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WO2007138568A2 - Dispositif pour expériences sous microscope sur des cellules vivantes non adhérentes avec renouvellement continu - Google Patents

Dispositif pour expériences sous microscope sur des cellules vivantes non adhérentes avec renouvellement continu Download PDF

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Publication number
WO2007138568A2
WO2007138568A2 PCT/IL2007/000570 IL2007000570W WO2007138568A2 WO 2007138568 A2 WO2007138568 A2 WO 2007138568A2 IL 2007000570 W IL2007000570 W IL 2007000570W WO 2007138568 A2 WO2007138568 A2 WO 2007138568A2
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WO
WIPO (PCT)
Prior art keywords
cells
interest
microscopic device
supporting member
flow
Prior art date
Application number
PCT/IL2007/000570
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English (en)
Other versions
WO2007138568A3 (fr
Inventor
Cher Ashtamker
Robert Fluhr
Original Assignee
Yeda Research And Development Co. Ltd.
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 Yeda Research And Development Co. Ltd. filed Critical Yeda Research And Development Co. Ltd.
Publication of WO2007138568A2 publication Critical patent/WO2007138568A2/fr
Publication of WO2007138568A3 publication Critical patent/WO2007138568A3/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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • 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
    • B01L2300/0822—Slides
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Definitions

  • the present invention relates to slides used in microscopic applications and particularly to those used for examining specimens containing cells or other particles of interest suspended in a liquid medium, in which the medium is exchanged or so- called flow-through experiments.
  • the invention also relates to methods of malting such devices, and further, to a method of using such devices in flow-through experiments.
  • Optical tweezers use the electromagnetic force of light in order to create "light traps" for particles and cells in liquids [1, 2]. This method was successfully used for cell sorting and for studying other cellular biological properties [3, 4]. Some modified methods use special materials that can switch their physical state from solution to gel, and vice versa, when they are exposed to certain light [5] or temperature [6], and allow entrapment of individual cells on the gel surface. Agarose gel surfaces were also applied to embed live bacterial cells for imaging [7]. Other methods use different surface coatings in order to attach cells to the slide surface.
  • One example is the mussel adhesive protein [8]; other known adhesion substrates suitable for a large variety of cell types are collagen and poly-Lysine.
  • a microscopic device for examining specimens containing cells or other particles of interest suspended in a liquid medium, in flow-through experiments.
  • the new device comprising: a supporting member; and a coverslip to be applied over the supporting member; characterized in that the device further comprises an inert open mesh to be located between the supporting member and the coverslip for receiving the specimen of interest; the open mesh being constructed and dimensioned to include a plurality of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving the specimen and effective in restricting the movement of cells or particles of interest therein, without unduly affecting the viability of the cells or disturbing their surrounding medium.
  • the device further comprises the specimen containing cells or other particles of interest on the supporting member.
  • a microscopic device for examining specimens containing cells or other particles of interest suspended in a liquid medium, in flow-through experiments.
  • the new device comprising: a supporting member; and a coverslip to be applied over the supporting member with a specimen of interest to be observed inbetween; characterized in that the device further comprises an inert open mesh to be located between the supporting member and the coverslip for receiving the specimen of interest; the open mesh being constructed and dimensioned to include a plurality of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving the specimen and effective in restricting the movement of cells or particles of interest therein, without unduly affecting the viability of the cells or disturbing their surrounding medium.
  • the small mesh openings of the open mesh define a two-dimensional array of "physical traps" (as distinguished from the "light traps” created by optical tweezers) for receiving the specimen.
  • the physical traps are interconnected by narrow passageways that while effective in imposing a restriction of cell movement, do not affect the cells viability nor confine the surrounding medium, facilitating free diffusion of chemicals and solutions used in flow-through experiments.
  • Such a device is particularly useful for examining live cells suspended in a liquid medium, such as non-adherent cells, sperm cells, blood cells, plant cells in suspension, embryonic cells, prokaryote cells and cancer cells, and is therefore described below with respect to this application, but it will be appreciated that such a device could be used for investigating cell aggregates, cell clusters such as e.g., pollen or other particles, such as microbeads, viral particles, subcellular structures, cell organelles e.g. nuclei, etc. in flow— through experiments.
  • a liquid medium such as non-adherent cells, sperm cells, blood cells, plant cells in suspension, embryonic cells, prokaryote cells and cancer cells
  • the open mesh includes interwoven filaments defining the two-dimensional array of interconnecting physical traps.
