Today, the majority of cell biology operations are carried out manually, even though the core steps are highly reproducible. Staff are expected to conduct repetitive tasks for hours each day, and often have to come in outside of normal working hours and adapt their biology to meet working weeks. This emphasis on manual steps limits both the throughput and the traceability of the workflow. In this webinar Russell Green, Automata's Director of Product Growth, talks about how reshaping cell biology experiments for automation will enable labs to overcome these challenges.

Webinar talking points:

- Reasons to drive more automation in tissue culture & cell biology.
- What this means for your lab in terms of time, space + labware.
- Handling cells with care. 
- Approaches at Automata to build scalable cell biology automation solutions. 

In vitro cell models constitute a crucial step in the preclinical research. Their wide applications in basic research, toxicology, immunology, and drug-development studies make them indispensable in biomedicine. This webinar will provide a comprehensive overview of the different cell-based models used in biomedical research, focusing on modeling human diseases using next-generation cell culture systems: organoids and iPSCs. Additionally, it will explore the plethora of tools available to leverage the wealth of information attainable from these in cellulo models. Finally, the session will highlight how Sino Biological leads in facilitating next-generation cell culture systems through its premium-quality products and services.

Learning Objectives:

1. Outline the different cell-based models used in biomedical research.

2. Review modeling human diseases using next-generation cell culture systems.

3. Discuss research tools for advanced cell models.

Extracellular vesicles (EVs) are lipid-bilayer enclosed nano-sized vesicles that are released by all cell types and found in all bodily fluids. EVs are emerging as important regulators of both physiological and pathological processes thanks to their ability to actively deliver a cargo of bioactive molecules (e.g., proteins, nucleic acids). Several functions have been attributed to EVs, including but not limited to intercellular signaling, mediation of the cross talk between cells and the microenvironment, modulation of angiogenesis, formation of the pre-metastatic niche and remodeling of the extracellular matrix. EVs are currently under investigation globally, in pre-clinical and clinical trial settings, for potential clinical applications, such as endogenous vehicles for drug delivery, and as biomarkers of disease (i.e., liquid biopsy). Notably, in the field of regenerative medicine stem cell-derived EVs are being tested in cell-free tissue regeneration applications. Because of the rapid expansion of the EV research field, the Extracellular Vesicle Core was founded at CHLA in 2018, as the first in the country to provide the research community with expertise, optimized tools and up to date technologies dedicated to the field of EVs. The core provides isolation, characterization, and analysis of EVs in an effort to facilitate EV research by adhering to the ever-evolving guidelines for standardization developed by the International Society of Extracellular Vesicles. The EV core assists researchers in the discovery and validation of vesicle-linked biomarkers and in the preparation of purified extracellular vesicles for functional analysis with the potential to facilitate future clinical developments.

Learning objectives:

1. Define the main types of Extracellular Vesicles.

2. Outline three methods utilized for the isolation of Extracellular Vesicles.

3. Outline the areas of potential therapeutic applications of Extracellular Vesicles.

Current methods used to predict safety and efficacy of candidate drugs accounts for as much as 90% failure rate. Approximately 30% of drugs have failed in human clinical trials due to adverse reactions, and another 60% fail due to lack of efficacy. A number of these failures can be attributed to poor predictability of human response from animal and 2D in vitro models currently being used in drug development. Recent systematic studies on the predictive value of animal models have demonstrated a poor correlation between animal data and human outcomes owing to substantial interspecies differences in key disease pathways and disease-induced changes in gene expression profiles, highlighting the critical need for alternative methods to model complex human-relevant conditions. To address this challenge, the NIH Tissue Chips or Microphysiological Systems (MPS) program https://ncats.nih.gov/tissuechip is developing better risk assessment tools that can provide more reliable readouts of toxicity and efficacy of candidate therapies. Tissue chips are bioengineered 3D microfluidic platforms utilizing chip technology and human-derived cells and tissues that are intended to mimic tissue cytoarchitecture and functional units of human organs and systems. In addition to drug development, these microfabricated devices are useful for modeling human diseases, and for studies in precision medicine and environmental exposures. By emulating human physiology, these chips have the potential to increase the predictive power of preclinical modeling, which in turn will move the pharmaceutical industry closer to its aspiration of clinically relevant and ultimately animal-free drug discovery. Tissue chips as novel preclinical modeling platforms offer improved clinical predictions of human response, provide a more efficient approach to mechanistic investigation, early safety liability screening and even more translationally relevant modeling of drug distribution and metabolism. Near term regulatory approval of these human-relevant platforms will fill gaps in current capabilities for assessing important properties of disposition, efficacy, and safety liabilities. The recent passage of the FDA Modernization Act 2.0 will only hasten this process. Towards this end, NCATS is also establishing Translational Centers for MPS to qualify tissue chips as drug development tools.

