WO2002101001A1 - Dispositif et procede pour manipuler des vesicules - Google Patents

Dispositif et procede pour manipuler des vesicules Download PDF

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Publication number
WO2002101001A1
WO2002101001A1 PCT/EP2002/006218 EP0206218W WO02101001A1 WO 2002101001 A1 WO2002101001 A1 WO 2002101001A1 EP 0206218 W EP0206218 W EP 0206218W WO 02101001 A1 WO02101001 A1 WO 02101001A1
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WO
WIPO (PCT)
Prior art keywords
vesicles
pores
sieve element
holes
sieve
Prior art date
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PCT/EP2002/006218
Other languages
German (de)
English (en)
Inventor
Martin Jenkner
Roland Thewes
Original Assignee
Infineon Technologies Ag
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 Infineon Technologies Ag filed Critical Infineon Technologies Ag
Publication of WO2002101001A1 publication Critical patent/WO2002101001A1/fr

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Classifications

    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • 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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • 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

Definitions

  • the invention relates to a device and a method for manipulation, in particular for positioning, selecting, transporting and / or fixing vesicles.
  • Vesicles i.e. biological objects from a closed, biological membrane, such as individual cells, represent a frequently manipulated, biological object. This is particularly true in the field of artificial insemination, biosensors and transfection of genes. With such methods and applications, individual cells have to be sorted out, transported and, if necessary, held or positioned. Due to the small size of the vesicles, typically 10 ⁇ m to 50 ⁇ m, technical aids such as micromanipulators or laser tweezers are used. In all of these methods, however, individual cells are always manipulated. This leads to problems in particular in the case of gene amplification and in the contacting of biological, neural networks by active array semiconductor structures, since the arrangement and positioning of the individual vesicles or cells is very time-consuming.
  • a micro-contact array is to be understood as a two-dimensional, field-like arrangement of micro-elements which come into contact with or interact with vesicles. If vesicles or cells have to be positioned on such microcontact arrays, correct positioning of the vesicle on the respective sensors, electrodes or other microelements is very important for successful extracellular detection or stimulation of biological signals in the vesicles or cells or an exchange of substances with the vesicles.
  • Micro contact arrays have an ever increasing number of individual contacts or electrodes. For micro contact arrays with 10 to 100 micro elements individual manual manipulation or positioning of the vesicles is still possible. For larger micro contact arrays with more than 10,000 electrodes, possibly more than 100,000 electrodes or elements in the future, this seems impossible.
  • Vesicles are understood here to mean objects, in particular biological objects from a closed lipid membrane, which can be handled in the liquid phase, preferably water. Generally, these are particles which comprise a lipid membrane with at least one lipid layer. Examples of such vesicles are cells, in particular nerve cells, genetically manipulated cells with voltage-dependent ion channels, giant cells fused from erythrocytes, so-called “ghosts” or spheres made of lipid bilayers.
  • the vesicles for the handling of which the invention is suitable preferably have a size between 1 and 500 ⁇ m.
  • the invention can also be used for manipulating and positioning other particles, such as latex beads, which have a corresponding size.
  • the device according to the invention has a sieve element with a multiplicity of pores for receiving or retaining preferably a single vesicle in each case, the pores having a predetermined size and being arranged at predetermined distances from one another, ie at predetermined positions.
  • a sieve element with a multiplicity of pores for receiving or retaining preferably a single vesicle in each case, the pores having a predetermined size and being arranged at predetermined distances from one another, ie at predetermined positions.
  • the pores can be arranged in the sieve element in desired positions, in particular in the form of a pattern or grid, which enables the vesicles to be arranged in precisely predetermined positions.
  • a grid or arrangement of the pores can in particular be matched to a desired arrangement of vesicles on a carrier or substrate such as, for example, a microcontact array.
  • the vesicles positioned and arranged in the individual pores can thus be arranged or deposited at predetermined positions, for example at the locations of electrodes on the substrate.
  • the individual pores have a predetermined cross-section, ie cross-sectional size.
  • the pores are preferably matched to the size of the vesicles, ie their diameter is preferably in the range from 1 to 500 ⁇ m.
  • the size or the cross section of the pores can determine that a maximum of one vesicle can be accommodated in one pore.
  • the size of the vesicles can be selected because, for example, the pore can be made so small that certain vesicles cannot penetrate it, ie are retained at the opening of the pore.
  • the individual pores are preferably at a distance from one another which corresponds approximately to twice the pore diameter.
  • the distance between the pores is preferably determined by the arrangement of the vesicles to be produced, ie the pores are spaced apart in the same way as the vesicles are to be positioned, for example, on a substrate.
