Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5058—Neurological cells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00364—Pipettes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00364—Pipettes
  • B01J2219/00367—Pipettes capillary
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00378—Piezoelectric or ink jet dispensers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
  • B01J2219/0065—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of liquid beads
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
• • 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
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N2035/1027—General features of the devices
  • G01N2035/1034—Transferring microquantities of liquid
  • G01N2035/1046—Levitated, suspended drops

Definitions

• the present invention relates to a method and a device making it possible to carry out reactions on one or more cells or on tissues of cells or networks of cells or between cells.
• Document WO01 / 07159 discloses a device making it possible to carry out biochemical protocols in series in microreactors. However, this device does not allow working on cells.
• the document WO95 / 34374 describes a device and a method making it possible to carry out microreactions in series. However, this device and this method do not envisage implementing so-called transfection reactions, in which a reagent enters a cell. Indeed, the cell is a living, heterogeneous organism, whose survival and reaction require special conditions, particularly with regard to gas exchange. These parameters are not taken into account or even considered in this document.
• Attachment with gelatin does not control the attachment of the transfected DNA. It also does not improve the efficiency of transfection. By this method the expression or blocking of the expression of a sufficient amount of protein can hardly be obtained. In addition, only one kind of cell can be used for each glass slide.
• the subject of the invention is therefore a process for reacting a reagent R with at least one cell C, said process being characterized in that:
• - cell C is deposited on a support S comprising a substantially flat surface, in the form of an aqueous drop on said surface; the substantially flat surface of the support S on which the aqueous drop containing the cell C has been deposited is covered by a separating film F, allowing the passage of gases and preventing the evaporation of the aqueous drops deposited on the support S, F being immiscible with reagent R.
• the reaction between reagent R and cell C is triggered by the introduction of reagent R into the aqueous drop containing cell C.
• an aqueous drop containing the cell C is deposited on the support S, a second aqueous drop containing the reagent R is injected, using any suitable injection means, directly into the drop containing the cell C
• a first variant is illustrated in FIG. 1.
• a first aqueous drop is deposited on the support S then a second aqueous drop is deposited on the same support near the first, one of these drops contains the cell C, the other the reagent R, the reaction of reagent R with cell C, and possibly its transfection into cell C, is triggered by the fusion of the two drops.
• the displacement and the fusion of the drops can be obtained by vibration within the support, by electrophoretic displacement of the electrically charged drops or by mechanical or optical tweezers. They can also be obtained by a modification of the surface properties of the support under the effect of an electric, magnetic field, by an optical or thermal treatment.
• FIG. 2 Such a variant is illustrated in FIG. 2.
• the reagent R is fixed to the support S or to the film F, the cell C is deposited in the form of an aqueous drop on the support S and the reagent R is then removed from the support S or from the film F in order to allow its reaction with the cell and possibly its transfection into the cell.
• This variant is illustrated in FIGS. 3 and 7.
• transfection is used to denote the penetration of any molecule of a reagent into a cell.
• the invention also relates to a device allowing the reaction of a reagent R with a cell C, this device being characterized in that it comprises: a support S comprising a substantially flat surface covered with a separation film F allowing the passage of gases and preventing the evaporation of the aqueous drops deposited on the support S, F being immiscible with the reagent R,
• the support S consists of a plate which can be made of silicon, glass or polymer, such as for example polyurethane, nylon, polyester, polyethylene, polypropylene, polyfluorocarbon, polymethylmefhacrylate (PMMA), polycarbonate, polyvinyl chloride (PVC), polydimethylsiloxane (PDMS) or polysulfone.
• a plate which can be made of silicon, glass or polymer, such as for example polyurethane, nylon, polyester, polyethylene, polypropylene, polyfluorocarbon, polymethylmefhacrylate (PMMA), polycarbonate, polyvinyl chloride (PVC), polydimethylsiloxane (PDMS) or polysulfone.
• the attachment of the drops to the support is done by capillarity, thanks to the surface tension forces.
• the support S has a substantially flat surface comprising at least one means intended for the reception of the aqueous drops.
• the means intended for the reception of the aqueous drops consists of zones of the substantially flat surface of the support S with a size ranging from 5 ⁇ m 2 to 5 mm 2 .
• the support S can have a hydrophobic character on its flat surface and to include one or more hydrophilic zones constituting said receiving means.
