Perfusion scaling slideshare

Upstream Processing: Dimensionality and Perfusion

Add Extra Dimension to Your Innovation™ © 3D BIOTEK, LLC

2D Is Easier, at What Cost?

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1960’s 1900’s 2000’s

Glass-‐Based Tissue Culture

Polystyrene-‐Based Tissue Culture

DissociaBon/Maintenance of Cells/Vaccine ProducBon

2D

2D

2

www.Pubmed.gov

Scale-‐up ProducBon: Cell lines and Biologics

Fermenter/2D

2

1. Lack of Dimensional Relevance

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Brain

Bone, CarBlage

Lung, Breast

Brain

Bone, CarDlage

Lung, Breast

Liver Liver

Petri Dish

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2. Lack of 3D Perfusion

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2D Surface

ConvecBve Flow

3D Scaffold

Perfusion Flow

UpregulaBon of global gene expression e.g. ECM, proliferaBon, and differenBaBon

DownregulaBon of hypoxia-‐related genes 4

Uncorrelated Gene Expression

3. Lack of volumetric Growth

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2D Surface 3D Scaffold

Monolayer

Longer ProliferaDon Profiles Shorter ProliferaDon Profiles

Tissue-‐Like

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4. Lack of Scalability

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2,809.0

Total Area in cm^2

6

3D 35-‐mm Chambers

T-‐175 Flask

35-‐mm Chamber

4 16

1 Loaded Chamber = 4 T175 Flasks

3D Technology: Process-‐Friendly and Biologically Relevant

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 Polymer Scaffold Prototyping  3D Perfusion and Seeding OpDmizaDon

3D Insert™ PlaWorm

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

Pore Size Scaffold Volume

3D Geometry 8

3D Insert™ Series

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3D Insert™-‐Polystyrene

3D Insert™-‐Poly-‐ε-‐Caprolactone

  ConfiguraBons: PS(1520) and PS(3040)  PS(1520) = 150-‐um Fiber/200-‐um Spacing  PS(3040) = 300-‐um Fiber/400-‐um Spacing  Fit 96 to 6 well plate formats  Total number of layers: 4

  ConfiguraBons: PCL(3030) and PS(3050)  PCL(3030) = 300-‐um Fiber/300-‐um Spacing  PCL(3050) = 300-‐um Fiber/500-‐um Spacing  Fit 96 to 6 well plate formats  Total number of layers: 6 9

3D Insert™: Tissue-‐like Behavior

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Fiber 2

Fiber 1 H. Astrocytes hMSCs

NIH 3T3 MCF-‐7

3D Insert™

Pore Structure

Fiber Curvature

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Ample Surface Area in Small Volume

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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


4 cm2 1520 21.08 cm2 3030 39.27 cm2

3040 19.65 cm2 3050 27.90 cm2


1.9 cm2 1520 10.20 cm2 3030 18.28 cm2

3040 9.56 cm2 3050 13.74 cm2


1 cm2 1520 4.28 cm2 3030 7.74 cm2

3040 3.78 cm2 3050 6.08 cm2


0.32 cm2 1520 1.36 cm2 3030 2.03 cm2

3040 1.21 cm2 3050 1.53 cm2 11

3D Technology: Perfusion

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3D Perfusion: Large-‐Scale Perfusion

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3D Chambers

Media Reservoir

CO2/Humidifier

Pump

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Parts and Features

3D Perfusion Bioreactor: 1. Hardware

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•  Durable Metal Frame   The metal frame is made of strengthened aluminum (anodized) for prolonged durability during autoclaving.   Low humidity peristalBc pump with maximum 60 rpm

3D Perfusion Bioreactor: 2. Liquid Handling

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Media Bo)les Humidifier Bo)les

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•  Economic and Re-‐usable   Media and humidified boeles and accessories are autoclavable, e.g. glass boeles, stainless steel, silicon gaskets, and polycarbonate caps. Total Media Capacity: 400-‐ml and 2-‐liters

3D Perfusion Bioreactor: 3. Tubing System

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(C)

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•  Media and Gas Transport   Medical grade clear silicon and beige peristalBc tubing

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

(B)

(A)

Features: 1. Color-‐Coded Assembly

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G B

R O Y

G

Media Reservoir

CO2/O2 Humidifier

CO2/O2 Humidifie

r

Media Reservoir

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Features: 2. ConDnuous Perfusion

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3D Bioreactor Perfusion Circuitry 19

Feature: 3. Variable Velocity Range

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Bioreactor Chamber Format 24-‐Well Insert 12-‐Well Insert 6-‐Well Insert

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 SpecificaDons)

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Features: 4. Streamlined Process

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For Perfusion Bioreactor, please visit us at h\p://www.3dbiotek.com/web/prod_3dbioreactor.aspx

Cell Seeding

3D Insert™ O-‐Ring

3 h incubaDon 37º C, 5% CO2

2.0 rpm, 24h Slow Perfusion

For 24 well format

Media In

Media Out

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3D Perfusion Bioreactor

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•  An innovaBve product for scale-‐up cell culture and bioprocessing

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

•  Simple design and least number of parts for faster assembly and beeer asepBc condiBons