Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.

Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.

A methodology for analysis of sugarcane productivity trends - 2. Comparing variety trials with commercial productivity

Previous research has reported both agreements and serious anomalies in relationships between production attributes of sugarcane varieties in variety trials (VTs) and commercial production (CP). This paper examines VT and CP data for tonnes of cane per hectare (TCH) and sugar content (CCS). Data, analysed by REML, included 107 VTs and 54 CP mill years for 9 varieties from the mill districts of Mulgrave, Babinda, and Tully for harvest years 1982-99. Important consistencies included high TCH of Q152, high CCS of Q117 and Q120, and low CCS of H56-752. Significant anomalies existed with respect to TCH for Q113, Q117, Q120, Q122, Q138, and H56-752 and to CCS for Q113 and Q124. Investigation of these anomalies was assisted by access to independent REML analyses of CP data for 65692 individual Tully cane blocks from 1988 to 1999 and by the knowledge of persons familiar with the preferential uses of varieties by farmers. Minor anomalies were due to limited year or mill area data. Q124 TCH was deemed to be decreased and its CCS increased by severe disease in Babinda CP in the extremely wet 1998 and 1999 seasons. Other serious anomalies have credible but unsubstantiated explanations. The most convincing, for Q113, Q117, Q138, and H56-752, are that these varieties were deployed unevenly with regard to late season harvesting, predominant use or avoidance on high fertility soils, or use confined to low fertility sandy soils, respectively. Uneven deployment results in confounding of these effects in the varietal CP statistics at mill area level. It is concluded that VTs cannot be enhanced to anticipate or evaluate most effects of uneven deployment. They give adequate predictions of relative CP performance for varieties deployed evenly across confounding influences. Routine analyses of individual block CP data would be useful and enhanced by addition of relevant information to the block records.