Reaction receptacle unit, method for selectively removing liquid and introducing liquid containing a target substance from / to a reaction receptacle of the reaction receptacle unit - Patents.com

Abstract

The present application discloses a reaction receptacle unit (20) having at least one reaction receptacle (1) having a receiving chamber (2) for receiving a liquid (40), the receiving chamber (2) having a retention area having a surface texture and / or shape that causes the retention area to have an increased retention effect relative to the liquid (40) on the surrounding areas due to adhesion forces between the liquid and the retention area and cohesion within the liquid, such that when the liquid (40) is removed from the receiving chamber (2) by centrifugation, a predetermined small amount of the liquid is retained at or within the retention area. Further, a method for selectively removing a liquid (40) from a reaction receptacle (1) of the reaction receptacle unit (20) by centrifugation is disclosed. Further, a method for introducing a liquid (40) containing a target substance (41) into a reaction receptacle (1) of the reaction receptacle unit (20) is disclosed.

Description

[Technical field]

[0001] The present invention relates to a reaction receptacle unit comprising at least one reaction receptacle, a method for selectively removing liquid from a reaction receptacle of the reaction receptacle unit, and a method for introducing a liquid containing a target substance into a reaction receptacle of the reaction receptacle unit. [Background technology]

[0002] It is a well-known phenomenon in laboratory work that when a reaction vessel is emptied, small amounts remain or are retained. With pipetting devices and pipetting robots, the problem of so-called residual volume frequently occurs. It is often desirable to determine the residual volume accurately. For example, when cells are present in microtiter plates and, more recently, also organoids or spheroids as cell complexes, the phenomenon of residual volume poses particular challenges when changing the culture medium. In these cases, the cell structures should usually remain in the reaction vessel, but the supernatant is, for example, removed and replaced with fresh medium.

[0003] For series tests such as flow cytometry, the cells suspended in the nutrient solution must be purified before testing so that metabolites, dyes, markers, etc. present or accumulated in the nutrient solution do not interfere with the test. In flow cytometry, fluorescently labeled molecules in suspension form are subjected to multiparametric analysis by a fluidic system in which the cells pass a laser beam and qualitative / quantitative data are obtained from the detection of light scattering and excitation bands. Further information can be found, for example, in Non-Patent Document 1. For purification purposes, it is common to pellet the cells, for example by centrifugation at 300 g for 5 min. Pelleting represents a high stress factor for the cells. This can lead to undesired reactions or activations, such as changes in gene expression, which can negatively affect subsequent measurements or lead to erroneous results.

[0004] Patent Document 1 discloses the structure of a middle part in a microtiter plate that acts as a mechanical barrier for the organoids located at the bottom of the plate. This makes it possible to remove the culture medium using a pipette without the risk of removing the organoids, i.e., aspirating the organoids.

[0005] US Pat. No. 5,399,633 describes a double-sided microtiter plate with cylindrical wells that can retain fluids by surface tension, capillary action or by suitable treatment, coating or texturing of the walls.

[0006] US Pat. No. 5,399,433 shows a suspension vessel in a microtiter plate that can serve as a natural barrier.

[0007] US Pat. No. 5,399,633 discloses a microtiter plate in which outwardly facing reaction vessels release their liquid during centrifugation and the reaction vessels are emptied accordingly.

[0008] US Pat. No. 5,399,633 and US Pat. No. 5,499,663 disclose centrifugation devices for cleaning reaction vessel units. In these centrifugation devices, the reaction vessels are arranged with their openings facing away from the axis of rotation, so that the liquid contained in the reaction vessels is discarded during centrifugation. It has been shown that the liquid can be removed from the reaction vessels without leaving any residues, and a high degree of purity can be achieved. The reaction vessels thus purified can be reused for biological reactions in which single molecules, in particular DNA or RNA strands, may represent unacceptable contaminations. The centrifugation of liquid from the reaction vessels by centrifugation is hereinafter also referred to as "cleaning by centrifugation", and such a cleaning process can be carried out with or without the addition of a cleaning solution. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent Application Publication No. 3633022 [Patent Document 2] US Patent Application Publication No. 2008 / 0003670 [Patent Document 3] US Patent Application Publication No. 2011 / 0278304 [Patent Document 4] U.S. Pat. No. 8,602,958 [Patent Document 5] US Patent Application Publication No. 2021 / 138485 [Patent Document 6] U.S. Pat. No. 11,117,142 [Patent Document 7] International Publication No. 2017 / 125598 [Patent Document 8] DE 102021124023 [Non-patent literature]

[0010] [Non-Patent Document 1] Fortis Life Sciences, “Flow Cytometry Protocols for Extracellular & Intracellular Targets”https: / / www.fortislife.com / products / documents / flow-cytometry-protocols-for-extracellular-intracellular-targets / appnote002 Summary of the Invention [Problem to be solved by the invention]

[0011] One object of the present invention is to create a reaction vessel unit having at least one reaction vessel which allows a reliable separation of the two volumes contained in the reaction vessel and also allows a defined residual volume to remain in the reaction vessel when the reaction vessel is washed by centrifugation.

[0012] It is a further object of the present invention to provide a method for removing liquid from a reaction vessel of a reaction vessel unit which is selective in the sense that a defined volume of fluid remains in the reaction vessel during centrifugation washing.

[0013] It is a further object of the present invention to provide a method for introducing a liquid containing a target substance into a reaction vessel of a reaction vessel unit, which allows at least a defined volume of fluid containing the target substance to remain in the reaction vessel when the reaction vessel unit is washed by centrifugation.

[0014] A further object of the present invention is to provide a method for purifying target substances, in particular cells, cell clusters, cell aggregates or organisms, dispersed or suspended in a liquid, which is efficient, cheap and rapid, in which the target substances are treated gently, in particular the cells or organisms are not activated.

[0015] It is a further object of the present invention to provide a method for performing tests on target substances dispersed or suspended in a liquid, in particular cells, cell clusters, cell aggregates or organisms, which is efficient, cheap and rapid, in which the target substances are treated gently, in particular the cells, cell clusters, cell aggregates or organisms are not activated.

[0016] One or more of the problems of the present invention are solved, at least in part, by the features of the independent claims. Preferred and advantageous further embodiments are set out in the dependent claims. [Means for solving the problem]

[0017] One aspect of the invention relates to a reaction receptacle unit having at least one reaction receptacle with a receiving chamber for receiving a liquid. According to the invention, the receiving chamber has a retention area, which has a surface texture and / or shape that causes the retention area to exert an increased retention effect on the liquid compared to the surrounding areas due to adhesion forces between the liquid and the retention area and cohesion within the liquid, so that when the liquid is removed from the receiving chamber by centrifugation, a predetermined amount, in particular a small amount of liquid compared to the receiving chamber, is retained at or in the retention area.

[0018] For the purposes of the present invention, a reaction vessel is understood to be a vessel that can be used in a laboratory environment and in which a chemical reaction or a biological or microbiological process occurs or can be carried out. A reaction vessel unit may include a single reaction vessel or may combine several reaction vessels in a fixed arrangement. The liquid may be any non-gaseous fluid, which may also include gelatinous or gel-like fluids. In particular, in a small amount compared to the receiving chamber. The holding area has a surface texture and / or shape that exerts an increased holding effect on the liquid compared to the surrounding area due to adhesion forces between the liquid and the holding area and cohesion within the liquid, so that a predetermined small amount of liquid can be held in or within the holding area when the liquid is removed from the receiving chamber, and also a defined residual volume can be held exactly in or within the holding area when the reaction vessel is emptied. The liquid and the holding area can be considered as a system for the retention effect, i.e. for which the properties of the liquid (e.g. density, surface tension, polar or non-polar) and the properties of the holding area (e.g. shape, roughness, chemistry, critical angle with the liquid) interact in the formation of adhesive and cohesive forces to form the retention effect. This means that the holding area can be specifically adapted to the fluid used and the desired application. Since for a specific system of holding area and liquid a limit value for the acceleration acting in the direction away from the holding area can be determined, the invention is particularly suitable for use with a centrifuge device for emptying the reaction vessel while retaining the residual volume in or at the holding area by setting the speed of the centrifuge device to a value safely below the limit value. However, the invention is also suitable for other manual, mechanical or partly mechanical methods for removing the liquid in the receiving chamber, for example by a pipetting device or simply by inverting the reaction vessel unit by hand or in a tilting device. Depending on the system, the acceleration of gravity may be sufficient to empty the receiving chamber, or emptying of the receiving chamber may be assisted by a controlled interruption or impact against a surface to generate a defined acceleration below a limit value.

[0019] In an embodiment, the receiving chamber may be bounded by a circumferential side wall and a bottom wall, and the holding area may be formed in or on the bottom wall of the receiving chamber. The side wall may have a single side wall with a circular or oval or elliptical or otherwise curved cross section, or several side walls forming a polygonal, e.g., square or rectangular or diamond or hexagonal, cross section with sharp or rounded edges, the rounded edges may facilitate emptying of the receiving chamber. The bottom wall may be flat or conical or U-shaped with sharp or rounded edges to the side wall, the rounded edges may facilitate emptying of the receiving chamber.

[0020] In an embodiment, the retention area may have a capillary cavity that opens into the receiving chamber, the walls of the capillary cavity being spaced very close together so that the liquid is retained in the capillary cavity by capillary action. In the capillary cavity, there is a large surface area between the liquid and the capillary cavity, which creates a corresponding high adhesive force due to interfacial tension. The cohesion in the liquid due to surface tension also holds the liquid molecules tightly together so that they are not carried away when the rest of the receiving chamber is emptied. Thus, the capillary effect results from specific adhesive and cohesive forces. In particular, when the liquid is released, the volume of liquid in the receiving chamber above the capillary cavity separates from the volume of liquid in the capillary cavity and the atmosphere flows into the receiving chamber, and thus an interface is formed at the opening of the capillary cavity, and the surface tension at this interface contributes significantly to the retention effect. Since the limit value of the acceleration acting away from the opening of the capillary cavity, below which the capillary effect dominates, can be determined for a particular system of capillary cavities and liquids, the invention is particularly well suited for use of a centrifuge device for emptying a reaction vessel while maintaining a residual volume in the capillary cavity by setting the speed of the centrifuge device to a value safely below the limit value. The capillary cavity can be formed as a recess in the bottom or side wall of the receiving chamber. The opening of the capillary cavity may be formed with a sharp or rounded edge relative to the bottom wall of the receiving chamber, either in the center or off-center, which may facilitate the retention of the liquid in the capillary cavity by capillary action. In an alternative embodiment, the capillary cavity may also open into the side wall of the receiving chamber, which allows higher speeds when emptying the receiving chamber. However, the handling of the liquid volume may be easier if the capillary cavity opens into the bottom wall of the receiving chamber.

