Tissue Engineering

Animal cells 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 mm 3 ) agitated in trypsin (~0.25%), buffered saline for 2h at 37 o C 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 37 o C 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 = 37 O C t d ~ h (20h typical) 5% CO 2 enriched air (buffers pH) HCO 3 2- /H 2 CO 3 - controls pH at 7.3 HEPES (N-[2- hydroxyethel]piperazine-N’-[2- ethanesulfonic acid]) buffer CHO cells

Other cell lines insect cells grow at 28 o C and pH 6.2 fish cells at 25 to 35 o C, 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.

O 2 requirements 0.06 to 0.2 x mol O 2 /h/cell 5 times less than plant cells and much lower than for microbial cells suspension culture: g/L (5 x 10 5 – 5 x 10 6 cells/mL 10 times higher for immobilized cells k L a values necessary h -1 (10 6 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, O 2, redox homogeneous CO 2 enriched air microcarriers (large surface) toxic product removal

Lab scale T-flasks ( mL) spinner flasks (100 mL – 1L) with paddle- type magnetic agitators roller bottles (50 mL – 5L), 1-5 rpm shallow trays incubator, 5% CO 2, 37 o C

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% CO 2 commercial erythropoietin and vaccine production

Microcarriers DEAE-Sephadex or DEAE- polyacrylamide high surface area (70,000 cm 2 /L) high cell density (10 7 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