  • the filaments create an open cage of selected depth depending on the thickness of the filaments used.
  • the coverslip is attached to the mesh and the supporting member by adhesive applied along opposed edges of the coverslip, leaving opposite edges unbounded to permit applying the flow-through solution to one of the unbounded edges and to be drawn from the other side of the unbounded edges.
  • the coverslip is of rectangular configuration, having a pair of opposed longitudinally- extending edges attached to the open mesh and supporting member, and a pair of opposed transversely-extending edges unbounded to the open mesh and supporting member. It will be appreciated, however, that the coverslip could be of different configuration then described.
  • the two-dimensional array of interconnecting physical traps is in the form of rectangular, or other geometrical weave pattern matrix.
  • the supporting member is a slide plate. It will be appreciated, however, that the supporting member could be another coverslip.
  • the open mesh is defined by a fabric of mono or multi-type filaments.
  • the fabric includes mesh openings of up to 325 microns.
  • the fabric is made of monofilaments of 28-122 micron diameter.
  • the fabric includes mesh openings of 200-5,000 microns.
  • the fabric is made of monofilaments of 150-1,000 microns diameter. According to yet another described preferred embodiment, the monofilaments are of nylon.
  • the monofilaments are of polyester.
  • the fabric is a braid of said monofilaments.
  • the laboratory device is made by applying the open mesh to the coverslip; applying the specimen containing the cells or other particles of interest to the open mesh; applying the coverslip, with the open mesh and the specimen therein, to the supporting member; and then attaching two opposed edges of the coverslip to the supporting member (slide plate or another coverslip), leaving two other opposed edges unbounded for the application of the flow-through solution application.
  • the device is made by applying the open mesh to the supporting member; applying the specimen containing the cells or other particles of interest to the open mesh; applying the coverslip over the open mesh and the specimen therein on the supporting member; and then attaching two opposed edges of the coverslip to the supporting member, leaving two other opposed edges unbounded for the flow-through solution application.
  • a method of using the above-described device for flow-through experiments comprising: applying the flow-through solution to one of the unbounded edges, while applying an absorbing material to the other of the unbounded edges to draw the flow- through solution through the interconnected physical traps defined by the open mesh, while the cells of interest are trapped therein.
  • the absorbing material is an absorbent paper sheet.
  • Automatic regulated pumps can replace the mechanical procedure described for obtaining flow-through.
  • the flow— through experiments comprise real time analysis.
  • such a device provides a number of important advantages over the previously-described devices for examining non-adherent live cells or other particles of interest, in flow— through experiments.
  • Fig. 1 is an exploded view illustrating one form of the device constructed in accordance with the present invention
  • Fig. 2 is an enlarged fragmentary view illustrating the construction of the open mesh in the device of Fig. 1;
  • FIG. 3 illustrates one manner of using the device of Figs. 1-2 for performing a flow-through experiment in order to examine live non-adherent cells of interest
  • Fig. 4 is an exploded view of Figure 3 that schematically illustrates the mechanism of action involved in the flow-through experiment using the described device
  • Fig. 5 is a confocal laser scanning microscopy (CLSM) image, which illustrates the results of two flow-through experiments using live BY-2 cells and performed using such a device constructed according to Figure 3.
  • CLSM confocal laser scanning microscopy
  • Fig. 6 is a CLSM image which illustrates the results of a flow-through experiment using live cells and performed using such a device constructed according to Figure 3. The same cells were stained with two probes, AR and DCF, after the addition of HaO 2 .
  • Figs. 7a-e illustrate results of flow-through experiments using live cells and performed using such a device constructed according to Figure 3.
  • Figure 7a depicts a cell stained in a basal level, and 13 minutes following addition of an elicitor (cryptogein) and following H 2 O 2 production.
  • Figure 7b is a graph depicting level of emission with and without an inhibitor which reduces H 2 O 2 production.
  • Figure 7c depicts two cells, showing the periphery and nuclear region of the cells (top panel), the same cells stained with DCF in a basal state (middle panel) and following the addition of the elicitor (lower right panel) or H 2 O 2 (lower left panel).
  • Figure 7d depicts graphs which show the emission of the probes through time, in the cell periphery and nucleus, and following the addition of the elicitor (right panel) or H 2 O 2 (left panel).
