The Ultimate Low Cell Binding Surface

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1 The Ultimate Low Cell Binding Surface Thermo Scientific Nunc HydroCell Surface SINGLE CELL CULTURES Grow cells that are sensitive to activation and differentiation signals arising from cell adhesion CELL CLUSTER CULTURES Higher yield and less unwanted adhesion-induced differentiation

2 Working with Single Cells and Cell Clusters in Suspension Single Cell Cultures Thermo Scientific Nunc HydroCell Surface enables cultivation of cells that are sensitive to unwanted activation and differentiation signals arising from cell adhesion. Suspension cultivation of cells that adhere to most surfaces, such as macrophages (Moriguchi et al., Mol. Cell. Biol. 2006, 26: ), and very sensitive cells, such as basophilic leukocytes (Yanase et al., Biosens. Bioelectron. 2007, 23: ) is possible in cultureware with Nunc HydroCell Surface. In traditional cultureware, whether tissue-culture treated or non-treated, suspension cultures are often heterogeneous. Some cells attach to the surface of the cultureware, while the remaining cells grow in suspension. With the Nunc HydroCell Surface, the number of cells that attach to the cultureware is minimal, resulting in a higher yield and a more homogeneous suspension culture. Single-cell suspensions harvested from Nunc cultureware with Nunc HydroCell Surface can be: re-seeded in cultureware with Nunc HydroCell Surface or traditional cell cultureware as part of a passaging procedure analyzed in vitro, for example, by flow cytometry manipulated to purify certain cell types used in cell transplantation research Seed cells in medium in cultureware with Nunc HydroCell Surface Place cells at 37 C Grow cells until desired density of cells is reached Harvest cells and medium Cell Cluster Cultures In cultureware with Nunc HydroCell Surface, certain cell types form cell clusters or spheroids. Cluster cultures are frequently used in stem cell biology and cancer biology. Examples of cluster cultures in stem cell biology are: Neural stem cells can be propagated as neurospheres, which are cell clusters containing stem cells as well as differentiated neural cell types (see Application Note). Embryonic stem cells can be differentiated in cluster cultures, termed embryoid bodies, where the stem cells differentiate into cell types of the three germ layers. The problem with using traditional cultureware (tissueculture treated or non-treated) for cell cluster cultures is that cells in the periphery of the cluster can adhere to the the cultureware. This can result in unwanted differentiation and lower yield. In cultureware with Nunc HydroCell Surface, the number of cell clusters attaching to the cultureware is minimal, resulting in a higher yield and more homogeneous cell clusters. Cell clusters harvested from Nunc cultureware with Nunc HydroCell Surface can be: dissociated and re-seeded in cultureware with Nunc HydroCell Surface or in traditional cell cultureware as part of a passaging procedure analyzed in vitro, for example, by immunohistochemistry used in different transplantation models Seed cells in medium in cultureware with Nunc HydroCell Surface Place cells at 37 C Grow cells until desired size of cell clusters is reached Harvest cell clusters and medium

3 Thermo Scientific Nunc HydroCell Surface Designed to Prevent Cell Attachment Cultivation of cells in suspension in traditional cultureware often leads to loss of cells and cell-secreted proteins due to attachment to the surface of the cultureware. The Nunc HydroCell Surface is designed to prevent cell attachment. A covalently immobilized super hydrophilic polymer forms a thin, even layer on cultureware with Nunc HydroCell Surface, resulting in minimal cell adhesion and protein adsorption. Depending on cell type, cells can be grown as a suspension of single cells or as a suspension of cell clusters. The Nunc HydroCell Surface enables cultivation of cells and cell clusters in suspension with maximum recovery of sample: Minimal cell adhesion get high yield of cells and eliminate unwanted adhesion-induced differentiation Minimal protein adsorption get high yield of cell-secreted proteins The Nunc HydroCell Surface is available on MicroWell plates, dishes and multidishes. Cultureware with Nunc HydroCell Surface Other brand of low cell binding cultureware Immediately after seeding One week after seeding 0.4 Low Cell Binding Surface Only Nunc HydroCell Surface prevents cell attachment completely. 0.3 Normal Rat Kidney (NRK) cells in medium with serum were seeded in cultureware with Nunc HydroCell Surface or in a low cell binding product of brand A, B or C. Degree of cell adhesion was determined using a TetraColor ONE assay system (Seikagaku Corp.; Tokyo, Japan) at seeding and after 5 days of incubation (bars). Optical Density (OD) at seeding 0.0 Nunc HydroCell Surface Brand A Brand B Brand C 1 Nunc HydroCell Surface 2 Brand A (low cell binding cultureware) 3 Non-treated cell cultureware Macrophages from mouse peritoneum in medium with serum were incubated for 3 days in (1) cultureware with Nunc HydroCell Surface, (2) another brand of low cell binding cultureware (Brand A), and (3) non-treated cell cultureware. Only the Nunc HydroCell Surface allowed uniform suspension culture (more than 99% of the cells remained in suspension) and simple cell dissociation and harvesting by pipetting.