  • the minimum distance is essentially determined by the mechanical stability of the screen element and can vary depending on the material used and the geometric shape of the screen element.
  • the sieve element also has the advantage that it can be removably positioned and fixed on a substrate on which the vesicles are to be arranged. In particular in the case of a micro contact array, it can therefore be based on a three-dimensional one Surface design of this is dispensed with, since the positioning of the vesicles is carried out solely by the sieve element.
  • the surface of the substrate or micro contact array can be made easy to clean.
  • the screen element can be removed and cleaned separately.
  • cleaning methods that are specifically tailored to the sieve element and, for example, a microcontact array can also be used, ie the sieve element can be cleaned, for example, by means of ultrasound, while the microcontact array can be cleaned by polishing.
  • the sieve element is preferably designed as a flat disk, preferably made of silicon or silicon oxide, and the pores are designed in the form of through holes.
  • the screen element or the disk preferably have a thickness of 100 ⁇ m to 600 ⁇ m and particularly preferably of approximately 500 ⁇ m.
  • Silicon or silicon oxide or silicon dioxide are particularly preferred materials because the pores or through holes can be produced in them by means of known photolithographic processes. Accordingly, other semiconductors can be used.
  • a method for producing the pores or through holes is known from WO 99/58746.
  • silicon or silicon oxide is particularly suitable since a sieve element made of this material is inert to electrolytic solutions, but is compatible with biological tissues. Furthermore, their surface properties can be compared with a wide range of reagents such as e.g.
  • Silanes can be defined temporarily or permanently.
  • silicon or silicon dioxide has the advantage that it is very easy to integrate electrically active structures which, for example, can take over sensor or actuator functions.
  • an actuator can be used to achieve a dielectrophoretically controlled entry of vesicles or particles into the pores.
  • the sieve element can also be made from other materials, such as glass, zirconium oxide, titanium oxide, boron oxide, aluminum oxide, ITO.
  • the material must be sufficiently stable and allow the formation of through holes with a high aspect ratio, ie ratio of length to diameter.
  • the material can preferably be structured photolithographically and with anisotropic etchable to form the pores.
  • the design of the pores as through holes has the advantage that when the vesicles, which are in a solution, are flushed in or introduced, the solution or the carrier fluid can flow out through the bores.
  • Through holes also allow individual vesicles, which are larger in cross section than the through holes, to be positioned on the surface of the sieve element in or on the individual holes. The individual vesicles can each be held at the entry opening of a pore or a through hole, in particular by negative pressure.
  • the through holes are further preferably cylindrical and preferably circular-cylindrical.
  • the pores or through holes are preferably adapted to the cross-sectional shape or outer shape of the vesicles to be positioned.
  • a round cross-sectional shape is particularly suitable for this, the diameter depending on the intended use being able to be matched to the vesicles to be positioned or manipulated.
  • the through holes have a cross-sectional area or a diameter which is slightly larger than the cross-section or diameter of the vesicles to be positioned. The size design ensures that the vesicles can enter the through holes and can move along them without adhering to the side walls of the holes.
  • vesicles size selection of the vesicles can take place, since larger vesicles which have a diameter which is larger than that of the through holes or pores cannot enter them. This effect is also used to position vesicles, which are larger than the pores or through holes, in their inlet opening and, if necessary, to fix them, for example by means of negative pressure.
  • the through holes preferably expand in a funnel shape to one side of the disk.
  • This funnel-shaped extension can be conical, for example.
  • the funnel shape creates a larger opening, which allows the vesicles to easily enter the through hole. An even easier automatic positioning in the pores or through holes can be achieved in this way.
  • this configuration is preferred if larger vesicles are to be fixed or positioned at the entry opening of the through holes. The vesicles can then enter the funnel-shaped part and are positioned and held in it.
  • the funnel-shaped part is advantageously dimensioned so that the depth of the funnel is greater than the vesicle diameter. As a result, the vesicle can be kept in the transport fluid, in particular water, without it coming into contact with air.
  • the largest diameter of the funnel must not be so large that the surface of the fluid or water bulges inwards in such a concave manner that the vesicle comes into contact with the surface. This depends on the ratio of the depth to the diameter of the funnel.
  • channels are formed on one side of the screen element between adjacent pores, which connect these to one another.
  • the channels preferably extend essentially transversely to the individual pores or through holes.
  • these channels have the advantage that, when the sieve element is placed on a substrate, they can serve to discharge a solution or a carrier fluid with which the vesicles are rinsed into the individual pores.
  • the screen element is arranged so that the side of the screen element in which the channels are formed comes to rest on the top of the substrate.
