Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
  • A61B10/02—Instruments for taking cell samples or for biopsy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
  • A61B10/02—Instruments for taking cell samples or for biopsy
  • A61B10/0291—Instruments for taking cell samples or for biopsy for uterus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
  • A61B10/02—Instruments for taking cell samples or for biopsy
  • A61B2010/0216—Sampling brushes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods
  • A61B17/32—Surgical cutting instruments
  • A61B2017/320004—Surgical cutting instruments abrasive
  • A61B2017/320008—Scrapers

Definitions

• the invention relates to the field of clinical diagnosis and / or noninvasive therapeutic monitoring. More particularly, the invention relates to a microtechnological device for collecting cells of biological interest by contact.
• spatula-type cell harvesting tools such as the cytological cell collection devices described in US Pat. No. 6,607,494 and in PCT Patent Application 99/25251. Such devices are intended to collect cells on the surface of a cellular tissue directly accessible by natural means.
• the invention in one aspect aims to overcome the disadvantages of existing sampling devices.
• An object of the present invention is in particular to perform non-invasive samples of small amounts of cells.
• the present invention thus provides a microtechnological device for collecting cells by contact with a tissue or other body element comprising a support having at least one face of interest on which is present at least one capture zone consisting of a bottom wall provided with a plurality of protuberances.
• the height of the protuberances of a device according to the invention is between 10 ⁇ m and 400 ⁇ m and the surface of the protuberances is between 3 x 3 ⁇ m and 80 x 80 ⁇ m; the protuberances, for example hexagonal or octagonal section, can be separated by spaces whose width is less than 50 microns.
• a capture zone comprises a bowl, said bottom wall corresponding to the bottom of the bowl.
• the bottom wall and / or the protuberances of a capture zone are functionalized, for example by adhesion molecules and / or by anti-bodies, the functionalized surfaces can be of anionic type. or cationic.
• bottom wall and / or the protuberances of a capture zone may be covered with microbeads, which may be functionalized.
• the functionalization may in particular comprise the presence of ligands joined to the surface by a silylated function.
• the device according to the invention comprises a plurality of capture zones on the same face of interest, the capture zones being separated by first interval zones.
• Means may be present in the gap areas to separate the capture areas, for example notches on one side of said support.
• the support of the device is in the form of a plate; it comprises first and second main planar faces with the exception of capture areas and possible means for separating them.
• the support may be plastic or be a microtechnological substrate, especially silicon.
• the invention may further comprise a handling rod whose end is associated with the support.
• a guide sleeve may also be provided.
• the invention relates to a method of removing cells from or in a tissue or other body element using a microtechnological device comprising a support having at least one face of interest on which there is at least one capture zone consisting of a bottom wall provided with a plurality of protuberances, the collection of cells being carried out by placing said at least one capture zone in contact with the tissue or the body element.
• the sample is not of the surgical type, that is to say for example that it relates to a tissue that has been previously removed, for example by biopsy, or that it relates to a dead body.
• the method can also be adapted and used in a patient or a living animal.
• the invention relates to a method for diagnosing and / or analyzing a tissue or other body element comprising a sampling step as defined above.
• a step of observation, by means of a microscope, of the cells present on the device after sampling may be provided, and / or a step of culturing the cells present on the device after sampling.
• Figure 1 shows a contact pickup system according to an embodiment of the invention.
• Figure 2 shows an embodiment of a capture area for a device according to the invention.
• FIG. 3 illustrates an example of functionalization of the surface of the device according to the invention.
• Figure 4 illustrates a functionalization by beads.
• FIGS. 5A to 5D illustrate different embodiments of means for separating the capture zones of a device according to the invention.
• Figure 6 shows a method of using a device according to the invention.
• the present invention aims to collect a small amount of cells from a tissue or a body element by means of a microtechnological sampling device described below.
• tissue or other body element any structural or functional entity of a human or animal body. It is for example an organ, that is to say a differentiated functional and structural entity that is specialized for a particular function (brain, lungs, ).
• a fabric is by example a tumor.
• other body element refers, among other things, to the skin, the venous-cardiac system and the digestive system.
• the collection of cells from a tissue or a body element by means of a device according to the present invention can be carried out in vivo or ex vivo, for example in situ.
• microtechnological sampling device Since the microtechnological sampling device according to the invention is very small, millimetric or less, it is preferably associated with handling means. Various sampling systems including such a microtechnological sampling device can be envisaged depending on the type of sampling planned.
