Cell Biology 2018

Room air conditions are hypothermic, hypocapnic and hyperphysioxic from the cells’ point of view. Yet we stress cells with these uncontrolled conditions routinely. It is no wonder that we see problems with the reproducibility and translatability of in vitro cell data. The nascent field of immunometabolism is revealing that metabolic processes, responsive to oxygen, do more than just support critical cell processes like proliferation and differentiation, but actually control them. In this talk we will dive into basic environmental conditions for cells in research, particularly physiologic oxygen. We will talk about the physiology behind different cellular oxygen levels in vivo and how to match those conditions in vitro for both cell incubation and cell handling.  We will also present data showing that constant, physiologically relevant conditions for both incubation and cell handling can improve cell growth in vitro. It is all part of establishing basic cell conditions that are Cytocentric, putting the cells’ needs first. 
 

Learning Objectives: 

1. Participants will be able to discuss physiologic oxygen knowledgably, both in vivo and in vitro.
2. Participants will be able to distinguish between Cytocentric and Peoplecentric cell culture conditions.

Cellular processes are orchestrated by a large number of biomolecules in a spatially and temporally coordinated manner within a tiny volume. To uncover the underlying organizational principles and their functional relevance, we take microscopy visualization as the primary approach to systematically map the spatial localization, temporal dynamics and activity profiles of proteins and nucleic acids. For this purpose, we have developed a fragment-tagging approach which fluorescently labels target proteins using our engineered split fluorescent proteins. This approach has enabled systematic tag knock-in in cell lines through CRISPR/Cas9-medied gene editing. Our method paves the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.
 

Learning Objectives: 

1. Methods to labeling endogenous proteins in cells by gene editing
2. New fluorescent label for cellular proteins

Cell motility and migration are of great interest to many researchers, e.g. in the fields of cancer metastasis and immunology. Using digital holographic microscopy (DH), cell movement can be investigated in great detail, as well as connected to other cellular parameters such as cell size and shape. DH results are both qualitative and quantitative, resulting in absolute measurements of cell movement and morphology. The technology allows for both cell population studies and for detailed analysis of the movements of individual cells. In addition, DH is label free and cell friendly, and will minimize the impact of the experiment on the cells. Moreover, the technology allows for a robust digital autofocus. Altogether, this makes DH very well suited for long term live cell imaging. In this talk, the DH technology and the potential applications in cell biology will be discussed, and results on cell motility studies from researchers all over the world will be presented.

Learning Objectives: 

1. To understand the technology and the potential of digital holographic microscopy in live cell imaging.
2. To understand the possibilities and challenges of measuring cell movement using digital holographic microscopy

Holotomography (HT) uses laser interferometry to measure 3-D refractive index (RI) distribution. HT serves as a powerful tool for imaging small transparent objects, such as biological cells and tissues. HT is an optical analogous to X-ray computed tomography (CT); HT measured multiple 2-D holograms of a sample with various illumination angles, from which a 3-D RI distribution of the sample is reconstructed by inversely solving the wave equation. Unlike conventional fluorescence-based imaging techniques, HT provides label-free 3-D imaging capability. Without any fixation or labeling, 3-D images of live cells can be obtained with high spatial resolution (down to 110 nm). Furthermore, HT provides quantitative imaging capability: RI maps of a cell are precisely and quantitative measured, from which various cellular analysis can be followed. 

We present the principle and applications of HT in various fields, including cell biology, microbiology, and hematology. Also, we also present the recently developed multimodal system which simultaneously measures the 3-D RI tomogram and 3-D fluorescence image of a sample, and enables unprecedented correlative and quantitative bioimaging capability. 

Learning Objectives: 

1. To understand the working principle of holotomography 
2. To learn direct applications of holotomography in the field of biology and medicine

There is considerable excitement surrounding the treatment potential of T cell immunotherapies. Amongst these, CAR-T cells have demonstrated impressive therapeutic efficacy in a subset of hematological malignancies, leading to the approval of KymriahTM and YescartaTM, and continue to be the focus of a growing clinical trial pipeline tackling a spectrum of liquid and solid tumor indications. Despite the clinical success of CAR-T cells, there remain challenges associated with routinely offering these products as treatment alternatives, including a costly manufacturing process that relies on lengthy and complex open workflows with high manual labor requirements that influence product variability. To address these challenges, we have investigated individual CAR-T unit operations to identify commercially available reagents and modular equipment that drive process closure and automation as a method to improve workflow efficiency and product consistency. Here an update is presented on development of our semi-automated closed CAR-T process, which includes the Smart-Max, SepaxTM C-Pro, XuriTM W25 bioreactor, SefiaTM and VIA FreezeTM equipment platform and achieves greater than 1.0E10 expanded T cells with upwards of 80% eGFP transduction efficiency across an 8-day manufacturing process. 