  • the vesicles then enter the pores with the solution or the fluid from the opposite side.
  • the solution flows through the pores and through the channels to the outside of the sieve element.
  • the channels are preferably formed so that they are smaller than the vesicles to be manipulated so that they are retained in the pores. Furthermore, the channels can be used to build up neuronal structures between the vesicles, which are arranged in individual pores, since they enable neuronal growth between the vesicles or cells, in particular nerve cells. The neural growth is guided or directed through the channels. This is particularly advantageous because the geometric relationships in neural networks are an important factor for neural information processing.
  • the parts of the sieve element which come into contact with vesicles, and in particular the pores are expediently designed to be passive, ie preferably with surface-active substances, so that they have no attachment points for vesicles.
  • the vesicles adhere to the sieve element, which is only intended to serve as a positioning or manipulation aid.
  • This is particularly advantageous if the vesicles are to be held on the pores only by, for example, negative pressure and are subsequently to be deposited on a substrate such as, for example, a microcontact array.
  • Such surface properties can be achieved in particular by appropriate coatings.
  • the hydrophilicity of the sieve element surfaces must be adapted to the desired uses in order to ensure their function and to prevent vesicles from adhering. This can be done, for example, by coating with silanes.
  • a surface modification of the side of the screen element may be desired, which should be brought into contact with a substrate such as a micro contact array.
  • the surface can be adjusted so that the screen element can be held or fixed by adhesion to a substrate such as a micro contact array. This prevents accidental displacement of the screen element after it has been placed on a substrate.
  • a substrate such as a micro contact array.
  • the adhesion via the substrate or micro contact array can preferably also be adjusted electrically.
  • Optical and / or mechanical positioning aids are further preferably formed on the screen element. These serve to place the sieve element with a predetermined pore grid precisely positioned on a support or substrate in order to ensure that the vesicles which are arranged on or in the pores are positioned exactly at the desired positions on the substrate.
  • these positions at which the vesicles are to be positioned are electrodes or micro elements.
  • the sieve element should be positioned on the micro-contact array in such a way that the individual micro-elements or electrodes are each exactly aligned with an associated pore, so that the vesicles are exactly centered on the Electrodes can be arranged.
  • An easy positioning of the sieve element on the substrate can be achieved, for example, by arranging the microelements in a grid, preferably at the edge of a micro contact array, the grid width of which deviates slightly from the grid width of the pores on the sieve element.
  • additional patterns or screens can be applied to the screen element and the substrate, which differ slightly in terms of their screen width. In this way, when the screen element is placed on the microcontact array, a moire effect is created by the raster with different raster width, which allows the screen element to be precisely aligned without further optical aids.
  • a purely mechanical positioning aid in the form of pins and recesses can be provided.
  • the recesses can advantageously be formed in the screen element and this can then be used as a molding tool for forming the pins on the substrate, for example in a not yet cured polymer.
  • Such a procedure corresponds essentially to micro-contact printing ( ⁇ CP).
  • ⁇ CP micro-contact printing
  • the sieve element can be attached to a pipette. It is arranged in such a way that a solution with the vesicles is sucked from the pipette through the sieve element, ie the through holes or pores formed therein. Depending on the cross section of the pores, only vesicles of a predetermined maximum size can enter the pipette, larger vesicles are retained on the sieve element at the openings of the pores. They can be held in position at the openings of the pores in order to transport them or to deposit them in a specific pattern or grid according to the arrangement of the pores.
  • the openings of the pores are preferably funnel-shaped in such a way that the vesicles are completely absorbed in the funnels and can be held there in the solution or in water without coming into contact with air. There is thus a closed transport of the vesicles Water poles possible. Furthermore, the sieve element enables the size of the vesicles to be selected.
  • the device described above is preferably used to manipulate vesicles, in particular to position, select, transport and / or fix them.
  • a use is particularly preferred in which the arrangement of the pores of the sieve element corresponds to a grid of microelements of a microcontact array and the sieve element is used to position individual vesicles on the microelements.
  • a micro-contact array is to be understood as a two-dimensional, field-like arrangement of micro-elements which come into contact with or interact with vesicles.
  • the microelements are, for example, electrodes on which individual vesicles are to be arranged.
  • the electrodes should preferably produce non-invasive contacts, for example in order to derive electrical potentials or to measure the pH. However, the electrodes can also cause invasive contacts, whereby a better contact is created, but the service life is shortened.
  • the screen element has a pore or hole pattern, which corresponds to the arrangement of the electrodes.