• the support S has on its flat surface cavities, with a depth ranging from 1 micron to 1 millimeter, and constituting said receiving means. It is also possible to provide for the support S to be a plate provided with protrusions of small thickness, from 1 micron to 1 millimeter, arranged on its surface and intended to favor the attachment of the drops.
• the support S has on its flat surface a hydrophobic character and comprises one or more hydrophilic zones constituting the receiving means.
• a hydrophobic character it is preferably covered with a hydrophobic material such as a polyfluorocarbon, such as for example polytetrafluoroethylene or Teflon®, a silane such as for example perfluorosilane.
• the hydrophobic zone of the support can consist of a jagged surface structure on a nanometric scale like “black silicon” used in optics. Examples of commercial slides of this type are the 40 well D2 mm super teflon immunofluorescence slides, sold by the company MERCK EUROLAB POLYLABO division. Even more preferably, the support further comprises a second means for receiving the drops superimposed on the first, such as for example a hydrophobic flat surface and hydrophilic small growths, or a hydrophobic flat surface and hydrophilic wells, or a hydrophobic flat surface and a hydrophilic yarn.
• the support is provided with a surface whose hydrophilicity / hydrophobicity properties can vary under the influence of a parameter such as temperature, an electric field, a magnetic field, an irradiation.
• the support can thus be active, to make the drops evolve on its surface using the principles of microfluidics in drop. This amounts to dynamically modifying the surface properties of the support (for example the variations in energy / surface tension) to make the drops move in a controlled manner.
• the drops of cell cultures can go through different stages of reactions conducted within the support: one can merge two drops which come together (one with the reagent and another with the cells for example).
• temperature can be used as a means of changing the surface properties of a support.
• Liang et al. (“Preparation of Composite-Crosslinked Poly (N-Isopropylacrylemide) Gel Loyer and Characteristics of Reverse Hydrophilic-Hydrophobic Surface” Journal of Applied Polymer Science 72: 1, 1999) describes a polymer that is hydrophilic at low temperatures ( ⁇ 30 ° C) and hydrophobic above. By integrating a localized temperature control system under the substrate, one can control the properties of the surface.
• the reagent R is fixed to the support S before the deposition of the aqueous drop containing the cell C.
• Such devices are known to a person skilled in the art for other uses: these are DNA chips as described by:
• Protein microarrays for highly parallel detecting and quantitation of specifies proteins and antibodies in complex solutions Genome Biol. 2001 Jan 22; 2 (2): RESEARCH 0004.1-0004.13; - Livache T., Bazin H., Caillât P., Roget A., Electroconducting polymers for the construction of DNA or peptide arrays on silicon chips, Biosens Bioelectron. 1998 Sep 15; 13 (6): 629-34. The same principle can be applied to molecules other than polynucleotides. Molecular chips are described in: Kuruvilla et al, Glucose signaling with small molecule microarrays, Nature (2002), 416 p. 653.
• the reagent molecule is first hung on the chip (for example by covalent hooking on a glass slide).
• the molecule can optionally be detached after depositing the aqueous drops containing cells on the molecule chip.
• the detachment of the reagent molecule can be done in a known manner by one of the following means:
• the reagent is a polynucleotide only: - cutting of the double stranded DNA by restriction enzymes, or by other nucleases,
• the substantially flat surface of the support S is covered with a separation film which fulfills three functions:
• the film F can be of different natures: - it can be a liquid immiscible with water such as for example an oil. Until now, it was known to use oil to preserve certain cells, however, it had never been used to carry out reactions on cells.
• oils which can be used in the process and the device according to the invention, mention may in particular be made of mineral oils and silicone oils.
• a light mineral oil is used.
• the rigid honeycomb cover may be a rigid honeycomb cover made of porous material, the size of the cells being adapted to be able to contain the drop of cell (s) and possibly of reagent.
• the rigid cellular cover can be functionalized, in each cell, by a reagent molecule and thus constitute a molecule chip or a nucleotide chip called to come into contact with the support on which drops of cells were deposited symmetrically with respect to the alveoli. This variant of the invention is illustrated in FIG. 7.
• the separation film is a gas or a liquid
• the deposition of the aqueous drops containing one or more cells or a reagent on the support S and under the separation film is done by means of capillaries, as illustrated in FIG. 1.
• these capillaries are connected to a pump or syringe pump making it possible to control the volume of the drops.
