Issue 1 - Thermo Fisher

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Take Your Cell-based Assays to the Edge Application Note In cell culture, a volume loss as small as 10% concentrates media components and metabolites enough to alter cell physiology, in some cases, severely. With this in mind, Thermo Scientific recently unveiled its latest advancement in plates for cell culture. The Nunc Edge 96-well plates for cell-based assays incorporate large perimeter evaporative buffer zones that eliminate well-to-well variability, while dramatically reducing the overall plate evaporation rate to lower that 2% after seven days of incubation. And what’s the result? Tests have shown more viable and healthy cell yields are obtained. The unique buffer zones enable the use of all 96 wells on the plate and greatly reduce the edge effect commonly experienced in cell culture and maintaining data consistency throughout the plate. The implications of volume loss can concentrate media components and metabolites enough to alter cell physiology, a phenomena which is more pronounced in the outer and corner wells. The Nunc Edge plates significantly reduce this occurrence, effectively maintaining sample concentrations over long periods of incubation. The prevention of cell death and toxicity in the plates’ outer wells allows results to remain more true to the population phenotype for more efficient, high throughput analysis. In cell-based assays where multiple targets are simultaneously imaged, all the fluorescence from a single location needs to focus at the same point within the imaging system. The flatness of the plates eliminates the occurrence of chromatic aberrations, enabling efficient automated imaging. Furthermore, varying reagent concentrations from assay washing/aspiration steps are also dramatically reduced. Advantages of Thermo Scientific Nunc Edge Plate • Low-evaporation “moat” • 96 well format - follows ANSI standard footprint and is amenable to automation • Simply fill moat with sterile H 2 O and eliminate edge effects • Cell culture treated or non-treated • Low fluorescence • Custom barcoding available – the safest way to track your samples • Designed for both automated or manual use • Superior imaging For more information please contact: Tina McLure (AU) tina.mclure@thermofisher.com or Ph: +61 3 9757 4382 Dr Jerry Wong (NZ) jerry.wong@thermofisher.com or Ph: +64 9 980 6768 Vita means Life : NEW Thermo Scientific Nunc Nunclon Vita Surface Animal component-free surface for growth of stem cells & other fastidious cells. Nunclon Vita Surface supports attachment, colony formation and growth of human ESC, human IPS cells and other fastidious cell lines in the absence of feeder cells and matrix coatings. In media supplemented with ROCK inhibitor, human ESC can be cultured on the Nunclon Vita Surface for at least 10 passages without loss of pluripotency. Human ESC has successfully been expanded for more than 10 passages without changes to the karyotype. The unique Nunclon Vita surface helps remove variability, requires less work, and allows for the treatment surface to be adaptable to scalable cell expansion. Human Embryonic Stem Cells (human ESC) • No matrix coating or feeder cells necessary • Expand for more than 10 passages in conditioned media with a ROCK inhibitor while maintaining pluripotency • Passage with enzymes or by mechanical selection • Alternatively, passage by withdrawing ROCK inhibitor followed by a short incubation and gentle pipetting 7
Application Note A Novel Feeder-Free Embryonic Stem Cell Culture System that Supports Mouse Embryonic Stem Cell Growth and Proliferation Kalle Johnson 1 , Robin Wesselschmidt 2 , Mark Wight1, Thomas I. Zarembinski 3 Figure 1: Phase contrast photographs of mESC colonies over the course of the study. Photos of colonies grown on HyStem-C (top Row) and photos of colonies co-cultured with MEF feeders (bottom row). Morphologies bear slight differences between HyStem-C and MEF co-cultures. However, cultures grown on both substrates were able to maintain colonies that generally exhibited relatively regular, phasebright, well defined borders, characteristic of undifferentiated mESC colonies. HyStem iMEF Passage 1 Passage 2 Passage 3 Passage 4 Passage 5 1 Thermo Fisher Scientific Inc., 925 West 1800 South, Logan, Utah 84321 2 Primogenix Inc., 165 Missouri Blvd, Laurie, MO 65038 3 Glycosan BioSystems, Inc., 675 Arapeen Drive, Suite 302, Salt Lake City, Utah 84108 References: 1) Gerecht S et al, Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells PNAS 2007 vol 104: 11298–11303. 2) Engler et al, Matrix Elasticity Directs Stem Cell Lineage Specification Cell 2006 vol 126: 677-689. For more information please contact: Peter Chisholm (AU) peter.chisholm@ thermofisher.com Ph: +61 3 9757 4457 Jerry Wong (NZ) jerry.wong@ thermofisher.com Ph: +64 9 980 6768 Standard embryonic stem cell culture systems require co-culture with mitotically inactive mouse embryonic fibroblast feeder cells (iMEFs) to maintain their undifferentiated proliferative state. While iMEFs provide a suitable attachment surface and crucial soluble factors promoting embryonic stem cell (ESC) growth and proliferation, iMEFs are timeconsuming to prepare. More importantly, iMEFs vary from lot-to-lot and contaminate ESCs with carry over iMEFs from previous culture passages. These latter shortcomings confound basic research attempting to dissect the culture components or underlying gene and protein expression patterns important for ESC proliferation and differentiation. Development of a feeder-free system for embryonic stem cell culture using a synthetic matrix would provide a ready-to-use substrate which is consistent from lot to lot without iMEF carry over. iMEFs make abundant amounts of hyaluronic acid (HA) which is important for not only embryogenesis but also for human embryonic stem cell culture (1) . Using mouse embryonic stem cells (mESCs) as a model system, we reasoned that the use of a commercially available HA-rich matrix would provide a suitable starting point for preparing a novel feeder-free substrate for undifferentiated growth. Here we report the use of a crosslinkable HA-based substrate (HyStem-CTM) for feeder-free propagation of mESCs in the presence of FBS. mESCs plated on HyStem-C maintain excellent morphology and offer comparable plating efficiency to those grown on iMEFs. Data from FACS analysis as well as immunocytochemistry to confirm the presence of key recognised pluripotency markers will be presented. (Figure 1) This novel feeder-free cell culture system has potential uses in proteomic analysis since no carryover iMEF proteins will be present in embryonic stem cell extracts. In addition, statistics from high content analysis and high-throughput screening efforts employing mouse embryonic stem cells will be improved due to increased consistency during the screening campaign. Finally, the possibility exists to alter the stiffness or composition of the matrix in a manner that may enhance efforts to drive the pluripotent cells down desired lineages (2) . Three types available • HyStem Hydrogel - Chemically modified hyaluronan crosslinked with PEFDA • HyStem-C – HyStem with chemically modified gelatin added • HyStem-HP – HyStem-C with chemically modified heparin Hydrogels can be easily customised • by adding ECM proteins • by varying the Hydrogel compliance to match the stiffness of the native tissues • easy control over the amount and type of ECM protein incorporated • no autofluorescence • ideal for non-adherent cells – easy to mobilise • for adherent cells, simply add choice/selective cell attachment peptide • allows encapsulation and implantation of cells 8
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Issue 1 - Thermo Fisher

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