WO2008092607A1 - Dispositif formant pipette, dispositif de manipulation et procédé de manipulation de cellules biologiques - Google Patents

Dispositif formant pipette, dispositif de manipulation et procédé de manipulation de cellules biologiques Download PDF

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Publication number
WO2008092607A1
WO2008092607A1 PCT/EP2008/000597 EP2008000597W WO2008092607A1 WO 2008092607 A1 WO2008092607 A1 WO 2008092607A1 EP 2008000597 W EP2008000597 W EP 2008000597W WO 2008092607 A1 WO2008092607 A1 WO 2008092607A1
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WO
WIPO (PCT)
Prior art keywords
pipette
cell
vibration
pipette tip
tip
Prior art date
Application number
PCT/EP2008/000597
Other languages
German (de)
English (en)
Inventor
Steffen Hering
Original Assignee
Universität Wien
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Universität Wien filed Critical Universität Wien
Publication of WO2008092607A1 publication Critical patent/WO2008092607A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • 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
    • B01L3/02—Burettes; Pipettes
    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components

Definitions

  • the invention relates to a pipette device which is set up for manipulating biological cells, in particular under the action of ultrasound, a manipulation device which is equipped with such a pipette device, and methods for manipulating biological cells.
  • the perfusion ring is placed with the pipette tip on the bottom of a culture vessel, so that the perfusion ring limits a region of the surface to be treated.
  • the cells can be detached by the enzyme solution and by shearing forces in the liquid.
  • a disadvantage of the method known from DE 197 42 163 may be that the detachment process is substantially dependent on the reaction time of the enzyme solution (incubation time).
  • the required incubation time is variable for different cell types, which makes fast removal of cells difficult. Long exposure times of enzyme solutions can also lead to cell damage.
  • a disadvantage may also be that the detachment process can not be standardized by the variable incubation times (for different cell types).
  • the application of the technique described in DE 197 42 163 is further limited to the fact that only a removal of cells is provided. Further processing of the cells is possible only with a considerable additional effort.
  • DE 101 08 799 A1 describes a process for the introduction of biological or pharmacological material into biological cell material under the action of ultrasonic vibrations.
  • the ultrasonic vibrations induce cavitations, which in cell membranes lead to pores through which the biological or pharmacological material is introduced.
  • the ultrasonic vibrations are generated by a vibrating glass fiber, which runs in a catheter tube and protrudes at the end of the catheter tube. Upon exposure of the glass fiber to ultrasonic vibration generated with an ultrasonic generator separate from the catheter tube, the ultrasonic vibrations propagate in the vicinity of the projecting end of the glass fiber.
  • the technique described in DE 101 08 799 A1 has, as a first disadvantage, that a local action on individual cells or cell groups is virtually impossible, since the ultrasonic waves are radiated on all sides from the end of the glass fiber into the environment.
  • Another disadvantage is the low efficiency of ultrasound generation. Much of the vibrational energy is lost along the length of the fiber.
  • the efficiency of generating ultrasonic waves on the biological material may depend on the position of the catheter tube relative to the vibration generator.
  • the operation of the conventional device is disadvantageous, as an automation, especially under sterile conditions is excluded.
  • the application of the technique according to DE 101 08 799 A1 is restricted to transfection methods.
  • Another general disadvantage of techniques known locally for local perfusion of cells is the occurrence of undesirable dead volumes in the devices used for perfusion. For perfusion, z.
  • dosages in the ⁇ l For treating or detaching cells, dosages in the ⁇ l
  • the invention has for its object to provide improved devices for manipulating biological cells, with which disadvantages in the prior art can be overcome.
  • a further object of the invention is to provide improved methods for manipulating biological cells.
  • the invention is based on the general technical teaching to provide a pipette device comprising a pipette tube and a vibrator, wherein a vibration source of the vibrator is attached to the pipette device and a vibrating element of the vibrator in the pipette tube, in a pipette tip, on the pipette tube is provided and / or arranged in a attached to the pipette tip or the pipette perfusion ring.
  • a compact construction of the vibration device is provided according to the invention, which is used to generate at least one vibration, preferably at least one vibration. raschallschwingung is set up.
  • Both the vibration source (oscillator) and the vibrating element which is arranged to introduce the vibration into an environment of the pipette device and / or into the pipette tip, form a composite with the pipette tube and the pipette tip or with the pipette tube, the pipette tip and the perfusion ring.
  • the composite is advantageously characterized by a simplified handling.
  • the conditions of generation and transmission of the oscillation are constant, so that the pipette device is characterized by stable oscillation parameters.
  • the path between the source of vibration and the location of the transfer of the oscillation into the environment is limited to the dimension of the pipette device, whereby an increased effectiveness of the oscillation generation is achieved.
