tissue engineering. animal cells 10-30 μm diameter spherical, ellipsoidal no cell wall fragile...
TRANSCRIPT
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