Corning Scientific Seminar Series

Advances in the understanding of the behavior and function of biological systems are dependent on the study of cells and tissues. An essential tool in this research is the use of in vitro cell culture. Many variables contribute to providing a physiological environment for the cell in the laboratory, a few examples include cell source, isolation techniques, growth conditions such as matrix proteins and soluble factors, and cell age. Basic laboratory practices are sometimes overlooked as a source of discrepancy in data; however the application of fastidious and reproducible technique can reduce cell culture as a source of data variation. As specialized techniques have been developed to modulate cells and tissues in vitro, the importance of reproducible data has become paramount. In this presentation, we will identify and discuss basic principles of in vitro mammalian cell culture that influence the quality of experimental results.

In recent decades, the nasal and bronchial mucosa has become an established administration site for systemic as well as local drug delivery. For developing novel drugs intended for this route, reliable methods are needed for assessing the rate and extent of absorption across the nasal and bronchial epithelium. Epithelix has developed a novel in vitro cell model of the human airway epithelium named MucilAir™. MucilAir™ maintains the fully differentiated, morphologically and functionally, characteristics of the native tissues for more than one year. Using this system, the typical characteristics of the airway epithelium are observed (e.g. tight junctions, cilia beating, ciliated cells, basal cells, mucous cells, cytokine/chemokine/metalloproteinase release, active ion transport and CypP450s activity). Epithelia from several pathologies can be reconstructed (e.g. Asthma, Allergic Rhinitis, COPD, CF, etc.). Due to its unique long shelf-life, this model is used for studying the human respiratory diseases, and for testing the long-term/chronic effects of drugs candidates on respiratory tract. Several applications of MucilAir™ relevant to drug delivery, inhalation efficacy and toxicity assessment will be presented. For each application, specific examples of in vitro tests will be selected to highlight the Pros and Cons of using in vitro human airway epithelia for development of new drug candidates for respiratory diseases.

Glucuronidation is as a major pathway for the metabolism and elimination of drugs in humans. It is second only to P450 for the metabolic clearance of currently marketed drugs. The glucuronidation reaction is catalyzed by the Phase 2 enzyme UDP-glucuronosyltransferase (UGT), which like the P450s exists as a superfamily of enzymes with broad and overlapping substrate specificities. Identification of the UGT isoforms important for the glucuronidation of new chemical entities is essential for predicting and avoiding the risk of adverse reactions due to either drug-drug interactions or genetic polymorphisms. This webinar will provide an in depth overview of in vitro methods for studying UGT pathways. Important parameters for setting up UGT assays using tissue fractions and recombinant UGT enzymes will be discussed, as well as the methods and tools currently available for conducting UGT reaction phenotyping studies.

Recent guidance from the FDA and EMA advocates the use of human hepatocytes as the test system for evaluating cytochrome P450 induction potential. Both agencies indicate that use of hepatocytes that have been prequalified for response to known clinical inducers and non-inducers are acceptable and/or preferred for certain model applications. In addition, the use of mRNA alone is suggested as the necessary endpoint to evaluate induction response (except in cases where enzyme stabilization is suspected as a mechanism). However, the applicability of using prequalified hepatocytes as the test system, and the use of mRNA alone as a response endpoint, has not been widely demonstrated. To this end, we have examined the induction response from a set of 20 compounds, including established clinical inducers and non-inducers of CYP3A4/5, in cryopreserved hepatocytes from three donors. The endpoint examined was CYP3A4 mRNA, typically over 8 concentration points in an effort to enable calculation of EC50 and Emax. Results were used to determine relative induction scores (RIS) and R3 values that may classify a drug candidate as a potential clinical inducer. The results and merits of these and other parameters for evaluating the induction potential of drug candidates will be discussed.

Culturing and producing healthy cells takes more than good sterile technique. Having a clear understanding of a cell culture’s requirements and history is a must. This knowledge is required to facilitate the development of the necessary conditions that will provide an environment for the cell culture to thrive . This webinar will cover the following topics:
• What are the conditions which make happy cultures and what can be done to maintain these conditions – tips and techniques
• Selecting the correct culture environment is critical to growing happy cells
• Understand the impact of stress on cultures and provide some ideas on to reduce stressful conditions

Transfection and transduction of eukaryotic cells are powerful techniques employed to manipulate and study gene expression. Reports have indicated that the use of viruses for the delivery of transgenes is one of the most commonly used tools in research. Additionally, virus transductions have been recently used at both the preclinical and clinical stages in cell therapy as well as with vaccine production leading to an increase in demand to produce more virus as efficiently as possible. This webinar will focus on Adenovirus and Lentivirus vector production scale up and will include optimizations techniques that may improve viral yields.

Dr. Turner’s research is focused on intestinal epithelial biology, with particular emphasis on tight junction regulation and inflammatory bowel disease. His group takes a multidisciplinary approach that integrates cell biology, transport physiology, electrophysiology, structural biology, molecular biology, and mucosal immunology to define fundamentals of structure and function; understand mechanisms of regulation in vitro and in vivo models; determine the contributions of barrier dysfunction to gastrointestinal disease; understand the role of the epithelial barrier in regulating other mucosal processes, e.g. immune responses; and develop novel means to correct barrier function and restore health.

In vitro cell-based assays have become a mainstay in many laboratories. The live cell provides a model far superior to that using purified components to build an assay.. Cell-based assays present particular challenges for the experimenter, however, primarily with maintaining cellular responses in microplates that reflect what occurs naturally (i.e. in tissue in a living organism). In this webinar, we will provide helpful advice and insights for setting up and optimizing cell-based assays using automation.

You will learn:
• Understanding how cell-based assays benefit from automated liquid handling
• The importance of choosing the correct microplate, instrumentation and settings to run your cell-based assay.
• Tools, tips and tricks for optimizing cell-based assays when using liquid handling instrumentation.

Tumor cells must detach from the primary tumor and invade through extracellular matrix to metastasize. In particular, the cells must attach, degrade, and migrate through the basement membrane matrix underlying blood vessels. A simple, rapid, reliable, flexible, and quantitative in vitro Invasion Assay was developed more than 25 years ago and is widely used today.
This webinar will cover how the assay is performed, provide some tips for optimal and reproducible results, and discuss various uses of the assay with examples. The emphasis will be on using basement membrane extract (BME) and tumor cells but other matrices and endothelial cells will be mentioned.

Contamination of cell cultures, whether from bacteria, fungus, virus or other cell line is of critical importance to laboratory personnel. Studies have shown that at least 20% of the animal cultures in existence today are contaminated. Cell line contamination can result in decreased productivity, loss of critical study tools, impact data quality and reduce bioprocessing capacity. In hopes of helping to reduce this common problem, this tutorial will focus on:
• Understanding the nature of contamination and its consequences.
• Understanding the major sources and causes of cell culture contamination.
• Discussing aseptic technique and its use in reducing day-to-day contamination.
• A discussion on the use of antibiotics and their potential hidden dangers.
• Simple methods to monitor for contamination and the importance in the use of cryopreserved cell lines.