Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing gas sample
Reexamination Certificate
2000-04-26
2002-03-19
Warden, Jill (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing gas sample
C435S283100, C435S297100, C435S267000, C435S270000, C435S286400, C210S236000, C210S087000, C210S669000, C215S247000
Reexamination Certificate
active
06358474
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus for as well as to a method of isolating cell material from a tissue system and/or a liquid.
PRIOR ART
Individual cells or cell systems are analysed for the medical diagnosis of clinical conditions and for scientific research applications, particularly in the field of bioengineering. The prerequisite for the analytical methods to be performed on cells is the isolation of individual cells from tissue systems or from liquids, respectively, where the individual cells occur in their natural form. In many cases, cells are sampled for the cell analysis from biological soft tissue such as from the spleen, the liver, and also from harder tissue such as the skin or hair. As part of the analysis of blood or urine, cells are frequently isolated from liquids, which are then analysed, for instance, to detect pathogenic agents. Such analyses also cover cells contained in body secretions, smears or other endogenous liquids, which cells, however, must be isolated from the individual tissue systems or liquids like in all the other aforementioned cases.
So far, however, standardised cell isolation methods have not become known. Predominantly, the manual separation of individual cells or cell agglomerates from tissue systems and/or liquids has been employed so far so that various, individually developed manual methods are applied for extracting cells from tissue, e. g. the spleen, which are adapted to the skill of the respective analysing operator. In this manner, different cell-isolating techniques have been devised in the course of time, which went then through different developments. Moreover, any helpful hints cannot be derived from the pertinent literature from which generally common or even standardised methods of universal cell isolation could be derived.
The production of a single-cell suspension for the analysis of a specific cell type require a number of operating steps to be performed in succession, which have been performed by hand exclusively to date.
For the analysis of cells occurring in tissue systems first of all a piece of tissue, e. g. from the spleen or the liver, must be severed in the desired size. The removed tissue sample is usually placed into a medium which is introduced, together with the sample, into a flat laboratory dish. Subsequently, the tissue sample contained in the medium is mechanically crushed and comminuted by means of suitable tools so that the cells to be analysed can be better extracted from the tissue. The cells or cell agglomerates, which are concentrated in the medium and are isolated from the individual crushed tissue particles can now be removed as a suspension from the medium contained in the laboratory dish. Using a pipette whereof the opening can be easily clogged, however, by extracted minor tissue particles usually performs this separation. The pipetting of the cell suspension therefore requires the respective operator to apply a particular manual skill because the isolated cells are present in the medium together with the tissue particles comminuted by the operation of mechanical isolation.
The cell suspension obtained by pipetting is introduced into a vessel again where the minor tissue fragments, which are removed together with the cells, can settle on the bottom of the vessel by sedimentation processes due to their greater mass. The afore-described pipetting operation is repeated again in order to obtain in this manner a suspension enriched with the cells. Depending on the type, size and percentage of extracted small tissue fragments present in the suspension, the extracting operation, which is based on sedimentation, must be repeated rather frequently. This operation can also be assisted by the application of centrifuges.
For the dissolution and separation of cell systems present in the suspension into individual cells, the suspension enriched with cell material is extracted by pipetting several times so that as many cells as possible will be present in an isolated condition in the suspension. The suspension of individual isolated cells from the tissue system, which is obtained in this manner, is usually transferred into another vessel into which ammonium chloride is added, for instance, for removing the red blood cells which are equally present in the suspension.
The afore-described methodology of cell isolation, e.g. of cells from a tissue fragment, e. g. of the liver, has gone to show that a great number of isolating steps is required which must be performed by hand in succession until individual isolated cells can be obtained. With each of the individual isolating steps, however, residual waste is produced which is difficult to discharge, such as the suspension liquids, the tools required for the isolating process like pipetting tips or isolating apparatuses and also the vessels used.
In an approach' to cut the expenditure entailed by the isolating operation many laboratories such as diagnostic PCR laboratories desist from the specific production of isolated cell suspensions and rather digest the complete tissue system. Even though this involves savings in terms of time it results in the disadvantage that inhomogeneous homogenisation products with a plurality of cell types, tissue fragments and frequently inhibiting substances for the subsequent analysis are produced.
BRIEF DESCRIPTION OF THE INVENTION
The present invention is therefore based on the problem of providing an apparatus as well as a method of isolating cell material from a tissue system and/or a liquid in such a way that the number of the manual operating steps to be performed will be reduced and that the cell isolation operation will be largely standardised so as to be able to obtain a reliable reproducibility of the analysis results linked up with the cell isolation. The waste resulting from each separate isolation operation, such as the flat laboratory dish as well as general vessels and tools, should be moreover reduced. In particular, the contamination of the operating place, which inevitably occurs with the conventional method, should be largely avoided.
The solution to the problem is solved by the apparatus and method of isolating cell material from a tissue system and/or a liquid according to the present invention.
The invention is based on the idea of providing a sample carrier system, which integrates all the functions for cell isolation from a tissue as well as for accumulation of cells from a liquid.
In accordance with the invention, the apparatus for isolating cell material from a tissue system and/or a liquid comprises a vessel open at the top, into which the cell material to be isolated can be introduced in the tissue system and/or in the liquid. Moreover, an isolating device having a die-shaped configuration is provided which comprises a flat isolating disk having a peripheral edge which is fluid-tight and flush with the inside walls of the vessel and presents at least one passage opening covered by a filter membrane. The isolating device can be inserted into the vessel from the top so that the isolating disk pressurises the cell material inclusive of the tissue system and/or the liquid and subjects it, by rotation, to shearing forces. In this system the isolating disk is so designed—and preferably provided with grinding elements on its underside—so that the cell material will be comminuted inside the vessel as the isolating disk rotates.
The application of the inventive apparatus makes it possible that following the sampling operation, e. g. in a physician's office, the taken sample as well as reagents assisting the process, if this is possible, can be transferred into the inventive container for transport. With an appropriate dimensioning of the vessel it is possible, at the same time, to determine the size of the tissue sample to be taken so that in this manner markings to be applied appropriately on the vessel will signal to the physician which tissue volume is required for the respective analysis.
This provision equally satisfies the demand for standardisation in the performa
Bernhagen Jürgen
Dobler Hannes
Kiesewetter Stefan
Kuhn Claus
Lindner Hans
Crowell & Moring LLP
Fraunhofer Gesellschaft zur Förderung der Angewandten Forschung
Sines Brian
Warden Jill
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