bioreactor design issues for cell cultures. cell culture - an engineering perspective
TRANSCRIPT
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Bioreactor design Issues for cell cultures
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Cell Culture
- An engineering perspective
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by Genentech, Corporate Communication
A Fermenter / Bioreactor And Its Parts
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Single System for Anchorage-Dependent and Suspension CulturesNew Brunswick Scientific Company
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BioFlo® Pro Customizable Cell Culture Bioreactors
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Fig. 2. Influenza production plant (6000 liter vessel for cultivating Vero cells on Cytodex™). Courtesy of Baxter Biosciences.
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• Nutrient Considerations• Environment Considerations• Common Culturing Systems
1. Spinner flasks2. Continuous stirred bioreactors3. Air (Liquid) lifted bioreactors4. Hollow-fibers bioreactors5. Microcarriers6. Perfusion systems7. Rotating wall bioreactors
• Examples
• Type of cultures
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Type of cultures
• Suspension cultures
• Anchorage dependent cultures
• monolayer
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Bioreactor: Advantages
Controlled environment:1. Mixing2. pH3. Dissolved oxygen4. Temperature
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pH probe
1. Steam sterilizable2. Combination electrode
1. Two major typesa. Galvanic b. Polargraphic
Dissolved oxygen probe
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Galvanic and Polargraphic Probes
Cathode 0.5 O2 + H2O + 2e- 2OH-
Pt
Anode (galvanic) Pb Pb2+ + 2e-
Anode (polargraphic) Ag + Cl- AgCl + e-
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Nutrient considerationsTwo major classes
• serum supplemented• serum-free (or low serum)
Major functions of serum- basic nutrients- hormone and growth factors- binding proteins carrying hormone,
vitamins, minerals, lipids, etc- non-specific protective functions- protease inhibitors- pH buffer
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Environment considerations- nutrient supply
- mixing
- oxygen supply
- pH- carbon dioxide- NaHCO or NaOH3
- temperature- waste accumulation
- lactate- ammonia
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Other considerations- inoculum
- growth phase (late exponential phase)- density (varies, as a guide ~5x104 to
2x105 cells/ml)
- mixing- shear
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Kolmogorov length scale (microns)
Relative net growth rate versus Kolmogorov eddy length scale for FS-4 cultures with 0.2 g/l microcarriers
Rel
ativ
e sp
ecif
ic g
row
th r
ate
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Nucleic acid synthesis
glutamine
glutamateglycine
alanine asparatate
TCA cycle
citrate malate
oxaloacetate
phosphoenolpyruvateglycolysis
glucose
pyruvate
lactate
-ketoglutarate
Schematic representation of some of the interrelationships of glucose an glutamine metabolism in mammalian cells
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Oxygen supply(a challenging problem since oxygen is sparsely soluble in water)
OTR = kla (C*-C)
OTR: oxygen transfer rate
kla: mass transfer coefficient
C*: saturated dissolved oxygen concentration
C: dissolved oxygen concentration in themedium
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Methods for O2 supply- direct sparging
- cell damage- pluronic F-68 supplement
- surface aeration- limited surface area
- silicon tubing supplement- to increase surface area
- perfusion
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Examples of performance of various aeration methods
Methods of oxygenating a 40 liter Bioreactor (30 liter working volume with a 1.5: 1 aspect ratio)
Oxygenating method Oxygen delivery(mg/l/h)
No. cells x106/mlsupported
AIR (10 ml/l/min at 40 r .p.m.)Surface aeration 0.5 0.08Direct sparging 4.6 0.76Spin filter sparging 3.0 0.40Perfusion (1 vol/h) 12.6 2.10
Perfusion (1 vol/h) + Spin filter sparging
15.9 2.65
OXYGEN (10 ml/min at 80 r .p.m.)Spring filter sparging 51.0 8.50+ Perfusion (1 vol/h) 92.0 15.00
(assuming oxygen utilization rate of 2-6 g/1 06 cells/h)
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Cultivation methods for anchorage dependent cells
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Commercially available spinner cultures. (A) LH Fermentation Biocul (1-20L);(B) Bellco and Wheaton Spinner Flasks (25 ml-2 liters); (C) Bellco and Cellon uspinner (25 ml-2 liters); (E) Techne (25 ml-5 liters); (E) Techne Cytostat (1 liter);(F) Techne BR-06 Bioreactor (3 liters).
