Respiratory and Cardiovascular Research Tools for Biologically Relevant Results

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1 BioResearch Respiratory and Cardiovascular Research Tools for Biologically Relevant Results Respiratory and Cardiovascular Research Tools

2 Avoid Research Roadblocks and Take a Direct Route to Results I m uncertain of the results that I get from genetically modified cell lines. SOLUTION: Simulate in vivo research in culture by choosing from oucomprehensive selection of primary cells. Page 5 Primary Cells and Media I can t transfect my primary cells or cell lines of interest efficiently. SOLUTION: Our Nucleofector Technology delivers up to 90% gene transfection efficiency, at high viability and maintenance of functionality. Page 11 Transfection 2 BioResearch Respiratory and Cardiovascular Research Tools

3 I can t find reliable assay tools for mrna, protein expression, cell proliferation, or cytotoxicity. SOLUTION: Now you can choose from a range of assays developed specifically for respiratory and cardiovascular disorders. Page 14 Cell Analysis I want to build better in vitro models to optimize and improve my drug targets. SOLUTION: Lonza can ease your drug discovery research with our advanced cell culture tools such as 3D culture systems and Cells on Demand Cell Culture Services. Page 16 Drug Discovery Tools Respiratory and Cardiovascular Research Tools BioResearch 3

4 Your Respiratory and Cardiovascular Toolkit Streamline your workflow by choosing from convenient, innovative research tools that have been designed and tested to work together. Lonza gives you what you need for biologically relevant results, from high-quality primary cells, through efficient transfection technology, to a wide range of analysis tools. Primary Cells and Media Transfection Drug Discovery Cell Analysis Normal and disease state cryopreserved Clonetics Primary Human and Animal Cells: Respiratory: Normal, Asthmatic, COPD, Cystic Fibrosis cell types: Human bronchial epithelial cells Human lung fibroblasts Human bronchial smooth muscle cells Human small airway epithelial cells Lung microvascular endothelial cells Normal and Diabetic donor cell types: Human cardiac fibroblasts; atrial and ventricle Rat primary cardiomyocytes Human umbilical vein endothelial cells Human aortic endothelial and smooth muscle cells Cardiac microvascular endothelial cells Coronary artery endothelial and smooth muscle cells Pulmonary artery endothelial cells Pulmonary artery smooth muscle cells Nucleofector Technology with optimized protocols for normal and diseased cells from various tissues and species. Primary cells, including: Endothelial cells Epithelial cells Smooth muscle cells Fibroblasts Adult and embroynic stem cells Cell lines, including: A549 Calu-6 WI-38 HMEC-1 and many more RAFT 3D Cell Culture System Cancer Research Airway Research Blood Brain Barrier Models Cells on Demand Services: Custom transfection services Custom cell isolation services Protein Expression Analysis: PAGEr Precast Protein Gels for Western blotting Cell Proliferation and Cytotoxicity BioAssay Kits: ViaLight Kit for measuring cell proliferation and cytotoxicity ToxiLight Kit for non-destructive measurement of cytotoxicity Living Cell Monitoring CytoSMART System: small, easy and affordable live cell imaging system for documentation or monitoring of cell cultures Optimized Clonetics Primary Cell Growth Media: EGM 2 Endothelial Growth Media BEGM Bronchial Epithelial Growth Media SmGM 2 Smooth Muscle Growth Media FGM 2 Fibroblast Growth Media FGM 3 Cardiac Fibroblast Growth Media SAGM Small Airway Growth Media B-ALI and S-ALI Epithelial Airliquid Interface Media EGM -2MV Microvascular Endothelial Growth Media SCGM Stromal Cell Growth Media 4 BioResearch Respiratory and Cardiovascular Research Tools

5 Primary Cells and Media Now you can take your choice of high-quality primary, non-transformed, non-immortalized cells from Lonza. They have all been tested and matched with media to save you the time and effort of optimization. More than 20% of cell culture studies are based on misidentified or cross-contaminated cell lines. Lonza s products have been developed to give you complete confidence in your results. Our cells have been sourced from a variety of donors, including those with diseased airways such as asthma and chronic obstructive pulmonary disease (COPD), Cystic Fibrosis, idiopathic pulmonary fibrosis and Diabetic donors. Day 25 post air lift normal bronchial epithelial cells cultured in B-ALI Media. Asthmatic, COPD and Cystic Fibrosis Primary Diseased Airway Cells Enhance the relevance of your results by working with human primary cells that have been isolated from asthmatic, chronic obstructive pulmonary disease (COPD), Cystic Fibrosis and Diabetic donors: Compare diseased cells to normal primary cells for a better understanding of effects To obtain extensive donor information on diseased cells, contact our scientific support team Cystic Fibrosis genotyping data now available. Contact customer service to obtain data Pre-screened and guaranteed-to-perform airway cells in Air-Liquid Interface Media All cells test negative for bacterial, fungal, and mycoplasma contamination Human cells test negative for HIV-1, Hepatitis-B, and Hepatitis-C New, Lung fibroblasts available from donors diagnosed with idiopathic pulmonary fibrosis Respiratory disease-relevant cells include: Diseased and normal human bronchial epithelial cells Diseased and normal human lung fibroblasts Diseased and normal human bronchial smooth muscle cells Diseased and normal small airway epithelial cells Pre-screened and guaranteed to perform bronchial/small airway epithelial cells in air-liquid interface media Diseased and normal pulmonary artery endothelial and smooth muscle cells Diseased and normal proliferating cells for all cell types listed above. For best results, match these cells with our optimized Clonetics BulletKit Growth Media. Clonetics Airway BulletKit Growth Media I m uncertain of the results that I get from genetically modified cell lines. SOLUTION: Simulate in vivo research in culture by choosing from our comprehensive selection of primary cells. Bronchial Air-Liquid-Interface BulletKit Growth Media Bronchial Epithelial BulletKit Growth Media Fibroblast BulletKit Growth Media Smooth Muscle BulletKit Growth Media Small Airway BulletKit Growth Media Endothelial Cell Growth Media Kits Respiratory and Cardiovascular Research Tools BioResearch 5