• the reagents or cells can also be dispensed by a conventional system such as those used for the manufacture of DNA chips.
• a conventional system such as those used for the manufacture of DNA chips.
• piezoelectric systems making it possible to compress a cavity and to eject a drop by a nozzle.
• the ejected drops pass through the film of liquid or gas thanks to their ejection speed and / or by gravity, this liquid or this gas being lighter than the solution to be deposited.
• the separation film is a solid film or a rigid cover, the latter is deposited on the support, after depositing the aqueous drops of cells and optionally of reagents by the same means as described above.
• the displacement and the fusion of the drops can be obtained by vibration within the support, by electrophoretic or electromagnetic displacement of the electrically charged drops or by mechanical or optical tweezers. It can also be obtained by a modification of the surface properties of the support caused by the application of an electric, magnetic field or a thermal or optical treatment.
• the support S of the device is mobile, so as to allow its displacement from a first deposition means to a second deposition means, and possibly to other deposition means.
• the support S can in certain cases consist of a solid film fixed to rollers at its two ends, the rollers being provided with winding means so as to allow the movement of the film and therefore the displacement of the drops which have been deposited thereon. .
• the method according to the invention provides for the displacement of the support S after the deposition on the support S of the first series of drops, whether these are cell drops or reagent drops.
• the support S is placed in an enclosure with a controlled atmosphere, the temperature, hygrometry and CO content of which are adjusted so as to allow cell survival.
• a controlled atmosphere the temperature, hygrometry and CO content of which are adjusted so as to allow cell survival.
• Such devices are in particular ovens with controlled atmosphere.
• the temperature in such a device can vary from 35 to 42 ° C, a preferred temperature being between 36.5 and 37.5 ° C.
• the temperature variation can in particular be used to induce cellular differentiations.
• the C0 rate is preferably maintained between 3 and 5%.
• the oxygen level O is preferably that of the ambient air.
• the aqueous drops containing one or more cells, a cellular tissue or a cellular network comprise a culture medium.
• the aqueous drops of cells will comprise MEM or "minimal essential medium” marketed by the company GIBCO BRL under the reference Cat. No. 12000-022.
• the culture medium can also contain other constituents such as calf serum, one or more antibiotics intended to control the sterility of the medium, such as for example penicillin.
• the use in the culture medium of chemical agents which induce the differentiation of the cells such as for example bromodeoxyuridine. It is also possible to provide that the aqueous drops in which the C cells are in culture are gelled, using any known gelling agent, such as for example agar or gelatin.
• the aqueous drops containing the cell or cells or the cellular tissue or the network of cells, and / or the aqueous drops containing the reagent comprise one or more constituents intended to promote transfection, such as for example liposomes.
• transfection agents are described in particular in documents WO 01/20015 and WO 98/33932.
• transfection methods can also be used in the device of the invention, such as: electroporation or microprecipitation. These transfection methods, well known to those skilled in the art, are described in particular at http://opbs.okstate.edu/ ⁇ melcher/MG/MGW4/MG43.html.
• the device comprises:
• the means used in the devices according to the invention will preferably be connected to a control device allowing the automation of the device and of the method according to the invention.
• heat treatment means which may consist, for example, of a heating device capable of being placed near the support S or fixed to this support and intended to carry the droplets at an appropriate temperature.
• the way heating may consist of electrically conductive wires also serving as a means for receiving the drops.
• the detection means are in particular devices intended to measure the fluorescence or the radioactivity of one or more drops or of the cells contained in one or more drops.
• the optical processing means are in particular means for processing with ultraviolet rays, the latter being known to induce crosslinking between complementary strands of DNA and between DNA and proteins.
• the use of the device and / or of the method according to the invention has numerous advantages: very small quantities of materials can be used: a single cell per drop makes it possible to carry out a transfection experiment.
• very small drop volumes less than 1 micro liter, preferably from 0.1 to 1000 nanoliters containing 1 to 500 cells, even more preferably from 0.1 to 10 nanoliters containing 1 to 10 cells.
• the separation film F makes it possible to control the gaseous exchanges of the culture medium of the cell and its sterility.
• each cell involved in the process can be transfected.
• the cell cultures in the form of drops under the separation film can be stored for at least 24 hours and up to several days without any significant changes in their cell activity being observed (without notable influence on the proliferation and growth of cells).
