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

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

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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.

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Company Information Founded in 2007, 3D Biotek, LLC is located in New Jersey’s Commercialization Center for Innovative Technologies.

Business

Stem Cells, Tissue Engineering, Medical Devices, Engineered Disease Model

Core Technology

Precision 3D Micro-Fabrication, Advanced BioManufacturing Coating Process; Porous Tubular Implant Fabrication Patents: USA (4), China (2), International (2)

Accomplishments

Two product lines launched in 2008; 3D Cell Transfection Kit launched 4/2010; Bone defect repair and peripheral vascular stent product under development

Cell Culture History and Trends

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History of Cell Culture

1907, Harrison 1838, Schleiden & Schwann “cell theory”

Inventor of tissue culture

1955, Eagle defined medium

1981, Martin & Evans Mouse ES cells

1885, Wilhelm Roux

1952, Gey

1965, Ham

Cells can live outside the body

HeLa cells

Colonial growth of mammalian cells

3D

1998, Thomson & Gearheart Human ES cells

1665, Hooke discovered “cells”

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Currently Available 3D Systems

Matrigel / PuraMatrix / Coatings

Ready to use

The Ideal Scaffold Gel Matrices PLA foam CaP foam Alginate Foam

Compatible Not Compatible

AlgiMatrix

100% interconnected pores

3D Calcium Phosphate Scaffold

High surface to volume ratio

Variable configurations (customizable)

3D Collagen Scaffold

Easy cell recovery

3D OPLA Scaffold

Plate reader compatible

Transparency (direct observation with light microscope)

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

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3D InsertTM Series

3D InsertTM-PCL

3D InsertTM-PS

• Well-defined pore size and porous structure • Organic solvent free • Custom design and fabrication • Compatible with current 2D assays • Reproducible from batch to batch • Non-toxic • Free of animal-derived material • 100% open connectivity

3D InsertTM-PCL Polycaprolactone (PCL) is a biodegradable polymer used in FDA approved medical devices. 3D InsertTM-PS Polystyrene (PS) is a transparent plastic/material used in traditional tissue culture plates. 9

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3D InsertTM-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 InsertTM-PS A

C

B

Four-layer structural design of a PS scaffold. Four distinct layers are visible from (A) sideangle, (B) side, and (C) top.

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Average Cell Growth Area: 2D versus 3D Average Total Cell Growth Area 2D

3D InsertTM-PS

3D InsertTM-PCL

6 well

6 well

6 well

9.6

cm2

1520

54.02 cm2

3030

99.21 cm2

3040

52.10 cm2

3050

75.62 cm2

12 well 4

12 well

12 well

1520

21.08 cm2

3030

39.27 cm2

3040

19.65 cm2

3050

27.90 cm2

cm2

24 well 1.9

cm2

24 well 1520

10.20 cm2

3030

18.28 cm2

3040

9.56 cm2

3050

13.74 cm2

48 well 1 cm2

48 well

0.32

48 well

1520

4.28 cm2

3030

7.74 cm2

3040

3.78 cm2

3050

6.08 cm2

96 well cm2

24 well

96 well

96 well

1520

1.36 cm2

3030

2.03 cm2

3040

1.21 cm2

3050

1.53 cm2

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

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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

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Materials and Methods Cell Seeding and Culture Example: 96-well compatible scaffolds and 2D 96-well plates  1x104 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 cm2 growing area) 3h incubation 37 º C, 5% CO

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 1x104 cells were seeded in a 200 µl volume (media + cells) into 2D 96-well tissue culture wells (0.32 cm2 growing area)

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3D Cell Seeding Video

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Results

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Research Areas

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

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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 96well 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 (150 µm pore size, 200 µm fiber diameter). At day 5, viable cells and their secreted extracellular matrix were stained for nuclei (DAPI, blue) and Fibronectin (primary mouse antibody and secondary rabbit-anti-mouse AlexaFluor 594, red).

Human mesenchymal stem cells (hMSCs) were seeded on PCL scaffolds (300 µm pore size, 300 µm fiber diameter) and cultured under osteogenic conditions. At day 7, fluorescent imaging shows that osteoblastic cells are viable (A-C) and extend into pores of the PCL scaffold (B) (F-actin: green, DAPI: blue, A: 40X, B-C: 200X).