  • the oscillation is localized to a narrow space.
  • the vibration is limited to an area whose dimension corresponds to the diameter of an outlet opening of the pipette tube or the pipette tip or the diameter of the perfusion ring.
  • a pipette device with a vibrating device results from the possibility, after the detachment of the cells from a cell carrier, of receiving them with the pipette device.
  • the pipette device can advantageously be used both as a separating tool and as a cell receptacle for transferring the cells for a further application or processing.
  • the oscillation is supplied to the surface of the cell carrier in a locally limited subregion (so-called effective region). In the effective range, the cells are exposed to vibrations during operation of the vibration device, while outside the effective range, the cells of the
  • Cell carrier is carried out with at least one component of the pipette device, preferably with the pipette tip or optionally provided perfusion ring.
  • a manipulation device which is set up for manipulating at least one biological cell and comprises the pipette device according to the first aspect of the invention and a positioning device, with which the pipetting device moves with respect to the at least one biological cell, in particular is positionable with respect to a cell carrier for receiving the cell.
  • the combination of the pipette and positioning means allows an accurate and reproducible adjustment of the position of the pipette device and thus the range of the action of the vibration on the cell carrier.
  • the at least one biological cell to be treated is arranged on a cell carrier in a conditioning and / or culture liquid (hereinafter generally referred to as treatment liquid).
  • the cell carrier comprises a solid surface such as a cultivation substrate or the bottom of a culture vessel.
  • the cell carrier may be part of the pipette device, in particular the pipette tip.
  • the invention is based on the general technical teaching to provide a method for manipulating at least one biological cell which is arranged in a liquid on a cell carrier and is treated under the action of a vibration associated with the pipette device according to the above-mentioned first Ge - Viewpoint is generated in a localized portion of the liquid.
  • a treatment of a single cell, a cell within a cell group, a single cell group or a cell group within a larger cell group on the cell carrier may be provided.
  • the invention enables a targeted treatment of one or more cells on surfaces with vibrations, in particular ultrasound.
  • a pipette device is equipped with an additional tool, with which the use of conventional manipulators for the treatment of cells with vibrations is considerably expanded.
  • pipette device (or: “pipette”) is generally referred to a device which is adapted for receiving and / or delivery of fluids from or into open fluid reservoirs.
  • the pipette device allows the recording, the temporary storage and the subsequent delivery of a liquid, which optionally contains at least one suspended cell.
  • the pipette device has a pipette tube and a liquid line (pipette tip), by means of which a fluidic connection with the liquid reservoir can be formed.
  • the pilot Petten having a plurality of pipette tips, which are connected to a common pipette tube.
  • a vibrating element of the vibrating device is preferably arranged in at least one of the pipette tips.
  • vibration is generally referred to herein as an apparatus for generating at least one mechanical vibration.
  • the vibration device has a vibrating element, via which the vibration is transmitted to the environment.
  • Oscillating element is adapted to stimulate a mechanical vibration in a surrounding liquid.
  • the mechanical vibration may generally include vibration at a frequency chosen in the range of infrasound, sound or ultrasound.
  • the vibrating element is arranged to excite an ultrasonic vibration in the surrounding liquid, since with this a particularly high efficiency of the cell treatment can be achieved.
  • the ultrasonic vibration preferably has a frequency in the range of 1 kHz to 1 GHz.
  • the vibration device preferably comprises an ultrasound source.
  • the vibrator is z. B. a piezoelectric vibrator, wherein the vibration source is formed by a piezoelectric body and the vibrating element by a vibration transmitter connected to the piezoelectric body.
  • the vibration transmitter is z. B. a pin for introducing the sound into a liquid or a solid material).
  • the vibrating element is positioned in the region of the pipette tip.
  • the positioning of the vibrating element in the region of the pipette tip becomes a geometric Arrangement referred to, in which the vibrating element is positioned in an immediate vicinity of the wall material of the pipette tip.
  • the vibrating element may contact the wall material of the pipette tip in order to improve the coupling of the vibrations into the wall material.
  • Oscillating element be arranged at a distance from the wall material of the pipette tip, preferably to couple the vibration in the liquid.
  • the vibration can be conducted through the liquid and / or the wall material of the pipette tip at its free end and its surroundings to the cells to be treated.
  • the cells are preferably applied outside the pipette tip with the vibration.
  • the invention makes it possible for the cells in the pipette tip to be subjected to the vibration.
  • the vibration source is arranged on the pipette tube or on the pipette tip or on the optionally provided perfusion ring.
  • the connection of the vibration source with the pipette tube or the perfusion ring is particularly preferred, as this allows an optimal alignment of the vibrating element, which is connected to the vibration source, with respect to the pipette tip.
  • the oscillating element it is possible for the oscillating element to be arranged in the region of the pipette tip at a distance from the pipette tip.
  • the vibrating element When the vibrating element is arranged in the pipette tip, the vibrating element according to a preferred embodiment of the invention is completely received by the pipette tip.
  • the oscillating element is arranged without moving in an axial direction from a free end (pipette outlet opening). stand out) of the pipette tip.
  • the arrangement of the vibrating element in the interior of the pipette tip, the vibrations can be conducted locally in the treatment liquid and to the cells.
  • the effective range is delimited by the damping of the oscillations in the vicinity of the pipette tip, a wall of the pipette tip placed on the cell carrier and / or the perfusion ring attached to the pipette tip.
  • this improves the localization of the vibration effect.
  • the vibration device in particular the vibration source or the vibration element is connected to a coupling device which connects the pipette tip with the pipette tube.
  • the vibration device can thus be arranged in the pipette device so that the vibration source in the pipette tube and the vibration element are positioned in the pipette tip.
  • the source of vibration such as the piezoelectric crystal
  • the vibration device is connected to a holding device which is arranged in the pipette tube.
  • a holding device which is arranged in the pipette tube.
  • the pipetting device can be flexibly adapted to different tasks.
  • Another advantage is the variability of the design of the holding device, the structure of which can be selected depending on the vibration source used.
  • the holding device may preferably comprise one of the following parts or a composition of these parts.
  • the holding device comprises a holding rod, which is fastened in the pipette tube and extends in an axial direction of the pipette tube.
  • the use of the holding rod has the advantage that with this the positioning of the vibration source can be easily adapted to the length of the pipette tube.
  • the holding device comprises a retaining plug.
  • Retaining plug extends in the radial direction over the inner cross section of the pipette tube.
  • the retaining plug in the pipette tube may be located near its distal end, i. H. be positioned near the pipette tip. This allows a reduction of the dead volume in the pipette tube.
  • the holding device may comprise a retaining ring, the inner diameter of which is set up for receiving the vibration source and whose outer diameter corresponds to the inner diameter of the pipette tube. The combination of retaining ring and vibration source can replace a retaining plug accordingly.
  • a composition of said parts may comprise, for example, a retaining plug with a pipette tip holding rod or a retaining plug with a retaining ring or a combination of all three parts. Furthermore, a plurality of said parts, such as a plurality of support rods or a plurality of retaining rings may be provided.
  • the vibration source is vibration-damped attached to the pipette device.
  • a greater proportion of the vibrations generated by the vibration source is conducted from the vibration source via the vibrating element in the vicinity of the pipette device, while only a small fraction Part is transferred to the pipette tube.
  • the oscillation source vibration damped attached to the pipette device in particular in the pipette tube and / or the perfusion ring.
  • an elastically deformable material may be used which is provided on the holding device, in particular on the holding bar, the holding plug and / or the retaining ring.
  • the vibration-damped fixation of the vibration source in the pipette tube by a retaining ring made of elastically deformable material, for. As provided rubber.
  • the pipette device when the pipette device has an inner shape by which the vibration from the vibrating element is directed to the pipette tip and its opening, the effectiveness of the vibration generation and the localization of the vibration effect can be advantageously improved.
  • the directed conduction of the oscillation to the pipette tip is preferably achieved by a tapering inner shape of the pipette tip, the pipette tube and / or the optionally provided perfusion ring.
  • the pipette tube has a conical view approach.
  • the conical projection includes a portion of the pipette tube in which the inner and outer diameters of the pipette tube decrease toward the pipette tip. This allows a simplified recording of the pipette tip on the end of the pipette tube and the provision of the tapering towards the pipette tip inner shape.
  • the focusing of the vibration can be further improved if, according to a further variant of the invention, the pipette device is equipped with a focusing device. equipped.
  • the focusing device With the focusing device, the oscillation in the pipette tip can be focused on its opening.
  • the focusing device comprises, for example, mechanical elements in the pipette tip that are set up for directional conduction of the vibration.
  • the focusing device comprises a surface region of the vibrating element. For example, a sound focusing with a parabolic or spherical surface of the vibrating element can be achieved.
  • the pipette tip of the pipette device according to the invention can optionally be equipped with a perfusion ring.
  • the term "perfusion ring" generally refers to an annular component which radially surrounds the pipette tip and has a sealing bearing surface which can be placed on the cell carrier in the direction of the free end of the pipette tip
  • the perfusion ring is preferably formed as the perfusion ring described in DE 197 42 163 A1 DE 197 42 163 A1 is incorporated by reference into the present description with regard to the construction and handling of the perfusion ring.
  • the perfusion ring advantageously allows a further delimitation of the vibration effect in the vicinity of the pipette tip.
  • the combination of the pipette tip with the perfusion ring makes it possible to simplify the production of cell arrays, since the perfusion ring improves local ultrasound action.
  • the cells can be transfected with the ultrasound treatment according to the invention to a cell carrier locally limited ultrasound-supported.
  • the perfusion ring is releasably attached to the pipette tip.
  • the perfusion ring can be firmly connected to the pipette tip.
  • a detachable compound is PHg, such. B.
  • the advantage of the separability of the perfusion ring from the pipette tip is the flexibility in adapting the pipette device to a specific one
  • the perfusion ring preferably has an inner holding region (tip receptacle) into which the pipette tip can be force-fitted.
  • tip receptacle an inner holding region into which the pipette tip can be force-fitted.
  • the vibrating element can be arranged outside the pipette tip in the perfusion ring. In this case, advantageously results in an enlarged, but sharply demarcated area of the vibration effect on the cell carrier.
  • the vibration source of the vibrator may be attached to the perfusion ring, so that the vibrating element projects into the free interior of the perfusion ring.
  • the vibrating element has a distance both to the pipette tip and to the perfusion ring, so that the vibration is preferably transferred into the liquid on the cell carrier and is supplied to the at least one biological cell on the cell carrier.
  • the pipette outlet opening a lateral opening, such as. B. have a notch, which can fulfill several functions.
  • the lateral opening when the pipette tip is placed with the pipette outlet opening on a cell carrier, cells are taken into the pipette tip and exposed there to the vibration effect.
  • the vibration can preferably be conducted through the lateral opening into the environment.
  • the pipette outlet opening can be obliquely formed relative to the axial longitudinal direction of the pipette tip.
  • the pipette outlet opening has a peripheral edge which extends in a plane which forms an angle not equal to 90 ° with the axis of the pipette tip.
  • the oblique peripheral edge advantageously allows an inclined placement of the pipette device on the cell carrier and thus a directed transmission of the vibration to cells on the cell carrier and a simplified recording of cells that have been detached under the action of the vibration from the cell carrier.
  • the oblique marginal edge at the free end of the pipette tip forms a tip region, with which the pipette tip can be placed on the cell carrier so that no or only a few cells on the cell carrier are injured.
  • the pipette device is equipped with a spring device and / or a metering device.
  • the spring device is preferably attached to the pipette tube.
  • One part of the spring device is firmly connected to the pipette tube, while another, elastically displaceable relative to the pipette tube. res part forms a stop for a force effect, such as a manual actuation force or a driving force of the positioning.
  • a placement force of the pipette device in particular the pipette tip and the optionally provided perfusion ring on the cell carrier can be adjusted.
  • the metering device is set up to fill an interior of the pipette tube and the pipette tip with a working fluid.
  • the volume of the supplied operating fluid can be adjusted.
  • the operating fluid preferably comprises a physiological saline solution, a liquid with at least one enzyme, in particular for the enzymatic degradation adherent to the cell carrier grown cells, and / or a liquid with at least one Schallkontraststoff.
  • this is equipped with a camera device.
  • the camera device is set up to record the pipette device, in particular the pipette tip and / or the optionally provided perfusion ring, and / or the cell carrier.
  • the camera device simplifies the setting of the pipette device with the positioning device.
  • a control device actuates the positioning device as a function of images of the pipette tip, the perfusion ring and / or the cell carrier.
  • Treatment of the cells comprises introduction of at least one active component into the cells (transfection) and / or detachment of the cells Cells from the cell carrier or a cell culture formed on the cell carrier.
  • the invention not only enables the detachment process of cells from the surfaces to be accelerated, but also predetermines arrangements (in particular microarrays) of specifically treated, adherent cells.
  • active component generally refers to a material (eg substance, substance composition or gas bubble) which can be introduced into the cell through a cell membrane and has a predetermined effect in the cell for the further state of the cell (eg, metabolism or cell division) or labeling (detectability) of the cell.
  • the introduction of the active component into the cell preferably takes place by means of a temporary, reversible interruption of the cell membrane (poration).
  • a sound contrast agent is introduced as an active component into the cells.
  • a “sound contrast agent” is generally a material that forms interfaces within the cells with respect to the cell constituents at which a reflection of sound waves occurs
  • Sound contrast agent in the cells adhering to the cell carrier has the advantage that the cells treated with the vibrations can be observed during further manipulation with sound-based detection methods.
  • the sound contrast contains at least one active substance, advantageously in addition to the said marking function of the contrast agent, a second effect, such as. B. an effect on the cell components or metabolism in the cell can be achieved.