  • a solution preferably water
  • the vesicles can easily be introduced into the pores or arranged on or in these. This arrangement then corresponds exactly to the desired arrangement or the desired grid of the microelements. In this way it is possible to arrange a large number of vesicles very easily and quickly in a desired pattern or grid.
  • the method according to the invention preferably uses a device as described above.
  • the vesicles to be positioned or manipulated are in a solution and are applied together with this to a sieve element.
  • Individual pores for accommodating preferably individual vesicles are formed in the sieve element.
  • the vesicles are introduced or flushed into the pores by means of the solution and thus arranged or fixed in them. Since the individual pores have a predetermined
  • the size and position of the sieve element are the vesicles. arranged through the pores in predetermined positions. In this way it is possible to arrange a large number of vesicles in predetermined positions very quickly.
  • the vesicles no longer have to be arranged individually at predetermined positions using appropriate aids. By applying them in a solution, they are deposited essentially automatically in the individual pores and are thus arranged at predetermined positions.
  • the pores are preferably designed as through holes and each have a cross section which corresponds to the size of the vesicles to be manipulated or positioned. This ensures that the vesicles of a certain size, which are to be manipulated or positioned, can enter the through holes and are thereby fixed in them and thus arranged at a defined position.
  • the cross section or the size of the through holes can preferably be slightly larger than the vesicles, so that the vesicles have little play in the holes and can move in their longitudinal direction without getting stuck or jamming.
  • the pores of the sieve element are arranged so that the arrangement of the pores corresponds to a desired arrangement of attachment points of the vesicles on a substrate, the sieve is positioned on the substrate such that the pores are aligned with the desired attachment points a solution with the vesicles applied to the sieve element.
  • the attachment points on the substrate are, for example, individual electrodes or micro-elements which are arranged in a predetermined grid and are to be contacted with individual vesicles. Because the pores in the sieve element are arranged in a grid corresponding to the attachment points, the individual vesicles are positioned in such a way that they each come to rest on an attachment point or a microelement.
  • the screen element is preferably placed flat on the substrate, so that one pore is centered above each attachment point. Then the solution with the vesicles is applied to the sieve element, the vesicles penetrate into the pores and thus come to rest on the individual attachment points.
  • the size of the pores is preferably chosen so that the pores are only slightly larger than the individual vesicles. In this way it can be ensured that only one vesicle comes to lie exactly on each attachment point.
  • the substrate is a micro contact array and the attachment points are micro elements according to which the vesicles are positioned.
  • the vesicles are positioned precisely on the micro elements, for example electrodes, in order to ensure a uniform strength of the signal transmission between vesicle and electrode.
  • the strength of the signal transmission decreases with the covering of an electrode or a sensor by the vesicle. For example, vesicles lying next to a sensor or an electrode cannot be detected or contacted.
  • the sieve element is preferably detachably fixed to the substrate by adhesion. This enables the sieve element to be fixed very easily to the substrate, and an exact positioning of the sieve element can be ensured and maintained so that the pores lie precisely at predetermined positions above the substrate. It is expedient that the screen element can be separated from the substrate again, since screen element and substrate can then be cleaned independently of one another. On the one hand, this enables the substrate and screen element to be cleaned more easily and, moreover, enables a suitable cleaning process to be used for each element.
  • channels formed on the screen element between the pores are brought into contact with the substrate and the solution is discharged through the channels.
  • the channels which are formed on one side of the screen element, lie on the surface of the substrate. This makes it possible for the solution in which the vesicles are located and which enters the pores from the top of the sieve element at the lower end of the pores or through holes through the channels dissipate. In this way it is achieved that the vesicles, which enter the pores at the upper end, sink very quickly into the pores and come to rest on the substrate surface.
  • the channels are preferably made smaller than the vesicles so that the vesicles cannot pass through the channels and are consequently retained in the pores.
  • nerve cells can be arranged as vesicles in the pores, which form a neuronal growth in the channels which are arranged between the pores in the sieve element.
  • the channels are preferably formed on one side of the screen element in the form of grooves or grooves and connect the individual pores to one another in a grid-like manner. In this way, a directed and geometrically defined neuronal growth can take place in order to build up a neural network.
  • the sieve element with the channels can preferably be arranged on the surface of a substrate, for example a silicon chip, so that a geometrically predetermined neural network is formed on the silicon chip.
  • the pores have a size which is smaller than the size of the vesicles to be manipulated or positioned.
  • the sieve element is suitable for selecting vesicles of a certain size, in particular if vesicles of different sizes are in the same solution.
  • vesicles that are larger than the pores can be positioned and held on the surface of the sieve element in the inlet openings of the sieve element.
  • the sieve element thus forms a transport sieve for the transport of vesicles.
