Post on 15-Jan-2016
Bioreactor design Issues for cell cultures
Cell Culture
- An engineering perspective
by Genentech, Corporate Communication
A Fermenter / Bioreactor And Its Parts
Single System for Anchorage-Dependent and Suspension CulturesNew Brunswick Scientific Company
BioFlo® Pro Customizable Cell Culture Bioreactors
Fig. 2. Influenza production plant (6000 liter vessel for cultivating Vero cells on Cytodex™). Courtesy of Baxter Biosciences.
• 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
Type of cultures
• Suspension cultures
• Anchorage dependent cultures
• monolayer
Bioreactor: Advantages
Controlled environment:1. Mixing2. pH3. Dissolved oxygen4. Temperature
pH probe
1. Steam sterilizable2. Combination electrode
1. Two major typesa. Galvanic b. Polargraphic
Dissolved oxygen probe
Galvanic and Polargraphic Probes
Cathode 0.5 O2 + H2O + 2e- 2OH-
Pt
Anode (galvanic) Pb Pb2+ + 2e-
Anode (polargraphic) Ag + Cl- AgCl + e-
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
Environment considerations- nutrient supply
- mixing
- oxygen supply
- pH- carbon dioxide- NaHCO or NaOH3
- temperature- waste accumulation
- lactate- ammonia
Other considerations- inoculum
- growth phase (late exponential phase)- density (varies, as a guide ~5x104 to
2x105 cells/ml)
- mixing- shear
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
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
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
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
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)
Cultivation methods for anchorage dependent cells
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).
Hollow fiber reactors- consists of ultrafiltration capillary fibers
- porous to macromolecules
- thin wall- provide large surface area
oxygenator
wastefreshm ed iu m
A ir(oxyg en )
c e ll c u ltu re
Flow diagram of a typical hollow fiber reactor
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
ri
ro
rc
[O2]
[O2]c
ri ro rc
fibre
[O2] – oxygen conc
[O2]c – critical oxygen conc
FiberCell Systems, Inc.
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.
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
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
Source: GE Healthcare – Microcarrier Cell Culture: Principles and Methods
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
Typical cell growth on microcarriers
Typical cell growth on microcarriers
FibraCel® DisksA Solid Support Growth Material for Mammalian, Animal & Insect Cells
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
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
Perfusion system- to provide fresh nutrient- to remove waste (especially toxic byproducts - mechanical signal
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)
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.
Large 3-D Cellular Aggregates
Hydrodynamic Focusing Bioreactor
BHK-21 Cell Culture Forms 2,000 m 3-D Cellular Aggregates within Two Days
Questions?
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
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.
A Microfabricated Array Bioreactor for Perfusion 3-D Liver Culture
Mark J. Powers et. al
Bioengineering & Biotechnology, 2002, 78:257-69
Examples
Cultivation of Cell-PolymerCartilage Implants in Bioreactors
LE. Freed, G. Vunjak-Novakovic, and R. Langer
J ournal of Cellular Biochemistry 51 :257-264 (1993)
Cell-polymer implants
Isolated chondrocytes
Cartilagebiopsy
In vitro tissue culture
Polymer scaffold
Petri dish Bioreactor
In vivo implantation
Implant
Proposed Therapy
Fig 3. Effects of scaffold thickness and implant cultivation time on cell growth rate
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
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
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
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.
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.
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
Table II. Biochemical compositions of cell–polymer constructs.
Table III. Cell metabolism in cell–polymer constructs.
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)
Figure 1. Schematic representation of the perfusion bioreactor system assembly.
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
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)
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).
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).
Questions?
Extras
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
“Protection” Property of Pluronic F-68
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
Photograph of BHK-21 cells om CMSM-GG microcarriers (200X)
Photograph of hepa-1,6 cells on magnetite microcarriers cultured in a MSFB bioreactor (400X)
Photograph of hepa-1,6 cells on magnetite microcarriers cultured in a MSFB bioreactor (400X)