3D BIOTEK PRESENTS 3D CELL CULTURE WORKSHOP November 17, 2010 1 - PowerPoint PPT Presentation

3D BIOTEK PRESENTS 3D CELL CULTURE WORKSHOP November 17, 2010 1

Overview  Introduction to 3D Biotek and its Products - Irina Briller, MBA, Marketing Associate  3D Cell Seeding Protocol, Routine Cell Culture and Stem Cell Research in 3D - Nobel Vale, M.S., Research Scientist  Cancer Research in 3D - Carlos Caicedo, Ph.D., Research Scientist  Tissue Engineering, Biomimetic Coatings - Chris Gaughan, Ph.D., Research Scientist  Summary, Product Pipeline - Irina Briller, MBA, Marketing Associate 2

Introduction Mission Provide innovative biomedical research products in order to accelerate the discovery and development process. Short-Term Goal Provide innovative yet easy to use research tools to enable the transition of cell culture from 2D to 3D. 3

Company Information Founded in 2007, 3D Biotek, LLC is located in New Jersey’s Commercialization Center for Innovative Technologies. Stem Cells, Tissue Engineering, Medical Devices, Business Engineered Disease Model Precision 3D Micro-Fabrication, Advanced Bio- Manufacturing Coating Process; Porous Tubular Core Technology Implant Fabrication Patents: USA (4), China (2), International (2) Two product lines launched in 2008; 3D Cell Transfection Kit launched 4/2010; Bone defect Accomplishments repair and peripheral vascular stent product under development

Cell Culture History and Trends 5

History of Cell Culture 1981, Martin & 1907, Harrison 3D 1955, Eagle Evans 1838, Schleiden & Schwann Inventor of tissue defined medium Mouse ES cells “cell theory” culture 1885, Wilhelm Roux 1952, Gey 1965, Ham 1998, Thomson & Gearheart Cells can live outside HeLa cells Colonial growth Human ES cells the body of mammalian cells 1665, Hooke discovered “cells” 6

Currently Available 3D Systems AlgiMatrix 3D Collagen 3D OPLA Matrigel / 3D Calcium Scaffold Scaffold PuraMatrix / Phosphate Scaffold Coatings High Variable 100% Transparency (direct Ready surface to Easy cell Plate reader configurations interconnected observation with to use volume recovery compatible pores light microscope) (customizable) ratio The Ideal Scaffold Gel Matrices PLA foam CaP foam Alginate Foam Compatible Not Compatible

Development Of Novel 3D Scaffolds • Non-toxic • Well-defined pore size and fiber diameter • Free of animal-derived material • Reproducible from batch to batch • Compatible with current 2D assays 8

3D Insert TM Series • Well-defined pore size and porous structure • Organic solvent free • Custom design and fabrication • Compatible with current 2D assays 3D Insert TM -PCL • Reproducible from batch to batch • Non-toxic • Free of animal-derived material • 100% open connectivity 3D Insert TM -PS 3D Insert TM -PCL Polycaprolactone (PCL) is a biodegradable polymer used in FDA approved medical devices. 3D Insert TM -PS Polystyrene (PS) is a transparent plastic/material used in traditional tissue culture plates. 9 9

3D Insert TM -PCL Evaluated and chosen by the National Institute of Standards and Technology (NIST) to be the standard scaffold A C Controlled pore size: 200 ~ 500 µm B Controlled strut: 200 ~ 500 µm PCL scaffold (A-B) and Scanning Electron Microscopy (SEM) characterization of PCL scaffolds (C) . 10

Uniqueness of 3D Insert TM -PS A C B Four-layer structural design of a PS scaffold. Four distinct layers are visible from (A) side- angle, (B) side, and (C) top. 11

Average Cell Growth Area: 2D versus 3D Average Total Cell Growth Area 3D Insert TM -PS 3D Insert TM -PCL 2D 6 well 6 well 6 well 54.02 cm 2 99.21 cm 2 1520 3030 9.6 cm 2 52.10 cm 2 75.62 cm 2 3040 3050 12 well 12 well 12 well 21.08 cm 2 39.27 cm 2 1520 3030 4 cm 2 19.65 cm 2 27.90 cm 2 3040 3050 24 well 24 well 24 well 1520 10.20 cm 2 3030 18.28 cm 2 1.9 cm 2 9.56 cm 2 13.74 cm 2 3040 3050 48 well 48 well 48 well 1520 4.28 cm 2 3030 7.74 cm 2 1 cm 2 3.78 cm 2 6.08 cm 2 3040 3050 96 well 96 well 96 well 1520 1.36 cm 2 3030 2.03 cm 2 0.32 cm 2 3040 1.21 cm 2 3050 1.53 cm 2

Wide Range of Research Applications with 3D Biotek’s Cell Culture Inserts • Stem Cell Research • Drug Discovery • In Vitro Normal/Diseased Models • Cell Biology • Tissue Engineering 13 13

Materials and Methods Precision Microfabrication Technology  Fiber diameter is controlled by nozzle diameter  Spacing between fibers (pores) is controlled by a motion control system  Plasma treatment  Gamma radiation  Scaffolds are compatible with 6-well to 96-well tissue culture plates Example: 96-well compatible PS scaffolds 14