  • Figure 7e is a graph depicting the maximal rate of signal acquisition as measured in the periphery or nucleus expressed as the time gap. Negative values were defined when the nuclear compartment reacted before the periphery compartment.
  • Figs. 9a-b are CLSM images which illustrate results of flow-through experiments using live cells and performed using such a device constructed according to Figure 3.
  • Figure 9a shows 3D reconstructions of BFA treated (lower panels) or untreated (control, upper panels) BY-2 cells stained with subcellular-specific stains. Cells were stained separately with the ER marker DiOC 5 (3) (right inserts) or DCF (left inserts).
  • Figure 9b depicts cells double-stained with DCF and the ER-specific marker DPX.
  • Upper insert shows non-treated cells showing DCF, DPX and merge images; Lower inserts, showing DCF, DPX and merge images were collected at 30 min (middle insert) and 60 min (lower insert) after BFA addition.
  • Scale bar 10 ⁇ m.
  • FIGs. 10a-e illustrate results of several flow-through experiments using live cells and cellular organelles and performed using such a device constructed according to Figure 3.
  • Figure 10a shows a confocal and transmission microscope image of the same purified nucleus triple stained with the DCF, the membrane specific dye FM A- 64 and with the DNA specific dye DAPI (green, red and blue colors, respectively).
  • Figure 10b is a CLSM image showing the same cell stained with DCF before and after the addition of 1 mM Ca 2+ .
  • Figure 10c shows confocal and transmission microscope images of the same isolated nuclei stained with DCF (upper panel) or AUR (lower panel) before and after the addition of Ca 2+ .
  • Figs. 1-2 illustrate a preferred embodiment of a device constructed in accordance with the present invention for examining cells of interest in flow-through experiments.
  • the illustrated device can be used to observe any particle of interest, is particularly useful for observing live, non-adherent cells when subjected to the flow-through solution in a manner wherein movement of the cells is restricted by the device without duly affecting the viability of the cells of interest, and allowing free diffusion of the flow-through solutions.
  • the specimen comprises cells (i.e., cellular specimen).
  • the cells may be prokaryotic or eukaryotic cells.
  • the cells are viable cells.
  • Prokaryote cells can be for example, plant or animal pathogen, an effect of which on a specific cell may be observed using the device of the present invention.
  • the cells may be directly isolated from an organism and subject to analysis " using the device of the present invention.
  • primary cultures or cell lines may be included in the specimen. Cells may be unaffected healthy cells or diseased cells such as cancer cells, pathogen infected cells and the like.
  • Cells of the present invention may be intact cells or subcellular particles or fragments isolated therefrom (homogeneous or heterogeneous preparations thereof). Examples include, but are not limited to membranes, nuclei, mitochondria and the like. Specimens of the present invention which comprise non-adherent cells are of specific interest in accordance with the present invention since changing of solutes and washing procedures require the separation of cells from medium, which is particularly difficult when cells are not adhered to a surface. Thus, examples of non- adherent cells which may be analyzed using the teachings of the present invention include, but are not limited to, sperm, blood, stem and plant cells (in suspension). Non adherent polyploidy cells, cell aggregates or clusters can additionally be observed using the device of the present invention. For example, pollen and developing first stage zygotes may be observed and analyzed according to the present invention.
  • the illustrated device includes a slide plate 2 and a coverslip 3 to be applied over the slide plate with a specimen of the cells of interest inbetween.
  • Slide plate 2 and coverslip 3 are of transparent materials, as in conventional constructions, to enable examination of the specimen by a microscope.
  • the illustrated device further include an open mesh 4 to be located between slide plate 2 and coverslip 3 for receiving the specimen to be examined.
  • open mesh 4 is constructed of a plurality of interwoven filaments 4a, 4b to produce a plurality of small mesh openings defining a two-dimensional array of physical traps for receiving the specimen to be examined.
  • each filament 4a, 4b alternate in contact with, and spaced from, the slide plate 2 and the coverslip 3, such that the spaces between the alternating contact points of the filaments defines narrow passageways interconnecting the physical traps 5 for receiving the specimen. This will be more clearly described below with respect to Figs. 2 and 4, showing the passageways at 6.
  • the narrowing of the passageways 6 interconnecting the physical traps 5 are effective in restricting movement of the cells of interest 8 without unduly affecting the viability of such cells of interest, or disturbing the flow-through medium used in the flow— through experiments.
