There was still the possibility that a virus was dooming the WI-3 cells. Hayflick examined them under his microscope for telltale signs of viral invasion. Viruses produce typical microscopic signs when they sicken cells. Cells become strangely shaped. They bloat. They may detach from the glass bottles that they normally adhere to. They can develop “inclusion bodies,” which are clumps of abnormal protein in the nucleus or cytoplasm. Hayflick saw none of these signs in the WI-3 cells. Of course, no matter how hard he looked, he couldn’t prove a negative—that is, he couldn’t prove that an undiscovered, undetectable virus wasn’t lurking in the cells. This was a reality that would eventually come to dog him.
Hayflick did all he could to revive WI-3. He kept splitting the cells into new bottles. There they continued to languish. Then he tried crowding several bottles’ worth of degenerating cells into one bottle. Nothing changed.
Had Hayflick not been so industrious and launched twenty-five cell lines from the organs of nineteen fetuses over the course of months and months—rather than, say, deriving just two or three cell lines—he might at this point have concluded, like so many scientists had before him, that he was simply falling short of the high standard set by the illustrious, Nobel Prize–winning Carrel. He might have concluded that his own ignorance was the culprit and the WI-3 cells were its victim.
However, as WI-3 languished in front of him, Hayflick was watching over his other cell lines. They were growing well until one day a few weeks later, when he again visited his incubation room and noticed that some other Blake bottles were becoming cloudy with debris. They were the bottles bearing WI-4, a cell line launched from the kidneys of fetus number 3 that had been doing very well—until recently, when the cells had begun multiplying more slowly. Now, like the WI-3 cells before them, they were grinding to a halt, succumbing to something. What was more, his bottles of WI-5, derived from the muscle of fetus number 3, were beginning to take longer to grow to confluence. By the time these muscle cells stopped dividing, the next group of cells, WI-7, from the thymus and thyroid glands of fetus number 4, had begun to grow sluggishly.
As Hayflick watched his newer cell lines flourish in their bottles while the cells from the first fetuses languished and finally died around them, he took his perplexity to several Wistar colleagues. One of them was Lionel Manson, a portly, avuncular immunologist with a dry, self-deprecating sense of humor and a razor-sharp intellect.
“I was telling Lionel what I found and I said, ‘I’m weighing several different explanations,’” Hayflick recalled in a 2014 interview. “And he just said cavalierly: ‘Have you thought about it having to do with aging?’ And I said: ‘No. But,’ I said, ‘aging is a wastebasket’—at that time it was; it still is to some extent—‘a trash basket into which you put everything you can’t explain.’”8
Still, Hayflick took Manson’s flippant suggestion back to his lab and pondered it. And the more he thought about it, the more convinced he became of its merit. It was the only theory that was supported by his now-voluminous data and his months of observations. And it led him inescapably to one conclusion: there was nothing wrong in his methods. What was wrong was the scientific faith in the immortality of cells in lab dishes that dated back nearly fifty years, to Carrel and his never-dying chicken heart. True, cancer cells like Wilton Earle’s mouse cells and Gey’s HeLa cells had been living in labs for years now, and it seemed they would go on doing so. But normal, noncancerous cells in a lab bottle were not immortal. They aged and died, just like human beings. The truth of it was staring him in the face.
Hayflick rehearsed the objections that he could already hear a chorus of critics raising. True, he couldn’t explain how it was that Carrel’s chicken heart tissue had continued to live for decades. But wasn’t the standard of scientific credibility the repeatability of an experiment? No one had been able to repeat Carrel’s experiment. The reason, he was now increasingly certain, was because it wasn’t repeatable.
There was also the matter of WI-1, the first cell line he had launched, back in September of 1959. It was still going strong, dividing energetically, six months later. But in this waning winter of 1960, he was now almost certain that this could be explained: WI-1 was a particularly hardy, long-lived line, but not an immortal one. He would bet money that sooner or later it would die too.
