Reduce Animal Usage Research Articles

Animal researchers' views on the publication of negative results and subsequent policy adoptions.

For over a decade, the non-publication of negative results from preclinical studies has been identified as a significant concern in biomedical research. Such underreporting is considered a contributor to the reproducibility crisis in the field and has been recognized by significant journals such as Science and Nature. In response to the consistently high non-publication rates of preclinical animal research in Europe, a survey was conducted among the biomedical research community to gather their views on publishing negative results. Using the EUSurvey platform, over 200 researchers directly working with animals were surveyed. The study aimed to understand the frequency of negative results, the reasons behind their non-publication, and the perceived pros and cons of making such results public. Insights from the survey could guide steps toward promoting transparency in science, refining research methodologies, reducing animal usage in experiments and minimizing research waste.

Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

Rapid generation of single‐insertion transgenics by Tol2 transposition in zebrafish

AbstractBackgroundThe Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F1 fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single‐insertion transgenic lines and the need to complete experiments within a reasonable timeframe.ResultsIn this article, we introduce a simple competitive dilution strategy for rapid generation of single‐insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single‐insertion F1 generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single‐insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects.ConclusionsWide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research.

Analysing the Potency of a Seasonal Influenza Vaccine Using Reference Antisera from Heterologous Strains.

The potency of inactivated seasonal influenza vaccine is harmonised by establishing the haemagglutinin (HA) content using the compendial single radial diffusion (SRD) method. SRD reagents (antigens and antisera) are prepared, calibrated and distributed by regulatory agencies as standards for potency testing, following the biannual World Health Organization (WHO) announcements of the virus strains suitable for inclusion in the vaccine. The generation of a homologous hyperimmune sheep antiserum constrains the time to vaccine release. This study tests the application of heterologous antisera to determine the potency of influenza vaccine compared to that of a standard homologous antiserum. The results indicate that the selected heterologous sheep antisera directed to seasonal H1N1, H3N2 or B Victoria virus strains can be used to determine the accurate potency of inactivated seasonal influenza vaccines. Individually selected antisera could be useful for two to fourteen seasons. A limitation to the heterologous antiserum approach is the diversity of each individual serum, indicating that the empirical determination of a specific serum is required. This application has the potential to enable the earlier availability of a seasonal vaccine and reduce animal usage.

Impact of quality and transparency in scientific writing on the reduction of animal usage in experimental protocols: a review based in pertinent literature.

The irreproducibility in scientific research has become a critical issue. Despite the essential role of rigorous methodology in constructing a scientific article, more than half of publications, on average, are considered non-reproducible. The implications of this irreproducibility extend to reliability problems, hindering progress in technological production and resulting in substantial financial losses. In the context of laboratory animal research, this work emphasizes the importance of choosing an appropriate experimental model within the 3R's principle (Refine, Reduce, Replace). This study specifically addresses a deficiency in data specification in scientific articles, revealing inadequacies in the description of crucial details, such as environmental conditions, diet, and experimental procedures. For this purpose, 124 articles from journals with relevant impact factors were analyzed, conducting a survey of data considered important for the reproducibility of studies. Important flaws in the presentation of data were identified in most of the articles evaluated. The results of this study highlight the need to improve the description of essential information, standardizing studies, and ensuring the reproducibility of experiments in areas such as metabolism, immunity, hormones, stress, among others, to enhance the reliability and reproduction of experimental results, aligning with international guidelines such as ARRIVE and PREPARE.

Systematic Investigation of the Effect of Lactobacillus acidophilus TW01 on Potential Prevention of Particulate Matter (PM)2.5-Induced Damage Using a Novel In Vitro Platform.

Exposure to ambient particulate matter (PM) and cigarette smoking (CS) is a risk factor for respiratory/lung infections and metabolic disorders. Lung-gut axis disruption involving the upregulation of oxidative stress, systemic inflammation, and gut barrier dysfunction by PM is one of the potential mechanisms. Thus, we designed a novel in vitro platform for pre-selecting probiotics with potentially protective effects against PM-induced lung damage through the lung-gut axis to reduce animal usage. The results showed that a high dose of Lactobacillus acidophilus TW01 (1 × 108 CFU/mL) inhibited reactive oxygen species (ROS) production. This strain could also reduce respiratory epithelial cell death induced by cigarette smoke extraction (CSE), as well as promoting Caco-2 cell migration in 1 × 106 CFU/mL. Although further animal experiments are needed to validate the in vitro findings, L. acidophilus TW01 is a promising probiotic strain for the potential prevention of PM2.5-induced damage.

