4D-Nucleofector ® – Stretching Transfection Dimensions Dr. Nazim El-Andaloussi Lonza Cologne, Germany

slide 2 n Introduction n The Principle of the Amaxa ® Nucleofector ® Technology n Available platforms n Details about the 4D-Nucleofector® n The Core Unit n The X-Unit n The Operation Software n Consumables n Protocol Transfer n Nucleofection and Stem Cells n Summary: System key benefits Agenda

slide 3 Notebook Control Unit Nucleofection ® – The Principle Other transfection methods  High transfection efficiency combined with low mortality  DNA is directed into the nucleus giving faster gene expression Nucleofection ®

slide 4 Primary NHDF-neo cells were transfected with TMR-labeled plasmid DNA encoding GFP, fixed after 2h in 3.5% PFA and analyzed by confocal microscopy. (R)R-DNA GFPDAPI Merge DNA is Delivered Into Cytoplasm and Nucleus 2 hrs

slide 5 Simple Handling – Optimized Protocols step 1 Harvest cells of interest step 3 Nucleofector ® Solution with Supplement Mix cells DNA or siRNA transfer to an amaxa certified cuvette Select Nucleofector ® program Insert cuvette Press start button “X” Rinse cuvette with culture medium and transfer to culture dish expression detected as early as 3-8 hours & combine with: step 2 step 4

slide 6 Device Nucleofector ® 2b* 96-well Shuttle ® 4D- Nucleofector ® HT Nucleofector ® Throughput Very low (1)Medium (96)Low (1-16)High (384) Reaction volume 100µl20µl100µl and 20µl20µl Electrode material Aluminum Conductive polymer Conductive Polymer Cell numbers ( orders of magnitude ) 10 5 to 10 7 (2)10 4 to 10 6 (2)10 4 to 10 7 (3)10 4 to 10 6 (2) Adherent Nucleofection NoYes Shuttle compatibility No-YesNo Our Product Family – A Technical Comparison * Nucleofector without Shuttle connectivity

slide 7 Notebook Control Unit The Amaxa ® Nucleofector ® Technology n Efficient transfection of primary cells & cell lines n Applications in Neurology, Metabolic diseases, Cancer, Immunology, Cardiology and other Therapeutic areas n Versatility in substrate delivery n DNA, siRNA, miRNA, mRNA, morpholinos n Peptides, proteins, antibodies n Small molecules n Applicable for n Assay development n RNAi and cDNA screening n Target validation

slide 8 n Introduction n The Principle of the Amaxa® Nucleofector® Technology n Available platforms n Details about the 4D-Nucleofector® n The Core Unit n The X-Unit n The Operation Software n Consumables n Protocol Transfer n Nucleofection and Stem Cells n Summary: System key benefits Agenda

slide 9 Overview 4D-Nucleofector ® System A new, modular system offering advanced performance and convenience. Comprising one Core Unit and several Functional Modules the system is designed for maximum flexibility. Features n 1D Easy n Transfection of various cell numbers with same conditions n 2D Fast n Different throughput, from one to 16 wells in 10 seconds n 3D Flexible n Nucleofection of cells in adherence for assays at various stages n 4D Future-proof n Modular system for upcoming transfection challenges

slide 10 Straight, very elegant, valuable and distinguishable n Fold-away Graphical User Interface n Electrically driven drawer for cuvette/sample retainer n Could be assembled side by side or on top of each other n Noble housing, shell made of metal Core Unit X-Unit 4D-Nucleofector ® System Y-Unit

slide 11 Notebook Control Unit Amaxa ® Nucleofector ® II.S Amaxa ® Nucleofector ® 96-well Shuttle ® Amaxa ® Nucleocuvette ® Plate n Intuitive Software n 96 different programs / plate n 3-4 min / plate n Connection via USB Amaxa ® Nucleofector ® 96-well Shuttle ® System USB 4D-Nucleofector® Core and X-Unit +

slide 12 4D-Nucleofector ® System – The Core Unit The Control Unit of the 4D-Nucleofector ® System n 5,7’’ foldable touch screen to operate the system (A) n Operated by intuitive operation software n Controls several functional units n Comprises USB and serial connectivity for the 96-well Shuttle ® (B) n USB port for software update and data transfer (C) A B C

slide 13 4D-Nucleofector ® System – The X-Unit The Unit supporting Nucleofection ® of various cell numbers n Features positions for Nucleocuvette ® Strips (A) and 100µl CP-cuvettes (B) n Comprises HV connectivity for the Shuttle (C) n Seamless transfer of conditions between Nucleofection ® Vessels n Suited for transfection of cells in adherence (ACT-Strips) A B C

slide 14 4D-Nucleofector ® System – Operation Software The intuitive tool operating the 4D-Nucleofector ® System n Easy-to-use through up-to-date touch screen interface n Comes with predefined Nucleofection ® Parameters and Experiments n Supports data transfer / software update via USB storage device n PC-Editor software included to predefine experiments off-line

slide 15 4D-Nucleofector ® System — Consumables Consumables tailored to customer needs n Kits supporting Nucleofection in 16-well Nucleocuvette Strips (20µl) (A) n Kits supporting Nucleofection in single CP-cuvettes (100µl) (B) n Three kits for cell lines; five kits for primary cells n Cell line and primary cell optimization kits n Kits for adherent cell Nucleofection B A

slide 16 Reduction of Specific Kits 4D-Nucleofector ® Kit Concept: n 5 Primary Cell Kits (P1-5) n 1 Primary Cell Optimization Kit n 3 Cell Line Kits (SE, SF, SG) n 1 Cell Line Optimization Kit 4D-Nucleofector OPs: n Modified 96-well Shuttle OPs with ordering information for 20µl and 100µl Kits.

