Device and method for suspending cells from a solid sample

Abstract

The present invention relates to a device for the collection, pre-analytical treatment, transport and / or grinding of a solid sample, comprising a flask for receiving the solid sample, a pre-analytical medium comprised in the flask and a plurality of beads also comprised in the flask, each bead having a diameter equal to or greater than 5 mm and a density of at least 5.0 g.cm-3. The present invention also relates to the use of said device and to a method for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample.

Description

DEVICE AND METHOD FOR SUSPENDING CELLS FROM A SOLIDSAMPLEFIELD OF INVENTION

[0001] The present invention relates to the field of preanalytical treatment of a solid biological sample; the sample may be a biopsy of soft tissue, or it can be hard tissue, such as bone, for example. More specifically, this invention relates to a device and a method for gently suspending entire eukaryotic or prokaryotic cells from a solid biological sample, in a pre-analytical medium, without compromising the integrity of the cell wall or the plasmic membrane of the cell. The suspension is then recovered for analytical purposes of the cells of the sample.BACKGROUND OF INVENTION

[0002] The analysis of solid samples or samples containing at least one solid element often occurs: the samples may be soil samples, plant samples or solid tissue in human or veterinary medicine. The treatment of a solid sample in order to perform an analysis is greatly facilitated by the sample grinding until a suspension or a solution is obtained. This is particularly the case for surgical operations and the diagnosis of infection on an implanted material; the problem is particularly sensitive in the diagnosis of infections on orthopedic devices. Indeed, infections on orthopedic material and especially on articular implants are difficult to diagnose because of the small number of microorganisms and because the metabolism of these microorganisms adapts to survive within a biofilm on the surface of the implant or inside the cells present at the site of the infection.

[0003] In the past, the preparation of a solid sample for the analysis of one or more elements involved several successive steps with repeated manipulations and multiple reagents whose traceability was difficult to establish. These successive steps could therefore lead to degradation of the sample and eventually to its contamination. In theprior art, WO2013179232 discloses a device for the collection, the preanalytical treatment, the transport and the grinding of solid samples, aiming at limiting as much as possible the handling of a solid sample between its collection and the analysis of the element(s) to be analyzed.

[0004] However, the grinders commonly used to perform the pre-analytical grinding step are not optimal and do not allow the dissociation of solid samples in a suitable manner to then extract the element(s) to be analyzed: the cells of the sample may be destroyed or damaged, and the internal content of the cells be mixed in the medium. Moreover, the samples to dissociate can comprise hard tissue (such as cortical and cancellous bone, tendon), soft tissue (such as muscle, fat) or elastic tissue (such as ligamentous, vascular tissue) from which the cells might not be adequately released due to insufficient grinding.

[0005] The purpose of the present invention is to remedy this problem by providing a device and method for treating a solid sample and individualizing the cells thereof, without degrading the cells to be analyzed. More particularly, the device shall further preserve the integrity of the cells of the sample, and their preservation, i.e. it shall be configured to prevent the sample and / or the resulting suspension from any contaminations.SUMMARY

[0006] Considering the issues of the prior art in obtaining entire cells from a solid sample, the Applicant felt that the technical issue at stake was not a mere selection of bead size. The Applicant felt a need in modifying the blending / grinding / milling devices from the prior art. This research program did not really succeed, and the inventors turned their mind into a multifactorial approach: bead size and density was at stake, but other factors as well, such as for example the means for agitating, the frequency of the agitation, the medium where the sample was placed. For a long time, the Applicant failed to find a reproducible process able to convert a solid sample into a suspension of cells whose integrity was preserved, so that they can be further analyzed.

[0007] Thus, one obj ect of the present invention refers to a device for the collection, pre- analytical treatment, transport and / or stirring of a solid sample, comprising, with combination to external agitation means at a frequency ranging from 10 to 50 Hz. a flask in which was introduced, simultaneously or sequentially, a pre-analytical medium and a at least one bead having a diameter ranging from 5 to 12 mm and a density of at least 5.0 g.cm-3 according to the ISO 3290-1 (2008) standard or the DIN 5401 (2000) standard , the device further comprising means for sealing the flask after introduction of the sample, medium and bead(s).

[0008] In one embodiment, the present invention refers to a device for suspending, in a pre-analytical medium, entire eukaryotic or prokaryotic cells from a solid biological sample, comprising a flask in which is introduced simultaneously or sequentially: a pre-analytical medium having a pH ranging from 6 to 9, and an osmolality ranging from more than 0 to 800 mOsm / kg of water, in an amount and a composition suitable to suspending the solid sample upon agitation, at least one bead (A), each bead (A) having a diameter ranging from 5 to 12 mm and a density of at least 5.0 g.cm'3, said flask being configured to collect or transport a biological solid sample in a ratio of the total mass of the beads to the mass of the solid sample ranging from 2 to 10, the device further comprising sealing means for the flask to be sealed at the place of sampling, after collection of the sampling, and the sealed flask is air- and water-tight, until the cells are analyzed, said flask being deprived from any internal agitation means, said sealed flask being configured to physically resist beads impact on the walls, bottom and seal of the flask, due to beads movement triggered by external agitation, said external agitation being in a range of at least 10-50 Hz, preferably at least 25 Hz, more preferably 26-35 Hz, preferably for 30-300 s, suitable for separating the cells from the sample and retaining integrity of at least one cell wall or plasmic membrane.external agitation ranging from 10-50 Hz, such that, with combination to the medium and beads, which separates the cells from the sample and retains integrity of at least one cell wall or plasmic membrane.

[0009] According to one embodiment, the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 8.0 g.cm'3, even more preferably of about 8 g.cm'3.

[0010] According to one embodiment, the diameter of each bead (A) ranges from 6 mm to 10 mm, preferably from 6 mm to 8 mm, more preferably from 6 mm to 7 mm, even more preferably the diameter of each bead (A) is of about 6.35 mm.

[0011] According to one embodiment, the number of beads (A) comprised in the flask is of at least two beads, preferably from 3 to 10 beads, more preferably the number of beads is of 4 or 5 or 6 beads.

[0012] According to one embodiment, the beads (A) are made of a material selected from the group consisting of glass, steel, ceramic and combinations thereof, preferably the beads are made of steel, and more preferably of stainless steel.

