Webinar Q&A — Controlling the Macroenvironment: A Novel Approach to Germ-free Derivations

Dr. Cristina Weiner recently presented a webinar featuring a novel approach to germ-free derivations. She discussed the importance of understanding baseline performance and making incremental process improvements as well as how Taconic Biosciences developed and implemented a new facility design which supports improved germ-free derivation success.

We present the full webinar Q&A here.

• Q: You stated during the presentation that, on balance, Taconic judges that performing derivations outside of an isolator is better than performing derivations within an isolator.

Dr. Cristina Weiner: While recognizing that the germ-free integrity of the animal is most protected and maintained when the animal remains within an isolator, the volume and nature of the projects that we conduct mandates several different inputs into our process. A great example is the source of our embryos, which is an entirely different facility than where the embryo transfer surgical suite is located. As a result of these inputs, process-wise it makes the most sense for Taconic to conduct these procedures outside of an isolator. While recognizing that the greatest risk to the microbial status of the animal is when you remove the animal from the isolator, touch it and manipulate it, rigor and vigilance are required to maintain the integrity of the animal at this point.

• Q: What sterilants does Taconic use for germ-free and gnotobiotic work?

CW: For germ-free work, we use MB10. We use different contact times depending on whether it's an animal entry or a material entry as well as the starting sterility of the material going into the isolator. Contact times range from five minutes to upwards of an hour. We use MB10 in the dunk tank in the gnotobiotic surgical biosafety cabinets. One of the benefits of using a dunk tank is ensuring that the chemical sterilant comes into contact with the material in toto. It's not a spray. With a dunk tank, the item is fully dunked and submerged. The benefit of a dunk tank is that it's fully contained. When you're thinking of particulates in the air, a dunk tank is really successful at minimizing risk not only to the animal, but also to personnel working in that location.

For gnotobiotic animals, we use 1:3:1 of clidox. The contact time ranges from five minutes to upward of 40 minutes, again depending on what process and material you are accommodating to gain access into the isolator.

• Q: What sort of respiratory personal protective equipment (PPE) is appropriate for gnotobiotic/germ-free work?

CW: Taconic has experience with all ranges, starting from virtually no respiratory PPE up to something like a MaxAir or Versaflow. If there is a concern or a need for using a higher degree of PPE, likely due to air changes per hour in your facility or any sort of filtration that may be supplied through the supply or exhaust air in your location, consider something like a dunk tank, as controlling the delivery of the chemical is can limit the exposure to personnel in that location. Much like humans have requirements for limiting exposure to chemical sterilant, our rodents are critically sensitive to that also, particularly in a reproductive context like a derivation.

• Q: Describe the training process for staff to learn the embryo transfer process at Taconic.

CW: Within our new facility, process revisions have been instituted which permit us to be more successful in maintaining the germ-free status of the animals. The training process for germ-free embryo transfers is not unlike that for other surgical procedures. It's really dictated by both didactic as well as practical, hands-on experience. Taconic's training program is very heavily SOP-based, but we also have competency and proficiency assessments as well as the support of trainers and other competent surgeons. In adapting our training in this new facility to existing protocols, it was just a matter of going through and rewriting those protocols, and then adapting them to metrics reporting systems that we use to monitor our training.

• Q: How can this approach to derivations be applied to a typical academic gnotobiotic setup?

CW: First and foremost, establish what your baseline is. If you do not currently have a surveillance program in place, consider implementation of one. Even if it's as simple and straightforward as a spot check. Particulate counts are relatively easy to collect, and TSA and RODAC plates are relatively easily accessible. When we first initiated this process several years ago, we started by collecting samples on TSA and RODAC plates in and around the biosafety cabinet used for surgery and surgical suite — areas we considered as the macroenvironment of the procedure. We learned a lot during that process. It validated what we knew intuitively. Take a look at the microbial integrity of that workspace because you may be shocked, even if you had decontamination protocols in place.

Another big thing we did which had a huge impact on process success was to thoroughly decontaminate the biosafety cabinets between use. When I say between use, it's up to your institution to dictate what that means. Is it a few surgeries? Is it one animal at a time? Is it between cage changes? That's up to you. But considerations for decontamination protocols should reflect the severity of contamination as established by your baseline sample collections. An example of decontamination process may include a thorough gas decontamination of your biosafety cabinet. Much like other equipment, a spray within your biosafety cabinet may not be sufficient to ensure sterility. Taconic often times will decontaminate our biosafety cabinets between procedures. Most facilities don't have clean rooms in which their biosafety cabinets are located, so if that describes your institution, you are not alone. There are other methods that you can use to ensure that the cabinet serves its purpose. That might be as simple and straightforward as giving it a good cleaning in between uses.

• Q: The Emtek air sampler is a piece of technology that comes from the cleanroom industry and so laboratory animal professionals may not be familiar with it. Can you describe the cost and ease of use of this equipment?

CW: I refer you to Paula Roesch and Jenn Brown's AALAS 2019 poster. The cost is not prohibitive. [Ed. note: Instrument cost is approximately $8000, plus labor time for sampling and costs for plates. If you want to identify agents found, costs vary depending on system used for ID.] It's super easy to use. You hold it up in the air, collect a sample and plate it out. It's a fundamental piece of equipment within the cleanroom environment for exactly that reason. It can not only help you establish your baseline, but also track and monitor results over time. Taconic's Biosecurity team just initiated use of this equipment, but even in a relatively short period of time, it's helped us learn a lot about our HVAC systems and whether something like heightened PPE or something like an air shower provides a bigger bang for your buck.

• Q: Can you recommend certain techniques to increase breeding success within isolators?

CW: There are a number of practices, and it really depends on your colony overall and how your colony overall is set up. For example, environmental enrichment, the number of breeders you have set up, and so on. Basic breeding technique applies.

• Q: Many academic facilities are not able to completely redesign their facilities. What is the single most impactful improvement a small facility can make to improve their germ-free and gnotobiotic derivation success without totally redesigning their facility and process?

CW: Establish the baseline cleanliness of your workspace. Based on those results, then evaluate the efficacy of your decontamination protocol. Before we brought our new derivation facility online, we were able to make significant improvements in our break rates over time simply by doing exactly that — establishing baseline and making continuous, stepwise improvements in decontamination and aseptic technique process. This must include a strong commitment to asepsis at every level of the process, based on what the riskiest part of the procedure is. We know that at least around the derivation, the likeliest risk for contamination happens when the animal is outside of the isolator and outside of the microisolator cage and is having an embryo transfer surgery performed on it. If it's in a biosafety cabinet, regardless of the status or type of cabinet, as long as your cabinet is clean, you have a better chance of staying clean than if it's not. So standardized procedure, commitment to asepsis — anybody can do that. You don't need a special facility for that. Start sterile, stay sterile.

• Q: What do you think the future of gnotobiotic production and research looks like?

CW: The future for me is always at the leading edge of how we do our work. Let's start with where we are. The animal is always most protected when it's contained within an isolator and you are as close as possible to assuring that 100% of the materials moving into the isolator are sterile and, upon exit, that you are preserving the biocontainment of that animal. But there's only so much that you can do with an animal that is contained within an isolator. So I'm really excited, as we continue to move forward with principles and practices such as these: the adherence to the very basic principles of asepsis and not relying on things like chemicals to clean up an environment that isn't otherwise controlled or sterile. To really push the envelope in terms of the cleanliness of both the macro and micro environment within which these animals are housed. I believe it will open a lot of doors for the use application of these animals.