  • Open mesh 4 is preferably a fabric of monofilaments formed in a braid so as to produce the two-dimensional array or matrix of mesh openings defining the interconnecting physical traps 5.
  • One example is a fabric of nylon monofilaments having a filament diameter of 28-122 microns, and mesh openings of 1-325 microns, supplied by A.D. Sinun, of Israel, under the trademark "Nitex”.
  • a second example is a fabric supplied by the same company under the same trademark but made of nylon monofilaments of 150-1,000 microns, and having mesh openings of 200-5,000 microns.
  • a third example is a fabric supplied by the same company under the trademark "Petex” and constituted of polyester monofilaments having a diameter of 32-1,000 microns and mesh openings of 1-5,000 microns. All the foregoing materials are inert and stable under heat and solvent conditions.
  • coverslip 3 is preferably of substantially rectangular configuration, having a pair of opposed longitudinally- extending edges 3a, 3b and a pair of opposed transversely-extending edges 3c, 3d.
  • Coverslip 3 is attached to the open mesh 4, and also to the slide plate 2, along the pair of opposed longitudinally-extending edges 3a, 3b; the pair of opposed transversely- extending edges 3c, 3d are unbounded.
  • the flow-through solution is applied to the unbounded transversely-extending edge 3 c and, after being drawn across the full length of the open mesh 4 between the coverslip 3 and slide plate 2, is removed from the unbounded transversely-extending edge 3d.
  • edges 3a, 3b of coverslip 3 is achieved by the use of an adhesive.
  • Any suitable adhesive may be used which does not release toxic substances to the specimen.
  • the diameter of the monofilaments in the open mesh 4, and the size of the mesh openings defining the physical traps 5, depend on the type of cells (or other particles) to be examined, e.g., fungi, bacteria, yeast, blood cells, microbeads, nuclei, etc.
  • Other compatible materials may be used for open mesh 4, preferably having non- fluorescent properties, such as polydimethylsaline (PDMS), etc.
  • PDMS polydimethylsaline
  • 1-2 may thus be made by applying the open mesh 4 either to the coverslip 3 or the slide plate 2; applying the specimen to the physical trap 5 in the open mesh 4; and applying the coverslip 3 to the slide plate 2 with the open mesh 4, containing the specimen, inbetween.
  • the coverslip 3 may then be attached to the slide plate 2, with the specimen-containing open mesh 4 inbetween, along the two longitudinally-transversely-extending edges 3a, 3b of the coverslip, leaving the two transversely-extending edges 3c, 3d unbounded.
  • open mesh 4 is applied to the coverslip 3 it will be appreciated that the open mesh 4 may be applied to the slide plate 2 instead.
  • Fig. 3 illustrates a preferred method of using the above-described laboratory device for performing flow-through experiments
  • Fig. 4 schematically illustrates the manner in which the interconnecting physical traps produced by the open mesh 4 are effective to restrict the movement of the cells of interest 8 in the specimen without unduly affecting the viability of the cells of, or unduly disturbing the progress of the flow-through solution.
  • the flow-through solution used in the experiments is applied from an applicator 10 to the slide plate 2 along the unbounded transversely- extending edge 3 c of the coverslip 3, while a liquid-absorbing material 11, such as an absorbent paper sheet, is applied to the opposite unbounded edge 3d of the coverslip.
  • the absorbent material thus draws the liquid through the full length of the open mesh 4.
  • the physical traps 5 defined by the filaments 4a, 4b of the open mesh restrict movement of the cells of interest within the specimen previously applied to the open mesh.
  • the interconnecting passageways 6 defined by the alternating contact points of the filaments 4a, 4b of the open mesh with the slide plate 2 and the coverslip 3 permit the flow-through solution to flow the complete length of the open mesh as shown in Fig. 4, such that the cells of interest can be observed while the flow-through solution is substantially undisturbed.
  • the articles provided herein may be used for any cellular or subcellular visual detection assays of interest, preferably in real time analyses such as for assaying cellular responses to different agents, cellular manipulation (e.g., genetic modification, cellular treatment with exogenous factors) which may be useful for drug screening, personalized medicine and research.
  • cellular manipulation e.g., genetic modification, cellular treatment with exogenous factors
  • a device generated according to the teachings of the present invention has been successfully used to follow changes in hydrogen peroxide (H 2 O 2 ) production in tobacco BY-2 suspension cells, in response to different elicitors. It also allowed a simpler and quicker execution of all the preparation procedures, such as probe incubation and washing steps, using minimal reaction volumes on slides. Materials and Experimental procedures
  • BFA brefeldin A
  • Figure 5 demonstrates the change in fluorescent emission of H 2 O 2 probes, DCF and AUR, before (control) and after W7 application.
  • Cells maintained their spatial position using the device of the present invention, allowing simple monitoring of signals changes through time.
  • Figure 6 shows complete co-localization of two fluorescent probes, AR and DCF, following the addition of H 2 O 2 . The same cell could be stained with different probes and held such that cellular organelles can clearly be defined. .
  • Figures 7a-e further demonstrate using the device of the present invention for monitoring H 2 O 2 elicitation and production in subcellular compartments.
  • Figures 7a-b show monitoring of stained cells in basal level and 13 minutes after addition of cryptogein.
  • Figures 7c-d show the kinetics of H 2 O 2 accumulation in the cellular periphery and nuclear regions. Quantitative analysis of five independent experiments showed an average of 2.6 ⁇ 0.3 second delay between the signal measured at the periphery, that occurred first, and the signal measured at the nuclear region, that followed ( Figure 7e).
  • Figures 8a-c show double staining of specific cells with DCF and specific markers for different subcellular compartments. All staining, washing, reagent supplementation, and monitoring procedures were done directly on the device.
  • Figure 9 shows cells examined after the application of BFA, which modifies intracellular protein traffic from the ER to the Golgi apparatus. Results show that BFA influenced the DCF signal, therefore indicating that induction OfH 2 O 2 with DCF is associated with the ER. In this case, signaling was monitored for time frames of 30 to 60 minutes, indicating that the device can be used for following single immobilized cells in real time for periods of at least 1 hour.
  • Figures 10a-e show the use of the device for monitoring real time processes in the nucleus.
  • Figure 10a shows a fluorescent emission and transmission microscope images of the same purified nucleus triple stained with DCF, the membrane specific dye FM 4-64, and with the DNA specific dye DAPI (green, red and blue colors, respectively).
  • DCF was shown to stain subnuclear components, and a complex of unstained substructures within and around the nucleolus could clearly be detected and documented, using the device of the present invention.
  • Figure 10b shows the results of addition of 1 mM Ca 2+ , which was found to induce the signal in B Y-2 cells. The generation of H 2 O 2 in response to calcium addition, suggests that plant nuclei are capable of generating their own calcium currents.
  • Figure 10c shows a nucleolus localized reaction stained with DCF (upper panel) or AUR (lower panel).
  • Figure 1Od shows that the rate of the H 2 O 2 -generated signal, as measured by DCF, correlated with Ca 2+ concentrations but not with the addition of the electron donor NADPH.
  • Figure 1Oe shows that the signal was partially inhibited by DPI (60 % inhibition) and by exogenous catalase (30 % inhibition).
  • H 2 O 2 kinetics could be continuously monitored in single specific cells and cellular compartments and organelles, using various probes and detection devices, for the duration of at least an hour and in time frames shorter than a second.
  • AU staining, washing, reagent supplementation, and monitoring procedures were done directly on the device without needing to move or pellet the cells under observation.
  • the real time results presented in the examples given above could not have been achieved with such precision and ease, with the former methods used for examining non-adherent live cells in flow-through experiments.
  • Optical Tweezers provide an excellent tool to study non-adherent cells by immobilizing them using electromagnetic properties of light in solution.
  • optical tweezers are fragile systems that need a strong laser setup (minimum 100 mW) and specialized instruments (including infrared capability, and specialized microscopes) that are not readily available in most labs. If one would like to study the reactions in cells in response to light, it is impossible as the immobilization is done with light. The addition of external solutions can interfere with the trapping of the cells, so this method cannot be used for flow-through experiments.
  • Optical tweezers are also not generally used in experiments utilizing fluorescent probes (because of the nature of the method, which requires light).
  • the MeshSlide does not require special equipment, can work with a wide variety of microscopes, does not interfere with fluorescence, and allows flow-through experiments.
  • Agarose gel Agarose gel has auto— fluorescent emission that interferes when using fluorescent probes on cells. It has an added disadvantage of coating the cell, therefore possibly interfering with some of the natural cellular processes. Cells can also react to the oligosaccharides present in the agar.