[0021] Preferably, there is only a single capillary cavity, which simplifies access for pipetting or rinsing and also ensures that a particular amount of target material in a liquid can be reliably allocated to a particular capillary volume, however, in other applications there may also be several capillary cavities, which can be individually rinsed to remove target material such as cells collected therein.

[0022] Furthermore, it is preferred that the capillary cavity is substantially free of partitions so that the contents of the capillary cavity can be completely removed or rinsed out of the capillary cavity by introducing a fluid jet. Partitions or other protruding structures are lateral barriers, which can form dead zones in which target material can remain when rinsing the capillary cavity. Such dead zones are an obstacle when emptying the capillary cavity and should therefore be avoided. However, to aid the rinsing process, a conical or pyramidal central ridge can be formed at the bottom of the capillary cavity, which can also act as a jet splitter or jet deflector for the fluid jet, but does not form a lateral barrier.

[0023] In an embodiment, the capillary cavity may have a rectangular, circular or elliptical cross section. The wall distance between the opposing side walls or wall sections of the wall of the capillary cavity may be dimensioned to hold the liquid by capillary action between the opposing side walls or wall sections of the wall of the capillary cavity. Depending on the nature of the liquid and the wall, the wall distance (d) may be, for example, at most 2.0 mm or at most 1.8 mm or at most 1.6 mm or at most 1.4 mm or at most 1.2 mm or at most 1.0 mm or at most 0.8 mm. Furthermore, the wall distance may be, for example, at least 0.1 mm or at most 0.3 mm or at most 0.5 mm or at most 0.8 mm. The wall distance may be advantageously dimensioned to optimize the capillary effect for the distance between the side walls of the receiving chamber, when the wall distance is at most 30%, or at most 25%, or at most 20%, or at most 15%, or at most 10%, or at most 5% of the wall distance between the side walls of the receiving chamber.

[0024] With regard to the capillary effect, it is also advantageous for the capillary cavity to have a depth that is at least 0.5 times, or at least 1 time, or at least 1.5 times, or at least 2 times the wall distance between opposing side walls or wall sections of the capillary cavity.

[0025] To create an effective capillary effect, the side walls of the capillary cavity are advantageously vertical or substantially vertical with a deviation from the vertical of at most 5°, or at most 3°, or at most 2°, or at most 1°, or at most 0.5°.

[0026] In an embodiment, the bottom wall of the receiving chamber can be conically inclined towards the opening of the capillary cavity or can be U-shaped. As a result, the bottom wall of the receiving chamber can form an introduction funnel for the target material entering the capillary cavity. This avoids obstacles that act perpendicular to the centrifugal force, especially during centrifugation, and the target material, such as cells, can slide along the bottom wall into the capillary cavity. Such a shape also makes it easier to remove the liquid from the receiving chamber and tear the volume in the capillary cavity. The steeper the introduction funnel, the more pronounced this positive effect is. This must be balanced with other effects. The steeper the bottom wall, the greater its surface area. This also increases the chances that target material, such as cells, cell clusters, cell aggregates or organisms, will attach to the reaction vessel. Also, as the gradient increases, the volume of the receiving chamber available as reaction volume decreases. Depending on the application, the ideal angle of inclination must be determined between these effects, which may depend on parameters such as the viscosity of the liquid or target material, the surface properties of the bottom wall, the attachment potential of the target material, etc. For example, the slope angle of the bottom wall can be at least 5°, or at least 15°, or at least 25°, or at least 35°, or at least 45°, and / or at most 75°, or at most 65°, or at most 55°, or at most 45°. The slope angle is measured relative to the plane of the opening of the capillary cavity. If the retention area is not a capillary cavity, the slope angle is measured relative to the tangent plane of the wall area in which the retention area is formed.

[0027] To increase the capillary effect of the capillary cavity on the receiving chamber, the sidewalls of the receiving chamber can also be designed to widen towards the opening with a deviation of at least 2°, or at least 3°, or at least 5°, or at least 10° from the vertical.

[0028] In an embodiment, the capillary cavity may have two opposing side walls dimensioned such that the distance between them exerts a capillary effect, and a bottom wall extending between the side walls, the bottom wall being continuously curved or also provided with two end walls extending between the side walls, the end walls rising from the bottom wall to the edge of the capillary cavity at an angle, in particular at a flat angle or with a concave curvature. Such a design allows a jet of liquid or compressed air along the bottom wall or one of the inclined end walls to easily flush the fluid volume held in the capillary cavity by capillary action.

[0029] In an embodiment, the walls of the capillary cavity can widen towards the opening of the capillary cavity. The widening makes it possible to control the volume removed from the cavity via centrifugal force during centrifugation, since the balance of forces between the centrifugal force and the retention force depends on the distance between the walls and the angle of widening between the walls. The widening can be cone / wedge / funnel shaped and can be straight or curved, especially in vertical cross section. A similar effect can be achieved if the walls of the capillary cavity have a coating with a hydrophilic or oleophilic effect that decreases towards the opening of the capillary cavity. In this case, the balance of forces between the centrifugal force and the retention force depends on the wall distance and the hydrophilic / oleophilic effect, and therefore it is also possible to control the volume removed from the cavity via centrifugal force during centrifugation. The opening angle of the opening is preferably 10° or less, 5° or less, 2° or less, 1° or less, 0.5° or less. The two variants of widening and decreasing hydrophilic / oleophilic effect can also be used in combination.

[0030] In an embodiment, the reaction vessel unit may comprise a plurality of reaction vessels, and the capillary cavities may be angled progressively away from the center of a line, in particular a centerline, that divides the reaction vessel unit or the reaction vessel arrangement into two halves, relative to a perpendicular line on the plane on which the reaction vessels are arranged. When a reaction vessel unit with capillary cavities arranged parallel to each other is centrifuged in a centrifuge, the centrifugal force also acts increasingly on the side walls of the cavities from the centerline towards the edge of the plate, due to the increasing angle relative to the radius from the rotation axis. This reduces the drainage effect. This can be compensated for by adjusting the alignment of the cavities as described above.

[0031] In an embodiment, the holding area can have a surface structure that increases the adhesive effect on the liquid and a size such that due to cohesion in the liquid, a molecular agglomeration is formed, so that a defined amount of liquid is held at or in the holding area. In principle, such a holding area can also be formed on a flat surface, i.e. independent of the capillary cavity, but also combined with the capillary cavity. The holding area must not be too large, since a droplet covering the entire holding area is formed on the holding area by cohesion (surface tension), so that a defined amount of liquid is held. If the holding area is too large, the droplet that spreads beyond the holding area by cohesion becomes too large and heavy, so that it cannot be held by cohesion and adhesion forces. As a result, only a part of the holding area is held by one or more small droplets with an undefined volume. This should be avoided. Holding areas with such surface structures can also be shaped like a trough. The more pronounced the trough, the better the ratio between adhesion and weight of the droplet, so that the droplet is held.

[0032] In an embodiment for aqueous solutions, the holding area may have a hydrophilic coating to improve the holding effect. In a further embodiment, the receiving chamber may have a hydrophobic coating to facilitate emptying the receiving chamber. A combination of both measures may be particularly effective, whereby the hydrophobic coating of the receiving chamber may be complementary to the hydrophilic coating of the holding area. To handle non-polar solutions such as oily and oleaginous solutions, the capillary cavity may have an oleophilic coating and / or the receiving chamber may have an oleophilic coating, which may be complementary. The holding area may also be defined independently of the shape or capillarity, solely by the surface chemistry (hydrophilic / hydrophobic, oleophilic / oleophobic, rough / smooth, etc.) compared to the surrounding area. This makes it possible, for example, to achieve the same effect by a two-dimensional spot on the flat base of a plate, i.e. to hold a defined volume for subsequent reactions.

[0033] An example of a reaction vessel within the meaning of the present invention is micro-vessels, which are often combined, especially in the form of a microtiter plate (MTP). The individual reaction vessels of a microtiter plate, also called wells, are usually arranged in a predefined grid of rows and columns, which specifies the numerical deviation and thus determines the possible filling volume. Cells are grown individually or in cell clusters in such microtiter plates in the context of cell-based assays. As mentioned at the outset, the cell structure in the reaction vessel should usually remain intact, for example when washing the microtiter plate, while the supernatant is removed and replaced, for example, with fresh medium. This can be achieved particularly well with the reaction vessel unit according to the present invention by providing a microtiter plate in which a holding area according to the present invention is formed at the bottom of each well.

[0034] This is particularly advantageous if all reaction vessels have openings at the top of the microtiter plate, which makes it easy to fill and empty the receiving chambers.

[0035] The reaction vessel unit can advantageously be made of plastics such as polystyrene (PS), polypropylene (PP), polyolefin carbonate (POC), cycloolefin copolymer (COC) or polyvinyl chloride (PVC). This, in combination with the properties that allow optical analysis of the cavities or capillaries, allows a particularly simple and dimensionally accurate production using known manufacturing processes such as injection molding, injection blow molding, thermoforming, etc. However, other materials are also possible, in particular glass. The use of photosensitive glasses or glass ceramics is known, for example, for the production of picotiter plates.

