Centrifuge or ultracentrifuge with centrifugal rotor, sample extraction needle, and method for extracting sample from 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 ultracentrifuge, more particularly to a centrifuge or ultracentrifuge rotor for use in a method for separating liquid substances of cellular organelles, viruses, virus-like particles, bacteriophages, exosomes and nanoparticles using said centrifuge or ultracentrifuge.Furthermore, the present invention describes a sample extraction needle, a method for in situ extraction of samples contained in a centrifuge tube bed of a centrifuge rotor or ultracentrifuge rotor, and the use of the centrifuge rotor and the sample extraction needle for carrying out the method for in situ extraction of samples.

[0002] The present invention describes an ultracentrifuge or centrifuge rotor that allows for in situ liquid sample collection and liquid sample fractionation in ultracentrifugation and centrifugation processes without removing the centrifuge tubes from the centrifuge tube rotor assembly or centrifuge tube buckets. Additionally, a self-sealing design of the sample withdrawal needle is described to allow for automatic sample withdrawal from centrifuge tubes that are still housed within the rotor assembly of the centrifuge rotor. [Background technology]

[0003] Centrifugation and ultracentrifugation are among the fundamental techniques for separating particulate fractions such as cell 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). Centrifugation and ultracentrifugation techniques include pelleting, where the particulate fraction is gradually removed from the suspension by forming a pellet at the bottom of the tube. Gradient separation, where a tube is filled from top to bottom with increasing concentrations of a high-density substance in the solution, is also often used to separate particulate fractions based on particulate fraction density and particulate fraction sedimentation coefficient. The sample is spun at high speed for a time sufficient to achieve separation. After centrifugation or ultracentrifugation, the rotor is brought to a smooth stop and the gradient is gently pulled from each tube in order to isolate the separated components by using different approaches.

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

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

[0006] Density gradient preparation in tubes and withdrawal / extraction of samples from the tubes after centrifugation or ultracentrifugation represent a particular bottleneck in these processes. One option for withdrawing samples from centrifuge test tubes is by puncturing the tube with a hypodermic needle (CN 208147961 U), and this approach has several drawbacks. If the plastic material for the manufacture of the centrifuge tubes is tough or the tube walls are of a certain thickness, the hypodermic needle can easily bend during the process of puncturing the tube. Furthermore, when the needle is forcibly inserted into the centrifuge tube, this procedure generates vigorous shaking, which upsets and partially mixes the layers of generated gradient and separated components. Also, leakage of liquids often occurs during needle withdrawal of samples from the centrifuge tubes. Leakage is often prevented by smearing a thin layer of Vaseline beforehand at the puncture site, but it poses a risk of contamination of the extracted sample. The area of ​​interest or density gradient layer is often visually located, which introduces additional errors during sample recovery.

[0007] When puncturing the tube wall, the soft plastic material of the tube can clog the needle tube of the extraction needle, so that a needle exchange is often required before material collection. Also, the vent hole is punctured manually with a beveled steel hypodermic needle in the upper part of the centrifuge tube. Sample collection cannot be automated by using this approach, and therefore the operation time and operation costs cannot be reduced. The manual insertion of the needle as well as the personal visual identification of the area of ​​interest is very inaccurate, resulting in unproductive results. Furthermore, the fractionation of the material is not controlled. The fractions are collected by volume and their characterization requires auxiliary analytical procedures. If fractions of larger size are obtained during fractionation, the risk of dilution of the fraction or of contamination with adjacent components increases.

[0008] In some cases, the withdrawal of samples 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 to the bottom of the centrifuge tube (CN 101875921 A) or specially designed centrifuge tubes that already contain a siphon tube are used for this purpose (US 5,866,071 A). The withdrawal of samples from centrifuge tubes by siphoning or suction has further disadvantages: the sample can be substantially disturbed when the tube is inserted at the bottom of the centrifuge tube. If the diameter of the siphon tube is too large, the difference in liquid densities will cause mixing of liquids of different densities in the siphon tube. That is, the densest liquid will be forced to move upwards in the siphon tube first. When the less dense liquid enters the tube, the denser liquid will start to spontaneously drop towards the bottom of the tube, resulting in a mixed solution.

[0009] Alternatively, the sample can be extracted from the centrifuge tube by displacing it upwards using a low-viscosity, dense, water-immiscible liquid such as perfluorodecalin. As with siphoning, the tube needs to be inserted at the bottom of the centrifuge tube, inducing disturbance of the separated layers. The introduction of the displacing liquid also risks material contamination. Decanting devices can also be implemented for layered sample extraction (US 3,682,305 A).

[0010] Centrifuge tubes with multiple closed chambers may be used to prevent mixing of components after the centrifugation step (US 4,511,349 A). This approach requires that the location of the molecules of interest within the density gradient be determined in advance. Centrifuge tubes must be adapted to the separation characteristics of the specific application, precluding a universal design of centrifuge tubes.

[0011] Careful manual handling is required to place tubes in and out of the centrifuge tube bucket or rotor, due to the high risk of perturbing the density gradient or the layers of separated components within the density gradient. Perturbation of the density gradient can occur by shaking the tube, inverting the tube, or placing the tube in an improper orientation (lying flat on a table). A centrifuge tube protection system has been proposed for sample recovery from centrifuge tubes. The centrifuge tube is enclosed in a protective bracket, a hypodermic needle is inserted into the bottom of the centrifuge tube, and the contents of the centrifuge tube are eluted and fractionated. A large number of centrifuge tubes can be processed simultaneously in a controlled environment (CN 210207236 U), but that approach requires transferring the tube from its position in the centrifuge rotor or bucket to the protective bracket, which may disturb the sample.

[0012] Sterility is difficult to establish and maintain with manual withdrawal of material from tubing because sterile connections between equipment cannot be verified. Beveled steel hypodermic needles pose a safety risk to workers because their sharp tips can easily penetrate workers' protective clothing. Injuries from beveled steel hypodermic needles pose a two-way risk of contamination: workers could be exposed to potentially harmful substances, or the product could become contaminated with the worker's tissues or body fluids. Summary of the Invention [Problem to be solved by the invention]

[0013] Starting from the aforementioned disadvantages, among the known prior art relating to centrifuges, ultracentrifuges and sample extraction devices and methods, the task of the present invention is to provide an improved centrifuge or ultracentrifuge that allows a more reliable and robust extraction method and the respective device to support such an improved method, which allows reproducible extraction results and, moreover, allows the use of automated processes without the need for manual intervention by the user, thus improving the safety and efficiency of the method and device. [Means for solving the problem]

[0014] The problem according to the invention is solved by a centrifuge rotor according to independent claim 1, a centrifuge or ultracentrifuge according to independent claim 11, a sample extraction needle according to independent claim 12, an in situ sample extraction method according to independent claim 18 as well as the use of a centrifuge rotor according to claim 22.

[0015] According to a first aspect of the present invention, a centrifuge or ultracentrifuge rotor is provided, comprising a rotor assembly comprising a plurality of centrifuge tube beds each having a longitudinal axis and adapted for centrifugally rotating about a central axis of rotation. Each of said centrifuge tube beds comprises a tubular cavity defined by a cavity side wall and a cavity bottom, the cavity side wall and the cavity bottom together acting as a support surface for an outer surface of a centrifuge tube when the centrifuge tube is received in said centrifuge tube bed. At least one of the plurality of cavity bottoms comprises at least one extraction opening connecting the tubular cavity to the exterior of the rotor assembly. The internal volume formed by the tubular cavity of the centrifuge tube bed may be connected to an external atmosphere surrounding the rotor assembly by providing at least one extraction opening in the cavity bottom. Furthermore, at least one closure device removably fixed to the rotor assembly is conceivable for hermetically sealing the at least one extraction opening.

[0016] The centrifuge rotor may be adapted for ultracentrifugation.

[0017] At least one extraction opening connecting the tubular cavity to the exterior of the rotor assembly provides the advantage that the centrifuge tubes housed and supported in the centrifuge tube bed of the centrifuge rotor can be reached and accessed by an extraction device in the region of the bottom of the centrifuge tube. The arrangement according to the invention allows an extraction device, such as a sample puller, to be inserted through the at least one extraction opening and to pierce the housed centrifuge tubes while still housed in the rotor assembly to 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 tubes, which allows in situ sample collection and sample fractionation from the ultracentrifugation and centrifugation process without the need to remove the centrifuge tubes from the centrifuge tube bed in the centrifuge rotor. The design of the ultracentrifuge and centrifuge rotor can be varied to accommodate centrifuge tubes with different sizes, materials and sealing means.

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

[0019] If a plurality of centrifuge tube beds are provided in the rotor assembly, it is preferred that at least one extraction opening is provided for each of the number of centrifuge tube beds, respectively, thereby allowing extraction from each of said plurality of centrifuge tube beds. Nevertheless, it is also possible that only a certain number of the plurality of centrifuge tube beds are provided with said at least one extraction opening at their cavity bottom.

[0020] The at least one closure device allows sealing the at least one extraction opening, whereby the at least one closure device can be formed as a single, independent device that can be fixed or released, preferably independently, from the rotor assembly, thereby allowing access to a given centrifuge tube bed of the plurality of centrifuge tube beds and allowing extraction from a selected specific centrifuge tube bed.

[0021] Nevertheless, in alternative embodiments of the invention, at least one closure device may be formed as a single device that allows for simultaneously sealing or unsealing each of a plurality of extraction openings, thereby allowing, for example, access to all of a plurality of centrifuge tube beds via the multiple extraction openings.

[0022] The cavity bottom of each of the centrifuge tube beds typically has a shape that narrows towards the bottom of the centrifuge rotor, and by way of example, the cavity bottom may be formed as a hemisphere, a cone, a cylindrical cone, or a truncated cone extending from the lower end of the cavity side wall in the direction of the longitudinal axis of the centrifuge tube bed. At least one closing device allows each of the multiple extraction openings to be sealed liquid-tight and thereby gas-tight.

[0023] Preferably, the rotor assembly is formed as a fixed angle rotor defined by a rotor body, the plurality of centrifuge tube beds being formed as tubular cavities within the rotor body, and the at least one extraction opening extending through the rotor body.

[0024] The extraction opening is located at the lowest point of the centrifuge tube bed with respect to the axis of rotation, whereby the extraction opening is located in the region of the cavity bottom forming the lowest point of the centrifuge tube bed. The longitudinal axis of each of the centrifuge tube beds is inclined to the central axis of rotation of the centrifuge rotor, the lowest point of the centrifuge tube bed having a greater distance in the horizontal direction to said central axis of rotation than the upper part of the centrifuge tube bed in the region of the cavity side wall. In the present invention, near-vertical rotors are also considered as fixed-angle rotors and fall within the scope of protection of the present invention.

[0025] In another embodiment, the rotor assembly is formed as a swinging bucket rotor comprising a plurality of rotor buckets pivotally connected to a rotating stem, the plurality of rotor buckets comprising at least one tubular cavity forming a centrifuge tube bed, and at least one extraction opening extending through the body of each rotor bucket. The dimensions of the centrifuge tube buckets can be varied to accommodate centrifuge tubes having different sizes, materials, and sealing means.

[0026] Each of the rotor buckets is pivotally connected to the rotating stem so as to swing out relative to the rotating stem when the centrifuge rotor rotates about the central axis of rotation. The centrifuge tube beds are formed as cavities in the rotor buckets, and the longitudinal axes of the centrifuge tube beds extend parallel to the central axis of rotation in a non-rotating and non-swinging state. When the swinging bucket rotor is rotated, the rotor buckets swing relative to the rotating stem and the central axis of rotation, whereby the longitudinal axes of each of the centrifuge tube beds are inclined relative to the central axis of rotation. When in a non-rotating state, the extraction opening is located at the lowest point of the centrifuge tube bed with respect to the central axis of rotation, whereby the extraction opening is located in the region of the cavity bottom forming the lowest point of the centrifuge tube bed. When in a non-rotating state, the longitudinal axis of each of the centrifuge tube beds extends parallel to the central axis of rotation. In the swing-out position of the rotor buckets during rotation of the centrifuge rotor, the longitudinal axis of each centrifuge tube bed is inclined relative to the central axis of rotation of the centrifuge rotor, and the lowest point of the centrifuge tube bed has a greater horizontal distance to said central axis of rotation in the region of the cavity side wall than the upper part of the centrifuge tube bed.

[0027] Preferably, the closure device comprises a shaft extending along an axis from a first end to a second end, the shaft adapted to the geometric shape of the extraction opening, the first end forming a partial area of ​​the cavity bottom when the closure device is fixed to the rotor assembly to seal the respective extraction opening, the shape of the first end 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] If several extraction openings are formed on the centrifuge rotor, several closing devices are of course provided, each extraction opening being capable of being closed or sealed in a fluid-tight manner by a respective closing device.

[0029] Preferably, the closure device is provided with a limit stop at the second end for limiting movement of said closure device in the axial direction when the closure device is fastened to the rotor assembly to ensure correct alignment of the first end with the peripheral surface of the cavity bottom when the closure device is in the fastened / sealed position.

[0030] More preferably, each of the plurality of closure devices includes at least one resilient seal that is received in a groove of the closure device and that 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 resilient seal improving the sealing of the centrifuge tube bed within the rotor assembly against the periphery of the centrifuge rotor.

[0031] At least a partial area of ​​the shaft of the closure device is provided with an external thread and the extraction opening is provided with an internal thread adapted to the shape and position of the external thread, so that the closure device can be fastened to the rotor assembly by adapted threading. The threading of the closure device with the extraction opening firstly establishes a fastening means of the closure device to the centrifuge rotor, but also makes it possible to establish an airtight seal. It can be foreseen that the threading extends in the counter-rotation direction of the centrifuge rotor in order to avoid unthreading of the closure device during rotation of the centrifuge rotor.

[0032] The second end of the shaft may include at least one interlocking element protruding at least partially in a radial direction of the shaft, and the rotor assembly may include at least one notch and / or pocket adapted to receive the at least one interlocking element and form an interlocking structure with the closure device to fasten the closure device to the rotor assembly. The interlocking structure may provide a secure fastening of the closure device to the rotor assembly.

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

[0034] It can be seen that more preferably, the cavity bottom is formed by a surface narrowing in the direction towards the lower end of the rotor assembly, and at least one extraction opening is located at the lowest point of the centrifuge tube bed with respect to the central axis of rotation. In case a swinging bucket rotor is provided, a narrowing surface of the cavity bottom is found in each of the rotor buckets, where the extraction opening is located at the lowest point of the centrifuge tube bed in the case of static non-rotating and non-swinging of the rotor buckets with respect to the rotating stem of the swinging bucket rotor.

[0035] According to a second aspect of the present invention, there is provided a centrifuge or ultracentrifuge comprising a centrifuge rotor or 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, said proximal end being formed by a sharp closed tip to seal the proximal end of the sample extraction needle with at least one drain hole arranged in a lateral side wall of the cannula. The sharp closed tip of the sample extraction needle allows the needle to penetrate the centrifuge tube by piercing the wall of the centrifuge tube. The drain hole of the extraction needle is arranged on the side / lateral side wall of the needle cannula, immediately below the needle tip at the proximal end. Such positioning of the drain hole prevents clogging of the needle by small particles generated during puncturing of the centrifuge tube. Furthermore, the drain hole can be arranged at a desired height position relative to the lowest point of the centrifuge tube to allow immediate extraction of a specific centrifugal fraction from the centrifuge tube, improving the flow of fluid during extraction by the extraction needle. Alternatively, other designs of non-coring needle tips (pencil tips, deflected tips) are preferably used for sample withdrawal from centrifuge tubes.

[0037] Preferably, the sample extraction needle comprises a needle cannula having a conical shape along the axial axis, the outer diameter of the cannula decreasing in the direction towards the sharp closed tip. The extraction needle has a conical shape that ensures a sealed connection between the centrifuge tube and / or rotor assembly and the extraction needle. The conical shape with a cross shape decreasing in the direction of the sharp closed tip allows easy insertion of the extraction needle into the extraction opening and realizes self-alignment of the needle upon insertion relative to 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 centrifuge tube. The vent hole at the top of the centrifuge tube can be made with the same sample extraction needle used to make the drain hole at the bottom of the centrifuge tube to extract the sample from the centrifuge tube. The vent hole at the top of the centrifuge tube is preferably made before the extraction hole at the bottom of the centrifuge tube. The vent and extraction needles are precisely inserted into the centrifuge tube using a manually operated lever or a robotic arm.

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

[0040] The cannula may be formed by a hollow stainless steel tube, preferably having a circular or elliptical cross section.

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

[0042] It is preferred that the distal end of the cannula is connected to a needle base, wherein the needle base has a diameter larger than the opening of the extraction opening of the centrifuge rotor according to the second aspect of the present invention and can act as a limiting device to limit the maximum insertion depth of the sample withdrawal needle into the centrifuge tube bed.

[0043] According to a fourth aspect of the present invention, there is provided a method for in situ sampling from centrifuge tubes contained in a centrifuge tube bed of a centrifuge rotor according to the first aspect of the present invention, the method comprising the steps of: A) performing centrifugation of samples contained in centrifuge tubes and accommodated in a centrifuge rotor; B) Transfer of the centrifuge rotor to the rotor stand and disassembly of the centrifuge rotor; C) selecting a first centrifuge tube from which contents are to be drawn from a plurality of centrifuge tubes contained in the centrifuge rotor; D) forming a vent hole in a top region of the first selected centrifuge tube; E) opening each extraction opening of the centrifuge tube bed in which the first centrifuge tube is located by removing the respective closing device; F) introducing an extraction needle, preferably according to the third aspect of the invention, through the extraction opening towards the outer wall of the centrifuge tube, piercing the wall of the centrifuge tube and inserting the extraction needle into the first centrifuge tube to create a fluid connection to the internal volume of the centrifuge tube; G) The contents of the centrifuge tube are withdrawn through the withdrawal needle; H) Preferably, steps C) to G) are repeated on at least a further centrifuge tube contained in the centrifuge rotor.