6 Human and Animal Cardiac Cells Streamline your cardiac cell function analysis with our characterized and easy-to-culture primary and embryonic stem cell-derived cells. They are ideally suited for electrophysiology, calcium flux analysis, hypertrophy, pathophysiology (such as hypertension and myocardial response to injury), and extracellular maintenance during growth. Choose from our range of: Human primary cardiac fibroblasts (atrial and ventricle) Human large vessel and microvascular endothelial cells (normal and diabetic donors) Human smooth muscle cells (normal and diabetic donors) Rat primary cardiomyocytes and aortic smooth muscle cells All cells test negative for bacterial, fungal, and mycoplasma contamination. Human cells test negative for HIV-1, Hepatitis-B, and Hepatitis-C. Human and Animal, Airway and Cardiac Media For best results, culture your human and animal cardiac cells with our optimized media. You can find recommended media with the ordering information on pages 21 and 22. Primary Cells for Validation of Cell Line Results Find the respiratory or cardiovascular cell line you are working with below and the related primary cell type to validate those results. Cell Line EA.hy926, ECV304, HUVEC-C (human umbilical vein cell lines) HBE4-E6/E7 (lung/bronchus epithelial cell line) HL-1 (C57BL/6J mouse cardiac muscle cell line) L-132 (This line was originally thought to be derived from embryonic lung tissue, but was subsequently found, to have been established via HeLa cell contamination) Others Recommended Primary Cell from Lonza Primary Human Umbilical Vein Endothelial Cells (HUVEC) Primary Human Bronchial Epithelial Cells (NHBE) Primary Rat Cardiomyocytes(rCM) Primary Small Airway Epithelial Cells (SAEC), Normal Human Lung Fibroblasts (NHLF), Bronchial Smooth Muscle Cells (BSMC) Contact Lonza Scientific Support for additional primary cell types that match your need for biological relevance 6 BioResearch Respiratory and Cardiovascular Research Tools

7 Asthma- and COPD-related Differential Gene Expression in Primary Human Lung Fibroblasts By Rochelle Myers 1, Lubna Hussain 1 and Ludger Altrogge 2 1 Lonza Walkersville, Inc., Walkersville, MD, USA; 2 Lonza Cologne GmbH, Koeln, Germany Introduction With the prevalence of asthma and chronic obstructive pulmonary disease (COPD) growing worldwide, the availability of primary human cells from these respiratory diseases is critical to increase research and knowledge about the disease at a cellular level. In this study, we sought to identify genes differentially regulated in asthma and COPD lung fibroblasts (DHLF-asthma and DHLF-COPD). Fibroblasts are the most common cells of connective tissue in humans. Insufficiency of tissue repair by lung fibroblasts may contribute to the decrease in elastic fibers in COPD 1. Lung fibroblasts are also the major producers of extracellular matrix (ECM) components within the lung and may initiate, regulate and contribute to the airway remodeling in asthma 2. Primary lung fibroblasts are isolated from normal, asthma and COPD human donors in accordance with all informed consent rules and regulations. The cells were cultured for seven passages in optimized media. Gene expression analysis was performed using the Human Immune System Phenotyping 96 StellARray qpcr Array (Bar Harbor BioTechnology, Inc.). The StellARray Gene Expression System is a quantitative polymerase chain reaction (qpcr)-based method and provides reliable profiling of biologically focused gene sets. This article summarizes a study comparing normal versus COPD lung fibroblasts and normal versus asthma lung fibroblasts to explore differences in gene expression among the donor samples. Medium (Lonza, cat. no. CC-3132) and then cryopreserved after the second passage. The vials were stored in liquid nitrogen until further use. Cell Harvesting and Cell Lysis Each lot of normal and diseased lung fibroblasts was thawed and plated at a density of 2,500 cells/cm 2 in FGM -2 Growth Medium (Lonza, cat. no. CC-3132). Growth media were changed after 24 hours and the cells were subcultured through seven passages. At P7, cells were pelleted and cell lysates were obtained using the QIAshredder column (Qiagen, cat. no ). qpcr Experimental Design To generate data with biologically relevant variance, three replicate samples were independently assayed for each cell type. RNA Isolation and cdna Synthesis Each cell lysate was transferred to the Qiagen RNeasy Mini Kit (cat. no ) and RNA was extracted. cdna was synthesized with 2 µg of RNA per sample using SuperScript II Reverse Transcriptase and dntp mix (Life Technologies, Inc., cat. no and , respectively). For primers, random decamers and oligo dt primers (Life Technologies, Inc., cat. no. AM5722G and ) were used. cdna synthesis reactions were performed according to the specifications of the supplier Materials and Methods In this study, we grew primary lung fibroblasts from normal, asthmatic and COPD tissues in standard submerged culture (see Table 1) to assess gene expression changes associated with diseased states. Cell Isolation Fibroblasts were isolated from normal and diseased lung tissues. Isolated cells were expanded in standard submerged culture in FGM -2 Growth NHLF: Normal Human Lung Fibroblasts DHLF-As: Diseased (Asthma) Human Lung Fibroblasts DHLF-COPD: Diseased (COPD) Human Lung Fibroblasts Cat. no. CC Lot no F3100 Age 45 Years 27 Years 55 Years Sex Male Male Female Race Caucasian Caucasian Caucasian Table 1 Donor characteristics of primary cells used. Lane Sample 1 Reference RNA 2 Marker 3 NHLF Normal Prep 1 4 NHLF Normal Prep 2 5 NHLF Asthma Prep 1 6 NHLF Asthma Prep 2 7 NHLF COPD Prep 1 8 NHLF COPD Prep 2 Respiratory and Cardiovascular Research Tools BioResearch 7

