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

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

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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 -

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3D BIOTEK PRESENTS 3D CELL CULTURE WORKSHOP

November 17, 2010

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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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Mission Provide innovative biomedical research products in

  • rder 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.

Introduction

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Business Stem Cells, Tissue Engineering, Medical Devices, Engineered Disease Model Core Technology Precision 3D Micro-Fabrication, Advanced Bio- Manufacturing 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

Company Information

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

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Cell Culture History and Trends

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History of Cell Culture

1665, Hooke discovered “cells” 1838, Schleiden & Schwann “cell theory” 1885, Wilhelm Roux Cells can live outside the body 1907, Harrison Inventor of tissue culture 1952, Gey HeLa cells 1955, Eagle defined medium 1965, Ham Colonial growth

  • f mammalian

cells 1981, Martin & Evans Mouse ES cells 1998, Thomson & Gearheart Human ES cells

3D

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Ready to use 100% interconnected pores High surface to volume ratio Variable configurations (customizable) Easy cell recovery Plate reader compatible Transparency (direct

  • bservation with

light microscope) The Ideal Scaffold Gel Matrices PLA foam CaP foam Alginate Foam 3D Collagen Scaffold 3D OPLA Scaffold 3D Calcium Phosphate Scaffold AlgiMatrix Matrigel / PuraMatrix / Coatings

Compatible Not Compatible

Currently Available 3D Systems

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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

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

3D InsertTM-PCL 3D InsertTM-PS

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3D InsertTM-PCL

Controlled pore size: 200 ~ 500 µm Controlled strut: 200 ~ 500 µm

PCL scaffold (A-B) and Scanning Electron Microscopy (SEM) characterization of PCL scaffolds (C).

A B C Evaluated and chosen by the National Institute of Standards and Technology (NIST) to be the standard scaffold

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Uniqueness of 3D InsertTM-PS

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

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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 12 well 12 well 4 cm2 1520 21.08 cm2 3030 39.27 cm2 3040 19.65 cm2 3050 27.90 cm2 24 well 24 well 24 well 1.9 cm2 1520 10.20 cm2 3030 18.28 cm2 3040 9.56 cm2 3050 13.74 cm2 48 well 48 well 48 well 1 cm2 1520 4.28 cm2 3030 7.74 cm2 3040 3.78 cm2 3050 6.08 cm2 96 well 96 well 96 well 0.32 cm2 1520 1.36 cm2 3030 2.03 cm2 3040 1.21 cm2 3050 1.53 cm2

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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)

  • 1x104 cells were seeded in a 200 µl volume (media + cells) into 2D 96-well

tissue culture wells (0.32 cm2 growing area)

3 h incubation 37º C, 5% CO 2

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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 tissue-like structures

NIH-3T3 cells cultured in 96-well 2D TCPs and on 96- well compatible PS scaffolds. Dapi: blue, F-actin: green, Fibronectin: red.

2D TCP 3D PS

pore pore

3D PS Scaffolds For Routine Imaging

  • Routine imaging techniques can be used to monitor cells growing on PS scaffolds

Fluorescence Confocal

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3D Cell Sheets

3D Scaffolds For Cell Proliferation

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 extra- cellular 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

  • n 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

Hematopoietic Stem Cells (blood) Mesenchymal Stem Cells

  • 1. Bone

Osteoblasts Osteoblastogenesis

  • 2. Fat

Adipocytes Adipogenesis

  • 3. Cartilage

Chondrocytes Chondrogenesis

Bone marrow derived stem cells are multipotent

Differentiation Process Lineage Cell Type

  • The differentiation process is initiated by the introduction of various growth factors and

differentiation-promoting factors into cell culture media

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3D PS Scaffolds For Stem Cell Research

Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured using

  • steoblastic conditions and stained for mineralized nodule formation with Von

Kossa assay.

Bone: osteoblasts

Day 14 Day 21

Stereo Microscope 2D 3D 2D 3D

A B C D E F

2D 3D 2D 3D

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3D PS Scaffolds For Stem Cell Research

Fat: adipocytes

2D 3D Lipid Droplets

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Week 1 Week 2 Week 3 Week 4 2D 3D

OD560

Oil-Red-O Staining for Lipid Droplets

* * * *

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

3D PS Scaffolds 2D TCP

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 1 2 3 4

Time (Weeks) Collagen mg/ml

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

Cartilage: chondrocytes

Week 1 Week 2 Week 3 Week 4

Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured using chondrocytic conditions and stained for collagen formation.

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World’s First 3D Transfection Kit

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27 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 (NIH- 3T3 cells, A-C) and 48 h (SH5Y cells, D) post-

  • transfection. A, D: 10X, B-C: 20X.

A B C D

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.

