3d biotek presents 3d cell culture …3dbiotek.com/documents/presentation.pdf · 2 overview...

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3D BIOTEK PRESENTS3D CELL CULTURE WORKSHOP

November 17, 2010

2

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 inorder to accelerate the discovery and developmentprocess.

Short-Term Goal

Provide innovative yet easy to use research tools toenable the transition of cell culture from 2D to 3D.

Introduction

BusinessStem Cells, Tissue Engineering, Medical Devices,

Engineered Disease Model

Core Technology

Precision 3D Micro-Fabrication, Advanced Bio-Manufacturing Coating Process; Porous Tubular

Implant FabricationPatents: 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

6

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 of mammalian

cells

1981, Martin & Evans

Mouse ES cells

1998, Thomson & Gearheart

Human ES cells

3D

Ready to use

100% interconnected

pores

High surface to

volume ratio

Variable configurations (customizable)

Easy cell recovery

Plate reader compatible

Transparency (direct observation with light microscope)

The Ideal Scaffold

Gel Matrices

PLA foam

CaP foam

Alginate Foam

3D Collagen Scaffold

3D OPLA Scaffold

3D Calcium Phosphate Scaffold

AlgiMatrixMatrigel / PuraMatrix /

Coatings

Compatible

Not Compatible

Currently Available 3D Systems

8

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

9

9

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 ScanningElectron 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

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

Average Cell Growth Area: 2D versus 3D

Average Total Cell Growth Area2D 3D InsertTM-PS 3D InsertTM-PCL

6 well 6 well 6 well

9.6 cm21520 54.02 cm2 3030 99.21 cm2

3040 52.10 cm2 3050 75.62 cm2


4 cm21520 21.08 cm2 3030 39.27 cm2

3040 19.65 cm2 3050 27.90 cm2


1.9 cm21520 10.20 cm2 3030 18.28 cm2

3040 9.56 cm2 3050 13.74 cm2


1 cm21520 4.28 cm2 3030 7.74 cm2

3040 3.78 cm2 3050 6.08 cm2


0.32 cm21520 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 MethodsCell 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) onto96-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-welltissue culture wells (0.32 cm2 growing area)

3 h incubation

37º C, 5% CO

2

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 stemcells (hMSCs) were cultured on PS scaffolds(150 µm pore size, 200 µm fiber diameter). Atday 5, viable cells and their secreted extra-cellular matrix were stained for nuclei (DAPI,blue) and Fibronectin (primary mouseantibody and secondary rabbit-anti-mouseAlexaFluor 594, red).

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

2121

Research Areas


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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

DifferentiationProcess

Lineage Cell Type

The differentiation process is initiated by the introduction of various growth factors anddifferentiation-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 usingosteoblastic conditions and stained for mineralized nodule formation with VonKossa assay.

Bone: osteoblasts

Day 14

Day 21

Stereo Microscope

2D 3D2D 3D

AB C

DE F

2D 3D2D 3D

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3D PS Scaffolds For Stem Cell ResearchFat: adipocytes

2D

3D

Lipid Droplets

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

Week 1 Week 2 Week 3 Week 4

2D 3D

OD

560

Oil-Red-O Staining for Lipid Droplets

**

* *

p≤0.05

Human mesenchymal stem cells (hMSCs) on PS scaffolds cultured usingadipocytic conditions and stained for lipid droplet formation using Oil-Red-Ostaining.

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3D PS Scaffolds For Stem Cell Research

3D PS Scaffolds2D TCP

00.5

11.5

22.5

33.5

44.5

5

1 2 3 4Time (Weeks)

Col

lage

n m

g/m

l

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

Cartilage: chondrocytes

Week 1

Week 2

Week 3

Week 4

Human mesenchymal stem cells (hMSCs) onPS scaffolds cultured using chondrocyticconditions and stained for collagen formation.

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World’s First 3D Transfection Kit

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3D Transfection. Using the 3D Cell TransfectionKit, 2x105 NIH-3T3 fibroblastic (A-C) and SH5Yneuronal (D) cells were simultaneously seeded andtransfected with EGFP. 3D EGFP expression wasmonitored 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

CD

Greater and extended IL-2 cytokine secretionin 3D. HEK293T were seeded and transfected in2D (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 µl3D Transfection Reagent). IL-2 secretion wasmeasured 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 usinga light microscope in real-time. (A-B: 100X, C: 200X)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Day 1 Day 4 Day 7 Day 14

2D3D

0

0.05

0.1

0.15

0.2

0.25

Day 1 Day 4 Day 7

2D3D

Abs

orba

nce

(570

nm

)

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

MTT assay

Abs

orba

nce

(570

/405

nm

)

Alamar Blue**

*

*

* * *

p≤0.05

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Breast Cancer Cells - 2D Versus 3D Resistance

0

0.2

0.4

0.6

0.8

1

4 7 10 13 4 7 10 13 4 7 10 13


0

5000

10000

15000

20000

25000

DN

A (n

g p

er w

ell)

2D3D

control + E2 + E2 + FUL

Enhanced MCF proliferation in 3Dafter estrogen stimulation. DNA assaywas performed to determine proliferationresponse.