[0013] According to one embodiment, the flask further comprises at least one additional bead different from the beads (A), said additional beads having a diameter strictly lower than 2 mm, preferably strictly lower than 1 mm, more preferably a diameter ranging from 0.05 to 1 mm, even more preferably from 0.08 to 0.5 mm.

[0014] According to one embodiment, the osmolality of the pre-analytical medium ranges from 40 to 500 mOsm / kg, more preferably from 100 to 400 mOsm / kg, and better still from 150 to 350 mOsm / kg of water.

[0015] According to one embodiment, the redox potential of the pre-analytical medium ranges from -200 to 200 mV, preferably from -150 to 0 mV and more preferably from - 100 to -50 mV.

[0016] According to one embodiment, the pH of the pre-analytical medium ranges from 6 to 9.

[0017] According to one embodiment, the flask is sterilized and packaged in an air-tight packaging, prior to introduction of the preanalytical medium, the beads and the sample.

[0018] The present invention further refers to a method for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample, said method comprising a step of agitating said solid sample in a pre-analytical medium with at least one bead set in motion by agitation at a frequency of 10 to 50 Hz, preferably 15 to 35 Hz, more preferably 18-26 Hz each bead having a diameter greater than 5 mm and a density of at least 5.0 g.cm'3, the ratio of the total mass of the beads to the mass of the solid sample being strictly greater than 1.

[0019] According to one embodiment, the solid sample is selected from the group consisting of a biological tissue, a medical device, and combinations thereof, preferably selected from the group consisting of: osteoarticular tissue, vascular tissue, cardiac tissue, skin tissue, lipidic tissue, muscular tissue, nervous tissue, pancreatic tissue, hepatic tissue, prostatic tissue, renal and urinary tract tissue, hematopoietic tissue, glandular, lymphatic tissue, immunogenic tissue, intestine tissue and digestive tract tissue, respiratory tissue, reproductive tissue, sutures or staples, grafts, fixation devices, parts of, or whole, prosthetic joint devices, vascular devices, mechanical or xenobiotic or native cardiac valves, atrial patches, drainage tubes, pacemakers including the probes and wires, and any combination thereof.

[0020] According to one embodiment, the total mass of the beads to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the beads to the mass of the solid sample is of about 5.

[0021] According to one embodiment, the all or part of at least one eukaryotic or prokaryotic cell is selected from the group consisting of: all or part of a bacterium and all or part of a mammalian cell.

[0022] According to one embodiment, the all or part of at least one eukaryotic or prokaryotic cell is selected from the group consisting of: a protein and a nucleic acid.DEFINITIONS

[0023] In the present invention, the following terms have the following meanings:

[0024] “About”, before a figure or number, refers to plus or minus 10% of the face value of that figure or number. In one embodiment, “about”, before a figure or number, refers to plus or minus 5% of the face value of that figure or number.

[0025] “stirring” refers to mixing in view of turning a solid + liquid mixture into a suspension.

[0026] “At least” means “greater than or equal to” and is thus different from “strictly less than” or “strictly greater than” which do not comprise “equal to”.

[0027] “Bead” or “Ball” refers to a solid object, most frequently (but not necessarily) in the form of a sphere, wherein “sphere” refers to a geometrical object that is a three- dimensional circle; otherwise expressed, a sphere is a surface made up of all the points located at the same distance r from a point called center of the sphere, r being the sphere's radius. However, for practical purposes, the skilled artisan appreciates that a bead may be geometrical objects not spherical in nature. Polyhedral, elliptical, or randomly shaped geometric objects such a garnet or pebbles can be used in the framework of this invention, in an equivalent fashion. Preferably, the beads or balls used according to the present invention respect the ISO 3290-1 (2008) standard or the DIN 5401 (2000) standard.

[0028] “Biological solid sample” refers to any solid sample comprising or consisting of a tissue made of or containing at least one eukaryotic or prokaryotic cell.

[0029] “Comprising” or “comprise” is to be construed in an open, inclusive sense, not limited to the features following this term. “Comprising” or “comprise” also encompasses narrower expression such as “substantially consist of’.

[0030] “Consisting of’ or “consist” is to be construed in a close, non-inclusive sense, limited to the features following this term.

[0031] “Flask” and “vial” are used interchangeably and design a small container.

[0032] “From X to Y” refers to the range of values between X and Y, the limits X and Y being included in said range.

[0033] “Gel” refers to a three-dimensional network of covalently or non-covalently bound molecules (forming a matrix) in a liquid (that acts as a swelling agent). The gel exhibits the properties of a solid ranging from ductile to brittle. A gel has a viscosity equal to or greater than 5.1 O’3Pa.s at a temperature of 25°C and a pressure of 1 atm.

[0034] “Liquid” refers to a nearly incompressible fluid that conforms to the shape of its container but retains a constant volume independent of pressure. It is one of the four fundamental states of matter (the others being solid, gas, and plasma) and is the only state with a definite volume but no fixed shape. A liquid has a viscosity lower than 5.1 O’3Pa.s at a temperature of 25°C and a pressure of 1 atm.

[0035] “Viscosity” or “dynamic viscosity” refers, for a laminar flow of a fluid, to the ratio of the shear stress to the velocity gradient perpendicular to the plane of shear. Viscosity may be measured by methods well-known to a skilled person in the art. Unless otherwise indicated, viscosity, in the present application, is measured by any viscosity measurement method well known to person skilled in the art. In particular, viscosity may be measured using a TA Instruments AR-G2 rheometer equipped with a 3 cm diameter, 2-degree angle cone with a plate geometry and a temperature control cell set at 25°C, the viscosity value being read at a shear rate equal to 10 s'1.DETAILED DESCRIPTION

[0036] Because the milling beads act as vehicles to transfer the kinetic energy generated by the agitation to the specimen to disrupt it, the kinetic energy transferred to the bead in the course of one cycle is a key parameter to both the effectiveness of the beadmilling and the mechanical resistance of the vial. Because the literature commonly considers the totality of the beads contained in a vial as a single entity, it fails to address the fact that one hundred low energy impacts are not mechanically equivalent to a single impact ofone hundred times higher energy. This confusion has greatly hindered the development of effective beadmilling devices. Without willing to be linked to any theory, the Applicant hightlights that beyond the frequency of the agitation of the beadmill (agitator), a key parameter is the course of the movement achieved by the vial as it determines the speed of the bead. The average kinetic energy is Ek= l / 2m.v2(m: mass; v: average speed). Considering that v=2.c.f (c: course or distance of ’A oscillation, f: frequency of the agitation), Ek= l / 2m.(2.c.f)2= 2.m.c2.f2.