8 For Each qpcr Plate, a Reaction Mix Was Prepared in the Following Manner 2 x SYBR Green Master Mix (Fast SYBR Green Master Mix; Life Technologies, Inc.) 1031 µl H 2 O RPMI, 10% FBS cdna template: 40 μl cdna synthesis reaction mix μl H 2 O DMEM, 10% FBS Real-time qpcr. GPR fold change CD14 IL7R CD22 CD40 20 μl of the reaction mix was distributed into Human Immune System Phenotyping 96 StellARray qpcr Array Plate. The master mixes contained AmpliTaq Fast DNA Polymerase (Life Technologies, Inc.), designed to allow instant hot start. Arrays were run on the BioRad CFX 96 using a standard qpcr program. Post-run data collection involved the setting of a common threshold (Ct) across all arrays within an experiment, exportation and collation of the Ct values, and analysis via GPR. GPR Algorithm Data input for GPR consists of a list of Ct values, derived directly from real-time PCR instruments, for each sample (normal or diseased). After designating the control and experimental sets of Ct values, GPR filters expression data to separate genes into two groups genes considered for analysis (G) and genes that can be used as potential normalizers (N). Genes that are not expressed in either sample are not considered further. After filtering the data into two sets, GPR performs a proprietary global normalization and statistical analysis by comparing each G to each N. The magnitude of change in expression ( fold change ) for each gene is subsequently determined using the ten best N genes, as defined within each experiment. Results and Discussion In each diseased cell type, statistically relevant differences in gene expression were detected in the diseased cells compared to the normal cells. Genes with P-values of <0.05 or better are reported Genes up- or downregulated in asthmatic sample vs. normal cells. COPD The COPD sample had several upregulated genes compared to the normal sample. Two of these genes, TNFSF4 and TNFSF18, encode for proteins that are cytokines and part of the tumor necrosis factor (TNF) ligand family. TNFSF4, upregulated 15.1 fold, is involved in T cell antigen-presenting cell interactions and is reported to mediate adhesion of activated T cells to vascular endothelial cells 7. TNFSF18, upregulated 10.4 fold, has been observed to modulate T lymphocyte survival in peripheral tissue and is expressed in endothelial cells. It is thought to be important for interactions between the two cell types 8. CD40 was upregulated 4.2 fold and encodes for a protein belonging to the TNF-receptor family. The receptor is critical in mediating a broad variety of immune and inflammatory responses 6. CD36 was upregulated 15.1 fold and the resulting protein of this gene serves as a receptor for thrombospondin in platelets 9. One study found that long-term cigarette smoke exposure is associated with fibrosis and inflammation via increased levels of thrombospondin 10. KLRD1, expressed by natural killer cells that mediate cytotoxic activity and secrete cytokines upon immune stimulation, was upregulated 7.1 fold 11. One significantly downregulated gene in the COPD donor sample was CD24 (25.3 fold) which encodes a sialoglycoprotein that is expressed on mature granulocytes and also in many B cells 12. Other downregulated genes included CD22 (7.9 fold) and LY75 (8.7 fold). Asthmatic The asthmatic fibroblast cells had two upregulated genes as compared to the normal. CD14 was upregulated 4.97 fold and IL7R was upregulated 2.8 fold. Both of these genes play a role in the innate immune system which is activated in response to stimuli in asthmatic patients 3. CD14 is a surface antigen that is expressed on several immune cells and IL7R is critical in the development and activation of lymphocytes 4,5. The two downregulated genes in the asthmatic donor as compared to the normal donor were CD22 and CD40 (3.2 fold and 4.6 fold respectively). CD40 encodes for a protein involved in a variety of immune and inflammatory responses 6. GPR fold change CD36 CD22 TNFSF4 CD24 IL7R TNFSF18 LY75 KLRD1 CD Genes up- or downregulated in COPD sample vs. normal cells. 8 BioResearch Respiratory and Cardiovascular Research Tools

9 Summary We have described a method in which normal, asthmatic, and COPD diseased lung fibroblasts were expanded and gene expression was analyzed using an immune system qpcr array. The resulting data revealed differences in gene expression between the normal and diseased cell types. Several of the detected genes were implicated in previously published work relating to asthma and COPD research which indicates that although this study involved cells from single donors, the resulting data is valuable and relevant. Both asthma and COPD are characterized by inflammation of the lungs and involve immune responses. Most drug targets are enzymes or receptors aimed at regulating these pathways. Access to these diseased cell types provides a convenient, biologically relevant model to assess the genetic pathways involved in the disease and may help to indicate other potential drug targets. References 1. Zhang J, Wu L, et al. Respir Physiol Neurobiol Aug 15;177(3): Lizbet Todorova. Effects of Asthma Combination Therapy on Extracellular Matrix Remodeling in Human Lung Fibroblasts. (Doctoral dissertation). ISSN: Klaassen et al. Allergy, Asthma & Clinical Immunology, 9:10, Pubmed GENE ID 929 description, (11 May 2013). 5. Pubmed GENE ID 3575 description, (12 May 2013). 6. Pubmed GENE ID 958 description, (14 May 2013). 7. Pubmed GENE ID 7292 description, (28 April 2013). 8. Pubmed GENE ID 8995 description, (26 Feb 2013). 9. Pubmed GENE ID 948 description, (12 May 2013). 10. Ishikawa T. et al. Correlation Between Thrombospondin-1 And Pulmonary Function After Long-Term Cigarette Smoke Exposure. Am J Respir Crit Care Med. 18:5, Pubmed GENE ID 3824 description, (11 May 2013). 12. Pubmed GENE ID description, (05 May 2013). Lonza currently offers a selection of normal, asthmatic, and COPD human bronchial epithelial cells, human lung fibroblasts, and human bronchial smooth muscle cells. The cells are tested and guaranteed to perform with the suggested Clonetics Media Kits and Reagents. More in-depth donor information can be obtained through Lonza s Scientific Support Team. Respiratory and Cardiovascular Research Tools BioResearch 9

10 Biologically Relevant Cells Case Study: Characterizing Respiratory Syncytial Virus (RSV) Infection Characterization of RSV in primary human airway epithelial cells in air-liquid interface systems. S. Krishnan, et al American Journal of Respiratory and Critical Care Medicine;181: A6419 Summary Researchers used primary human airway epithelial cells grown in an airliquid interface system to evaluate the characteristics of RSV infection. Methods and Materials Normal and diseased frozen primary human bronchial epithelial cells were obtained from Lonza and seeded in vitro, in air-liquid interface (ALI) format. They subsequently grew into a multi-layered, polarized, and differentiated tissue. Cells were stained for ß-tubulin to determine ciliated morphology and then infected with RSV A Long or A2, at multiplicities of infection (MOI) of and Following incubation for 0 24 hours or 1 5 days, RSV F protein expression was assayed by Western blot. Results After days in Bronchial-ALI (B-ALI ) Medium, about 75% of the upper columnar bronchial epithelial cells demonstrated cilia formation and mucus production: F protein was expressed after 3 and 5 days at MOI 1.0, and after day 1 at MOI 10.0 RSV replication increased significantly within hours Viral output from the apical surface of the epithelial cells also increased The primary bronchial epithelial B-ALI Media system provided a valuable model for characterizing aspects of RSV infection, such as viral replication, expression of viral proteins, and viral budding/secretion. Primary human bronchial epithelial cells stained for ZO-I tight junction protein (red) co-localized with ß-tubulin (green) under fluorescence microscopy. Conclusions The primary bronchial epithelial cells grown in B-ALI Media can be used as an effective tool in evaluating anti-viral biologics or molecules against RSV, and potentially other respiratory pathogens. Primary human bronchial epithelial cells stained for cilia with ß-tubulin at day 25 post air lift. Primary human bronchial epithelial cells stained for mucin production with Alcian blue at day 21 post air lift. 10 BioResearch Respiratory and Cardiovascular Research Tools