• FIG. 1 illustrates an example of a sampling system 1 making it possible to take cells inside a tissue or a body element; the sample may concern a body, human or animal, living or dead, or even a biopsy extracted from an animal or a patient, or any other tissue of interest.
• the system comprises a guide sleeve 2, for example a catheter: the guide 2 makes it possible, among other things, to define the passageway for the sampling device. In particular, it may be put in place, possibly under optical or radiological control, beforehand in a target zone 3 of a tissue or other body element.
• the end portion 4 of the guide 2 is provided with closure means 5 which protect the sampling device 6 during its insertion and make it possible to put it in contact with the tissue or the bodily element of interest 3 once in place.
• the sealing means 5 are preferably located along the longitudinal axis of the guide 2, the distal end is closed.
• the sealing means 5 may for example be a rotary or sliding window, or a partially absorbable membrane.
• the sampling device 6 advantageously comprises a handling rod 7, the length of which depends on the use and the depth of insertion, and which can slide in the guide 2.
• the end portion 8 of the rod 7 is intended to to the sample itself.
• the guide 2 and thus the manipulation rod 7 have a very small diameter so as not to alter the tissue or the body element 3, and so as to allow non-invasive procedures.
• the rod 7 may have a diameter restricted to a few millimeters, or even 100 microns; the guide 2 has an outer diameter close to the diameter of the rod 7.
• the rod may, for example, be of surgical stainless steel.
• the end portion 8 of the sampling device 6 has at least one capture zone 10 whose developed surface is much greater than the normal surface, from 3 to more than 20 times.
• the sampling device 6 thus comprises a support 12 which is preferably independent of the handling rod 7 at the end of which it can be secured, for example by gluing preferably with a biocompatible glue. This makes it possible in particular to separate the manufacturing processes of the two handling and sampling parts, and to use a biocompatible rod 7 conventional low cost.
• the support 12 is preferably made of a biocompatible material, in particular silicon as specified below; the different elements making up the guide sleeve 2 are also compatible with a biological and / or medical use, for example in gold or plastic, ....
• the support 12 may be of any shape, but advantageously it is plane, in the form of a plate, as will appear in the description of the manufacturing processes. Whatever the case, it is possible to define on the support 12 a first face 14 and a second opposite face 16: in the case of a non-planar support 12, the terms “face” and “opposite face” designate portions of the outer surface of the support 12 which are symmetrical with respect to a secant plane of the support 12.
• the faces 14, 16 are included in a support 12 which is of the order of 1 to 3 cm long (in the direction of the rod) over a width of 300 to 800 ⁇ m, for a thickness of the order of 200 to 400 ⁇ m.
• the first face 14 of the support 12 is provided with a capture zone 10; it is preferable that the capture zone 10 leaves a proximal end portion 8 sufficiently long, for example from 2 to 5 mm, to allow easy attachment to the rod 7.
• a preferred embodiment P relates to a support 12 rectangular silicon of dimensions 300 ⁇ m ⁇ 600 ⁇ m ⁇ 2 cm, the structured zone 10 starting at 3.2 mm from the edge secured to the rod 7.
• several capture zones 10a, 10b, 10c, 10d are present on the face 14 of the support 12, separated by zones of gap 18.
• the interval zones 18 may be only "virtual”, that is to say that the capture zones 10 are a priori confused at the macroscopic level, but that means make it possible to distinguish them at the microscopic level. or even to separate them.
• capture zones 20 are placed on the second face 16.
• the second capture zones 20 are aligned and in opposition with the first zones 10.
• the second capture areas 20 may be identical in nature and geometry to the first zones 10, or different, as shown diagrammatically in FIG. 1: the various embodiments presented below may be combined.
• At least one capture zone of the sampling device comprises a set of protuberances or pins.
• the pins are intended to come into contact with the cells to be taken, the cells removed being trapped between the pins of the device.
• FIG 2 illustrates an example of a sampling device including a set of protuberances on a capture area.
• the capture zone 10 comprises a bottom wall 22.
• the bottom wall 22 can be placed at the bottom of an open cavity formed on the surface of the support 12 or can be an "open" surface laterally, as this is shown in Figure 2.
• the bottom wall 22 has a surface s_, and has a plurality of protuberances 24.