4 Application Note Neurosphere Initiation and Propagation on HydroCell Surface Stem cells derived from exfoliating human deciduous teeth have the ability to differentiate into neuronal and glial cells in vitro. Dental pulp is derived from the neural crest and odontoblasts, and expresses high levels of nestin, a marker for neural progenitors. Dental pulp may, therefore, be a potential source of easily obtainable cells that can be used in cell replacement therapies for various neurological disorders. This application note demonstrates that adult rat dental pulp cells can be propagated as neurospheres in cultureware with HydroCell Surface. It is based on Sasaki et al., Neurosphere Generation from Dental Pulp of Adult Rat Incisor, European Journal of Neuroscience 2008; 27: Methods Cells from dissociated adult rat dental pulp were suspended in Dulbecco s Modified Eagle s Medium/ F12 supplemented with B27, penicillin-streptomycin (all Invitrogen, CA, USA), and growth factors bfgf and EGF (both at 20 ng/ml; PeproTech, NJ, USA), and seeded in a 24-well Multidish with HydroCell Surface. The cells were incubated at 37 C in a humidified atmosphere of 5% CO2 in air. Fresh culture medium was added to the culture 4-5 days after seeding. Level of attachment of the dental pulp cells to the HydroCell Surface and the formation of spheres were examined using phase-contrast microscopy. The presence of differentiated neural cells in the dental pulp-derived spheres was examined by fluorescence microscopy. After 8 days of culture, spheres were fixed in formalin, washed, treated with Triton X-100, and blocked by incubation in phosphatebuffered saline containing 3.3% goat serum. Spheres were then incubated with a monoclonal antibody against nestin (1:500 dilution; BD Transduction Laboratories, NJ, USA), a monoclonal antibody against Tuj1 (1:500 dilution; Covance, NJ, USA), or a polyclonal antibody against S100 (1:2000 dilution; DakoCytomation, Denmark) for 60 min. at room temperature or overnight at 4 C. Spheres were washed and incubated with secondary antibody for 60 min. at room temperature. Finally, cell nuclei of the spheres were stained with Hoechst Results Figure 1. Sphere generated from dissociated adult rat dental pulp during 7 days of culture on HydroCell Surface. Within a few days of culture, adult rat dental pulp cells formed small spheres, which after 7 days of culture grew into spheres with a diameter of more than 50 μm. Scale bar, 20 μm. Printed with kind permission from European Journal of Neuroscience. A nestin B Tuj1 C S100 Figure 2. Differentiated neural cells in dental pulpderived spheres grown for 8 days on HydroCell Surface. Spheres were immunostained for (A) nestin (red), (B) Tuj1 (red), and (C) S100 (green). Nuclei were stained with Hoechst (blue). The spheres contained nestinpositive neuronal progenitors, Tuj1-positive neuronal cells and S100-positive oligodendrocytes, indicating that the dental pulp-derived spheres were neurospheres. Scale bars, 10 μm. Printed with kind permission from European Journal of Neuroscience.

5 Quality Assurance Quality is inherent in our culture. From product development and sourcing raw materials to manufacturing and customer service, quality is reflected in every Thermo Scientific Nunc product. A certificate of quality is packed in each box of cultureware treated with Nunc HydroCell Surface. This certificate is your guarantee that the product has been validated according to the following tests: Cell adhesion Each manufacturing lot is sampled and subjected to performance testing for low cell binding properties with the A549 cell line (derived from a human lung carcinoma) in accordance with standard operation procedures. The number of adhered cells is counted. Acceptance level: adhesion of less than 1% of seeded cells. Sterility Sterility is obtained by ethylene oxide gas according to ISO (Sterilization of health care products, ethylene oxide, Part 1: Requirements for development, validation and routine control of a sterilization process for medical devices). Non-pyrogenic Representative samples of products with Nunc HydroCell Surface are tested according to the principles of the LAL-test described in the FDA guidelines and certified non-pyrogenic with a documented endotoxin level of less than 20 endotoxin units/device (0.5 Endotoxin units/ml) as stated in the USP. Toxicity The material has successfully passed the USP biological reactivity class VI test - 50 C (7 days implant). Cytotoxicity test according to ISO : Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity. Products Cultureware with Nunc HydroCell Surface With lid. Sterile. Cat. No Format 96 MicroWell Plate w/flat bottom 96 MicroWell Plate w/round bottom 24 well Multidish 12 well Multidish 10 cm Dish Number of wells Culture area, cm²/ well Max. external dimensions, mm Suggested working volume, ml/well 6 cm Dish cm Dish 128x86 128x86 128x86 128x86 92x17 60x15 40x Airvent Units per pack/ case 1/8 1/8 1/6 1/6 1/6 5/30 5/30 Cultureware with Nunc HydroCell Surface is intended for research purposes only and single-use only. Any other use is not warranted by Thermo Fisher Scientific. Do not use the product for clinical or diagnostic purposes. HydroCell surface is licensed from CellSeed Inc. Made in Japan.

6 Cell Culture Excellence Essential products for the cell culture laboratory Our comprehensive portfolio includes advanced tools designed to help you achieve excellence at every stage of your cell culture process from growth and passage to experimentation through characterization, analysis and storage. To learn about our full array of cell culture products and services, go to: North America - Tel: technical.nunc@thermofisher.com Asia Pacific - Tel: intlmktg@thermofisher.com China - Tel: info.nnichina@thermofisher.com Europe (Nalgene) - Tel: +44 (0) sales@nalgene.co.uk Europe (Nunc) - Tel: info.nunc@thermofisher.com India - Tel: Toll Free: contact.lpg.in@thermofisher.com Japan - Tel: info@nalgenunc.co.jp - All other locations (USA, International Department) - Tel: intlmktg@thermofisher.com Thermo Fisher Scientifi c Inc. All Rights Reserved /N20231 Ver / YNI. HydroCell is the trademark of CellSeed Inc. All other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries.