Centrifuge or ultracentrifuge equipped with a centrifugal rotor, sample extraction needle, and method for extracting a sample from a centrifuge tube in situ.

Abstract

A centrifuge rotor including a rotor assembly adapted for centrifugally rotating about a central axis of rotation, the rotor assembly comprising a plurality of centrifuge tube beds each having a longitudinal axis, each centrifuge tube bed comprising a tubular cavity defined by cavity side walls and a cavity bottom which together act as a support surface for an outer surface of a centrifuge tube when the centrifuge tube is received within the centrifuge tube bed, at least one of the plurality of cavity bottoms comprising at least one extraction opening connecting the tubular cavity to an exterior of the rotor assembly, and preferably at least one closure device removably fixed to the rotor assembly to hermetically seal the at least one extraction opening.

Description

Technical Field

[0001] The present invention relates to a centrifuge or an ultracentrifuge, and more specifically, to a centrifuge rotor or an ultracentrifuge rotor used in a method for separating liquid substances of cell organelles, viruses, virus-like particles, bacteriophages, exosomes and nanoparticles using the centrifuge or the ultracentrifuge. Further, the present invention relates to the Location where it was contained ( in-situ ) sampling method of a sample contained in a centrifuge tube bed of a sampling needle, a centrifuge rotor or an ultracentrifuge rotor, and the use of a centrifuge rotor and a sampling needle for carrying out the method of sampling a sample in-situ.

[0002] The present invention describes an ultracentrifuge rotor or a centrifuge rotor that enables in-situ liquid sample collection and liquid sample fractionation in an ultracentrifugation process and a centrifugation process without removing the centrifuge tube from a centrifuge tube rotor assembly or a centrifuge tube bucket. Further, the self-sealing design of a sampling needle is described to enable automatic sample extraction from a centrifuge tube still housed within the rotor assembly of a centrifugal separator rotor.

Background Art

[0003] Centrifuge and ultracentrifugation are among the fundamental techniques for separating particulate fractions such as organelles (US 2006 / 266715 A1), viruses (US 10066213 B2), virus-like particles (US 6,077,662 A), bacteriophages (AU 680279 B2), exosomes (WO 2015 / 048844 A1), and nanoparticles (WO 2006 / 069985 A2). Centrifuge and ultracentrifugation techniques include pelletization, in which the particulate fraction is gradually removed from the suspension by forming a pellet at the bottom of the tube. Gradient separation, in which the tube is filled from top to bottom with increasing concentrations of the denser substance in the solution, is also often used to separate particulate fractions based on the particulate fraction density and the particulate fraction sedimentation coefficient. The sample is spun at high speed for a sufficient time to achieve separation. After centrifugal separation or ultracentrifugation, the rotor is brought to a smooth stop, and the gradient is gently pulled out from each tube to isolate the separated components using different approaches.

[0004] Centrifuges and ultracentrifuges are equipped with a wide variety of rotors suitable for a wide range of experiments. Recently, centrifuge development has mainly focused on automated sample loading (US 2008 / 318755 A1) and automated operation of centrifuges (US 8,795,144 B2).

[0005] Centrifugal and ultracentrifugation tubes are available in different sizes and formats to accommodate different sample volumes and different centrifugation and ultracentrifugation techniques for maintaining liquid samples within the tubes.

[0006] The preparation of density gradients in tubes and the withdrawal / extraction of samples from tubes after centrifugation or ultracentrifugation represent specific bottlenecks in these processes. One option for withdrawing samples from centrifugal test tubes is to puncture the tube with a subcutaneous needle (CN 208147961 U), but this approach has several drawbacks. If the plastic material used to manufacture the centrifuge tubes is tough or the tube walls are of a certain thickness, the subcutaneous needle can easily bend during the process of puncturing the tube. Furthermore, forcing the needle into the centrifuge tube causes violent shaking in this procedure, which upsets and partially mixes the layers of the generated gradient and separated components. Also, when needle-withdrawing the sample from the centrifuge tube, liquid leakage often occurs. Leakage is often prevented by pre-smearing a thin layer of Vaseline at the puncture site, but this poses a risk of contamination of the extracted sample. The area or density gradient layer of interest often visually positions elements that introduce additional errors during sample retrieval.

[0007] When puncturing the tube wall, the soft plastic material of the tube can clog the needle tube of the extraction needle, thus often requiring needle replacement before material collection. Additionally, the vent hole is manually punctured with a beveled steel subcutaneous needle located at the top of the centrifuge tube. Sample collection cannot be automated using this approach, and therefore, operational time and costs cannot be reduced. Manual needle insertion, along with personal visual identification of the region of interest, is highly inaccurate, resulting in unproductive outcomes. Furthermore, material fractionation is uncontrolled. Fractions are collected by volume, and supplementary analytical procedures are required for characterization. If larger fractions are obtained during fractionation, the risk of dilution of the fraction or contamination with adjacent components increases.

[0008] In some cases, sample extraction is performed by using the siphon effect or by aspirating the sample from the centrifuge tube using a dedicated sample pump. The tube is either pushed into the bottom of the centrifuge tube (CN 101875921 A) or a specially designed centrifuge tube that already contains a siphon tube is used for this purpose (US 5,866,071 A). Extraction of samples from centrifuge tubes by siphon or aspiration has further disadvantages. When the tube is inserted into the bottom of the centrifuge tube, the sample can be substantially disturbed. If the diameter of the siphon tube is too large, the difference in liquid density will cause mixing of liquids of different densities within the siphon tube. That is, the denser liquid is first forced to move upward within the siphon tube. When a less dense liquid enters the tube, the denser liquid begins to spontaneously descend towards the bottom of the tube, creating a mixed solution.