• the capture area comprises a cavity
• the height of the protuberances 24 is identical to the depth of the cavity but it is possible that they are prominent.
• the surface of the support 12 is uniform, preferably flat, with the exception of the capture zones 10, and any separation means (described later).
• the developed area _S of the capture zone 10 is therefore equal to the surface s_ of the bottom wall 22 to which is added the surface of each of the lateral walls of the protuberances 24.
• the surfaces satisfy the relation: S> 3 -s, the factor 3 may advantageously take the values 5 or 10 for example.
• the protuberances 24 can take any desired geometry, for example square columns or hexagonal section. Preferably, the protuberances 24 are arranged in a regular manner, for example in a square or hexagonal mesh network. According to the mode preferred embodiment P, the surface patterning is in the form of octagonal pins 24 of silicon, 50 ⁇ m high and 20 or 80 ⁇ m wide. Different manufacturing processes can be envisaged for such capture zones 10: for example, if the support 12 is made of plastic, it is possible to use injection or hot-stamping techniques ("hot embossing”), which make it possible to obtain, by replication, complementary pieces of molds previously produced.
• hot embossing injection or hot-stamping techniques
• the removal of cells from or into a tissue or other body element by means of a microtechnological sampling device involves contacting the capture areas of the device with the tissue or body component. This contact can be more or less pronounced. By a micro-abrasion effect, it is possible to recover cells between the protuberances or pins of the sampling device.
• the capture zones are preferably covered with a coating having an "attractive” power on the cells.
• This attractive power can be electrical, chemical, physical ...
• the capture areas covered with such a coating are called “functionalized”. Examples of coating are described below. Among these examples we can define two families coating.
• the first family includes coatings able to react or interact directly with cells.
• the second family includes coatings capable of reacting or interacting with smaller elements, such as proteins, present around cells or in the extracellular matrix of cells. In the case of a coating of the second family, the attraction of the cells is via the attraction between the device and the smaller target cells of the cell.
• coatings consisting of adhesion molecules such as adhesion proteins or peptides, coatings consisting of antibodies or anionic or cationic coatings.
• an electrically conductive coating possibly covered with a thin, biocompatible insulating layer.
• Such an electrical coating can be polarized positively or negatively by means of an electric polarization device.
• Such an electrical polarization device could be "embedded" on the sampling device and possibly be realized monolithically with the latter.
• a capture zone 10 In order to increase the attractive power of a capture zone, it is also possible to increase the developed area of a capture zone 10 by covering the bottom wall 22 and possibly the protuberances 24 with microbeads. It should be noted that the microbeads must not completely fill the spaces 26 between the protuberances 24 in order to maintain a sufficient space between the protuberances for the sampled cells.
• Microbeads are commonly used in microbiology; they conventionally have a diameter of the order of ten nanometers up to a hundred microns, and may be composed of glass, porous or not, which allows them to be functionalized and remain biocompatible.
• the coupling function A corresponds to all existing organic and inorganic functions such as the functions: CH 3 , alkenes, alkynes, aryl derivatives, halogens (Br, Cl, I, F), organometallic derivatives, alcohols, phenols, diols , ethers, epoxies, carbonyl derivatives (aldehydes, ketones, carboxylic acids, carboxylates, esters, amides, acid chlorides, acid anhydrides), nitrogen derivatives (amines, nitro derivatives, diazos derivatives, imines, enamines, oximes, nitriles ), phosphorus derivatives (phosphines, phosphites, phosphates, phosphonates), silicon derivatives, sulfur derivatives (sulphides, disulphides, thiols, thioethers, sulphones, sulphites, sulphates, sulphonic acids, sulphonates, azasulfonium
• a spacer group E, used between the two functions A, Y of the coupling agent, makes it possible to confer particular properties on the film obtained by silanization.
• the group E is chosen from among the radicals making it possible to obtain an organized monolayer: a long chain alkylene radical E allows interchain interaction (among the radicals E of the alkylene type, those having from 8 to 24 atoms are particularly preferred.
• a radical E comprising two triple bonds -C C- allows crosslinking; a radical E comprising a conjugated aromatic chain confers nonlinear optical properties (for example, mention may be made of phenylene-vinylene and phenylene-acetylene radicals); a radical E of the pyrrole, thiophene or polysilane type confers an electronic conduction; a radical E of the heterosubstituted polyaromatic type confers photo / electroluminescence properties (for example, mention may be made of quinones and diazo compounds); a group E of the alkyl or fluoroalkyl type, in particular an alkyl or fluoroalkyl group having from 3 to 24 carbon atoms, makes it possible to use the layers obtained by chromatography or electrophoresis.
