifferentiated colonies to a fresh plate of MEFs, using a process called "pick-to-keep."
10. AVOID PICK-TO-KEEP
If you're picking-to-keep often, says Manning, you're probably doing something wrong. "We generally say that you should find yourself picking to remove about once every other passage, give or take. So therefore, you should never hit over half being differentiated. ... If you find yourself picking to keep, say more than once every other month, maybe once every three months ... you're neglecting your cells. You should be doing pick-to-remove, not pick-to-keep."
11. DON'T BE A PERFECTIONIST
Picking too frequently can cause abnormal cell outgrowth, so it's important not to be a perfectionist, says Manning. "If you see a few patches of differentiation, maybe 2% to 3% of the culture, that's not that much and you can be sure those colonies are probably normal." But if, month after month, you constantly pick to produce a pure, undifferentiated culture, you can promote an abnormality – trisomy 12 or 17, for instance. "So don't be overzealous. We've learned that ourselves by picking too much, and suddenly it's 40% trisomy 17 in the culture." Plus, there's no better way to contaminate your cells than picking.
12. DON'T USE ANTIBIOTICS
"We find that if you have perfect cell culture technique and perfect sterile technique, there's no need for antibiotics," says Manning. Besides, they could adversely affect your culture. "You never know when you're adding another element to your media how it's going to affect your embryonic stem cells. Those antibiotics could select for abnormal cells for all you know."
13. DON'T FORGET TO KARYOTYPE
Not Just for WiCell Lines
Though WiCell's protocols pertain directly only to its lines, chances are good they pertain to you, too. Of the 22 federally approved hESC lines, five come from the Wisconsin Alumni Research Foundation, which established WiCell in 1999 to, among other things, distribute those lines to researchers around the world. WiCell spokesperson Andy Cohn says the Institute has fulfilled 300 requests to date, at $5,000 each. That represents some 40% of all hESCs shipped, according to NIH statistics.
WiCell distributes cells between passage 20 and 25 that have been karyotyped to ensure they are genetically normal. But you should have them karyotyped, too, says Manning. "We recommend you get them karyotyped before you start experiments, just to make sure they are normal. Then, when you start getting really great data, it's good to check before you finish, and again before you publish. We also recommend you karyotype every 10 passages after passage 50."
14. FREEZE EARLY, FREEZE OFTEN
Build up an emergency supply of frozen cells as soon as possible after thawing. Says Manning, "once you're at the point of expanding, start freezing, a few vials at a time. You want to start freezing as soon as possible, because you want the lowest passage cells. Don't wait until you have lots of cells to start freezing." Cells are ready to freeze about two days before they are ready to split, at about day 5, says Manning. That's when the cells are growing most rapidly and will be thaw most robustly.
15. IT'S A FULL-TIME JOB
Manning stresses that hESC culture cannot realistically be a side project. Between culturing MEFs and splitting and maintaining the hESCs, the culture work consumes more than 20 hours per week. As a result, it's critical that you train another person to cover your culture needs in the event you are sick or just need a vacation. "It's too much of a commitment for one person to do alone," she says, "so always have a backup."
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