EP2599548B1 - Support d'échantillon pour le positionnement d'un échantillon organique, biologique et/ou médical - Google Patents

Support d'échantillon pour le positionnement d'un échantillon organique, biologique et/ou médical Download PDF

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Publication number
EP2599548B1
EP2599548B1 EP13156764.6A EP13156764A EP2599548B1 EP 2599548 B1 EP2599548 B1 EP 2599548B1 EP 13156764 A EP13156764 A EP 13156764A EP 2599548 B1 EP2599548 B1 EP 2599548B1
Authority
EP
European Patent Office
Prior art keywords
sample
sample carrier
structural element
bottom plate
magnetic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP13156764.6A
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German (de)
English (en)
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EP2599548A1 (fr
Inventor
Roman Zantl
Helga Wagner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ibidi GmbH
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Ibidi GmbH
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Filing date
Publication date
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Priority to EP13156764.6A priority Critical patent/EP2599548B1/fr
Publication of EP2599548A1 publication Critical patent/EP2599548A1/fr
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Publication of EP2599548B1 publication Critical patent/EP2599548B1/fr
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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D25/00—Details of other kinds or types of rigid or semi-rigid containers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B5/00—Packaging individual articles in containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, jars
    • B65B5/04—Packaging single articles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces

Definitions

  • the invention relates to a method for positioning an organic, biological and / or medical sample in a desired surface area of a sample carrier.
  • the invention relates to a method for positioning a sample by means of a magnetic device.
  • sample carriers are used for the examination of organic, biological and / or medical samples.
  • US2003003571 shows a sample carrier from a bottom plate and a lid, wherein the sample carrier includes a structural element in the bottom plate. The structural element serves to position the sample in an observation channel. It is an object of the invention to provide a method for positioning an organic, biological and / or medical sample, which allows to position the sample in a desired surface area of the sample carrier.
  • This method enables a precise positioning of the sample in a desired surface area of the sample carrier.
  • the organic, biological and / or medical sample may be a biological cell.
  • the method may be performed for a plurality of cells.
  • a desired cell distribution in a desired surface area of the sample carrier can be achieved.
  • the cells can be introduced in the form of a suspension in the sample carrier.
  • the sample may also be a microorganism or DNA.
  • the sample carrier may comprise a plastic, in particular COC (cycloolefin copolymer), COP (cycloolefin polymer), PS (polystyrene), PC (polycarbonate) or PMMA (polymethyl methacrylate).
  • the sample carrier may be formed as an injection molded part.
  • the sample carrier may comprise a bottom plate, in particular wherein the sample carrier rests on the bottom plate during operation, and wherein the bottom plate may comprise a plastic and / or glass.
  • the bottom plate can be thin, for example between 1 .mu.m and 300 .mu.m. This allows high-resolution microscopy through the bottom plate.
  • the sample carrier may be dimensioned such that the volume of a cavity is in the range of 5 .mu.l to 1000 .mu.l, in particular between 100 .mu.l and 500 .mu.l.
  • the sample carrier for Mikrofluiduntersuchungen is usable.
  • the sample carrier may comprise a cover plate, wherein the cover plate with the bottom plate is liquid-tight, in particular directly connected.
  • the bottom plate and / or top plate may have a predetermined intrinsic fluorescence, which is in particular less than or equal to the intrinsic fluorescence of COC or COP or a conventional cover glass, and / or a predetermined refractive index, in particular> 1.2 and / or ⁇ 1.7.
  • the intrinsic fluorescence may be less than or equal to the intrinsic fluorescence of a conventional coverslip (for example, pure white glass of hydrolytic class 1 (such as Menzel coverslip, in particular having a starch # 1.5.)
  • the predetermined refractive index may be> 1.2 and / or ⁇ 1.7, with such a high-quality optical material microscopy investigations can advantageously be carried out, for example, the birefringence can be so low that DIC (Differential Interference Contrast) is possible.
  • the bottom plate and / or cover plate can be antireflective in a frequency range of the electromagnetic radiation used for microscopy. This allows the Transmission through the bottom plate and / or cover plate can be increased so that single molecule measurements are possible with the help of fluorescence.
  • the sample carrier may comprise at least one surface region for arranging a sample, in particular wherein the surface region is arranged on the bottom plate.
  • the sample carrier may include a cavity for receiving a sample. At least one opening for filling and / or emptying the cavity with the sample and / or a liquid can lead into the cavity.
  • the cavity may be formed by recesses in the cover plate and / or in the bottom plate.
  • samples which do not have their own magnetic moment can also be positioned with the aid of the magnetic device.
  • the magnetic particles may be made of a material that does not have a toxic effect on the cell.
  • Arranging the sample may include aligning the sample in the desired magnetic field arrangement.
  • the sample can align itself by the magnetic force effect of the desired magnetic field arrangement.
  • the sample can move as a result of the magnetic force action and thereby an arrangement of the sample can be achieved.
  • the placement of the sample may include moving the magnetic device relative to the sample carrier.
  • a sample which has been introduced in a predetermined area of the sample carrier may be arranged in a desired surface area. This may be particularly advantageous if the desired surface area is located in an inaccessible or hard to reach area of the sample carrier.
  • the sample can align in the desired magnetic field arrangement and thereafter be moved by moving the magnetic device by magnetic force action in the desired surface area.
  • the predetermined area of the sample carrier may comprise the desired surface area.
  • the placement of the sample can only be done by aligning the sample in the desired magnetic field arrangement.