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Research Areas

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

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Mesenchymal Stem Cells Bone marrow derived stem cells are multipotent Hematopoietic Stem Cells (blood)

Lineage

Mesenchymal Stem Cells

Cell Type

Differentiation Process

1. Bone

Osteoblasts

Osteoblastogenesis

2. Fat

Adipocytes

Adipogenesis

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 3D

2D

E

F

D Day 21

2D

3D 2D

Stereo Microscope

3D

Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured using osteoblastic conditions and stained for mineralized nodule formation with Von Kossa assay.

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3D PS Scaffolds For Stem Cell Research Fat: adipocytes Lipid Droplets

Oil-Red-O Staining for Lipid Droplets

2D

0.5 0.45

2D

3D

OD560

0.35

*

0.3 0.25

*

*

0.4

*

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 staining.

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3D PS Scaffolds For Stem Cell Research Cartilage: chondrocytes 2D TCP

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3D PS Scaffolds

Chondrogenesis (3D) Chondrogenesis (2D) Control (3D) Control (2D)

4.5

Week 1

Collagen mg/ml

4 3.5 3 2.5 2 1.5 Week 2

1 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

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World’s First 3D Transfection Kit

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One Step Transfect And Seed 3D Cell Transfection Kit

A

B

C D 3D Transfection. Using the 3D Cell Transfection Kit, 2x105 NIH-3T3 fibroblastic (A-C) and SH5Y neuronal (D) cells were simultaneously seeded and transfected with EGFP. 3D EGFP expression was monitored by fluorescence microscopy 24 h (NIH3T3 cells, A-C) and 48 h (SH5Y cells, D) posttransfection. A, D: 10X, B-C: 20X.

Greater and extended IL-2 cytokine secretion in 3D. HEK293T were seeded and transfected in 2D (10x103 cells, 0.25 µg IL-2 cytokine plasmid, 0.5 µl commercial transfection reagent) and 3D (200x103 cells, 0.5 µg IL-2 cytokine plasmid, 3 µl 3D Transfection Reagent). IL-2 secretion was measured by ELISA assay at each time-point.

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3D PS Scaffolds Support In Vitro Cell Transfection 3D InsertTM-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

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Research Areas

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

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2D Cell Culture As Disease Models Limitations  Limited cell-cell interaction  Disrupted cellular organization and polarity  Inaccurate representation of the cellular environment experienced by cells in vivo  Disconnect between cellular behavior in vitro and in vivo

Debnath J, et al. 2003

Fishbach C, et al. 2007

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3D Cell Scaffold As Disease Models Advantages Dimensionality  Realistic signaling from microenvironment to cells  Better representation of in vivo drug resistance  Maintenance of true cancer phenotype Biphasic Cellular Systems  Fiber to pore distribution mimicking medullar structures  Introduction of stroma compartments  Integration of crucial cellular components

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3D PS Scaffolds For Disease Models Breast Cancer Cells - Morphology and Viability

Absorbance (570 nm)

0.25 0.2

2D 3D

MTT assay * *

*

0.15 0.1 0.05

Absorbance (570/405 nm)

MCF-7 cells imaged using a light microscope in realtime. (A-B: 100X, C: 200X)

0.7 2D 3D

0.6

Alamar Blue *

*

0.5

*

0.4 0.3

*

0.2 0.1 0

0 Day 1

Day 4

Day 7

Day 1

Day 4

Day 7

Day 14

Sustained cell viability in cells cultured on 3D PS scaffolds. MCF-7 human breast cancer cells were cultured in 2D and on 3D PS scaffolds. Cell viability was measured by (A) MTT and (B) Alamar blue assay. 32

p≤0.05

3D PS Scaffolds For Disease Models Breast Cancer Cells - 2D Versus 3D Resistance

1

0.6

25000

*

2D 3D

0.8

DNA assay

*

0.4

*

*

*

*

*

*

*

0.2

*

2D 3D

*

20000 15000 10000

&

^

5000

0 Day

DNA (ng per well)

Absorbance (570 nm)

MTT assay

4

7

con

10

13

4

7

10

10-6 M

13

4

7

10

13

10-5 M

Effects of tamoxifen on MCF-7 cells grown in 2D and 3D. Cell viability after tamoxifen treatment was measured by MTT assay.

