“I remember receiving whole fetuses at three, four months’ gestation. A baby this big,” Hayflick said during a 2012 interview, holding his hands about six inches apart. “I remember distinctly not being disturbed by that—don’t ask me why—to dissect that. I can’t explain it. But my constitution was such that it didn’t affect me.
“It was definitely going to end up in an incinerator,” he added. “If it was used for research purposes, some good possibly could come out of it for people.”41
The first fetus that arrived in his lab was a male. After several hours’ work dissecting the lungs, breaking up the lung tissue with the enzyme trypsin, and spinning the resulting cells in a centrifuge, Hayflick planted the cells in four rectangular Pyrex flasks known as Blake bottles.42 He poured in nourishing medium, carefully plugged each glass bottle with an amber-colored silicone stopper, put the bottles on a tray, and walked them into the 96.8-degree heat behind the heavy door of the incubation room beside his lab. He placed the bottles on their flat sides on a wooden shelf. This allowed the maximum amount of “floor space” for the cells, which didn’t float in the fluid medium but sank to the bottom. They would multiply until they covered the floor in a single layer.
It was about three days later that Hayflick spotted signs of growth in the bottles of cells from that first fetus: a near-transparent haze on the bottom of the bottle where the cells, once attached to the glass floor of the bottle, had begun dividing.43 He poured off the growth medium in each bottle, replaced it with a fresh batch, closed the door, and waited. By ten days later the cells covered the bottom of each bottle in a single confluent layer. (Normal, noncancerous cells stop dividing at this point, rather than continuing to multiply and pile up on top of one another. This property is called “contact inhibition.”)
Now Hayflick delegated to his technician, Fred Jacks—a young military veteran who would eventually earn his MD and become the first African American resident at Abington Memorial, a suburban Philadelphia hospital—the tedious job of “splitting” the bottles so that the cells coating the bottom of one bottle were halved and half of them were placed in a new Blake bottle. This involved a long series of steps that required Jacks first to use trypsin, the “jackhammer” digestive enzyme, to loosen the cells from the side of the bottle, where they were firmly stuck. Later he would bathe the loosened cells in growth medium and redistribute half of them to a new bottle by sucking them up into and then shooting them out of a glass pipette. His mouth was protected only by a wad of cotton placed in the upper end of the pipette. Labs all over the country used this technique, occasionally leading to unsavory accidents. Today most pipettes are operated by thumb-controlled pistons, much like syringes.
Not long after the initial split, the lung cells seemed to kick into much higher gear, multiplying fast enough that Hayflick soon instituted a strict schedule of splitting the bottles every third or fourth day. (The frugal Hayflick would take the spent culture medium home in lab bottles and use it to fertilize his roses, daffodils, and tulips.)
As two bottles became four, and four became eight, and eight became sixteen, and sixteen became thirty-two, it became clear to Hayflick that the bottles would soon take over the communal incubation room next door to his lab. So he set Jacks to freezing the cells, transferring them first into tiny wine bottle–shaped glass ampules not quite two inches high, which he would seal by melting closed the neck of the ampule with a quick pass through a Bunsen burner. Each ampule contained a fraction of an ounce of growth medium. And floating in each were between three million and four million cells.44
Living human lung contains many types of cells, even just sixteen weeks after conception. In a fetus of this age, there are cubelike endothelial cells lining blood vessels; taller, columnlike epithelial cells lining the thousands of tiny airways called bronchioles; and underlying smooth-muscle cells giving them support, to name a few. But holding the lung together is connective tissue, and its components are made by fibroblasts: long, spindly cells with tapered ends, shaped something like a compass needle. Fibroblasts, it turned out, were the fittest when it came to survival in the lab. They were the only cells still living in Hayflick’s bottles after a few weeks of life in the lab.
