Mannitol cell culture

Hyperosmolar medium is known to be advantageous for the production of proteins such as antibodies in animal cell cultures [70, 71]. Likewise, raising the osmolarity of MS medium using mannitol was found to improve the accumulation of foreign pro-... 

Rebif is produced via recombinant DNA technology in a CHO cell line. It displays an identical amino acid sequence to that of native human IFN-P-la and, like the native product, is glycosylated. After cell culture the interferon is purified using a series of chromatographic steps (affinity, ion-exchange, gel-filtration and reverse-phase liquid chromatography). It is formulated as a sterile solution in pre-filled syringes and contains mannitol, HSA, sodium acetate, acetic acid and sodium hydroxide as excipients. It is administered subcutaneously three times weekly. 

An alternative method for assessing cell layer integrity is through the use of hydrophilic paracellular transport markers (e.g., radiolabeled D-mannitol or fluorescein-Na+), which passively traverse cells by the paracellular route. Small amounts of compound required for in vitro conjunctival cell culture transport experiments make this approach well suited for screening purposes. Relative absorption index of a series of pharmacologically active molecules can be ranked against known markers for the identification of candidates with potential absorption problems, which is a reliable tool to select drug candidates with optimal characteristics. 

Composition of electroporation buffer is an important factor affecting electroporation yields. Ionic strength of cell suspension medium needs control, which determines resistance of the cell suspension and resultant RC time constant of the field pulse. Medium supplemented with Ca and Mg in mM concentration range is found to promote efficiency of transformation and cell viability. Erythrocytes electroporated in isotonic buffer in the presence of EDTA or membrane specific drugs showed significant modification in hemolysis response to electroporation [33,34]. Use of square wave pulse removes the medium conductivity mediated effects on cell/tissue electroporation outcome. Generally, cells are pulsed in suspensions of sucrose, mannitol, or sorbitol. Electroporation as well as incubation of pulsed cells can be carried out in medium containing usual cell culture recipes. 

Kainate-induced death of murine cerebellar neurones in culture was prevented by inhibiting the enzyme xanthine oxidase, a cellular source of cytotoxic superoxide radicals (Dykens et al. 1987). Moreover, neurones were also protected from exitotoxin-induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenged superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalled formation of xanthine oxidase. In rat cerebellar granule cell cultures melatonin was found to be beneficial in preventing kainate-induced excitotoxicity (Giusti et al. 1995). 

Figure 14 Observed permeability coefficients of urea and mannitol across monolayers of rat alveolar epithelial cells in primary culture in the Transwell system are correlated with transepithelial electrical resistance and days in culture.

Culture protocols have been published which describes an accelerated differentiation process where monolayers are ready to be used after 3-7 days of culture [90-92]. One of these systems, the so-called BD BioCoat Intestinal Epithelium Differentiation Environment, is commercially available through BD Bioscience. This system is described to produce monolayers of a quality that are comparable with the typical Caco-2 cells with respect to permeability for drugs transported transcellularly. The paracellular barrier function is however low, as indicated by high mannitol permeability and low TER. The functional capacity for active uptake and efflux is not as thoroughly characterized as for the standard Caco-2 mono-layers. 

Figure 9.1 Relationship between the transepithelial electrical resistance (TEER) value of the passage-cultured human nasal epithelial cell layer and permeability of 14C-mannitol (o, passage-2 A, passage-3 , passage-4) and budesonide ( , passage-2 , passage-3 , passage-4). (Data from Ref. [40]).

Table 9.1 Apparent permeability coefficients (Papp) of 14C-mannitol and budesonide across the passage-cultured human nasal epithelial cell layer grown under AIC or LCC conditions for 7 days.

Enhancement of metabolism of " C-toxaphene by several added cofactors was studied in 8 hrs aerobic cultures of washed Pseudomonas putida cells. The results of these experiments are summarized in Table 6. In the NAD group, NADH showed the greatest stimulatory effect on the production of total polar metabolites (1.2 times) over that of the 8 hrs incubate value. Similarly, FAD was responsible for a modest increase (1.1 times). The combination of NADH-FAD showed stimulation above that of either of the unpaired cofactors (2.0 times). Addition of mannitol to NADH-FAD stimulated this combination s total polar metabolite value from 2.0 to 2.3 times that of the 8 hrs incubate. In the sequence NADH to NADH-FAD to NADH-FAD-mannitol, the increase in the radioactivities in the ethyl acetate phase (moderately polar metabolites)... 

---