  • the sound contrast agent with biological macromolecules, such. As DNA or RNA molecules or components, proteins, peptides, etc. loaded with which advantageously directly cell biological processes can be influenced in the cell.
  • ultrasound contrast agents used according to the invention are: free gas bubbles, precursors of gases, gas bubbles encapsulated by organic or inorganic substances, solutions, colloidal solutions, suspensions,
  • Dispersions ionophores, dissolved microparticles, dissolved polymer spheres, dissolved organic and / or inorganic molecules, sugar-containing solutions, microparticles, ferro- or paramagnetic metals, microaggregates, porous particles of organic and / or inorganic material, lipid-containing
  • Microspheres and / or emulsions wherein the gas bubbles preferably contain air, nitrogen, oxygen, carbon dioxide, fluorinated gas and / or another biocompatible gas.
  • the gas bubbles preferably contain air, nitrogen, oxygen, carbon dioxide, fluorinated gas and / or another biocompatible gas.
  • carbohydrate-based, bubble-forming ultrasound contrast agent such as Levovist (trade name) or Optison (trade name, octafluoropropane gas encapsulated in a human albumin sphere).
  • Levovist trade name
  • Optison trade name, octafluoropropane gas encapsulated in a human albumin sphere.
  • the gas inclusion in these ultrasound contrast agents advantageously leads to an improved power transmission.
  • the particles introduced into the cells as such can have a cell biological effect.
  • the particles carry an active substance, such as. B. the above-mentioned biological macromolecules.
  • at least one active substance can be bound as a layer on the surface of the nanoparticles in order to get into the interior of the cells with the nanoparticles.
  • the at least one vibration in the liquid surrounding the biological cell is generated with an energy such that cavitations occur in the liquid.
  • at least one perforation is formed in the cell membrane, which allows introduction of the sound contrast agent and / or an active substance from the liquid into the cell.
  • a physiological solution containing an enzyme for promoting cell detachment is used as the treatment liquid.
  • the enzyme preferably comprises trypsin or another proteolytic enzyme, such as e.g. Collagenase.
  • the detachment of the cells from the cell carrier takes place without contact, ie exclusively under the effect of the treatment liquid covering the cells on the cell carrier and without contact with solid surfaces of separating tools.
  • the detached cell is free of contact with mechanical tools.
  • the treatment liquid is used to transfer the vibrations to the cells. Under the effect of the vibrations, a separation of the adherent cell contacts with the cell carrier is promoted.
  • the effect The treatment liquid can optionally be increased by creating a perfusion flow.
  • the oscillation element In order to direct the oscillation, in particular the ultrasound, directly from the oscillation element of the oscillation device to the cells, the oscillation element according to a particularly preferred embodiment of the invention is arranged in the pipette device such that the oscillation element is spaced from the pipette tip walls and from the treatment liquid surrounded by the pipette tip.
  • the oscillations can be directed to the cells with the pipette tip placed on the cell carrier.
  • the cells are removed from the cell carrier by the mechanical vibration of the pipette tip.
  • a particularly advantageous embodiment of the invention is characterized by the inclusion of the at least one detached biological cell in the pipette device.
  • a vacuum is formed on the pipette tube and / or the pipette tip, preferably with the metering device, through which liquid with the detached cell is drawn into the pipette tip through the pipette outlet opening.
  • the pipette device thus fulfills a dual function, namely the function of localized application of the biological cell to a vibration and the function of recording the detached cell for further manipulations, such as, for example, transmission to another cell carrier or perfusion.
  • FIG. 1 shows a first embodiment of a pipette device according to the invention
  • FIGS. 2 and 3 variants of the pipette tip of the pipette device according to FIG. 1;
  • FIGS. 4 to 6 further embodiments of the pipette device according to the invention.
  • FIG. 7 shows an embodiment of a manipulation device according to the invention.
  • FIG. 1 shows, in a schematic perspective view, a first embodiment of the pipette device 10 according to the invention with the pipette tip 11, the pipette tube 13 and the pipette tip
  • Vibration device 20 which is arranged in the interior of the pipette device 10, in particular in an inner volume within the pipette tip 11 and / or the pipette tube 13.
  • the pipette tip 11 is connected to the pipette tube 13 by a coupling device 12.
  • the components 11, 12 and 13 in the illustrated embodiment are constructed substantially as known from conventional liquid pipettes.
  • the pipette tip 11 has a shape that tapers from the coupling device 12 to the free end of the pipette tip 11 with the pipette outlet opening 14.
  • the wall material of the pipette tip 11 is z. B. plastic.
  • Typical dimensions of the pipette tip 11 include, for. B: axial length: 5 cm, diameter of the pipette outlet opening 14: 0.1 mm to 2 mm, and diameter at the coupling device 12: 6 mm.