  • the desired magnetic field arrangement may have a magnetic field strength, a magnetic force flux and / or a magnetic field line distribution.
  • the magnetic device can provide a magnetic field, wherein the magnetic field, in particular the desired arrangement of the magnetic field or desired magnetic field arrangement in predetermined range can be characterized by a magnetic field strength, a magnetic flux and / or a magnetic field line distribution.
  • the magnetic field strength corresponds to a vectorial quantity and is also referred to as magnetic induction or magnetic flux density.
  • the magnetic field strength is proportional to the magnetic excitation.
  • the predetermined region of the sample carrier may comprise a surface region of the sample carrier and the amount of the magnetic field strength in the predetermined region, in particular in the surface region, may have at least one local extremum, in particular a local maximum, and / or at least one saddle point. In this way, the sample can be moved away by magnetic force to the local extremum or away from the local extremum. By selecting the strength and / or the position of the local extremum, a targeted arrangement of the sample is possible.
  • the surface area may comprise a partial area in which the magnetic field lines condense.
  • the desired magnetic field arrangement has a local maximum in the amount of the magnetic field strength. This also means that the magnetic flux through the surface in this subregion can have a local maximum.
  • the magnetic field strength of the desired magnetic field arrangement may have a magnetic field component parallel and / or perpendicular to the surface area at any point or in a plurality of points of the predetermined range.
  • the sample can be moved parallel and / or perpendicular to the surface area by magnetic force.
  • in combination with a local extremum can be achieved in this way, the placement of the sample by aligning the sample in the desired magnetic field arrangement.
  • the magnetic device may provide a dipole field or a quadrupole field.
  • the magnetic device may also provide a combination of multiple dipole fields and / or quadrupole fields.
  • the magnetic field arrangement can thereby be varied or predetermined in the predetermined range.
  • the desired magnetic field arrangement in particular its magnetic field line distribution, may be radially symmetrical with respect to a predetermined axis. This can be achieved, for example, when the magnetic device provides a dipole field.
  • the be predetermined axis perpendicular to the surface area of the sample carrier. This allows the sample to be arranged in a radially symmetric surface area.
  • the bonding of the sample to one or more magnetic, in particular paramagnetic, particles may include adhering a particle to the surface of the sample and / or picking up or introducing a particle into the sample.
  • the sample does not have its own magnetic moment, by connecting the sample with a magnetic particle, the placement of the sample can be realized by means of a magnetic force effect of the desired magnetic field arrangement on the magnetic particle.
  • the particle can be taken up by the cell.
  • the magnetic particle is smaller than the cell, in particular, the volume and the maximum spatial extent of the magnetic particle is smaller than the volume and the maximum spatial extent of the cell.
  • the magnetic, in particular paramagnetic, particle can be attached to the surface of the cell. This can be achieved by positively charged end groups.
  • the particle can then be taken up (phagocytosed) by the cell.
  • the particle can be stored in particular in vesicles in the cytosol.
  • the magnetic, in particular paramagnetic, particles can be coated with a polymer matrix, in particular wherein the polymer matrix is provided with a coating which can adhere to a surface of the sample.
  • a particle can be connected to the surface of the sample.
  • the particle may be larger than when it is to be introduced into the sample.
  • a particle one-fiftieth of cell size may be used.
  • the coating of the polymer matrix may comprise surface proteins, in particular CD molecules or activated tosyl groups. The coating can be chosen such that the particle can adhere to a desired cell type, especially only to the desired cell type.
  • the magnetic device may comprise a permanent magnet and / or an electromagnet.
  • the permanent magnet may in particular be a neodymium iron boring magnet.
  • a particularly high field strength can be achieved.
  • the maximum amount of magnetic field strength may be between 0.5 Tesla and 1.4 Tesla.
  • An electromagnet may include a coil having one or more turns.
  • the electromagnet may comprise an iron core.
  • the magnetic device may comprise at least one tip, in particular wherein the tip comprises a magnetic, in particular ferromagnetic, material.
  • the tip comprises a magnetic, in particular ferromagnetic, material.
  • Arranging the magnetic device relative to the sample carrier may include placing the tip relative to the predetermined region. Since a high magnetic force flux is provided in the area of the tip, the position of the tip can be used to determine the strength and position of the local extremum in the surface area.
  • the magnet device may comprise a conically shaped element, in particular wherein the conically shaped element comprises the tip.
  • an iron core of an electromagnet may comprise a tip and / or correspond to a conically shaped element.
  • the conically shaped element may be connected to a permanent magnet or an electromagnet, be a permanent magnet, or be partially disposed inside a coil of an electrically conductive material, in particular wherein the coil is part of an electromagnet.
  • an electromagnet may be advantageous since the magnetic field, in particular the magnitude of the magnetic field strength, can be varied in this case.
  • an electromagnet can be switched off and on. This may be particularly advantageous in the case of automation of the method.
  • the conically shaped element may have at the base a diameter which corresponds to the maximum spatial extent of a permanent magnet.
  • the conical shaped element may have at the base a diameter which corresponds to the diameter of a cylindrical permanent magnet or a cylindrical iron core of an electromagnet.
  • the opening angle of the conically shaped element can be between 30 ° and 90 °, in particular 60 °.
  • the sample carrier may comprise an observation area, wherein the observation area is designed such that a sample arranged in the observation area can be observed by means of an optical device, for example a microscope.
  • the desired surface area may correspond to an observation area of the sample carrier or an observation area may comprise the desired surface area.