[0044] In particular, the order of steps D) and E) may be changed, for example so that step E) is performed before step D).

[0045] Preferably, the sample extraction from the centrifuge tube occurs by displacing the contents of the centrifuge tube with compressed gas under pressure. Alternatively, the sample can be displaced from the centrifuge tube by feeding a low density, preferably water-immiscible liquid through the vent of the centrifuge tube. Preferably, an HPLC-type pump or a peristaltic pump or a syringe pump that allows good flow control is used for sample displacement from the centrifuge tube. Alternatively, the sample extraction from the centrifuge tube is performed by sample aspiration by using a suitable pump.

[0046] According to a preferred method, the contents extracted by the withdrawal needle are fed to at least one of the devices indicated in the group of an ultraviolet-visible detection system, a fluorescence detection system, a light scattering detection system, a device for performing high performance liquid chromatography (HPLC) and / or an automatic fraction collector.

[0047] The first centrifuge tube can be placed in a centrifuge tube stand prior to step D) and steps following step D).

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

[0049] According to a sixth aspect of the present invention there is provided an automated system for in situ sample extraction, the system comprising: A centrifuge rotor according to a second aspect of the present invention; comprising at least an extraction needle according to the third aspect of the invention; The system is preferably configured to carry out a method for in situ sampling according to the fourth aspect of the invention. [Brief description of the drawings]

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

[0051] [Figure 1] 1 shows a cross-section of a centrifuge rotor in a fixed angle rotor configuration, with a cut through an exemplary centrifuge tube bed having an extraction opening that is closed by a closure device. [Diagram 2] FIG. 2 shows a cross section of a centrifuge rotor in a swinging bucket rotor configuration, showing a partial cut through the swinging bucket centrifuge tube floor, with an extraction opening visible at the bottom within the centrifuge tube floor sealed by a closure device. [Figure 3A] 1 shows a first exemplary embodiment of a closure device. [Figure 3B] 1 shows a second exemplary embodiment of a closure device. [Figure 3C] 3C shows the closing device of FIG. 3B during the process of installation onto the rotor assembly of the centrifuge rotor. [Figure 4] FIG. 1 illustrates an exemplary embodiment of a sample withdrawal needle. [Diagram 5] 1 shows a schematic diagram of a sample withdrawal needle being inserted into a centrifuge tube contained within a centrifuge rotor to extract a sample from said centrifuge tube. [Figure 6] A high performance liquid chromatography pump evenly dispenses low density liquid through the vent of a centrifuge tube and delivers the displaced sample to an ultraviolet-visible detection system, a fluorescence detection system, a light scattering detection system, and a conductivity detection system. [Figure 7] A high performance liquid chromatography pump was used to draw the sample through the extraction port of the centrifuge tube and deliver the displaced sample to an ultraviolet-visible detection system, a fluorescence detection system, a light scattering detection system, and a conductivity detection system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] With reference to FIG. 1, an exemplary embodiment of a centrifuge rotor 1 in a fixed-angle rotor configuration is shown. The centrifuge 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 adapted for centrifugal rotation about a central axis of rotation 14, shown in FIG. 1 by a dashed line. As will be understood by those skilled in the art, the rotor body 12 is rotationally symmetrical about the central axis of rotation 14. In FIG. 1, a cross section of one of a plurality of centrifuge tube beds 16 is shown with a longitudinal axis 160 of the centrifuge tube bed 16. The centrifuge tube bed 16 is defined by a tubular cavity 162 adapted to receive and support the centrifuge tubes 4 during the centrifugation process. The tubular cavity 162 is formed in the material of the rotor body 12 and is bounded by a cavity side wall 162a and a cavity bottom 162b. The cavity side wall 162a and the cavity bottom 162b together act as a bearing surface for the outer surface of the centrifuge tube 4 which can be accommodated in the tubular cavity 162 of the centrifuge tube bed 16. As can be seen in Fig. 1, the cavity bottom 162b comprises a single extraction opening 18 which in the example of Fig. 1 is closed in Fig. 1 by the closing device 2. In the exemplary embodiment according to Fig. 1, the cavity bottom 162b is formed to have a shape which narrows towards the bottom 10b of the centrifuge rotor assembly 10. The top of the rotor assembly 10 is marked with the reference sign 10t.

[0053] The cavity bottom 162b is formed by a hemisphere in the example of FIG.

[0054] The tubular cavity 162 of the centrifuge tube bed 16 of the centrifuge rotor 1 is formed in 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 with the surroundings of the rotor assembly 10.

[0055] FIG. 2 shows an alternative configuration of the centrifuge rotor 1, in which the rotor assembly 10 is formed by a swinging bucket rotor and comprises a plurality of rotor buckets 13 pivotally connected to a rotating stem 11, the plurality of rotor buckets 13 having at least one tubular cavity 162 to form at least one centrifuge 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 FIG. 2, only a cross-sectional view of one of the rotor buckets 13 is shown, in which a single exemplary centrifuge tube 4 is received. The swinging bucket rotor as shown in FIG. 2 is configured to rotate about a central rotation axis 14, and each of the plurality of swinging buckets 13 is pivotally connected to the rotating stem 11 via a swing-out axis 15. The longitudinal axis 160 of the centrifuge tube bed 16 in FIG. 2 extends substantially parallel to the central rotation axis 14 of the rotating stem 11 in a non-swinging or static state.

[0056] An extraction opening 18 formed in the housing or material defining the swinging bucket 13 is closed and sealed by a closure device 2 .

[0057] 3A shows a first exemplary embodiment of the closure device 2 formed as a capping screw with 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 partial area of ​​the cavity bottom 162b in the situation when the closure device 2 is fastened to the rotor assembly 10, as derived from the examples shown in FIGS. 1 and 2. As can be seen from FIG. 2, the first end 221 fits the shape of the cavity bottom 162b and forms a smooth transition of the surface of the first end 221 and the peripheral surface of the cavity bottom 162b. The closure device 2 as shown in the configuration of FIG. 3A further comprises a limit stop 24 at the second end 222 for limiting the movement of the closure device 2 in the direction along the shaft axis 22a when the closure device 2 is fastened to the rotor assembly 10, in order to ensure a correct alignment of the first end 221 with the peripheral surface of the cavity bottom 162b. The closure device 2 shown in Fig. 3A comprises a groove 26 for receiving one elastic seal 28 configured as an O-ring. The elastic seal 28 is received in said groove 26 of the closure device and is deformed when the closure device 2 is fastened to the rotor assembly 10. When the closure device 2 is fastened to the rotor assembly 10, the closure device 2 of Fig. 3A engages with the outer surface of the rotor body 12 or the body of the rotor bucket 13 at least in the region of the limit stop 24, elastically deforming the elastic seal 28 to define an airtight seal. Also, as can be seen from the exemplary embodiment of the closure device 2, a partial region of the shaft 22 comprises an external thread 27, which comprises an internal thread adapted to the shape and position of the external thread 27, so that the closure device can be fastened to the rotor assembly 10 by the adapted internal-external thread 27.

[0058] FIG. 3B shows a second exemplary embodiment of the closing device 2, in which the closing device 2 is configured to form an interlocking structure with the rotor assembly 10 to achieve the fixation. The closing device 2 as shown in FIG. 3B differs from the device as shown in FIG. 3A in that it is not provided with an external thread 27 in the area of ​​the shaft 22 to achieve the fixation of the closing device 2 to the rotor assembly 10. The closing device 2 according to FIG. 3B comprises two interlocking elements 29 that allow the closing device 2 to be fixed to the rotor assembly 10. The interlocking elements 29 can be provided in addition to or as an alternative to the external thread 27, as explained with reference to FIG. 3A. The closing device 2 according to FIG. 3B comprises two elastic seals 28, one of which is arranged to be received in a groove 26 in the area of ​​the shaft 22 and a further elastic seal 28 is arranged in a groove 26 located in the area of ​​the second end 222 of the joint surface of the limit stop 24 with the rotor assembly 10. The second end 222 of the closure device 2 has a recess 222a on its face facing away from the rotor housing, in the area of ​​the limit stop 24, which makes it possible to rotate the closure device 2 around the shaft axis 22a to fasten and unfasten the closure device 2 to the rotor housing 10.

[0059] As shown in FIG. 3C, the closing device 2 is detailed during the fastening process to the rotor assembly 10. FIG. 3C shows a top view on the second end 222 of the closing device 2. During fastening of the closing device 2, the closing device 2 is placed in the area of ​​the extraction opening 18, where the interlocking element 29 is placed in the respective cutout 19a of the rotor assembly 10. To fasten the closing device 2 to the rotor assembly 10, the closing device is rotated about the axis 22a, moving the interlocking element 29 into the adapted pocket 19b of the rotor assembly 10. The interlocking element 29 and the pocket 19a as well as the pocket 19b form a mutually interlocking structure, which is adapted to achieve a secure fastening of the closing device 2 to the rotor assembly.

[0060] 4 shows an exemplary embodiment of a sample extraction needle 3, in which the sample extraction needle 3 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 sharpened closed tip 31 to seal the proximal end 30p of the cannula 30 of the sample extraction needle 3. In the embodiment shown in FIG. 4, the sample extraction needle 3 comprises four drainage holes 32 arranged in the region of the lateral sidewall 33 of the cannula 30. In the schematic view of FIG. 4, only three of the four drainage holes 32 are shown evenly distributed in the circumferential direction of the cannula 30 in the lateral sidewall 33 around the axis 300.

[0061] 4, needle cannula 30 has a conical shape along axis 300, with the outer diameter of cannula 30 decreasing in the direction toward sharp closed tip 31. Sharp closed tip 31 is formed by a circular cone, where the front surface of 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 by a hollow stainless steel tube, which can have an outer diameter in the range of 0.7-1.5 mm in the region of the distal end 30d. The distal end 30d of the cannula 3 is connected to a needle base 34, which has a diameter 34d dimensioned to be larger than the opening of the extraction opening 18 of the centrifuge rotor 1 according to the invention. The needle base 34 is thereby configured to act as a limiting device limiting the maximum insertion depth I of the sample extraction needle 3 in the centrifuge tube bed 16.

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

[0064] FIG. 6 shows an example of sample displacement from a centrifuge tube by dispensing low density liquid evenly through the vent of the centrifuge tube with a high performance liquid chromatography pump.

[0065] Density gradient fractionation was performed in a T890 fixed angle rotor using 11.5 mL polyethylene UltraCrimp centrifuge tubes (Thermo Scientific) on a Sorvall® WX 90+ ultracentrifuge (Thermo Scientific). Adeno Associated Virus (AAV) samples were mixed with concentrated cesium chloride to obtain AAV samples in 3M cesium chloride. Centrifugation was performed at room temperature at 53,500 RPM x 24 h. The tubes were then secured to a stand and a vent hole was pierced near the top with a hypodermic needle (23 gauge, 70 mm, B Braun). Another hypodermic needle was used to pierce an extraction opening at the bottom of the centrifuge tube. The tube contents were displaced from the centrifuge tube by pumping water at a constant flow rate of 1 mL / min using the HPLC pump of a PATfix® LPG HPLC system (BIA separation) through the vent hole at the top of the centrifuge tube. The extraction opening at the bottom of the centrifuge tube was directly connected to the monitor array of a PATfix® LPG HPLC system, which evacuated the tubes in order of decreasing density. UV absorbance was monitored at 260 nm (solid trace). Intrinsic fluorescence was monitored with a fluorescence detector (Shimadzu dash trace) at an excitation wavelength of 280 nm and an emission wavelength of 348 nm. Light scattering was monitored at a 90° angle with 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). The higher the conductivity, the higher the density of cesium chloride.

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

[0067] Density gradient fractionation was performed in a T890 fixed angle rotor using 11.5 mL polyethylene UltraCrimp centrifuge tubes (Thermo Scientific) on a Sorvall® WX 90+ ultracentrifuge (Thermo Scientific). Adeno Associated Virus (AAV) samples were mixed with concentrated cesium chloride to obtain AAV samples in 3M cesium chloride. Centrifugation was performed at room temperature at 53,500 RPM x 24 h. The tubes were then secured to a stand and a vent hole was pierced near the top with a hypodermic needle (23 gauge, 70 mm, B Braun) and left open to allow normal air pressure. Another hypodermic needle was used to pierce an extraction opening at the bottom of the centrifuge tube. The tube contents were drawn from the centrifuge tube through the extraction opening at the bottom of the centrifuge tube at a constant flow rate of 1 mL / min using the HPLC pump of the PATfix® LPG HPLC system (BIA separation). An HPLC pump directed the contents of the centrifuge tubes to a monitor array of a PATfix® LPG HPLC system. This evacuated the tubes in order of decreasing density. UV absorbance was monitored at 260 nm (solid trace). Intrinsic fluorescence was monitored with a fluorescence detector (Shimadzu dash trace) at an excitation wavelength of 280 nm and an emission wavelength of 348 nm. Light scattering was monitored at a 90° angle with 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). The higher the conductivity, the higher the density of cesium chloride.

Claims

1. A rotor assembly (10) adapted for centrifugal rotation about a central axis of rotation (14) comprising: a plurality of centrifuge tube beds (16), each of said plurality of centrifuge tube beds (16) having a longitudinal axis (160); The plurality of centrifuge tube beds (16) each include a tubular cavity (162) defined by a cavity sidewall (162a) and a cavity bottom (162b); At least one of the plurality of cavity bottoms (162b) includes at least one extraction opening (18) connecting the tubular cavity (162) to an exterior of the rotor assembly (10); at least one closure device (2) removably fixed to said rotor assembly (10) for sealing at least one extraction opening (18) in an airtight manner; The cavity side wall (162a) and the cavity bottom (162b) together form a support surface for the outer surface of the centrifuge tube (4) when the centrifuge tube (4) is received in the centrifuge tube bed (16).