• reaction batteries The method and the device according to the invention also make it possible to produce reaction batteries:
• aqueous drops each comprising at least one cell can be deposited on the support S, said drops being isolated from each other.
• each of these drops is placed in a separate receiving means.
• All the cells can be identical, but we can also plan to place different cells (at least two different kinds of cells) in the different drops.
• Drops containing the reagent (s) are deposited, near each drop containing a cell, so as to allow the fusion of a drop containing the appropriate reagent with the drop containing the target cell.
• a support is advantageously provided comprising receiving means regularly arranged in the form of matrices, so as to allow the automation of the process.
• the support and the capillaries intended for depositing the aqueous drops of cells and reagents are connected to control means so as to allow the automation of the process.
• the method and the device according to the invention therefore make it possible to carry out simultaneously and in an automated manner a large number of reactions of a reagent on a cell by varying the nature of the reagent and of the cell, while working on extremely reduced volumes. .
• - cell lines cells can perpetuate themselves eternally and thus form lines
• - stem cells they are obtained from a sample taken from animals or from biopsies
• the cells are cultured in culture medium (aqueous) in a known manner. You can also grow heterogeneous cells for several days and use this mixture.
• the support is a hydrophobic plate comprising hydrophilic zones, it is immersed in an aqueous solution containing the cells and then it is removed from this solution, allowing the excess liquid to flow out.
• the medium drops containing the cells are retained in hydrophilic areas.
• This step is followed by the deposition of a layer of separation film F and the deposition of the drops containing a reagent or other cells.
• the film F fluid or solid
• it is deposited before or after the deposition of the reagent drops or of other cells.
• the reagents R capable of being used in the process and the device according to the invention, there may be mentioned:
• RNA molecules single strand and double strand, in particular siRNA molecules (small RNA for interference); DNA molecules, single strand and double strand; PNA molecules (from the English “peptidic nucleic acid” or peptide nucleic acid) which are peptide-nucleic acid chimeras; ribozymes; RNA double strand interference or proteins and peptides.
• siRNA molecules small RNA for interference
• DNA molecules single strand and double strand
• PNA molecules from the English “peptidic nucleic acid” or peptide nucleic acid
• ribozymes RNA double strand interference or proteins and peptides.
• transcription factors mention may be made most particularly of transcription factors.
• the reagent molecules can be formulated in solution ready to be deposited. They can also be prepared directly after deposition on the support, for example by synthesis, in particular by organic synthesis, in situ, or by in vitro transcription in gout. Prion-type molecules can also be obtained in a drop by polymerase chain reaction or PCR (from the English "polymerization chain reaction") peptide before their transfection into cells. When nucleic acid molecules are used, their preparation can be done by nucleic PCR. As already explained above, the reagent can also be attached to the support.
• DNA precipitation can also be carried out in the aqueous drop deposited on the support by fusion with a drop of the appropriate reagent.
• the transfection of reagents into a first type of cell can be used so as to trigger a cellular reaction, such as the production of a recombinant protein, and then react this first cell population with a cell population of another type by merge with another drop.
• a cellular reaction such as the production of a recombinant protein
• the support is provided with separation means making it possible to separate two distinct types of cells but allowing the passage of small molecules between these cells.
• Such a separation means is intended to mimic a biological barrier, such as for example the barrier existing between the blood and the cervical cells.
• separation means are arranged at the level of the reception means, on the support.
• aqueous drop comprising at least one cell of a first type on one side of the separation means and an aqueous drop comprising at least one cell of a second type on the other side.
• separation means The fusion of the drops on either side of the separation means allows communication between the cells by means of molecules capable of diffusing through the separation means. This communication can then be studied by any means, in particular by adding reagents in the form of an aqueous drop before or after the fusion of the cell drops. By the automated transfection of these drops, it is possible to analyze the biological role of the factors transfected in a biological multilayer.
• the separation means which can be used according to this variant of the invention are artificial membranes such as for example a nitrocellulose filter, silicon pierced with nano-holes, a paper blotter, a fabric filter; one can also plan to use a solid gel such as an agarose gel, collagen or gelatin.
• the device and the method according to the invention make it possible to automate the expression of recombinant proteins obtained by the entry of DNA coding in the cells, to carry out the screening of nucleic molecules intended to modify (block or on the contrary increase) l expression of genes in cells and search for promoter genomic sequences.