Learning Objectives: 

1. Understand CAR-T manufacturing challenges
2. Identify closed automated solutions for commercial manufacturing of CAR-T therapies

Lowe Syndrome (LS) is a devastating genetic disease characterized by abnormalities in the eyes, brain and kidneys that unfortunately leads to the premature death of affected children due to renal failure. Despite being described more than 60 years ago, this condition lacks a clear delineation of its mechanism and no specific cure is available. One contributing cause to this slow progress has been the absence of proper disease models for this condition and the inaccessibility of patient cells from the major affected organs. 

However, using patient skin fibroblasts we recently reported the first successful preparation of Lowe syndrome induced Pluripotent Stem Cells (iPSCs) and their reprogramming as renal cells1. This work not only represents a technological advance for the LS research field, but also provided insight as to how the patient’s kidney complications develop.

Specifically, monitoring the process of in vitro kidney cell differentiation provided clues as to how renal deficiency arises in patients. Specifically, we found that in LS kidney cells the transcription factor Six2 (crucial for renal development) was abnormally retained outside the cell nucleus impairing its gene regulatory function. This deficient Six2 activity caused decreased production of the so-called proximal tubular cells which are involved in critical functions of the kidney, such as avoiding the excretion of important serum proteins. 

Misregulation of a differentiation pathway is a novel LS phenotype that is predicted to have great impact in patients’ renal function. Further, this work suggests that developing strategies directed to enhance proper Six2 function or to prevent its retention outside the nucleus constitute viable options to maintain renal function in LS patients.

Learning Objectives: 

1. Understand how Lowe Syndrome can lead to premature death of affected children.
2. Understand how patient skin fibroblasts are helping those affected with Lowe Syndrome.

Room air conditions are hypothermic, hypocapnic and hyperphysioxic from the cells’ point of view. Yet we stress cells with these uncontrolled conditions routinely. It is no wonder that we see problems with the reproducibility and translatability of in vitro cell data. The nascent field of immunometabolism is revealing that metabolic processes, responsive to oxygen, do more than just support critical cell processes like proliferation and differentiation, but actually control them. In this talk we will dive into basic environmental conditions for cells in research, particularly physiologic oxygen. We will talk about the physiology behind different cellular oxygen levels in vivo and how to match those conditions in vitro for both cell incubation and cell handling.  We will also present data showing that constant, physiologically relevant conditions for both incubation and cell handling can improve cell growth in vitro. It is all part of establishing basic cell conditions that are Cytocentric, putting the cells’ needs first. 
 

Learning Objectives: 

1. Participants will be able to discuss physiologic oxygen knowledgably, both in vivo and in vitro.
2. Participants will be able to distinguish between Cytocentric and Peoplecentric cell culture conditions.

Cell motility and migration are of great interest to many researchers, e.g. in the fields of cancer metastasis and immunology. Using digital holographic microscopy (DH), cell movement can be investigated in great detail, as well as connected to other cellular parameters such as cell size and shape. DH results are both qualitative and quantitative, resulting in absolute measurements of cell movement and morphology. The technology allows for both cell population studies and for detailed analysis of the movements of individual cells. In addition, DH is label free and cell friendly, and will minimize the impact of the experiment on the cells. Moreover, the technology allows for a robust digital autofocus. Altogether, this makes DH very well suited for long term live cell imaging. In this talk, the DH technology and the potential applications in cell biology will be discussed, and results on cell motility studies from researchers all over the world will be presented.

Learning Objectives: 

1. To understand the technology and the potential of digital holographic microscopy in live cell imaging.
2. To understand the possibilities and challenges of measuring cell movement using digital holographic microscopy

Holotomography (HT) uses laser interferometry to measure 3-D refractive index (RI) distribution. HT serves as a powerful tool for imaging small transparent objects, such as biological cells and tissues. HT is an optical analogous to X-ray computed tomography (CT); HT measured multiple 2-D holograms of a sample with various illumination angles, from which a 3-D RI distribution of the sample is reconstructed by inversely solving the wave equation. Unlike conventional fluorescence-based imaging techniques, HT provides label-free 3-D imaging capability. Without any fixation or labeling, 3-D images of live cells can be obtained with high spatial resolution (down to 110 nm). Furthermore, HT provides quantitative imaging capability: RI maps of a cell are precisely and quantitative measured, from which various cellular analysis can be followed. 

We present the principle and applications of HT in various fields, including cell biology, microbiology, and hematology. Also, we also present the recently developed multimodal system which simultaneously measures the 3-D RI tomogram and 3-D fluorescence image of a sample, and enables unprecedented correlative and quantitative bioimaging capability. 