11 Transfection of Primary Cells and Cell Lines You can deliver almost any substrate such as plasmid DNA, sirna, shrna, or peptides to primary cells and hard-to-transfect cell lines with our non-viral Nucleofector Technology. This improved technique will play an increasingly important role in delivering genes to primary fibroblasts smooth muscle, epithelial, and endo-thelial cells. Nucleofector Technology I can t transfect my primary cells or cell lines of interest efficiently. SOLUTION: Our Nucleofector Technology delivers up to 90% gene transfection efficiency, at high viability and maintenance of functionality. Transfect primary respiratory or cardiovascular cells with up to 90% efficiency while maintaining excellent cell viability. Nucleofection allows you to: Use validated transfection protocols for 10 Clonetics Respiratory and Cardiovascular Primary Cells Select optimized transfection conditions for more than 20 respiratory and cardiovascular cell lines Easily establish Nucleofection Conditions for other mammalian cells using our cell group-specific basic protocols Preserve cellular function and reduce toxicity Use the same transfection conditions for different cell numbers and volumes Select from a range of platforms to suit your cell number and throughput needs Choose the Nucleofector Platform that Suits Your Research Needs Advanced Platform 96-well Add-on High-throughput Platform Basic Device Device 4D-Nucleofector System 96-well Shuttle System 96-well Shuttle System Nucleofector 2b Device Unit Throughput (samples per run) Low to medium (1 16) Low to high (1 96) High (384) Low (1) Reaction volume 20 μl µl 20 µl 20 µl 100 µl Electrode material Conductive polymer Conductive polymer Conductive polymer Aluminum Low cell numbers (20 µl) to to to High cell numbers (100 µl) to to DNA Vector amount/sample μg (20 µl) μg μg 1 5 μg 1 5 μg (100 µl) sirna amount/sample pmol (20 µl) pmol pmol pmol (concentration 2 nm 2 µm) pmol (100 µl) Compatibility with 96-well Shuttle System To find transfection data for your cell type of interest. Respiratory and Cardiovascular Research Tools BioResearch 11

12 Transfection Efficiency and Cell Viability with Nucleofector Technology Cardiac Cells Efficiency* Viability* Kit for 4D-Nucleofector and 96-well Shuttle Systems Kit for Nucleofector II/2b Device Cardiomyocyte, rat 7580 % 5060 % Primary Cell Optimization Rat Cardiomyocyte Cardiomyocyte, ESC-derived (Cor.At ), mouse 5565 % 8090 % P4 Primary Cell (protocol via Scientific Support) Contact Scientific Support for guidance Endothelial Cells Endothelial, umbilical vn.(huvec), human 8090 % 5575 % P5 Primary Cell (HUVEC) HUVEC Endothelial, aortic (HAEC), human 5575 % 4560 % P5 Primary Cell (Endothelial Cell, Basic) Endothelial Cell, Basic Endothelial, coronary artery (HCAEC), human 5060 % 4050 % P5 Primary Cell (Endothelial Cell, Basic) HCAEC Endothelial, microvascular, lung (HMVEC-L), human 5080 % 4555 % P5 Primary Cell (Endothelial Cell, Basic) HMVEC-L Epithelial Cells Epithelial, bronchial (NHBE), human 5065 % 5060 % P3 Primary Cell (NHBE) NHBE Epithelial, small airway (SAEC), human 6070 % % P3 Primary Cell (Epithelial Cell, Basic ) Epithelial Cell, Basic Smooth Muscle Cells SMC, aortic (AoSMC), human 7580 % 5595 % P1 Primary Cell (AoSMC) AoSMC SMC, coronary artery (CASMC), human 6070 % 8090 % P1 Primary Cell (Smooth Muscle Cell, Basic) Smooth Muscle Cell, Basic SMC, pulmonary artery (PASMC), human 7585 % 6070 % P1 Primary Cell (Smooth Muscle Cell, Basic) Smooth Muscle Cell, Basic Stem Cells Mesenchymal stem cells (MSC), human 5590 % 5085 % P1 Primary Cell (Human MSC) Human MSC Cardiovascular Cell Lines** A % 7080 % Cell Line Optimization Cell Line L EMC 8090 % 9095 % Cell Line Optimization Cell Line L H9c2(2-1) 8090 % 8595 % Cell Line Optimization Cell Line L HL % 5070 % Cell Line Optimization Cell Line V HMEC % 7585 % Cell Line Optimization Cell Line R HUV-EC-C 7080 % 7080 % Cell Line Optimization Cell Line V T/G HA VSMC 5060 % 7080 % Cell Line Optimization Cell Line V Lung Cell Lines** A % 7585 % Cell Line Optimization Cell Line T Calu % 5590 % SE Cell Line Cell Line V BEAS-2B 3585 % Cell Line Optimization Cell Line T Calu % 5090 % Cell Line Optimization Cell Line L IMR % 6575 % Cell Line Optimization Cell Line R NCI-H929 (H929) 1530 % 5080 % Cell Line Optimization Cell Line T R9ab 6575 % 7590 % Cell Line Optimization Cell Line L V % 4555 % Cell Line Optimization Cell Line V WI % 8595 % Cell Line Optimization Cell Line V Primary cells marked blue have Lonza-validated optimized protocols. *Approximate ranges extrapolated from larger result collections, including Lonza and customer data ** This is only a selection of cell lines. For further cell lines of respiratory or cardiac origin, and protocol guidance: 12 BioResearch Respiratory and Cardiovascular Research Tools