  • the sample carrier may comprise a bottom plate, wherein the sample carrier in operation rests on the bottom plate and wherein the magnetic device is arranged so that it is arranged in operation under the bottom plate, in particular wherein the tip of the magnetic device is disposed directly under the bottom plate.
  • the bottom plate may include the desired surface area.
  • the sample can be positioned in a desired surface area of the bottom plate.
  • the desired magnetic field arrangement can be designed in such a way that a magnetic force acts on the introduced sample, in particular on the particles connected to the sample, so that the sample can be moved in the desired magnetic field arrangement by a magnetic force.
  • the magnetic force may be greater than a frictional force between the sample and a surface of the sample carrier.
  • a liquid may be disposed in the sample carrier and, if the sample is in the liquid, the magnetic force may be greater than a viscous frictional force between the sample and the liquid.
  • the sample can align in the desired magnetic field arrangement and be moved along the magnetic field lines.
  • the magnetic force can be smaller than the force with which the sample and the at least one magnetic particle are connected to each other. As a result, the sample can be moved by force transmission with the particle.
  • the step of placing the sample may include moving the sample carrier.
  • the sample carrier may be moved such that, when the sample is in contact with a surface of the sample carrier, the sample is released from the surface. This may be advantageous when the magnetic force is less than a frictional force between the sample and a surface of the sample carrier.
  • the movement of the sample carrier may include a periodic or aperiodic movement, such as jarring or pivoting of the sample carrier or vibrations by ultrasound.
  • a sample in particular in a difficult to reach cavity of a sample carrier, can be positioned effectively and precisely.
  • sample carrier may comprise one or more of the features described above.
  • the second liquid may have a higher viscosity, a lower density and / or a higher hydrophobicity than the first liquid. Due to the higher viscosity can be achieved that the pulse of the introduced sample is aligned parallel to the direction of gravity effect.
  • the viscosity of the second liquid can be ten times to 10 6 times the viscosity of the first liquid, in particular 10 to 1000 times or 1000 to 10 6 times.
  • the second liquid Due to the lower density of the second liquid can be achieved that the second liquid floats on the first liquid, and thereby remains arranged in the through hole. In particular, this can reduce or avoid contact instabilities at the boundary layer between the first and the second liquid, for example Rayleigh-Taylor instabilities.
  • the density of the second liquid may be between 70% and 95% of the density of the first liquid.
  • an arrangement of the second liquid in the through hole can be achieved by capillary forces.
  • the first and / or second liquids may be chosen so that they do not have a toxic effect on the sample. This may be particularly advantageous when the sample is a living biological cell.
  • the first liquid may comprise water and / or the second liquid may comprise an oil, in particular a mineral oil and / or a silicone oil.
  • the sample may be introduced in the form of a suspension in the second liquid, in particular wherein the suspension comprises a third liquid, wherein the third liquid is a has stronger hydrophilicity than the second liquid.
  • the second liquid may be a two-component liquid, wherein the second liquid is solidifiable after the step of introducing the sample by cross-linking or polymerizing. This allows the sample chamber to be closed. In particular, contamination of the first liquid from the outside and / or evaporation of the first liquid can thereby be avoided or reduced.
  • the through hole may be formed so as to taper toward the cavity.
  • the taper can be conical.
  • the first liquid After filling with the first liquid, the first liquid may be arranged in the sample carrier such that no liquid is inside the through-hole.
  • the second liquid may be disposed completely in the through hole after insertion.
  • the second liquid can be introduced into the through hole such that the second liquid does not protrude beyond the outer opening of the through hole. As a result, a safe introduction of the sample into the second liquid can be achieved.
  • the invention also provides a positioning system for positioning an organic, biological and / or medical sample in a desired surface area of a sample carrier, comprising a magnetic device, a sample carrier holder, and a device for placing the sample carrier relative to the magnetic device.
  • the positioning system can be used in particular in a method described above. With the help of such a positioning system, the placement of the sample can be done precisely.
  • sample carrier and / or the magnetic device may comprise one or more of the features described above.
  • the magnetic device may comprise a permanent magnet and / or an electromagnet.
  • the magnetic device may comprise a conically shaped, in particular magnetic or ferromagnetic element, in particular wherein the conically shaped element comprises a tip. In the area of the tip, a high magnetic flux of force can be provided.
  • the positioning system may also include means for automatically moving the magnetic device relative to the sample carrier.
  • the means for automatically moving may be used to position the magnetic device relative to the sample carrier such that a desired magnetic field arrangement is provided in a predetermined region of the sample carrier.
  • the device for automatically moving the magnetic device can be used for arranging the sample in a desired surface area of the sample carrier with the aid of the magnetic device, in particular wherein the arrangement of the sample comprises moving the magnetic device relative to the sample carrier.
  • a more precise positioning of the sample can also be achieved by the device for automatic movement than when the method steps are carried out manually.
  • the positioning system may include means for automatically moving the sample carrier holder, whereby the sample carrier holder may be moved therewith such that when a sample is in contact with a surface of the sample carrier, the sample may detach from the surface.
  • This may be particularly advantageous when arranging the sample comprises aligning the sample in the desired magnetic field order, the magnetic force being less than a frictional force between the sample and a surface of the sample carrier.
  • the device for automated movement of the sample carrier holder may in particular comprise an ultrasonic element and / or a pivoting element. The ultrasound element can set the sample carrier holder, in particular with the sample carrier fixed therein, in vibration.
  • the positioning system may further comprise a pipetting device for, in particular automated, filling a sample carrier fixed in the sample carrier holder, wherein the pipetting device may comprise one or more pipettes.