• • •
In the summer of 1960 Hilary Koprowski published a sleek, eight-by-eleven-inch booklet that featured write-ups on the work of all the labs at his rejuvenated institute. The Wistar Institute’s biennial report for 1958 and 1959 featured just one photo on its cover. It showed Hayflick’s fibroblasts. Inside, a description of the photo read: “These cells are from a normal fetal human lung with a [normal] number of chromosomes.”45
Hayflick’s excitement comes through between the lines of his written report explaining the significance of the cover photo. It was possible, he wrote, to grow from human fetuses cells that had not—not yet, anyway—turned cancerous in lab bottles. The fetal cell line pictured on the cover had been growing in such bottles in his incubator for six continuous months. It was named WIHL, for “Wistar Institute Human Lung,” and while it had repeatedly outgrown its bottles, so that it had had to be halved and transferred to new bottles dozens of times, “it still retains the diploid number of chromosomes,” Hayflick wrote, referring to the normal number of human chromosomes: forty-six.46
What was more, he went on, these WIHL cells didn’t have any of the telltale microscopic hallmarks of cancer cells: disorganization, irregular sizes, and bloated nuclei. In fact, they looked no different under a microscope from how they had when freshly harvested from the fetus months earlier. They were classical fibroblasts: elongated cells with slightly thicker middles and tapered ends. The WIHL cell strain “is an extremely important tool in this investigation and has very important implications in other fields as well,” Hayflick reported. “It is, from all indications, a normal human cell.” That, he added, made it an ideal cell for use in his quest to discover cancer-causing viruses.
But Hayflick’s research into whether viruses caused cancer was about to get left behind, supplanted by a discovery that he didn’t anticipate and that would stand scientific wisdom on its head.
CHAPTER FIVE
Dying Cells and Dogma
Philadelphia, 1960–61
Science, my boy, is made up of mistakes; but they are mistakes which it is useful to make, because they lead little by little to the truth.
—Jules Verne, Journey to the Centre of the Earth1
Biologists had been growing cells in culture since 1907, when Ross Harrison, the workaholic embryologist at Johns Hopkins, first coaxed those bits of frog brain to grow in a lab dish. For virtually all of that time, they had operated under this simple piece of received wisdom: cells grown in lab dishes, properly treated, should live indefinitely. If they died, the fault lay not with the cells but with the scientist. His glassware wasn’t clean, or her medium didn’t contain just the right mix of nutrients, or a sloppy technician had sneezed on a plate of cells, launching a fatal infection. This faith in the open-ended life of cells in the lab grew out of the work of a bald, bespectacled, publicity-seeking French scientist named Alexis Carrel. In 1912 Carrel was awarded the Nobel Prize for inventing a much-needed method for surgically joining the two ends of a severed artery. But for Hayflick and other cell culturists, Carrel’s half century of influence resulted from an entirely different experiment.
In the early years of the twentieth century, the charismatic Carrel worked at the Rockefeller Institute in New York City, then the pinnacle of American medical science. One mid-January day in 1912, Carrel took a snippet of tissue from the heart of an eighteen-day-old chick fetus and put it in a lab dish, where he began nourishing it with diluted chicken blood plasma. (“Plasma” is another word for blood serum.) Two months later, having grown so “abundantly” that it had been split and planted in new vessels eighteen times, the heart tissue was still alive and even, he reported, beating in the lab dish. Carrel published a paper reporting on his f
indings, entitling it “On the Permanent Life of Tissues Outside of the Organism.”2
When, later that same year, he won the Nobel Prize for his artery-joining advance, many scientists and journalists mistakenly thought that Carrel was being honored for the launch of the undying chicken heart. The miraculous beating tissue “was the leading topic of discussion by medical men the world round,” the New York Times reported in an article on Carrel’s Nobel Prize that consumed the entire front page of the broadsheet’s Sunday magazine section.3
Carrel soon handed off the work of maintaining the beating bit of chicken heart to a laboratory colleague, Albert Ebeling, who looked after it for the next thirty-four years, feeding it with medium, dividing the cells when they outgrew their space, and dispensing with most of them while keeping a residual piece of heart always going in a dish. The undying chicken heart became a favorite with the popular press. “Isolated Tissue Holds Life 12 Years in Test . . . Growth Continues as in Body,” the New York Tribune proclaimed in 1924, as the chicken heart approached its twelfth “birthday.”4 So did Carrel himself, who at one point landed on the cover of Time magazine—although he likely hastened his retirement from the Rockefeller Institute when in 1935 he published a book, Man, the Unknown, propounding the use of gas to euthanize criminals, both sane and insane.5 The heart-in-a-dish outlived him; Carrel died two years before Ebeling finally disposed of the culture in 1946.