[0036] In an embodiment, the reaction vessel unit may include a plurality of reaction vessels, and the retention effect of the retention area of ​​at least two reaction vessels may be different. The difference may preferably be such that the retention effect increases from a line, in particular a center line, that divides the reaction vessel unit or the arrangement of reaction vessels into two halves, so that the retention force of the retention area remains constant or nearly constant across the reaction vessels when the reaction vessel unit rotates about an axis that is parallel to the center line and whose radius through the center line is perpendicular to the plane in which the reaction vessels are arranged. When a reaction vessel unit (e.g. a microtiter plate) is centrifuged in a centrifuge, the centrifugal force increases from the center line towards the edge of the plate, due to the increasing distance from the axis of rotation. This can be compensated for by adjusting the retention effect of the retention area. For example, the wall spacing of the capillary cavities may narrow towards the edge of the plate, and / or the capillary cavities may be angled gradually towards the center, and / or the hydrophilic / lipophilic effect of the coating may be increased. Apart from this special design, the variable retention effect may also be advantageous for different separation volumes or special test devices. For example, one can consider an experimental arrangement in which volume is removed from the holding area for only some of the reaction vessels to achieve a time series on a single plate.

[0037] Similarly, a concentration series can be achieved by repeatedly retaining a defined volume of target liquid in the holding area while removing the residual volume in the receiving chamber, then adding (dispensing) a defined volume of dilution liquid into the receiving chamber after the target liquid has mixed with the dilution liquid, and emptying the receiving chamber. A defined volume of diluted target liquid at this point then remains in the holding area. For example, if the holding area has a volume of 1% compared to the receiving chamber, dispensing will result in a dilution of the target liquid in the holding area initially of 1:100 in a first step. If the receiving chamber is emptied and a defined volume of the now diluted target liquid is retained in the holding area, after repeating the process, the dilution will be 1:100 x 1:100 (=10 4 For example, if the procedure is applied line by line in a microtiter plate, this results in a dilution of 1:10 2 , 1:10 4 , 1:10 6 etc. This method is particularly advantageous when the concentration series is displayed on a microtiter plate without the use of pipetting tips or other consumables, but simply by repeatedly evacuating the receiving chamber. The dilution can also be set very accurately by the defined volume, especially the holding area, and therefore lower requirements can be imposed on the dispensing accuracy when dispensing the liquid.

[0038] For practical realization of mixing, a wide range of variations are conceivable. For example, the dispensing process can be carried out, for example, by combining dispensing with rinsing of the holding area with the diluent, so that the target liquid is mixed directly with the diluent during dispensing. Alternatively, the target liquid in the holding area can be brought into the receiving chamber together with the diluent by centrifugation so that the two liquids are mixed in the receiving chamber, and then, if necessary, centrifuged in reverse to ensure that the holding area is filled again with the diluted target liquid. The reverse case is also conceivable, i.e., the diluent is forced into the holding area by centrifugation. These mixing considerations are particularly important when the holding area is a cavity, in particular a capillary cavity. Mixing is particularly easy when the holding area is determined solely or mainly by the structure or properties of the wall, because mixing can be realized by shaking or stirring or by the introduction of gas, or even by the dispensing process alone.

[0039] In an embodiment, a collecting device may be provided, which is arranged opposite an opening of a receiving chamber or openings of a receiving chamber of at least one reaction vessel, and is designed to capture liquids leaking or expelled from the receiving chamber or chambers. The collecting device may have one or more compartments, preferably in the form of a microtiter plate, with an opening of a compartment of the collecting device or openings of a compartment facing an opening of a receiving chamber or openings of a receiving chamber of at least one reaction vessel. The number of compartments of the collecting device may be equal to, greater than or less than the number of reaction vessels. For example, a second plate may be attached to the first plate (reaction vessel unit) as a collecting device, and the supernatant may be collected from the second plate. The collected fluids can be reused, which can significantly save the cost of using the often expensive fluids used. The collecting device may be compartmentalized like the reaction vessel unit itself. Thus, for example, if the reaction vessel unit comprises a microtiter plate with 96 or 384 or another number of reaction vessels, the collection device can also be provided as a microtiter plate with 96 or 384 or another number of compartments. The collection device can also have more or less compartments than the reaction vessels provided. For example, the collection device can also be designed as a simple dish. The supernatants can then be combined and aliquots thereof can be analyzed, for example, by NGS (next generation sequencing). The collection plate can also have some compartments offset upwards or downwards from the number of reaction vessels of the reaction vessel unit.

[0040] Another aspect of the invention is a method for selectively removing liquid from a reaction receptacle of a reaction receptacle unit, the liquid being contained within a reaction receptacle unit as described above, the method comprising: - Centrifuging the reaction vessel unit with the opening of the receiving chamber facing radially away from the centrifugation axis at a speed just below the threshold speed at which the retention effect of the retention area is overcome, so that a partial volume of liquid located in or at the retention area remains therein and the remaining liquid located in the receiving chamber is removed.

[0041] For this purpose, the reaction vessel units can be arranged, for example, in a centrifuge device in a position where the openings of all reaction vessel units face away from the centrifugation axis. The centrifuge device can then be controlled with a time-speed profile suitable for centrifuging the liquid in the receiving chamber while the liquid in or on the holding area, for example in the capillary cavities, remains there. This process is suitable both for emptying the reaction vessel of nutrients or reaction liquid and for separating any volume of liquid while retaining the target substance, such as an array of cells. This method also makes it possible not to use special devices in the centrifuge device, such as magnetic devices that can retain substances mixed with magnetic beads, etc., at the bottom of the reaction vessel during centrifugation. This is possible in principle, but such magnetic devices require a higher level of technical equipment, which entails structural modifications to the centrifuge device and the movement of additional masses in the centrifuge device, since they must of course rotate together with the reaction vessel units.

[0042] In embodiments, there may be provision for collecting the remaining liquid in a collection device, particularly as described above, which allows the remaining removed volume to be used for other purposes, which may help to reduce costs and conserve resources.

[0043] Another aspect of the invention is a method for introducing a liquid containing a target substance into a reaction vessel of a reaction vessel unit, the method comprising the steps of: using a reaction vessel unit as described above for containing a liquid; Guiding the target material to the holding area by a guiding system, the guiding system comprising: - an arrangement of the centrifuge device and the reaction vessel unit in which the holding area is radially outward or substantially outward with respect to the centrifugation axis with respect to the remainder of the receiving chamber; a magnetic device arranged to interact with the magnetic properties of a magnetic auxiliary material or a target material; or an electrostatically charged auxiliary material or electrostatic properties of the target material, and an electrode device arranged to interact with the electrostatically charged auxiliary material or electrostatic properties; Includes.

[0044] A target substance can be any substance, including chemical and biological substances, that is a component of a solution and is to be specifically separated from the bulk of the solution. In particular, a target substance is a substance in which a reaction is initiated or observed in a reaction vessel. In the context of the present invention, a guiding system is a system designed to move a target substance in a desired direction, in particular towards a holding area, for example into a capillary cavity. The guiding system can react to the target substance itself and / or to auxiliary substances contained in or in a matrix in which the target substance is embedded, to form a target substance system.

[0045] Centrifugation is particularly suitable for target substances or target substance systems that have a higher density than liquids. If the retention area is a capillary cavity, the process can be carried out such that the opening of the capillary cavity is oriented radially relative to the centrifugation axis. This means that the centrifugal acceleration acting towards the opening of the capillary cavity ensures that the liquid is guided towards the opening of the capillary cavity and forces the liquid into the capillary cavity.

[0046] The magnetic auxiliary material can be, for example, a magnetic bead. The electrostatically charged auxiliary material can be any charge carrier. Certain target substances, such as DNA molecules, can be charged so that they can be attracted to the electrode device. The electrode device can be provided in the reaction vessel unit, in the individual reaction vessels, or externally.

[0047] Subsequent removal of the liquid in the receiving chamber by the above-mentioned process can then also ensure separation of the target substance or target substance system from the remaining liquid. In particular, this can be achieved by centrifuging a reaction vessel unit having a retention area located radially inward or essentially inward with respect to the centrifugation axis with respect to the remaining part of the receiving chamber at a speed below the threshold speed at which the retention effect of the retention area is just overcome, whereby a partial volume of liquid passing into or up to the retention area and containing the target substance remains therein and the remaining liquid located in the receiving chamber is removed.

[0048] Again, the remaining liquid can be collected in a collection device, in particular as described above. This allows, for example, an aliquot of a valuable reagent to be placed in a holding area, for example in a capillary cavity, the receiving chamber can be emptied by centrifugation, and the remainder of the reagent can be collected and used for further aliquots. For this purpose, a collection device such as a catcher plate is positioned "upside down" opposite the opening of the receiving chamber of the reaction vessel prior to centrifugation.

[0049] Another aspect of the invention is a method for purifying a target substance dispersed in a liquid, such as a cell, cell cluster, cell aggregate or organism in a reaction vessel of a reaction vessel unit, the liquid being within said reaction vessel unit, the method comprising: - collecting the target substance in a holding area, in particular by centrifuging using an arrangement of reaction receptacle units, so that the holding area is located radially outward or substantially outward with respect to the centrifugation axis with respect to the remainder of the receiving chamber; removing liquid from the receiving chamber by centrifuging the reaction vessel unit with the opening of the receiving chamber facing radially away from the centrifugation axis at a speed just below the threshold speed at which the retention effect of the retention area is overcome, so that a partial volume of liquid located in or at the retention area remains therein and the remaining liquid located in the receiving chamber is removed; Introducing a further liquid into the receiving chamber, in particular by dispensing or pipetting, mixing the target substance with another liquid in a receiving chamber; Includes.

[0050] With such a purification process, sufficient purification of interfering components can be achieved by collecting the target material in the capillary cavity and exchanging the liquid, thus avoiding pelleting with its associated contamination.

[0051] In the method of this aspect, the mixing step comprises: leaving it for a predetermined period of time; - shaking the reaction vessel unit; introducing additional liquid into the receiving chamber by dispensing or pipetting so as to wash or flush the target substance from the holding area; pipetting the target material out of the holding area and back into further liquid introduced outside the holding area; may include at least one of the following:

[0052] In an exemplary embodiment of the method, the receiving chamber may have a volume on the order of 200-400 μl, the holding area is a capillary cavity at the bottom of the receiving chamber and has a volume of about 5 μl, and collection of the target material is performed by centrifugation of at least 2 g or at least 5 g or at least 10 g or at least 20 g and / or up to 1000 g or up to 500 g or up to 200 g or up to 100 g or up to 50 g or up to 40 g or up to 30 g or up to 20 g for a time of at least 1 second or at least 2 seconds or at least 5 seconds or at least 10 seconds or at least 10 seconds or at least 30 seconds or at least 60 seconds or at least 90 seconds or at least 2 minutes or at least 5 minutes and / or 60 minutes or less or 30 minutes or less or 20 minutes or less or 15 minutes or less or 10 minutes or less or 5 minutes or 2 minutes or less or 1 minute or less or 30 seconds or less or 20 seconds or less or 10 seconds or less or 5 seconds or 2 seconds or less. It is understood that impossible time ranges in which the maximum duration is less than the minimum duration are excluded. For example, it is conceivable to work in high-throughput applications with very high centrifugal acceleration (e.g. 1000 g) but very short exposure times (e.g. 1 s). This allows very fast and reliable collection or concentration of target substances in the capillary cavities. In order to avoid aggregation or other disturbances of the target substances, very strict limitations must be observed with respect to the exposure time. Conversely, in applications with very sensitive cells, centrifugation can be performed with very low centrifugal acceleration, e.g. 2 g, but for very long times (e.g. 1 h). The exposure time limits can be defined more broadly here. As a rule, the product of exposure time and centrifugal acceleration should not exceed 72,000 gs or 20,000 gs, in particular 1000 gs.

[0053] In a 96 microtiter plate format reaction vessel unit, the volume of the receiving area is typically about 200-400 μl. The load can be an order of magnitude lower than when pelleting at about 300 g, which proves to significantly reduce stress on the cells or organisms. In reaction vessel units with smaller working volumes of the receiving chamber (e.g., 384 format, 1536 format), the dimensions of the capillary cavity may be changed accordingly and the g-force may be larger. In reaction vessel units with larger working volumes of the receiving chamber (e.g., 48 format), the dimensions of the capillary cavity may be changed accordingly and the g-force may be smaller.

[0054] In this process, the removed liquid may also be collected in a collection device as described above.

[0055] Another aspect of the invention is a method for testing for target substances dispersed in a liquid, such as cells, cell clusters, cell aggregates or organisms in a reaction vessel of a reaction vessel unit, the liquid being placed in the reaction vessel unit as described above. The method comprises the steps of: - purifying the target substance according to the method described above; - withdrawing a predetermined amount of liquid from a reaction vessel in which the target substance is dispersed or suspended; - supplying a predetermined amount of the target substance to a test device, such as a flow cytometry device; Includes. Effect of the Invention

[0056] This method makes it possible to carry out tests with the advantages of the purification process described above. The cells or organisms, being protected, do not react or only react slightly, significantly reducing the influence of the process itself on the measurement, making it much more reliable and reproducible. Of course, in all other test methods on target substances dispersed or suspended in a liquid, the target substances may contain impurities or undesirable accompanying or auxiliary substances, which can benefit from this new method.

[0057] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. [Brief description of the drawings]

[0058] [Figure 1] FIG. 2 is a side cross-sectional view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a side cross-sectional view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Diagram 3] FIG. 2 is a top view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Figure 4] FIG. 2 is a top view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Diagram 5] FIG. 2 is a top view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Figure 6A] 6 shows the reaction vessel of FIG. 5 in a side view taken along line VI-VI. [Figure 6B] 6 shows the reaction vessel of FIG. 5 in a modified version, in a side view cut along line VI-VI. [Figure 7] FIG. 2 is a top view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Figure 8] FIG. 2 is a top view of a reaction vessel unit in the form of a microtiter plate according to one embodiment of the present invention. [Figure 9] To illustrate a process according to an embodiment of the present invention, a centrifuge apparatus having two reaction vessel units is shown in FIG. 8 in partial cross-sectional side view. [Figure 10] To illustrate a method according to one embodiment of the present invention, a filled reaction vessel and a magnetic device according to FIG. 1 in a reaction vessel unit are shown in a partially cross-sectional side view. [Figure 11] FIG. 2 is a side cross-sectional view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Figure 12] FIG. 2 is a side cross-sectional view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. [Figure 13A] To illustrate a method according to one embodiment of the present invention, a filled reaction vessel is shown in cross-sectional side view. [Figure 13B] To illustrate a method according to one embodiment of the present invention, a filled reaction vessel is shown in cross-sectional side view. [Figure 13C] To illustrate a method according to one embodiment of the present invention, a filled reaction vessel is shown in cross-sectional side view. [Figure 14] FIG. 2 is a side cross-sectional view of a reaction vessel of a reaction vessel unit according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] All graphic representations should be understood generally. Size ratios may be distorted for clarity. Unless otherwise indicated, designations of directions and positions refer to customary usage for the purposes of the present invention. Unless otherwise indicated, the designation "horizontal" refers to the plane of the opening 6 in which the reaction vessel 1 of the reaction vessel unit 20 is to be filled or emptied, and the designation "vertical" refers to a direction perpendicular to the horizontal.

[0060] The reaction vessel unit 20 has at least one reaction vessel 1 (FIGS. 1-7, 10). When used alone, the reaction vessel 1 can also be understood as a reaction vessel unit within the meaning of the present invention. The reaction vessel 1 has a receiving chamber 2 for receiving a liquid and a capillary cavity 3 opening into the receiving chamber 2. The receiving chamber 2 has a peripheral side wall 4 and a bottom wall 5. The upper edge of the side wall 4 can form an opening 6 of the receiving chamber 2. The capillary cavity 3 opens into the receiving chamber 2 through an opening 7 in the bottom wall 5. The capillary cavity 3 has walls 8 extending from the opening 7, which are closely spaced such that the liquid in the capillary cavity 3 is held in the capillary cavity 3 by capillary action.

[0061] The capillary effect occurs in narrow blood vessels and can be understood as a balance of forces between the surface tension of the liquid and the interfacial tension between the liquid and the vessel surface. In principle, if the liquid wets the wall material, it can rise between the narrow walls by capillary action to form a concave surface, and if the liquid does not wet the wall material, it can fall to form a convex surface. In addition to the surface tension and the contact angle, the capillary effect also depends on the density of the liquid, the radius of the container or the wall distance, and the acceleration of gravity. For example, the rising height h of a column of liquid in a cylindrical container can be calculated by the following formula:

number

[0062] The capillary cavity 3 opens into the receiving chamber 2 and the walls 8 of the capillary cavity 3 are closely spaced such that the liquid is held in the capillary cavity 3 by capillary action, thereby ensuring that the receiving chamber 2 is emptied whilst a precisely defined residual volume is maintained in the capillary cavity 3 when the reaction vessel 1 is emptied. Emptying can be done for example by centrifugation as described in more detail below or by other means such as aspiration / pipetting.

[0063] The bottom wall 5 of the receiving chamber 2 can be flat (Fig. 1). To facilitate both emptying the receiving chamber 2 and collecting in the capillary cavity 3, the bottom wall 5 can also be conically inclined towards the opening 7 of the capillary cavity 3 (Fig. 2, Fig. 13A-13C, Fig. 14) or U-shaped (not shown in detail) to form a kind of funnel towards the capillary cavity 3. The inclination angle γ of the bottom wall 5 relative to the plane of the opening 7 of the capillary cavity 3 (this plane is perpendicular to the vertical V and therefore a horizontal plane) must be adapted to the application and can advantageously be between 5° and 75°. The edge 9 between the bottom wall 5 and the side wall 4 of the receiving chamber 2 can be sharp or rounded, a rounded edge allowing easier emptying of the receiving chamber 2. In the illustrated embodiment example, the side wall 4 of the receiving chamber 2 forms a square cross section (FIGS. 3, 4, 5, 7) and therefore has in particular several side walls 16, which meet at an edge 17 to form a generally closed side wall 4 (see FIG. 3). However, this does not limit the invention. In a variant, the cross section of the receiving chamber 2 can also be rectangular or approximately polygonal, for example diamond-shaped or hexagonal, or circular, oval, elliptical or curved, in any desired manner. The edges 17 between the individual side walls 16 can be sharp or rounded, the rounded edges 17 making it easier to empty the receiving chamber 2.

[0064] The capillary cavity 3 may be circular (FIG. 3), square (FIG. 4), elliptical (FIG. 7) in cross section, or may have any other suitable cross-sectional shape. The opening 7 of the capillary cavity 3 may be located in the center of the bottom wall 5, as in the embodiment shown in FIGS. 1 to 8. In a variant, the opening 7 of the capillary cavity 3 may also be located off-center in the bottom wall 5 of the receiving chamber 2.

[0065] The edge 10 (see Figures 1 and 2) between the wall 8 of the capillary cavity 3 and the bottom wall 5 of the receiving chamber 2 can be sharp or rounded. In this case, the sharp edge 10 can facilitate retention of liquid in the capillary cavity 3 by capillary action.

[0066] The capillary effect is determined by the wall distance between the opposing side walls 18 or wall sections 19 of the wall 8 of the capillary cavity 3; the surface properties and surface tension of the liquid also have a significant influence on the capillary effect. The wall distance between the opposing side walls 18 or wall sections 19 of the capillary cavity 3 is therefore dimensioned such that the liquid is held by capillary action between the opposing side walls 18 or wall sections 19 of the capillary cavity 3. Depending on the nature of the liquid and the wall 8 of the capillary cavity 3, the wall distance may be, for example, approximately 0.1 mm to 2.0 mm. Suitable upper and lower limits have been mentioned above, the specifically selected upper limit being determined by the expected or desired capillary effect, and the specifically selected lower limit being determined by the application. The ratio of the wall distance of the capillary cavity 3 to the wall distance of the receiving chamber 2 itself is also decisive for the application. If the capillary cavity 3 is too wide relative to the receiving chamber, it may be difficult to realize a technically usable difference in the capillary effect. The capillary effect in the capillary cavity 3 is therefore rather small, although it must always be understood in relation to the capillary effect in the receiving chamber 2 which is considered in principle. The capillary effect in the receiving chamber 2 can also be further reduced by widening the side wall 4 of the receiving chamber 2 towards the opening 6 by an opening angle β with respect to the vertical line V (FIG. 14). The opening angle β can be, for example, 0.5° to 5° in order to significantly reduce the capillary effect.

[0067] In one example embodiment, the capillary cavity 3 is formed from two opposing side walls 18 dimensioned such that the distance between them provides a capillary effect, and a bottom wall 12 extending between the side walls 18, the bottom wall 12 being curved and terminating at an edge 11 in an opening 7 in the bottom wall 5 of the receiving chamber 2 (FIGS. 5, 6A). In cross section, the bottom wall forms a smooth continuous arc (FIG. 6A).

[0068] Thus, the capillary cavity 3 in this example embodiment has an elongated cross-sectional shape, especially at the opening 7. Sometimes it may be desirable to first empty the receiving chamber 2 and then also remove any residual volume remaining in the capillary cavity 3. In this example embodiment, this is particularly easy by flushing out the fluid volume held in the capillary cavity 3 by capillary action using a jet of liquid or compressed air along the curved bottom wall 12. In an alternative example embodiment, the capillary cavity 3 may have two opposing side walls 18 dimensioned such that the distance between them exerts a capillary effect, a flat bottom wall 13 extending between the side walls 18, and two end walls 14 extending obliquely from the flat bottom wall 13 at an edge 15 and between the side walls 18, where the inclined end walls 14 terminate at an edge 11 at the opening 7 of the bottom wall 5 of the receiving chamber 2 (FIGS. 5, 6B). An effect of facilitating the emptying of the capillary cavity 3 corresponds to the variant having a curved bottom wall 12, and this effect is more pronounced the more the end wall 14 has a flatter angle.

[0069] When the reaction vessel 1 is emptied by centrifugation, the orientation of the capillary cavity 3 with respect to the direction of rotation determines whether the residual volume is retained in the capillary cavity 3 or is expelled by centrifugation. If the side wall 18 of the capillary cavity 3 is aligned perpendicular to the direction of rotation, the residual volume is retained in the capillary cavity 3. If the side wall 18 of the capillary cavity 3 is aligned with the direction of rotation, the residual volume is expelled from the capillary cavity 3. Therefore, the reaction vessel unit is preferably designed such that the side wall 18 of the capillary cavity is aligned transversely to the direction of rotation during centrifugation.

[0070] In a variant, the capillary cavity can also be formed by a structure located anywhere in the receiving chamber, protruding from the bottom or side wall. Such a capillary cavity can be realized, for example, by a ring structure with a small diameter or by parallel walls forming a capillary gap.

[0071] This can be used, for example, to advantageously form a release system, i.e. a system for time-delayed release of a target substance into a liquid in a receiving chamber. The capillary cavity is filled as described above, and then the receiving chamber is filled with another liquid. Instead of immediately mixing with the liquid, the target substance can slowly pass into the liquid, for example by diffusion. One application is, for example, a series of tests on the long-term effect of a drug on a cell culture. The target substance in the capillary cavity is a drug or reagent that interacts with the cells and whose effect on, for example, cell growth is being tested. A nutrient solution containing the cell culture fills the receiving chamber. The cells may be adherent, i.e. adhere to the surface of the receiving chamber. Alternatively, the cells can be held in a second capillary cavity. The reagent should be in contact with the cells during the experiment. Depending on the design of the capillary, this release system can release the reagent immediately or over a longer period of time. The release system and the release of the reagent can be controlled by applying centrifugal force. For this purpose, it is advantageous for the capillary cavity with the reagent to be open (radially outward) at the top, such as at two vertical ribs in the side wall. If the cells are retained in the second capillary cavity, it is advantageous if the retention effect is greater than in the capillary cavity with the reagent. To prevent leakage of the nutrient solution from the receiving chamber during centrifugation, a lid can be provided which tightly closes the receiving chamber at least during this time.

[0072] To improve the capillary effect when handling aqueous liquids, the capillary cavity 3 can have a hydrophilic coating. To facilitate emptying of the receiving chamber 2, the receiving chamber can have a hydrophobic coating. This can be achieved, for example, by using PTFE, or generally by using a material that has a contact angle of about 90° or more in the presence of water.

[0073] FIG. 11 shows a variant of the reaction vessel 1 in which the wall 8 of the capillary cavity 3 widens towards the opening 7. In other words, the wall 8 has a distance d2 in the region of the opening 7, which is greater than the distance d1 in the region of the bottom wall 13. The widening makes it possible to control the volume removed from the capillary cavity 3 via centrifugal forces during centrifugation, since the force balance between the centrifugal forces and the retention forces depends on the distance between the walls and the angle of widening between them. Preferably, the capillary cavity is designed to widen gradually or almost evenly from the bottom wall 13 to the opening 7, so that the horizontal cross-sectional area of ​​the capillary cavity 3 becomes gradually larger in the direction of the opening 7. This allows the capillary cavity to be emptied to different extents using different centrifugal forces.

[0074] A similar effect can be achieved if the walls of the capillary cavity have a coating with a decreasing hydrophilic or oleophilic effect towards the opening of the capillary cavity. In a variation of the above variation, the wall 8 can be curved to achieve the expansion (FIG. 12). The radius of curvature r of the wall 8 can be constant in the middle of the wall 8 or can be variable.

[0075] The capillary cavity 3 is an example of a retention area, the shape and / or surface properties of which, due to the adhesive forces between the liquid and the retention area and the cohesion within the liquid, the retention area exerts an increased retention effect on the liquid compared to the surrounding areas and thus is able to retain a predefined small amount of liquid when the liquid is removed from the receiving chamber. It is also conceivable to form such a retention area on the bottom or side wall of the receiving chamber only by increasing the adhesive effect with the liquid compared to the surrounding areas of the bottom or side wall, for example by forming a different hydrophilic / hydrophobic or oleophilic / oleophobic, by different coating, roughening, smoothing, etc. It is advantageous, for example, to design the size of such an area according to the desired residual volume, so that a droplet of a defined size is formed, which adheres to the area and remains as compact as possible without breaking up into several partial droplets.

[0076] It is also conceivable to provide several holding areas in one receiving chamber. The holding areas can be of the same design to accommodate several partial volumes of the same or different types, for example to accommodate or hold partial volumes or target substances of different types. For example:

[0077] One embodiment of the present invention is a reaction vessel unit 20 in the form of a microtiter plate having a frame 21 containing a plurality of individual reaction vessels 1 (FIG. 8). The individual reaction vessels 1 of the microtiter plate 20, also called wells, are usually arranged in a predefined regular grid of rows and columns. The grid specifies the staggered number of reaction vessels 1 and therefore also determines the possible filling volume. Cells are cultured individually or in cell clusters in such microtiter plates in the context of cell-based assays. As mentioned at the beginning, the cellular structures should usually remain in the reaction vessels when the microtiter plate is washed, but for example the supernatant is removed and replaced with fresh medium. This can be achieved particularly well with the reaction vessel unit according to the invention by providing a microtiter plate in which a capillary cavity is formed at the bottom of each well.

[0078] Microtiter plates are available in a variety of sizes and designs. The base area usually corresponds to the ANSI standard recommended by the Society for Biomolecular Screening (SBS) of L=27.76 mm x W=85.48 mm x H=14.35 mm, the height can vary. Common sizes are listed in the table below: [Table 1] As shown in.

[0079] The name of the microtiter plate depends on the number of wells. For example, a microtiter plate with 24 wells is called a 24-well microtiter plate, a microtiter plate with 384 wells is called a 384-well microtiter plate, etc. The wells can have different bottom shapes such as F-bottom (flat bottom), C-bottom (flat bottom with minimally rounded edges), V-bottom (tapered bottom) and U-bottom (U-shaped well). The wells can have different cross-sectional shapes such as circular, oval, elliptical, square, diamond, hexagonal, and polygonal shapes can also have rounded edges.

[0080] In the illustrated embodiment example, the reaction vessel unit 20 is a 96-well microtiter plate, with 8 rows arranged in the direction of width B and 12 columns arranged in the direction of width B. The spacing x of the reaction vessels 1 is the same in the length direction and in the width direction, but the invention is not limited to this. In the illustrated embodiment example, the reaction vessels 1 and their receiving chambers 2 are each of circular cross section, and the capillary cavities 3 embedded in the bottom walls of the receiving chambers 2 are also of circular cross section. However, the invention is not limited to this. For the sake of simplicity of illustration, the reaction vessel unit 20 is marked only partially complete, the contours beyond the crack line are only dashed lines, and the reaction vessels 1 are only indicated by their centers.

[0081] Known centrifuges for washing microtiter plates are designed such that the rotation axis of the centrifuge is parallel to the longitudinal direction of the respective microtiter plate, and therefore the above-mentioned elongated capillary cavities are advantageously also aligned parallel to the longitudinal direction of the microtiter plate.

[0082] Cells are grown in microtiter plates in the context of cell-based assays. The cells bind to a surface (become adhesive) and sooner or later the medium must be changed, because otherwise the culture medium will have a negative effect on the cells or will make them unusable. There are inventive methods that allow for centrifugation-based washing of microtiter plates, for example those described in US Pat. No. 5,399,433 and US Pat. No. 5,499,446. In recent years, cell assemblies are increasingly used in the pharmaceutical industry for testing purposes. These cell assemblies are spheroids or organoids, i.e. clusters (up to several hundred) of cells. These cell assemblies are also grown in microtiter plates, the smooth surface of which often prevents cell adhesion. This means that spheroids and organoids float freely in the solution. In order to perform a medium change, it is desirable to fix the cell clusters so that the medium can be changed without losing cells. Various matrices can be used for this purpose, which are poured into the plate to allow the cells to be captured and allowed to grow. An example is the matrix GrowDex (www.Growdex.com). The wells are filled one third with matrix and two thirds with culture medium. The matrix has almost gel-like properties and adheres to the plate, but is not very strong. In centrifugation-based washing, it is only desired to wash the medium off the plate without washing away the cells (including the matrix). However, above a certain centrifugal acceleration, the gel will also be released from the plate.

[0083] The invention is very well applicable to such reaction vessel units 20 in the form of microtiter plates that are cleaned by means of a centrifuge. For a given system of capillary cavities 3 and liquid, a limit value can be determined for the acceleration acting in the direction away from the opening 7 of the capillary cavities 3, below which the capillary effect becomes dominant. If the microtiter plate is emptied as a reaction vessel unit 20 while retaining a residual volume in the capillary cavities 3, the speed of the centrifuge can be set to a value safely below the limit value. The capillary cavities 3 of the reaction vessels 31 have an equally advantageous effect if the reaction vessels 1 are emptied by other means, such as a pipetting device.

[0084] Prototypes have demonstrated that the limiting speed of aqueous solutions with uncoated polycarbonate microtiter plates is typically in the range of several hundred rpm, preferably below 1000 rpm, especially below 800 rpm or 500 rpm or 300 rpm.

[0085] Both the microtiter plate and also other types of reaction vessel units or reaction vessels can advantageously be made of plastic and can be manufactured using known manufacturing processes such as injection molding. In particular, the reaction vessel unit 20 in the form of a microtiter plate can be formed integrally with all the reaction vessels 3. However, the invention is not limited to the choice of material nor to the one-piece design.

[0086] A method for selectively removing liquid from reaction vessels 1 of a reaction vessel unit 20 using a centrifuge 30 is another embodiment of the present invention (FIG. 9).

[0087] The centrifuge 30 is shown only in the parts essential for the understanding of the invention. It has a frame-like rotor 31 fixed in rotation to a shaft 32. The shaft 32 can be set to rotate by a motor (not shown in detail) for rotating at an angular velocity ω or a corresponding rotational speed n about a centrifugation axis 33, which can be controlled and regulated by a control unit (not shown in detail), which also rotates the rotor 31. A cover 34, which can be in the form of a cylindrical tube or be part of a housing not shown in detail, surrounds the frame 31. A carrier 35 for receiving the reaction vessel units 20 is arranged in a receiving section of the rotor 31. In particular, the carrier 35 together with the reaction vessel units 20 in the form of microtiter plates can be pressed into said receiving section from an end face of the rotor 31 by a loading unit (not shown in detail), which can receive the carrier 35 in the manner of a rail and hold the reaction vessel units 20 in the manner of a clamp radially from the outside.

[0088] To apply this method, first, liquid is placed in the reaction vessel 1 of the reaction vessel unit 20, as described above. Then, the reaction vessel unit 20 is placed on a carrier 35 and loaded into the centrifuge 30, with the opening 6 of the receiving chamber 2 of the reaction vessel 1 facing away from the centrifugation axis 33 (top (A) of FIG. 9). Finally, the rotor 31 is set to rotate together with the reaction vessel unit 1, the speed n being controlled to be less than the limit speed at which the capillary effect of the capillary cavity 3 is just overcome. In this way, it is possible to ensure that the liquid in the capillary cavity 3 remains therein and that the remaining liquid in the receiving chamber 2 is centrifuged, i.e. removed. For this purpose, the centrifuge 30 can be controlled with a suitable time-speed profile. The centrifuged liquid is collected inside the cover 34 and can be drained. It can also be captured in a catcher plate or other collection device, which is placed on the reaction vessel unit 20 with at least one of its openings before centrifugation so that the centrifuged liquid can be reused. The carrier 35 with the washed reaction receptacle units 20 can be removed and the reaction receptacle units 20 with the residual volume held in the capillary cavities 3 can be reused. When target material, e.g. a cell array in a matrix, has been received in the capillary cavities 3 and the used nutrient solution has been removed from the receiving chamber 2 by the methods described above, the receiving chamber 2 can be filled with fresh nutrient solution and the cells can be further grown.

[0089] Of course, the process is also suitable for separating any volume of liquid.

[0090] The method of introducing the liquid 40 containing the target substance 41 into the reaction vessel 1 of the reaction vessel unit 20 is another embodiment of the present invention and can also be carried out using a centrifuge device 30 (FIG. 9).

[0091] To carry out the method, first the liquid 40 is held in one of the reaction vessels 1 with the reaction vessel unit 20 together with the target substance 41 (FIG. 10). The target substance 41 may or may not be embedded in a matrix 42. The auxiliary substance 43 can also, but does not have to, be embedded in the matrix 42. The reaction vessel unit 20 is then placed on the trough 35 and loaded into the centrifuge 30, with the opening 7 of the capillary cavity 3 of the reaction vessel 1 oriented radially relative to the centrifugation axis 33 (lower part (B) of FIG. 9). The rotor 31 with the reaction vessel unit 1 then rotates, where a centrifugal acceleration a acts in the direction of the bottom wall 5 of the receiving chamber 2, which separates the target substance 41, possibly together with the matrix 42 and any auxiliary substance 43, from the rest of the liquid 40 and forces it into the capillary cavity 3 of the reaction vessel 1. In this case, the speed n of the centrifuge device 30 can be controlled to be greater than a second limit speed, at which speed it is ensured that any resistance that may arise from the volume of air or liquid present in the capillary cavity 3 is pushed out of the capillary cavity 3. For this purpose, the centrifuge device 30 can be controlled with a suitable time-speed profile. The trough 35 with the reaction vessel unit 20 can then be removed and the reaction vessel unit 20 with the target substance 41 held in the capillary cavity 3 can continue to be used. If the target substance 41, for example a cell array in a matrix 42, is contained in the capillary cavity 3 and the remaining liquid 40 is a nutrient liquid, the reaction vessel 1 or the reaction vessel unit 20 can be used to attract the cells. The nutrient liquid can be constantly removed and replaced with new nutrient liquid using the above method as necessary, until the reaction vessel 1 is completely empty, ensuring that the target substance 41 remains in the capillary cavity 3.

[0092] By positioning the reaction vessel unit 1 so that the opening 7 of the capillary cavity 3 is oriented radially relative to the centrifugation axis 33, the centrifuge 30 forms a guiding system designed to move the target material in a desired direction within the meaning of the present invention.

[0093] A further method for introducing a liquid 40 containing a target substance 41 into a reaction vessel 1 of a reaction vessel unit 20 is a variant of the previously described embodiment of the invention, which differs in the guiding system used. This variant uses as guiding system an auxiliary material 43 which is magnetic and a magnetic device 44 which is arranged to interact with the magnetic auxiliary material 43 in order to carry out the process (Figure 10).

[0094] Again, the liquid 40 containing the target substance 41 can be filled and received in the reaction vessel 1 of the reaction vessel unit 20. The target substance 41 can be, for example, a cell structure such as a spheroid or an organoid, but the invention is not limited thereto. The auxiliary material 43 can, for example, comprise magnetic beads. The magnetic device 44 can comprise an electromagnet 45 with a ferrite core 46 and a winding 47, and a current source 48. To carry out the method, the electromagnet 45 can be placed directly below the capillary cavity 3 of the reaction vessel 1. In practice, the magnetic device 44 can be stationary and the reaction vessel 1 can be positioned relative to the magnetic device 44, so that the capillary cavity 3 of the reaction vessel 1 is located directly above the electromagnet 45. In particular, if the reaction vessel 1 is part of the reaction vessel unit 20, such as a microtiter plate, the magnetic device can have a number of electromagnets 45 that are precisely positioned in the grid wells of the microtiter plate. When the power supply 48 is switched on, the ferrite core 46 becomes polarized and can exert a magnetic attraction on the magnetic auxiliary material 43. The magnetic auxiliary material 43 is thus forced into the capillary cavity 3 and pulls the matrix 42 together with the target material 41. Instead of one or more electromagnets, one or more permanent magnets can also be used.

[0095] Instead of the magnetic auxiliary material 43, the magnetic properties of the target material 41 or matrix 42 itself can also be utilized to interact with the magnetic device 44. For example, the target material 41 can include ferrite materials or biomagnetically active cells. Biomagnetic phenomena are often very weak, so the magnetic device 44 may need to be designed accordingly to achieve the required interaction.

[0096] A further variant of the method for introducing a liquid containing a target substance into a reaction receptacle 1 of a reaction receptacle unit 20 again differs in the guiding system used. This variant uses as guiding system an electrostatically charged auxiliary material 43 and an electrode device 49 arranged to interact with the auxiliary material 43 for carrying out the method (FIG. 10).

[0097] Again, the liquid 40 with the target substance 41 can be filled and received in the reaction vessel 1 of the reaction vessel unit 20. The target substance 41 can be, for example, a cellular structure such as a spheroid or an organoid, but the invention is not limited thereto. The auxiliary material 43 can, for example, comprise ionized particles. The electrode device 49 can comprise an electrode 50 extending below the capillary cavity 3 of the reaction vessel 1 and a current source (not shown in detail). The electrode 50 can be integrated into the bottom of the reaction vessel 1 or the bottom of the reaction vessel unit 20, or can be provided externally, in the latter case the electrode 50 must be placed below the capillary cavity 3 of the reaction vessel 1 to carry out the process. When the power supply is switched on, the electrode 50 exerts an attractive force on the charged auxiliary material 43. This causes it to push into the capillary cavity 3, pulling the matrix 42 with the target material 41.

[0098] The electrode may have a flat extension in the region of the capillary cavity 3, whereas in the region away from the capillary cavity 3 the electrode becomes thinner and the electrostatic attraction is concentrated below the capillary cavity 3. In particular, if the reaction vessel 1 is part of a reaction vessel unit 20 such as a microtiter plate, the electrode 50 may have a grid-like design, the grid nodes being precisely located within the grid of the wells of the microtiter plate and may have the above mentioned flat extension.

[0099] Instead of an electrostatically charged auxiliary material 43, it is also possible to utilize the electrostatic properties of the target material 41 or matrix 42 itself to interact with the electrode device 49. For example, the target material 41 can carry charged cells or molecules. Since such charges are often very weak, a corresponding design of the electrode device 49 may be necessary to achieve the required interaction.

[0100] In this embodiment with all modifications, after successful introduction of the target substance, the supernatant, i.e. the residual volume not received in the capillary cavity, can be removed from the receiving chamber 2 using the methods described above for separation or selective removal. Again, a collection device can be advantageously used to collect the residual volume, for example so that expensive reactants can be reused or they can be utilized elsewhere.

[0101] The basic selective removal process has other possible applications.

[0102] For example, it is possible to empty the reaction vessels of a reaction vessel unit differently or to introduce the target substance differently into the reaction vessels of a reaction vessel unit when different g-forces act during centrifugation (this is especially true for small radii, since the reaction vessels at the outer radial positions are accelerated more). It is also possible to imagine a series of titrations along a g-force gradient.

