perfusion scaling slideshare
TRANSCRIPT
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
≠
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
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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
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 11
3D Perfusion: Large-‐Scale Perfusion
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3D Chambers
Media Reservoir
CO2/Humidifier
Pump
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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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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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