• This invention also makes it possible to study the interactions between cells of different types, this interaction being triggered by the mixture of the drops.
• the device and the method according to the invention make it possible to obtain a global view of the biological effects of the reaction of molecules of all kinds with cells, and in particular of the automated entry of molecules of all kinds into cells.
• the global detection of the cell phenotypes generated by the entry of the molecules into the cells will be carried out using labeled molecules, that is to say molecules which can be detected without compromising the integrity of the medium. that contains them.
• labeled molecules that is to say molecules which can be detected without compromising the integrity of the medium. that contains them.
• One of the advantages of the method and of the device according to the invention resides in the fact that all of the steps of transfection and manipulation of cellular interactions are carried out in the liquid phase which promotes cell culture in nutritive medium and the enzymatic reactions.
• the method and the device according to the invention have the following advantages: - Improvement of the efficiency of the transfection, compared to the transfection made in the conventional way in a culture well;
• the reagents can be obtained directly in the drop before fusion
• oligonucleotides used did not have sufficient affinity for the target RNA and did not allow its translation to be blocked in the cells. Synthetic DNA molecules being toxic to the eukaryotic cell, we try to use minimal quantities. To block the expression of a target gene in a cell, it is necessary to intervene at four levels:
• the first two steps can be carried out using a conventional oligonucleotide chip on which is tested the hybridization of a family of oligonucleotides to target RNA rendered fluorescent (we can for example refer to the work of Olejnik et al., NAR, 26, 3572 (1998)).
• the method according to the invention will make it possible to test the penetration of the oligonucleotides into the cell and their stability.
• modified oligonucleotides such as for example phosphorothioate derivatives, will be used, this modification conferring on the oligonucleotide concerned resistance to nucleases of several days.
• Antisense molecules can also be made up of RNA duplexes, called interference RNA (RNAi), which hybridize to messenger RNAs by forming triple RNA helices (for this approach, we can refer to Elbashir and al., Nature, 411, 494-498 (2001)).
• RNAi interference RNA
• the method and the device according to the invention also make it possible to screen long (plasmid) and short (synthetic) RNAi sequences.
• Another application of the method and the device according to the invention relates to the automated production of recombinant proteins in drops. Automatic transfection should make it possible to test the expression of different DNA fragments coding as well as that of different mutants of this same DNA.
• the automated expression of recombinant proteins on the devices according to the invention can be an alternative to the protein chip. It is then no longer necessary to produce the proteins, to purify them and to hang them on a solid support, the proteins are manufactured de novo on the device.
• RNAsi 'RNA small interference' or small RNA for interference
• RNAsi are double stranded RNA molecules capable of specifically and effectively blocking the expression of genes in cells in culture and in animals.
• the RNAsi were elected 'molecules of the year 2002' by the magazine Science: these molecules could allow in particular innovative therapies in oncology and virology. They have also already made it possible to characterize the function of previously unknown genes (T. Tuschl, Nature Biotechnology, vol.20, May 2002, p.446-448)
• RNAsi To manufacture these RNAsi, four routes have been listed. The first uses chemistry, the two strands of siRNA are each made in a nucleic acid synthesizer and then are combined after synthesis (synthetic molecule made from phosphoramidite RNA described in: SMElbaschir et al, Nature, 411, 24 May 2001, 494-498). The second uses an RNA polymerase in vitro, each strand of siRNA is produced in a complementary manner with respect to a DNA strand then the two strands are combined after synthesis (example: commercial kit pSilencer sold by the company Ambion and commercial kit HiScrib marketed by the company NEB Biolabs).
• the third uses in vivo synthesis in eukaryotic cells or bacteria, both strands are made using the RNA polymerases present in vivo.
• the fourth uses long, single or double stranded RNAs (produced in vitro or in vivo) which will be transformed into small molecules (RNAsi) by the nucleases of the transfected cells (R. Agami, Current Opinion in Chemical Biology, 2002, 6, 829-834).
• RNAsi the second path that can be applied, that which attempts to manufacture RNAsi from DNA molecules contained in a tube.
• This route is used in particular because it is less expensive than that which consists in making RNAsi chemically.
• the structure of RNAsi synthesized in vitro is different from that of RNAsi obtained chemically, it makes it possible to obtain a greater affinity in the case of hybridization of RNAsi obtained in vitro with the target RNAs of the transfected cells. .