13 Transfection Case Study: Biological Pacemakers Can a biological pacemaker be delivered to the heart? I. Potapova, A. Plotnikov, et al Circulation Research; 94: (Extracted with permission of Lippincott Williams & Wilkins, 2004, American Heart Association) Summary Researchers from State University of New York, Stony Brook, and Columbia University set out to create and deliver a biological pacemaker. They chose Poietics Human Mesenchymal Stem Cells (hmscs) and Nucleofector Technology for their research model. Methods and Materials hmscs were cultured up to passage four in Poietics Mesenchymal Stem Cell Growth Medium. They were then transfected with either egfp control vector or the vector for egfp plus the pacemaker gene (mouse HCN2). To avoid the use of viruses, this was performed using Nucleofector Technology. Transfected cells were plated onto a small, localized region of a coverslip. Neonatal rat ventricular myocytes were subsequently plated over the entire coverslip. Action potentials of the myocytes were later recorded. Results egfp expression was found to be around 45% within hours of transfection. Pacemaker function in the in vitro model was achieved. The genetically engineered hmscs increased the spontaneous beating rate of co-cultured rat neonatal myocytes from 93 beats per minute to 161 beats per minute. Conclusions Nucleofector Technology can transfect primary hmscs to achieve the pacemaker phenotype in vitro. This has potential future clinical applications. Action Potential Recording A) Vm (mv) B) Vm (mv) s +EGFP 93 ± 16 bpm +EGFP+mHCN2 161 ± 4 bpm Spontaneous electrical activity of neonatal rat ventricular myocytes that were co-cultured with (A) hmscs transfected with EGFP or (B) hmscs transfected with mhcn2 and EGFP. Respiratory and Cardiovascular Research Tools BioResearch 13

14 Cell Analysis Tools for Respiratory and Cardiovascular Research CytoSMART System Your Cell Culture is Just One Click Away Monitor your cell culture anytime using our new CytoSMART System your personal live cell monitoring device. The System consists of a mini-microscope and the CytoSMART Device that is linked to an accompanying tablet outside the incubator. Via this tablet you start your CytoSMART Projects and capture the images of your cell culture in defined intervals. All images are transmitted to the CytoSMART Connect Cloud via wireless transmission (WiFi), meaning you can monitor your cell culture remotely and in real-time with any browsercapable device, be it your smartphone, tablet or PC. I can t find reliable assay tools for mrna, protein expression, cell proliferation, or cytotoxicity. SOLUTION: Choose from our assays developed specifically for respiratory and cardiovascular disorders. The Smarter Way for Precious Cell Cultures With the CytoSMART System your cells can remain in their defined conditions in the incubator while you monitor the cell culture online. Easy documentation and video-taping of your cell culture Remote monitoring of cultures grown under defined conditions (e.g. hypoxia) Cell culture standardization through operator independent determination of confluency with automatic alerts for cell culture functionality Easy and immediate download of cell culture images or videos from CytoSMART Connect Cloud Figure 1. Image of human skeletal muscle cells (SkMC, CC-2561) cultured in SkGM using the CytoSMART Lux 10X System. Please refer to our website to view the video of a SkMC culture recorded with the CytoSMART System. 14 BioResearch Respiratory and Cardiovascular Research Tools

15 Precast PAGEr Protein Gels for Consistent, Reliable Western Blotting ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit PAGEr Gels are easy-to-use precast protein mini-gels that offer sharp resolution and consistent protein transfer. Each lot is functionally tested and shipped fresh every time, for guaranteed shelf life and performance. PAGEr Gels give you: Faster migration and longer shelf-life Sharp resolution for crisp separation of proteins 5 kda 300 kda Marked sample lanes and simple twist open design Compatibility with most chambers Multiple well formats and gel concentrations Tris-Glycine buffer for traditional Laemmli separation Applications SDS-PAGE Native PAGE Western blotting Antibody screening Comb Options and Well Volumes The ViaLight Assay incorporates bioluminescent detection of cellular ATP as a measure of viability. Measurement of ATP is the most accurate, effective, and direct way of determining the number of living cells in culture: Process and analyze a 96-well plate in less than 15 minutes Detect as few as ten cells; allowing for lower seeding densities and more assays Easily perform scalable automation with our simple, no-shake protocol Add two reagents directly to your culture and read luminescence Expand your dynamic range to five decades, in adherent or suspension cultures Run this test on a variety of luminometers or scintillation counters Avoid radioactive or toxic materials ToxiLight Non-destructive Cytotoxicity BioAssay Kit 2D well 550 μl sample, or 7 cm IPG strip, 12 μl marker 10 well 32 μl *Compatible with multichannel pipettes 8+1 well* 30 μl sample 12 μl marker 16 well 14 μl 12 well 20 μl 17 well* 14 μl Check a compound s cytotoxic effects non-destructively by measuring adenylate kinase (AK) leakage from damaged cells: Generate results from as few as 10 cells Eliminate the need to lyse by monitoring cytotoxicity from supernatant Simply add a single reagent directly to cells, or to a supernatant aliquot Process and analyze a 96-well plate in less than 10 minutes Freeze supernatants and return to your work later without losing AK activity Respiratory and Cardiovascular Research Tools BioResearch 15