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

3. 2. The centrifuge rotor (1) of claim 1, wherein the rotor assembly (10) is a swing bucket rotor including a plurality of rotor buckets (13) pivotally connected to a rotating stem (11), the plurality of rotor buckets (13) including at least one tubular cavity (162) forming the centrifuge 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. 4. The centrifugal rotor (1) according to claim 1, 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) adapted to a shape of the extraction opening (18), the first end (221) forming a partial area 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) adapted to a shape of the cavity bottom (162b) forming a smooth transition between a surface of the first end (221) and a peripheral surface of the cavity bottom (162b).

5. 5. The centrifugal rotor (1) according to claim 4, wherein each of the plurality of closing devices (2) is provided with a limit stop (24) at the second end (222) for limiting the axial (22a) movement of the closing device (2) and ensuring that the first end (221) is correctly aligned with the peripheral surface of the cavity bottom (162b) when the closing device (2) is fixed to the rotor assembly (10).

6. 6. The centrifugal rotor according to claim 1, wherein each of the plurality of closure devices (2) comprises at least one elastic seal (28), which is received in a groove (26) of the closure device (2) and deforms when the closure device (2) is fastened to the rotor assembly (10) to form an airtight seal with the rotor assembly (10).

7. 7. The centrifugal rotor according to claim 4, wherein at least a portion of the shaft is provided with an external thread (27), the extraction opening (18) is provided with an internal thread (27) that matches the shape and position of the external thread (27), and the closure device (2) is fastened to the rotor assembly (10) by the matching external and internal threads (27).

8. 8. The centrifugal rotor according to claim 4, wherein the second end (222) of the shaft (22) comprises at least one interlocking element (29) protruding at least partially in a radial direction of the shaft (22), and wherein 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 closure device (2) is fastened to the rotor assembly (10).

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

10. 10. The centrifugal rotor according to claim 1, wherein the cavity bottom (162b) is formed by a narrowing surface in a direction 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) relative to the central axis of rotation (14).

11. A centrifuge or ultracentrifuge (1) comprising a centrifuge rotor (1) according to any one of the preceding claims.

12. A method for in situ sampling of centrifuge tubes (4) stored in a centrifuge tube bed (16) of a centrifuge rotor (1), said centrifuge rotor (1) having the features according to any one of claims 1 to 10, said method comprising: A) centrifuging the sample contained in the centrifuge tube (4) contained in the centrifuge rotor (1); B) transferring the centrifuge rotor (1) to a rotor stand and disassembling the centrifuge rotor (1); C) selecting a first centrifuge tube (4) from which the contents are to be extracted from among a plurality of centrifuge tubes (4) contained in said centrifuge rotor (1); D) forming at least one vent hole in the upper region of the selected first centrifuge tube (4); E) opening the extraction opening (18) of the centrifuge tube bed (16) in which the first centrifuge tube (4) is placed by removing the closing device (2); F) introducing a sample withdrawal needle (3) through the extraction opening (18) towards the outer wall of the centrifuge tube (4), piercing the wall of the centrifuge tube (4) and inserting the sample withdrawal needle (3) into the first centrifuge tube (4) to create a fluid connection to the internal volume of the centrifuge tube (4); G) Extracting the contents of the centrifuge tube (4) through the sample needle (3); method

13. H) repeating steps C) to G) on another centrifuge tube (4) contained in the centrifuge rotor (1); The method of claim 12.

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

15. 13. The method of claim 12, wherein the sample is displaced from the centrifuge tube (4) by evenly dispensing a low density, water-immiscible liquid through the vent of the centrifuge tube.

16. 16. The method according to claim 15, using a high performance liquid chromatography, HPLC type pump, a peristaltic pump or a syringe pump, enabling sample displacement from the centrifuge tube (4).

17. 17. The method according to any one of claims 12 to 16, wherein the contents extracted by the sample withdrawal needle (3) are fed to at least one device selected from the group of a UV-VIS detection system, a fluorescence detection system, a light scattering detection system, a high performance liquid chromatography and / or an automated fraction collector.

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

19. Use of a centrifuge rotor according to any one of claims 1 to 10 or a centrifuge or ultracentrifuge according to claim 11 and use of a sample withdrawal needle for carrying out the method according to any one of claims 12 to 18.

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

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