• the surface ester functions located on the tool will react with functionalized beads bearing primary hydroxyl function. After immobilization of the balls, the tool has a hydrophilic developed surface (FIG. 4).
• a sampling device makes it possible, thanks to the pins present on the cell capture zones, to recover one or more cell layers of the tissue or of the body element of interest. In the case of pins of a few tens of microns in height, it is possible to recover up to ten cell layers, depending on the size of the cells collected. Once the cell collection is done, it is possible to analyze the spatial composition of the sampled cell layers. For example, a microscope can be used to visually identify the nature of the cells removed.
• An advantage of the sampling device according to the present invention is that the presence of cell multilayers makes it possible to obtain a histological section of a tissue or other body element. After collection, the recovered cells can be cultured in order to have a larger number of cells.
• a sampling device makes it possible to recover "aggregates" of cells and not “dissociated” cells.
• the culturing of cell aggregates makes it possible to obtain better yields and a better quality of the cultured cells. It is thus possible to envisage practicing cellular therapies from cells, and in particular cell aggregates, taken by a device according to the present invention.
• Such a therapy would consist, for example, of taking "sick" autologous cells, of amplifying them (in particular by culture), of modifying their genome and then of reintroducing them into the tissue or the body element that one wishes to heal. Moreover, from the cells taken by means of a device according to the present invention, it is possible to carry out various analyzes of the molecules present in the cells or generated by them.
• the capture zones are covered with a coating of the second family, it will also be possible to eliminate the cells present between the protuberances of a capture zone, and then carry out analyzes of the molecules "trapped" by the coating.
• sampling device 6 has several capture areas 10i (see FIG. 1), it is possible to use the same functions on each capture zone, or to perform a spatial differentiation, for example by the known localized spotting ("spotting") for DNA chips.
• spotting localized spotting
• the supports 12 may be divisible for each of the preceding embodiments.
• the interval zones 18 between the capture zones 10 are provided with separation means. For example, notches may have been etched at the same time as the realization of the protuberances 24 and / or walls: FIGS. Interval areas 18 can then easily be sectioned.
• cleavage primer 42 by etching the support 12 on the face 16 opposite to the face 14 comprising the capture zones 10, by mask and etching for example (FIG. 5A ). It is also possible to make this notch 44 on the "front" face 14, or to choose, for example, an isotropic chemical etching, for example KOH (FIG. 5B).
• capture zones 10, 20 are present on each face 14, 16, it is possible to position separation means only on one of the faces (FIG. 5C), or both (FIG. 5D).
• two embodiments can be noted for the devices comprising capture zones 10, 20 on each of their opposite faces 14, 16: a support 12 (FIG. 5C) or bonding of two supports 12, 12 '(FIG. 5D ).
• notches 42, 44 can be used interchangeably and in combination.
• a silicon wafer 100 mm in diameter is machined to obtain 142 end devices after cutting.
• the support 12 of silicon is advantageously marked: in particular, the name of the device, alignment crosses, cutting marks, etc. are etched, for example at 500 nm, by photolithography with mask and dry etching.
• the rear face undergoes a similar treatment (photolithography with mask aligned with the previous one, dry etching of 5 to 10 ⁇ m, removal of the resin from the mask) to form notches.
• the front face is then drawn and engraved for microstructuring, with photolithography with aligned mask, 50 ⁇ m deep dry etching and removal of the resin.
• the surfaces are then prepared to allow their biological and / or medical use: in particular the polymer (for example C 4 F 8 ) formed on the flanks of the cavities during etching is removed, by total deoxidation, followed by wet oxidation. over 100 nm, then total deoxidation; a final SiO 2 layer is obtained by wet oxidation over 500 nm.
• the polymer for example C 4 F 8
• the guide 2 is first put in place, preferably under control in the target zone 3; the support 12 is glued at the end of the rod 7.
• the rod 7 is inserted into the guide 2, under optical control also to ensure the accuracy of its positioning, and in particular to determine the areas A, B, C, D of the tumor 3 corresponding to each of the capture zones 10a-10d.