The lesson from Carrel’s chicken heart was not lost on the biologists who struggled through the first half of the twentieth century both to repeat his chicken-heart experiment—no one could—and to keep other kinds of cells alive indefinitely in culture. When they failed, as they did repeatedly, it was surely their incompetence that was to blame. That belief was only strengthened when, in 1943, a round-faced, bespectacled cell culturist at the National Cancer Institute coaxed a single cancerous mouse cell into unending lab-dish life.6 Wilton Earle’s mouse cells became the first demonstrably immortal cell line—one that scientists could obtain from Earle and observe for themselves replicating endlessly in the lab.
It was 1951—the year that Hayflick earned his undergraduate degree—before the same feat was repeated with human cells. That year an innovative and determined cancer researcher and cell culturist named George Gey, working at Johns Hopkins University, launched the first human cells to survive indefinitely in the lab, by taking the cells of an extremely aggressive cervical cancer from the womb of a dying woman named Henrietta Lacks. Within a few years the HeLa cell line was being studied and used in biology labs all over the world.
With the perspective of hindsight, it’s easy to say that Earle’s mouse-tumor cells, Gey’s HeLa cells, and other undying cell lines that soon followed in the 1950s were able to live endlessly in the lab only because they were cancerous. After all, it is the very definition of cancer that cells escape the normal constraints on growth and divide uncontrollably and endlessly. But for Hayflick, working nearly sixty years ago, things were not so clear-cut. Indeed, there was no reason to believe that normal, noncancerous fetal cells—just like Carrel’s normal chicken heart cells—shouldn’t also live open-endedly in lab dishes.
So when one day in the winter of 1960 Hayflick noticed something amiss in the incubation room, he started looking for his own mistakes. By now there were several other fetal cell lines in addition to WIHL—the lung cells from the first fetus he had received—multiplying in glass bottles on the wooden shelves.
During late 1959 he had continued to receive fetuses at random intervals from the Hospital of the University of Pennsylvania. With them he had developed cell lines from several fetal organs: skin and muscle; thymus and thyroid glands; kidneys and heart. He found that when the bottles were first planted with cells, it took about ten days for the cells to establish themselves, growing to cover the floor of a bottle in a semiopaque sheen that was visible to a practiced eye. (Hayflick labeled this initial stage of the cells’ growth, when they grew to first cover the bottom of the bottle, phase I.)
Once he had split the contiguous cells, putting half of them into a new bottle, all of the cells began dividing much more rapidly and needed to be split again every three to four days. (He called this period of rapid division, which went on for months, phase II.) He found that no matter how large the variety of cell types in the organ he started with, all of the cultures ended up consisting of only one cell type: the long, tapered fibroblasts that spin the connective tissue that holds organs and cells together. For whatever reasons, other kinds of cells did not thrive in his bottles.
As he planted these cell lines, Hayflick became systematic about naming them. Since he soon developed a second cell line from fetal lungs, he needed to rebrand the first line something other than Wistar Institute Human Lung. He decided to keep it simple and name the lines in numerical order. WIHL he renamed WI-1. The next line he named WI-2, and so on. By September 1960 he would launch WI-25, the last in the series. It came from the lungs of fetus number 19, a female. (There were fewer fetuses than cell lines because Hayflick derived more than one cell line from several of the fetuses, using different organs. For instance, the second line he developed, WI-2, was grown from the skin and muscle of fetus number 1.)7
As was normal in science, there had been mishaps. WI-2 had been lost to bacterial contamination after growing for several months. And cells from some organs, he was learning, grew better in the lab than others. Cell lines from the heart were sluggish: his WI-6 cells had given up and died after not even three months. But the kidney cells seemed to be doing well, and his WI-1 lung cells were star growers. He had begun to suspect that lung fibroblasts were the best suited to life in the lab.
