Transcript 08.Tissue Engineering Animal Cells.web

Tissue Engineering
Animal cells
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10-30 μm diameter
spherical, ellipsoidal
no cell wall
fragile plasma membrane
shear sensitive
generally negatively charged
hybridomas are nonanchorage dependent
Guts
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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
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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
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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-[2hydroxyethel]piperazine-N’-[2ethanesulfonic 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
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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.
1 dp
 q p    
X dt
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
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large cells
slow growing
shear sensitive
anchorage dependent or suspension
culture
• product titer low (μg/mL)
• ammonium, lactate toxic metabolites
Design approach
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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 paddletype magnetic agitators
• roller bottles (50 mL – 5L), 1-5 rpm
• shallow trays
• incubator, 5% CO2, 37oC
Industrial scale
• anchorage dependent
cells
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microcarriers
hollow fiber reactors
ceramic matrix
porous beads
• suspension culture
– stirred reactors
– airlift or bubble column
Perfusion reactors
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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 DEAEpolyacrylamide
• 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