3d perfusion bioreactor technical presentation

3D Perfusion Bioreactor™

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Agenda/Learning Objec0ves

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•  Introduc*on • Product Descrip*on • Opera*on Principle • Applica*ons

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Introduc>on

3D Perfusion Bioreactor™

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•  An innova*ve product for cell culture and bioprocessing

•  The only polymer scaffold-‐based perfusion bioreactor for 3D dynamic cell culture

•  Simple design and minimal parts for faster assembly and beAer asep*c condi*ons

3D Perfusion Bioreactor Adds Value

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•  Proven 3D InsertTM technology, scaffolds with 100% open pore structure and pore Interconnec*on

•  Choice of both biodegradable and non-‐biodegradable scaffolds.

3D Perfusion Bioreactor Adds Value, Cont

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• Process can be standardized, scaffolds were selected as Reference scaffold by NIST

• Adjustable dynamic perfusion velocity range

• BoAom to top flow to maintain even wePng of stacked scaffolds

Fields of Applica0on

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•  Stem cell/*ssue engineering  Bone, car*lage, liver, etc.

•  In vitro *ssue model for drug discovery  Tumor and liver models

•  Bioprocessing   Cell expansion (enhanced prolifera*ve capacity)   Recombinant protein   Hybridoma-‐based an*body produc*on

Product Descrip>on

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Product Specifica0on

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•  Autoclavable System (Except Pump) •  Low Humidity Working Pump •  Color Coded Tubing for Easy Assembly •  Op*mized Cell Seeding in Scaffolds •  Adjustable Perfusion Veloci*es •  3D Inserts™   wide range of pore sizes   Any polymer of interest

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3D Perfusion Bioreactor™ Diagram

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3D Perfusion Bioreactor: Hardware

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•  Durable Metal Frame   The metal frame is made of strengthened aluminum (anodized) for prolonged durability during autoclaving.

  Low humidity peristal*c pump with maximum 60 rpm.

3D Perfusion Bioreactor™: Perfusion Circuit

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3D Perfusion Bioreactor: Liquid Handling

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Media BoJles

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•  Economic and Re-‐usable   Media and humidified boAles and accessories are autoclavable, e.g. glass boAles, stainless steel, silicon gaskets, and polycarbonate caps.

3D Perfusion Bioreactor: Tubing System

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(C)

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•  Media and Gas Transport   Medical grade clear silicon and beige peristal*c tubing

Tubing and Transport   (A) Perfusion Chamber   (B) Media System   (C) CO2/O2 System

(B)

(A)

3D Perfusion Bioreactor: Color -‐coded Assembly

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G B

R O Y

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Media Reservoir

CO2/O2 Humidifier

CO2/O2 Humidifie

r

Media Reservoir

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3D Insert™ Scaffold Geometry

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3D Insert™-‐Polystyrene

3D Insert™-‐Poly-‐ε-‐Caprolactone

Poly-‐ε-‐Caprolactone (PCL) is a biodegradable polymer used in FDA approved medical devices

Polystyrene (PS) is a transparent polymer used in tradi*onal cell culture.

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3D Insert™ Scaffold Specifica0ons

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  Configura*ons: PS(1520) and PS(3040)  PS(1520) = 150-‐um Fiber/200-‐um Spacing  PS(3040) = 300-‐um Fiber/400-‐um Spacing  Total number of layers: 4

  Configura*ons: PCL(3030) and PS(3050)  PCL(3030) = 300-‐um Fiber/300-‐um Spacing  PCL(3050) = 300-‐um Fiber/500-‐um Spacing  Total number of layers: 6

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3D Insert™-‐Polystyrene

3D Insert™-‐Poly-‐ε-‐Caprolactone

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3D Insert™ Technology

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Fiber Diameter Fiber-‐to-‐Fiber Spacing Scaffold Layering

Pore Size Scaffold Volume

3D Geometry

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3D Perfusion: Specific Product Features

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•  Easy –Handle 3D Insert™   Scaffolds adapted for beAer handling   Increase loading precision   Faster loading *me

•  Chamber Lateral Micro Channels   Polycarbonate chamber   0.6-‐mm channels inside the chamber   Prevent bubbling during ini*al media flow   Helps maintain op*mum ini*al seeding density

3D Insert™: Ideal Scaffold for Perfusion Bioreactor

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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 observation with light microscope)

3D Insert™

Gel Matrices

PLA foam

CaP foam

Alginate Foam

Alvetex®

Compa>ble

Not Compa>ble

3D Collagen Scaffold

3D OPLA Scaffold

3D Calcium Phosphate Scaffold

AlgiMatrix Matrigel / PuraMatrix / Coa>ngs

Alvatex®

Product Comparison: Bioreactors

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Easy Cell

Recovery

100% interconnect

ed pores

High surface to

volume ratio

Variable configurations (customizable)

Continuous Shear

3D Dynamic Perfusion

3D Perfusion

Roller Bottle

Fiber Cell

Stirrer Flask

BioLevitaror

Compa>ble Not Compa>ble

3D Perfusion Bioreactor™: Cell Seeding

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Cell Seeding

3D Insert™ O-‐Ring

3 h incuba*on 37º C, 5% CO2

1.93 rpm, 24h Slow Perfusion

Media In

Media Out

3D Seeding 3D Perfusion 23

3D Perfusion: Cell Seeding Volumes

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Insert™ Format

Seeding Volume (ul)

PS(1520) PS(3040) PS(3030) PS(3050)

24-‐well 60 120 130 160

12-‐well 120 240 270 340

6-‐well 300 600 690 880

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3D Perfusion: Cell Culture Media Volumes

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Bioreactor Format and Tubing

Volume to Fill (ml)

6 –Well Chamber 45-‐ml

12-‐Well Chamber 15-‐ml

24-‐Well Chamber 7.0-‐ml

Media Tubing 5.0-‐ml

Reservoir BoEle Max. volume 90-‐ml 25

3D Perfusion: rpm and Velocity

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Bioreactor Chamber Format

24-‐Well 12-‐Well 6-‐Well Velocity(cm/min) rpm rpm rpm

0.0 0 0 0

0.2 1.93 4.84 12.08

0.4 3.86 9.69 24.15

0.6 5.8 14.53 36.23

0.8 7.73 19.38 48.31 *Pump maximum rpm capacity is 60 rpm (Manufacturer Specifica*ons)

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3D Perfusion: Increased Cell Viability

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Neonatal Fibroblats Calcein Viability

BoAom

Top 2

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10

0

1

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5

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1 3 5 7 9

Cell Fold In

crease

Scaffold Posi>on (Top (Lec) to Bodom (Right))

Dynamic Sta>c

Neonatal Fibroblats Growth in Perfusion 28

3D Perfusion: Superior Cell Prolifera0on

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3D Perfusion Causes Early Prolifera0on of Osteosarcoma 29

300-‐um

Fiber

3D Perfusion

3D Sta>c

n = 3 (STDEV)

Day 5

3D Perfusion: Enhances in vivo-‐like Behavior

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Total Collagen Staining Shows Increasing Deposi0on of ECM 30