Laboratory Animal Sciences 2021

The concept of the 3Rs remains more prevalent than ever in global discussions related to humane animal use in aspects of research, teaching and testing. The 3Rs serve as a touchstone, in terms of refinement, reduction, and replacement, for veterinary staff, ethical review committees and scientists. Russell and Burch established these words as part of a new applied science in 1959, aiming to improve animal welfare with the 3Rs as a launchpad for future development of scientific methods and medical discovery. In all aspects of animal use, the anticipated benefits of scientific inquiry are to be weighed against the potential detriments borne by the animals themselves; this ethical balance is referred to as the ‘harm-benefit’ analysis. Over time, aspects of the use of the 3Rs in study design (refinement), statistical analysis (reduction), and selection of alternatives to live animals (replacement), have strayed into the potential for abuse by groups that seek to constrain the spirit of their interpretation. For example, with the generation and availability of novel techniques (e.g., CRISPR) and development of human disease (e.g., COVID-19) in animal models, any rigid 3Rs interpretation of ‘reduction’ to minimal numbers until we achieve ‘replacement’ of all animals is incompatible with the necessity to support science and production of reliable and reproducible results. The 3Rs have remained ubiquitous in large part because of their benefit to animals, although importantly they are also of benefit to those stakeholders invested in the animal research enterprise.  The exquisiteness of the 3Rs is that, much like a classical piece of art, they are inherently subject to broad interpretation across the contemporary biomedical community. Reconsidering the foundational 3Rs with progressive interpretations, in combination with other ethical tools of analysis and validity assessments, will be essential in the ongoing intricate considerations of appropriate and future animal use.

Learning Objectives:

1. Review the foundational 3Rs for animal use and welfare and discuss limitations of rigid interpretation of the terminology

2. Provide expanded definitions of the 3Rs that better address the complexity of appropriate animal use and welfare in contemporary society

Primates have been essential in unlocking novel immune mechanisms that have driven development of groundbreaking new drugs in infectious disease and transplantation, amongst others related to the complexity of the primate immune system that is most similar to humans. As a result, primates have served as critical ‘gatekeepers’ in safety and efficacy evaluations of vaccines and immunotherapies.  Behind this success is a path with considerable examples of where primates have overestimated the effect, with an exaggerated immune response that is not present in clinical trials. The explanation for translational failures may partially relate to model-imposed stressors that affect the immune system. Common sampling practice in nonhuman primate (NHP) safety and toxicology studies involves serial sampling from a chemically or capture (mechanically) restrained animal. While sedation events may be required to protect researcher safety during studies of infectious disease and promote ease of sample collection, this comes at a cost to animal welfare and introduces confounding that reduces our ability to interpret data accurately. Study specific endpoints related to stress, immune response, and safety that are critical for vaccine development and toxicity studies are confounded by the effects of restraint stress and sedation on the research subjects. For example we have observed significant differences in cytokine levels by sex and cytokine levels were significantly blunted in sedated NHPs when compared to awake cooperating NHPs, highlighting the importance of model design on translational relevance for clinical settings. Acute and chronic stress alters immune responses by shifting immune cell populations or altering signaling, such that the model design must be carefully considered to be representative of the typical Phase I/II clinical studies in healthy immunocompetent adults to avoid misinterpretation of immune-based study endpoints. At the conclusion of this session, participants will develop an improved understanding of how model-related parameters affect study validity by influencing background immunologic and metabolic processes, impacting accurate disease characterization, and defining our ability to recapitulate the target disease state in ways that may confound outcome measures and impact translatability.

Learning Objectives:

1. An overview of the role of experimental design factors from animal selection to model handling in influencing animal welfare and scientific outcomes

2. Example strategies using behavioral management as a strategy to intentionally introduce variation for generalizability or reduce confounding to make an experiment more robust.