Evaluation of silver bio-functionality in a multicellular in vitro model: towards reduced animal usage in implant-associated infection research.

Despite the extensive use of silver ions or nanoparticles in research related to preventing implant-associated infections (IAI), their use in clinical practice has been debated. This is because the strong antibacterial properties of silver are counterbalanced by adverse effects on host cells. One of the reasons for this may be the lack of comprehensive in vitro models that are capable of analyzing host-bacteria and host-host interactions. In this study, we tested silver efficacy through multicellular in vitro models involving macrophages (immune system), mesenchymal stem cells (MSCs, bone cells), and S. aureus (pathogen). Our model showed to be capable of identifying each element of culture as well as tracking the intracellular survival of bacteria. Furthermore, the model enabled to find a therapeutic window for silver ions (AgNO3) and silver nanoparticles (AgNPs) where the viability of host cells was not compromised, and the antibacterial properties of silver were maintained. While AgNO3 between 0.00017 and 0.017 µg/mL retained antibacterial properties, host cell viability was not affected. The multicellular model, however, demonstrated that those concentrations had no effect on the survival of S. aureus, inside or outside host cells. Similarly, treatment with 20 nm AgNPs did not influence the phagocytic and killing capacity of macrophages or prevent S. aureus from invading MSCs. Moreover, exposure to 100 nm AgNPs elicited an inflammatory response by host cells as detected by the increased production of TNF-α and IL-6. This was visible only when macrophages and MSCs were cultured together. Multicellular in vitro models such as the one used here that simulate complex in vivo scenarios can be used to screen other therapeutic compounds or antibacterial biomaterials without the need to use animals.

TransOrGAN: An Artificial Intelligence Mapping of Rat Transcriptomic Profiles between Organs, Ages, and Sexes.

Animal studies are required for the evaluation of candidate drugs to ensure patient and volunteer safety. Toxicogenomics is often applied in these studies to gain understanding of the underlying mechanisms of toxicity, which is usually focused on critical organs such as the liver or kidney in young male rats. There is a strong ethical reason to reduce, refine and replace animal use (the 3Rs), where the mapping of data between organs, sexes and ages could reduce the cost and time of drug development. Herein, we proposed a generative adversarial network (GAN)-based framework entitled TransOrGAN that allowed the molecular mapping of gene expression profiles in different rodent organ systems and across sex and age groups. We carried out a proof-of-concept study based on rat RNA-seq data from 288 samples in 9 different organs of both sexes and 4 developmental stages. First, we demonstrated that TransOrGAN could infer transcriptomic profiles between any 2 of the 9 organs studied, yielding an average cosine similarity of 0.984 between synthetic transcriptomic profiles and their corresponding real profiles. Second, we found that TransOrGAN could infer transcriptomic profiles observed in females from males, with an average cosine similarity of 0.984. Third, we found that TransOrGAN could infer transcriptomic profiles in juvenile, adult, and aged animals from adolescent animals with an average cosine similarity of 0.981, 0.983, and 0.989, respectively. Altogether, TransOrGAN is an innovative approach to infer transcriptomic profiles between ages, sexes, and organ systems, offering the opportunity to reduce animal usage and to provide an integrated assessment of toxicity in the whole organism irrespective of sex or age.

Bovine Rectoanal Junction In Vitro Organ Culture Model System to Study Shiga Toxin-Producing Escherichia coli Adherence.