[0037] In one embodiment, the kinetic energy of the agitated beads ranges from 0,2 to 100 mJ. In one embodiment, the kinetic energy of the beads ranges from 0,2 to 12 mJ. In one embodiment, the kinetic energy of the beads ranges from 0,5 to 30 mJ.

[0038] In one embodiment, the bead diameter ranges from 5.5 to 12 mm, the bead density is of 8.0 g / cm3, the frequency of agitation ranges from 25 to 35 Hz, and the kinetic energy of the beads range from 0,2 to 100 mJ.

[0039] Therefore the vial must sustain the mechanical stress generated by the impact of the beads. In the case of the common laboratory beadmills in which the vial is held by a clamp subjecting the cap and the botton to the impact of the beads, the average energy delivered to the sample or the vial may be a function of the mass of each bead, and the frequency of the agitation.

[0040] The effective extraction of analytes such as eukaryotic or prokaryotic cells from solid specimen such as bone, preferably cancellous bone, elastic tissues such as lung tissue, vascular tissues or ligaments, fibrous tissues such as connective tissues or tendons, or soft tissues such as muscle, skin, lymphoid, liver or other biological tissues requires sufficient energy to disrupt the tissular structure and recover the analyte in the form of a suspension or a solution. Contrary to commonly held beliefs, we observed that the grinding efficiency achieved by a vial placed on a beadmill is not dependent on the energy transferred to the plurality of beads in the vial, but to the energy transferred to individual beads in the device, providing a greater efficacy to the implementations of fewer beads of greater mass, over a greater number of beads of lower individual mass.

[0041] The configuration of the vial is such that the vial can withstand the energy transferred by a bead upon impact, so that the vial is not breached and neither the sample nor the environment are contaminated by the communication established between the outside and the inside of the vial

[0042] This invention encompasses the device of the invention before use, in use, and after use. In one embodiment, the device is a single-use device. In one embodiment, the flask is a single-use flask. In one embodiment, before use, the flask is a single-use sterile flask in which was introduced, or is suitable for introduction of, a pre-analytical medium and at least one bead, as described herein. In one embodiment, in use, the flask is a singleuse flask comprising a pre-analytical medium and at least one bead, and a solid sample, preferably a biopsy. In one embodiment, the device of the invention comprises a sealed flask, eventually sterile, having at least one beads and a suspension of loosen cells in a preanalytical medium, said cells being from a solid sample initially introduced in the flask.

[0043] In one embodiment, the device of the invention comprises both a single-use sterile flask for receiving the solid sample, and external agitation means. In one embodiment, the device of the present invention includes an external mechanical stirrer, with holding means adapted for holding at least one flask of the invention. In one embodiment, the stirrer includes holding means adapted for holding 2-24, preferably 4-8 flasks. In one embodiment, the stirrer agitates the flask at a frequency of 10 to 50 Hz, preferably 15 to 30 Hz, more preferably 18-26 Hz. In one embodiment, the flask includes grippable areas suitable for being held by an external stirrer.

[0044] In one embodiment, the device is a double wrapped sterile flask comprising a pre-analytical medium and at least one bead (A), preferably two beads (A) which may be the same or different, each bead (A) having a diameter greater than or equal to 5 mm, and a density of at least 5.0 g.cm'3, according to the ISO 3290-1 (2008) standard or the DIN 5401 (2000) standard.

[0045] In one embodiment, the device of the present invention does not include internal agitating means.

[0046] In one embodiment, the device of the invention is a sterile packaged flask suitable for suspending, in a pre-analytical medium, entire eukaryotic or prokaryotic cells from a solid sample, comprising a flask in which was introduced, simultaneously or sequentially: a pre-analytical medium having a pH ranging from 6 to 9, and an osmolality of ranging from more than 0 to 800 mOsm / kg of water, and an amount of liquid medium ranging from 1 to 10 ml; at least one bead (A), preferably two beads (A) which may be the same or different, each bead (A) having a diameter ranging from 5 to 12 mm and a density of at least 5.0 g.cm'3said flask being configured to collect a biological solid sample in a ratio of the total mass of the beads to the mass of the solid sample ranging from 2 to 10, said flask being also configured to be sealed at the place of sampling, after collection of the sampling, said flask and seal being configured to physically resist a stirring step of 50 Hz.

[0047] In one embodiment, said stirring step is such that, upon stirring, with combination to the pre-analytical medium and beads, it suspends individual cells from the sample and retains integrity of at least 80% of the cells, (meaning that it does not destroy cell walls or plasma membrane of said cells) until a suspension that can be extracted from the flask is obtained. In one embodiment, the resulting suspension may be extracted with a 18G pipet.

[0048] In one embodiment, the sterile wrapped flask is a single use device.

[0049] According to one embodiment, the density of each bead (A) is of at least 5.0 g.cm'3, preferably of at least 6.0 g.cm'3, more preferably of at least 7.0 g.cm'3, even more preferably of at least 8.0 g.cm'3. Advantageously, the density of each bead (A) is of about 8 g.cm'3. Advantageously, all the beads (A) comprised in the flask, have the same density.

[0050] According to one embodiment, the diameter of each bead (A) ranges from 5 mm to 12 mm, preferably from 5 mm to 10 mm, more preferably from 6 mm to 10 mm, even more preferably from 6 mm to 8 mm, better from 6 mm to 7 mm, still better from 6 mmto 6.5 mm. Even more advantageously, the diameter of each bead (A) is of about 6.35 mm.

[0051] According to one embodiment, the number of beads (A) comprised in the flask is of at least two beads, preferably from 3 to 10 beads, more preferably the number of beads is of 4 or 5 beads. In one embodiment, the at least two beads (A), corresponding to all the beads (A) comprised in the flask, have the same diameter.