Primates have been essential in unlocking novel immune mechanisms that have driven development of groundbreaking new drugs in infectious disease and transplantation, amongst others related to the complexity of the primate immune system that is most similar to humans. As a result, primates have served as critical ‘gatekeepers’ in safety and efficacy evaluations of vaccines and immunotherapies.  Behind this success is a path with considerable examples of where primates have overestimated the effect, with an exaggerated immune response that is not present in clinical trials. The explanation for translational failures may partially relate to model-imposed stressors that affect the immune system. Common sampling practice in nonhuman primate (NHP) safety and toxicology studies involves serial sampling from a chemically or capture (mechanically) restrained animal. While sedation events may be required to protect researcher safety during studies of infectious disease and promote ease of sample collection, this comes at a cost to animal welfare and introduces confounding that reduces our ability to interpret data accurately. Study specific endpoints related to stress, immune response, and safety that are critical for vaccine development and toxicity studies are confounded by the effects of restraint stress and sedation on the research subjects. For example we have observed significant differences in cytokine levels by sex and cytokine levels were significantly blunted in sedated NHPs when compared to awake cooperating NHPs, highlighting the importance of model design on translational relevance for clinical settings. Acute and chronic stress alters immune responses by shifting immune cell populations or altering signaling, such that the model design must be carefully considered to be representative of the typical Phase I/II clinical studies in healthy immunocompetent adults to avoid misinterpretation of immune-based study endpoints. At the conclusion of this session, participants will develop an improved understanding of how model-related parameters affect study validity by influencing background immunologic and metabolic processes, impacting accurate disease characterization, and defining our ability to recapitulate the target disease state in ways that may confound outcome measures and impact translatability.

Learning Objectives:

1. An overview of the role of experimental design factors from animal selection to model handling in influencing animal welfare and scientific outcomes

2. Example strategies using behavioral management as a strategy to intentionally introduce variation for generalizability or reduce confounding to make an experiment more robust.

Aseptic technique, including the use of sterile drapes, is essential to reduce microbial contamination to the lowest practical level and improve surgical outcomes. In biomedical research, surgeons are often responsible for simultaneously conducting rodent surgical procedures, monitoring anesthesia, and adjusting nonsterile equipment. This presentation will cover agile and innovative techniques on how to improve rodent aseptic surgery using common household products: cling film and foil. Recently, some institutions have approved the use of Press’n Seal® cling film and Reynolds Wrap® aluminum foil as practical, cost-effective alternatives to sterile drapes and barriers during rodent surgeries. This presentation will also cover recent studies and publications that evaluated sterility properties for both Press’n Seal® cling film® and Reynolds Wrap® aluminum foil and other potential benefits for each product in rodent surgery.

Learning Objectives:

1. Methods on how to aseptically use foil (Reynolds Wrap) in rodent surgery

2. Methods on how aseptically use cling film (Press N' Seal) in rodent surgery

3. Long-term and short-term benefits to using both cling film and foil in aseptic rodent surgery

Tissue models, including organoids, are proving valuable in modeling human tissue health and disease for a variety of applications. This is due, in part, to the dynamic cell-cell interactions important in tissue function fostered within the 3D tissue-like space. To this end, the more these models can recapitulate the different interactions found in native tissue, such as that between parenchyma and the microvasculature, the better the fidelity of the model. The microvasculature, which is comprised of a spectrum of cell types and matrix proteins, provides not only perfusion in its support of tissue health, but also cellular interactions and biochemical dynamics important in tissue phenotype and function. We have developed a generalized strategy for creating human, vascularized tissue models for research and therapeutic applications using isolated, intact human microvessel fragments to generate a neovasculature within the fabricated tissue bed. By isolating intact microvessels, instead of dissociated single vascular cells, we have retained the full complement of vascular and vascular-related cells intrinsic to the native microvasculature. With this strategy, we have fabricated vascularized thick tissue constructs and organoids useful in research, pharmaceutical and autologous therapeutic applications and demonstrate that the integrated microvasculature imparts new functionality to the constructs and organoids.

Learning Objectives:

1. Define an effective in vitro tissue model

2. Identify the features of a microvasculature

3. Explain the approaches for building in vitro tissue models

Traditional drug discovery often begins with simple biochemical and transformed cellular models, advances to more complex animal models in late discovery phases, and often does not ‘see’ appropriate human biology until clinical trials. While widely deployed, this process can result in initial hits that do not translate to the clinic and ultimately lead to late-stage failures while running counter to the emerging standard in animal use of replace, reduce, and refine.

