Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1995-01-17
1997-09-09
Gitomer, Ralph J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
422 71, 436804, C12Q 116, G01N 2306
Patent
active
056655622
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the study of cellular and biochemical processes in living cells or in components of cells. Specifically described are devices and methods for the study of cellular and biochemical processes, utilising the Scintillation Proximity principle.
BACKGROUND TO THE INVENTION
1. Studies of Cellular Processes in Living Cells
Mammalian cell culture is an essential tool for fundamental research in eukaryotic biology and it has contributed to advances in virology, somatic cell genetics, endocrinology, carcinogenesis, toxicology, pharmacology, immunology and developmental biology (McKeehah, W. I., In Vitro Cell Dev. Biol., 26, 9-23, (1990)). Classical cell culture technology is carried out in nutrient mixtures with cells usually cultured as a monolayer attached to a hydrophilic surface, commonly sterile treated polystyrene. Considerable progress has been made in developing cell culture systems for specific cell types, with the aim of reconstructing the cell and its environment into a defined unit for the study of responses and properties of cells in a dynamic context. However, experimentation on such culture systems using biological assays is often limited by the need to use invasive or disruptive processes that compromise the structural and functional integrity of the cells.
Certain types of investigations lend themselves particularly to studies with whole cells and inevitably require cell culture techniques as an essential step in the investigation. General areas of study include: p1 (i) intracellular activity, including the replication and transcription of nucleic acids, protein synthesis and lipid metabolism, cytoplasm, translocation of human receptor complexes, fluctuations in lipid and protein metabolic pools, transport of ions and other small molecules across membranes, chemically induced transformation, drug action and metabolism, response to external stimuli and secretion of specialised products, and kinetics, cell-cell adhesion and motility.
A vast array of radiolabelled ligands which are available commercially, has played a major role in the development of methods currently used to study intracellular activity, metabolism and cell-ligand interactions in cell culture assay systems. Particular examples relating to the study of cellular processes are:
Studies involving the measurement of [.sup.3 H] thymidine uptake currently suffer from an absolute requirement for cell disruption and consequently are prone to artifactual effects (Adams, R. L. P., Cell Culture for Biochemists, p181-192; Saegusa, Y. et al, J.Cell Physiol., 142, 488-495 (1990)). In addition to providing an assessment of cellular proliferation and growth in living cells, thymidine uptake studies are also used to study the extent of DNA repair and/or damage occurring during culture, in the presence or absence of external agents (McKeehan, W. et al, In Vitro Cell Dev. Biol., 26, 9-23, (1990)). Current methods however, require cell disruption and do not readily lend themselves to temporal studies. Thymidine uptake has been used more recently, in tandem with other potential markers, in the field of programmed cell death, or apoptosis, where there is currently considerable pharmacological and clinical interest (Tritton, T. and Hickman, J., Cancer Cells Quarterly Rev., 2, 95-105, (1990)). However, few of the current methods are able to explore and quantify spatial and temporal events occurring during apoptosis (Lock, R. B. and Ross, W. E., Proc. Amer. Assoc. Cancer Res., 30, 621, (1989)). [.sup.3 H] Thymidine uptake studies are also used in cell cycle studies in order to monitor regulation of this essential process (Studzinski, G. P.,Cell Tissue Kinetics, 22, 405-424, (1989)). However, there are currently no methods available for the direct measurement of thymidine uptake in living cells.
Most of the methods used in this field require binding of a radiolabelled ligand, followed by quantification of receptor number and affinity in competition studies at a fixed time (Goldstein, J. L. and Brown,
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Amersham International plc
Gitomer Ralph J.
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