[0052] In one embodiment, the mass of each bead (A), comprised in the flask, ranges from 0.1 g to 5 g, preferably 0.5 g to 4 g, preferably from 0.7 g to 2 g, more preferably from 0.8 g to 1.4 g, even more preferably the mass of each bead (A) is of about 1 g. In one embodiment, the at least two beads (A), corresponding to all the beads (A) comprised in the flask, have the same mass.

[0053] Advantageously, the total mass of the at least two beads (A), corresponding to the total mass of all the beads (A) comprised in the flask according to the present invention, ranges from 2 g to 10 g, preferably from 3 g to 10 g, more preferably from 3 g to 7 g, even more preferably the total mass of the at least two beads (A) is of about 5 g.

[0054] Advantageously, the at least two beads (A), comprised in the flask, are made of a material, which is chemically inert and which is not susceptible to corrosion in the pre- analytical medium used according to the present invention. Preferably, the beads (A) are made of a material selected from the group consisting of: glass, steel, ceramic, including zirconium, and combinations thereof. More preferably, the beads (A) are made of steel. Better still, the beads (A) are made of stainless steel, preferably of grades AISI 304 / 304L or more preferably AISI 316 / 316L.

[0055] The advantage of those beads, is that they convey the energy of the external agitation means to the content of the flask, and dissociate the solid sample. Functionally, the beads used in this invention are such that they are in a sufficient mass, diameter and density that they dissociate the solid sample without damaging the flask or the cap of the flask, and without destroying the cell walls or membranes.

[0056] The external agitation device transfers kinetic energy to the beads according to their speed and mass. The beads in turn transfer the energy to the tissue sample that contains the cells to be extracted depending on the material of the beads. Contrary to the general consensus that considers the mass, size and density of the totality of the beads included in the device, the invention implements combinations of beads where the efficacy is driven by the kinetic energy of each bead and not the kinetic energy of the combined beads. Kinetic energy is approximated using the average velocity over a cycle of agitation and is dependent on the geometry and frequency of each beadmill.

[0057] In one embodiment, the kinetic energy of each bead is comprised between 0.1 mJ and lOOmJ, preferably between 0.5 mJ and 50 mJ, even more preferably between 1.5 mJ and 6 mJ. Velocities are ranging from 1 m / s to 6 m / s, preferably from 1.6 m / s to 3.2 m / s, more preferably from 1.9 m / s to 2.8 m / s, even more preferably from 2.2 m / s to 2.5 m / s.

[0058] According to one embodiment, the flask further comprises at least one additional bead different from the beads (A), said additional beads having a diameter strictly lower than 2 mm, preferably strictly lower than 1 mm, more preferably a diameter ranging from 0.05 to 1 mm, even more preferably from 0.08 to 0.5 mm. According to a particular embodiment, the device of the present invention comprises a combination of four identic stainless-steel beads (A) having a diameter of 6.35 mm and at least one zirconium bead, having a diameter of 0.1 mm.

[0059] In one embodiment, the density of each bead (A) is of about 8.0 g.cm'3; and the number of beads (A) is of 4 or 5; and the diameter of each bead (A) ranges from 6 mm to 6.5 mm.

[0060] Advantageously, the pre-analytical medium is a medium suitable for the preservation, the transport, the survival and / or the growth of eukaryotic and / or prokaryotic cells.

[0061] Advantageously, the osmolality of the pre-analytical medium ranges from 20 to 800 mOsm / kg, preferably from 40 to 500 mOsm / kg, more preferably from 100 to 400 mOsm / kg, and even more preferably from 150 to 350 mOsm / kg of water. The osmolalityof a solution quantifies the osmotically active particles or ions contained in a solution. In contrast to molarity, which determines the number of particles or ions in a volume of fluid, osmolality refers to the mass weight. It is normally expressed as mOsm / kg (milliosmole / kg) of water or mmol / kg of water. The osmolality of a solution can be determined in accordance with methods well-known in the art. For example, osmolality can be measured by using a freezing point depression osmometer or a vapour pressure depression osmometer. Since the freezing point of a water-based composition is depressed dependent on the number of osmotically active particles or ions contained, it is possible to measure osmolality by determining the freezing point of the composition.

[0062] According to one embodiment, the redox potential of the pre-analytical medium ranges from -200 to 200 mV, preferably from -150 to 0 mV and more preferably from -100 to -50 mV.

[0063] According to an embodiment, the pH of the pre-analytical medium ranges from 4 to 11, preferably from 5 to 10, more preferably from 6 to 9. More preferably, the pH of the pre-analytical medium ranges from 7 to 8, and even more preferably from 7 to 7.4.

[0064] Advantageously, the pH of the pre-analytical medium ranges from 6 to 9, preferably from 7 to 8 and more preferably from 7 to 7.4; and the osmolality of the pre- analytical medium ranges from 0 to 800 mOsm / kg, preferably from 40 to 500 mOsm / kg, more preferably from 100 to 400 mOsm / kg, and even more preferably from 150 to 350 mOsm / kg of water.

[0065] Advantageously, the pH of the pre-analytical medium ranges from 6 to 9, preferably from 7 to 8 and more preferably from 7 to 7.4; and the redox potential of the pre-analytical medium ranges from -200 to 200 mV, preferably from -150 to 0 mV and more preferably from -100 to -50 mV.

[0066] Advantageously, the pH of the pre-analytical medium ranges from 6 to 9, preferably from 7 to 8 and even more preferably from 7 to 7.4;the osmolality of the pre-analytical medium ranges from 0 to 800 mOsm / kg, preferably from 40 to 500 mOsm / kg, more preferably from 100 to 400 mOsm / kg, and even more preferably from 150 to 350 mOsm / kg of water; and the redox potential of the pre-analytical medium ranges from -200 to 200 mV, preferably from -150 to 0 mV and more preferably from -100 to -50 mV.

[0067] According to one embodiment, the pre-analytical medium is in the form of a gel or a liquid. In one embodiment, the volume of the pre-analytical medium ranges from 5 mL to 20 mL.

[0068] The pre-analytical medium of the present invention preferably comprises water, an organic solvent and mixtures thereof. Preferably, the pre-analytical medium is a liquid and is selected from the group consisting of: an aqueous solution comprising 0.9 % w / v of NaCl relative to the total volume of said aqueous solution, chloroform, water and acetonitrile. The water may be ultrapure or sterile water.