Advances in human induced pluripotent stem cells (iPSCs), three-dimensional (3D) culture technologies, combined with advanced analytical methods offer solutions for these shortcomings. Initiating the drug discovery process with disease-based human iPSC-derived 3D cultures and high-throughput assays for rapid compound screening and multi-parametric analysis of biological signals enables a faster and more accurate drug discovery pipeline with novel target identification, improved hit efficacy and safety, and ultimately increased clinical success all while reducing animal use.

This presentation will:

1) discuss how a human iPSC-derived 3D-neurospheroid in vitro screening platform can reduce, replace, and refine animal use in pre-clinical research

2) utilize a case study with Rett Syndrome iPSC-derived neurospheroids to demonstrate rapid target and hit identification/validation

3) provide an example of clinical translation for the neurospheroid platform

Learning Objectives:

1. Deploying advanced human culture systems in phenotypic screening

2. Key elements for a successful phenotypic screen using human iPSC-derived neurospheroids

Since the first introduction of the 3R concept in 1959 by Russell and Burch, there has been an increasing interest in finding alternatives to animal testing for ethical and economic reasons. Only looking at the landscape in drug development, millions are invested every year but only a few of all drug candidates make their way to the clinics. Although the amount of knowledge generated from animal models is unquestionable, in terms of translation, rising evidence suggests that non-animal methods can be as valuable and even more predictive for human. Proving that a molecule is safe for the human respiratory tract and distal lung area is essential in the pipeline of drug and molecule development. Especially inhaled drugs are gaining more and more attention, and fitting in-vitro systems for testing them are missing. Simulating the lung environment in vitro poses a significant challenge, especially when it comes to distal airways. The lack of established cell models for the alveolus and its dynamic microenvironment have led to the generation of microphysiological systems (MPS). MPS gather key features of the organ microenvironment, including mechanical cues, human primary cells and extracellular matrix. In this talk, we will recapitulate the short but remarkable history of the “lung-on-chip”, its key elements and how this technology has changed the paradigm of in vitro testing whilst representing a promising 3R alternative to conventional animal-based research.

Learning objectives:

1. Key features of lung MPS systems – What makes a good in vitro model for the alveolus?

2. Lung-on-chip applications in toxicology, safety and efficacy testing: benefits, shortcomings and outlook

Remarkable progress in life science and technology in the past century has advanced our fundamental understanding of the human body beyond our imagination. The ever-increasing knowledge of human anatomy and biology, however, has done surprisingly little to improve the way we emulate and probe the complex inner workings of the human body. Even today, our ability to model human physiological systems relies on the century-old practice of cell culture or animal experimentation that has raised significant scientific and ethical concerns. The paucity of predictive and human-relevant model systems is emerging as a critical impediment to our scientific endeavors for a wide variety of biomedical applications. This talk will present interdisciplinary research efforts in my laboratory to develop microengineered in vitro models that can emulate the structural and functional complexity of human organs. Specifically, I will talk about i) bioinspired microsystems that mimic the alveoli and airways of the human lung during health and disease, ii) microengineering of vascularized and perfusable 3D human tissues, iii) a blinking eye-on-a-chip microdevice that emulates the ocular surface of the human eye, and iv) microengineered physiological models of human reproductive organs.

Learning Objectives:

1. Learn about the design principles of organ-on-a-chip technology

2. Discuss current state-of-the-art and recent trends in the field of organ-on-a-chip and microphysiological systems

Many of the most debilitating and life-threatening diseases are associated with the central nervous system. Some, such as neurodegenerative diseases, predominately afflict our aging population. Yet others such as brain cancers and ALS are prevalent at all ages. In this presentation, several models will be presented that attempt to recapitulate certain aspects of the central or peripheral nervous system, both to probe the disease process, and as a platform to screen for new therapeutics. Examples will be presented selected from the following: 1) A model for the healthy blood-brain barrier and neurovascular unit has been developed in order to capture the essential aspects of barrier impairment in disease. 2) The blood-brain barrier model is used to study metastasis of cancers to the brain, identifying the critical role of astrocyte-secreted CCL2 in tumor cell extravasation. 3) A model for the healthy and diseased neuromuscular junction will be presented as a step toward developing therapeutic strategies for treating ALS.  4) A model for cerebral amyloid angiopathy, often associated with Alzheimer’s disease, will be discussed as a disease model and its drug screening potential.  Time-permitting, translation of these models to screening platforms and potential barriers will be discussed.

