Stirred cultures

The conventional culture media can be used (DMEM, IMDM, etc.) but additional components can be added to increase the success of the culture if considered necessary. Pluronic F-68 (polyglycol) (BASF, Wyandot) can be added at 0.1% to protect the cells against mechanical damage, especially at reduced serum concentrations. Carboxymethyl cellulose (CMC) (15-20 cP) can also be used at 0.1% for this purpose. Antifoam (6 p.p.m.) (Dow Chemical Co.) is recommended if foaming occurs which usually happens if the serum concentration is above 5%. The use of Hepes buffer can also be considered to stabilize pH during set up and if gassing facilities are not available. 

Add 2-4 X 10 cells (1-2 X 10 /ml) harvested from a growing culture of cells (i.e. not a stationary or dying culture). NB this is a higher inoculum than used for stationary cultures. 

Place spinner vessel on magnetic stirrer at 37 °C and set at 100-200 r.p.m. (this is variable depending upon the vessel size, geometry, fluid volume, and individual cell. Set a speed which visually shows complete homogeneous mixing of the cells throughout the medium—150 r.p.m. is usually a safe choice). 

Monitor the growth daily by removing a small sample through the side-arm in a Class II cabinet and carrying out a cell count and visual inspection for ceU morphology and lack of microbial contamination. 

Monitor the pH (colour change), especially in closed (non gassed) systems, and should the culture become acidic (under pH 6.8) add sodium bicarbonate (5.5% stock solution) or re-gas with 5% COj. After three days a better option would be to allow the cells to settle out, remove 40-70% of the medium, add fresh pre-warmed medium to the culture, and continue stirring. 

---

The SF-837 strain, namely Streptomyces mycarofaciens identified as ATCC No. 21454 was inoculated to 60 liters of a liquid culture medium containing 2.5% seccharified starch, 4% soluble vegetable protein, 0.3% potassium chloride and 0.3% calcium carbonate at pH 7.0, and then stir-cultured in a jar-fermenter at 28°C for 35 hours under aeration. The resulting culture was filtered directly and the filter cake comprising the mycelium cake was washed with dilute hydrochloric acid. 

On the other hand, Cruz et al. (2000) reported that 28 mM lactate reduced the growth of a BHK cell line by 50%, and that lactate was consumed at concentrations above 30 mM. Increased concentrations of lactate reduced cell growth and specific ammonia production but increased specific glutamine and glucose consumption. The effect of lactate was at least partially due to an increase of osmolarity. An increase in lactate from 0 to 60 mM induced a 40% reduction in specific productivity of a recombinant fusion protein secreted by the BHK cells in both stationary and stirred cultures. 

Metabolism of Gibberella fujikuroi in stirred culture, A. Borrow, E.G. Jefferys,... 

Mass production of inoculum for nematode control has not yet been upscaled industrially. To assure optimal longevity and infectivity of the conidia, the fungi are generally grown in solid-state surface cultures see under Pochonia chlamydosporia. For the production of Hirsutella rhossiliensis inoculum, stirred cultures in 5-L containers were used (Patel et al., 2001). 

Harvest yeast at OD x) = 0.6-0.7. Do not exceed ODstx) = 0.8. Pool and stir culture in an ice-water bath while collecting cells in the next step. Because light scattering estimates from spectrophotometric optical density measurements are not very precise, different spectrophotometers will give different estimates of cell density. In our laboratory an ODsoo of 0.6 = 5 x 10 cells/ml. 

There is a range of porous microcarriers available (Table 3), which allow one to design their own fluidized system, or alternative some of the carriers are designed for stirred cultures. 

Microcarrier culture Microcarriers are small particles, usually spheres 100 to 300 fim in diameter that are suspended in stirred culture medium. The technique was initiated in 1967 but required considerable developmental work to produce a range of suitable microcarriers (e.g., the Cytodex series by Pharmacia). The first industrial process based on microcarriers was for FMDV. Subsequently, a wide range of microcarriers based on gelatin, collagen, polystyrene, glass, cellulose, polyacrylamide, and silica have been manufactured to meet all situations. The key criteria in the design of effective microcarriers were to make the surface chemically and electrostatically correct... 

Bacilysin is a hydrophilic substance formed by certain aerobic sporeforming bacteria which causes lysis in cultures of growing staphylococci. Abraham and his collaborators have described its production by aerated cultures of a strain of Bacillus subtilis, and they showed that on hydrolysis (6m HCl) it gave L-alanine and L-tyrosine, although it did not contain a tyrosine residue. On the basis of physiochemical measurements the unusual dipeptide structure (96) was assigned to bacilysin. In later work a further substance (A A 1), identical with the C-terminal amino acid of bacilysin, was isolated from the culture fluid of Bacillus subtilis strains and the same amino acid has been obtained from stirred cultures of Streptomyces griseoplanus by Neuss and his collaborators and named anticapsin. Detailed analysis of the products of acid hydrolysis of anticapsin or A A 1 showed them to contain both L-tyrosine and m-L-tyrosine in a ratio of 9 2. 

Fernandes, A. M., Fernandes, T. G., Diogo, M. M. et al. 2007. Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system. J Biotechnol 132(2) 227-36. 

Russian workers have been successful in obtaining griseofulvin in stirred culture using P. nigricans (Semkina et al.y I963 L vova et al., 1964) but the yields are relatively minute. Methylene chloride is preferred to butyl acetate or acetone for extraction of the antibiotic from the culture medium (Leningrad Antibiotic Institute, 1964). 

Yang, S., H. Cai, H. Jin et al. 2008a. Hematopoietic reconstitution of CD34-I- cells grown in static and stirred culture systems in NOD/SCID mice. Biotechnol Lett 30(1) 61-5. 

Collins, P. C., W. M. Miller, and E.T. Papoutsakis. 1998. Stirred culture ofperipheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications. Biotechnol Bioeng 59(5) 534-43. 

Large-Scale Expansion of Human Pluripotent Stem Cells. 37-10 Stirred Culture Vessels Rotary Cell Culture Systems Microfluidic Culture Systems... 

Stirred culture vessels, including stirred-tank bioreactors and spinner flasks, are widely used in the expansion of mammalian cells for protein production. Not surprisingly, stirred culture systems have been adapted to pluripotent stem cell expansion and differentiation. The expansion of undifferentiated... 

---