  • a single layer of vesicles, i.e. one vesicle can be transported per pore.
  • a defined arrangement of the vesicles is ensured, for example in order to place them on a substrate in the defined arrangement.
  • the sieve element can be arranged on a pipette and then a solution with vesicles can be taken up by the pipette, vesicles with a predetermined minimum size being fixed to the pores of the sieve element. Smaller vesicles, the diameter of which is smaller than the size of the pores, will enter the pipette through the pores. Vesicles with a larger cross-section or diameter stick to the entry openings of the pores or through holes and are thus positioned on the pores. As long as suction is maintained via the pipette, the vesicles are further held on the pores so that they can be easily transported and handled with the pipette and the sieve element attached to it.
  • the vesicles maintain a predetermined arrangement or positioning according to the arrangement of the pores in the sieve element. This makes it possible to transport a single layer of vesicles, which also have precisely defined positions.
  • the end regions of the through holes are funnel-shaped, so that the vesicles are completely absorbed into the larger funnel-shaped region before they are retained at the beginning or entrance of the narrower through hole.
  • the depth of the funnel area is selected so that the vesicle is constantly covered by the solution or water even during transport.
  • the vesicles can thus be transported in a closed liquid phase, such as a water phase, between two non-contiguous liquids, for example water in two separate vessels, without having to pass through the interface between air and the liquid.
  • the vesicles do not come into contact with air when they are transported.
  • the pores are preferably arranged in a predetermined grid, which corresponds to an arrangement of pores on a second sieve element, the pores in the second sieve element being larger than the pores in the first sieve element.
  • the second sieve element can be, for example, a sieve element which is arranged in the manner described above on a substrate such as a micro contact array in order to position individual vesicles on electrodes or micro elements. Because the grid on the first screen element corresponds to the grid of the pores in the second screen element, the two screen elements can be positioned one above the other in such a way that the individual pores on the first screen element are each arranged above a pore on the second screen element.
  • vesicles which are held in position on the first sieve element, can be deposited exactly at predetermined positions above the second sieve element, so that they enter the Pores enter the second screen element. Further, a maximum diameter at the second sieve can by setting 'be achieved that only vesicles of a certain maximum size enter the pores in the second screen element. The result is a method which represents a bandpass for the vesicle size. In this way, vesicles can be extracted from a heterogeneous dispersion, the diameter of which lies within freely adjustable limits which are predetermined by the pore sizes in the first and second sieve elements.
  • the pores are preferably designed as through holes, one end of which widens conically and which have a cross section which is smaller than the vesicles to be positioned, and the vesicles are positioned in the region of the conical widening and are preferably fixed to the through holes by negative pressure.
  • the vesicles enter the conical enlargement of each through hole and are positioned and held in this. Due to their larger size, they cannot enter the through hole any further.
  • the vesicles can be held in this position by negative pressure which is generated in the through holes.
  • Such a method is particularly suitable for the simultaneous handling and transport of a large number of vesicles.
  • the vesicles can be held in a fixed, defined position, e.g.
  • an entire field of egg cells are fertilized by an automated injection cannula.
  • the egg cells are fixed mechanically, preferably by negative pressure, to the sieve element, so that position evaluation using complex image recognition can be omitted. Similar applications arise in the field of molecular biology, where cellular injections are also used.
  • FIG. 1 shows a schematic cross-sectional view of the device according to the invention when used as a positioning screen
  • FIG. 2 shows a schematic top view of the application shown in FIG. 1
  • 3 shows a schematic cross-sectional view of a positioning screen with channels
  • FIG. 4 shows a schematic top view of the application shown in FIG. 3
  • FIG. 5 shows a schematic cross-sectional view of a positioning screen with channels for neuronal growth
  • FIG. 6 shows a schematic top view of the 5 the application shown
  • FIG. 7 shows a schematic cross-sectional view of the use as a transport sieve
  • FIG. 8 schematically shows the deposition of vesicles with a transport sieve according to FIG. 7,
  • FIG. 9 shows a detailed view of a transport screen
  • FIG. 10 shows a schematic cross-sectional view of an application as a fixing screen.
  • Fig. 1 shows schematically the use of the device according to the invention as a positioning screen.
  • a substrate 2 is provided in the form of a micro contact array, on which vesicles are positioned.
  • micro-elements 4 in the form of sensors or electrodes, for example, are arranged at regular intervals on a surface.
  • a sieve or sieve element 6 is arranged on the microcontact array 2.
  • the screen element 6 forms a positioning screen.