DNA assay

*

^

&

p≤0.05

Abs

orba

nce

(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 MTTassay.

MTT assay2D3D

*

*

*

**

* *

* **

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A B

HepG2 Cells - Potential For Drug Discovery


HepG2 cells imaged usinga light microscope in real-time. (A: 200X, C: 200X).

0

10000

20000

30000

40000

50000

60000

70000

2D 3D

Rifampicin

CYP3A Activity

RLU

0

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 withRifampicin and assayed for CYP3A induction and cell viability.

*

p≤0.05

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Blood Cancer Model - Potential For Drug Discovery


Non-Hodgkin Lymphoma Proliferation

+ Stroma

3D 2D

Day 0 1,000 1,000

Day 7197,222

+/- 23,94055,777

+/- 8.071

% Surplus 19,722 5,577

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Research Areas


What is it?Tissue Engineering is the combination of cells, engineering, andmaterials 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 ofMinnesota: Stem Cell Institute, 2008

“Ear Mouse”

Tissue Engineering

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3D Cell Sheets

3D PS Scaffolds For Normal Tissue Models

Human dermal fibroblastscultured on 96-wellcompatible PS scaffolds.DAPI: blue, F-actin: green,(A: 100x, B: 200X).

00.020.040.060.080.1

0.120.140.160.18

Day 3 Day 6

2D 3D

Neutral Red Assay

Abs

orba

nce

(560

nm

)

**

Human epidermal keratinocytes(neonatal) cultured in 96-well 2DTCPs and on 96-well compatible PSscaffolds.

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 durationof the experiment, hFB were cultured in fibroblasticmedia and hMSCs were cultured using osteoblasticconditions. At each time-point, hMSC and hFB wereassayed for alkaline phosphatase activity (A),calcium deposition (B), and stained for mineralizationwith Von Kossa (C).

A B

C

0

5

10

15

20

25

30

35

40

45

Week 4

hFBhMSC

Calcium Deposition

Abs

orba

nce

(OD

490

nm)

0

5

10

15

20

25

30

35

40

45

Week 4

hFBhMSC

Calcium Deposition

Abs

orba

nce

(OD

490

nm) *

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01


hFBhMSC

Alkaline Phosphatase Activity

uM/m

inut

e/ng

DN

A/w

ell

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01


hFBhMSC

Alkaline Phosphatase Activity

uM/m

inut

e/ng

DN

A/w

ell

*

* *

*

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

4141

Collagen Coated Scaffolds

4242

Culture Under Reduced-Serum Conditions

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

Hepatocytes

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Commonly Used Assays Compatible With 3D PS/PCL Scaffolds

Compatible AssaysCell 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 HepG2LYRH

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

FibroblastsHuman fibroblasts, adult NIH-3T3 L929

3D PS/PCL Scaffolds Support The Growth of Many Other Cells/Tissues

3D InsertTM-PS

3D InsertTM-PCL

Summary of 3D Insert Benefits

• 3D InsertTM-PCL and InsertTM-PS are compatible withcommonly used 2D assays

• 3D InsertTM-PCL and InsertTM-PS improve cell growth andfunction

• 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 PipelineCurrent 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 mmPlate 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

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

Current Customers & Distributors

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

- Distributors

- Customers

5050

Contact Information

3D Biotek, LLC675 US Highway One

North Brunswick, NJ 08902Phone: (732) 729-6270Fax: (732) 729-7270

[email protected]

Irina Briller, MBA Carlos Caicedo, Ph.D.Marketing and Sales Research Scientist732-729-6270, ext. 4105 732-729-6270, ext. [email protected] [email protected]

Chris Gaughan, Ph.D. Nobel Vale, M.S.Research Scientist Research Scientist732-729-6270 732-729-6270 ext. [email protected] [email protected]

mailto:[email protected]�





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3d biotek presents 3d cell culture …3dbiotek.com/documents/presentation.pdf · 2 overview...

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