16 Drug Discovery Tools Our dedicated drug discovery team gives you access to Lonza s expertise in many areas of cell biology. We trust that we have identified the products and services of value to drug discovery customers and we welcome the opportunity to discuss and develop them further with you. RAFT 3D Cell Culture System 3D cell culture differs significantly from the traditional 2D culture that most researchers have been using since the past decades. There is a trending shift both in academia and industry to personalized research solutions and more in vivo like models to understand cell behavior. This is fueling the growing market need for better solutions in 3D cell culture such as RAFT 3D Cell Culture System. " How can I optimize my diabetic drug leads?" SOLUTION: Lonza can ease your drug discovery research with our advanced cell culture tools such as 3D culture systems and Cells on Demand Cell Culture Services. An important differentiation of the RAFT System to other 3D platforms or to 2D culture methods is its ability to engineer tissue-like 3D cultures. This is especially needed for certain applications such as development of in vitro liver fibrosis models or corneal models. With RAFT System, cells can be cultured within a high-density collagen scaffold, or on top, or both. The addition of permeable membrane cell culture inserts provides other extensions to the system allowing the generation of barrier models including air-lift models. RAFT Cultures can be created in less than an hour using simple protocols and standard labware! RAFT Kits are available in a choice of 24-well, insert-well and 96-well formats. RAFT System has already been used to successfully generate 3D cultures in a number of research areas including oncology, toxicology, barrier modeling, dermal research and pulmonary research. Figure 1. RAFT 3D Culture System consists of RAFT Reagent Kit and Absorbers for 96-well, 24-well and trans-well inserts. Contact your local sales representative to learn how RAFT System can support your experiments. Cells on Demand Services The advancement of assay technologies has enabled the use of live cells in over 50% of high throughput screening. To meet this growing need, Lonza research solutions has leveraged its decades of cell culture and transfection experience to provide high quality cell products and services. Cells on Demand Transfection Services Access Lonza s transfection expertise to achieve high transfection efficiencies and viabilities in almost every cell. Benefits include: Receive validated stable clones quickly Stress-free optimization of Nucleofection Protocols for your cell type of interest Figure 2. Dermal fibroblasts fixed and stained after 11 days in RAFT System. Figure 3. Sample acellular RAFT Cultures in a vial showing high-density collagen scaffolds. Cells on Demand Cell Culture Services Avoid delays in obtaining vital cell cultures. Just select the cell types and format and our cell culture experts will do the rest. Easily move to primary cells with our primary cell expansion service Avoid the aggravation of tissue acquisition, failed isolations, and low yields with our primary cell isolation service We can establish RAFT 3D Cell Culture Systems tailored to your needs within 96-well or 24-well formats 16 BioResearch Respiratory and Cardiovascular Research Tools

17 Fluorescence Microscopy-based Characterization of Cells Grown in RAFT 3D Culture By Cecile Villemant 1, Grant Cameron 1, Lubna Hussain 2, Jenny Schroeder 3 1 TAP-Biosystems, Royston, UK; 2 Lonza Walkersville, Inc., Walkersville, MD, USA; 3 Lonza Cologne GmbH, Koeln, Germany Introduction In vitro assays typically use cells grown on two-dimensional (2D) hard plastic or glass substrates, which are not representative of the true in vivo cell environment. 1 In tissue, cells interact with neighboring cells and with the extracellular matrix (ECM). In a simplified in vitro 2D environment, most of the tissue-specific architecture, cell-cell communication and cues are lost. Therefore, the need exists for advanced culture methods that better mimic cellular function within living tissue. Three-dimensional (3D) cell culture methods, in comparison, provide a matrix that encourages cells to organize into structures more indicative of the in vivo environment, thereby developing normal cell-cell and cell-ecm interactions in an in vitro environment. The RAFT 3D Culture System (Figure 1) uses a collagen matrix at physiologically relevant concentrations. Cells and neutralized collagen are mixed and dispensed into wells of standard cell culture plates, and subsequently incubated at 37 C to allow the formation of a cell-seeded hydrogel. Specialized RAFT Absorbers are placed on top of the hydrogels. The RAFT Absorbers gently remove the medium, thus concentrating the cell/collagen hydrogel to a layer approximately 120 μm thick, mimicking physiological conditions. The cultures are then ready for use. Optionally, additional collagen layers or epithelial or endothelial cell overlays may be added to study co-cultures or more complex cultures. Assessing the viability of cells grown in a RAFT 3D Cell Culture, or performing immunofluorescence staining, may not seem as trivial as assessing the viability of a 2D cell culture. However, here we show that by adding some basic controls, the LIVE/DEAD Assay is a straightforward and reliable method to assess the viability of cells inside a RAFT 3D Cell Culture. In addition, we show that immunofluorescence microscopy can be performed easily and routinely on RAFT 3D Cell Cultures. RAFT TM Sequence Empty well or insert Cell and collagen mix forms hydrogel at 37 C Absorber contacts the hydrogel Liquid starts to be absorbed Process concentrates cells and collagen In less than 1 hour, culture formation complete Figure 1. Creation of 3D cell/collagen hydrogel using the RAFT System in standard cell culture plates. General Materials RAFT Absorbers and Reagent Kits, visit for a list of RAFT Products and RAFT Protocols For a list of recommended cell culture plates (either 96-well black wall or 24-well plate, not supplied with the kit), contact Lonza s Scientific Support Team Widefield fluorescence microscope with appropriate filters Methods Cell Culture Early passage primary human neonatal dermal fibroblasts (HDFs) were cultured in standard growth medium that supports dermal fibroblasts. Generation of RAFT Cultures RAFT Cultures were made according to the protocol supplied with the RAFT Kits in black-walled, μclear 96-well cell culture plates (Greiner, ). For the LIVE/DEAD Assay, 3,000 HDFs per well were seeded and incubated at 37 C with 5% CO 2, either overnight (1 day) or for 7 days. In addition, two acellular cultures were made for use as a background control. For immunocytochemistry, either 5,000 or 50,000 HDFs per well were seeded and then cultured for either 11 days or 3 days, respectively, prior to being fixed and stained. Respiratory and Cardiovascular Research Tools BioResearch 17