[0069] In one embodiement, the pre-analytical medium comprises divalent cations such as Mg2+and / or Ca2+in concentrations ranging from 0.5 mM to 20 mM. Preferably, the concentration of divalent cations is ranging from 1 mM to 5 mM. More preferably, the concentration of divalent cation is ranging from 1.5mM to 2.5 mM.

[0070] In one embodiment, the pre-analytical medium of the present invention preferably comprises water, an organic solvent and mixtures thereof.Examples of suitable pre-analytical medium that can be used according to the present invention are notably selected among water, BS, PBS, BIS-TRIS, BIS-TRIS Propane, HEPES, HEPES Sodium Salt, MES, MES Sodium Salt, MOPS, MOPS Sodium Salt, Sodium Chloride, Ammonium acetate solution, Ammonium formate solution, Ammonium phosphate monobasic solution, Ammonium tartrate dibasic solution, BICINE buffer Solution, Bicarbonate buffer solution, Citrate Concentrated Solution, Formic acid solution, Imidazole buffer Solution, MES solution, Magnesium acetate solution, Magnesium formate solution, Potassium acetate solution, Potassium acetate solution, Potassium acetate solution, Potassium citrate tribasic solution, Potassium formate solution, Potassium phosphate dibasic solution, Potassium phosphate dibasicsolution, Potassium sodium tartrate solution, Propionic acid solution, STE buffer solution, STET buffer solution, Sodium acetate solution, Sodium formate solution, Sodium phosphate dibasic solution, Sodium phosphate monobasic solution, Sodium tartrate dibasic solution, TNT buffer solution, TRIS Glycine buffer solution, TRIS acetate-EDTA buffer solution, Triethylammonium phosphate solution, Trimethylammonium acetate solution, Trimethylammonium phosphate solution, Tris-EDTA buffer solution, TRIZMA® Base, and TRIZMA® HCL.In one embodiment, the pre-analytical medium is selected from a transportation medium, such as for example: UNIVERSAL TRANSPORT MEDIUM preferably of the trademark (UTM ®), M4RT, Cary -Blair® PuritanMD, AMIES, STUART.

[0071] In one embodiment, the pre-analytical medium is a gel. Preferably, the pre- analytical medium is a gel and is selected from the group consisting of: an agarose gel, a collagen gel, a matrigel, an alginate gel, a biocompatible polymer gel (e.g., but not limited to, PG, PLGA and thermally-reversible polymer gel such as N-isopropylacrylamide), a hydrogel, gelatin, a fibrin gel, a hydragel, and any combinations thereof.

[0072] In one embodiment, the flask has a height ranging from 1 cm to 10 cm, preferably from 4 to 9 cm. In one embodiment, the flask is closed with a sterile cap. In one embodiment, before use, the flask is wrapped in a sterile double-pouch, preserving the flask from contamination until it is used.

[0073] Advantageously, the flask has the shape of a cylinder or of a parallelepiped (square or rectangular). More advantageously, the flask has the shape of a cylinder whose base radius ranges from 0.5 cm to 5 cm. More advantageously, the flask has the shape of a square parallelepiped whose base has a width ranging from 1 cm to 3 cm and a length ranging from 1 cm to 10 cm. In one embodiment, the flask has an internal diameter of 30 to 40 mm, preferably 32-35 mm.

[0074] Advantageously, the flask is made of a polymeric material. The polymeric material is preferably selected from the group consisting of: polystyrene, poly(carbonate), poly(ester), polyethylene terephthalate (PET), poly(amide), poly(ethylene) and poly(propylene), fluoropolymers (such as polytetrafluoroethylene (PTFE) or fluorinatedethyl ene-propylene (FEP)), silicones, hydrocarbons and derivatives, and mixtures thereof. More advantageously, the flask is made of polyethylene or polypropylene. Even more advantageously, the flask material is designed to ensure the absence of gaseous exchange through the polymeric material. This is achieved by the combination of multiple layered materials to achieve mechanical strength and gastight seal. In one embodiment, the flask is transparent or translucid, such that its content can be visually checked. This feature helps during collection, transportation and final pipetting for analytical purposes.

[0075] According to the invention, the two dimensional or three-dimensional agitation of the beads inside the vials produces a grinding effect releasing the cells from the solid specimen. The agitation delivers kinetic energy to the beads that are transferred to the specimen upon impact. According to the invention, the vial walls, cap and bottom are designed to resist the impact and remain watertight before, during and after the beadmilling stage. The vials must be capable of withstanding sterilization processes that may mighty alter their native properties. It is particularly well known that gamma radiation used for sterilization makes some polymer-based flasks such as polypropylene or polycarbonate flasks brittle and subject to breakage. Moist heat sterilization during autoclave cycles may also alters the mechanical performance of polypropylene and may also generates brittleness and susceptibility to breakage.

[0076] In one embodiment, the flask is sterilized. In another embodiment the flask is not sterilized. In a third embodiment, sterilization of the inner of the flask is ensured.

[0077] According to an embodiment where the flask has to remain sterile, the flask material of the invention is packaged in sterile packaging for medical devices having a high-level requirement of protection from contamination and creating an airtight barrier that protects the device from external contaminants. Various sterilization techniques may be used.

[0078] In a first embodiment, where the flask is sterilized by autoclave, the first wrap is a gas permeable pouch, in which the flask is autoclaved, and a second air-tight pouch is wrapped around the first wrap after autoclaving for ensuring air-tight barrier. In this embodiment, a third air-tight pouch may also be provided to secure airtightness.

[0079] In a second embodiment, where the flask is sterilized using ethylene oxide (EO) gas or ionizing radiation, the first pouch may be airtight, and a second air-tight pouch may be sufficient to secure airtightness.

[0080] In one embodiment, the material used to manufacture the flask itself may be designed for ensuring a gas barrier function, and improve the sterility of the inner volume of the flask. In one embodiment, the flask may be made, entirely or partially, of a multilayer material, typically but not limitatively, of polycarbonate / nylon / polycarbonate materials, that makes the flask airtight.of the device

[0081] The inventors have surprisingly discovered that a device according to the invention allows efficient grinding although it comprises beads larger than those used in the prior art for the grinding of a biological solid sample. In addition, the device according to the invention allows an efficient grinding while preserving the viability of all or part of the at least one eukaryotic or prokaryotic cell extracted from the solid sample. In particular, the device according to the invention allows an efficient grinding while preserving the viability of the bacteria extracted from the solid sample.