• blocking of gene expression is better with RNAsi obtained in vitro than with chemical RNAsi: better efficiency and need for lower siRNA concentrations.
• This variant of the invention is based on a process for manufacturing RNAsi on a solid substrate.
• the present invention proposes to manufacture
• RNAsi in drop using two DNA templates containing a promoter sequence for an RNA polymerase (of type SP6 or T7 for example) and to use this drop formatting to transfect, by drop fusion, the molecules of RNAsi double strand in cells cultured in a drop on a solid substrate.
• the DNA templates can be covalently or non-covalently attached to the solid substrate.
• RNAsi sequences have already been proposed concerning the percentage of GC nucleotides for example, or the T of the corresponding oligonucleotide, or even the size of the molecules (21 mer). However, nothing replaces experience, screening is necessary, not only to find the optimal sequence to block the expression of a gene but also to determine its concentration and its time of action in cell culture.
• Chemosensitivity can be analyzed on a chip.
• RNAsi N.S. Lee et al., Nature biotechnology, vol. 19, May 2002, 500-505
• the global view of fluorescence is made by bi-parametric detection:
• the cells can be analyzed alive in drops under the oil layer.
• - analysis of the fluorescence of fixed cells after fixing the cells by PFA or paraformaldehyde (see application No. 1) the glass slide containing the recombinant cells is used as a histology slide. It is possible to carry out radioactive labeling and fluorescent labeling experiments (see application No. 5.2).
• the total fluorescence of the slide can be analyzed using a conventional microscope.
• We also used a scanner conventionally used in DNA chip experiments (Scanner: GenePix 4000B, sold by the company Axon Instrument). The precision in this kind of device is 5 ⁇ m, it is therefore possible to visualize a cell using ten or so pixels.
• FIG. 4 illustrates an example of application of the device according to the invention: the expression of a recombinant protein in a suspension of glial cells and the activation of a suspension of neurons.
• the first two drops are first fused by mechanical displacement of the 2 drops using the end of a pipette to obtain the drop Gi of transfected cells.
• the glial cell thus expresses a recombinant protein.
• oligonucleotides capable of blocking the expression of a gene of interest called a target in this example.
• a target in this example.
• E-GFP Green Fluorescent Protein type E, sold by the company Clontech.
• the antisense activity is measured by reducing the fluorescence of the reporter proteins.
• it is essential to screen at least 50 oligonucleotides of different sequences for the same gene. During this screening in cells ,. we looked in particular for the oligonucleotide which has the greatest affinity for the target RNA and the greatest penetrating power in the cell.
• the transfection with calcium phosphate was chosen because it is very effective: using oligonucleotides labeled with cy5 (Cyanine 5), these oligonucleotides being marketed by the Company Eurogentec, 70% of HEK 293 cells (Human Embryonic Kidney) and 80% of COS cells (Chinese Ovary Sarcoma) become fluorescent after one day of culture (fluorescence measured by flow cytometry). Other transfection methods involving the formation of lipid droplets around the DNA can also be used.
• Experiment 1a transfection by drop fusion
• the oligonucleotides are synthesized in the form of residues from 18 to 21 nucleotides in length, and contain phosphorothioate bonds capable of limiting their degradation by nucleases. Each of these oligonucleotides is precipitated in the form of calcium phosphate (classic reaction: l ⁇ l of oligonucleotide resuspended in water at 1 mM is mixed with 100 ⁇ l of 0.25 M calcium chloride and 100 ⁇ l of HBS buffer (Chen and Okayama , Biotechniques 1988, 6, 632-638).
• a drop of 1 ⁇ l of each of these 50 precipitates is fused with a drop of 10 ⁇ l of cells in their culture medium (DMEM marketed by the Gibco Company) (approximately 5000 cells fibroblast 3T3).
• DMEM culture medium
• the drops are placed at the bottom of the box on the plastic support under the oil.
• the antisense activity is measured after 2 days of culture by decreasing the fluorescence of the GFP expressed in tandem with the target protein whose expression is sought to block.
• the fluorescence of the 50 drops is observed simultaneously under the microscope. This experiment is carried out several times in parallel to ensure the relevance of the results. It is important to do these 50 tests in parallel in order to compare the antisense activity of each of the oligonucleotides.