Learning Objectives: 

1. To understand the working principle of holotomography 
2. To learn direct applications of holotomography in the field of biology and medicine

Early adverse exposures, such as maternal stress during pregnancy and child abuse, are thought to result in long-lasting consequences on neural circuit function and stress hormone regulation and ultimately in an increased risk for psychiatric but also medical disorders later in life. Overall, this presentation will describe putative molecular mechanisms how genetic variants and exposure to adversity interact to shape risk and resilience for psychiatric disorders, with a focus on stress hormones. These glucocorticoids (GCs) have been shown to alter gene expression pattern and to induce long-lasting epigenetic changes in specific loci through binding of the glucocorticoid receptor (GR) to glucocorticoid responsive elements (GREs). 

The talk will first highlight data from a human hippocampal cell line that identify long lasting epigenetic changes in DNA methylation in response to GCs. These lasting epigenetic changes are located in brain development- and disease-relevant gene enhancer regions and lead to increased transcriptional sensitivity to future stress exposure. Data from human brain organoids and single cell sequencing will then delineate whether specific subtypes of cells show differential sensitivity to early GC exposure during brain development. The second focus will be on common genetic variants in long-range enhancer elements that can modulate the transcriptional and epigenetic response to GR activation and early life adversity. These functional genetic variants associate with increased risk for psychiatric phenotypes and differences in neural correlated of stress processing and are in turn enriched among the loci showing lasting DNA methylation changes with GC in the hippocampal cell line described above. 

Overall, the presentation will outline how stress-exposure can have lasting effects on cell and tissue function and how this relates to risk or resilience to stress-related disorders in the context of common genetic variation.

Learning Objectives: 

1. Understand how stress can lead to lasting epigenetic changes
2. Understand possible molecular mechanisms of gene x stress interactions

CRISPR has revolutionized how we do science in a similar way PCR changed the face of molecular biology. Learn how to take advantage of this phenomenal technology in the easiest way with Synthego, the leader in genome engineering. We have a full stack solution from design, edit and analyze that you can utilize to get the insights that you really want!

Learning Objectives: 

1. Introduction to CRISPR/Cas9 technology and how it can be utilized
2. How to design, edit and analyze your CRISPR experiment 

With the advent of cost-effective culturing approaches, 3D cell culture models (3D-CCMs) have been rapidly adopted for drug discovery since they provide a more physiologically relevant micro-environment; showing improved predictive utility for assessing drug efficacy and/or toxicity when compared to traditional 2D monolayer models. High-content analysis/screening (HCA/HCS) also plays a major role in drug screening, but one of the unique challenges of evaluating 3D-CCMs (i.e. spheroids, organoids, micro-tissues, organs-on-a-chip) is their opacity and thickness that limit optical imaging to only the outermost layers (~20=30 microns). Since the outermost cells are exposed to different physiological test conditions (oxygen, nutrients, media exchange, drug dosing), current image-based approaches induce a bias in the results since the outer layer of cells are found in a significantly different micro-environment than the interior. This shortcoming is particularly problematic when ascertaining the relative effectiveness of current therapeutic agents (e.g. immunoglobulin-based therapeutics, anti-proliferatives) since their effects are likely to be localized or concentrated to the surface while their efficacy within the interior are obfuscated and not fully resolved. Visikol™ has shown that through the addition of high content confocal microscopy with the Thermo Scientific™ CellInsight™ CX7 LZR High-Content Analysis Platform and the Visikol® HISTO-M™ reagent that the entire population of cells within 3D-CCMs can be characterized.

Learning objectives:

1. To understand the limitations of 3D cell culture models and considerations for using them in assays

2. To understand the challenges associated with imaging 3D cell culture models and designing HCS assays

When looking to answer complex biological questions, using in vitro techniques, you want the model that best reflects the in vivo condition.  For decades two-dimensional cell culture has dominated in this regard.  However, is it accurate to equate flat two-dimensional growing single monolayer cells to a complex three-dimensional tissue or organ system? Organs boast a unique three-dimensional cellular architecture, with cell-cell and cell-matrix interactions, creating a complex communication network through biochemical and mechanical signals.  However, important and distinct differences exist between 2D and 3D cell culturing, as well as the in vivo situation.  Differences between these two culturing extremes culminate into discrepancies in treatment responses.  Proof of concept suggests that 3D models may be more apt at providing information when evaluating new lead compounds.  Various types of 3D cell culture model systems are currently available and being explored.  The choice of system depends on the hypothesis, study design or target organ, and not one system is superior to the other with each offering various advantages and disadvantages.  The dynamic micro-gravity spheroid 3D system exhibits the ability to overcome many of the shortcomings of traditional 2D cell cultures.  In implementing this system in our laboratories, findings obtained from 2D and 3D in vitro hepatoxicity experiments showed significant discrepancies between the two systems.  While 3D experiments compared to an in vivo animal model, indicated that the 3D model is able to accurately predict possible hepatoxic events.  Looking at all the information provided, I put it to you, does dimension really matter?