Studies evaluating the interactions between Shiga toxin-producing Escherichia coli O157:H7 (O157) and the bovine recto-anal junction (RAJ) have been limited to either in vitro analyses of bacteria, cells, or nucleic acids at the RAJ, providing limited information. Alternatively, expensive in vivo studies in animals have been conducted. Therefore, our objective was to develop a comprehensive in vitro organ culture system of the RAJ (RAJ-IVOC) that accurately represents all cell types present in the RAJ. This system would enable studies that yield results similar to those observed in vivo. Pieces of RAJ tissue, obtained from unrelated cattle necropsies, were assembled and subjected to various tests in order to determine the optimal conditions for assaying bacterial adherence in a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Tissue integrity was assessed using cell viability, structural cell markers, and histopathology, while the adherence of bacteria was evaluated via microscopy and culture methods. DNA fingerprinting verified the recovered bacteria against the inoculum. When the RAJ-IVOC was assembled in Dulbecco's Modified Eagle Medium, maintained at a temperature of 39 °C with 5% CO2 and gentle shaking for a duration of 3-4 h, it successfully preserved tissue integrity and reproduced the expected adherence phenotype of the bacteria being tested. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.

Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach

To support registration of monoclonal antibodies (mAbs) for chronic indications, 6-month toxicity studies have historically been conducted. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class, with most toxicity findings anticipated based on pharmacology. We evaluated whether a 6-month toxicity study is necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. Opportunities to further optimize study designs to reduce animal usage were identified. For 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. An iterative, weight-of-evidence model which considers factors that influence the overall risk for a mAb to cause toxicity was developed. This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs.

Retrospective analysis of the potential use of virtual control groups in preclinical toxicity assessment using the eTOX database

Virtual Control Groups (VCGs) based on Historical Control Data (HCD) in preclinical toxicity testing have the potential to reduce animal usage. As a case study we retrospectively analyzed the impact of replacing Concurrent Control Groups (CCGs) with VCGs on the treatment-relatedness of 28 selected histopathological findings reported in either rat or dog in the eTOX database. We developed a novel methodology whereby statistical predictions of treatment-relatedness using either CCGs or VCGs of varying covariate similarity to CCGs were compared to designations from original toxicologist reports; and changes in agreement were used to quantify changes in study outcomes. Generally, the best agreement was achieved when CCGs were replaced with VCGs with the highest level of similarity; the same species, strain, sex, administration route, and vehicle. For example, balanced accuracies for rat findings were 0.704 (predictions based on CCGs) vs. 0.702 (predictions based on VCGs). Moreover, we identified covariates which resulted in poorer identification of treatment-relatedness. This was related to an increasing incidence rate divergence in HCD relative to CCGs. Future databases which collect data at the individual animal level including study details such as animal age and testing facility are required to build adequate VCGs to accurately identify treatment-related effects.

Immunocompetent 3D Follicular Lymphoma Model: A Preclinical Tool to Design Tailored Immunotherapies

Follicular Lymphoma (FL), the most common indolent non-Hodgkin Lymphoma, is a paradigm of the contribution of the immune tumor microenvironment (TME) to disease onset, progression, and therapy resistance. Moreover, macrophages, together with TFH and TREG, are fundamental players of FL immunosuppression. Patient-derived models are scarce and fail to recapitulate immune TME. Thus, we have established the first FL Patient Derived Lymphoma Spheroids (FL-PDLS) model that recapitulate the crosstalk between malignant B cells and the tumor immune microenvironment, and constitutes a pre-clinical tool to design tailored immunotherapies. Lymph node (LN) biopsies and peripheral blood (PB) samples were cultured in 96-well ultra-low-attachment plates within the context of an optimized cytokine cocktail representing the TME signals. FL-PDLS include tumor B cells and autologous T cells obtained from patient samples, together with healthy donors’ monocytes to complete the LN immune microenvironment. These populations self-organize in disc-shaped structures, as determined by light sheet microscopy, where B and T cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. Interestingly, RNA-seq analysis demonstrated that tumor cells in FL-PDLS recapitulate fundamental hallmarks of FL-LN (i.e. cell cycle, mTOR, EZH2, VEGF, IFNγ, IL2, IL10 and IL15, among others). Using public data basis, we have determined by differential expression analysis (FL-LN vs normal tonsils) an immune profile characteristic of FL-LN. Flow cytometry analysis of FL-PDLS determined that immune exhaustion profile is recapitulated in our model, including high expression of PD-1, TIM-3, LAG-3 and ICOS, and their corresponding ligands. Finally, we have validated FL-PDLS as a tool for immunotherapy drug screening. First, we have demonstrated that the combination of the standard of care rituximab (anti-CD20) with nivolumab (anti-PD1) reduce tumor load in a significant proportion of FL-PDLS, in accordance with previously published clinical results. Noteworthy, this combination leads to immune reactivation demonstrated by the increase of Granzyme B, IFNγ and TNFα secretion in FL-PDLS supernatants. Likewise, FL-PDLS has been useful to identify novel potential combinatorial immunotherapies (i.e. nivolumab + anti-ICOS, rituximab + anti-Tim3) that may be effective in specific set of patients. In summary, this novel FL-PDLS system represents a step towards personalized medicine, and contributes to reduce animal usage in biomedicine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Skin Sensitization Testing: The Ascendancy of Non-Animal Methods