• To observe the decrease in the fluorescence of the target protein it is possible either to observe the living transfected cells under a microscope, or to fix the cells with paraformaldehyde (conventionally 4% of PFA). To fix the cells, paraformaldehyde is added at the same volume as the cell drop for 10 minutes, then the drop + oil mixture is rinsed with PBS twice.
• the transfection is obtained after detaching the oligonucleotides from the solid support.
• the cells are cultured near the deposits of oligonucleotides on the glass slide.
• the oligonucleotides (marketed by the company Eurogentec) are hooked by their 5 ′ amine end on a silanized blade (for example a Surmodics blade marketed by the company Motorola).
• the DNA of the deposits is complexed with calcium phosphate salts (according to the same principle of DNA precipitation as that described in the application la) and kept wet on the slide.
• the adherent 3T3 cells are then deposited in a drop on the surface of the oligonucleotide spots, the whole is kept under 1 ml of mineral oil for one day in a cell incubator: the drops can be formed on the surface of the DNA deposits or formed using a composite surface (hydrophilic and hydrophobic, ProLabo blade described in application no. 5).
• the stall of the oligonucleotides, complexed with calcium phosphate and located under the cell cultures in a drop, is obtained as illustrated in FIG. 5 by illumination of the slide by UV at 365 nm which make it possible to cut the photocleavable bond introduced into 5 ′ of the oligonucleotides (3 ′ of the amino site). example of an oligonucleotide hooked, then unhooked by photocleavage, then finally transfected into adjacent HEK 293 cells:
• Application 2a Expression of recombinant proteins in a cell type.
• Kinesins form a family of proteins with similar biochemical properties, they are motor proteins associated with microtubules. These proteins are found in all eukaryotic cells. They transform the hydrolysis of ATP into mechanical energy and play a fundamental role in the transport of organelles, mRNAs and protein complexes along microtubules. They can move on the positive side of microtubules (N-kinesins) or on the negative side (C-kinesins). They also participate in chromosomal movements during mitosis and meiosis and play a role important during cell division. We can notably refer to the following publications:
• the device according to the invention we obtained the expression of about twenty DNA coding for these kinesins using expression plasmids making it possible to express in tandem the protein of interest and the GFP (pcDNA3.1 / CT-GFP-TOPO, sold by the company InVitrogen).
• the transfection was carried out as during application la: the drops were fused under 1 ml of oil, 10 ng of plasmid precipitated with calcium phosphate was brought into contact with a drop of 10 ⁇ l containing approximately 5000 HEK cells 293 (human embryonic kidney). After 1 day of transfection, the drops of HEK 293 cells are fluorescent by expression of the GFP protein in the cells.
• 10 ng of plasmid containing in tandem the genes coding for the factor Pax6 and for GFP (pcDNA3.1 / CT-GFP-TOPO, marketed by the company InVitrogen) precipitated with calcium phosphate (see precipitation of DNA with phosphate of calcium in application la) is brought into contact with a drop of 10 ⁇ l containing approximately 5000 glial cells (radial glial cells of cortex, post natal, 2 divisions). After 1 day of culture, the drop Gl of glial cells is fused to another drop G2 of 10 ⁇ l containing 5000 cortical cells (primary cortical culture isolated from post natal cerebral cortex).
• the recombinant Gl glial cells expressing the Pax6 factor are for example capable of inducing the neurogenesis of the astrocytocal cells contained in G2.
• This application is important for the screening of new drugs with neurotrophic potential, for example in the search for compounds capable of regenerating dopaminergic neurons in brains altered by Parkinson's disease.
• the system according to the invention can be easily confined: the formation of the drops can be carried out mechanically without the intervention of a user, and the latter can be kept safe from contaminants by being kept under a layer of oil for several days.
• Virus transfection can be achieved in two ways:
• the drops it is possible to produce the virus or the pathogenic agent (amplification and encapsulation) or to detect it (biological concentration and antigen-antibody reaction).
• reporter genes and more specifically of "promoter-reporter activity" constructions has been widely used for the characterization of regulatory regions upstream of genes.
• Microtechnologies today make it possible to considerably increase the number of promoters studied.
• the transcriptional regulation of these regions can be observed in real time thanks to the use of a reporter gene coding for GFP (Green Fluorescent Protein).
• GFP Green Fluorescent Protein
• the fluorescence of this protein can be observed without having to lyse the cells.
