TECHNOLOGY FEATURE

Frozen cells and empty cages: researchers struggle to revive stalled experiments after the lockdown

Abandoned experiments have to be restarted — sometimes from scratch.
Jyoti Madhusoodanan is a science writer based in Portland, Oregon.

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Two lab workers wearing gloves, surgical masks and lab coats work at distant benches

As labs reopen, researchers must navigate technical issues alongside new requirements for social distancing and personal protective equipment.Credit: Misha Friedman/Getty

In early March, developmental biologist Lee Niswander’s lab at the University of Colorado Boulder had just successfully generated strains of mice that lacked certain sections of a gene involved in brain development. Some strains had taken nearly a year and a half to develop, and preliminary data suggested that the gene was essential to survival. But the surging COVID-19 pandemic led the team’s institute, like others around the world, to abruptly shut down all research unrelated to the coronavirus. “We literally got the mice pups the day we shut down,” Niswander recalls.

Her lab members split up breeding pairs of mice, shelved reagents and froze or refrigerated biological materials. Generally, researchers cryopreserve mouse embryos to maintain mutant mouse lines in central repositories, but Niswander’s team hadn’t yet completed the necessary experiments. And the researchers weren’t even sure they would still be able use the mice they already had. Mice that have been separated for months might not want to mate any more. “My biggest worry is that we’re past that point now,” Niswander says. “If they don’t reproduce, that’s it — the resource might be lost.”

Niswander’s team is not the only one attempting to navigate the pandemic’s toll on months — and sometimes years — of labour. Coronavirus-related closures have forced researchers to downsize Drosophila colonies, kill laboratory animals and freeze delicate stem-cell lines or patient-derived samples. Getting those experiments up and running again will take time. Social-distancing guidelines will inevitably constrain the pace of research by limiting how many people can work at once, and strained supply chains and short-staffed shipping departments are likely to slow the flow of required materials.

For now, many researchers are easing back in simple ways: regrowing cell lines or animal colonies, or finishing off experiments that were nearly complete when they shut down. “I’ve been surprised at the number of things we take for granted in terms of lab maintenance,” says microbiologist Ami Bhatt at Stanford University in California. “It’s daunting to think of bringing all that back up.”

Sudden stops

Bhatt leads a multi-site clinical study that is analysing stool samples to study patients’ intestinal microbiota. When COVID-19 struck, research coordinators were no longer allowed to visit hospitals to collect the samples — and at that point, no one was certain whether the samples might transmit the virus. The team quickly assembled at-home mailer kits so that participants could ship their samples safely. As part of a different study, Bhatt and her team wanted to test stool samples collected using similar kits for the presence of RNA from SARS-CoV-2. But they couldn’t come up with ways to assess whether any viral RNA in the samples had been properly preserved. Mailer kits are optimized for two purposes: to preserve DNA from intestinal bacteria, and to be easy for participants to use at home. “We have no idea if the collection tubes can preserve viral RNA,” Bhatt says. “The kit has not been tested for that purpose.”

Although these kits are widely used in biomedical research, Bhatt and her colleagues will need to validate the samples against the ones collected with pre-pandemic protocols to confirm that they yield comparable results in terms of gene sequence, expression and metabolite concentration. “It’s a matter of making your best guess in the moment, but then recognizing that when we go back into the lab, we’ll need to do very rigorous testing to see if these choices were adequate,” Bhatt says.

Also having to do some very rigorous post-shutdown testing are stem-cell researcher Ru Gunawardane and her team at the Allen Institute for Cell Science in Seattle, Washington. Her team froze hundreds of cell lines when the pandemic shut her city down in March. Two months later, a few lab members have begun thawing the lines and resuming experiments to verify the integrity of the cultures, which are used by other researchers at their institute and around the world. The team needs to monitor cells for physical changes to their shape, behaviour, survival and growth rate and other features. These tests are crucial for confirming that the stem cells retain their stem-like properties, but the experiments can take weeks to run, Gunawardane says. And the people who usually perform them aren’t yet back in the lab, so less-experienced lab members are having to step in and learn how to run the tests. “If you miss the signs, it could affect downstream applications,” Gunawardane says. “For instance, it might mean the stem cells don’t differentiate into heart cells in a later experiment.”

For developmental biologist Jose Pastor-Pareja at Tsinghua University in Beijing, the challenge is restarting his fly lines. Pastor-Pareja was the only person allowed to enter his lab when the university shut down in mid-February, and he had no choice but to discard hundreds of transgenic fruit flies. If left to breed for more than 20 days, fruit flies start to cross-breed between generations, resulting in a genetic cocktail that’s useless for experiments. Pastor-Pareja focused on preserving stable genetic stocks that could be used to regenerate transgenic flies, a process that will take a few weeks — but only once students return to the lab. “A lot of work was lost,” he says, “but it’s nothing we can’t repeat because we have these stable lines.”

Neurobiologist Christopher Harvey at Harvard Medical School in Boston, Massachusetts, is bracing himself to repeat work of a different kind. Harvey’s team studies how mice learn and remember information as they navigate through a maze to a tasty treat. Training a mouse can take up to three months, but without daily practice, it retains the memories only for a week or so. Breaks in training can affect how a mouse’s brain works through the task. “We have no idea what happens if they go untrained for two months,” Harvey says. “We’ll probably have to start afresh with new mice.”

Delays and shortages

Researchers who need to order supplies or send samples out to other locations for processing could find themselves facing more delays. The timing of Beijing’s shutdowns wasn’t as bad as it could have been, because many Chinese labs were already winding down for the nation’s week-long spring festival, says epigenetics researcher Magdalena Koziol at the Chinese Institute of Brain Research in Beijing. But when she and her team returned to work in March, they found that they couldn’t order plasmids and other supplies because companies in the United States were still shut down.

If mice and other animals need to be reordered from central repositories, it can then take more than a month to rear the necessary pups, says veterinary pathobiologist Craig Franklin at the University of Missouri in Columbia. Thawing embryos, establishing a surrogate pregnancy and weaning pups requires several weeks at least. “If it all goes perfectly, it takes six weeks to have a three-week-old mouse available,” Franklin says.

Chemical reagents and personal protective equipment such as masks and gloves — which researchers often donated to local hospitals — might also be out of stock. The waits are likely to worsen as more labs reopen and place orders. Bhatt has experienced shortages in materials such as culture media in which to transport viruses, RNA extraction reagents and kits for building sequencing libraries, probably owing to demand from more-urgent COVID-19 research. Basic reagents such as TRIzol, a solvent used to extract RNA from cells, “have been difficult to get for us and others”, she says.

“I anticipate it’s going to be much worse in the coming weeks as labs open back up,” Bhatt adds. Commonly used reagents and enzymes will be in high demand. “Just like there was a rush on toilet paper, there’s going to be a rush on Taq polymerase.”

Gunawardane’s group is carefully juggling its orders. Growth media for stem cells have a short shelf life, making stockpiling difficult, but their use is currently reduced, because lab members are conducting fewer experiments, she says. Developmental biologist Romain Levayer at the Pasteur Institute in Paris noted a similar trend with the supplies needed for his fruit flies. In some regions, he says, yeast — used both in baking as well as to make fruit flies’ meals — was in short supply, spurring some Drosophila researchers to grow their own yeast.

Lab-maintenance issues, such as putting orders away or ensuring equipment is in working order, now fall on the shoulders of fewer people. In the past, “small maintenance issues would get noticed very quickly”, Bhatt says. “Now, if the microfuge is broken, it may not even be noticed for a few days, and may take another week to get resolved.”

Bhatt’s greatest worry is the team’s anaerobic chamber, which is used to cultivate gut microbes that won’t grow in the presence of oxygen. Restarting the chamber is a days-long process that will require ordering new gas canisters and regenerating a catalyst.

Microbiologist Yves Brun at the University of Montreal in Canada is already experiencing the fallout of such maintenance issues. When he and his team resumed work after 2.5 months away, they found one of the four –80 °C freezers switched off and mould on the boxes inside, which contained custom antibodies that they had developed over the past 20 years. It’s unclear why this freezer was turned off, Brun says. The freezers are fitted with alarms that should have alerted building security, but because there was no power supply to the unit, the alarm’s rechargeable batteries were dead.

It’s possible that the antibodies are still functional, say Brun and other researchers who offered help through Twitter. “My thought was that since antibodies are stable in the blood of animals, they might still be fine even after what we think was at least a week, probably more, at room temperature,” Brun says. “We are going to test our best antibodies first to see if it’s worth testing all the rest.”

If not, the team will have to recreate them in an expensive, months-long process, and resort to alternative techniques that don’t require antibodies to conduct their research. Colleagues have also stepped in to help. “A couple of colleagues have offered to send us antibodies to proteins we both study — a fantastic example of collaboration,” Brun says. “I’m lucky to be in a field where people are generous with such reagents.”

Psychologist Carly Demopoulos at the University of California, San Francisco, faces a different challenge. Demopoulos uses psychometric tests and magnetoencephalography (MEG) brain imaging to study neural responses in children with autism spectrum disorder . The MEG scanner is shared between several labs, and the kind of test she uses cannot be administered remotely. The children can’t wear masks because it would hide their facial expressions, and they are likely to feel uncomfortable with researchers whose faces are obscured by a mask or shield.

Demopoulos has been working with a Bay Area firm to develop a cloud-based version of one lab test that participants could complete from home. But how it will compare with lab-based tests remains to be seen. Once the test is validated, she will need to submit a new proposal for approval by her institutional review board. “It’s essentially proposing a new study,” Demopoulos says. “This experience has made me aware of how important it’s going to be to find flexible ways to collect data without compromising on its quality.”

New ways to work

‘Flexibility’ is the watchword for many labs. So, too, is safety: for most researchers, technical concerns are secondary to personnel issues.

Social-distancing requirements restrict the number of people who can be in a lab space at the same time, requiring careful planning and slowing productivity. Many researchers are in limbo waiting to start postdocs or other jobs. Others have been forced to shelter-in-place while in their home towns, on holiday or in the field. As they resume work, all these researchers will require support, both from their own groups and from their institutions to address practical concerns such as safely using public transport or caring for family members. “In my opinion, the first group of people [back in the lab] should be totally self-appointed,” Gunawardane says. “They should never feel that they have to come in to be employed or anything like that.”

Recommendations on the use of gloves, masks and other protective equipment can feel foreign to some researchers. But it’s commonplace for many, says molecular archaeologist Christina Warinner, who runs labs at Harvard University in Cambridge, Massachusetts, and the Max Planck Institute for the Science of Human History in Munich, Germany. Her team members have used many of these measures for years to avoid a problematic contaminant: lab-worker DNA can render ancient-DNA samples useless.

Warinner and her team change from street clothes into overalls when they enter, then don protective suits, masks and gloves. Nearly all their work is done individually in clean rooms. After a person uses the lab, they clean everything with bleach and a decontaminant, and the lab is irradiated with UV light for three hours every night. “We’ve done these for years, so we thought the only thing we’d need to limit was the number of people in the lab at one time,” Warinner says of labwork in the COVID-19 era. Yet she still finds herself juggling competing needs. Four technicians who don’t have responsibilities caring for children or elderly relatives are working longer hours than others, and the team has pooled resources so that they can be distributed across multiple projects.

Other researchers share the concerns about group dynamics. Levayer’s group, for instance, has very strict time constraints. His team’s experiments need to be timed precisely, because some genes are expressed at specific points during development, and the flies must be dissected a certain number of days later. Crosses to create or maintain genetic lines must be performed at specific times, too. Before the pandemic, lab members typically ‘owned’ their project. Now, each lab member keeps tabs on every other person’s ongoing crosses. If a certain experiment requires a heat shock to trigger gene expression, it can be done by whoever is in the lab.

Levayer also split the group based on expertise, such as pairing those who have experience working with fruit-fly larvae with those who know how to work with pupae. “What I’ve tried to do is always have the largest possible skill set in the lab at a given time, so there’s hopefully always one person who can work on each kind of experiment,” he says.

As for Gunawardane, her lab has turned to technology to ramp up its expertise, hooking computers up to microscopes so that those with more experience can remotely assess how the stem-cell cultures are faring. With labs continuing to open, expect such ingenuity to become ever more prominent. “In many ways, this situation is showing the best of science,” Warinner says. “There’s a tremendous sense that we’re all on the same team here. It’s really drawn people together.”

doi: 10.1038/d41586-020-01704-y

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