  • the screen element 6 is preferably formed from a silicon wafer or a silicon wafer in which pores or through holes 8 are formed. These through holes 8 are preferably circular cylindrical and extend substantially perpendicular to the surface of the screen element 6.
  • a method for forming such through holes 8 in a silicon wafer is described in WO 99/58746.
  • the through holes 8 are arranged in the screen element 6 at a distance from one another such that a grid is formed which corresponds to the arrangement or the grid of the micro elements 4 on the substrate 2.
  • the sieve element 6 is placed on the surface of the microcontact array 2 and is preferably held thereon by adhesion or fixed in another suitable manner.
  • the sieve element 6 is positioned such that each of the through holes 8 is arranged exactly above a microelement 4.
  • the diameter of the through holes 8 is selected such that it is the same or preferably slightly larger than the diameter of a vesicle 10.
  • the vesicle 10 In this way, it is possible for the vesicle 10 to enter the through holes 8 from the top and enter them to move to the underside of the screen element 6, so that they come to lie exactly positioned on the micro elements 4 of the micro contact array 2.
  • the vesicles 10, for example cells or nerve cells, are in a fluid or a solution, preferably water, with which they are applied to the sieve element 6.
  • the vesicles 10 enter the through holes 8 or are flushed into them.
  • the fact that the through holes 8 are only slightly larger than the vesicles 10 ensures that only one vesicle 10 comes to rest on a microelement 4.
  • Fig. 2 shows a top view of the corresponding arrangement. It can be seen that the through holes 8 in the screen element 6 lie precisely aligned above the micro elements 4 of the substrate 'or micro contact array 2. Vesicles 10 have already entered some of the holes or through-holes 8 in the sieve element 6 in FIG. 2, while other through-holes 8 are shown in such a way that no vesicles 10 have entered and the micro-elements 4 are visible.
  • FIG. 3 shows an alternative embodiment of the arrangement shown in FIG. 3.
  • the arrangement essentially corresponds to the arrangement shown in FIG. 1 with the difference that channels 12 are additionally formed in the sieve element 6, which connect the through bores 8 to one another.
  • the channels 12 extend as long channels over the entire length of the screen element 6 and are each connected to the adjacent through holes 8 via short, essentially transverse connecting channels 13.
  • the channels 12 open to the side of the screen element 6.
  • the channels 12, 13 are formed in the surface of the screen element 6 as grooves or channels. When the surface with the grooves is placed on the substrate 2, closed channels 12, 13 are formed between the sieve element 6 and the substrate 2 through the grooves together with the substrate surface.
  • the solution with the vesicles 10 When the solution with the vesicles 10 is supplied, such a flow can be generated in the through holes 8 that the solution enters the through holes 8 from the top and then through the lower opening of the through holes 8 into the connecting channels 13 and the channels 12 flows and is discharged through them to the outside.
  • the vesicles 10 in the solution are flushed into the through holes 8, and they quickly sink onto the microelements 4, since the solution is discharged through the channels 12, 13.
  • the channels 12 and in particular the connecting channels 13 have a cross section which is smaller than that of the through holes 8 and the vesicles 10, so that the vesicles 10 are retained in the through holes 8 on the microelements 4.
  • FIGS. 5 and 6 show in cross section and in plan view a further modification of the anomalous element shown in FIGS. 1 and 2.
  • the arrangement essentially corresponds to the arrangement shown in FIGS. 1 and 2, with the difference that additional channels 14 are arranged on the side of the sieve element 6 which bears on the surface of the substrate or micro contact array 2.
  • the channels 14 are arranged in such a way that they form a cross-shaped or checkerboard-like pattern, the through-bores 8 each lying at the crossing points of the channels 14.
  • the channels 14 thus always connect adjacent holes or pores 8 to one another.
  • the channels 14 are formed as grooves or grooves in the surface of the screen element 6. Together with the substrate surface, these grooves form the closed channels 14.
  • Such a grid of channels 14, which are formed between the sieve element 6 and the substrate 2, enables guidance for a directed neuronal growth of vesicles, in particular nerve cells 10, which are in the through holes 8 are arranged on the micro elements 4.
  • the positioning of the nerve cells 10 takes place via the through holes 8 in the sieve element 6.
  • a directed neuronal growth can develop in the channels 14.
  • a directional neural network can be formed on a substrate 2, for example in the form of a silicon chip.
  • the directional growth in given forms is advantageous because the geometric relationships in neural networks are an important factor for neuronal information processing.
  • Fig. 7 shows the use of the device according to the invention as a transport screen.
  • a sieve element 16 which corresponds in principle to the sieve element 6 explained above, is arranged at the end of a pipette 18.