  • the coupling device 12 comprises a connecting piece for the detachable connection of the pipette tip 11 to the pipette tube 13. It is, for example, a screw connection made of plastic provided.
  • the pipette tube 13 includes, for example, a plastic tube or a conduit having a diameter in the range of z. B. 1 mm to 0.3 cm.
  • the vibration device 20 includes as the vibration source 21 an ultrasonic vibrator for generating ultrasound and as a vibrating element 22 a pin or a needle, which is provided for transmitting the ultrasound to the liquid in the pipette tip 11.
  • the Schwingungsquel- Ie 21 is electrically connected via connecting lines 23 to a control device (not shown).
  • the vibration source 21 includes z. B. a piezoelectric crystal, which is adapted to generate ultrasonic vibrations in the range of 0.1 to 3 MHz.
  • the vibrating element 22 is fixedly connected to the vibration source 21.
  • Vibration device 20 is attached to the coupling device 12, so that the pin 22 projects into the inner volume of the pipette tip 11.
  • the oscillating element 22 can have as a focusing device a parabolic or spherical surface region 24 which forms a hollow shape at the tip of the oscillating element 22 (see enlarged partial image in FIG. 1).
  • the hollow surface portion 24 is preferably formed on a surface of the tip or a cylinder having a diameter of at least 10 mm.
  • a preferred variant of the method according to the invention comprises the following steps. First, the pipette device 10 is placed on a cell carrier 70 in the vicinity of the cells 1 to be treated. The cells 1 form on the surface of the cell carrier 70 an adherent cell culture which is covered with a liquid (eg treatment and / or culture liquid, not shown).
  • the pipette device 10 is arranged such that the pipette outlet opening 14 is positioned at a distance from the surface of the cell carrier 70. The distance is selected, for example, in the range of 50 microns to 1 mm. In this state, the pipette tip 11 is immersed in the liquid on the cell carrier 70.
  • the pipette device 10 is actuated in order to suck the liquid into the pipette tip 11.
  • a negative pressure is formed on the pipette tube 13, as is known from conventional pipettes.
  • the volume of the liquid sucked in is selected so that the vibrating element 22 of the vibrating device 20 is at least partially covered with the liquid.
  • the pipette tip 11 is not completely filled, but a distance of the surface 2 of the liquid from the coupling device 12 is formed.
  • the positioning and actuation of the pipette device 10 is carried out with a control device (not shown), as described for example in DE 197 42 163 as a manipulator.
  • DE 197 42 163 A1 is incorporated by reference into the present description with regard to the construction and handling of the manipulator.
  • ultrasonic waves 4 are passed through the liquid within the pipette tip 11 through the pipette outlet opening 14 to the cells 1 and transferred to the surface and its boundary to the cell carrier 70.
  • the pipette tip 11 Localized contact points between the cells and the surface of the cell carrier 70 are loosely localized to a portion of the liquid and the cell carrier 70 so that cells can move freely as detached cells 3 in the liquid layer above the cell carrier 40.
  • the detached cells 3 can be sucked up with the pipette device 10.
  • the pipette outlet opening 14 may be designed in accordance with FIGS. 2 and 3.
  • a lateral opening 15 is provided, through which, when a negative pressure is produced in the pipette device 10, the liquid is sucked into the pipette tip 11.
  • the liquid supply is made possible through the opening 15 without the pipette tip 11 having to be lifted off the surface of the cell carrier 70.
  • the pipette outlet opening 14 has a peripheral edge 16, which is inclined with respect to the axial extent of the pipette tip 11.
  • the peripheral edge 16 forms a tip portion, which may alternatively be used as a mechanical ultrasonic tool acting directly on the cells.
  • FIG. 4 shows a further embodiment of the pipette device 10 according to the invention with the pipette tip 11 and the pipette tube 13.
  • the pipette tube 13 has at its end a conical projection 17 on which the pipette tip 11 can be placed.
  • the pipette tip 11 can form a clamped fit with the projection 17 in the plugged state.
  • the projection 17 and the pipette tip 11 have correspondingly identical outer and inner cone angles and at least one of the parts has an elastic deformable angle. on.
  • the pipette tip 11 can be screwed onto the pipette tube 13 with a coupling device (see FIG. 1).
  • the pipette tip 11 and the pipette tube 13 are shown in the separated state. To connect both parts, the pipette tip 11 is pushed onto the pipette tube 13 (see arrow).
  • the vibration device 20 with the vibration source 21 and the vibration element 22 is arranged with a holding device 30 in the pipette device 10 such that a part of the vibration element 22 projects into the pipette tip 11.
  • the holding device 30 includes a holding rod 31 and a retaining plug 32.