A century ago, toxicology was an empirical science identifying substance hazards in surrogate mammalian models. Over several decades, these models improved, evolved to reduce animal usage, and recently have begun the process of dispensing with animals entirely. However, despite good hazard identification, the translation of hazards into adequately assessed risks to human health often has presented challenges. Unfortunately, many skin sensitizers known to produce contact allergy in humans, despite being readily identified as such in the predictive assays, continue to cause this adverse health effect. Increasing the rigour of hazard identification is inappropriate. Regulatory action has only proven effective via complete bans of individual substances. Since the problem applies to a broad range of substances and industry categories, and since generic banning of skin sensitizers would be an economic catastrophe, the solution is surprisingly simple—they should be subject to rigorous safety assessment, with the risks thereby managed accordingly. The ascendancy of non-animal methods in skin sensitization is giving unparalleled opportunities in which toxicologists, risk assessors, and regulators can work in concert to achieve a better outcome for the protection of human health than has been delivered by the in vivo methods and associated regulations that they are replacing.

Ecotoxicology assessments in avian species using cell-based models: A review

Cell-based models in avian species have historically focused on virology due to the demands of animal agriculture and vaccine production industries. Recent years have witnessed a gradual rise in the use of these models ( in ovo, cell lines, primary cell cultures, organ slices, and organ-on-a-chip) in ecotoxicological studies as scientists and governments begin the shift to new approach methodologies, a shift validated by the recent memo by the Environmental Protection Agency announcing the end of mammalian testing in the next two decades. This rise has been hindered by the limited standards available for avian species and the unknowns surrounding cell-based assay applicability in extrapolation to in vivo. Toxicologists have incorporated these models in many different studies, including maternal deposition, mechanistic, metabolic, and non-target analysis methods, demonstrating the broad utility of cell-based assays. In ovo methods are ideal for reproductive and early life stage development studies, primary cell cultures for metabolic analysis, cell lines for long term studies requiring culture, organ slices for metabolic research, and organ-on-a-chip models for predictive analysis. These models all have their limitations that researchers need to consider when choosing which is most appropriate for the intended research, however. The current indications are that future avian cell-based model testing would benefit from expanding the species diversity available in cell lines and increasing metabolic conservation in full replacement methods. In ovo and primary cell culture methods should also be examined to increase efficiency and further reduce animal usage. This review examines the use, limitations, and published applications of these models in an ecotoxicological context to understand the current state of avian cell-based models to explain what future directions should be taken and how best to apply the methods available to current problems that avian researchers are approaching.

Latest impact of engineered human liver platforms on drug development.

Drug-induced liver injury (DILI) is a leading cause of drug attrition, which is partly due to differences between preclinical animals and humans in metabolic pathways. Therefore, in vitro human liver models are utilized in biopharmaceutical practice to mitigate DILI risk and assess related mechanisms of drug transport and metabolism. However, liver cells lose phenotypic functions within 1–3 days in two-dimensional monocultures on collagen-coated polystyrene/glass, which precludes their use to model the chronic effects of drugs and disease stimuli. To mitigate such a limitation, bioengineers have adapted tools from the semiconductor industry and additive manufacturing to precisely control the microenvironment of liver cells. Such tools have led to the fabrication of advanced two-dimensional and three-dimensional human liver platforms for different throughput needs and assay endpoints (e.g., micropatterned cocultures, spheroids, organoids, bioprinted tissues, and microfluidic devices); such platforms have significantly enhanced liver functions closer to physiologic levels and improved functional lifetime to >4 weeks, which has translated to higher sensitivity for predicting drug outcomes and enabling modeling of diseased phenotypes for novel drug discovery. Here, we focus on commercialized engineered liver platforms and case studies from the biopharmaceutical industry showcasing their impact on drug development. We also discuss emerging multi-organ microfluidic devices containing a liver compartment that allow modeling of inter-tissue crosstalk following drug exposure. Finally, we end with key requirements for engineered liver platforms to become routine fixtures in the biopharmaceutical industry toward reducing animal usage and providing patients with safe and efficacious drugs with unprecedented speed and reduced cost.