  • the pipetting device may comprise one or more pipettes.
  • the invention further provides a sample carrier comprising a structural element, wherein the structural element is designed such that an organic, biological and / or medical sample introduced into the sample carrier can be arranged in a desired partial area, in particular in a desired surface area, of the sample carrier.
  • a structured sample carrier allows positioning of the sample in a desired surface area of the sample carrier.
  • Such a sample carrier may be used in particular in any of the methods described above.
  • sample carrier may comprise one or more of the features described above.
  • the sample carrier may comprise a predetermined surface area, wherein the predetermined surface area comprises the structural element, and wherein the structural element is configured such that the introduced sample is arranged in a desired portion of the predetermined surface area, in particular in the desired surface area.
  • the structural element may be designed in the form of an elevation and / or a depression.
  • the structural element may be in the form of a dome, a pyramid, a groove and / or a depression.
  • the desired subregion can adjoin the structural element or completely surround the structural element.
  • the structural element may comprise the desired partial area. This may be the case, for example, if the structural element is designed in the form of a groove or a depression or comprises a groove and / or a depression.
  • the structural element may comprise a curved surface area or an inclined plane, in particular, so that the introduced sample can be guided along the curved surface area or along the inclined plane into the desired partial area.
  • the sample when the sample is a biological cell, in particular a living biological cell, it can not grow or only with difficulty on an inclined plane or a curved surface area.
  • the sample which is arranged after insertion in the curved surface area or the inclined plane of the structural element, can be guided by moving the sample carrier into a desired partial area.
  • the sample carrier may comprise a bottom plate, wherein the sample carrier rests on the bottom plate during operation, and wherein the bottom plate comprises the predetermined surface area.
  • the desired partial area may be partially or completely flat.
  • the sample can stably adhere or be stably positioned in the planar region of the desired portion.
  • the sample carrier comprises a bottom plate and a cover plate wherein the cover plate and / or the bottom plate are liquid-tightly connected to each other so that a cavity is formed and wherein the structural member comprises a through hole through the bottom plate or cover plate, wherein the through hole is arranged such that the sample can be arranged in a desired portion of the cavity.
  • the structural element can be designed such that the sample can be fixed by capillary forces in the desired portion of the cavity.
  • the sample carrier may comprise a plastic, in particular COC (cycloolefin copolymer), COP (cycloolefin polymer), PS (polystyrene), PC (polycarbonate) or PMMA (polymethyl methacrylate).
  • the sample carrier may be formed as an injection molded part.
  • the bottom plate may comprise a plastic and / or glass. The bottom plate is thin between 1 ⁇ m and 300 ⁇ m. This allows high-resolution microscopy through the bottom plate.
  • the sample By moving the sample carrier, the sample can be arranged in the desired surface area.
  • the sample when placed in a curved portion or an inclined plane of the structural member, the sample may be directed by moving to the desired surface area.
  • sample carrier may comprise one or more of the features described above.
  • the structural element can be designed such that the sample can be held by capillary forces in the desired portion of the cavity.
  • a gel can be introduced into the desired portion of the cavity.
  • a collagen gel an agarose gel or a matrigel can be used.
  • the through-hole in particular with an optically transparent material, can be closed.
  • the organic, biological and / or medical sample may be a biological cell.
  • a plurality of cells can be positioned. Thereby, a desired cell distribution in a desired surface area of a sample carrier can be provided.
  • a random cell distribution occurs.
  • the cell distribution often depends on the type of filling, i. For example, how quickly the cell suspension is pipetted in and how the vessels are moved immediately after filling.
  • cell distribution often depends on the geometry of the structures that receive the cell suspension.
  • the local cell density can be defined in order to be able to compare results from different experiments sufficiently well, to carry out more economically and / or to facilitate the evaluation or to enable an automation of the evaluation.
  • a microscopic assay it is not always necessary for cells to colonize the entire surface area of a sample carrier, but it is sufficient for cells to be arranged only in the optically accessible area or in a part thereof. This can save you rare or expensive cell material.
  • the migration of adherent cells can be measured by observing the temporal evolution of the shape of an initial circular array of cells in a suitable gradient of concentration of a chemical. Remains the Form homogeneously circular over time, the cells show no directional movement, however, if the shape expands more in the direction of the gradient as in the direction perpendicular to it, a directed movement is close.
  • a sample carrier may comprise a plastic, in particular COC, COP, PS, PC or PMMA.
  • An example of a sample carrier is in DE 101 48 210 described.
  • the sample carrier may correspond to an injection molded part or comprise an injection molded part.
  • the sample carrier may comprise a bottom plate and a cover plate. By connecting the bottom plate with the cover plate, a cavity or an upwardly open area can be formed. In the cavity may lead an opening, in particular wherein the opening for filling or emptying of the cavity, for example with the sample, can be used.
  • the bottom plate can be connected, for example by means of fusion or gluing with the cover plate. In particular, glass can be attached by gluing.
  • Adhesives used are, for example, UV-curing adhesives, adhesive tapes or other adhesives. In particular, substances which do not have a toxic effect on the sample can be used. Suitable welding technologies are in EP 1 579 982 described.
  • the sample carrier in particular the bottom plate, may comprise a structural element, in particular a three-dimensional structural element.
  • the structural element may be formed in the form of a survey and / or depression.
  • the structural element can be used to position the sample, for example because a sample can not grow on a raised or rounded dome as it falls vertically or down an inclined plane.
  • a depression for example in the form of a groove, can be formed, in which the sample can be positioned.
