Advanced Granulation Technology (AGT(TM)). An alternate format for serum-free, chemically-defined and protein-free cell culture media.

To overcome limitations of conventional milling technology, we investigated the application of fluid bed granulation for the production of dry-form nutrient media. Serum-free, protein-free and chemically-defined specialty media were produced in granulated format and compared with identical formulations manufactured by conventional methods. HPLC analysis of multiple lots of granulated materials demonstrated that biochemical constituents were precisely and homogeneously distributed throughout the granules and that nutrient levels were comparable to conventional formats. Comparison of medium performance in cell proliferation and biological production assays demonstrated equivalence with reference media. The fluid bed granulation process meets pharmaceutical quality requirements and may be applied to a broad range of nutrient formulations required for bioproduction applications.

Simultaneous determination of deflazacort metabolites II and III, cortisol, cortisone, prednisolone and prednisone in human serum by reversed-phase high-performance liquid chromatography.

A new high-performance liquid chromatographic method for the analysis of human serum from subjects administered prednisolone or deflazacort has been established. Two deflazacort metabolites (metabolites II and III), prednisolone and its metabolite (prednisone), as well as cortisol and cortisone in human serum were analyzed simultaneously. These six compounds and the internal standard, betamethasone, were extracted from 1 ml of human serum using a Sep-Pak Plus Environmental C18 column and then separated on a Hypersil ODS (25 x 0.46 cm I.D.) column using a gradient system. The lower limit of quantitation for each compound was 10 ng/ml based on signal-to-noise ratio of 5 and calibration curve linearity. Within-day and between-day validations gave a coefficient of variation of less than 10% and a relative error of ca. 10% (n = 11).

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts.

Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity if the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing.

Simplified cell microelectrophoretic method applied to the macrophage electrophoretic mobility test for cancer diagnosis.

Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.

Zeta-protentials of intact cell monolayers determined by electro-osmosis.

By measuring the electro-osmotic flow velocity of buffer through a glass capillary, on the inner surface of which cells have been grown, the zota-protential of these cells can be determined without the changes in zota-potential, viability and morphology caused by the chemical, enzymatic or mechanical pretreatment hitherto necessary for cell dispersion in the microelectrophoretic method. Cells grown in a monolayer inside the capillaries were: BGM, HEP-2, and RPMI 1846 (the last one also could be grown in suspension).