[0082] Moreover, the device according to the present invention can be transported easily to collect solid samples in situ. The solid sample can then be directly grinded according to the method of the present invention, without additional intermediate step. The device of the present invention thus allows to preserve the integrity of the solid sample, while preventing it from contamination.

[0083] Another object of the invention refers to a method for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample, said method comprising a step of grinding said solid sample with a plurality of beads set in motion by agitation, each bead having a diameter equal to or greater than 5 mm and a density of at least 5.0 g.cm'3, the ratio of the total mass of the beads to the mass of the solid sample being strictly greater than 1.

[0084] According to an embodiment, the solid sample is selected from the group consisting of a biological tissue, a biological implant, and combinations thereof, preferably selected from the group consisting of: osteoarticular tissue, preferably selected from bone, cartilage, joint, ligament, or tendon, cardiac tissue, preferably selected from striated muscle tissue, vascular tissue including atheromatous tissue or valvular tissue, skin tissue preferably selected from epidermis, dermis, or hypodermis, lipidic tissue, muscular tissue, nervous tissue (in particular peripheral or central nervous), pancreatic tissue, hepatic tissue, prostatic tissue, renal and urinary tract tissue, hematopoietic tissue (in particular hematomas, coagulates, and bone marrow biopsies), glandular tissue (in particular exocrine and endocrine tissue), lymphatic tissue, immunogenic tissue (in particular spleen, thymus and lymph nodes), intestine tissue and digestive tract tissue, respiratory tissue (in particular lung, tracheal, bronchial, pleural tissue), reproductive tissue (in particular ovarian, fallopian, uterine, placental, testicular, prostatic, epididymal), and implants selected from a group consisting of sutures or staples, grafts (in particular vascular grafts, corneal implants, bone grafts, xenografts), fixation devices (in particular screws, plates, cement such as polymethylmethacrylate, rods, fixation pins), parts of, or whole, prosthetic joint devices (in particular hip, knee, shoulder, elbow, ankle, finger), vascular devices (in particular catheters, infusion chambers, other vascular lines, shunts, stents), atrial patches drainage tubes (in particular surgical wound drains, pleural drains, articular drains, pancreatic drains, cerebrospinal drains, peritoneal drains), pacemakers including the probes and wires, and any combination thereof.

[0085] In one embodiment, the implant is selected from a group consisting of sutures or staples

[0086] In one embodiment, the biological solid sample is a biological tissue, and more preferably an osteoarticular tissue.

[0087] In one embodiment, the biological sample is not a suspension of cells.

[0088] In one embodiment, the solid biological sample is not a dried sample. Dried sample may be cereals, corn, wheat, seeds, hemp and the like.

[0089] According to one embodiment, the ratio of the total mass of the beads to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the beads to the mass of the solid sample is of about 5.

[0090] According to an embodiment, the mass of the solid sample ranges from 0.1 mg to 10 g, preferably from 0.1 g to 5 g, more preferably from 0.5 g to 1.5 g, even more preferably the mass of the sample is of about 1 g.

[0091] According to an embodiment, the eukaryotic or prokaryotic cells are selected from the group consisting of: bacteria and mammalian cells.

[0092] According to an embodiment, the all or part of at least one eukaryotic or prokaryotic cell is selected from the group consisting of: a protein and a nucleic acid.

[0093] The invention further relates to a method for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample.

[0094] Said method comprises a step of grinding said solid sample with at least two beads (A), as defined previously, set in motion by agitation, each bead having a diameter equal to or greater than 5 mm and a density of at least 5.0 g.cm'3, the ratio of the total mass of the at least two beads (A) to the mass of the solid sample being strictly greater than 1.

[0095] Advantageously, the step of grinding of said solid sample is carried out in a pre- analytical medium as defined previously, and better still is carried out in a device according to the invention as described above.

[0096] All the elements defined previously for the device according to the present invention apply mutatis mutandis to the method.

[0097] According to one embodiment: the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 7.5 g.cm'3, even more preferably of about 8.0 g.cm'3; and the ratio of the total mass of the at least two beads (A) to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the at least two beads (A) to the mass of the solid sample is of about 5.

[0098] Advantageously, according to another embodiment: the mass of the solid sample ranges from 0.1 mg to 10 g, preferably from 0.1 g to 5 g, more preferably from 0.5 g to 1.5 g, even more preferably the mass of the solid sample is of about 1 g; the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 7.5 g.cm'3, even more preferably of about 8.0 g.cm'3; and the ratio of the total mass of the at least two beads (A) to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the at least two beads (A) to the mass of the solid sample is of about 5.

[0099] Advantageously, according to still another embodiment: the mass of the solid sample ranges from 0.1 mg to 10 g, preferably from 0.1 g to 5 g, more preferably from 0.5 g to 1.5 g, even more preferably the mass of the solid sample is of about 1 g; the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 7.5 g.cm'3, even more preferably of about 8.0 g.cm'3; the ratio of the total mass of the at least two beads (A) to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6,even more preferably the ratio of the total mass of the at least two beads (A) to the mass of the solid sample is of about 5; and the number of beads (A) is of at least two beads, preferably of at least three beads, more preferably ranges from 3 to 10 beads, even more preferably of 4 or 5 beads.

[0100] Advantageously, according to still another embodiment: the mass of the solid sample ranges from 0.1 mg to 10 g, preferably from 0.1 g to5 g, more preferably from 0.5 g to 1.5 g, even more preferably the mass of the solid sample is of about 1 g; the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 7.5 g.cm'3, even more preferably of about 8.0 g.cm'3; the number of beads (A) ranges from 2 to 10, preferably from 3 to 7, more preferably the number of beads (A) is of 4 or 5; the ratio of the total mass of the beads (A) to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the beads (A) to the mass of the solid sample is of about 5; and the diameter of each bead (A) ranges from 5 mm to 12 mm, preferably from 5 mm to 10 mm, more preferably from 6 mm to 10 mm, even more preferably from6 mm to 8 mm, better from 6 mm to 7 mm, still better from 6 mm to 6.5 mm.