  • a fluid or a solution and vesicles 10 located therein can be sucked in through the through holes 8 in the sieve element 16.
  • vesicles 10a which are smaller in size than the through holes 8, are sucked through them into the pipette 18.
  • Larger vesicles 10b which have a size that is larger than the diameter of the through holes 8, remain attached to the entry openings of the through holes 8 and are positioned and fixed thereon. This fixation takes place only by the suction generated by the pipette in the through holes 8.
  • the vesicles 10b are released again from the sieve element 16 and can be placed exactly in position.
  • the through holes 8 in the sieve element 16 are designed in such a way that their entry regions 20 are funnel-shaped or narrow conically in the direction of flow. This enables the vesicles 10a to enter the through holes 8 more easily. Furthermore, the larger vesicles 10b can be better held and positioned in the funnel-shaped or conical entry areas of the through holes 8, since they can enter this enlarged area of the holes 8.
  • FIG. 7 While the suctioning or picking up of the vesicles 10a, 10b by means of a pipette 18 is shown schematically in FIG. 7, the deposition of the vesicles 10b with the pipette 18 is shown in FIG. The suction in the pipette 18 is released or vice versa, so that a pressure is generated which pushes the solution in the pipette out through the through holes 8 in the sieve element 16. The vesicles 10b are thereby detached from the transport sieve or sieve element 16.
  • the sieve element 16 is preferably designed in such a way that it has a surface that is as non-adherent as possible, so that the vesicles 10b are held in the through holes 8 only by a negative pressure and do not adhere to the sieve element 16.
  • a second sieve element 6 is arranged on a substrate 2. This second screen element 6 corresponds essentially to the positioning screen explained with reference to FIGS. 1 to 4, which is why a detailed description is given here is waived.
  • 6 channels are shown in the sieve element, which can correspond to the channels 12 and 14 in FIGS. 3 and 5. Alternatively, however, an arrangement according to FIG. 1, ie without channels, can also be used.
  • the entry areas 24 of the through holes 8 in the sieve element 6 are also funnel-shaped, ie they widen towards the outside of the sieve element 6 in order to allow the vesicles 10b to enter more easily.
  • the vesicles 10b can still be positioned precisely through the sieve element 6 if the sieve element 16 with the pipette is not positioned exactly above the sieve element 6, so that the through holes 8 in the sieve element 16 are slightly offset from the through holes 8 in the Sieve element 6 are.
  • FIG. 9 shows a detailed view of a transport screen corresponding to FIG. 7.
  • a pore 8 is shown in the transport screen 16.
  • the funnel-shaped entry area 20 of the pore 8 is made larger.
  • the entry area 20 is so large that a vesicle 10b can be completely accommodated therein.
  • the pore or the through hole 8 has a cross section which is smaller than the cross section of the vesicle 10b.
  • the funnel-shaped entry region 20 of the pore is so large that the vesicle 10b, when it is held on the through hole 8, is covered with solution, preferably water 25.
  • the vesicle 12b is thus in a closed water phase. This makes it possible to transport the vesicle 12b through the large area of solution or water and air, for example from one container to another, since the vesicle can be prevented from coming into contact with air. It is important to adjust the ratio of the diameter to the depth of the funnel-shaped entry area 20 so that the surface of the solution or the water 25 does not bulge inwards so much concave that it comes into contact with the vesicle 12b.
  • This configuration of the through holes is preferably used in a transport screen 16 according to FIG.
  • a sieve element 26 is used as a fixing sieve for positioning and for fixing individual vesicles 10, such as cells.
  • the screen element or fixing screen 26 has a plurality of through holes 28 which are arranged at regular intervals in the form of a grid.
  • the upper opening areas of the through holes 18 are preferably flared, so that they form a funnel-shaped entry area.
  • the through holes 28 have a smaller diameter than the vesicles 10 to be positioned and fixed.
  • the vesicles 10 are preferably applied to the fixing screen 26 in a fluid or a solution, so that they are distributed in the individual through bores 28.
  • a vesicle 10 is preferably arranged in each through-hole 28 in such a way that it comes to lie in the funnel-shaped entry region of a through-hole 28 but cannot enter the through-hole 28 any further.
  • the vesicles 10 can then be fixed on the fixing screen 26, for example by negative pressure, which is generated in the through holes 28.
  • the vesicles 10 are positioned precisely because they lie in the entry areas of the through holes 28, which are arranged in precisely defined positions in the fixing screen 26.