  • biological cells can be locally concentrated in a desired partial area.
  • FIG. 1 shows a sample carrier comprising a bottom plate 101 and a cover plate 102, which are connected to each other such that an upwardly open area is provided.
  • An outburst on the side of the figure facing the observer serves as an illustration.
  • Structural element 103 is in the form of an elevation, in particular in the form of a dome.
  • the inner diameter of the sample carrier can be 7 mm.
  • the structural element may have a diameter of 2 mm, a radius of curvature of the upper edge of 0.5 mm and a height of 1 mm.
  • the structural element can be provided by deep drawing in the bottom plate 101, for example.
  • the sample carrier can be filled with 100 ⁇ l of cell suspension, in particular wherein the concentration or number density of the cells in the suspension can be selected such that a desired surface area 104 can be exposed to 100% confluence with cells. 100% confluent means that no free space is visible between the cells.
  • the sample carrier can be alternately moved obliquely in opposite directions, so that cells which have deposited on the structural element 103 are guided into the desired surface area 104.
  • a sample carrier according to FIG. 1 can be used for a migration assay.
  • a predetermined time for example after 2 hours
  • an image of the sample carrier can be taken and the confluence of the cells evaluated.
  • the percentage of confluency indicates the ratio of cell occupied area to the total area of the sample carrier surface area intended for the migration assay.
  • This migration assay has significant advantages over known migration assays.
  • a pipette tip is scraped into a cell-grown surface area to scrape a cell-free area and time measured by the cells until the scratch is closed again.
  • Problems for reproducibility can arise, among other things, that the stud generally does not have a well-defined width, and that scratching can destroy or damage possible surface coatings of the sample carrier.
  • a region may be kept free of a sample by covering it with a silicone part that is either mechanically pressed onto the growth surface or self-adhered to the growth surface by a tacky layer.
  • a silicone part that is either mechanically pressed onto the growth surface or self-adhered to the growth surface by a tacky layer.
  • a sample carrier as in FIG. 1 does not include any moving parts in contact with the sample.
  • the dimensioning of the structural element 103 can be reproducible, for example, by means of a correspondingly optimized thermoforming process.
  • FIGS. 2 to 5 each show a cross section through a sample carrier with a structural element 203, 303, 403 or 503.
  • the structural element 203 or 303 in FIGS. 2 and 3 is designed in the form of a pyramid.
  • the structural element 203 or 303 comprises a plurality of inclined planes.
  • the structural element 203 or 303 in FIG FIGS. 2 and 3 a dull pyramid, ie the tip is flattened.
  • FIG. 4 and FIG. 5 show a structural element 403 or 503 in the form of a dome with vertical walls.
  • FIGS. 3 and 5 For example, individual samples 305 and 505 are shown, which are arranged in a desired surface area.
  • the sample carrier comprises in each case a bottom plate 201, 301, 401 or 501 and a cover plate 202, 302, 402 and 502, respectively.
  • FIGS. 6 to 8 show a cross section through a sample carrier comprising a structural element 603, 703 or 803.
  • the samples 605, 705 and 805 are arranged in a medium 606, 706 or 806, in particular a liquid.
  • the filling height of the liquid 606 corresponds to the height of the structural element 603.
  • FIG. 7 shows the sample carrier FIG. 6 at a later time, with the samples 705 arranged in a desired surface area of the sample carrier. In other words, the samples 705 have dropped to the bottom of the sample carrier and adhere there.
  • FIG. 8 shows the sample carrier from the FIGS. 6 and 7 wherein the sample carrier is filled with the medium 806 up to a predetermined filling level.
  • the sample carrier each comprises a bottom plate 601, 701 or 801 and a cover plate 602, 702 and 802, respectively.
  • FIG. 9 shows a sample carrier comprising a cavity 907, wherein the cavity 907 comprises an observation channel 908.
  • a structural element 903 is arranged in the observation channel 908, a structural element 903 is arranged.
  • a sample carrier as in FIG. 9 can be used, for example, for a chemotactic experiment.
  • a gradient of a chemical substance between two subregions of the cavity 907 is built up, for example by filling only a portion of the cavity 907 with this chemical substance.
  • An optical system 909 in particular a microscope, can be used to monitor the movement of the samples 905, especially living biological cells.
  • the focus of the viewing optics 909 can be adjusted so that only samples located at the highest point of the feature 903 are sharply imaged.
  • the structural element 903 may comprise a round or flattened tip.
  • FIG. 10 shows a surface region of a sample carrier, in particular a surface region of a bottom plate 1001, comprising three structural elements 1003, wherein each of the structural elements 1003 is formed as an elongated elevation. It is also possible to use structural elements in the form of elongated depressions or to combine elongate depressions with depressions, for example with depressions of different diameters. Height and width of the strip-shaped structural elements can be varied.
  • FIGS. 11 to 13 show a sample carrier comprising a cavity 1107, 1207 or 1307, a bottom plate 1101, 1201 and 1301, respectively, and a cover plate 1102, 1202 or 1302 connected to the bottom plate 1101, 1201 and 1301, respectively.
  • a structural element 1103, 1203 or 1303 comprises an opening 1111 or 1211 in the cover plate 1102, 1202 or 1302.
  • the opening 1111 or 1211 is in particular conical, in particular wherein the opening 1111 or 1211 tapers towards the bottom plate 1101, 1201 and 1301, respectively.
  • a sample 1205 or 1305 can be introduced in the form of a suspension 1110 in the sample carrier (see Fig. 11 ).
