Freezing extracellular

Chitosan freeze-dried fleeces support chondrocyte attachment and synthesis of extracellular matrix [344]. Chitosan was used to assist the spontaneous tissue repair of the meniscus [345]. The repair of the cartilage and the prevention of its degradation in osteoarthritis is, however, possible with the association of glucosamine sulfate salt and chondroitine sulfate, the latter being particularly effective [346]. 

Figure 2. Computed kinetics of water loss from mouse ova cooled at 1 °C to 32 °C/min in 1M DMSO. The curve labeled EQ shows the water content that ova have to maintain to remain in equilibrium with extracellular ice. If ova or embryos contain more than equilibrium amounts of water when they cool to below -30 °C, they will undergo intracellular freezing. Usually such freezing is lethal, but if the quantity of ice is small, some internally frozen cells can be rescued by rapid warming. (From Mazur, 1990.)...

Fujikawa, S. Miura, K. (1986). Plasma membrane ultrastructural changes caused by mechanical stress in the formation of extracellular ice as a primary cause of slow freezing injury in fhiit-bodies of basidiomycetes (Lyophyllum ulmarium [Fr.J KOhner). Cryobiol. 23,371-382. 

Enbrel is a product now approved for medical use that is based upon this strategy. The product is an engineered hybrid protein consisting of the extracellular domain of the TNF p75 receptor fused directly to the Fc (constant) region of human IgG (see Box 13.2 for a discussion of antibody structure) The product is expressed in a CHO cell line from which it is excreted as a dimeric soluble protein of approximately 150 kDa. After purification and excipient addition (mannitol, sucrose and trometamol), the product is freeze-dried. It is indicated for the treatment of rheumatoid arthritis and is usually administered as a twice-weekly s.c. injection of 25 mg product reconstituted in WFI. Enbrel functions as a competitive inhibitor of TNF, a major pro-inflammatory cytokine. Binding of TNF to Enbrel prevents it from binding to its true cell surface receptors. The antibody Fc component of the hybrid protein confers an extended serum half-life on the product, increasing it by fivefold relative to the soluble TNF receptor portion alone. 

Streptokinase is a 48 kDa extracellular bacterial protein produced by several strains of Streptococcus haemolyticus group C. Its ability to induce lysis of blood clots was first demonstrated in 1933. Early therapeutic preparations administered to patients often caused immunological and other complications, usually prompted by impurities present in these products. Chromatographic purification (particularly using gel filtration and ion-exchange columns) overcame many of these initial difficulties. Modern chromatographically pure streptokinase preparations are usually supplied in freeze-dried form. These preparations (still obtained by non-recombinant means) often contain albumin as an excipient. The albumin prevents flocculation of the active ingredient upon its reconstitution. 

Although fusion between SFV and the plasma membrane normally does not occur, cell surface-bound viruses can be induced to fuse simply by decreasing the extracellular pH below 6 for a few seconds (White et al., 1980 Vaananen et al., 1981). As a result of its fusion activity SFV can hemolyze red blood cells at pH 5.8 (VaanSnen and Kaari inen, 1979, 1980). However, the lysis occurs only with virus damaged by freezing and thawing. Cells can also be made to fuse with each other using SFV at low pH (White et al., 1981). 

Practically all available iodinated extracellular X-ray contrast agents have been encapsulated into liposomes using different lipids and methods of preparation. Table 1 gives a short and intentionally incomplete overview of some of the approaches. The first liposomal contrast agent preparation that was tested in humans contained diatrizoate [48]. The injected dose was up to 0.5 ml kg k The preparation was effective even in plain radiography where lesions down to 0.8-1.0 cm could be detected in patients. However, adverse events such as fever and hyperthermia, which occurred in 30% of the patients, limited further use. We have incorporated iopromide into MLVs that were prepared from phosphatidyl choline (PC), cholesterol and stearic acid at a molar ratio of 4 5 1 using the ethanol-evaporation technique [44]. The liposomes can be stored freeze-dried and they are reconstituted before use by... 

Figure 8-15 Freeze-fractured membranes of two erythrocyte "ghosts." The upper fracture face (PF) shows the interior of the membrane "half" closest to the cytoplasm. The smooth region is lipid and contains numerous particles. The lower face, the extracellular half (EF), possesses fewer particles.

Standard chemical fixation fails to preserve extracellular materials. In contrast, Os04-microwave heating or the high-pressure freezing-freeze-substitution technique is able to preserve such materials (Eggli and Graber, 1994). The former technique is simpler... 

The culture medium containing the bioproduct should be processed as soon as possible, to avoid the possibility of degradation, for example, by oxidation or the action of proteases. When immediate processing of extracellular products is not possible, it is recommended to remove the cells and freeze the material, provided the bioproduct is stable to freezing. 

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