• • •
There are two subtle but important points worth making at this juncture about nomenclature; both will bear on this story as it moves forward. The first arises from what is actually happening in a bottle of fetal fibroblasts lying on its side incubating. If one takes a bottle, the flat side of which is covered with cells that have grown to confluence, and splits the cells in half, planting half of them in a new bottle of the same size, it might seem to make sense to conclude that, once the bottoms of the two bottles are covered with cells, every cell in that initial bottle (the original bottle is commonly called the “mother” and the new one the “daughter”) has divided once. One would be wrong.
Cells, like people, vary in their vigorousness. Some cells divide more sluggishly, while some are eager, rapid replicators. So over a given period of time, some cells will replicate fewer times than others, some perhaps not at all. Which means that the only conclusion that can be drawn when the floors of the two bottles are eventually covered with cells is that the initial population in the mother bottle has doubled in size. For it now covers twice the area that it did.
This is why biologists don’t speak about individual cells doubling or say that “these cells have now doubled in number five times.” Instead they refer to “population doubling levels,” which they describe with the acronym “PDL.” Given the inherent variability of individual cells, it’s the only accurate term to use.
There is a second issue with terminology that can be confusing. It is this: Whenever a biologist splits a confluent culture of cells and puts some of them in one or more new bottles, this is called a “passage,” because cells have “passed” from one bottle into another. However, scientists will often use the term “passage” as a synonym for a cell population’s doubling. This is accurate if, and only if, as the cells move through a sequence of passages, half of them are placed into just one new daughter bottle of the same size as the mother bottle. This is called a 2:1 split.
However, cells can be placed into any number of new bottles. For instance, if three quarters of the cells are removed from a confluent mother bottle and placed in equal portions into three additional daughter bottles of the same size, and then the cells in all four bottles are allowed to grow to confluence again, the original cell population will clearly have quadrupled in size. Yet it will have been through only one “passage,” in that the cells will have been “passed” into new bottles just once. The terms “passage” and “PDL” will be used interchangeably in this book—only because, in the experiments involved, the cells were routinely put through 2:1 splits.
• • •
As the winter of 1960 turned into spring, the U.S. Food and Drug Administration approved the world’s first officially sanctioned oral contraceptive pill. The University of Pennsylvania prepared to open its new Women’s Residence Hall. And in a speech to newspaper editors in Washington, DC, the Roman Catholic Democratic presidential candidate John F. Kennedy announced that “my religion is hardly, in this critical year of 1960, the dominant issue of our time.”9
The Hayflick family had recently moved into a modest, three-bedroom brick house in the leafy suburb of Penn Wynne, just northwest of the city on the edge of Philadelphia’s fashionable Main Line. Across the street was an expansive, hilly park where the Hayflick children spent hours playing hide-and-seek, hunting for frogs in a stream, and swinging on a rope swing with a wooden seat. The couple would soon add a fourth bedroom to accommodate their growing family: daughters Rachel and Annie were born in 1963 and 1965 respectively.
That summer and fall Hayflick’s fetal cell lines continued to age
and die. He dubbed the stage in which their dividing slowed and stopped and they degenerated and finally died “phase III.” And he wrestled with the question of how to prove that it was something intrinsic to the cells—some inherent property and not anything in their environment—that was the cause of their mortality. Hayflick’s former colleague and friend from Galveston, the chromosome expert Paul Moorhead, had since moved to the Wistar. In a 2012 interview Moorhead recalled that it was he who proposed the simple, elegant experiment that did the trick.10
Moorhead’s lab at the Wistar reflected his lowly designation as a postdoctoral fellow: it was a tiny cupboard of a place up on the third floor. But the chromosome aficionado from Arkansas had there what counted most: a Zeiss Jena made by the Reichert Company—in his opinion, the best microscope that money could buy. Leaning over the microscope’s sturdy black base, he could paste both eyes to the eyepieces and peer at chromosomes at eight hundred magnifications.
