Exponential growth in the use and development of genetically engineered rodent models during the last several decades has resulted in researchers at many institutions requiring ever-increasing amounts of vivarium space. However, new technologies will drive different design considerations and space planning in future rodent facilities, says Neil S. Lipman, professor and executive director of the Center of Comparative Medicine and Pathology at the Memorial Sloan Kettering Cancer Center and Weill Cornell Medicine.
“Looking forward, we are going to be using select models in a way that is much less efficient,” says Lipman. “For some models, you may need more space to house an equivalent number of animals. However, this may be countered by other technologies allowing for the development of genetically engineered models in a manner requiring fewer animals. The ultimate space requirements will be the net of these two opposing factors.”
Evolving Science and Animal Model Impact
Five notable, interdependent technological drivers and/or areas of scientific focus have emerged during the past five to 10 years that could lead to an increased, decreased, or level usage of rodent models:
First among the five is high-throughput sequencing technologies in which the genome can be sequenced cost effectively using technologies such as next generation sequencing (NGS). This technology can be applied to sequencing an entire organism, or more commonly individual cells, particularly cancerous cells, as well as microorganisms. Lipman notes the broad application of this technology will likely increase the use of animal models.
Gene editing, including the latest technology known as CRISPR, is the second driver. A bacterial system that allows scientists to carefully edit the genome, gene editing has streamlined and simplified the classic, more involved process by which genetically engineered rodents have been created over the last several decades. While Lipman concedes these technologies have driven the exponential rise in rodent census, he believes that gene editing’s more robust and more refined characteristics—which would decrease rodent census—coupled with increased overall usage by researchers, will result in level or possibly decreased mouse populations in vivaria.
In his work at Memorial Sloan Kettering Cancer Center and Weill Cornell Medicine, Lipman has witnessed what he calls remarkable progress in the third driver: oncology. The understanding that cancer is principally a genetic disease is creating robust growth in rodent model usage. “Once you understand particular molecular pathways that drive the abnormal growth of a cancer cell, one can target those pathways for therapy. We are also learning how the role of our genetic makeup impacts the drugs that we receive,” explains Lipman. “Now we are taking tumors that have specific genetic changes, putting them into (engrafting) animals, most commonly mice, and using them as avatars for evaluating specific therapies.” In this area, Lipman sees robust growth.
Increasingly, the development and use of highly immunocompromised mouse models is the fourth driver shaping future vivaria. These models allow scientists to engraft a greater number of cell types, as well as create humanized models. Those models recapitulate aspects of the human immune system, which are subsequently used to study a variety of diseases. As the ability to create mice with ever-greater immunodeficiencies expands, so does the need for pristine animal care and environments. Maintaining these models in the long term, stresses Lipman, requires more intensive husbandry, using methodologies such as complete sterile cage units that are only handled aseptically within HEPA-filtered animal change stations (ACS) or biological safety cabinets (BSC). Often there will be a need for multiple ACSs and/or BSCs in animal holding rooms maintaining these models, decreasing space efficiency significantly. This fact, as well as the increasing use of these models, will likely drive the need for additional vivarium space.
The final driver, the microbiome, has emerged as an increasingly important area of study, which is likely to increase significantly going forward. The re-emerging interest in the microbiome reflects the increasing understanding of the influence of microorganisms on all areas of biology. “All of us are colonized with large numbers of microorganisms, mostly bacteria,” explains Lipman. “Recognize that when you get on the scale, almost five pounds of your body weight is contributed by the bacteria that you harbor on your body, mostly in the GI tract, but also in your mouth, and on your skin among other sites. The challenge in studying the microbiome is the intensive husbandry that these gnotobiotic (known biology) models require.”
These models depend on the science of gnotobiology, in which sterile animals—those without any associated microorganisms—are then associated with a defined microbial population, with many micro-organisms or as few as a single micro-organism. Creation and maintenance of these models requires highly specialized equipment and techniques. The more traditional and most foolproof ways to house the animals is within flexible film or semi-rigid isolators. These are essentially glove boxes requiring all materials to be autoclaved in, says Lipman, resulting in labor-intensive handling and an inefficient use of space. The resulting cost of maintaining isolators is high, so scientists are looking for more efficient and easier-to-use options. Among newer, more sophisticated systems are a ventilated cage that is hermetically sealed and pressurized with HEPA-filtered air to ensure isolation, while another less expensive and sophisticated option uses a static isolator cage nested within another larger static isolator. Regardless of the system used—and in most institutions, it will likely include both the traditional and newer systems—there is a loss of housing efficiency. With this loss, as well as the increasing use of these models, additional vivarium space will be needed.
To Sterilize or Not to Sterilize: The Fundamental Question
How an organization answers the question of whether or not to routinely bulk autoclave rodent caging after sanitization (washing) is among the most important operational decisions driving vivarium design, asserts Lipman, as it has cost, space, equipment, and lifecycle implications.
“The principal advantage of bulk sterilizing all caging is that you have guaranteed decontamination,” he says. “You get peace of mind. Proper autoclaving is going to kill all microorganisms.”
When compared to using only cagewash sanitation, bulk autoclaving’s disadvantages—capital and maintenance expenses, energy consumption, additional labor, and reduced component life—are significant. Basing estimates on 2018 costs and a facility with a 20,000-cage average daily census, for example, he estimates a facility would need to make a capital investment of $15,000 per year for a floor-loading autoclave ($300,000 total cost amortized over 20 years) and would spend $7,000 each year on service and repairs. Using New York City utility rates, the electric, water, and steam to run the autoclave would cost approximately $41,000 annually. The biggest cost of bulk autoclaving, highlights Lipman, comes from the degradation of the plastic cage components and the resulting shortened lifecycle, down to as few as two years as compared to the 12-year lifecycle he observes in his facility, for a component subjected to only cagewash sanitization. The resulting annualized yearly replacement cost for a cage bottom with an initial purchase price of approximately $18, for example, is $9 per year for each cage subjected to bulk autoclaving, as compared to $1.50 per year for each cage that underwent only cagewash sanitization.
Additional, Pertinent Price Tags
Other considerations in future vivarium design are space utilization, cage metrics, and the resulting cost per square foot. Lipman encourages planners and researchers to remember that specialized studies are requiring reexamination of the ratios of cages to ancillary equipment, such as a change station or a biosafety cabinet. Where a typical animal holding room may contain 350 to 450 cages per change station or biological safety cabinet, models with heightened immune-deficiencies or the use of animals engrafted with human tumors, for example, result in a marked reduction in holding room capacity.
“I use about two cages per square foot of animal room space as a metric for mouse caging systems,” says Lipman. “That includes all of the ancillary equipment. When we compare that to the space needed to support highly immunocompromised mice used for patient-derived xenografts, we can house only about half the maximum capacity of cages.” As previously discussed, housing gnotobiotic rodent models in isolators results in a significant reduction in space utilization efficiency. Maintaining a cage in a more traditional flexible film isolator, says Lipman, can require as much as 3.6 sf per cage, costing as much as $5 per day. However, using the new ventilated caging systems for supporting gnotobiotic models improves both space utilization and daily cage maintenance costs, “while not as efficient as the space that we use in our standard mouse rooms. We are at about, say, 0.75 sf per cage, about one-and-a-half times higher than the most efficient operation, but we can drive daily maintenance costs down considerably to about $2 per cage.”
By Ellen Gamble