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

CellMax® artificial capillary cell culture system

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)