A primary cell culture, right, containing GFP-labeled "touch" neurons, which allow nematodes to sense gentle body touch. Cell nuclei in the cultures are stained with DAPI (blue). A C. elegans L1 larva, left, expressing a GFP-labeled protein in four "touch" neurons.
Advance in C. elegans research developed
Michael Christensen
Micrographs of cultured embryonic cells expressing cell-specific GFP reporters. Left: A neuron expressing a GFP-labeled protein common to all C. elegans neurons. Right: A GFP-labeled cholinergic motor neuron physically interacting with a body wall muscle cell.
For some people, the words “it can’t be done” pose an irresistible challenge. Graduate student Michael Christensen is one of those people.
Christensen, a doctoral student in the laboratory of Kevin Strange, Ph.D., had a question to answer and no good way to address it. Bringing dogged determination and an open mind to the fore, he developed a breakthrough technique that has not only helped him answer his own research questions, but may also help answer the questions of countless researchers worldwide.
Christensen and co-workers developed a method for large-scale culture of embryonic cells from the popularly used model organism C. elegans. The culture methods, published last month in the journal Neuron, allow the cells to undergo extensive differentiation within 24 hours of isolation, and keep the cells alive and functional for many days to weeks.
The availability of cell-specific green fluorescent protein (GFP) reporter genes facilitates the isolation and study of differentiated cells—such as neurons, muscle cells, or intestinal cells—from this cell culture preparation.
“We really didn’t set out to develop culture methods,” said Strange, professor of Anesthesiology, Pharmacology, and Molecular Physiology & Biophysics. “We set out to answer a scientific question. It turns out, though, that nematode cell culture is viewed widely as an extremely important methodological advance in the field.”
One of the scientists who reviewed the details of the paper before publication described the methodology as “the most important advance in the field [of C. elegans research] in a decade.”
C. elegans, a tiny, transparent roundworm made up of less than 1,000 cells, was first introduced as a model organism for genetic studies in the 1960s by bacterial geneticist Sydney Brenner. Others had attempted to culture C. elegans cells over the years, but they had little success.
“This organism was attractive to me as a way to study the genetic basis of physiological processes,” Strange said. “When I first started talking with people about studying C. elegans, I asked them how you make direct electrophysiological measurements in the worm and how you culture cells. The answer that I got was that it was too difficult or simply not possible.”
Strange and Christensen were undaunted by that response, however.
“Since we were new to the field, we had no preconceived notions of what was and was not possible,” Strange said. “We just went in and tried, and it turns out, you can do these things. You just have to look at the problem from a different perspective.”
Christensen wanted to perform two experiments. First, he wanted to determine whether a mechanically activated ion channel he had discovered in the developing worm embryo by electrophysiological measurements was also expressed in differentiated neurons and muscle cells. He also wanted to use RNA interference methods to knock out specific genes in the developing embryo and determine the effect on ion channel activity.
Though the location of specific cell types in the adult worm’s body is well-known, attempts to study the physiology of the individual cells—and the genetics underlying that physiology—have been hampered by C. elegans anatomy. The worm is very small—about one tenth as long as an eyelash—and a tough cuticle surrounds its pressurized body. These physical barriers make it difficult to bring the equipment necessary to measure electrical activity in direct contact with the target cells.
So the first hurdle, according to Christensen, was to gain access to the worm cells. Because of their large size, he chose to start with embryonic cells. But C. elegans embryos are surrounded by an eggshell—another physical barrier to overcome. Treating the eggshells with chitinase, a natural enzyme known to dissolve insect and crab shells, Christensen was able to remove the shell and create suspensions of the freed embryonic cells.
Calling on instincts developed from previous mammalian cell culture experience, he used trial and error to find the right ingredients to not only keep the cells alive and healthy, but also to allow them to develop into the specific cell types they were pre-programmed to become.
“It just worked out,” Christensen said. “I simply had to pay attention to what the properties might be of, say for example, the solutions surrounding the cells. As problems would arise, I would just address each one individually, one at a time. Eventually, we had a tried-and-true method that works, and it works every time.”
Strange said he didn’t really expect this level of success.
“I actually encouraged Michael to try it to see if we could keep some of these embryonic cells alive for a couple of days in culture to do some specific experiments,” he said. “After two days, he called me over to the microscope and, not only did they survive, but they were turning into neurons and muscle cells and epithelial cells.”
The researchers first introduced these cell culture methods to other scientists last June at the 13th International C. elegans Meeting in a number of poster presentations. According to Strange, the techniques generated considerable excitement among the other scientists there.
“We were mobbed,” he said. “We had a poster one day and you literally couldn’t walk down the aisle for several hours—you just couldn’t get close to it.”
At the request of one of the meeting organizers, Strange consented to lead a workshop on the culture methods. Arriving early to the designated room, he saw only about 40 chairs set up.
“I had to tell the organizer that I thought it might get a bit crowded, given the response we had seen at the poster sessions,” he said. “She scrambled around and found a 500-seat auditorium that ended up being filled almost to capacity for this workshop.”
Strange has fielded a steady stream of requests for the cell culture protocol, and has had almost a dozen researchers visit the lab to learn the techniques, including one from another country. The lab is trying to figure out the best way to handle the overwhelming response.
Lab manager Becky Morrison, who refined the method into a standard lab practice and who has taken on the yeoman’s job of teaching it to others, said she has enjoyed watching the process unfold.
“It’s really taken on a life of its own,” she said. “I lucked out because Michael had worked out most of the bugs, so I’ve just had to tweak it a bit for specific experiments. It’s been fun to share this with people who are excited about taking it in different directions in their labs.”
Several Vanderbilt researchers have also been trained and have incorporated the methodology into their research and grant proposals. Strange is principal investigator on a recently awarded R-21 exploratory grant from the National Institutes of Diabetes & Digestive & Kidney Diseases that is supporting further development of these methods. He also has a number of other nematode cell culture projects in the works that are supporting new grant applications.
Strange believes being able to study C. elegans in this new way will be attractive to other researchers who might not have considered using the model previously.
“C. elegans, which has a fully sequenced genome, is genetically tractable, and in which it is relatively easy to manipulate gene expression, can now be used to answer physiological and cell biological questions that it was not possible to address without direct access to individual cells,” he said. “Nematode cell culture will bring scientists in from outside the field, just like where we were three years ago.”
Morrison said that it was Christensen’s personality as much as anything that figured into the success of the methodology. “Michael’s just the type of person that, if you tell him something can’t be done, he thinks ‘Well, maybe not in your hands. Give me a try.’”
Christensen, who will soon defend his dissertation research, no doubt has plenty of “tries” ahead of him in his burgeoning career.
Other Vanderbilt researchers who participated in the characterization of the cultures and are co-authors on the Neuron paper include Ana Estevez, Xiaoyan Yin, Rebecca Fox, Maureen McDonnell, Christina Gleason, and David Miller.
The work was funded by several grants from the NIH, a NIDDK C. elegans program project grant headed by Strange, and support from a NSF post-doctoral fellowship.