[0103] Conversely, reaction vessel units are conceivable in which forces arise due to different adhesion or capillary action, whereby physically existing gradients in centrifugal acceleration (due to different radii) are precisely balanced by appropriate formation of the retention area, so that the retained partial volumes have the same size everywhere after centrifugation, despite this gradient.

[0104] The principles of the invention can also be used to form a system for controlled, time-delayed release of a target substance into a sample, a so-called release system. In a variant, the capillary space can also be formed by a structure located anywhere in the receiving chamber, protruding from the bottom or side wall and forming a capillary gap. This can be used to: A capillary located anywhere in the plate is filled as described herein; A reagent is a substance intended to interact with cells, for example its effect on cell proliferation is tested; In cell experiments, the reagent is usually brought into contact with cells in the receiving chamber; The cells may be adhesive, i.e. adhere to the surface of the receiving chamber; Depending on the design of the capillary, this release system can release the reagent immediately or over a longer period of time; The release system and the release of the reagent can be controlled by applying centrifugal force.

[0105] A particularly advantageous application of the invention is a method for purifying cells forming a target substance 41. The target substance 41 can equally also include particularly organic / biological substances such as cell clusters, cell aggregates or organisms, but in the following, by way of example, only cells are mentioned. The cells are located in a reaction vessel 1 of a reaction vessel unit suspended in a liquid 40. The reaction vessel 1 has a funnel-shaped bottom wall 5 opening into a capillary cavity 3. In a specific example, the reaction vessel unit can be designed, for example, in a 96 microtiter plate format. The reaction vessel 1 can have a receiving chamber 2 with a working volume of 200 μl and a capillary cavity 3 with a volume of 5 μl. The cells are initially distributed approximately uniformly in the receiving chamber 2 of the reaction vessel unit 1, which may also contain cells whose distribution essentially corresponds to that in the remaining receiving chamber 2.

[0106] At defined intervals or to test the cells in suspension, it is necessary to replace the liquid 40 with fresh liquid 40 or another liquid 40 without removing the cells from the reaction vessel 1. This replacement is called purification.

[0107] In this method, in a first step, the reaction vessel unit is removed to the centrifuge with the capillary cavity 3 facing away from the centrifugation axis 33 (see arrangement (B) in FIG. 9) and rotated around the centrifugation axis 33 with an angular velocity ω1. This causes an acceleration a to act on the suspension, which causes the cells to collect in the capillary cavity 3 supported by the inclination of the funnel-shaped bottom wall 5 (FIG. 13A). In this example, the angular velocity ω1 is set so that the acceleration a is 10-30 g and the centrifugation takes 2-4 minutes. At the end of this step, there are no or very few cells left in the receiving chamber 2 above the capillary cavity 3.

[0108] In a second step, the reaction vessel unit is removed to the same or another centrifuge (see arrangement (A) in FIG. 9) with the capillary cavity 3 facing the centrifugation axis 33 and rotated around the centrifugation axis 33 with an angular velocity ω2. The angular velocity ω2 is set such that the liquid 40 is released from the receiving chamber 2 of the reaction vessel 1 under the effect of an acceleration a, while the cells in the capillary cavity 3, still suspended but at a very high concentration, are held in the capillary cavity 3 by the capillary force K. The liquid 40 in the receiving chamber 2 separates from the liquid 40 containing the cells in the capillary cavity 3, and the atmosphere 130 flows into the space above the capillary cavity 3, resulting in the formation of a phase boundary 131 spanning the opening 7 of the capillary cavity 3, whose surface tension contributes to the capillary effect (FIG. 13B). At the end of this step, the receiving chamber 2 above the capillary cavity 3 is completely emptied of the liquid 40 and the cells are concentrated in the capillary cavity. The emptying process may take approximately 30 seconds in this example.

[0109] In a third step, the receiving chamber 2 of the reaction vessel 1 is refilled with liquid 40. In this embodiment, this is done by a dispensing device. The liquid 40 is dispensed into the receiving chamber 2 in a dispensing nozzle 132. A liquid flow 133 has a velocity v at the outlet of the dispensing nozzle 132. In this method, the dispensing nozzle 133 is directed towards the capillary cavity 3 in such a way that the cells (41) are washed or rinsed out of the capillary cavity 3 and distributed in the liquid 40 throughout the receiving chamber 2 (FIG. 13C). The velocity is such that the cells are washed out of the capillary cavity 3 but the liquid 40 does not fall out of the reaction vessel 1 and the cells always remain suspended in the liquid 40. Although shown as illustrated, the dispensing nozzle 132 does not have to protrude into the receiving chamber 2. Rather, the dispensing nozzle 132 can also end above the opening 6 of the receiving chamber 2. At the end of this step, the cells are again approximately evenly distributed in the liquid 40 in the receiving chamber 2. In this example, the filling process may take about 15 seconds (preferably for all 96 reaction vessels 1 in this example, which are filled specifically row by row).

[0110] Using this process, stress on cells during purification can be significantly reduced compared to conventional processes such as pelleting. Possible cell losses during emptying in the second step can be kept to a minimum (<10%) and can be replaced as needed before, after or during loading.

[0111] The dispensing device may be attached to the centrifuge and the reaction vessel unit may be moved by a loading and unloading device of the centrifuge, allowing a precise and efficient positioning of the reaction vessel unit with respect to the rotation space of the centrifuge and with respect to the dispensing device. The reaction vessel unit may remain on or in the centrifuge during the entire process. A centrifuge having a loading and unloading device with a rigid displacement rod for positioning the reaction vessel unit in the rotor of the centrifuge or for unloading the reaction vessel unit from the rotor of the centrifuge, the displacement rod being displaceably arranged by a linear motor so as to move in an unloading position, extending through the rotor in a discharge position in the rotor chamber and being withdrawn from the area of ​​the rotor chamber occupied by the rotor during at least one revolution in a loading position, is described in US Pat. No. 5,399,992, the disclosure of which is hereby incorporated by reference in its entirety. A centrifuge with such loading and unloading, in which a distribution unit is also attached to the outer wall of the centrifuge, by means of which reagent liquid can be supplied in the discharge position to a reaction vessel unit located below it, is described in the forthcoming US Pat. No. 5,399,433, the disclosure of which is hereby incorporated by reference in its entirety.

[0112] It will be appreciated that collection of the cells in the capillary cavity 3 may be achieved by means other than centrifugation, for example by magnetic, electrostatic means as described above. Removal of the liquid 40 may be performed by means other than centrifugation, such as by pipetting. Refilling may also alternatively be performed by pipetting.

[0113] The purification process described above can be advantageously used in connection with a test procedure in which cells are tested in suspension. A partial volume of the suspension in a purified state is removed for this purpose and fed into a test device. Cells lost due to testing can be optionally replaced.

[0114] The capillary cavity 3 is essentially free of walls to avoid shading by dispensing jets, rinsing jets etc., which may form dead zones where target material undesirably remains during cleaning. However, to facilitate rinsing or pipetting, a ridge 140 may be provided at the bottom of the capillary cavity 3, having a central, for example pyramidal or conical, tip 141 (FIG. 14). The remainder of the base of the capillary cavity 3 around the ridge 140 may be designed with as few edges as possible to allow as laminar flow as possible.

[0115] The present invention can be briefly summarized as follows: The present application discloses a reaction receptacle unit (20) having at least one reaction receptacle (1) having a receiving chamber (2) for receiving a liquid (40), the receiving chamber (2) having a retention area, the retention area having a surface texture and / or shape that exerts an increased retention effect on the liquid (40) compared to the surrounding areas due to adhesion forces between the liquid and the retention area and cohesion within the liquid, such that when the liquid (40) is removed from the receiving chamber (2) by centrifugation, a predetermined small amount of the liquid is retained at or in the retention area. Further, a method is disclosed for selectively removing a liquid (40) from a reaction receptacle (1) of the reaction receptacle unit (20) by centrifugation. Further, a method is disclosed for introducing a liquid (40) containing a target substance (41) into a reaction receptacle (1) of the reaction receptacle unit (20), the target substance (41) being guided to the retention area by a guidance system by centrifugation or by magnetic or electrostatic interactions. The present application further discloses a method for purifying a target substance (41) dispersed or suspended in a liquid (40), such as a cell, a cell cluster, a cell aggregate or an organism, and a method for performing tests on a target substance (41) dispersed or suspended in a liquid (40) using a reaction vessel unit (20) according to the present invention.

Claims

1. A reaction vessel unit (20) comprising a microtiter plate in which a plurality of reaction vessels (1) are arranged in a predetermined grid, or a microtiter plate, At least one of the reaction vessels (1) has a receiving chamber (2) for receiving a liquid (40), all of the reaction vessels (1) have a mouth (6) on the upper side of the microtiter plate, the receiving chamber (2) has a retention area, the retention area has a surface texture and / or shape that exerts an increased retention effect on the liquid (40) compared to surrounding areas due to adhesion forces between the liquid and the retention area and cohesion within the liquid, so that when the liquid (40) is removed from the receiving chamber (2) by centrifugation, a predetermined small amount of liquid is retained at or within the retention area, The holding area has a capillary cavity (3) that opens into the receiving chamber (2), and the side walls (8) of the capillary cavity (3) are spaced very closely together so that the liquid (40) is held within the capillary cavity (3) by capillary action.

2. 2. The reaction vessel unit (20) of claim 1, characterized in that the receiving chamber (2) is bounded by one or more circumferential side walls (4) and a bottom wall (5), and the holding area is formed in or on the bottom wall (5) of the receiving chamber (2).

3. 2. The reaction vessel unit (20) of claim 1, wherein the holding area comprises a single capillary cavity (3).

4. 4. The reaction vessel unit (20) according to claim 3, characterized in that the capillary cavity (3) is essentially free of dividing walls so that the contents of the capillary cavity (3) can be completely removed from the capillary cavity by introducing a fluid jet.

5. 5. The reaction receptacle unit (20) according to claim 3 or 4, characterized in that the capillary cavity (3) has a rectangular, circular or elliptical cross section, and a wall distance (d) between opposing side walls (8, 18) or wall sections (19) of the capillary cavity (3) is dimensioned to hold a liquid (40) by capillary action between the opposing side walls (8, 18) or wall sections (19) of the capillary cavity (3), the wall distance (d) being preferably at most 2.0 mm, or at most 1.8 mm, or at most 1.6 mm, or at most 1.4 mm, or at most 1.2 mm, or at most 1.0 mm or at most 0.8 mm, and the wall distance (d) is preferably at most 30%, or at most 25%, or at most 20%, or at most 15%, or at most 10%, or at most 5% of the wall distance between the side walls (4) of the receiving chamber (2).