[0009] Alternatively, the sample can be withdrawn from the centrifuge tube by displacing it upward using a low-viscosity, high-density, non-aqueous miscible liquid such as perfluorodecalin. Similar to siphoning, the tube must be inserted into the bottom of the centrifuge tube, inducing disturbance to the separated layers. The introduction of the displacement liquid also carries a risk of material contamination. Decanting equipment can also be implemented for layered sample withdrawal (US 3,682,305 A).

[0010] In some cases, centrifuge tubes with multiple sealed chambers are used to prevent mixing of components after the centrifugation procedure (US 4,511,349 A). This approach requires pre-determining the position of the target molecules within the density gradient. Centrifuge tubes that match the separation characteristics of the specific application are required, and a general-purpose design of centrifuge tubes is not possible.

[0011] Due to the high risk of perturbation of density gradients or layers of separated components within density gradients, careful manual handling is required when placing tubes into and removing them from centrifuge tube buckets or rotors. Perturbation of density gradients can occur by shaking, inverting, or improperly oriented tubes (such as laying them flat on a table). Centrifuge tube protection systems have been proposed for sample recovery from centrifuge tubes. Centrifuge tubes are sealed in protective brackets, and a subcutaneous needle is inserted into the bottom of the centrifuge tube to elute and fractionate its contents. While multiple centrifuge tubes can be processed simultaneously in a controlled environment (CN 210207236 U), this approach requires moving the tubes from the centrifuge rotor or bucket to the protective bracket, which can disturb the samples.

[0012] Sterility is difficult to establish and maintain through manual material extraction from tubes because sterile connections between devices cannot be verified. Beveled-tip steel subcutaneous needles pose a safety risk to workers because their sharp tips can easily penetrate worker protective clothing. Injuries from beveled-tip steel subcutaneous needles present two-way contamination risks: the worker may be exposed to potentially harmful substances, or the product may be contaminated with the worker's tissues or bodily fluids. [Overview of the project] [Problems that the invention aims to solve]

[0013] Starting from the aforementioned disadvantages, and considering the known prior art of centrifuges, ultracentrifuges, and sample extraction apparatuses and methods, the task of the present invention is to provide a more reliable and robust sampling method and an improved centrifuge or ultracentrifuge that enables the respective apparatus to support such an improved method, thereby enabling reproducible sampling results and further enabling the use of an automated process without requiring manual intervention by the user. Thus, the safety and efficiency of the method and apparatus are improved. [Means for solving the problem]

[0014] The problems of the present invention are solved by using the centrifugal rotor described in independent claim 1, the centrifuge or ultracentrifuge described in independent claim 11, the sample extraction needle described in independent claim 12, the in-situ sample extraction method described in independent claim 18, and the centrifugal rotor described in claim 22.

[0015] According to a first aspect of the present invention, a centrifugal rotor or ultracentrifugal rotor is provided, comprising a plurality of centrifugal tube beds, each having a longitudinal axis, and a rotor assembly adapted to centrifugal rotation about a rotational axis. Each of the aforementioned centrifugal tube beds comprises a tubular cavity defined by a cavity sidewall and a cavity bottom, both of which act as support surfaces for the outer surface of the centrifugal tube when the centrifugal tube is received in the centrifugal tube bed. At least one of the plurality of cavity bottoms comprises at least one extraction opening that connects the tubular cavity to the outside of the rotor assembly. The internal volume formed by the tubular cavity of the centrifugal tube bed can be connected to the external atmosphere surrounding the rotor assembly by providing at least one extraction opening in the cavity bottom. Furthermore, at least one closure device can be provided, which is removablely fixed to the rotor assembly, to hermetically seal at least one extraction opening.

[0016] The centrifugal rotor can be adapted for ultracentrifugation.

[0017] At least one extraction opening connecting a tubular cavity to the outside of the rotor assembly provides the advantage that an extraction device can reach and access the bottom region of the centrifuge tubes housed and supported in the centrifuge tube bed of the centrifuge rotor. The configuration according to the present invention allows an extraction device, such as a sample extractor, to be inserted through at least one extraction opening and, while still housed in the rotor assembly, the device to penetrate the housed centrifuge tube and extract the sample housed in the centrifuge tube. The centrifuge rotor assembly includes at least one access well / extraction opening located at the bottom of the centrifuge tube, enabling in-situ sample collection and sample fractionation from the ultracentrifugation and centrifugation process without the need to remove the centrifuge tube from the centrifuge tube bed in the centrifuge rotor. The design of the ultracentrifuge and centrifuge rotor can vary to accommodate centrifuge tubes of various sizes, materials, and sealing means.

[0018] The centrifuge tube is made from a material that allows the sample extraction device to puncture the centrifuge tube without damaging the material. In a preferred embodiment, the centrifuge tube is made from a material comprising polypropylene, polypropylene copolymer, polysulfone, Nalgene, or HDPE.

[0019] When multiple centrifuge tube beds are provided in the rotary assembly, it is preferable that at least one extraction opening is provided for each of the number of centrifuge tube beds, thereby enabling extraction from each of the multiple centrifuge tube beds. Nevertheless, it is also possible that only a certain number of the multiple centrifuge tube beds have the aforementioned at least one extraction opening at the bottom of their cavities.

[0020] At least one closure device allows for the sealing of at least one extraction opening, thereby enabling the at least one closure device to be formed as a single, independent device that can preferably be fixed to or detached from the rotor assembly independently. This allows access to a given centrifuge tube bed of multiple centrifuge tube beds and enables extraction from a selected specific centrifuge tube bed.

[0021] Nevertheless, in an alternative embodiment of the present invention, at least one closure device may be configured as a single device that allows simultaneous sealing or desealing of each of the multiple extraction openings, thereby enabling, for example, access to all of the multiple centrifuge tube beds through the multiple extraction openings.

[0022] Each cavity bottom of the centrifugal tube bed typically has a shape that narrows toward the bottom of the centrifugal rotor. For example, the cavity bottom may be formed as a hemisphere, cone, cylindrical cone, or frustocone extending from the lower end of the cavity sidewall in the direction of the longitudinal axis of the centrifugal tube bed. At least one closure device allows each of the multiple extraction openings to be sealed liquid-tight and thereby airtight.

[0023] Preferably, the rotor assembly is formed as a fixed-angle rotor formed by a rotor body, the plurality of centrifugal tube beds are formed as tubular cavities within the rotor body, and the at least one extraction opening extends through the rotor body.

[0024] The extraction opening is located at the lowest point of the centrifugal tube bed with respect to the axis of rotation, thereby positioning the extraction opening in the cavity bottom region that forms the lowest point of the centrifugal tube bed. Each longitudinal axis of the centrifugal tube bed is inclined with respect to the rotational axis of the centrifugal rotor, and the lowest point of the centrifugal tube bed is at a greater distance from the rotational axis in the horizontal direction than the upper part of the centrifugal tube bed in the region of the cavity sidewall. In this invention, near-vertical rotors are also considered fixed-angle rotors and fall within the scope of protection of this invention.

[0025] In another embodiment, the rotor assembly is formed as a swing-bucket rotor comprising a plurality of rotor buckets rotatably connected to a rotating stem. The plurality of rotor buckets each have at least one tubular cavity forming a centrifugal tube bed, and at least one extraction opening extends through the body of each rotor bucket. The dimensions of the centrifugal tube buckets can be varied to accommodate centrifugal tubes of various sizes, materials, and sealing means.

[0026] Each of the plurality of rotor buckets is pivotally connected to the rotary stem so as to swing out with respect to the rotary stem when the centrifuge rotor rotates about the rotation central axis. The centrifuge tube bed is formed as a cavity within the rotor bucket, and the longitudinal axis of the centrifuge tube bed extends parallel to the rotation central axis in a non-rotating and non-swinging state. When rotating the swing bucket rotor, the rotor bucket swings with respect to the rotary stem and the rotation central axis, whereby the longitudinal axis of each centrifuge tube bed is inclined with respect to the rotation central axis. The extraction opening is located at the lowest point of the centrifuge tube bed with respect to the rotation central axis when in the non-rotating state, whereby the extraction opening is located in the region of the cavity bottom that forms the lowest point of the centrifuge tube bed. The longitudinal axis of each centrifuge tube bed extends parallel to the rotation central axis when in the non-rotating state. At the swing-out position of the rotor bucket during rotation of the centrifuge rotor, the longitudinal axis of each centrifuge tube bed is inclined with respect to the rotation central axis of the centrifuge rotor, and the lowest point of the centrifuge tube bed has a greater distance with respect to the rotation central axis in the horizontal direction than the upper part of the centrifuge tube bed in the region of the cavity side wall.

[0027] Preferably, the closure device comprises a shaft extending from a first end to a second end along an axis, the shaft being adapted to the geometric shape of the extraction opening, and when the closure device is fixed to the rotor assembly to seal each extraction opening, the first end forms a partial region of the cavity bottom, and the shape of the first end is adapted to the shape of the cavity bottom so as to form a smooth transition between the surface of the cavity bottom and the surrounding surface of the cavity bottom.

[0028] When a plurality of extraction openings are formed on the centrifuge rotor, naturally, a plurality of closure devices are provided. Each extraction opening can be closed or sealed in a fluid-tight manner by its respective closure device.

[0029] Preferably, the closing device is provided with a limit stop at the second end to restrict the axial movement of the closing device when the closing device is fastened to the rotor assembly, in order to ensure proper alignment between the first end and the surrounding surface of the cavity bottom when the closing device is in the fastened / sealed position.

[0030] More preferably, each of the multiple closure devices comprises at least one elastic seal that is received in a groove of the closure device and deforms when the closure device is fastened to the rotor assembly to form an airtight seal between the rotor assembly and the closure device. The elastic seal improves the sealing of the centrifugal tube bed within the rotor assembly relative to the periphery of the centrifugal rotor.

[0031] The closing device can be fastened to the rotor assembly by adapted threading, with at least a portion of the shaft of the closing device having male threads and the extraction opening having female threads adapted to the shape and position of the male threads. The threading between the closing device and the extraction opening first establishes a means for fastening the closing device to the centrifugal rotor, and further, enables the establishment of an airtight seal. The threading may be expected to extend in the reverse direction of rotation of the centrifugal rotor to avoid unthreading of the closing device during the rotation of the centrifugal rotor.

[0032] The second end of the shaft may include at least one interlocking element projecting at least partially radially from the shaft, and the rotor assembly may include at least one notch and / or pocket, which receive at least one interlocking element to form an interlocking structure with the closing device and fasten the closing device to the rotor assembly. The interlocking structure ensures secure fastening of the closing device to the rotor assembly.

[0033] The shaft may have a diameter in the range of 0.5 mm to 8 mm, and more preferably in the range of 0.5 to 2.0 mm.

[0034] More preferably, the cavity bottom is formed by a surface that narrows toward the lower end of the rotor assembly, and at least one extraction opening is located at the lowest point of the centrifugal tube bed relative to the rotational axis. If a swing bucket rotor is provided, the narrowed surface of the cavity bottom is observed in each of the rotor buckets. Here, the extraction opening is located at the lowest point of the centrifugal tube bed relative to the rotational stem of the swing bucket rotor when the rotor bucket is static, non-rotating, and non-oscillating.

[0035] According to a second aspect of the present invention, a centrifuge or ultracentrifuge is provided that includes a centrifugal rotor or an ultracentrifuge rotor according to the first aspect of the present invention.

[0036] According to a third aspect of the present invention, a sample extraction needle is provided, comprising a cannula extending along an axial axis from a distal end to a proximal end, the proximal end of which is formed by a sharp closure tip that seals the proximal end of the sample extraction needle to at least one drainage hole located in the lateral side wall of the cannula. The sharp closure tip of the sample extraction needle allows the needle to penetrate the centrifuge tube by piercing the wall of the centrifuge tube. The drainage hole of the extraction needle is located on the side / lateral side wall of the needle cannula, just below the needle tip at the proximal end. Such positioning of the drainage hole prevents needle blockage by small particles generated during puncture of the centrifuge tube. Furthermore, positioning the drainage hole at a desired height relative to the lowest point of the centrifuge tube allows for immediate extraction of a specific centrifugal fraction from the centrifuge tube, thereby improving fluid flow during extraction by the extraction needle. Alternatively, other designs of non-coring needle tips (pencil tip, deflected tip) are preferably used for sample extraction from centrifuge tubes.

[0037] Preferably, the sample extraction needle comprises a needle cannula having a conical shape along its axial axis, with the outer diameter of the cannula decreasing toward the sharply closed tip. The extraction needle has a conical shape to ensure a sealed connection between the centrifuge tube and / or the rotor assembly and the extraction needle. The conical shape with a cross shape that decreases toward the sharply closed tip allows for easy insertion of the extraction needle into the extraction opening and enables self-alignment of the needle during insertion into the extraction opening and the centrifuge tube.

[0038] A vent hole is required at the top of the centrifuge tube to remove the sample from the sealed tube. The vent hole at the top of the centrifuge tube can be made with the same sample extraction needle used to create a discharge hole at the bottom of the centrifuge tube for extracting the sample. It is preferable that the vent hole at the top of the centrifuge tube be made before the extraction hole at the bottom of the centrifuge tube. The vent and extraction needle are precisely inserted into the centrifuge tube using a manually operated lever or a robotic arm.

[0039] Preferably, the sharp closure tip is formed by a cone, preferably a circular cone, the front surface of the proximal end is formed by the tip of the cone, and the ground surface of the cone is conformed to the shape of the cannula end.

[0040] The cannula can be formed from a hollow stainless steel tube. Preferably, the cross-section of the hollow stainless steel tube can be circular or elliptical.

[0041] The cannula may have an outer diameter in the distal end region ranging from 0.7 to 1.5 mm.

[0042] Preferably, the distal end of the cannula is connected to the needle base, where the needle base has a larger diameter than the opening of the extraction opening of the centrifugal rotor according to a second aspect of the present invention, and can act as a limiting device to limit the maximum insertion depth of the sample extraction needle in the centrifugal tube bed.

[0043] A fourth aspect of the present invention provides a method for in-situ sample extraction from centrifuge tubes housed in a centrifuge tube bed of a centrifuge rotor according to the first aspect of the present invention. The method includes the following steps: A) Performing centrifugation of a sample contained in a centrifuge tube and placed in a centrifuge rotor; B) Transferring the centrifugal rotor to the rotor stand, and disassembling the centrifugal rotor; C) Select a first centrifuge tube from which the contents should be extracted, from among the multiple centrifuge tubes housed in the centrifuge rotor; D) Forming a vent in the top region of the selected first centrifuge tube; E) Opening each extraction opening of the centrifuge tube bed in which the first centrifuge tube is located by removing each closure device; F) Preferably, an extraction needle according to a third aspect of the present invention is introduced through the extraction opening toward the outer wall of the centrifuge tube, piercing the wall of the centrifuge tube, and the extraction needle is inserted into the first centrifuge tube to create a fluid connection to the internal volume of the centrifuge tube; G) Remove the contents of the centrifuge tube through a needle; H) Preferably, steps C) through G) are repeated on a further centrifugal tube, which is housed in at least a centrifugal rotor.

[0044] Specifically, the order of steps D) and E) can be changed so that, for example, step E) is executed before step D).

[0045] Preferably, sample removal from the centrifuge tube is achieved by replacing the contents of the centrifuge tube under pressure with compressed gas. Alternatively, the sample can be replaced from the centrifuge tube by supplying a low-density, preferably water-miscible, liquid through the vents of the centrifuge tube. Preferably, an HPLC-type pump, peristaltic pump, or syringe pump that allows for good flow control is used for sample displacement from the centrifuge tube. Alternatively, sample removal from the centrifuge tube is performed by sample aspiration using a suitable pump.

[0046] According to a preferred method, the contents extracted by the extraction needle are supplied to at least one of the following apparatuses: an ultraviolet-visible detection system, a fluorescence detection system, a light scattering detection system, an apparatus for performing high-performance liquid chromatography (HPLC), and / or an automated fraction collector.

[0047] The first centrifuge tube can be placed on the centrifuge tube stand before step D) and the steps following step D).

[0048] According to a fifth aspect, the present invention provides the use of a centrifugal rotor according to a first aspect of the present invention or a centrifugal rotor according to a second aspect of the present invention, and preferably a method according to a fourth aspect of the present invention, with a sample drawing needle according to a third aspect of the present invention.

[0049] According to a sixth aspect of the present invention, an automated system for in-situ sample extraction is provided, the system: A centrifugal rotor according to a second aspect of the present invention, The invention comprises at least one extraction needle according to a third aspect of the present invention; The system is preferably configured to carry out a method for in-situ sample extraction according to a fourth aspect of the present invention. [Brief explanation of the drawing]

[0050] In the following, exemplary embodiments of the apparatus according to the present invention will be described with reference to the attached drawings.

[0051] [Figure 1] The image shows a cross-section of a centrifugal rotor in a fixed-angle rotor configuration, with a cut through an exemplary centrifugal tube bed having an extraction opening that is closed by a closure device. [Figure 2] The diagram shows a cross-section of the centrifuge rotor in the configuration of a swing bucket rotor, a partial cut through the centrifuge tube bed of the swing bucket, and an extraction opening visible at the bottom of the centrifuge tube bed, which is sealed by a closure device. [Figure 3A] A first exemplary embodiment of the closing device is shown. [Figure 3B] A second exemplary embodiment of the closing device is shown. [Figure 3C] Figure 3B shows the closing device during the installation process of the centrifugal separator rotor into the rotor assembly. [Figure 4] This figure shows an exemplary embodiment of a sample retrieval needle. [Figure 5] A schematic diagram of a sample return needle, which is inserted into the centrifugation tube housed in the centrifugation rotor, is shown for extracting a sample from the aforementioned centrifugation tube. [Figure 6] The results shown are obtained by uniformly dispensing a low-density liquid through the vent of a centrifugal tube using a high-performance liquid chromatography pump, and then supplying the displaced sample to a UV-Vis detection system, a fluorescence detection system, a light scattering detection system, and a conductivity detection system. [Figure 7] The results are shown below, obtained by using a high-performance liquid chromatography pump to aspirate a sample from the extraction port of a centrifuge tube and supplying the displaced sample to a UV-Vis detection system, a fluorescence detection system, a light scattering detection system, and a conductivity detection system. [Modes for carrying out the invention]

[0052] Referring to Figure 1, an exemplary embodiment of a centrifugal rotor 1 in a fixed-angle rotor configuration is shown. The centrifugal rotor comprises a rotor assembly 10 formed by a rotor body 12. The rotor assembly 10 in the form of a fixed-angle rotor body 12 is configured to centrifugally rotate about a rotational axis 14 shown by a dashed line in Figure 1. As will be understood by those skilled in the art, the rotor body 12 is rotationally symmetric with respect to the rotational axis 14. In Figure 1, a cross-section of one of several centrifugal tube beds 16 is shown along with the longitudinal axis 160 of the centrifugal tube bed 16. The centrifugal tube bed 16 is defined by tubular cavities 162 adapted to receive and support centrifugal tubes 4 during the centrifugal separation process. The tubular cavities 162 are formed within the material of the rotor body 12 and are separated by cavity sidewalls 162a and cavity bottom 162b. The cavity sidewall 162a and the cavity bottom 162b both act as support surfaces for the outer surface of the centrifugal tube 4 that can be housed in the tubular cavity 162 of the centrifugal tube bed 16. As can be seen in Figure 1, the cavity bottom 162b includes a single extraction opening 18, which in the example of Figure 1 is closed in Figure 1 by a closure device 2. In the exemplary embodiment according to Figure 1, the cavity bottom 162b is formed to have a shape that narrows toward the bottom 10b of the centrifugal rotor assembly 10. The top of the rotor assembly 10 is denoted by reference numeral 10t.

[0053] The cavity bottom 162b is formed by a hemisphere in the example shown in Figure 1.

[0054] The tubular cavity 162 of the centrifugal tube bed 16 of the centrifugal rotor 1 is formed within the rotor body 12 of the fixed-angle rotor, and at least one extraction opening 18 is formed as a hole extending through the rotor body 12 to fluidly connect the tubular cavity 162 to the periphery of the rotor assembly 10.

[0055] Figure 2 shows an alternative configuration of the centrifugal rotor 1, where the rotor assembly 10 is formed by a swing bucket rotor and comprises a plurality of rotor buckets 13 swivelably connected to a rotating stem 11, the plurality of rotor buckets 13 each having at least one tubular cavity 162 to form at least one centrifugal tube bed 16, and at least one extraction opening 18 extending through the body of each rotor bucket 13. The tubular cavity 162 is also formed in the material of the rotor bucket 13. In Figure 2, only one cross-sectional view of the rotor bucket 13 is shown, where a single exemplary centrifugal tube 4 is received. The swing bucket rotor as shown in Figure 2 is configured to rotate about a central rotation axis 14, and each of the plurality of swing buckets 13 is swivelably connected to the rotating stem 11 via a swing-out axis 15. In Figure 2, the longitudinal axis 160 of the centrifugal tube bed 16 extends substantially parallel to the rotational axis 14 of the rotating stem 11 in a non-oscillating or static state.

[0056] The extraction opening 18 formed within the housing or material defining the swing bucket 13 is closed and sealed by the closing device 2.

[0057] Figure 3A shows a first exemplary embodiment of a closing device 2 formed as a capping screw, comprising a shaft 22 extending along the shaft axis 22a from a first end 221 to a second end 222, the first end 221 forming a sub-region of the cavity bottom 162b when the closing device 2 is fastened to the rotor assembly 10, as can be seen from the example shown in Figures 1 and 2. As can be seen from Figure 2, the first end 221 conforms to the shape of the cavity bottom 162b, forming a smooth transition between the surface of the first end 221 and the surrounding surface of the cavity bottom 162b. The closing device 2 as shown in the configuration of Figure 3A further comprises a limit stop 24 at the second end 222 to restrict the movement of the closing device 2 in the direction along the shaft axis 22a when the closing device 2 is fastened to the rotor assembly 10, in order to ensure correct alignment between the first end 221 and the surrounding surface of the cavity bottom 162b. The closing device 2 shown in Figure 3A includes a groove 26 for receiving a single elastic seal 28 configured as an O-ring. The elastic seal 28 is received within the groove 26 of the closing device and deforms when the closing device 2 is fastened to the rotor assembly 10. Once the closing device 2 is fixed to the rotor assembly 10, the closing device 2 in Figure 3A engages with the outer surface of the rotor body 12 or the body of the rotor bucket 13 in at least the area of ​​the limit stop 24, causing the elastic seal 28 to elastically deform and define an airtight seal. Furthermore, as can be seen from the exemplary embodiment of the closing device 2, a portion of the shaft 22 is provided with an external thread 27, which is provided with an internal thread adapted to the shape and position of the external thread 27, thereby allowing the closing device to be fixed to the rotor assembly 10 by the adapted internal and external threads 27.

[0058] Figure 3B shows a second exemplary embodiment of the closing device 2, where the closing device 2 is configured to form an interlocking structure with the rotor assembly 10 to achieve fixation. Unlike the device shown in Figure 3A, the closing device 2 shown in Figure 3B does not have an external thread 27 in the region of the shaft 22 to achieve fixation of the closing device 2 to the rotor assembly 10. The closing device 2 according to Figure 3B includes two interlock elements 29 that enable the closing device 2 to be fixed to the rotor assembly 10. The interlock elements 29 can be provided in addition to or as an alternative to the external thread 27, as described with respect to Figure 3A. The closing device 2 according to Figure 3B includes two elastic seals 28, one of which is positioned to be received in a groove 26 in the region of the shaft 22, and the other elastic seal 28 is positioned in a groove 26 located in the region of the second end 222 at the mating surface of the limit stop 24 with the rotor assembly 10. The second end 222 of the closing device 2 has a recess 222a on the surface facing away from the rotor housing within the area of ​​the limit stop 24, thereby allowing the closing device 2 to be rotated around the shaft axis 22a to lock or unlock the closing device 2 from the rotor housing 10.

[0059] As shown in Figure 3C, the closing device 2 during the fastening process to the rotor assembly 10 is detailed. Figure 3C shows a top view of the second end 222 of the closing device 2. During the fastening of the closing device 2, the closing device 2 is positioned in the area of ​​the extraction opening 18, where the interlocking element 29 is positioned in the respective notches 19a of the rotor assembly 10. To fasten the closing device 2 to the rotor assembly 10, the closing device is rotated around the axis 22a, moving the interlocking element 29 into the fitted pockets 19b of the rotor assembly 10. The interlocking element 29 and the pockets 19a and 19b are fitted together to form an interlocking structure, thereby ensuring secure fastening of the closing device 2 to the rotor assembly.

[0060] Figure 4 shows an exemplary embodiment of a sample extraction needle 3, which includes a cannula 30 extending along an axial axis 300 from a distal end 30d to a proximal end 30p, the proximal end 30p being formed by a sharp tip closure 31 to seal the proximal end 30p of the cannula 30 of the sample extraction needle 3. In the embodiment shown in Figure 4, the sample extraction needle 3 has four drainage holes 32 located in the region of the lateral side wall 33 of the cannula 30. In the schematic diagram of Figure 4, only three of the four drainage holes 32 are shown evenly distributed in the circumferential direction of the cannula 30, in the lateral side wall 33 around the axis 300.

[0061] As can be seen in Figure 4, the needle cannula 30 has a conical shape along the axis 300, where the outer diameter of the cannula 30 decreases in the direction toward the sharp occlusal tip 31. The sharp occlusal tip 31 is formed by a circular cone, where the front surface of the proximal end 30p is formed by the tip of the cone. The ground surface of the cone is adapted to the shape of the cannula end.

[0062] The cannula 30 of the extraction needle 3 can be formed from a hollow stainless steel tube, and the cannula 30 may have an outer diameter in the range of 0.7 to 1.5 mm in the region of the distal end 30d. The distal end 30d of the cannula 3 is connected to a needle base 34, where the needle base 34 has a diameter 34d that is sized to be larger than the opening of the extraction opening 18 of the centrifugal rotor 1 according to the present invention. Thereafter, the needle base 34 is configured to act as a limiting device that limits the maximum insertion depth I of the sample extraction needle 3 in the centrifugal tube bed 16.

[0063] Figure 5 shows a schematic diagram of a sample extraction needle 3 according to the present invention, which is inserted into a centrifuge tube 4 housed in an exemplary embodiment of a rotor assembly 10 of a fixed-angle rotor, via an extraction opening 18 of the rotor body 12, and then inserted into the centrifuge tube 4 to extract the sample and / or fraction contained in the centrifuge tube 4 after the centrifugation process. Figure 5 shows the use of a limiting device 9 having a thickness S, in which case the limiting device 9 has a through hole through which the insertion needle 3 can be guided through the extraction opening 18, inserted into the centrifuge tube bed 16, and then inserted into the centrifuge tube 4. The use of the limiting device 9 allows the extraction needle 3 to be inserted into the centrifuge tube 4 to a desired and previously defined insertion depth I, such that the previously observed drain hole 32 is located at a desired height H above the lowest point of the centrifuge tube bed 16. The above configuration allows the extraction needle 3 to be inserted to a desired insertion depth I, thereby allowing the drain hole to be positioned at a desired height above the bottom of the centrifuge tube 4.

[0064] Figure 6 shows an example of sample displacement from a centrifuge tube by uniformly dispensing a low-density liquid through the vent holes of the centrifuge tube using a high-performance liquid chromatography pump.

[0065] Density gradient fractionation was performed on a Sorvall® WX 90+ ultracentrifuge (Thermo Scientific) using 11.5 mL polyethylene UltraCrimp® centrifuge tubes (Thermo Scientific) with a T890 fixed-angle rotor. Adeno Associated Virus (AAV) samples were mixed with concentrated cesium chloride to obtain AAV samples in 3 M cesium chloride. Centrifuge was performed at room temperature at 53,500 RPM × 24 h. The tubes were then fixed to a stand, and a vent hole was perforated near the top using a subcutaneous needle (23 gauge, 70 mm, B Braun). An extraction opening at the bottom of the centrifuge tube was perforated using another subcutaneous needle. The contents of the centrifuge tube were replaced by pumping water at a constant flow rate of 1 mL / min through the vent hole at the top of the centrifuge tube using an HPLC pump of a PATfix® LPG HPLC system (BIA separation). The extraction opening at the bottom of the centrifuge tube was directly connected to the monitor array of the PATfix® LPG HPLC system. The tube was evacuated in order of decreasing density. UV absorbance was monitored at 260 nm (solid trace). Intrinsic fluorescence was monitored at excitation wavelength 280 nm and emission wavelength 348 nm using a fluorescence detector (Shimadzu Corporation Dash Trace). Light scattering was monitored at a 90° angle using a DAWN® HELEOS II multi-angle light scattering detector (Wyatt Technology, Dash Dot Black Trace). The density of cesium chloride is represented by the conductivity profile (dotted trace). Higher conductivity was associated with a higher density of cesium chloride.

[0066] Figure 7 shows an example of sample aspiration through an extraction opening in a centrifuge tube equipped with a high-performance liquid chromatography pump.

[0067] Density gradient fractionation was performed on a Sorvall® WX 90+ ultracentrifuge (Thermo Scientific) using 11.5 mL polyethylene UltraCrimp® centrifuge tubes (Thermo Scientific) with a T890 fixed-angle rotor. Adeno Associated Virus (AAV) samples were mixed with concentrated cesium chloride to obtain AAV samples in 3 M cesium chloride. Centrifuge was performed at room temperature at 53,500 RPM × 24 h. Next, the tubes were fixed to a stand, and a vent was punctured near the top with a subcutaneous needle (23 gauge, 70 mm, B Braun) and left open to normal atmospheric pressure. An extraction opening at the bottom of the centrifuge tube was punctured using another subcutaneous needle. The contents of the centrifuge tube were withdrawn from the centrifuge tube at a constant flow rate of 1 mL / min through the extraction opening at the bottom of the centrifuge tube using an HPLC pump of a PATfix® LPG HPLC system (BIA separation). The HPLC pump guided the contents of the centrifuge tubes to the monitoring array of the PATfix® LPG HPLC system. The tubes were evacuated in order of decreasing density. UV absorbance was monitored at 260 nm (solid trace). Intrinsic fluorescence was monitored at excitation wavelength 280 nm and emission wavelength 348 nm using a fluorescence detector (Shimadzu Dash Trace). Light scattering was monitored at a 90° angle using a DAWN® HELEOS II multi-angle light scattering detector (Wyatt Technology, Dash Dot Trace). The density of cesium chloride is represented by the conductivity profile (dotted trace). Higher conductivity was associated with a higher density of cesium chloride.

Claims

1. The rotor assembly (10), which is adapted to rotate centrifugally around the central axis of rotation (14), The system comprises multiple centrifuge tube beds (16), each of which has a vertical axis (160). Each of the multiple centrifugal tube beds (16) includes a tubular cavity (162) defined by a cavity side wall (162a) and a cavity bottom (162b). At least one of the plurality of cavity bottoms (162b) includes at least one extraction opening (18) that connects the tubular cavity (162) to the outside of the rotor assembly (10), At least one closing device (2) is removably fixed to the rotor assembly (10) to seal at least one extraction opening (18) in an airtight manner. The cavity side wall (162a) and the cavity bottom (162b) together become support surfaces for the outer surface of the centrifuge tube (4) when the centrifuge tube (4) is received in the centrifuge tube bed (16). A combination of a centrifuge tube (4) and a centrifuge rotor (1) that allows a sample to be extracted from the centrifuge tube, which is received in the centrifuge tube bed (16), through the at least one extraction opening (18).

2. The combination of centrifugal tubes (4) and centrifugal rotor (1) according to claim 1, wherein the rotor assembly (10) is a fixed-angle rotor formed by a rotor body (12), the plurality of centrifugal tube beds (16) are formed as tubular cavities (162) within the rotor body (12), and the at least one extraction opening (18) extends through the rotor body (12).

3. The combination of centrifugal tube (4) and centrifugal rotor (1) according to claim 1, wherein the rotor assembly (10) is a swing bucket rotor comprising a plurality of rotor buckets (13) rotatably connected to a rotating stem (11), the plurality of rotor buckets (13) comprising at least one tubular cavity (162) forming the centrifugal tube bed (16), and the at least one extraction opening (18) extending through the body of at least one of the plurality of rotor buckets (13).