  • the amount of suspension may be such that, as in Fig.
  • the observation area 1208 is filled and a portion of the suspension 1110 is disposed in the opening 1211.
  • a liquid outlet from the observation area 1108, 1208 or 1308 in a first or second portion of the cavity 1207 is prevented by capillary effects.
  • the samples can sink in the region of the opening 1111 or 1211 to the bottom of the observation area 1108, 1208 or 1308 and adhere there. After adhering, the cavity 1107, 1207 or 1307 can be filled.
  • the opening 1111 or 1211 can be sealed and sealed with an optically transparent material, for example PDMS (polydimethylsiloxanes, eg Sylguard 184, Dow Corning Corporation).
  • PDMS polydimethylsiloxanes, eg Sylguard 184, Dow Corning Corporation
  • FIG. 14 shows a sample carrier comprising an observation area, wherein in the observation area a piece of gel 1412, for example Collagen1 gel, agarose gel or Matrigel (for example, by Becton Dickinson), is arranged.
  • gel 1412 for example Collagen1 gel, agarose gel or Matrigel (for example, by Becton Dickinson)
  • the sample 1505 in the form of a suspension 1410
  • the sample carrier as in FIGS. 15 and 16
  • the cells can be arranged in a spatial area above the desired surface area. In other words, for several samples, a three-dimensional distribution of the Samples in the gel can be achieved.
  • FIGS. 1412 for example Collagen1 gel, agarose gel or Matrigel
  • a sample carrier comprising a bottom plate 1401, 1501 or 1601, a cover plate 1402, 1502 or 1602, a cavity 1407, 1507 or 1607 and a structural element 1403, 1503 or 1603.
  • a piece of gel 1412, 1512 or 1612 arranged in FIGS. 14 and 15 an opening 1411 or 1511 is shown in the structural element 1403 or 1503 in the form of a through hole through the cover plate 1402 and 1502, respectively.
  • a sample carrier comprising a cavity and an opening which leads into the cavity
  • the cavity can be filled with a medium, in particular wherein the medium does not enter the opening via the height of the cavity.
  • the medium may comprise a nutrient medium for biological cells and in particular correspond to a first liquid.
  • the opening can then be closed with a second liquid, in particular a drop of oil, for example silicone oil or mineral oil, wherein only so much is filled that the oil surface does not bulge upwards.
  • the sample may be added to the oil in the form of a suspension.
  • the sample sinks through the oil to the desired surface area and can adhere or grow there.
  • the sample can be accurately positioned. In particular, multiple samples can be positioned, with the number of samples being precisely adjustable. As a result, it is possible to work with a smaller number of samples and the sample can also be positioned in hard-to-reach surface areas of the sample carrier.
  • experimental preparations may be made prior to introduction of the sample.
  • a concentration gradient can be established in the sample carrier before the sample is introduced into the sample carrier.
  • the idea is to introduce samples, in particular cells, into an experimental environment only when all or a majority of the experimental parameters, for example the gradient of a chemical substance, the temperature, the gas concentration in the medium and / or the ph value, are set.
  • the cells are not disturbed by the preparations of the experiment, which can happen, for example, by changes in solution, vibrations or temperature fluctuations.
  • the cells can be in a (maximally) comparable state at the beginning of the experiment.
  • the cells may be in the desired gradient so that the response of the cells can be observed without delay.
  • slightly or non-adherent cells ie cells that are not on a surface of the sample carrier can be examined with this method.
  • immune cells for example neutrophils and other leukocytes. Since the oil largely prevents evaporation of the first liquid, it is possible in particular to work with small amounts of medium.
  • a sample carrier comprising two reservoirs and an observation channel disposed therebetween, such as in FIG EP 1 741 487 described, filled with samples.
  • the sample carrier is first filled with a neutral medium.
  • a gradient of a chemical substance is built up between the reservoirs. Since this can take a certain time, in particular a few hours, it is possible by this method that the sample is introduced only in the fully established gradient.
  • the observation channel may be an opening, which is for example conical and closed with a hydrophobic liquid.
  • the hydrophobic liquid may be, for example, a silicone oil or a mineral oil, in particular where the oil is selected such that it does not act toxic to the sample and does not arouse or destroy the materials of the sample carrier.
  • a hydrophobic liquid As a hydrophobic liquid, a two-component liquid can be used, which is introduced into the filling opening shortly before the introduction of the sample and then polymerize or otherwise cross-link and solidify.
  • these are silicone oils mixed with crosslinkers or, for example, Sylguard 184 from Dow Corning (PDMS).
  • PDMS Dow Corning
  • a positioning of a sample in a desired surface area of a sample carrier can be carried out by means of a magnetic force.
  • the sample must have magnetic properties and be exposed to magnetic forces in a corresponding sample carrier.
  • Biological cells usually have no magnetic properties.
  • paramagnetic particles in particular paramagnetic nanoparticles are suitable.
  • the particles can be connected to the sample in a variety of ways. Small particles can be phagocyted by cells, that is, taken up. Prerequisite for the inclusion is an attachment of the particles on the surface of the cell.
  • positively charged end groups are suitable for attachment to the surface of the cell, since the cell membrane usually carries negative charges.
  • the particles can be stored, for example, in vesicles in the cytosol. With an appropriate amount of collected particles, the external action of a magnetic field may be large enough to move a non-adherent cell in a sample carrier.
  • the magnetic particles may be larger, i. be nearly as big as the cell itself or bigger.
  • the size of a particle may correspond to one-fiftieth of the cell size.