[0101] Advantageously, according to still another embodiment: the mass of the solid sample ranges from 0.1 mg to 10 g, preferably from 0.1 g to 5 g, more preferably from 0.5 g to 1.5 g, even more preferably the mass of the solid sample is of about 1 g; the number of beads (A) ranges from 2 to 10, preferably from 3 to 7, more preferably the number of beads (A) is of 4 or 5; the ratio of the total mass of the beads (A) to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the beads (A) to the mass of the solid sample is of about 5;the total mass of the beads (A) ranges from 2 g to 10 g, preferably from 3 g to 10 g, more preferably from 3 g to 7 g, even more preferably the total mass of the beads (A) is of about 5 g; and the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 7.5 g.cm'3, even more preferably of about 8.0 g.cm'3.

[0102] Advantageously, the all or part of at least one eukaryotic or prokaryotic cell is a biological analyte, i.e. it is intended to be analyzed. More advantageously, the all or part of at least one eukaryotic or prokaryotic cell is intended to be analyzed for a purpose selected from the group consisting of: clinical diagnostic purposes, research purposes, food industry purposes, phytosanitary purposes, and combinations thereof. Even more advantageously, the all or part of at least one eukaryotic or prokaryotic cell is intended to be analyzed for clinical diagnostic purposes.

[0103] Advantageously, the all or part of at least one eukaryotic or prokaryotic cell may be selected from the group consisting of: all or part of a bacterium and all or part of a mammalian cell. All or part of a mammalian cell may be selected from a protein and a nucleic acid. For example, the all or part of at least one eukaryotic or prokaryotic cell may be a biomarker.

[0104] Advantageously, when at least one nucleic acid is to be extracted from the solid sample, the pre-analytical medium may further comprise at least one detergent selected from the group consisting of: Triton X-100 and NP-40.

[0105] The invention also relates to the use of the device according to the invention for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample. The solid sample is those received in the flask and described previously.

[0106] According to a preferred embodiment, the present invention relates to the use of a device for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample, wherein the device comprises a flask for receiving the solid sample, said flask comprising a pre-analytical medium and at least two beads (A), which may be the same or different, preferably the same, each bead (A) having a diameter equal to or greater than5 mm and a density of at least 5.0 g.cm'3; the ratio of the total mass of the beads to the mass of the solid sample being strictly greater than 1.

[0107] All the elements defined previously for the device and the method according to the present invention apply mutatis mutandis to the use of the device. EXAMPLES

[0108] The present invention is further illustrated by the following examples.Example 1: Grinding efficiency as a function of diameter and number of beadsMaterials and Methods

[0109] Twelve samples of fragments of bone tissue (veal rib) have been grinded by the same total mass of beads (4.16 grams), but with various diameters and number of beads:2 beads having a diameter of 7.93 mm, 4 beads having a diameter of 6.35 mm, 8 beads having a diameter of 5.00 mm and 9 beads having a diameter of 4.76 mm. All the beads were of stainless steel AISI 316L.

[0110] For each of the 4 types of beads (considering diameter and number), 3 samples have been grinded and analyzed before and after grinding. The flask is transparent, which helps for examining the sample during the operation.Results

[0111] The results are presented in the following table:

[0112] This example evidences the fact that, in the device of the invention, the diameter of the beads at constant total mass plays a key role in the bacterial extraction: the larger the diameter the higher the bacterial extraction.Example 2: Grinding efficiency as a function of the total mass of the beads Materials and Methods

[0113] Twelve samples of fragments of bone tissue (veal rib) have been grinded by the same total mass of beads (6.29 grams, thus a higher total mass than those of Example 1 of the present application), but with various diameters and number of beads: 3 beads having a diameter of 7.93 mm, 6 beads having a diameter of 6.35 mm, 12 beads having a diameter of 5.00 mm and 14 beads having a diameter of 4.76 mm. All the beads were of stainless steel AISI 316L.

[0114] For each of the 4 types of beads (diameter and number), 3 samples have been grinded and analyzed before and after grinding.Results

[0115] The results are presented in the following table:

[0116] These results confirm those of Example 1 : the diameter of the beads at constant total mass (i.e. 6.29 grams) plays a key role in the bacterial extraction: the larger the bead diameter, the higher the bacterial extraction. In addition, these results illustrate that the increase in the total mass of the beads (compared to Example 1) does not increase theyield of the bacteria extraction. In addition, it appears that increasing the number of beads may be deleterious to the efficiency of the grinding system.Example 3: Grinding efficiency as a function of the number of beadsMaterials and Methods

[0117] 3 samples of fragments weighing about 1 gram have been grinded by a various number of beads (3 to 7). All beads had a diameter of 6.35 mm and were of stainless steel AISI 316L.

[0118] For each of the 5 different number of beads, 3 samples have been grinded and analyzed before and after grinding. The flask has an internal diameter of about 32 mm.Results

[0119] The results are presented in the following table:

[0120] These results show the existence of a maximum grinding efficiency under identical container and stirring conditions. It appears that, contrary to current recommendations, the optimum is not the mass equivalence between the mass of the grinding beads and the mass of the solid sample (the total mass of the beads was of 5.2 g when 5 beads were used, for 1 gram of solid sample).Example 4: Grinding efficiency as a function of the material and density of beadsMaterials and Methods

[0121] 9 samples of fragments have been grinded each by 5 beads. For 3 samples, the 5 beads were made of stainless steel, for 3 other samples, the 5 beads were made of zirconium and for the 3 last samples, the 5 beads were made of alumina ceramic. Allbeads had a diameter of 6.35 mm.

[0122] The samples were analyzed, before and after grinding.Results

[0123] The results are presented in the following table:

[0124] These results show that, while stainless steel beads with the highest density produce the highest grinding efficiency, the grinding efficiencies obtained with zirconium and alumina ceramic beads are not predicted by the mass. It appears that for the same size, the use of stainless-steel beads provides the best grinding efficiency.Example 5: Grinding efficiency as a function of the size of beads

[0125] Sections of veal ribs with cancellous bone inoculated with S. aureus have been ground with an identical total mass of 9 stainless steel beads of 4.76mm, 8 stainless steel beads of 5.0 mm, 6 stainless steel beads of 5.5 mm and 4 stainless steel beads of 6.35 mm in diameter at a frequency of 21 Hz for 150 s. Four to 5 specimen were ground in reductive buffer medium.