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Hollow fiber reactors- consists of ultrafiltration capillary fibers
- porous to macromolecules
- thin wall- provide large surface area
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oxygenator
wastefreshm ed iu m
A ir(oxyg en )
c e ll c u ltu re
Flow diagram of a typical hollow fiber reactor
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Hollow fiber culture reactor and a diagrammatic representation of the pressuredrop/nutrient gradient along the length of the cartridge. I, lumen of fibers;e, extracapillary space; h harvesting port; p, medium perfusion path
p p
hI e
h
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ri
ro
rc
[O2]
[O2]c
ri ro rc
fibre
[O2] – oxygen conc
[O2]c – critical oxygen conc
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FiberCell Systems, Inc.
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Cells grow on and around hollow fibers.♦ Fiber geometry is
optimized for both adherent and suspension cell types.
♦ Small molecules such as lactate, and glucose can easily cross the fiber.
♦ Large molecules such as mono clonal antibodies and proteins are retained and concentrated in the small volume of the extra capillary space.
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MicrocarriersMajor Advantages:
- possess high surface-to-volume ratio (as high as 2x107 cell/ml are achieved)- microcarriers can be settled easily- facilitate cell and product harvesting- cell propagation can be carried out in high
productivity reactors- enable control and monitoring of reactor
environment- possible to take representative sample for
monitoring purposes
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Desired properties- functional attachment group
- buoyant density of the bead- for mixing consideration ( ~ 1.03 to 1.10 g/l)
- size of the bead (100-200 m)
- size distribution
- smooth surface (allow cell spreading)
- transparency ( microscopic observation)
- toxicity
- rigidity
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Source: GE Healthcare – Microcarrier Cell Culture: Principles and Methods
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A sample listing of commercially available microcarriersTrade Name Manufacturer Material SG Diam (m) Area
(cm2/g)
Acrobead Galil Polyacrolein 1.04 150 5000Biosilon Nunc Polystyrene. 1.05 160-300 255Bioglas Solohill Eng. Glass. 1.03 150-210. 350Bioplas Solohill Eng. Polystyrene. 1.04 150-210 350(Biospheres Collagen. 1.02 150-210 350Biocarrier Biorad Polyacrylamide 1.04 120-180 5000Cellfast QDM lab. Silica/Chitosan 10000Cytodex 1 Pharmacia DEAE Sephadex 1.03 160-230 6000Cytodex 2 Pharmacia DEAE Sephadex 1.04 115-200 5500Cytodex 3 Pharmacia Collagen 1.04 130-210 4600Cytosphere lux Polystyrene 1.04 160-230 250Dormacell Pfeifer & Langen Dextran 1.05 140-240 7000OE-53 Whatman Cellulose 1.03 Fibres 4000Gelibead Hazelton lab. Gelatin 1.04 115-235 3800Mica Muller-Ueheim Polyacy(amide 1.04 350Micarcel G Reactifs IBF Polyacrylamide' 1.03 5000
Collagen/glucoglycanMicrodex Oextran Prod. DEAE Dextran 1.03 150 250Superbeads Flow lab. DEAE Sephadex 1.03 150-200 6000Ventreglas Ventrex Glass 1.03 90-210 300Ventregel Ventrex Gelatin 1.03 150-250 4300
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Typical cell growth on microcarriers
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Typical cell growth on microcarriers
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FibraCel® DisksA Solid Support Growth Material for Mammalian, Animal & Insect Cells
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Hybridoma Anchorage-Dependent Insect
DA4.4123A127A
GAMMA67-9-B
3T3, COS, Human OsteosarcomaMRC-5, BHK, VERO
CHO, rCHO-tPArCHO – Hep B Surface Antigen
HEK 293, rHEK 293rC127 – Hep B Surface Antigen
Normal Human FibroblastsStroma
Hepatocytes
Tn-368SF9rSF9Hi-5
FibraCel® Disks
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FibraCel® Disks
Yes Autoclavable
Yes Cytotoxicity tested
Yes Bioburden tested
Yes Endotoxin tested
3 x 105 cells/mL final volume Required inoculum
6 mmDisk diameter
1200 cm2Surface Area per gram
Specifications
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Perfusion system- to provide fresh nutrient- to remove waste (especially toxic byproducts - mechanical signal
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Fig. 1 Schematic diagram of the perfusion–bleeding culture system. The settler consists of a cylinder part and a cone part. Dimensions of the settler: height of the cylinder, 5.5 cm; height of the cone, 5.5 cm; internal diameter (i.d.) of the cylinder, 5 cm; i.d. of pipes number 1 and number 3, 3 mm; i.d. of pipe number 2, 5 mm. Pipe number 1 is connected to the settler in the middle part of the cylinder
(Z.-Y. Wen and F. Chen, Applied Microbiology and Biotechnology, 57: 316 – 322, 2001)
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S. Zhang, A. Handa-Corrigan,and R.E. Spier, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 41, NO. 7, MARCH 25, 1993
Figure 1. Schematic diagram of the perfusion culture system.