  • the particles may, for example, consist of a paramagnetic material in their core and may be coated with a polymer matrix. On this polymer matrix, the particles may have a coating that can adhere to a cell surface. Examples include surface proteins such as CD molecules or activated tosyl groups.
  • the binding of the particles to the cells may be specific or nonspecific by the choice of coating. In particular, the coating can be chosen so that it adheres to only one type of cells, that is specific. As a result, a desired type of cells can be filtered out of a multiplicity of cells.
  • a magnetic field can be applied, in particular perpendicular to the potential direction of movement, for example to the growth surface of the sample carrier.
  • a field can be created whose field lines condense to the desired surface area. If one strives for a circular cell spot, the field in this area can be strongest and in concentric circles around the desired surface area the field lines can become less dense. This can be achieved, for example, with an iron cone, the tip of which is placed directly below the desired surface area.
  • FIGS. 17 to 20 show a part of a sample carrier, in particular an observation channel 1708, 1808, 1908 or 2008, comprising a bottom plate 1701, 1801, 1901 and 2001 and a cover plate 1702, 1802, 1902 and 2002, respectively.
  • a cone or conically shaped element 1713, 1813 , 1913 or 2013 of a magnetic or magnetizable material is connected to a permanent magnet 1714, 1814, 1914 and 2014, respectively.
  • the permanent magnet 1714, 1814, 1914, and 2014 for example, may be a neodymium-iron-boron (NdFeB) magnet.
  • the amount of field strength of the permanent magnet 1714, 1814, 1914, and 2014, respectively, may be between 0.5 and 1.4 Tesla.
  • the magnetic field is collimated to the top of the conical element 1713, 1813, 1913, and 2013, respectively, and a magnetic field line distribution is formed in which the field lines at the tip of the conical shaped element 1713, 1813, 1913, and 2013, respectively, are highly compressed.
  • the permanent magnet 1714, 1814, 1914 and 2014 may have a diameter between 1 mm and 20 mm, in particular 3 mm to 10 mm.
  • the conically shaped element 1713, 1813, 1913, and 2013, respectively, may have a diameter at the base that corresponds to the diameter of the permanent magnet 1714, 1814, 1914, and 2014, respectively.
  • the opening angle of the conically shaped element 1713, 1813, 1913 and 2013 may between 30 ° and 90 °, in particular 60 °.
  • a conically shaped element 1713, 1813, 1913 or 2013 with a diameter of the base of 4 mm is suitable.
  • the conically shaped element 1713, 1813, 1913 and 2013 may taper to a flattened tip, wherein the flattened portion may have a diameter of 0.5 mm.
  • the opening angle of the conically shaped element 1713, 1813, 1913 and 2013 may be 60 °.
  • the permanent magnet 1714, 1814, 1914 and 2014 may have a diameter and a height of 4 mm.
  • an electromagnet can be used instead of a permanent magnet 1714, 1814, 1914 and 2014. This can be advantageous for automation of the method since the magnetic field of an electromagnet varies, in particular can be switched on and off.
  • the magnetic device can be positioned relative to the sample carrier.
  • the position of the magnetic device can be changed parallel to the sample carrier, as in Fig. 17 indicated, or perpendicular to it, as in Figures 18-20 shown.
  • the desired magnetic field arrangement in particular the strength of a local extremum of the magnitude of the magnetic field strength, can be varied by the perpendicular distance to the sample carrier.
  • FIGS. 18 to 20 show the magnetic device at different distances to the sample carrier.
  • the diameter of the desired surface area in which samples 1705, 1805, 1905 and 2005 are arranged can be varied.
  • FIGS. 21 and 22 show a magnetic device 2114 or 2214 and a part of a sample carrier, in particular an observation channel 2108 or 2208, comprising a bottom plate 2101 or 2201 and a cover plate 2102 or 2202.
  • the magnetic device 2114 or 2214 has a tip 2115 and 2215, respectively Shape of a cuboid extension on.
  • the magnetic device can be positioned relative to the sample carrier.
  • the size of the desired surface area can be determined by the perpendicular distance of the tip 2115 or 2215 from the observation channel 2108 or 2208. For example, shows Fig.
  • the samples can be introduced, for example, in a suspension into the sample carrier, wherein the number density of the samples in the suspension corresponds to the desired cell density.
  • the suspension can be introduced with a pipette, wherein the entire liquid of the suspension can flow over the position of the magnetic field peak.
  • the cells are held in the magnetic field, but not immediately concentrated at the top.
  • This method can be used for observation channels.
  • the liquid is necessarily rinsed past the desired magnetic field arrangement with suitable positioning of the magnetic device.
  • the samples before they can adhere to a surface region of the sample carrier, are caused by small shocks or vibrations in motion.
  • the samples, in particular the cells move in the direction of the reinforcing field lines. In other words, they can be gradually shaken to a maximum of magnetic field strength.
  • the movement or small impacts can be achieved by vibrations on a shaker, by ultrasound or by pivoting the sample carrier.
  • the entire experimental set-up in particular the sample-carrying sample carrier and the magnetic device, can be placed in an incubator for adhesion. This may take several hours. Only after this time, the magnetic device can be removed.
  • a sample carrier can be used for chemotactic assays, where migration of cells in a gradient is to be monitored.
  • the aim is to analyze whether cells migrate more or less in the direction of the increasing concentration of a substance.
  • closable reservoirs can be connected by an observation channel, the height of the observation channel being less than 10% of the height of the reservoirs, for example 70 ⁇ m at a reservoir height of 800 ⁇ m. Through openings, cells and solutions can be filled into the reservoirs.