Learning Objectives: 

1. What are the major differences between two-dimensional and three-dimensional cell culture, and how will this influence results.
2. The potential applications of novel three-dimensional cell culture models and techniques in cell biology.

To address the need for rapid and sensitive quantitation of miRNA and protein biomarkers from biological samples, we have developed the FirePlex® Technology Platform. Utilizing patented FirePlex hydrogel particles and a three-region encoding design, FirePlex allows for true, in-well multiplexing, providing flexible and customizable quantification of analytes directly from biofluids, without the need for RNA isolation or sample processing prior to analysis. 

In this seminar, we will highlight the unique features of the FirePlex's particle-based multiplexing, and present the FirePlex miRNA and Immunoassays which can be customized for a broad range of research areas, including organ injury, inflammation, and cancer immunology. We will also introduce the new FirePlex-HT, a two-step, no-wash immunoassay designed for high-throughput biomarker screening studies.

Learning Objectives: 

1. Profiling up to 65 miRNA directly in a single sample from biofluids without RNA extraction
2. Profiling up to 70 proteins directly in a single biofluid sample

Cellular processes are orchestrated by a large number of biomolecules in a spatially and temporally coordinated manner within a tiny volume. To uncover the underlying organizational principles and their functional relevance, we take microscopy visualization as the primary approach to systematically map the spatial localization, temporal dynamics and activity profiles of proteins and nucleic acids. For this purpose, we have developed a fragment-tagging approach which fluorescently labels target proteins using our engineered split fluorescent proteins. This approach has enabled systematic tag knock-in in cell lines through CRISPR/Cas9-medied gene editing. Our method paves the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.
 

Learning Objectives: 

1. Methods to labeling endogenous proteins in cells by gene editing
2. New fluorescent label for cellular proteins

There is considerable excitement surrounding the treatment potential of T cell immunotherapies. Amongst these, CAR-T cells have demonstrated impressive therapeutic efficacy in a subset of hematological malignancies, leading to the approval of KymriahTM and YescartaTM, and continue to be the focus of a growing clinical trial pipeline tackling a spectrum of liquid and solid tumor indications. Despite the clinical success of CAR-T cells, there remain challenges associated with routinely offering these products as treatment alternatives, including a costly manufacturing process that relies on lengthy and complex open workflows with high manual labor requirements that influence product variability. To address these challenges, we have investigated individual CAR-T unit operations to identify commercially available reagents and modular equipment that drive process closure and automation as a method to improve workflow efficiency and product consistency. Here an update is presented on development of our semi-automated closed CAR-T process, which includes the Smart-Max, SepaxTM C-Pro, XuriTM W25 bioreactor, SefiaTM and VIA FreezeTM equipment platform and achieves greater than 1.0E10 expanded T cells with upwards of 80% eGFP transduction efficiency across an 8-day manufacturing process. 

Learning Objectives: 

1. Understand CAR-T manufacturing challenges
2. Identify closed automated solutions for commercial manufacturing of CAR-T therapies

Lowe Syndrome (LS) is a devastating genetic disease characterized by abnormalities in the eyes, brain and kidneys that unfortunately leads to the premature death of affected children due to renal failure. Despite being described more than 60 years ago, this condition lacks a clear delineation of its mechanism and no specific cure is available. One contributing cause to this slow progress has been the absence of proper disease models for this condition and the inaccessibility of patient cells from the major affected organs. 

However, using patient skin fibroblasts we recently reported the first successful preparation of Lowe syndrome induced Pluripotent Stem Cells (iPSCs) and their reprogramming as renal cells1. This work not only represents a technological advance for the LS research field, but also provided insight as to how the patient’s kidney complications develop.

Specifically, monitoring the process of in vitro kidney cell differentiation provided clues as to how renal deficiency arises in patients. Specifically, we found that in LS kidney cells the transcription factor Six2 (crucial for renal development) was abnormally retained outside the cell nucleus impairing its gene regulatory function. This deficient Six2 activity caused decreased production of the so-called proximal tubular cells which are involved in critical functions of the kidney, such as avoiding the excretion of important serum proteins. 

Misregulation of a differentiation pathway is a novel LS phenotype that is predicted to have great impact in patients’ renal function. Further, this work suggests that developing strategies directed to enhance proper Six2 function or to prevent its retention outside the nucleus constitute viable options to maintain renal function in LS patients.

Learning Objectives: 

1. Understand how Lowe Syndrome can lead to premature death of affected children.
2. Understand how patient skin fibroblasts are helping those affected with Lowe Syndrome.