Learning Objectives:

1. Learn about what tissue chips/MPS are how they are being used as risk assessment tools for safety and efficacy studies.

2. Learn how Tissue chips/MPS are currently being used in industry, especially in drug development.

3. Learn about what it takes to get tissue chips/MPS into regulatory acceptance.

Cancer is a group of complex genomic diseases. Today still most clinicians, patients, and policymakers think that precision oncology is the sequencing of cancer genomes, which is for many patients a fatal mistake. Here we present how a combination of multiple-omics approaches including a Luminex based multiplexed proteomics approach can help to identify not only better treatment regimen for the individual patient, but also biomarkers that may change the way how future patients will be treated.

The capture of tumor-derived extracellular vesicles (TEVs) by cells in the tumor microenvironment (TME) contributes to metastasis and notably to the formation of the pre-metastatic niche (PMN). However, due to the challenges associated with modelling release of small EVs in vivo, the kinetics of PMN formation in response to endogenously released TEVs have not been examined. Here, we have studied the endogenous release of TEVs in mice orthotopically implanted with metastatic human melanoma (MEL) and neuroblastoma (NB) cells releasing GFP-tagged EVs (GFTEVs) and their capture by host cells to demonstrate the active contribution of TEVs to metastasis. Human GFTEVs captured by mouse macrophages in vitro resulted in transfer of GFP vesicles and the human exosomal miR-1246. Mice orthotopically implanted with MEL or NB cells showed the presence of TEVs in the blood between 5 and 28 days after implantation. Moreover, kinetic analysis of TEV capture by resident cells relative to the arrival and outgrowth of TEV-producing tumor cells in metastatic organs demonstrated that the capture of TEVs by lung and liver cells precedes the homing of metastatic tumor cells, consistent with the critical roles of TEVs in PMN formation. Importantly, TEV capture at future sites of metastasis was associated with the transfer of miR-1246 to lung macrophages, liver macrophages, and stellate cells. This is the first demonstration that the capture of endogenously released TEVs is organotropic as demonstrated by the presence of TEV-capturing cells only in metastatic organs and their absence in non-metastatic organs. The capture of TEVs in the PMN induced dynamic changes in inflammatory gene expression which evolved to a pro-tumorigenic reaction as the niche progressed to the metastatic state. Thus, our work describes a novel approach to TEV tracking in vivo that provides additional insights into their role in the earliest stages of metastatic progression.

Learning Objectives: 

1. Distinguish the different subtypes of extracellular vesicles based on features and composition.

2. Understand the role of extracellular vesicles in the preparation of the pre-metastatic niche.

3. Recognize their potential in diagnosis and therapy.

Acute T-cell mediated rejection (TCMR) is the most frequent complication after liver transplantation (LT), which can affect up to 50% of LT recipients in the first 5-year post-transplant. Little is known about the immunological factors and specific leukocyte subpopulations driving TCMR and its recurrence in LT. The introduction of spatial single-cell technologies such as Imaging Mass Cytometry (IMC), allow an in-depth immune cell identification, including detection of rare cell phenotypes, along with spatial resolution of cell-to-cell interaction. However, the analysis of those high dimensional data requires computational and immunology expertise to meaningfully characterize immune-reach microenvironments. IMC comes with certain images artifacts (e.g. noise, aggregates) that can deteriorate the quality of the data which lead to either discard part of data (in variable percentage) or acceptance of errors in cell classification due to the association of markers belonging to different cell lineage, thus creating non-biological phenotypes. Additionally, segmentation of raw images (training of classifiers) can be time consuming and operator dependent thus introducing additional data variability.  Several toolkits have been developed for images preprocessing (e.g MAUI) and segmentation including Mesmer and Ilastik. By using a multi-steps IMC pipeline comprehensive of images preprocessing and semi-supervised clustering, we analyzed 96 images obtained from liver biopsies of patients with no-rejection, TCMR and chronic rejection. We identified 32 unique immune subpopulations, including three specific PD1⁺ T-cell subclusters, with a peculiar spatial distribution, which are associated with TCMR in LT recipients. The PD1 coinhibitory pathway has emerged as an important mediator and therapeutic target in several diseases including cancer, but its role in TCMR in LT is unknown. Our findings provide new insights in the pathophysiology of TCMR which support further investigations of the role of PD1 in TCMR.