  • both reservoirs can be filled with a neutral liquid.
  • the neutral liquid can one Nutrient liquid for cells correspond.
  • a permanent magnet can be placed under the cell-filled reservoir. This magnet can then be moved in the direction of the second reservoir. The cells follow the movement of the magnetic device until they get stuck in the groove. There you can let the cells adhere. Subsequently, a gradient of a chemical substance can be built up in the observation channel.
  • the magnetic cells may be introduced into the wells by moving the sample carrier systematically relative to the magnet.
  • the maximum radii of the depressions may be, for example, 50 ⁇ m to 1 mm, and the maximum depth of the depressions may be about 5 ⁇ m to 100 ⁇ m.
  • the structural element may correspond to a rectangular barrier whose longitudinal direction is perpendicular to the connecting line of the two reservoirs.
  • the width of the barrier may correspond approximately to the maximum distance traveled by a cell during the observation period. Typical observation periods are for example 12 or 24 hours. In 12 hours, for example, human endothelial cells such as HU-VEC, on average, recover 200 ⁇ m in the direction of a well-defined gradient, or 400 ⁇ m in 24 hours. Over a length of about 200 ⁇ m to 400 ⁇ m, the migration of many cell types from mammals to chemotaxis needs to be analyzed and assessed. Therefore, a barrier width between 50 microns and 1000 microns can be selected.
  • the experiment can be carried out such that cells are introduced into the observation channel and removed by tilting the sample carrier from the barrier-shaped structural element.
  • cells can be positioned in a partial area or removed from a partial area which adjoins the barrier-shaped structural element.
  • the observation of the migration of the cells can be carried out by means of video microscopy. It can thereby be determined whether significantly more cells migrate in the direction of the increasing or decreasing concentration of the chemical substance.
  • the barrier width can also be less than 50 microns.
  • the structural element can not comprise a flat but, for example, only a curved region. If the cells are fluorescent, for example, by a GFP construct (Green Fluorescent Protein construct), one can cross the barrier Visualize cells by appropriate focusing when they are near the highest region of the feature.
  • GFP construct Green Fluorescent Protein construct
  • sample carriers can be combined with different methods for positioning an organic, biological and / or medical sample.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Mechanical Engineering (AREA)
  • Physics & Mathematics (AREA)
  • Fluid Mechanics (AREA)
  • Sampling And Sample Adjustment (AREA)

Claims (8)

  1. Porte-échantillon comportant un élément de structure (903), l'élément de structure (903) étant configuré de manière à ce qu'un échantillon (905) organique, biologique et/ou médical, introduit dans le porte-échantillon, puisse être agencé dans une zone partielle souhaitée du porte-échantillon, le porte-échantillon comprenant une plaque de fond et une plaque de couverture, et la plaque de couverture et la plaque de fond étant reliées l'une à l'autre de façon étanche aux liquides, de manière à ce que soit formée une cavité (907) qui comprend un canal d'observation (908), caractérisé en ce que l'élément de structure (903) est agencé dans le canal d'observation (908), le porte-échantillon repose sur la plaque de fond, en service, la plaque de fond comprend l'élément de structure (903), et la plaque de fond présente une épaisseur entre 1 µm et 300 µm.
  2. Porte-échantillon selon la revendication 1, dans lequel l'élément de structure est réalisé sous la forme d'une proéminence et/ou d'un creux, notamment sous la forme d'une coupole, d'une pyramide, d'une rainure et/ou d'un lamage.
  3. Porte-échantillon selon la revendication 2, dans lequel ladite zone partielle souhaitée est adjacente à l'élément de structure, ou bien entoure complètement l'élément de structure.
  4. Porte-échantillon selon l'une des revendications précédentes, dans lequel l'élément de structure comprend une zone de surface courbe et/ou un plan incliné.
  5. Porte-échantillon selon l'une des revendications précédentes, dans lequel la zone partielle souhaitée est en partie ou en totalité plane.
  6. Porte-échantillon selon l'une des revendications précédentes, dans lequel l'élément de structure est fabriqué par emboutissage ou par emboutissage à chaud.
  7. Porte-échantillon selon l'une des revendications précédentes, dans lequel l'élément de structure est fabriqué par emboutissage ou par emboutissage à chaud de la plaque de fond.
  8. Utilisation d'un porte-échantillon selon les revendications 1 - 7, pour un essai de migration.
EP13156764.6A 2009-05-13 2009-05-13 Support d'échantillon pour le positionnement d'un échantillon organique, biologique et/ou médical Active EP2599548B1 (fr)

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EP13156764.6A EP2599548B1 (fr) 2009-05-13 2009-05-13 Support d'échantillon pour le positionnement d'un échantillon organique, biologique et/ou médical
EP09006469A EP2253378A1 (fr) 2009-05-13 2009-05-13 Procédé de positionnement d'un échantillon organique, biologique et/ou médical

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DE102012108158B4 (de) * 2012-09-03 2016-03-17 Johann Wolfgang Goethe-Universität Kapillarzelle, Anordnung und Verfahren zur Aufnahme, zur Positionierung und zur Untersuchung einer mikroskopischen Probe
GB201219196D0 (en) * 2012-10-25 2012-12-12 Isis Innovation Droplet assembly method
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CN105188934B (zh) 2012-12-07 2018-12-04 牛津大学创新有限公司 3d打印的液滴组件
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US20100308945A1 (en) 2010-12-09
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US20130175195A1 (en) 2013-07-11

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