Abstract 2625: 3D in vitro prostate cancer PDX resource for studying cancer health disparities

Abstract Prostate cancer (PCa) incidence and mortality is nearly twice that in Black men than any other race (Non-Hispanic White, Asian/Pacific Islander, American Indian/Alaska Native, Hispanic (any race)). Characterization of the underlying biological factors that contribute to this cancer health disparity (CHD) is required to close the gap and improve patient outcome for Black men. In vitro PCa research tools which reflect the racial/ethnic diversity of this patient population are urgently needed, as most PCa cell lines are of Non-Hispanic White-origin. PCa patient-derived xenografts (PDXs) retain many of the characteristics of patient tumors and can be isolated from specific racial/ethnic groups to address this concern. Rodent PDX models provide a valuable resource for studying human cancer, however recent trends in reducing animal usage have instituted a search for alternative methods for studying PDXs that also maintain their complexity. While 2D in vitro culture of PCa PDXs has been largely unsuccessful, recent evidence suggests 3D in vitro culture of PCa PDXs may enable better long-term culture of the tumor cells. Here, we present a racially/ethnically diverse PCa PDX library of specimens (Black, Non-Hispanic White, Hispanic) developed at MD Anderson Cancer Center (the MDA PCa PDX series) compatible with 3D in vitro culture. In order to confirm self-reported race/ethnicity, each PCa PDX was characterized by whole-exome sequencing and compared to reference populations for a genetic ancestry estimation. For 3D in vitro culture, we utilized a high throughput microfluidic culture platform with 96 chips, the MIMETAS OrganoPlate® 2-lane. This platform suits both 3D tissue/cell model development and throughput needs required for drug discovery. MDA-PCa PDX cell clusters were suspended in hyaluronic acid-based hydrogel solutions, seeded into the OrganoPlate, and cultured under continuous perfusion. Genetic ancestry estimation studies revealed that patient-reported race/ethnicity often aligned with the same racial/ethnic population genetic signatures. For example, two self-reported Black PDXs were primarily of West African origin (74.6-84.6%). When cultured in 3D, PCa PDX cultures were stable and viable for at least 7 days, as determined by high content fluorescence imagining coupled with cell viability dyes. By immunofluorescent staining, PCa PDX cultures exhibited appropriate expression of phenotypic prostate-specific antigen (PSA) and androgen receptor (AR) which was maintained over the life of the culture. Studies are ongoing to screen a panel of chemotherapy drugs and determine the predictivity of the platform. This well characterized racially/ethnically diverse PCa PDX library, together with the 3D in vitro platform and methods is a valuable resource for evaluating population-based tumor response. Citation Format: Peter Shepherd, Andrei Bonteanu, Stanley Hooker, Kristin Bircsak, Divya Iyer, Dwayne Dexter, Daniel A. Harrington, Rick Kittles, Nora M. Navone. 3D in vitro prostate cancer PDX resource for studying cancer health disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2625.

Combination of cassette-dosing and microsampling for reduced animal usage for antibody pharmacokinetics in cynomolgus monkeys, wild-type mice, and human FcRn transgenic mice.