[0126] Bacteria recovered in the buffered medium were enumerated on selective chromogenic S. aureus agar plate after 24h of incubation at 35+ / -2°C. The 6.35mm beads achieved a significantly greater bacterial extraction..Example 6: Grinding efficiency as a function of the kinetic energy of beads

[0127] Sections of veal ribs with cancellous bone inoculated with S. aureus have been ground for 150 s with four stainless steel beads of 5.0 mm and 4 stainless steel beads of 6.35 mm in diameter at different frequencies of 18 Hz and 26 Hz. The calculated kinetic energy of each bead was calculated according to the mechanical characteristics of the beadmill beads. Four specimen were ground in 10 ml of reductive buffer medium.

[0128] Bacteria recovered in the buffered medium were enumerated by plating 500pl of the 10 ml suspension on selective chromogenic S. aureus agar plate. CFUs were enumerated after 24h of incubation at 35+ / -2°C. AISI 316L beads with the lower kinetic energies (5mm diam. at 18 Hz and 26 Hz and 6.35mm diam xl8 Hz ) achieved statistically similar extraction, while higher energy beads (6.35MM diam. at 26 Hz) achieved significantly higher extraction efficacy.

Claims

CLAIMS1. A device for suspending, in a pre-analytical medium, entire eukaryotic or prokaryotic cells from a solid biological sample, comprising, with combination to external agitation means, a flask in which is introduced simultaneously or sequentially: a pre-analytical medium having a pH ranging from 6 to 9, and an osmolality ranging from more than 0 to 800 mOsm / kg of water, in an amount and a composition suitable to suspending the solid sample upon agitation, at least one bead (A), each bead (A) having a diameter ranging from greater than 5 mm to 12 mm and a density of at least 5.0 g.cm'3, said flask being configured to collect or transport a biological solid sample in a ratio of the total mass of the beads to the mass of the solid sample ranging from 2 to 10, the device further comprising sealing means for the flask to be sealed at the place of sampling, after collection of the sampling, so that the sealed flask is air- and water-tight, until the cells are analyzed, said flask being deprived from any internal agitation means, said sealed flask being configured to physically resist beads impact on the walls, bottom and seal of the flask, due to beads movement triggered by external agitation, said external agitation being in a range from 10-50 Hz suitable for separating the cells from the sample and retaining integrity of at least one cell wall or plasmic membrane.

2. The device according to claim 1, wherein the density of each bead (A) is of at least 6.0 g.cm'3, preferably of at least 7.0 g.cm'3, more preferably of at least 8.0 g.cm'3, even more preferably of about 8 g.cm'3.

3. The device according to any one of claims 1 or 2, wherein the diameter of each bead (A) ranges from 6 mm to 10 mm, preferably from 6 mm to 8 mm, more preferably from 6 mm to 7 mm, even more preferably the diameter of each bead (A) is of about 6.35 mm.

4. The device according to any one of claims 1 to 3, wherein the number of beads (A) comprised in the flask is of at least two beads, preferably from 3 to 10 beads, more preferably the number of beads is of 4 or 5 or 6 beads.

5. The device according to any one of claims 1 to 4, wherein the beads (A) are made of a material selected from the group consisting of glass, steel, ceramic and combinations thereof, preferably the beads are made of steel, and more preferably of stainless steel.

6. The device according to any one of claims 1 to 5, wherein the flask further comprises at least one additional bead different from the beads (A), said additional beads having a diameter strictly lower than 2 mm, preferably strictly lower than 1 mm, more preferably a diameter ranging from 0.05 to 1 mm, even more preferably from 0.08 to 0.5 mm.

7. The device according to any one of claims 1 to 6, wherein the osmolality of the pre- analytical medium ranges from 40 to 500 mOsm / kg, more preferably from 100 to 400 mOsm / kg, and better still from 150 to 350 mOsm / kg of water.

8. The device according to any one of claims 1 to 7, wherein the redox potential of the pre-analytical medium ranges from -200 to 200 mV, preferably from -150 to 0 mV and more preferably from -100 to -50 mV.

9. The device according to any one of claims 1 to 8, wherein the pH of the pre- analytical medium ranges from 6 to 9.

10. The device according to any one of claims 1 to 9, wherein the flask is sterilized and packaged in an air-tight packaging.

11. A method for extracting all or part of at least one eukaryotic or prokaryotic cell from a solid sample, said method comprising a step of agitating at a frequency of 10-50 Hz for 30 s to 300 s said solid sample in a pre-analytical medium with at least one bead set in motion by agitation, each bead having a diameter greater than 5 mm and a density of at least 5.0 g.cm'3, the ratio of the total mass of the beads to the mass of the solid sample being strictly greater than 1.

12. The method according to claim 11, wherein the solid sample is selected from the group consisting of a biological tissue, a medical device, and combinations thereof, preferably selected from the group consisting of: osteoarticular tissue, vascular tissue, cardiac tissue, skin tissue, lipidic tissue, muscular tissue, nervous tissue, pancreatic tissue, hepatic tissue, prostatic tissue, renal and urinary tract tissue, hematopoietic tissue, glandular tissue, lymphatic tissue, immunogenic tissue, intestine tissue and digestive tract tissue, respiratory tissue, reproductive tissue, sutures or staples, grafts, fixation devices, parts of, or whole, prosthetic joint devices, vascular devices, mechanical or xenobiotic or native cardiac valves, atrial patches, drainage tubes, pacemakers including the probes and wires, and any combination thereof.

13. The method according to claim 11 or 12, wherein the ratio of the total mass of the beads to the mass of the solid sample ranges from 2 to 10, preferably from 3 to 7, more preferably from 4 to 6, even more preferably the ratio of the total mass of the beads to the mass of the solid sample is of about 5.

14. The method according to any one of claims 11 to 13, wherein the all or part of at least one eukaryotic or prokaryotic cell is selected from the group consisting of: all or part of a bacterium and all of part of a mammalian cell.

15. The method according to any one of claims 11 to 14, wherein the all or part of at least one eukaryotic or prokaryotic cell is selected from the group consisting of: a protein and a nucleic acid.

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