In a basement laboratory abutting an 1,800-acre wildlife park in San Diego, California, Marlys Houck looks up to see a uniformed man holding a blue insulated lunch bag filled with small pieces of eyes, trachea, feet and feathers.
“Ah,” she says, softly. “Here are today’s samples.”
The bag in question contains small bits of soft tissue collected from animals who have died of natural causes at the zoo. Today, the samples include a leaf frog and a starling.
The man holding the bag is James Boggeln, a volunteer with the zoo, who hands it off to Houck, the curator of this laboratory, known as the “Frozen Zoo”. She and her team will start the process of turning these bits of tissue into a bank of research and conservation for the future. They will put the tissue into flasks where enzymes digest them, then the lab members will slowly incubate them over the course of a month – growing an abundance of cells that can be frozen and eventually reanimated for future use.
At nearly 50 years old, the Frozen Zoo holds the world’s oldest, largest and most diverse repository of living cell cultures – more than 11,000 samples that represent 1,300 different species and subspecies, including three extinct species and more that are very close to extinction.
Today the Frozen Zoo is operated by an all-female team of four, who watch over a vast collection of hand-marked vials with labels such as “giraffe”, “rhino” and “armadillo”, all stored in massive circular tanks filled with liquid nitrogen. In a world suffering from a climate and biodiversity crisis, putting species on ice offers one way to be hopeful about the future.
The work done here has always felt meaningful, but an accelerating extinction crisis has put mounting pressure on Houck and her team. It’s a race against time to put samples into the Frozen Zoo before they slip away from the world outside the lab. The women who hold these jobs see it as their duty to hold the future in place.
The work can be painstaking – samples from birds, mammals, amphibians and fish all require different processes, for example. But with stakes so high, Houck describes it with a kind of sacred reverence.
She feels the pressure of the role – when her predecessor was in charge, a mechanical failure led to the loss of 300 samples, a year’s work. So her mind is focused on keeping the samples the zoo has frozen safe, Houck says – “but then combined with the excitement and this joy, because it’s an honor to be able to do this”.
‘Collect things for reasons you don’t yet understand’
The zoo was founded by a German American pathologist named Kurt Benirschke in 1972, who started a collection of animal skin samples in his lab at the University of California at San Diego and then moved it to the San Diego Zoo a few years later. At the time there was no technology to use it beyond basic chromosome research, but Benirschke often quoted the American historian Daniel Boorstin: “You must collect things for reasons you don’t yet understand.”
That quote still hangs on a poster in the Frozen Zoo, where Houck pulls vials from tanks of liquid nitrogen that resemble giant silver thermoses the size of a human. The tanks are pressurized at -320F, a temperature that stops cells from moving or changing – keeping them alive but in suspended animation. From this temperature, the cells can be revived and continue living as if decades – or centuries – hadn’t passed.
No species is exactly the same, and some groups are more challenging to preserve than others. The Frozen Zoo started with mammals, then expanded to cryobanking birds, reptiles, and amphibians. The success rate with mammals is close to 99%, Houck says. “With amphibians, it was close to 1% for a number of years, and now I think maybe we’re 20 to 25%. Birds are pretty high.”
Racks in the nitrogen tanks hold 100 vials each, and each vial contains 1m to 3m living cells. Those cells – a giraffe, a lemur, or something more endangered like a vaquita – hold possible solutions for an array of existing and future problems.
Eventually, the cells could be used to bring back fully extinct species – but that’s not the main goal. Instead, the material is generally used to rescue existing species that are struggling. In 2020, the Frozen Zoo used cryopreserved DNA to clone a black-footed ferret, the first endangered species in the United States to be cloned. Last year, frozen cells cryopreserved 42 years ago were used to clone two critically endangered Przewalski’s wild horses, bringing valuable genetic diversity back to the living population that will make it more resilient to new illnesses or environmental threats. One of the foals was named Kurt, after the Zoo’s founder.
The work that San Diego’s Frozen Zoo is part of a global movement to cryobank everything from animals to seeds. Today there are around a dozen wildlife-based cryobanks around the world, mostly located in North America and Europe.
The work done in San Diego has been particularly groundbreaking, says Sue Walker, head of science at Chester Zoo, and co-founder and vice-chair of the UK nonprofit Nature’s Safe, a cryobank that collects live cells and sperm and eggs. She says that in a few more decades, it might be possible to turn these cells into pluripotent stem cells, which can be reprogrammed to produce sperm and eggs.
In an ideal world, species could be conserved in the wild – but in reality, that’s not the case. “We’re losing species too fast for science to keep up,” she says. “So the least we can do is try and bank that material down.”
It’s tricky to get permits to bring in tissues from animals in other countries, so the hope is to grow the capacity in other places to cryobank locally, particularly near conservation hubs in Africa, South America and south-east Asia. But that means building up the capacity to process and preserve the population of cells in a uniform way. It’s expensive and complex work – but also necessary, Walker says.
“I think we have to throw everything at it in order to save some of these species that are on the brink of extinction,” she says. “It’s about banking hope.”
Time machines to past and future
Working on the cell cultures can be like operating a time machine. Houck was once studying rhino chromosomes and she opened a vial with her predecessor Arlene Kumamoto’s handwriting on it. Kumamoto had put the cells into a deep freeze the same month Houck graduated from high school. “I just thought, oh my gosh … she was freezing cells that I’m now using for my studies. If she hadn’t done that, I wouldn’t be doing what I’m doing,” Houck says. “What are we doing today that will be used in the future?”
Julie Fronczek, who has worked at the zoo for 24 years, looks up from her microscope to offer a theory about why a group of women is leading this work at the Frozen Zoo. “We’re nurturing the cells. They’re living creatures and they have to be fed and taken care of, then you have to know what they want when they want it,” she says. “It’s kind of like babies.”
The team adds around 250 to 350 specimens to the zoo each year. The leaf frog that arrived today is a high priority, Houck says. The starling that also arrived? Less critical. Such decisions weigh heavily on her. Since each new animal that arrives needs to be cultured and preserved, and takes up space in the giant containers, she has to consider how many of that species are already represented, and how many possibilities there are to get more of them. “I would like to not have to turn anything away. It would be better if we could accept every sample that came in because they’re all important.”
The collection includes three extinct species: the po’ouli or Hawaiian honeycreeper, Rabbs’ tree frog, and the Saudi gazelle. They’re holding a collective breath as they watch for more species in their collection to go extinct. “The next will probably be the white rhino and the vaquita,” Houck says.
The room holding the tanks is full – but the tanks aren’t yet at capacity, so the team continues its work of culturing and preserving the cells that could mean life or death for endangered animals in the future.
In the future, the lab needs to update its methods – those handwritten vials will eventually be scannable barcodes. It also needs fresh scientists to come and keep a mindful watch over the growing zoo on ice.
“I think we’re all very protective of the frozen zoo and the legacy and Dr Benirschke’s legacy,” Houck says. “And I hope that we can instill a generation who will carry it forward from us.”