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Large 3-D Cellular Aggregates
Hydrodynamic Focusing Bioreactor
BHK-21 Cell Culture Forms 2,000 m 3-D Cellular Aggregates within Two Days
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Questions?
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Transport in a Grooved Perfusion Flat-Bed Bioreactor for Cell Therapy Applications
Marc Horner, William M. Miller, J. M. Ottino, and E. Terry Papoutsakis
Biotechnol Prog 1998 Sep-Oct;14(5):689-98
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Figure 1. Model of the perfusion chamber, a flat-bed bioreactor in which a series of 190 grooves at the chamber bottom (shown in figure) retains cells in the presence of constant medium perfusion. This is a closed system, with no headspace when the lid is placed on top. Medium flows in the z-direction across the chamber. yand zrepresent the local coordinate system in a cavity.
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A Microfabricated Array Bioreactor for Perfusion 3-D Liver Culture
Mark J. Powers et. al
Bioengineering & Biotechnology, 2002, 78:257-69
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Examples
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Cultivation of Cell-PolymerCartilage Implants in Bioreactors
LE. Freed, G. Vunjak-Novakovic, and R. Langer
J ournal of Cellular Biochemistry 51 :257-264 (1993)
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Cell-polymer implants
Isolated chondrocytes
Cartilagebiopsy
In vitro tissue culture
Polymer scaffold
Petri dish Bioreactor
In vivo implantation
Implant
Proposed Therapy
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Fig 3. Effects of scaffold thickness and implant cultivation time on cell growth rate
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6
2
4D
oub
ling
tim
e (d
ays)
0.088 0.116 0.168 0.307 0.384
Fig. 4 Effect of scaffold thickness on cell doubling time
Scaffold thickness
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TABLE II. Chondrocyte Growth on Microcarriers in Bioreactors
Cell density Doubling time (cells/cm3 reactor volume) (days)
Group Bioreactor 2 days 8 days 2 days 8 daysA Magnetically stirred
flask (75 rpm)1.30 x. 105 1.58 x 106 1.67 1.67
B Shaking flask(140 rpm)
1.49 x 104 1.54 x 105 1.78 1.78
C Unmixed test tubes 1.98 x 105 2.96 x 105 4.91
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Hi Me Lo Hi Me LoCell Density
Petri dish Bioreactor
Dou
blin
g ti
me
(day
s)
6
2
4
Fig. 6 Effects of Cell density on cell doubling time
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Gas Exchange is Essential for Bioreactor Cultivation of Tissue Engineered Cartilage
Bojana Obradovic, Rebecca L. Carrier, Gordana Vunjak-Novakovic, Lisa E. Freed
Biotechnology and Bioengineering, 63: 197–205, 1999.
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Figure 1. Model system. Isolated primary chondrocytes are seeded onto fibrous, biodegradable PGA scaffolds and cultured in vitro for 5 weeks in rotating bioreactors under different conditions of gas and medium exchange.