  • the retaining plug 32 is seated in the pipette tube 13, wherein a form or interference fit is formed.
  • the holding rod 31, to which the oscillation source 21 is fixed, is located on the side of the holding stopper 32 facing the pipette tip 11.
  • the holding rod 31 is made, for example, of a plastic or another vibration-damping material, such as silicone rubber, in particular hardened silicon. gum rubber.
  • the retaining plug 32 can perform a multiple function.
  • the pipette tube 13 is sealed at its rear end with the retaining plug 32.
  • the retaining plug 32 can be set up for vibration damping, so that the vibration transmission from the vibration device to the pipette tube 13 is reduced and preferably to the liquid in the pipette tip 11 takes place.
  • the retaining plug 32 preferably consists of an elastically deformable material, for example of foam or silicone rubber. Since the pipette tube 13 is closed on the back by the holding device 30, the embodiment of the pipette device 10 shown in FIG. 4 requires a further component for supplying liquid in the inner volume of the pipette device 10.
  • the pipette device 10 is preferably equipped with a metering device 50. comprising a syringe plunger with a liquid reservoir.
  • the metering device 50 is mounted laterally on the pipette tube 13 and adapted to supply liquid in the pipette tube 13 and the pipette tip 11 upon actuation of the syringe plunger or to form a negative pressure in the inner volume of the pipette device 10.
  • the metering device 50 is shown schematically reduced in size.
  • the syringe piston of the metering device 50 has a volume of, for example, 0.5 ml.
  • the metering device 50 can be operated manually or by a drive device (not shown).
  • the metering device 50 can be actuated automatically, in particular during operation of the manipulation device according to the invention (see FIG. 7).
  • the integration of the holding device 30 in the pipette tube 13 has the further advantage that the dead volume in the inner volume of the pipette device 10 can be reduced. With the metering device 50 smallest fluid volumes can be transferred to the cells to be treated with high accuracy and reproducibility.
  • the holding device 30 may be arranged adjustably in the pipette tube 13.
  • the retaining plug 32 may be connected to the pipette tube 13 via a screw connection.
  • the position of the vibrating element 22 can be changed. It can in particular an immersion depth of the vibrating element 22 varies in a liquid in the pipette tip and the dead volume in the pipette device are minimized.
  • FIGS. 5A and 5B show a further embodiment of the pipette device 10 according to the invention, which is advantageously distinguished by a further reduced dead volume.
  • the pipette device 10 comprises the pipette tip 11 and the pipette tube 13, which form a plug-in or screw connection via the conical projection 17.
  • the vibration device 20 with the vibration source 21 and the vibration element 22 is arranged in the assembled state of the pipette device 10 in the pipette tip 11.
  • the vibrating element 22 comprises a pointed needle, e.g. made of stainless steel.
  • the holding device 30 for positioning the vibrator 20 comprises a holding plug 32 which extends in the pipette tube 13 into the conical projection 17.
  • the retaining plug 32 is fixed in the pipette tube 13 by two retaining rings 33.
  • the retaining rings 33 serve to seal the pipette tube 13 and the vibration damping.
  • the retaining plug 32 includes a plurality of channels 34, 35, as shown schematically in the longitudinal view of Figure 5A and the cross-sectional view of Figure 5B.
  • the channels include the axially extending channels 34, which receive the connecting lines
  • the seals 36 comprise, for example, adhesive, with which the interior of the channels 34 is liquid-tightly separated from the environment.
  • the channels include a radial channel 35 which is adapted for distribution of liquid from the metering device 50 into the pipette tip 11 or to form an air cushion.
  • the retaining plug 32 has at least one lateral recess 37 (see FIG. 5B), with which advantageously the mechanical contact of the retaining plug 32 with the pipette tube 13 is minimized. Accordingly, the vibration transmission to the pipette tube 13 can be minimized.
  • the pipette device 10 which is constructed in accordance with the embodiment with FIG. 4, additionally comprises a spring device 60 with which a placement force can be set with which the pipette device 10 can be placed on the cell carrier 70.
  • the spring device 60 comprises a guide rod 61, which is connected to the rear side of the retaining plug 32 and is designed to guide a propulsion plate 62.
  • the propulsion plate 62 is supported by the retaining plug 32 via a coil spring 63.
  • the propulsion force is partially absorbed by the coil spring 63, so that the force with which the pipette device 10 is placed on the cell carrier 70 is reduced accordingly.
  • damage to the pipette outlet opening of the pipette tip 11 or the optionally provided perfusion ring is thus avoided.
  • a perfusion ring 40 is additionally arranged on the pipette tip 11.
  • the perfusion ring 40 comprises an annular wall 41 with an inner support region 42, which is connected to the annular wall 41 via webs 43.