Learning Objectives:

1. Understand how primary and iPS cell-derived cells can be used to develop 3D in vitro models of organ function and disease

2. Learn about the different types of microphysiological systems using 3D hydrogels and relying on cellular self-organization and self-assembly

3. Identify some of the barriers to generating fully-functional, user-friendly models in moderate throughput platforms

Handling laboratory animals can stimulate stress and anxiety that negatively impacts animal welfare and introduces a substantial confounding factor in many areas of scientific research. However, the method used to pick up laboratory mice is critical in determining whether handling induces stress and anxiety. While the traditional method of picking up mice by the tail induces strong aversion and high anxiety, picking up mice in a handling tunnel or scooping them on the open hand (known as cupping) is non-aversive and substantially reduces anxiety and handling stress. Differences between tunnel and tail handled mice are particularly dramatic. Importantly, mice familiarised with non-aversive handling methods continue to interact readily with handlers and show reduced anxiety even after routine procedures that may induce some pain or discomfort, such as scruff restraint, subcutaneous or intraperitoneal injection, oral gavage, anaesthesia, tattooing or ear-tagging. This underlines the very strong impact that non-aversive handling methods have in taming animals to accept human contact and restraint, providing a major refinement over picking up mice by the tail. Concern about practicability can be a major barrier to implementing non-aversive handling methods by those already trained to pick up mice by the tail. However, mice can be picked up just as quickly with non-aversive methods once the handler has developed good skills and I will share some tips for gaining good technique. New handlers find tunnel handling the easiest method to learn - mice are easier to catch, less likely to bite and easier to restrain. Replacing tail handling wherever possible with non-aversive methods avoids unnecessary handling stress and anxiety in animals, makes handling easier, reportedly improves job satisfaction for the handler, and removes an important confound in many areas of animal research.

Learning Objectives:

1. Scientific evidence that picking up mice by the tail induces strong handling aversion, stress and anxiety that negatively impacts animal welfare, makes animals harder to handle and can be a major confounding factor in a wide range of research that is sensitive to handling stress

2. Scientific evidence showing the benefits of using non-aversive methods to pick up mice, such as use of a handling tunnel or scooping on the open hand (cupping)

3. How to pick up mice efficiently using non-aversive methods and tips for implementation in animal facilities

Organoids are emerging tools to study homeostatic function and disease of human organs. This technology has now for the first time been extended to reptiles by establishing cultures of the snake venom gland. Venom gland organoids from a panel of species produce active toxins and allow for the study of multiple aspects of venom such as gene regulation and evolution.  These cultures could form the basis for a novel strategy of anti-venom production and bioprospecting.  In the second part of the talk, a co-culture of human intestinal organoids with genotoxic Escherichia coli is presented. Long-term exposure of organoids to these bacteria leads to accumulation of a specific mutational signature in the human cells. These specific mutations are also found in the genomes of colorectal tumours, highlighting for the first time the induction of specific mutations by bacteria in human cancer.

Learning Objectives:

1. Define adult stem cell-based organoid systems

2. Identify current approaches for increasing the complexity in vitro cell culture systems

Key to the development of the HUB Organoid Technology was the discovery of LGR5+ intestinal adult stem cells by the Clevers’ lab. With the identification of these stem cells, we were able to develop a culture system that allowed for the virtually unlimited, genetically and phenotypically stable expansion of the epithelial cells from animals including humans, both from healthy and diseased tissue (Sato et al., Nature 2009, 2011; Gastroenterology 2011; Huch et al., Nature 2013, Cell 2015; Boj et al., Cell 2015). Since the initial development, we have generated adult stem cell (ASC)-derived organoid models of different tissue origin like colon, lung, breast, pancreas and ovary for the study of Cancer, Cystic Fibrosis and other diseases (Dekkers, Nat Med 2013; van de Wetering, Cell 2015; Drost et al Nature 2015). These organoids have been characterized at the genetic and transcriptional level and provide an unique in vitro system for screening the efficacy of both approved drugs and compounds in preclinical development. Furthermore, ASC-derived organoids can serve as a patient avatar in clinical development and/or diagnostics. More recently, we were able to demonstrate that the in vitro response of ASC-derived organoids directly correlates with the clinical outcome of the patient from which the organoid was derived (Dekkers et al., Sci Trans Med 2016; Sachs et al., Cell 2018).