  • the MeshSlide allows one to work with cells in their native media, with minimal interference with the natural surroundings, and also does not interfere with fluorescence emissions.
  • each step involves spin-down of the cells by centrifugation, removal of the supernatant, and re- suspending of cells in the next solution.
  • the steps include: (1) initial spin down of cells; (2) addition of probe solution; and (3) several washes.
  • Each step in the process requires a few minutes. Any time one wants to change reagents, the whole process has to be started again.
  • everything can be done in the unit itself, drastically cutting the time needed to take measurements from the time a change in conditions is made.
  • the MeshSlide also requires a smaller reaction volume and results in a major reduction of costs. Summary of Advantages
  • the discussed device is compatible for all non-adherent cells visualization experiments; it restricts cell movement thereby simplifying the surveillance, even with the addition of different solutions; it minimizes the time lapse between sample preparation and detection methods, thereby allowing retrieval of data points at time scale previously not attainable; it is a fast and easy way of microscopy sample preparation; because it enables the use of low sample and reaction volumes, it saves reagents; it enables all manipulation procedures (pre-incubation, staining, washing) to be performed on the device; it maintains the natural media conditions of cells and enables their refreshment with minimum interference; it enables the mesh also to be applied as a grid for cell counting; it eliminates the need for additional accessories or cells modifications; and it enables volume production of the laboratory device at low cost.

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Abstract

La présente invention concerne un dispositif utilisé dans les applications de microscopie, et particulièrement celles utilisées pour examiner des spécimens contenant des cellules vivantes non adhérentes ou d'autres particules étudiées en suspension dans un milieu liquide, dans des expériences avec renouvellement continu. Ce dispositif comporte un réseau maillé ouvert se plaçant entre lame et lamelle de façon à recevoir un spécimen des cellules étudiées. De par sa construction et ses dimensions, ce réseau maillé ouvert, qui constitue une multitude de petites ouvertures de mailles définissant une matrice bidimensionnelle de pièges physiques reliés les uns aux autres de façon à recevoir le spécimen, est capable de limiter le déplacement des cellules étudiées à l'intérieur du réseau sans affecter de façon indue la viabilité des cellules étudiées, ou de limiter l'écoulement du milieu environnant, ce qui permet de conduire des expériences avec renouvellement continu.
PCT/IL2007/000570 2006-05-25 2007-05-10 Dispositif pour expériences sous microscope sur des cellules vivantes non adhérentes avec renouvellement continu WO2007138568A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US80811106P 2006-05-25 2006-05-25
US60/808,111 2006-05-25

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WO2007138568A2 true WO2007138568A2 (fr) 2007-12-06
WO2007138568A3 WO2007138568A3 (fr) 2009-04-23

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CN103105670A (zh) * 2013-02-06 2013-05-15 李宏 镜下观察用细胞载片
WO2013093507A1 (fr) * 2011-12-21 2013-06-27 University Of Leeds Dosage de la viabilité cellulaire fonctionnelle
CN109351377A (zh) * 2018-11-28 2019-02-19 浙江警察学院 一种内设交叉阵列可单向拦截杂质的微流控芯片
US20190056296A1 (en) * 2016-02-23 2019-02-21 Noul Co., Ltd. Blood staining patch, method and device for blood test using the same

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US3768914A (en) * 1972-09-18 1973-10-30 T Kinney Electron microscopy tissue grid staining and storing rack and method
US5257128A (en) * 1988-06-22 1993-10-26 Board Of Regents, The University Of Texas System Freezing/perfusion microscope stage
DE4132379A1 (de) * 1991-09-28 1993-04-08 Kernforschungsz Karlsruhe Substrat fuer zellkulturen und kultur von zellen oder zellaggregaten

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013093507A1 (fr) * 2011-12-21 2013-06-27 University Of Leeds Dosage de la viabilité cellulaire fonctionnelle
CN103105670A (zh) * 2013-02-06 2013-05-15 李宏 镜下观察用细胞载片
US20190056296A1 (en) * 2016-02-23 2019-02-21 Noul Co., Ltd. Blood staining patch, method and device for blood test using the same
CN109351377A (zh) * 2018-11-28 2019-02-19 浙江警察学院 一种内设交叉阵列可单向拦截杂质的微流控芯片

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