• the sealing means 5 are open, and the sample is taken by apposition; no manipulation of the device itself is necessary, the contact area of the capture areas 10 being directly accessible (without cover for example). This also allows a miniaturization of the whole, and In particular, the support 12.
• the closure means 5 can then optionally be closed again.
• the rod 7 is then removed from the guide 2, the support 12 is detached, and the capture areas 10a-10d can be analyzed.
• each zone 10a-10d is treated independently.
• the support 12 is broken and the different zones 10a-10d can be processed, analyzed separately.
• the cells A, B, C, D are extracted and introduced into tubes 50a-50d and "rinsed" with solutions for extracting the cells from the capture zones.
• the support 12 which thus retains the definition of the active zones AD stepped within the tumor 3. It is the support 12 itself which serves as a substrate for the device 60 of final analysis, for example for microscopic observation or cell culture. Whichever approach is chosen, one can obtain a map of the tissue of interest, and results concerning the cellular and protein composition as a function of the depth in the target zone 3.
• the sampling device according to the invention thus has particularly advantageous characteristics: the sample is not very invasive: in particular, the apparent diameter of the device 6, and even of the system 1, is reduced, in particular to a few millimeters, preferably 1 mm,
• the sample is not aggressive: it is done by contact (or "apposition") without tissue section 3,
• the portion of the machined device actually used for sampling is reduced and covers only the support 12, which can be associated with a low-cost handling rod, the machining of the sampling portion 12 is reduced to the manufacture of the contact zones 10, 20, without other mechanical elements or additional steps of sealing or gluing, the device 6 can be used in surgical procedure in vivo or post-operatively, or be used in vitro on a tissue removed and requesting cellular and possibly molecular analysis,
• staged capture zones 10a-IOd makes it possible to analyze, after imprinting, the distribution of the cells of interest in the sampling zone 3, each capture zone 10 can be functionalized according to the targeted cells and / or the type of analysis or subsequent treatment, each capture zone 10a-10d can be separated from the others and be analyzed by a clean technique, the support of the device 12 can be compatible with any equipment for analysis or subsequent treatment, mapping along the depth axis of the zone 3 analyzed can be established according to the successive active zones DA differentiated along the device; the stereoscopic method of operation makes it possible to precisely guide the device 6 and to know exactly which region AD has been probed.
• anionic type coating is described below.
• the previous device P Si support 600 ⁇ 300 ⁇ m 2 , with 24 octagonal protuberances was silanized and then functionalized to give the carboxylate function. Indeed, at physiological pH, biological systems are naturally charged; ionic interactions (based on the principles of chromatography) can be used to specifically absorb protein markers. For anionic surfaces (negatively charged), the carboxylate derivatives are the most commonly used.
• the acid function is protected in the form of a methyl ester after reaction of undecenoic acid with sulfuric acid and methanol; the incorporation of the silyl group is conventionally carried out by a hydrosilylation reaction.
• 10-undec-1-ene is manufactured to form the methyl ester of trimethoxysilylundecan-10-oic acid by the following method:
• the hydroxylation of the silicon substrate coated with a thermal oxide layer of 500 nm is carried out in a 3.5 M sodium hydroxide solution for 2 hours, with a silanizing solution of concentration 10 -2 M in anhydrous trichlorethylene, the silanization reactions being carried out at a controlled temperature of 2 ° C for 24 h.
• the modified support is brought into contact with a solution of aluminum iodide in order to release the carboxylic acid function, which will in turn react with an aqueous sodium hydroxide solution to give the corresponding carboxylate function.
• a solution of aluminum iodide in order to release the carboxylic acid function, which will in turn react with an aqueous sodium hydroxide solution to give the corresponding carboxylate function.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Surgery (AREA)
• Animal Behavior & Ethology (AREA)
• Biomedical Technology (AREA)
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• Medical Informatics (AREA)
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• Public Health (AREA)
• Veterinary Medicine (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Sampling And Sample Adjustment (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2006
  • 2006-07-13 FR FR0652975A patent/FR2903590B1/fr not_active Expired - Fee Related
• 2007
  • 2007-07-12 WO PCT/EP2007/057147 patent/WO2008006871A2/fr not_active Ceased
  • 2007-07-12 JP JP2009518891A patent/JP5415944B2/ja active Active
  • 2007-07-12 EP EP07787418A patent/EP2040623A2/de not_active Withdrawn
  • 2007-07-12 US US12/307,195 patent/US20090317835A1/en not_active Abandoned

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