Learning Objectives:

1. Identify the advantage of spatial single-cell technology (IMC), in the study of liver allograft rejection.

2. Use of images preprocessing to enhance data analysis

3. Discuss the identification of biomarker of rejection.

Healthy human red blood cells (RBCs) gradually degrade over their lifespan of ~120 days. However, sickle cells - RBCs from sickle cell disease (SCD) patients - degrade much faster within their much-shortened lifespan of ~10-20 days. Understanding the underlying mechanisms of RBC senescence in health and disease is crucial for studying many RBC diseases. Both normal RBCs and sickle cells experience repeated cycles of hypoxia and mechanical fatigue. To investigate the impact of these challenges on RBC damage and degradation accumulation, we developed in vitro microfluidic assays for testing RBCs under cyclic hypoxia loading and/or cyclic mechanical loading. Moreover, spleen plays a crucial role in maintaining the balance between RBC formation (erythropoiesis) and removal. Altered RBCs are cleared from circulation through splenic RBC retention and elimination, which mainly occur in open circulation, where RBCs flow through macrophages and inter-endothelial slits (IESs). Specifically, we developed an in-vitro oxygen-mediated spleen-on-a-chip platform, combined with in-silico simulations and in-vivo/ex-vivo observations to study spleen function and antisickling drug efficacy. Our results provide mechanistic insights into how spleen maintains its homeostatic balance between splenic RBC retention and elimination, and how disruptions in this balance could lead to anemia, splenomegaly, and acute splenic sequestration crisis in SCD.

Learning Objectives:

1. Summarize the root cause of sickle cell disease and the importance of studying sickle cell (i.e., red blood cell from sickle cell disease patient) behavior under hypoxia.  

2. Explain why the microfluidic assays developed for testing accumulated red blood cell degradation due to the challenges of cyclic hypoxia and mechanical fatigue: device setup and key results.

3. Summarize the oxygen-mediated spleen-on-a-chip platform for studying the retention of sickle cells by splenic interendothelial slits and the adhesion and elimination of sickle cells by macrophages: device setup and key results.

The global incidence of cancer is on a steady rise, with a projected annual diagnosis rate of nearly 30 million cases by 2040. Despite this alarming trend, our understanding of the intricate mechanisms underlying carcinogenesis and cancer invasion into vasculature and bone remains limited due to a dearth of in vitro and ex-vivo models that faithfully recapitulate the complexities of biological tissues while maintaining precise experimental control.

Furthermore, the arduous process of drug development, characterized by its complexity and high attrition rate, imposes substantial financial and temporal burdens in bringing new cancer therapies to market. To address these challenges, there is an imperative to develop preclinical platforms that not only meet the functional standards of traditional animal models but also surpass them, thereby enhancing our comprehension of diverse cancer types and expediting the pace of drug discovery.

This presentation aims to elucidate the utility of microphysiological systems featuring engineered vasculature and bone in systematically investigating cancer invasion into these critical tissues. Our findings include compelling evidence of the pivotal role played by perivascular cells in fostering a pro-tumoral microenvironment, as well as insights into the paracrine mechanisms through which bone cells induce epithelial mesenchymal transition in head and neck cancer. These discoveries hold promise for advancing cancer research and therapeutic development.

Learning Objectives: 

1. Discover Innovative Microphysiological Testing Platforms: Present novel perspectives on the development of functional and standardized microphysiological testing platforms that not only match but surpass the capabilities of preclinical animal models.

2. Evaluate Perivascular Cell Functionality: Examine the regulatory role of perivascular cells in modulating the responses of tumoral vasculature to extracellular mechanical changes, shedding light on their significance in the context of cancer progression.

3. Analyze Epithelial Mesenchymal Transition in Cancer-Bone Interaction: Delve into the mechanisms underlying epithelial mesenchymal transition in head and neck cancer within the context of interactions with bone tissue, fostering a deeper understanding of this critical aspect of cancer biology.

In recent years in vivo CNS dosing has gone from being a relatively uncommon modality to one that is fairly standard. However, bringing this administration route into maturity requires an understanding of the nuances and unknowns to be resolved. This presentation will discuss several of the challenges and a promising approach to filling in the gaps in our knowledge and creating true best practices that provide reproducible high quality data that will be reliable over time.

Learning objectives:

1. Understand the current gaps in our knowledge of the mechanics of in vivo CNS dosing

2. Design a method to increase out understanding of this modality.

3. Discuss the human factors which will allow us to solve these problems and leverage the expertise of a holistic project group.