0

control

+ E2

+ E2 + FUL

Enhanced MCF proliferation in 3D after estrogen stimulation. DNA assay was performed to determine proliferation response. p≤0.05

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3D PS Scaffolds For Disease Models HepG2 Cells - Potential For Drug Discovery A

B HepG2 cells imaged using a light microscope in realtime. (A: 200X, C: 200X).

Viability

CYP3A Activity 300000

70000 Rifampicin

250000

50000

RLU

RLU

60000

40000 30000

Rifampicin

*

200000 150000 100000

20000 10000

50000

0

0

2D

3D

2D

3D

HepG2 cells cultured on 2D TCP and in 96-well compatible PS scaffolds were treated with Rifampicin and assayed for CYP3A induction and cell viability.

p≤0.05

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3D PS Scaffolds For Disease Models Blood Cancer Model - Potential For Drug Discovery

Non-Hodgkin Lymphoma Proliferation

+ Stroma

3D

2D

Day 0

1,000

1,000

Day 7

197,222 +/- 23,940

55,777 +/- 8.071

% Surplus

19,722

5,577

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Research Areas

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

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Tissue Engineering What is it? Tissue Engineering is the combination of cells, engineering, and materials for the purpose of improving or replacing biological functions.

Applications  Organ transplants  Disease models  Medical devices Dr. Anthony Atala, Wake Forest University, 2006

Doris Taylor, University of Minnesota: Stem Cell Institute, 2008 “Ear Mouse”

3D PS Scaffolds For Normal Tissue Models 3D Cell Sheets

Human dermal fibroblasts cultured on 96-well compatible PS scaffolds. DAPI: blue, F-actin: green, (A: 100x, B: 200X).

PS fiber

PS fiber

Absorbance (560 nm)

Neutral Red Assay 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

2D

3D

*

*

Human epidermal keratinocytes (neonatal) cultured in 96-well 2D TCPs and on 96-well compatible PS scaffolds. p≤0.05

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Day 3

Day 6

3D PCL Scaffolds For Tissue Models Biodegradable polycaprolactone scaffolds A

Alkaline Phosphatase Activity

B

hFB

0.008

hMSC

*

0.007

*

0.006 0.005

*

0.004 0.003

Calcium Deposition 45

0.009

Absorbance (OD490 nm)

uM/minute/ng DNA/well

0.01

*

0.002 0.001

40 35

*

hFB hMSC

30 25 20 15 10 5

0

0

Week 1

Week 2

Week 3

Week 4

Human mesenchymal (hMSC) and fibroblastic (hFB) cultured on PCL scaffolds. For the duration of the experiment, hFB were cultured in fibroblastic media and hMSCs were cultured using osteoblastic conditions. At each time-point, hMSC and hFB were assayed for alkaline phosphatase activity (A), calcium deposition (B), and stained for mineralization with Von Kossa (C). p≤0.05

Week 4

C

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• Compatible with any sized bioreactor and can be used for in vivo work

Biomimetic Coatings • Enhance attachment of specific cell-types • Facilitate culture under low serum or serum free conditions • Enable isolation of primary cells

Collagen Poly-D-Lysine Fibronectin 3D InsertTM-PCL

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Collagen Coated Scaffolds

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Culture Under Reduced-Serum Conditions

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Hepatocytes Huihui Mou, Yannan He, Kanghong Hu, State Key Laboratory of Virology, Institute of Virology, Chinese Academy of Sciences, 430071, Wuhan, PR China

Commonly Used Assays Compatible With 3D PS/PCL Scaffolds Compatible Assays Cell lifting and tissue digestion (Trypsin/Trypsin-EDTA, Collagenase)

RNA isolation (Tri-Reagent)

Protein Assays (Western Blot, ELISA)

Proliferation Assays (DNA Assay [fluorescent detection], Alamar Blue, MTT, Neutral Red)