18 Methods continued LIVE/DEAD Assay The LIVE/DEAD Assay was carried out, using at least one dead control and two background controls (acellular cultures). The LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies) was used according to the fluorescence microscopy protocol provided up to point 3.5. However, with the following adjustments: 1. To get a thorough wash of the whole 3D cell culture, the medium on the culture is aspirated and replaced with 100 μl PBS and the plate left for 510 minutes on a rocker (while preparing the combined LIVE/DEAD Assay Reagents). 2. To prepare the dead cell sample, we incubate the cells with 1% w/v saponin for at least 30 minutes. If you have added 100 μl of medium onto your RAFT Cultures, just add 25 μl of 5% w/v saponin 30 min-nutes before performing the assay. 3. Due to the presence of the collagen matrix, it is advised to include acellular RAFT 3D Cultures to act as a control for background noise. Instead of point 4.1, onward, in the supplied LIVE/DEAD Viability/Cytotoxicity Kit Protocol, we used the following protocol: Aspirate the PBS, added previously from the wells, and replace it with 100 μl of the combined LIVE/DEAD Assay Reagents. We have found that in the case of HDFs, final concentrations of 0.4 μm for Calcein AM and 4 μm for Ethidium homodimer 1 (EthD-1) were optimal. Between 10 minutes and 1.5 hours after adding the reagents, z-series at 5 µm intervals were captured using a fluorescence microscope fitted with a z-focus drive. The numbers of live cells (stained with Calcein AM) and dead cells (stained with EthD-1) were counted from three separate images over two wells for the live samples. The background noise for EthD-1 was counted in two acellular cultures (bckgdethd-1 and bckgdethd-1 ). The background noise for Calcein AM was assessed in the acellular cultures and in the dead control; however, there was no visible background detected. Therefore, for each image, the percentage of viability was calculated as follows: Number of live cells %viability d=1 or d=7 = Number of live + dead cells average (bckgd EthD-1 & ) Immunocytochemistry On the day of assay, the following protocol was used: 1. Each RAFT Culture was washed three times over a 15-minute period with 100 μl PBS. 2. The PBS was replaced with 100 μl of 3.7% formaldehyde solution to fix the cells, and the plate incubated at room temperature for 30 minutes. 3. The formaldehyde solution was replaced with 100 μl of quenching solution (1 mm Tris-HCl and 20 mm Glycine in PBS) to quench the formaldehyde cross-linking, and the plate incubated at room temperature for 10 minutes. 4. The RAFT Cultures were washed as in point The PBS was replaced with 100 μl of 0.1% Triton X-100 solution to permeabilize the cells, and the plate was incubated at room temperature for 4 minutes. 6. The RAFT Cultures were washed as in point The primary rat anti-tubulin antibody (YOL1/34; Abcam) was diluted 1:100 in 1% w/v bovine serum albumin in PBS (which was the same dilution that was optimal for cells cultured in 2D) and 50 μl of this solution was added in each well. 8. The plate was then incubated overnight at 4ºC. 9. The RAFT Cultures were washed as in point The secondary antibody Cy3-AffiniPure Goat Anti-Rat IgG (H+L) (Stratech Scientific), phalloidin (the recommended 1/40 dilution was used) and DAPI were diluted in 1% w/v bovine serum albumin in PBS and 50 μl of this solution was added in each well. 11. The plate was then incubated at room temperature for 2.5 hours. 12. The RAFT Cultures were washed as in point The wells were imaged on a fluorescence widefield microscope with ms exposure times for the anti-tubulin antibody and the phalloidin. Confocal imaging or the use of a high content imaging device would also be possible. 18 BioResearch Respiratory and Cardiovascular Research Tools

19 Results LIVE/DEAD Assay In Figure 2, we show the typical images that can be obtained after culturing HDFs in RAFT 3D Cell Cultures for 1 and 7 days and staining the cultures with the combined LIVE/DEAD Assay Reagents. We also show, in Figure 2, some examples of the Calcein AM and EthD-1 stain on an acellular construct and a dead control. Immunocytochemistry After 3 days in culture, HDFs have elongated within the collagen matrix and display a typical actin and microtubule cytoskeleton as can be seen in Figures 4 and 5. The presence of the collagen matrix has little impact on the background fluorescence of the culture, in particular, when the antibodies were diluted in a BSA-containing blocking buffer. Figure 2. Examples of images that can be taken after staining RAFT Cultures with the combined LIVE/DEAD Assay Reagents. HDF live cells are displaying Calcein AM staining (green) while dead cells display EthD-1 staining (magenta). Each image is a projection on the z-axis of a whole z-series. Day 1 and Day 7: HDFs were cultured for 1 or 7 days, respectively, in a RAFT Plate before being treated with the combined LIVE/DEAD Assay Reagents. Dead control: HDFs cultured in RAFT Plate for 1 day were killed using 1% w/v saponin before being stained with the combined LIVE/DEAD Assay Reagents. Acellular culture: RAFT Acellular Culture stained with the combined LIVE/DEAD Assay Reagents. Figure 3 shows that the mean percentage viability of HDFs is 92% at Day 1 and 94% at Day 7 using the methods described above. For comparison, we have added the viability observed with the same cells cultured at the same time in a 2D planar environment, which is at 95% and 98% at Day 1 and Day 7, respectively. Figure 4. HDF cells fixed and stained for tubulin and actin after being cultured in the RAFT System for 3 days. A series of z-planes, taken at 0.5 μm intervals, was imaged on a widefield microscope after staining the RAFT Culture for tubulin (red), actin (green) and nucleus (blue). The z-stack from each channel was projected onto one plane using the maximum z-projection function of ImageJ software and the merge of all channels is shown in the large bottom panel. Across the top panel, one frame of the z-stack is shown with each individual channel represented separately in grayscale. This represents the area boxed in the large lower panel, to better show the detail of each staining. % Viability Figure 3. Comparison of the viability of HDFs after 1 and 7 days in a 2D or in a RAFT 3D Cell Culture. The percentage viability was determined as explained in the methods above and the average for two separate experiments is shown in this graph. The standard deviation is shown for each sample. 0 Day 1 Day 7 Day 1 Day 7 2D 3D (RAFT ) Day of Assay and Type of Culture Day of Assay and Type of Culture Respiratory and Cardiovascular Research Tools BioResearch 19

20 Results continued As shown in Figure 5, using a simple type of image deconvolution, such as ImageJ 3D parallel spectral deconvolution ( nih.gov/ ij/plugins/), can help improve the sharpness of the signal and remove background noise from the cells surrounding the cell of interest (such as the blurry, out of focus cell observed above the fibroblast imaged in Figure 4). However, with our anti-tubulin antibody, images taken with our widefield microscope, and not subjected to deconvolution, were defined enough to observe microtubules within the cells in the RAFT 3D Culture (Figure 4). Actin fibers can also be seen clearly in cells embedded in the RAFT Collagen Matrix (Figure 4). Conclusion The LIVE/DEAD Viability/Cytotoxicity Kit is an easy and rapid assay that can be used to assess cell viability in a RAFT 3D Cell Culture, provided that controls are included in the test to be able to take into account the possible background noise. In this experiment, we show that HDFs display a mean viability between 92% and 94% when cultured in RAFT 3D Culture from Day 1 and for at least 7 days, which is comparable to the viability observed for cells cultured in a 2D environment. Cells cultured in the RAFT 3D Cell Culture Collagen Matrix can be fixed and stained using standard immunofluorescence protocols. The presence of the collagen matrix has little impact on the background fluorescence of the culture, if an appropriate blocking solution is used. The resulting images were defined enough to observe microtubules and actin fibers within the cells in the RAFT 3D Culture. Image quality can be further enhanced by using simple types of image deconvolution software. In conclusion, with its easy-to-follow protocols, the RAFT System allows researchers to set up biologically relevant 3D cell cultures quickly and reproducibly. Many 2D cell analysis methods can be easily applied to RAFT Cultures, often requiring no, or only minor modifications of exist-ing protocols. This empowers researchers to generate more biologically meaningful data from their cell culture studies in multiple areas of basic research and drug discovery. References Figure 5. HDF cell fixed and stained for tubulin after being cultured in the RAFT System for 11 days. One z-plane location was imaged here on a widefield microscope, after staining the RAFT Culture for tubulin (red) and the nucleus (blue). The image from each channel was deconvolved using the 3D spectral deconvolution software from ImageJ (generalized Tikhonov) and the merge of the two channels is shown here. 1. Pampaloni, F.; Reynaud, E.G.; Stelzer, E.H.K. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007, 8, BioResearch Respiratory and Cardiovascular Research Tools