But what was wrong with the WI-3 cells on this day? The WI-3 line had been grown from the lung cells of fetus number 2 and, if his hunch was right, these cells should have been dividing and thriving, just like the lung cells of WI-1. But he had noticed during the past several weeks that the WI-3 cells were taking longer than they had in the past to grow to confluence on the bottom of their bottles. What was more, their culture medium wasn’t turning from pink to yellow as quickly as it once had. The change in the fluid’s color resulted from acid production and meant that the cells were metabolizing actively. A slower progression from pink to yellow meant that the cells were slowing down in their activities of daily living—acting, in oversimplified terms, a lot more like seventy-year-olds than twentysomethings. And now, five months after the launch of the third line, WI-3, there was a new, worrisome sign. The culture medium was normally clear. But today it was cloudy with what he feared was the debris of dying cells.
Hayflick took the bottle of WI-3 cells next door to the lab and peered at the cells through his inverted microscope. He saw what he had expected he might see: there were grainlike bits of debris scattered around. What was more, when he scrutinized the tangled, dense, dark chromosomes that are visible at high magnification in actively dividing cells, they were very few and far between. The cells were slowing, perhaps stopping, their division.
Over the coming months the WI-3 cells would completely degenerate. Healthy fibroblasts tend to line up like soldiers in parallel formations, with each cell immediately next to its neighbors, their finely tapered ends pointing in the same direction. Instead, the WI-3 cells would become spread out in no discernible pattern, pointing in random directions. Black bits of debris, the detritus of dying cells, would litter the white spaces between the cells on Hayflick’s microscope field and cling to the cells’ surfaces like so much washed-up driftwood.
The changes would come on slowly, but at this first sign of the cells’ deterioration, Hayflick began trying to figure out where he was going wrong with WI-3. He adjusted the components of the culture medium—perhaps it was deficient in some essential nutrient—and poured the rejiggered fluid over the lagging cells. But even as he did so, he felt that it wouldn’t make a difference. The medium he used was made by technicians in the basement media room, in large batche
s that lasted for weeks. If WI-3 was struggling, then the other cells that he had bathed with precisely the same medium should be struggling too. Yet the rest of the cell lines were thriving. His suspicion was confirmed when the readjusted medium failed to resuscitate the WI-3 cells, whose bottles continued to grow cloudier with debris.
Perhaps he was screwing up in some other way. Could dirty glassware be the culprit? But the lab had been using the same glassware, scrubbed and sterilized by the same people working in the same glassware-washing facility in the bowels of the building. Perhaps, occasionally, dirty bottles slipped through due to some oversight, but what were the odds of those random dirty bottles always being used for WI-3 and no other cell lines? Virtually nil.
The only really plausible explanation was that WI-3 had become infected. It was easy enough to rule out bacterial contamination: he planted some sluggish WI-3 cells on plates of agar and left the plates incubating. No bacteria grew. There was no bacterial contamination stunting these cells.
Other microbes were trickier to detect. One key group of suspects was the PPLOs—those nuisance organisms that were smaller than bacteria but larger than viruses and that Hayflick had studied as a graduate student. They were a bane of cell culturists: always popping up like weeds where they weren’t wanted, even after a scientist thought they’d been eliminated with antibiotics. Fortunately for Hayflick, he had made himself into an expert on identifying them microscopically. Colonies of PPLOs had a “fried egg” appearance, but it took a practiced eye to pick this up. He checked the ailing cultures and found no signs of PPLOs.
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