  • the positioned vesicles 10 can be processed or manipulated, for example by means of a microelectrode 30 for injection. Because the position of the vesicles 10 via the through holes 28 is precisely predetermined, position detection of the vesicles 10 for controlling a micromanipulator or a microelectrode can be dispensed with, since only the center of the holes of the through holes 28 can serve as their target coordinates. Such an arrangement can be used in particular in the area of intracellular mass injection. A single arrangement and positioning or fixation of the vesicles 10 can be dispensed with, since a large number of vesicles 10 can very easily be arranged in predetermined positions by the device or the method according to the invention.
  • the sieve element according to the invention makes it very easy to arrange a very large number of vesicles at predetermined positions.
  • the vesicles are simply applied to the sieve element in a solution. They arrange themselves automatically or automatically in the pores in the sieve element, which define defined positions for the vesicles.

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Abstract

L'invention concerne un procédé pour manipuler, en particulier positionner, sélectionner, transporter et/ou fixer des vésicules (10) au moyen d'un élément tamis (6 ; 16 ; 26) comportant une pluralité de pores (8 ; 28) pour recevoir respectivement de préférence une vésicule individuelle (10), ces pores (8 ; 28) présentant une taille déterminée et étant disposés à une certaine distance les uns des autres. L'invention concerne en outre un procédé de manipulation et de positionnement de vésicules (10), selon lequel les vésicules (10) en solution sont placées sur un élément tamis (6 ; 16 ; 26) dans lequel sont formés des pores (8 ; 28) pour recevoir de préférence des vésicules individuelles (10), les différents pores (8 ; 28) présentant une taille déterminée et une position déterminée dans l'élément tamis (6 ; 16 ; 26), et les vésicules (10) sont déposées dans les pores (8 ; 28).
PCT/EP2002/006218 2001-06-13 2002-06-06 Dispositif et procede pour manipuler des vesicules WO2002101001A1 (fr)

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DE2001128574 DE10128574A1 (de) 2001-06-13 2001-06-13 Vorrichtung und Verfahren zur Manipulation von Vesikeln
DE10128574.4 2001-06-13

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1998022819A1 (fr) * 1996-11-16 1998-05-28 Nmi Naturwissenschaftliches Und Medizinisches Institut An Der Universität Tübingen In Reutlingen Stiftung Bürgerlichen Rechts Systeme de microelements, procede de mise en contact de cellules situees dans un environnement liquide et procede permettant de realiser un systeme de microelements
WO1999055826A1 (fr) * 1998-04-24 1999-11-04 Genova Pharmaceuticals Corporation Dispositif de micro-compartimentalisation et ses utilisations
WO2001059447A1 (fr) * 2000-02-11 2001-08-16 Yale University Electrodes patch-clamp planes
WO2002024862A2 (fr) * 2000-09-19 2002-03-28 Cytion S.A. Systeme de positionnement et d'analyse d'echantillons

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DE2722586C3 (de) * 1976-05-31 1979-10-04 Olympus Optical Co., Ltd., Tokio Pipette für eine Kulturflüssigkeit für Gewebe- und Zellkulturen
US5310674A (en) * 1982-05-10 1994-05-10 Bar-Ilan University Apertured cell carrier
JP2662215B2 (ja) * 1986-11-19 1997-10-08 株式会社日立製作所 細胞保持装置
DE19528662C2 (de) * 1995-08-04 2003-04-10 Nmi Univ Tuebingen Interphase-Kultur auf Multifunktionsarray (MFA)
DE19712309A1 (de) * 1996-11-16 1998-05-20 Nmi Univ Tuebingen Mikroelementenanordnung, Verfahren zum Kontaktieren von in einer flüssigen Umgebung befindlichen Zellen und Verfahren zum Herstellen einer Mikroelementenanordnung
US6048457A (en) * 1997-02-26 2000-04-11 Millipore Corporation Cast membrane structures for sample preparation
GB9925904D0 (en) * 1999-11-03 1999-12-29 Univ Belfast Cell migration and chemotaxis chamber

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022819A1 (fr) * 1996-11-16 1998-05-28 Nmi Naturwissenschaftliches Und Medizinisches Institut An Der Universität Tübingen In Reutlingen Stiftung Bürgerlichen Rechts Systeme de microelements, procede de mise en contact de cellules situees dans un environnement liquide et procede permettant de realiser un systeme de microelements
WO1999055826A1 (fr) * 1998-04-24 1999-11-04 Genova Pharmaceuticals Corporation Dispositif de micro-compartimentalisation et ses utilisations
WO2001059447A1 (fr) * 2000-02-11 2001-08-16 Yale University Electrodes patch-clamp planes
WO2002024862A2 (fr) * 2000-09-19 2002-03-28 Cytion S.A. Systeme de positionnement et d'analyse d'echantillons

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