6. 5. The reaction vessel unit (20) according to claim 3 or 4, characterized in that the capillary cavity (3) has a depth which is at least 0.5 times, or at least 1 time, or at least 1.5 times, or at least 2 times the wall distance (d) between the opposing side walls (8, 18) or wall sections (19) of the capillary cavity (3).

7. 5. The reaction vessel unit (20) according to claim 3 or 4, characterized in that the side walls (8, 18) of the capillary cavity (3) are formed vertically or substantially vertically with a deviation from the vertical of at most 5°, at most 3°, at most 2°, at most 1° or at most 0.5°.

8. 5. The reaction vessel unit (20) according to claim 3 or 4, characterized in that the bottom wall (5) of the receiving chamber (2) is conically inclined or U-shaped towards the opening (7) of the capillary cavity (3), the inclination angle (γ) of the bottom wall (5) being at least 5°, or at least 15°, or at least 25°, or at least 35°, or at least 45°, and / or at most 75°, or at most 65°, or at most 55°, or at most 45°.

9. 5. The reaction vessel unit (20) according to claim 3 or 4, characterized in that the side wall (4) of the receiving chamber (2) is formed so as to widen towards the opening (6) with a deviation of at least 2°, or at least 3°, or at least 5°, or at least 10° from the vertical.

10. The capillary cavity (3) has two opposing side walls (18) dimensioned such that a wall distance (d) provides a capillary effect, and a bottom wall (12; 13) extending between the side walls; 5. A reaction vessel unit (20) according to claim 3 or 4, characterized in that the bottom wall (12) is continuously curved or is further provided with two end walls (14) extending between the side walls (18) and rising from the bottom wall (13) to the edge of the capillary cavity (3) at an angle, in particular at a flat angle or with a concave curvature.

11. 5. A reaction vessel unit (20) according to claim 3 or 4, characterized in that the side walls (8) of the capillary cavity (3) have a widening and / or a coating which reduces the hydrophilic or oleophilic effect towards the opening (7) of the capillary cavity (3), the opening angle at the opening (7) being preferably 10° or less, or 5° or less, or 2° or less, or 1° or less, or 0.5° or less.

12. The reaction vessel unit (20) according to any one of claims 3 to 4, characterized in that the reaction vessel unit (20) comprises a plurality of reaction vessels (1), and the capillary cavities (3) are angled progressively away from the centre of a line, in particular a centre line, dividing the reaction vessel unit (20) or the arrangement of reaction vessels (1) into two halves, with respect to a perpendicular line on a plane in which the reaction vessels (1) are arranged.

13. The reaction vessel unit (20) of any one of claims 1 to 4, characterized in that the holding area has a surface structure that enhances adhesion to the liquid (40) and a size that allows molecular aggregation to form by said aggregation in the liquid, and a predetermined amount of the liquid is held in or within the holding area.

14. The reaction receptacle unit (20) according to any one of claims 1 to 4, characterized in that the holding area has a hydrophilic or oleophilic coating.

15. The reaction vessel unit (20) according to any one of claims 1 to 4, characterized in that the receiving chamber (2) is coated hydrophobically or oleophobically in the surrounding area of ​​the holding area, in particular in the area complementary to the holding area.

16. 5. The reaction receptacle unit (20) according to claim 1, wherein the reaction receptacle unit (20) comprises a plurality of reaction receptacles (1), and the holding effect of the holding areas of at least two reaction receptacles (1) is different, preferably such that the holding effect increases from a line, in particular a centerline, dividing the reaction receptacle unit (20) or the arrangement of the reaction receptacles (1) into two halves, so that when the reaction receptacle unit (20) is rotated about an axis that is parallel to the centerline and whose radius extends through the centerline at a right angle to the plane in which the reaction receptacles (1) are arranged, the holding force of the holding areas remains constant or nearly constant across the reaction receptacles (1).

17. 2. The reaction vessel unit (20) according to claim 1, characterized in that it is provided with a collecting device arranged opposite one opening of one of the receiving chambers (2) of at least one reaction vessel (1) or opposite the openings of the plurality of receiving chambers (2) and designed to capture liquid (40) leaking or being discharged from the one receiving chamber (2) or the plurality of receiving chambers (2).

18. 18. The reaction vessel unit (20) of claim 17, characterized in that the collection device has one or more compartments, preferably in the form of a microtiter plate, and one opening of one compartment of the collection device or multiple openings of multiple compartments faces one opening of one receiving chamber (2) of at least one reaction vessel (1) or multiple openings of multiple receiving chambers (2), and the number of compartments of the collection device is equal to, greater than, or less than the number of reaction vessels (1).

19. 1. A method for selectively removing a liquid (40) from a reaction vessel (1) of a reaction vessel unit (20), the liquid (40) being in a reaction vessel unit (20) according to claim 1, the method comprising: centrifugally separating the reaction vessel unit (20) with the opening of the receiving chamber (2) facing radially away from the centrifugation axis (33) at a speed just below the threshold speed at which the retention effect of the retention area is overcome, so that a partial volume of the liquid (40) located in or at the retention area remains therein and the remaining liquid (40) located in the receiving chamber (2) is removed.

20. 20. The method according to claim 19, wherein the remaining liquid (40) is collected in a collection device, in particular as defined in claim 17.

21. A method for introducing a liquid (40) containing a target substance (41) into a reaction vessel (1) of a reaction vessel unit (20), comprising: - using a reactor vessel unit (20) according to claim 1 to contain said liquid (40); - guiding the target material (41) to the holding area by a guidance system, the guidance system comprising: - a centrifuge device (30) and an arrangement of the reaction vessel unit (20) in which the holding area is radially outward or substantially outward with respect to the centrifugation axis (33) with respect to the remainder of the receiving chamber (2); a magnetic device (44) arranged to interact with the magnetic properties of the magnetic auxiliary material (43) or the target material (41), or an electrostatically charged auxiliary material (43) or electrostatic properties of the target substance (41) and an electrode device arranged to interact with the electrostatically charged auxiliary material (43) or electrostatic properties of the target substance (41); A method comprising:

22. Centrifuging the reaction receptacle unit (20) with the holding area being radially inward or substantially inward with respect to the centrifugation axis (33) with respect to the remainder of the receiving chamber (2) at a speed just below the threshold speed at which the retention effect of the holding area is overcome, so that a partial volume of the liquid (40) that has passed into or up to the holding area and contains the target substance (41) remains therein and the remaining liquid (40) located in the receiving chamber (2) is removed.

22. The method of claim 21 further comprising:

23. 23. A method according to claim 22, wherein the remaining liquid (40) is collected in a collection device, in particular as defined in claim 17.

24. A method for purifying a target substance (41), such as a cell, a cell cluster, a cell aggregate or an organism, dispersed or suspended in a liquid (40) in a reaction vessel (1) of a reaction vessel unit (20), said liquid (40) being located in a reaction vessel unit (20) according to claim 1, said method comprising the steps of: - collecting the target substance (41) in the holding area, in particular by centrifuging using the arrangement of the reaction receptacle units (20), so that the holding area is located radially outward or substantially outward relative to the centrifugation axis (33) with respect to the remainder of the receiving chamber (2); - removing the liquid (40) from the receiving chamber (2) by centrifuging the reaction receptacle unit (20) with the opening of the receiving chamber (2) facing radially away from the centrifugation axis (33) at a speed below the threshold speed at which the retention effect of the retention area is just overcome, so that a partial volume of the liquid (40) located in or at the retention area remains therein and the remaining liquid (40) located in the receiving chamber (2) is removed; - introducing a further liquid (40) into said receiving chamber (2), in particular by dispensing or pipetting; - mixing the target substance (41) with another liquid (40) in the receiving chamber (2); A method comprising:

25. The mixing step comprises: - leaving it for a predetermined time; - shaking the reaction vessel unit (2); - introducing the further liquid (40) into the receiving chamber (2) by dispensing or pipetting so as to wash or flush the target substance (41) from the holding area; - pipetting the target substance (41) out of the holding area and back into the further liquid (40) introduced outside the holding area; 25. The method of claim 24, comprising at least one of:

26. 25. The method of claim 24, wherein the receiving chamber (2) has a volume of the order of 200-400 μl, the holding area is a capillary cavity (3) at the bottom of the receiving chamber (2) and has a volume of about 5 μl, and the collection of the target substance is carried out by centrifugation of at least 2 g, or at least 5 g, or at least 10 g, or at least 20 g, and / or up to 1000 g, or up to 500 g, or up to 200 g, or up to 100 g, or up to 50 g, or up to 40 g, or up to 30 g, or up to 20 g, for a time of at least 1 second, or at least 2 seconds, or at least 5 seconds, or at least 10 seconds, or at least 10 seconds, or at least 30 seconds, or at least 60 seconds, or at least 90 seconds, or at least 2 minutes, or at least 5 minutes, and / or up to 60 minutes, or up to 30 minutes, or up to 20 minutes, or up to 15 minutes, or up to 10 minutes, or up to 5 minutes, or up to 2 minutes, or up to 1 minute, or up to 30 seconds, or up to 20 seconds, or up to 10 seconds, or up to 5 seconds, or up to 2 seconds.

27. 25. Method according to claim 24, characterized in that the remaining liquid (40) is collected in a collecting device, in particular as defined in claim 17.

28. 10. A method for performing a test on a target substance (41) dispersed or suspended in a liquid (40), such as a cell, cell cluster, cell aggregate or organism placed in a reaction vessel (1), said liquid being located in a reaction vessel unit (20) according to claim 1, said method comprising the steps of: - purifying the target substance (41) according to the method of claim 24; - withdrawing a predetermined amount of the liquid (40) from the reaction vessel (1) with the target substance (41) dispersed or suspended therein; - supplying the target substance (41) contained in the predetermined amount to a testing device such as a flow cytometry device; A method comprising:

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