slide 17 Cell Optimization Strategy FINE TUNE Cell Line Optimization Easy Optimization n Test three solutions for cell lines or five solutions for primary cells each with 15 different programs n Based on best solution and program combination, fine tune transfection efficiency or viability FINE TUNE Primary Cell Optimization P1P2P3P4 P5 SE SF SG

slide 18 Cell Line Optimization Strategy Sol.SESFSG Row ACA-137DS-150CA-137DS-150CA-137DS-150 BCM-138DS-120CM-138DS-120CM-138DS-120 CCM-137EH-100CM-137EH-100CM-137EH-100 DCM-150EO-100CM-150EO-100CM-150EO-100 EDN-100EN-138DN-100EN-138DN-100EN-138 FDS-138EN-150DS-138EN-150DS-138EN-150 GDS-137EW-113DS-137EW-113DS-137EW-113 HDS-130ControlDS-130ControlDS-130Control Strip 1 Strip 2 Strip 3

slide 19 4D-Nucleofector ® System – Optimized Protocols Excellent performance on various cell numbers n 4D-Nucleofector ® performs equally compared to the 96-well Shuttle ® % Transfection efficiency 4D-Nucleofector ® 96-well Shuttle ® JurkatHUVECT CellsNIH-3T3K562

slide 20 Same conditions for different volumes / cell numbers n 20µl and 100µl Nucleocuvettes ® working with the same parameters – no additional optimization required 4D-Nucleofector ® System – Protocol Transfer TE VIA %

slide 21 n Introduction n The Principle of the Amaxa® Nucleofector® Technology n Available platforms n Details about the 4D-Nucleofector® n The Core Unit n The X-Unit n The Operation Software n Consumables n Protocol Transfer n Nucleofection and Stem Cells n Summary: System key benefits Agenda

slide 22 Transfection Efficiency * Viability Adipose Stem Cells (human) 76 % CD34 + (human) 71% – 83%62% MSC (human) 47% – 78%65 – 78% ES Cells (human) 45% – 78%50 – 98% ES Cells (mouse) 77% - 90%68 – 95% NSC (cow) 65%75% NSC (human) 90% NSC (mouse) 60%80% NSC (rat) 42% – 90%75% Nucleofection of Stem Cells * pmaxGFP

slide 23 Nucleofection™ of human Embryonic Stem Cells H1 H9 H9.2 Hues-9 BG01V Customer Efficiency Viability Data for Nucleofection ™ of human stem cells are compiled from experiments performed by leading stem cell research customers

slide 24 Data kindly provided by Jennifer Moore, Rutgers University, Piscataway, USA GFP SSEA4 OCT4 Nuclei H9 cells express pluripotency markers SSEA4 and OCT4 24 hours post Nucleofection ™ of pmaxGFP ™ reporter vector Human ES cells maintain pluripotency SSEA4GFPSSEA4/GFP % Positive cells (flow cytometry)

slide 25 Nucleofection™ for generation of iPS cells High transfection efficiencies for human chondrocytes, dermal fibroblasts (NHDF) or mouse embryonic fibroblasts – potential for generation of iPS via Nucleofection ™ Mouse Embryonic Fibroblast Photograph courtesy of Dr. H. Hermanns and Prof. P.H. Heinrich, University of Aachen, Germany Transfection Efficiency and viability (%) NHDF-adult* NHDF-neo* Human chondrocytes* Transfection EfficiencyViability

slide 26 n Virus-free induction of pluripotency and subsequent excision of reprogramming factors; Nature, 9 Apr 2009; 458 (7239) n Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector PNAS June 2, 2009 vol. 106 no n Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures Cell Research (2011) 21: n Efficient Feeder-Free Episomal Reprogramming with Small Molecules (2011) PLoS ONE 6(3): e Nucleofection™ for generation of iPS cells

slide 27 n Introduction n The Principle of the Amaxa® Nucleofector® Technology n Available platforms n Details about the 4D-Nucleofector® n The Core Unit n The X-Unit n The Operation Software n Consumables n Protocol Transfer n Nucleofection and Stem Cells n Summary: System key benefits Agenda

slide 28 Major Benefits of the 4D-Nucleofector ® n Easy-to-use through intuitive software and touch screen interface n Upgradeable by adding new Functional Modules n Customizable through its modular architecture n Uses existing 96-well Shuttle ® Protocols n The use of CP electrodes allows fewer solutions for primary cells n Supports Nucleofection ® of various cell numbers with same conditions n Allows Nucleofection ® of cells in adherence n Excellent technical and application support by Nucleofection ® Experts System Key Benefits

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