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Group 1 (control) — regular medium replacement (50%v/v, 3 times per week), continuous gas exchange
Group 2 (infrequent gassing) — regular medium replacement(50% v/v, 3 times a week), periodic gas exchange (3times per week for 5 h, after medium replacement)
Group 3 (no gassing) — regular medium replacement(50% v/v, 3 times per week), no gas exchange
Group 4 (infrequent feeding) — Infrequent medium replacement(50% v/v, once per week), continuous gas exchange
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Table II. Biochemical compositions of cell–polymer constructs.
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Table III. Cell metabolism in cell–polymer constructs.
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Comparison of Chondrogensis in Static and Perfused Bioreactor Culture
David Pazzano,† Kathi A. Mercier,†,| John M. Moran,†,‡ Stephen S. Fong,†,‡ David D. DiBiasio,‡ Jill X. Rulfs,§ Sean S. Kohles,| and Lawrence J. Bonassar*,†
Biotechnol Prog. 16(5):893-6 (2000)
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Figure 1. Schematic representation of the perfusion bioreactor system assembly.
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Figure 3. (A) Static sample at 2 weeks stained with safranin-O/fast green revealed light staining and no discernible orientation (400, bar ) 10 Ìm). (B) Bioreactor sample at 2 weeks stained with safranin-O/fast green (400, bar ) 10 Ìm). Intense staining was observed, as well as alignment of cells in the direction of media flow.
A
B
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Cardiac Tissue Engineering: Cell Seeding, Cultivation Parameters, and Tissue Construct Characterization
Rebecca L. Carrier, Maria Papadaki, Maria Rupnick, Frederick J. Schoen, Nenad Bursac,5 Robert Langer, Lisa E. Freed, Gordana Vunjak-NovakovicBiotechnol Bioeng. 64(5):580-9 (1999)
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Figure 1. Effect of seeding vessel on the cellularity and metabolic activity of 3- day constructs. (a) DNA content (mg/construct) (*) significantly greater than mixed flask group, p < 0.05 (n44). (b) Medium LDH content (total U over 3 days of seeding) (*) significantly greater than all other groups, p < 0.05 (n 4 4). (c) Tetrazolium conversion (MTT assay OD units/mg DNA) (*) significantly greater than all other groups, p < 0.05 (n 4 4).
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Figure 4. Cardiac-specific features: Constructs cultured for 1 week in a HARV (a, c, d) or a flask mixed at 50 rpm (b) and immunohistochemicallylabeled for (a) muscle desmin, (b) cardiac myosin, (c) cardiac troponin-T, and (d) sarcomeric tropomyosin. The arrow denotes a polymer fiber. (e)Transmission electron photomicrograph from a cardiac construct cultured for 1 week in a HARV demonstrating several adjacent cardiac myocytes with intercellular desmosome-like junctions (small arrows), myofibrils with sarcomeric organization highlighted by z lines (broad arrow), and compact mitochondria (open arrow). The nucleus of one cell is designated by the asterisk. Scale bars are 25 mm in a–d and 2 mm in e (original magnification 12,000).
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Questions?
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Extras
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Typical oxygen consumption rate
assume1. oxygen utilization rate = 6 g/1 06 cells/h2. oxygen satuaration = 1.09 mmol/l3. cell density = 1 07 cells
oxygen will be consumed in ~0.5 h
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“Protection” Property of Pluronic F-68
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D O , Te m p. and pH C o ntro l le r
D ata Ac quis i t io n
CO
2
O2
N2
G ro wthc ham be r
F ie ld c o ils to ge ne ra te m a gne tic f ie ld
T e mp e ra tu re P ro b e
p H P ro b e
D O Pro b eF low m e te r
M ic roc om pute r
S ole noid V a lve
E nviro nm e nt c o ntro l c ham be r
G as c yl inde rs
F luid f lo w c o ntro l lo o p
D is t r ib u t o r
Schematic of a magnetically stabilized bioreactor system
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Photograph of BHK-21 cells om CMSM-GG microcarriers (200X)
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Photograph of hepa-1,6 cells on magnetite microcarriers cultured in a MSFB bioreactor (400X)
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Photograph of hepa-1,6 cells on magnetite microcarriers cultured in a MSFB bioreactor (400X)
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