Tissue Engineering

Animal cells

• 10-30 μm diameter

• spherical, ellipsoidal

• no cell wall

• fragile plasma membrane

• shear sensitive

• generally negatively charged

• hybridomas are nonanchorage dependent

Guts

• endoplasmic reticulum (protein synthesis)

• mitochondria (respiration)

• lysosomes (digestion)

• Golgi body (secretion)

• nucleus (chromosomal DNA)

• cytoskeleton (strength, shape, response)

Typical medium

• glucose (C source)• glucosamine (C source – ammonia and

glutamate... other amino acids)• amino acids• horse or calf, fetal bovine serum (5-20%)• growth factors, vitamins• mineral salts, buffer• Dulbecco’s modified Eagle’s medium (DME)

Cultivation method

• tissues excised aseptically from lung, kidney (~2 mm3)

• agitated in trypsin (~0.25%), buffered saline for 2h at 37oC

• cell suspension filtered and centrifuged to wash cells

• primary culture formed in T-flasks or roller bottles

• medium contains serum, antibiotics• normally form monolayers on surface

(anchorage dependent)• trypsin (protease) separates tissue into

single cell culture (suspension culture)

Cultivation

• secondary culture established from primary culture

• cells removed from flask surface using EDTA, trypsin, collagenase or pronase

• 5-30 min at 37oC• serum added and suspension centrifuged,

washed with buffered saline• many secondary cultures are suspension

culture (nonanchorage dependent)

Mortal versus immortal

• most differentiated mammalian cell lines are mortal

• divide for only limited number of generations (eg. 30 generations)

• human fibroblasts (WI-38 or MRC-5)• immortal cells are “continuous” or

“transformed”• cancer cells are immortal/transformed

naturally

• reluctant to approve products from transformed (cancer) cells

• often become attachment independent

• cultured indefinitely in suspension culture

• early 90’s, started approval of therapeutic proteins, tissue plasminogen activator from immortalized cells

Mortal (normal) Transformed

• anchorage dependent (except blood cells)

• finite number of divisions• monolayer culture• dependent on growth

factor signals for growth• better retention of

differentiated cell function• typical cell surface

receptors

• nonanchorage dependent (suspension culture)

• immortal• multilayer cultures• growth factors may not be

needed• loss of differentiation

• cell surface receptors may be altered

Other considerations

• insect cells are naturally continuous

• senescence observed in many fish cell lines

Serum based media

• serum costs $100-$500/L• complicates purification (serum proteins)• filter sterilized• potential for virus, mycoplasma

contamination common• potential contamination by prions• prone to foaming• inherent variability and instability

Serum-free alternative

• reduced cost

• simplifies product purification

• improved reproducibility

• reduced contamination

• not all cell lines have adapted

Examples

• Eagle’s minimal essential medium (MEM-FBS)

• MCDB 170MDS (serum free – see Sigma catalogue)

Mammalian cell growth

• pH ~ 7.3, T = 37OC

• td ~ 10 - 50h (20h typical)

• 5% CO2 enriched air (buffers pH)

• HCO32-/H2CO3

- controls pH at 7.3

• HEPES (N-[2-hydroxyethel]piperazine-N’-[2-ethanesulfonic acid]) buffer

CHO cells

Other cell lines

• insect cells grow at 28oC and pH 6.2

• fish cells at 25 to 35oC, pH 7 – 7.5

Kinetics

• short lag

• rapid drop in viable cells

• peak 3 – 5 days

• MAb production continues

• most products mixed growth associated

from Reuveay et al., J. Immunol. Meth. 86: 53-59, 1986)

MAb kinetics

• Luedeking-Piret eqn. pqdt

dp

X

1

O2 requirements

• 0.06 to 0.2 x 10-12 mol O2/h/cell• 5 times less than plant cells and much lower

than for microbial cells• suspension culture: 0.1-1 g/L (5 x 105 – 5 x 106

cells/mL• 10 times higher for immobilized cells• kLa values necessary 5 - 25 h-1 (106 cells/mL)• cells are shear sensitive

Bioreactor design considerations

• large cells

• slow growing

• shear sensitive

• anchorage dependent or suspension culture

• product titer low (μg/mL)

• ammonium, lactate toxic metabolites

Design approach

• gently agitated and aerated

• T, pH, O2, redox homogeneous

• CO2 enriched air

• microcarriers (large surface)

• toxic product removal

Lab scale

• T-flasks (25-100 mL)

• spinner flasks (100 mL – 1L) with paddle-type magnetic agitators

• roller bottles (50 mL – 5L), 1-5 rpm

• shallow trays

• incubator, 5% CO2, 37oC

Industrial scale

• anchorage dependent cells– microcarriers– hollow fiber reactors– ceramic matrix– porous beads

• suspension culture– stirred reactors– airlift or bubble column

Perfusion reactors

• membrane reactor

• microencapsulation

• cells retained

• product and toxic metabolites removed

Roller bottles

• not practical for large scale (few exceptions)

• liquid covers 25% of bottle surface

• 1-5 rpm• 75% time, cells exposed to

5% CO2

• commercial erythropoietin and vaccine production

Microcarriers

• DEAE-Sephadex or DEAE-polyacrylamide

• high surface area (70,000 cm2/L)• high cell density (107 cells/mL)• also dextran, hollow glass• surface collagen coat promotes cell

adhesion• mono- or multilayer cell growth• macroporous carriers increase SA and

protect cells, but diffusion problems