4. A combination of a centrifugal tube (4) and a centrifugal rotor (1) according to any one of claims 1 to 3, wherein at least one closure device (2) comprises a shaft (22) extending along a shaft axis (22a) from a first end (221) to a second end (222), the shaft (22) conforming to the shape of the extraction opening (18), the first end (221) forming a partial region of the cavity bottom (162b) when the closure device (2) is fastened to the rotor assembly (10), and the shape of the first end (221) conforming to the shape of the cavity bottom (162b) to form a smooth transition between the surface of the first end (221) and the surrounding surface of the cavity bottom (162b).

5. The combination of a centrifugal tube (4) and a centrifugal rotor (1) according to claim 4, wherein each of the multiple closing devices (2) is provided with a limit stop (24) at the second end (222) to restrict the movement of the closing device (2) in the axial direction (22a), and to ensure that the first end (221) is properly aligned with the surrounding surface of the cavity bottom (162b) when the closing device (2) is fixed to the rotor assembly (10).

6. A combination of a centrifugal tube (4) and a centrifugal rotor (1) according to any one of claims 1 to 5, wherein each of the plurality of closing devices (2) comprises at least one elastic seal (28), the elastic seal (28) being received in a groove (26) of the closing device (2), and deforming when the closing device (2) is fastened to the rotor assembly (10) to form an airtight seal together with the rotor assembly (10).

7. The combination of a centrifugal tube (4) and a centrifugal rotor (1) according to any one of claims 4 to 6, wherein at least a portion of the shaft is provided with a male thread (27), the extraction opening (18) is provided with a female thread (27) that conforms to the shape and position of the male thread (27), and the closing device (2) is fastened to the rotor assembly (10) by the conforming male and female threads (27).

8. A combination of a centrifugal tube (4) and a centrifugal rotor (1) according to any one of claims 4 to 7, wherein the second end (222) of the shaft (22) comprises at least one interlocking element (29) projecting at least partially radially from the shaft (22), and the rotor assembly (10) comprises at least one notch (19a) and / or at least one pocket (19b) configured to receive the at least one interlocking element (29), forming an interlocking structure when the closing device (2) is fastened to the rotor assembly (10).

9. A combination of a centrifugal tube (4) and a centrifugal rotor (1) according to any one of claims 4 to 7, wherein the diameter of the shaft (22d) is in the range of 0.5 mm to 8.0 mm, and preferably in the range of 0.5 mm to 2.0 mm.

10. The combination of a centrifuge tube (4) and a centrifuge rotor (1) according to any one of claims 1 to 9, wherein the cavity bottom (162b) is formed by a constricting surface toward the lower end of the rotor assembly (10), and the at least one extraction opening (18) is located at the lowest point of the centrifuge tube bed (16) with respect to the rotational axis (14).

11. A centrifuge or ultracentrifuge (1) comprising a combination of a centrifugal tube (4) and a centrifugal rotor (1) as described in any one of claims 1 to 10.

12. A method for extracting a sample from a centrifuge tube (4) stored in a centrifuge tube bed (16) of a centrifuge rotor (1) at its stored position, wherein the centrifuge rotor (1) has the features described in any one of claims 1 to 10, and the method is: A) The sample contained in the centrifuge tube (4) housed in the centrifuge rotor (1) is centrifuged; B) Remove the centrifugal rotor (1) from the rotational axis (14), and transfer the centrifugal rotor (1) to the rotor stand. C) Select a first centrifuge tube (4) from which to extract contents from a plurality of centrifuge tubes (4) housed in the centrifuge rotor (1); D) Form at least one vent in the upper region of the selected first centrifuge tube (4); E) By removing the closure device (2), the extraction opening (18) of the centrifuge tube bed (16) in which the first centrifuge tube (4) is placed is opened; F) The sample extraction needle (3) is introduced through the extraction opening (18) toward the outer wall of the centrifuge tube (4), perforating the wall of the centrifuge tube (4), and the sample extraction needle (3) is inserted into the first centrifuge tube (4) to create a fluid connection to the internal volume of the centrifuge tube (4); G) Extract the contents of the centrifuge tube (4) through the sample extraction needle (3); method

13. H) Repeat steps C) to G) in another centrifugal tube (4) housed in the centrifugal rotor (1); The method according to claim 12.

14. The method according to claim 12 or 13, wherein the sample is removed from the centrifugal tube (4) by replacing the sample under pressure with compressed gas, or by aspirating the sample from the centrifugal tube (4) using a suitable pump.

15. The method according to claim 12, wherein the sample is displaced from the centrifuge tube (4) by uniformly dispensing a low-density, water-miscible liquid through the vent holes of the centrifuge tube.

16. The method according to claim 15, wherein a high-performance liquid chromatograph, an HPLC-type pump, a peristaltic pump, or a syringe pump is used to enable sample displacement from the centrifugal tube (4).

17. The method according to any one of claims 12 to 16, wherein the contents extracted by the sample extraction needle (3) are supplied to at least one of the devices selected from the group consisting of a UV-VIS detection system, a fluorescence detection system, a light scattering detection system, a high-performance liquid chromatography system and / or a self-moving particle collector.

18. The method according to any one of claims 12 to 17, wherein the first centrifuge tube (4) is placed on a centrifuge tube stand before step D).

19. Using a centrifugal rotor according to any one of claims 1 to 10, or using a centrifuge or ultracentrifuge according to claim 11, and using a sample extraction needle for carrying out the method according to any one of claims 12 to 18.

20. An automated system for sample extraction: A centrifuge or ultracentrifuge according to claim 11, and Equipped with at least an extraction needle; The system is configured to perform the sample extraction method described in any one of claims 12 to 18.

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