Cell Transfections (Transient [baculovirus], and Stable)

Differentiation Assays (ALP Activity, In Situ Collagen Content, GAG Characterization)

Characterization Stains (Von Kossa, Oil-Red-O, Alcian Blue, Sirius Red, Albumin)

Immunofluorescence and Immunohistochemistry *readily compatible with inverted light and fluorescent microscopes

Viability and Toxicity Assays (Multiplexing Assays, ADME/Tox Assays) *readily compatible with microplate readers

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3D PS/PCL Scaffolds Support The Growth of Many Other Cells/Tissues Cell Lines Cultured on 3D InsertTM-PS/PCL Tumor cells

MCF-7 MCF-7:WS8 ECC1 HepG2 LYRH

Stem Cells

Human Mesenchymal Stem Cells (hMSCs) Mouse bone marrow stromal stem cells (mBMSSCs)

Hepatocytes

Huh-7 HepG2

Osteoblasts

7F2 hMSC-derived osteoblasts

Chondrocytes

hMSC-derived chondrocytes

Adipocytes

hMSC-derived adipocytes

Neural cells

SH5Y U87

Cardiomyocytes

H9c2 Rat primary cardiomyocytes

Keratinocytes

Human keratinocytes, neonatal (HEKn)

Epithelial cells

MCF-10A HEK293T

Fibroblasts

Human fibroblasts, adult NIH-3T3 L929

3D InsertTM-PS

3D InsertTM-PCL

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Summary of 3D Insert Benefits • 3D InsertTM-PCL and InsertTM-PS are compatible with commonly used 2D assays • 3D InsertTM-PCL and InsertTM-PS improve cell growth and function • PS scaffolds create superior in vitro tissue/disease models • PS scaffolds can be used for drug studies • 3D InsertTM-PCL and InsertTM-PS support superior stem cell expansion and differentiation • 3D InsertTMs are applicable for tissue engineering applications • 3D InsertTM pore and fiber size can be custom configured to better suit various cell lines 46

Product Pipeline Current Products

Future Products

• 3D Tissue Culture Plates with PS Inserts (Clear/Black/White)

• 3D Tissue Culture Flask with Insert

• 3D Differentiation Kit

• 3D Tissue Culture Plates with PCL Inserts

• 384-Well Plates

• 3D Cell Transfection Kit

• Scaffold Coating (standard and custom)

• 3D Tissue Culture 100 mm Plate with PCL Insert

• Nanofiber Technology

• Custom Products (PLGA, etc.) • 3D Bioreactor

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Awards and Collaborations Awards: 2010  Tax Grant, Stent  3D Biotek received five Life Science Technology Fellowhsip Awards from Bio-1  STAR award from the Society for Biomaterials: Dr. Marika Bergenstock's paper, entitled “Engineered Polystyrene Scaffolds For In Vitro Three-Dimensional Disease Models,” was nominated as an outstanding contribution to the Society For Biomaterials 2010 Annual Meeting

Awards: 2009  2009 Incubator Company to Watch, New Jersey Technology Council  Edison Innovative R&D Grant, NJ Commission on Science & Technology  SBIR Phase I Grant, NIH  3 Fellowship Awards, NJ Commission on Science & Technology

Awards: 2008  3D Biotek received an incubator seed award from the NJCST  1 Fellowship Award, NJ Commission on Science & Technology

Collaborations   

Stem Cell Research Facility BioCellChallenge Celltreat Scientific Products 48

Current Customers & Distributors

Isn’t it time YOU see the world in 3D?

- Distributors - Customers

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Contact Information 3D Biotek, LLC 675 US Highway One North Brunswick, NJ 08902 Phone: (732) 729-6270 Fax: (732) 729-7270 [email protected] Irina Briller, MBA

Carlos Caicedo, Ph.D.

Marketing and Sales 732-729-6270, ext. 4105 [email protected]

Research Scientist 732-729-6270, ext. 4106 [email protected]

Chris Gaughan, Ph.D. Research Scientist 732-729-6270 [email protected]

Nobel Vale, M.S. Research Scientist 732-729-6270 ext. 4108 [email protected]

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Questions?

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