• the variations in fluorescence will allow us to study the kinetics of the transcriptional regulation of a large number of promoters in parallel and in real time.
• the reporter activity of known genes induced by ionizing radiation was used to validate the microarray and the experimental model.
• the sequences of interest upstream of the P53 and c-myc genes were amplified and then cloned upstream of the GFP gene in the phrGFP vector (Genentech).
• the transfection was carried out as during application la: the drops were fused under oil, 10 ng of plasmid (phrGFP containing the promoter DNA sequences) precipitated with calcium phosphate was brought into contact with a drop of 10 ⁇ l containing approximately 5000 human keratinocyte cells.
• the keratinocyte By its location in the skin, the keratinocyte represents one of the cell types most exposed to irradiation in vivo. After 1 day of transfection, the drops of the keratinocytes are fluorescent by expression of the GFP protein in the cells. The measurement of the GFP reporter activity is made by global fluorescence reading by microscopy coupled to a CCD camera (Charge Coupled Device).
• FIG. 6 Study model: making the cell drops This experiment is illustrated in FIG. 6.
• the living cells are deposited in homogeneous drops on a solid support.
• the production of this cellular device can be facilitated by the use of a composite surface (hydrophilic and hydrophobic), glass slide sold by the company ProLabo, covered with a teflon® film and containing 3 mm hydrophilic circular wells. of diameter.
• a large number of homogeneous drops can be produced by simply dipping the support in a cell suspension.
• the drops are then covered with a layer of mineral oil to prevent them from drying out and to promote cell survival until the screening test.
• the cell drops can be stored on this type of support for several days in an incubator intended for cell culture.
• n-drops of test compounds are deposited individually on the cell drops previously formed. It can also be provided that chemical compounds are grafted onto the surface of the support (as for the oligonucleotides, in application 1b) and then released together (for example by UV illumination). The insertion of a photocleavable site in the molecules can be obtained by combinatorial chemistry.
• FRET fluorescence resonance by electron transfer or fluorescence resonance by electron transfer
• Example of screening measurement of the activity of the adrenergic receptor: We can refer to the following publication: ref. 7: Ghanouni et al, Agonist induces conformational changes in the G protein coupling domain of the beta2 adrenergic receptor, PNAS (2001) 98, 11, 5997-6002.
• the recombinant cells can be fixed on the support using PFA (see application No. 1).
• an antibody to reveal the expression of the recombinant protein in 3T3 cells.
• antibody marketed by the company Upstate Biotechnology Rabbit polyclonal anti-CK2 beta antibody.
• FIG. 7 illustrates this variant: drops of cells in a culture medium are deposited in the form of a matrix on a Teflon support.
• a PDMS cover onto which organic molecules from a screening bank are grafted and comprising cells of a volume substantially equal to that of the drops is placed on the support.
• the spacing of the drops has been planned for an exact correspondence between the support and the cover.
• the molecules are removed from the cover by any appropriate treatment so as to transfect cells.
• Trypan blue is a marker of cell viability. This reaction of mixing trypan blue and cells made it possible to verify that the electric field did not kill the cells since the% of colored cells did not exceed 2%.
• the transfections on droplet cell chips carried out manually on commercial slides, can be transferred to a micro-battery manufacturing machine and thus allow a particular mode of automated DNA transfection. For this, three successive series of deposits of three drops are carried out on the same chip, therefore on the same pad of the commercial slides, the drops will merge to allow the mixing of the reagents and the cells.
• a 96-well microplate containing the various samples to be deposited is prepared: the DNA solutions, the transfection medium, the cell media.
• DNAs single-stranded oligonucleotides, PCR products or plasmids
• the robot takes from the 96-well plate 2 ⁇ L of a solution containing one of the DNAs and then deposits a drop of 10 nL on one of the studs of the blade. And so on for the different DNAs.
• FIG. 9 is a photograph representing the plate on which the first three drops have been deposited.
• the slides After depositing the cells, the slides are placed in a petri dish containing PBS (to avoid evaporation of the cell medium). They are then transferred to a cell incubator (37 ° C, 5% CO 2) for 48 hours to allow the expression of the EGFP protein. After this culture step, the cells are fixed in a paraformaldehyde solution (4% in PBS) for 20 minutes and then rinsed twice with PBS. The slides are then mounted in PBS, then the fluorescence is observed under a microscope.

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