Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Patent
1987-03-25
1990-05-22
Rosen, Sam
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
424529, 424534, 424561, 435243, 435261, A01N 102, C12N 102
Patent
active
049277502
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a novel composition for the separation and purification of biological cells and subcellular components. In particular, this invention relates to a novel composition for isolating specific cell types or specific subcellular components based upon their buoyant density. In still another aspect, this invention relates to a novel method and means for use in the isolation of specific cell types and specific subcellular components which provides improved purity of recovered cells and subcellular components.
BACKGROUND OF THE INVENTION
Separating components of biological fluids and tissues is often necessary for clinical diagnostic procedures, scientific research, and occasionally treatment of patients. In the clinical diagnostics field, for example, there is a need for compositions and methods which permit rapid isolation of purified lymphocytes for tissue typing procedures, immunologic function tests, and various other procedures. Basic research also requires purified lymphocytes as well as other cell types from blood. In addition, studies on cultured cells and subcellular components such as plasmids, DNA, chromosomes, mitochondria and other subcellular components also require highly purified preparations.
Separation and purification might be effected in several ways. However, since the isolated cells are often used in procedures which require viable cells, it is important that the functions of the cells so isolated be unimpaired. To insure viability of the cells and unimpaired biological function of cells and subcellular components, it is important to avoid introducing possible interfering substances in the course of the separation procedure. For example, isolated lymphocytes used in histocompatibility tests are stimulated with various mitogens. The medium used must not in itself be a mitogen since this will affect the validity of the measurements of DNA synthesis.
Density gradient centrifugation is one technique used for separation of biological cells and subcellular components. It is highly desirable that the material chosen for formation of the gradient have certain characteristics which will impart compatability with sensitive biological materials. Gradient materials which have been employed in the art include sucrose, dextran, bovine serum albumin (BSA), Ficoll (registered trademark of Pharmacia), iodinated low molecular weight compounds such as Metrizamide and heavy salts such as cesium chloride. Most of these materials, however, have undesirable characteristics which potentially may impair the biochemical functions of the desired isolated fractions. For example, some currently available blood separation media contain erythrocyte aggregation polmers which may decrease mitogen responsiveness of isolated lymphocyte preparations (J. Immunol. Meth. 38:43-51, 1980). These materials may also form solutions of undesirably high osmolality or viscosity. Cell aggregation is often caused by BSA at physiological pH (7.4) and it is undesirable to employ reduced pH (5.1) because it introduces other problems such as cell swelling (which alters cell density) and possible impairment of cell function. It is also expensive for use in large scale separations. Ficoll may similarly cause cellular aggregation which can be remedied only by use of a dispersing agent, or undesirably, lowering of the pH. Ficoll is also highly viscous, making it difficult to generate a linear isoosmotic gradient with it. It is also difficult to separate cells with very similar buoyant densities with any of these materials, even with the use of discontinuous gradients.
A density gradient material which has been used with some success for cell separation is colloidal silica. Colloidal silica is an aqueous suspension of colloidal particles formed by polymerization of monosilicic acid from SiO.sub.2 dissolved in water. Individual particles average 130-140 .ANG. in size and generally range from about 30 to 220 .ANG.. The colloidal suspension is most stable for storage at pH 8-10 at which the colloidal particl
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Gallard-Schlesinger Che
Rosen Sam
Simpson Jeanette
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