One Step Transfect And Seed 3D Cell Transfection Kit

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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

3D PS Scaffolds Support In Vitro Cell Transfection

  • 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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Breast Cancer Cells - Morphology and Viability

3D PS Scaffolds For Disease Models

MCF-7 cells imaged using a light microscope in real-

  • time. (A-B: 100X, C: 200X)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 Day 1 Day 4 Day 7 Day 14 2D 3D 0.05 0.1 0.15 0.2 0.25 Day 1 Day 4 Day 7 2D 3D

Absorbance (570 nm) 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.

MTT assay

Absorbance (570/405 nm)

Alamar Blue * * * * * * *

p≤0.05

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Breast Cancer Cells - 2D Versus 3D Resistance

0.2 0.4 0.6 0.8 1 4 7 10 13 4 7 10 13 4 7 10 13

3D PS Scaffolds For Disease Models

5000 10000 15000 20000 25000 DNA (ng per well) 2D 3D

control + E2 + E2 + FUL

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

DNA assay * ^

&

p≤0.05

Absorbance (570 nm) con 10-6 M 10-5 M

Day

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

MTT assay

2D 3D

* * * * * * * * * *

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A B

HepG2 Cells - Potential For Drug Discovery

3D PS Scaffolds For Disease Models

HepG2 cells imaged using a light microscope in real-

  • time. (A: 200X, C: 200X).

10000 20000 30000 40000 50000 60000 70000

2D 3D

Rifampicin

CYP3A Activity RLU

50000 100000 150000 200000 250000 300000

2D 3D

Rifampicin

RLU Viability 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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Blood Cancer Model - Potential For Drug Discovery

3D PS Scaffolds For Disease Models

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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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

  • f

Minnesota: Stem Cell Institute, 2008 “Ear Mouse”

Tissue Engineering

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3D Cell Sheets

3D PS Scaffolds For Normal Tissue Models

Human dermal fibroblasts cultured

  • n

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

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18

Day 3 Day 6 2D 3D

Neutral Red Assay

Absorbance (560 nm)

* *

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

p≤0.05

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3D PCL Scaffolds For Tissue Models

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

Biodegradable polycaprolactone scaffolds

Human mesenchymal (hMSC) and fibroblastic (hFB) cultured on PCL scaffolds. For the duration

  • f 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).

A B C

5 1 1 5 20 25 30 35 40 45

Week 4 hFB hMSC

Calcium Deposition Absorbance (OD490 nm)

5 1 1 5 20 25 30 35 40 45

Week 4 hFB hMSC

Calcium Deposition Absorbance (OD490 nm)

*

0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01

Week 1 Week 2 Week 3 Week 4 hFB hMSC Alkaline Phosphatase Activity uM/minute/ng DNA/well

0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01

Week 1 Week 2 Week 3 Week 4 hFB hMSC Alkaline Phosphatase Activity uM/minute/ng DNA/well

* * * *

p≤0.05

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Biomimetic Coatings

  • Enhance attachment of specific cell-types
  • Facilitate culture under low serum or serum

free conditions

  • Enable isolation of primary cells

3D InsertTM-PCL

Collagen Poly-D-Lysine Fibronectin

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Collagen Coated Scaffolds

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Culture Under Reduced-Serum Conditions

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Huihui Mou, Yannan He, Kanghong Hu, State Key Laboratory of Virology, Institute

  • f Virology, Chinese Academy
  • f Sciences, 430071, Wuhan,

PR China

Hepatocytes

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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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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 PS/PCL Scaffolds Support The Growth of Many Other Cells/Tissues

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

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Product Pipeline

Current Products

  • 3D Tissue Culture Plates

with PS Inserts (Clear/Black/White)

  • 3D Tissue Culture Plates

with PCL Inserts

  • 3D Cell Transfection Kit
  • 3D Tissue Culture 100 mm

Plate with PCL Insert

  • Custom Products (PLGA,

etc.)

  • 3D Bioreactor

Future Products

  • 3D Tissue Culture Flask

with Insert

  • 3D Differentiation Kit
  • 384-Well Plates
  • Scaffold Coating (standard

and custom)

  • Nanofiber Technology
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Awards and Collaborations

Collaborations

  • Stem Cell Research Facility
  • BioCellChallenge
  • Celltreat Scientific Products

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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
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Current Customers & Distributors

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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 info@3dbiotek.com

Irina Briller, MBA Carlos Caicedo, Ph.D.

Marketing and Sales Research Scientist 732-729-6270, ext. 4105 732-729-6270, ext. 4106 ibriller@3dbiotek.com ccaicedo@3dbiotek.com Chris Gaughan, Ph.D. Nobel Vale, M.S. Research Scientist Research Scientist 732-729-6270 732-729-6270 ext. 4108 cgaughan@3dbiotek.com nvale@3dbiotek.com

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Questions?