The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce the number of animals used. Cetuximab, denosumab, infliximab, and a mixture of the three antibodies, i.e., cassette-dosing, were administered intravenously to cynomolgus monkeys, C57BL/6J mice, and homozygous human neonatal Fc-receptor transgenic (Tg32) mice. Mouse blood was collected from one animal continuously via the jugular vein at nine points. In cynomolgus monkeys, infliximab showed faster elimination in the cassette-dosing group than in the single-dose group. Anti-drug antibody production was observed, but the PK parameters of the clearance and distribution volume were similar in both groups. In C57BL/6J and Tg32 mice, each of the plasma concentrations-time profiles after cassette-dosing were similar to those after single dosing. PK evaluation using a combination of cassette-dosing and microsampling in mice may reduce the number of mice used by approximately 90% compared with the conventional method. The combination of antibody cassette-dosing and microsampling is a promising PK evaluation method as a high-throughput and reliable with reduced numbers of mice and cynomolgus monkeys.

Biologia Futura: the importance of 3D organoids-a new approach for research on neurological and rare diseases.

3D cell cultures and organoid approach are increasingly being used for basic research and drug discovery of several diseases. Recent advances in these technologies, enabling research on tissue-like structures created in vitro is very important for the value of the data produced. Application of 3D cultures will not only contribute to advancing basic research, but also help to reduce animal usage in biomedical science. The 3D organoid approach is important for research on diseases where patient tissue is difficult to obtain. Therefore, this review aims to show recent advances in the 3D organoid technology in disease modeling and potential usage in translational and personalized medicine of diseases with limited patient material such as neurological diseases and rare diseases.

Volumetric absorptive microsampling (VAMS®) in therapeutic protein quantification by LC-MS/MS: Investigation of anticoagulant impact on assay performance and recommendations for best practices in method development

Microsampling techniques have been employed as an alternative to traditional serum/plasma sampling because of their inherently proven and desirable advantages across the pharmaceutical industry. These include reduced animal usage in pre-clinical studies, as well as, permitting the collection of samples that would otherwise be inaccessible in clinical studies. The application of volumetric absorptive microsampling (VAMS®) technology, a second-generation dried microsampling method, coupled with LC–MS, has been extensively explored for small molecule drugs at various drug development stages. However, the potential of using VAMS technology and LC–MS analysis for biological therapeutic development has yet to be well-established. In this work, we describe the method development, validation, and a proof-of-concept non-human primate study of a LC–MS/MS method for VAMS utilized to obtain pharmacokinetic (PK) data for a therapeutic monoclonal antibody. A good correlation between VAMS data and data from conventional serum samples was established in rhesus monkeys and indicated the possibility of using of this novel sampling technology in clinical studies. However, during the initial clinical study, a significant difference in internal standard (IS) response between the patient fingerstick samples and the standard/QC samples was observed, which posed a question on the accuracy of the clinical results. A comprehensive investigation confirmed that the EDTA anticoagulant used in the standard/QC samples was the root cause of the observed anomalous IS responses. Special considerations and corresponding best practices during method development and validation are proposed to ensure early detection of potential issues and appropriate implementation of VAMS technology in clinical studies in the future.

Exploratory analysis of the application of animal reduction approaches in proteomics: How much is enough?_suppl

Animal testing has been the corner stone for chemical safety assessments, but fish embryo assays now represent an alternative. Increases in omics studies offers unparalleled access to examine early molecular responses in organisms in response to environmental stressors and yet reduction in animal usage within this context has been overlooked. For proteomics, there is significant disparity and variability in organismal pool size ranging from 1-2000 embryos per replicate for zebrafish alone. However, it is unknown if varying sample pool size results in higher protein identifications. To examine whether the proteome changes are dependent on this variable, 3 pool sizes (5, 10 or 20 embryos per replicate) were examined using the two most common fish models with appropriate biological replicate number determined based on statistical power analysis (n=7). Samples were analysed by data-independent acquisition (DIA), resulted in 1,946 and 2,770 protein groups identified (1 % FDR) for the fathead minnow and zebrafish, respectively. Proteins were not differentially expressed among pool sizes, and no significant difference was observed among the protein groups identified. However, for the fathead minnow, a decrease in the number of proteins identified was observed with increasing pool size, while only a modest increase of 110 protein identifications was observed in zebrafish between the lowest and highest pool size. Taken together, our observations suggests that a proteome characterization experiment using these fish models can achieve comparable protein identifications using pool sizes of less than 5 per replicate assuming a protein requirement of 50µg or less.