Learning Objectives:

1. Why HUB Organoids™ are considered to be superior 3D preclinical model systems

2. How to apply HUB Organoids to drug discovery

3. Impact and possibilities of HUB Organoids in (oncology and immuno-oncology) drug discovery

The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Microphysiological Systems (MPS) Affiliate is a group of pharmaceutical industry scientists who seek to expedite appropriate characterization and incorporation of MPS to potentially improve drug safety assessment and provide safer and more effective medicines to patients.  The IQ MPS Affiliate (formed in June of 2018), was formerly a Working Group (WG) of the IQ Consortium (https://iqconsortium.org) created at the request of the NIH National Center for Advancing Translational Science (NCATS) with a mission for pharmaceutical companies to work collaboratively with NCATS funded tissue chip developers to advance MPS, for improving the understanding of human pharmacology and the predictivity of safety and toxicology translation. In order to broaden impact and provide a venue for appropriate cross-pharma collaboration and data sharing to facilitate industry implementation and qualification of MPS models, the IQ MPS WG transitioned to an IQ Affiliate in 2018. This session will provide an update on IQ MPS Affiliate’s accomplishments and share future aims.

Learning Objectives:

1. Learn about the current status of microphysiological systems in drug discovery and development

2. Learn about collaborative efforts within Pharma

3. Learn about importance of human and animal cell based microphysiological systems in drug discovery and development

Pain management includes the choice of analgesia agents, their dose, administration method, duration and frequency of treatment, and a pain-monitoring scheme for each individual animal. Insufficiently treated pain or inadequately chosen or insufficient reported anesthesia or analgesia protocols may carry the potential to hamper animal welfare and the reproducibility of animal experiments substantially. Respective protocols are therefore an important part of experimental design, and determining an optimal protocol is mandatory when planning animal experiments. Unfortunately, there is evidence that especially the most widely used laboratory species, the mouse, is often treated insufficiently or that protocols are applied that may not reflect the recent state of the art of veterinary analgesia/anesthesia. In this talk requirements for a good analgesia protocol in general, but also the challenges that we face when applying analgesia to small patients like laboratory mice are discussed. Important concepts of (mouse) analgesia including preventive and multimodal approaches and strategies to overcome specific challenges will be discussed. The focus of this talk will lie on stress-free or stress reducing, sustained release or voluntary oral administration protocols for analgesic substances that have a great potential to improve animal welfare in animal research.

Learning Objectives:

1. Define the requirements of a good analgesia protocol

2. Identify various challenges in designing an appropriate analgesia protocol

3. Identify and choose potential refinement opportunities for analgesia protocols

There is growing concern about the reliability of biomedical research results. Poor experimental design, inappropriate analysis methods and incomplete reporting have all contributed to inconsistent results. The NC3Rs has developed resources to assist researchers in designing rigorous experiments, selecting the appropriate analysis methods and reporting experiments thoroughly. One such resource is the Experimental Design Assistant (EDA). The EDA is free online software with a supporting website to help researchers design more rigorous in vivo experiments. Experiments designed in the EDA are represented as machine readable diagrams, which the system analyses using computer-based logical reasoning to provide feedback. This feedback highlights the implication of particular design features and enables users to make informed choices, tailored to the objective of their own experiments. The system also suggests statistical analysis methods compatible with the design of the experiment. It includes inbuilt power calculators to determine the appropriate sample size, and features to facilitate randomisation and blinding. The EDA is also a very useful tool to communicate experimental plans as the diagram contains detailed information for each step of the experiment and provides a visual overview, which is helpful to support discussions. The EDA is endorsed by major biomedical research funders worldwide, to help researchers design experiments that are more likely to yield robust and reproducible data while using the minimum number of animals consistent with the scientific objectives.

Learning Objectives:

1. Understand the impact of subjective bias on the reliability of animal research results

2. Identify the experimental design features that can be used to reduce effects of bias and make results more reliable

3. Understand how the EDA can be used to design rigorous in vivo experiments that generate reliable results