  • the perfusion ring 40 is z. B. plastic.
  • the pipette tip 11 is inserted into the holding region 42, as described in DE 197 42 163 Al.
  • the oscillation device 20 ' can be arranged such that the oscillation element 22' is arranged outside the pipette tip 11 in the perfusion ring 40 (shown in dashed lines).
  • the vibrating device 20 is attached to the perfusion ring 40 or introduced into the perfusion ring 40 with an additional actuating device (not shown) as a separate component separated from the remaining parts of the pipette device 10.
  • the vibration device may be arranged such that the vibration element is in direct mechanical contact with the wall of the pipette tip 11.
  • FIG. 7 illustrates a preferred embodiment of the manipulation device 100 according to the invention, which comprises the pipette device 10, the positioning device 200 and optionally the camera device 300.
  • the pipette device 10 shown by way of example in connection with the manipulation device 100 in FIG. 7 comprises the pipette tip 11 and the pipette tube 13, which is compactly formed in the region of the conical projection 17, and an axial passage opening 18 for receiving the vibrating element 22 and a radial passage opening 19 for feeding of liquid from the metering device 50. Due to the compact construction of the pipette tube 13 in the region of the conical projection 17, the dead volume is advantageously further reduced.
  • the vibrator 20 includes the vibration source 21, which is mounted with retaining rings 33 in the pipette tube 13.
  • the pipette device 10 comprises, for example, the following dimensions: axial length a of the pipette tip 11: 35 to 40 mm, distance b of the tip of the oscillating element 22 from the pipette outlet 16: 25 to 30 mm, and distance c of the pipette outlet 16 from the cell carrier 70 (operating position for manipulation of biological cells): 0.5 to 1 mm.
  • the positioning device 200 comprises a carrier with which the pipette device 10 is adjustable relative to the cell culture carrier 70. Furthermore, the positioning device 200 may include a drive for the metering device 50. The components 200, 300 and 50 may be controlled by a common controller (not shown).

Abstract

L'invention concerne un dispositif formant pipette (10) comprenant une pointe de pipette (11), un tube de pipette (13) et un dispositif d'oscillation (20), qui est agencé pour produire une oscillation, et qui présente une source d'oscillation (21) et un élément d'oscillation (22), la source d'oscillation (21) étant fixée au dispositif formant pipette (10), et étant mobile avec ce dernier, et l'élément d'oscillation (22) étant disposé dans le tube de pipette (13), dans la pointe de pipette (11) et/ou dans un anneau de perfusion (40) prévu au niveau de la pointe de pipette (11). L'invention concerne également un dispositif de manipulation (100) pour une manipulation de cellules biologiques, et un procédé pour une manipulation de cellules biologiques (1) qui sont disposées dans un liquide sur un support de cellules (70).
PCT/EP2008/000597 2007-01-31 2008-01-25 Dispositif formant pipette, dispositif de manipulation et procédé de manipulation de cellules biologiques WO2008092607A1 (fr)

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DE102007004856A DE102007004856A1 (de) 2007-01-31 2007-01-31 Pipetteneinrichtung, Manipulationseinrichtung und Verfahren zur Manipulation biologischer Zellen
DE102007004856.6 2007-01-31

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US10016756B2 (en) * 2014-09-24 2018-07-10 Duke University Disposable pipette tip and methods of use
CN108395982A (zh) * 2018-05-30 2018-08-14 厦门市科环海洋生物科技有限公司 一种菌落喷布装置
CN110643489A (zh) * 2019-10-20 2020-01-03 刘晓 一种移液器移卵针适配器
CN112014429A (zh) * 2019-05-30 2020-12-01 华东理工大学 一种基于超微电渗流调控的细胞膜振动检测方法

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US9017991B2 (en) 2009-03-13 2015-04-28 Tufts University Methods tip assemblies and kits for introducing material into cells
DE102011006581A1 (de) * 2011-03-31 2012-10-04 Hamilton Bonaduz Ag Zustandsüberwachung einer Pipettenspitze mit von innen angekoppelten piezoelektrischen Elementen
WO2021086550A1 (fr) * 2019-10-28 2021-05-06 Siemens Healthcare Diagnostics Inc. Pointes de pipette vibrantes et procédés de prévention de frottement de pointe de pipette
CN113578407A (zh) * 2021-07-12 2021-11-02 广东省科学院健康医学研究所 一种自动化抽吸装置

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CN112014429A (zh) * 2019-05-30 2020-12-01 华东理工大学 一种基于超微电渗流调控的细胞膜振动检测方法
CN112014429B (zh) * 2019-05-30 2024-01-30 华东理工大学 一种基于超微电渗流调控的细胞膜振动检测方法
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