21 Ordering Information Cat. No. Description Size Primary Human Airway Research-Related Cells CC-2527 HMVEC-L Human Lung Microvascular Endothelial Cells 500,000 cells CC-2530 HPAEC Human Pulmonary Artery Endothelial Cells 500,000 cells CC-2581 HPASMC Human Pulmonary Artery Smooth Muscle Cells 500,000 cells CC-2576 BSMC Normal Human Bronchial Smooth Muscle Cells, in SmGM 2 Growth Media, cryopreserved 500,000 cells CC-2540 CC-2540S CC-2541 NHBE Normal Human Bronchial/Tracheal Epithelial Cells, with retinoic acid, in BEGM Growth Media, cryopreserved NHBE Normal Human Bronchial/Tracheal Epithelial Cells, with retinoic acid, in BEGM Growth Media, cryopreserved, guaranteed in B-ALI Media NHBE Normal Human Bronchial/Tracheal Epithelial Cells, without retinoic acid, in BEGM Growth Media, cryopreserved 500,000 cells 500,000 cells 500,000 cells CC-2512 NHLF Human Lung Fibroblasts, in FGM 2 Growth Media, cryopreserved 500,000 cells CC-2547 SAEC Human Small Airway Epithelial Cells, in SAGM Growth Media, cryopreserved 500,000 cells CC-2932 D-SAEC-As Diseased Small Airway Epithelial Cells Asthma 500,000 cells CC-2933 D-SAEC Diseased Small Airway Epithelial Cells Cystic Fibrosis 500,000 cells CC-2934 D-SAEC Diseased Small Airway Epithelial Cells COPD 500,000 cells CC-2923 D-HPAEC Diseased Human Pulmonary Artery Endothelial Cells Diabetes Type I 500,000 cells CC-2924 D-HPAEC Diseased Human Pulmonary Artery Endothelial Cells Diabetes Type II 500,000 cells CC-2913 D-PASMC Diseased Human Pulmonary Artery Smooth Muscle Diabetes Type II 500,000 cells CC-2915 D-PASMC Diseased Human Pulmonary Artery Smooth Muscle Cells Diabetes Type I 500,000 cells DHBE-As Asthmatic Human Bronchial Epithelial Cells, with retinoic acid, in BEGM Growth Media, 500,000 cells cryopreserved S D-HBE-As Diseased Human Bronchial/Tracheal Epithelial Cells 500,000 cells Asthma for B-ALI Bronchial Air Liquid Interface DHLF-As Asthmatic Human Lung Fibroblasts, in FGM 2 Growth Media, cryopreserved 500,000 cells DBSMC-As Asthmatic Human Bronchial Smooth Muscle Cells, in SmGM 2 Growth Media, cryopreserved 500,000 cells DHBE-COPD, Human Bronchial Epithelial Cells, with retinoic acid, in BEGM Growth Media, cryopreserved 500,000 cells S D-HBE-COPD Diseased Human Bronchial/Tracheal Epithelial Cells 500,000 cells COPD for B-ALI Bronchial Air Liquid Interface DHLF-COPD Human Lung Fibroblasts, in FGM 2 Growth Media, cryopreserved 500,000 cells DBSMC-COPD Human Bronchial Smooth Muscle Cells, in SmGM 2 Growth Media, cryopreserved 500,000 cells DHBE-CF Cystic Fibrosis Human Bronchial Epithelial Cells, with retinoic acid, in BEGM Growth Media, 500,000 cells cryopreserved DBSMC-CF Cystic Fibrosis Human Bronchial Smooth Muscle Cells, 500,000 cells in SmGM 2 Growth Media, cryopreserved DHLF-CF Cystic Fibrosis Human Lung Fibroblasts, in FGM 2 Growth Media, cryopreserved 500,000 cells CC-7231 DHLF-iPL Diseased (Idiopathic Pulmonary Lung Fibrosis) Lung Fibroblasts 500,000 cells Primary Human Cardiac Research-Related Cells CC-2914 D-AoSMC Diseased Human Aortic Smooth Muscle Diabetes Type I 500,000 cells liquid nitrogen (-180 C) CC-2916 D-AoSMC Diseased Human Aortic Smooth Muscle Diabetes Type II 500,000 cells, liquid nitrogen (-180 C) CC-2917 D-CASMC Diseased Human Coronary Artery Smooth Muscle Diabetes Type I 500,000 cells liquid nitrogen (-180 C) CC-2918 D-CASMC Diseased Human Coronary Artery Smooth Muscle Diabetes Type II 500,000 cells liquid nitrogen (-180 C) CC-2919 D-HAEC Diseased Human Aortic Endothelial Diabetes Type I 500,000 cells CC-2920 D-HAEC Diseased Human Aortic Endothelial Diabetes Type II 500,000 cells CC-2927 D-HAEC Diseased Human Aortic Endothelial Diabetes Type II 500,000 cells CC-2928 D-HMVEC Diseased Cardiac Microvascular Endothelial Cells Diabetes Type II 500,000 cells CC-2903 NHCF-A Normal Human Cardiac Fibroblasts - Atrial in FGM 3 Growth Media, cryopreserved 500,000 cells CC-2904 NHCF-V Normal Human Cardiac Fibroblasts - Ventricular in FGM 3 Growth Media, cryopreserved 500,000 cells Respiratory and Cardiovascular Research Tools BioResearch 21