About the time that U.S. voters went to the polls and elected the forty-three-year-old Kennedy to replace the seventy-year-old Dwight D. Eisenhower, Hayflick took his oldest and his youngest fetal cell lines and mixed them. The first were the now-elderly WI-1 cells, which, as he had expected, had stopped dividing in late summer, after eleven months. They were now in phase III—still alive, still metabolizing, but ever so slowly. They had been split in their bottles forty-nine times and might perhaps divide once or twice more, if they could screw up the energy. But basically, they were reaching the end.
To these WI-1 cells Hayflick added youthful WI-25 cells that he had launched just weeks earlier and that were vigorously replicating. They would do so, he expected, for months to come, for the WI-25 bottles had been split a mere thirteen times. Hayflick then left the mix of young and old cells, both in the same bottle, being nourished by the same medium, to incubate.11
Moorhead’s stratagem relied on the fact that, apart from their ages, there was a singular difference between the two groups of cells. The WI-1 cells had come from a male fetus and thus bore one X chromosome and one Y chromosome. The WI-25 cells were from a female fetus, so they bore two X chromosomes. Both kinds of chromosome would be visible under the microscope to Moorhead’s expert eye.
After the cells had been incubating for about two months and had been split into new bottles seventeen times, Hayflick handed the mixture to Moorhead. The Arkansan expert carefully prepared the cells, using techniques that stained and spread the chromosomes so that they were individually visible, rather than messed together in a tangled pile. Then he studied them at hundreds of magnifications. He saw virtually only X chromosomes. The younger, female cells, now having undergone about thirty total divisions, were thriving. As for Y chromosomes, Moorhead spotted vanishingly few. The male cells, which had already been elderly at the start of the mixing experiment, were gone. Dead.
The mixing experiment had clinched the case. If the cell-killing factor had been in the glassware or in the medium, or if it had been some other technical slip, all of the cells would have been exposed to the problem and all of them would have been dead. What was more, as Hayflick and Moorhead wrote archly in a paper that would be named a “Citation Classic” because other papers referred to it so frequently, “If a latent virus had been responsible . . . it seems unlikely that it would have been able to discriminate between male and female cells.”12
Hayflick could now confidently assert that something intrinsic to the cells was behind what he was seeing over and over again with his own eyes. Something inside them was causing them to die. Admittedly, WI-25, the last cell line he had launched, was still dividing, as were several other more recently derived lines. But he was now sure that these too would eventually slow their replicating and then stop. Then they would degenerate and die. Not one of the twenty-five cell lines he had launched was immortal.
Hayflick was thirty-two years old and a virtual unknown in the rarefied universe of top biologists that he would have loved to inhabit. He was faced with the prospect of making an audacious claim. A claim that would challenge fifty years of received wisdom, along with the reputation of the Nobel Prize–winning Carrel. A claim that normal cells aged and, finally, died in their lab dishes. He was nervous. And his confidence wasn’t helped by a warning from one of the most respected cell biologists of the time. Gey, the talented developer of the HeLa cells, was visiting the Wistar Institute one day when Hayflick confided in him his new findings. In a 2012 interview Hayflick recalled Gey’s response: “Be careful, Lenny. You’re going to ruin your career.”13
Gey couldn’t have been more mistaken. Hayflick’s finding would one day make his name and distinguish him as the man who opened the door to a whole new realm—the study of cellular aging. It is an area of huge relevance to two of our top health preoccupations today: aging and cancer. But Hayflick had a long road to travel before recognition came.