Materials and Methods Cell Seeding and Culture Example: 96-well compatible scaffolds and 2D 96-well plates  1x10 4 cells were seeded in a 20 µl suspension droplet (media + cells) onto 96-well compatible PS scaffolds (150 µm fiber and 200 µm pore size, 1.4 cm 2 growing area) 3 h incubation 37º C, 5% CO 2  1x10 4 cells were seeded in a 200 µl volume (media + cells) into 2D 96-well tissue culture wells (0.32 cm 2 growing area) 15

3D Cell Seeding Video 16

Results 17

Research Areas • Routine Cell Culture • Stem Cell Research • Cancer Models • Tissue Engineering 18 18

3D PS Scaffolds For Routine Imaging 3D tissue-like structures 2D TCP 3D PS pore Fluorescence Confocal pore NIH-3T3 cells cultured in 96-well 2D TCPs and on 96- well compatible PS scaffolds. Dapi: blue, F-actin: green, Fibronectin: red. • Routine imaging techniques can be used to monitor cells growing on PS scaffolds 19

3D Scaffolds For Cell Proliferation 3D Cell Sheets Proliferating human mesenchymal stem cells (hMSCs) were cultured on PS scaffolds Human mesenchymal stem cells (hMSCs) were seeded (150 µm pore size, 200 µm fiber diameter). At on PCL scaffolds (300 µm pore size, 300 µm fiber day 5, viable cells and their secreted extra- diameter) and cultured under osteogenic conditions. At cellular matrix were stained for nuclei (DAPI, day 7, fluorescent imaging shows that osteoblastic cells blue) and Fibronectin (primary mouse are viable (A-C) and extend into pores of the PCL scaffold antibody and secondary rabbit-anti-mouse (B) (F-actin: green, DAPI: blue, A: 40X, B-C: 200X). AlexaFluor 594, red). 20

Research Areas • Routine Cell Culture • Stem Cell Research • Cancer Models • Tissue Engineering 21 21

Mesenchymal Stem Cells Bone marrow derived stem cells are multipotent Hematopoietic Lineage Cell Type Differentiation Stem Cells Process (blood) 1. Bone Osteoblasts Osteoblastogenesis Mesenchymal 2. Fat Adipocytes Adipogenesis Stem Cells 3. Cartilage Chondrocytes Chondrogenesis  The differentiation process is initiated by the introduction of various growth factors and differentiation-promoting factors into cell culture media 22

3D PS Scaffolds For Stem Cell Research Bone: osteoblasts B C A Day 14 2D 3D 2D 3D E F D Day 21 2D 3D 2D 3D Stereo Microscope Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured using osteoblastic conditions and stained for mineralized nodule formation with Von Kossa assay. 23

3D PS Scaffolds For Stem Cell Research Fat: adipocytes Lipid Droplets Oil-Red-O Staining for Lipid Droplets 2D 0.5 2D 3D * 0.45 * 0.4 0.35 * 0.3 OD 560 * 0.25 0.2 0.15 0.1 0.05 0 3D Week 1 Week 2 Week 3 Week 4 p ≤0.05 Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured using adipocytic conditions and stained for lipid droplet formation using Oil-Red-O 24 staining.

3D PS Scaffolds For Stem Cell Research Cartilage: chondrocytes 5 2D TCP 3D PS Scaffolds Chondrogenesis (3D) 4.5 Chondrogenesis (2D) Control (3D) 4 Control (2D) 3.5 Week 1 Collagen mg/ml 3 2.5 2 1.5 1 Week 2 0.5 0 1 2 3 4 Time (Weeks) Week 3 Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured using chondrocytic conditions and stained for collagen formation. Week 4 25

World’s First 3D Transfection Kit 26 26

One Step Transfect And Seed 3D Cell Transfection Kit A B Greater and extended IL-2 cytokine secretion C in 3D. HEK293T were seeded and transfected in 2D (10x10 3 cells, 0.25 µg IL-2 cytokine plasmid, D 0.5 µl commercial transfection reagent) and 3D (200x10 3 cells, 0.5 µg IL-2 cytokine plasmid, 3 µl 3D Transfection. Using the 3D Cell Transfection Kit, 2x10 5 NIH-3T3 fibroblastic (A-C) and SH5Y 3D Transfection Reagent). IL-2 secretion was neuronal (D) cells were simultaneously seeded and measured by ELISA assay at each time-point. transfected with EGFP. 3D EGFP expression was monitored by fluorescence microscopy 24 h (NIH- 3T3 cells, A-C) and 48 h (SH5Y cells, D) post- transfection. A, D: 10X, B-C: 20X. 27

3D PS Scaffolds Support In Vitro Cell Transfection 3D Insert TM -PS/Transfection Reagent: Cell Lines Used HEK293 (Kidney Cells) NIH3T3 (Fibroblast) MCF-7 (Breast Cancer) MEF (Embryonic Fibroblast) SH5Y (Neuroblastoma) U87 (Glyoblastoma astrocytoma) VERO (Monkey kidney cells) 1 ˚ Rat Fibroblast 1 ˚ H. Neuroblastoma • New Products/Directions 28

Research Areas • Routine Cell Culture • Stem Cell Research • Cancer Models • Tissue Engineering 29 29