And how to explain Alexis Carrel’s normal chicken-heart cells, replicating faithfully in the lab from their launch in 1912 until Carrel’s colleague Ebeling finally dispensed with them in 1946? Years later, in the 1960s, a woman who had worked as a technician for Carrel in the 1930s approached Hayflick after he gave a talk at a scientific meeting. In his lecture Hayflick had speculated that Carrel’s method had a fatal flaw related to the fluid that he and his technicians extracted from chick embryos and used to “glue” the chicken-heart cells to the floor of a new culture vessel whenever they overgrew their current dish and needed to be divided. This fluid extract from chick embryos was also used daily to feed the cells. In preparation for this, it was spun in an antiquated centrifuge, a process that was supposed to remove cells, leaving only nutritious fluid. Hayflick proposed that, with or without Carrel’s knowledge, the fluid extract actually contained errant fresh cells from the chick embryos; that the culture stayed alive for decades because it was frequently replenished with these new young cells. The woman told Hayflick that he was on the mark; that in the 1930s she had raised questions with Carrel’s chief technician indicating that she thought this might be happening. She had been told to forget what she was seeing or risk losing her job. In the midst of the Depression, that was not something she was eager to do.14
• • •
It is a measure of Hayflick’s productivity, energy, and ambition that in the autumn of 1960, as he and Moorhead conducted the male-and-female-cell-mixing experiment, he was also using his new human fetal cells to develop a first-of-its-kind polio vaccine—more on this will follow—as well as running several studies that he knew would be crucial to the successful reception of the landmark paper that he and Moorhead were now putting together.
(Hayflick also, at about this time, codiscovered the cause of walking pneumonia. The culprit was a species of Mycoplasma, the tiny microbes that he had studied as a graduate student. Mycoplasma pneumoniae was the first of these microbes discovered to cause disease in humans, and the New York Times splashed the discovery at the top of its front page.)15
That paper would boldly hypothesize that the cells’ deaths in their bottles were the outcome of “[aging] at the cellular level.”16 And it would define what later came to be known as “the Hayflick limit”—the number of divisions that a normal cell in culture can undergo before it ceases to divide. Based on the data from his twenty-five fetal cell lines, Hayflick estimated this number at fifty divisions, plus or minus ten. Importantly, freezing the cells didn’t affect the Hayflick limit. For instance, when Hayflick froze some WI-1 cells that had divided just nine times, and then thawed them months later and put them in the incubation room, they began dividing again and, seeming to “remember” their age, went through forty-one more divisions over five months before dying in their bottles. What was more, this fact—that the cells would commence dividing again after being frozen and thawed—meant that freezing appropriate numbers of them at young ages could ensure an all-but-endless supply of cells into the future.
But for what practical purpose would such an e
ndless supply of cells ever be needed? It’s in the answer to this question that the enormous and lasting public-health impact of Hayflick’s work rests. For in his groundbreaking paper with Moorhead, Hayflick went beyond an iconoclastic assault on the immortality of normal cells. He also suggested that the new cells could make a big contribution to vaccine making.17 He had conducted the experiments to prove it.
• • •
In 1960 the making of new viral vaccines was a top priority for virologists and a goal that was eminently within reach, thanks to the technical breakthroughs of the previous two decades. The fight against polio, fresh in everyone’s minds, had shown as much. It had been a terrifying bane—the disease stirred something like the fear that Ebola does today. But in the space of five years, since the launch of Jonas Salk’s killed vaccine in 1955, it had been reduced to a preventable disease. What was more, it was becoming clear that a stronger, longer-lasting live polio vaccine was within a year or two of approval by U.S. regulators.
Now the prospect of developing vaccines against other viral diseases beckoned to scientists. Measles, mumps, and German measles, also called rubella, were regular childhood afflictions. Hepatitis was rarer, and gravely serious. Chicken pox was a particular bane for children with weakened immune systems. For some diseases, like rubella and hepatitis, vaccines couldn’t yet be made, because the viruses hadn’t yet been captured in lab dishes. Virologists set out to hunt them down. For others, like measles and mumps, the viruses had been isolated, and scientists were hurrying to develop vaccines. The U.S. government soon provided the money and muscle to make sure that the new vaccines were used. In 1962 President John F. Kennedy signed the Vaccination Assistance Act, allowing the Communicable Disease Center (CDC) to support mass immunization campaigns and ongoing maintenance programs and to funnel vaccine money and resources directly to state and local health departments.
The Vaccine Race Page 9