WO2006016044A1 - Nouveau systeme microfluidique et procede de capture de cellules. - Google Patents
Nouveau systeme microfluidique et procede de capture de cellules. Download PDFInfo
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- WO2006016044A1 WO2006016044A1 PCT/FR2005/001753 FR2005001753W WO2006016044A1 WO 2006016044 A1 WO2006016044 A1 WO 2006016044A1 FR 2005001753 W FR2005001753 W FR 2005001753W WO 2006016044 A1 WO2006016044 A1 WO 2006016044A1
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- 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
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- 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
- G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
-
- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
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- 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
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00099—Characterised by type of test elements
- G01N2035/00158—Elements containing microarrays, i.e. "biochip"
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- 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
Definitions
- the subject of the invention is a novel microfluidic device and its use for capturing subpopulations of cells within a biological fluid. More particularly, the invention relates to a device comprising a fluid circulation chamber whose wall is provided with a graft for capturing a sub-population of cells in a biological sample circulated in this chamber. This device allows the capture of cells present in a very small amount in this biological sample. The invention also relates to a method for capturing cells in a biological sample, this method being characterized in that it comprises a step of passing the biological sample in such a device.
- the device of the invention can be used for the purification and characterization of circulating tumor cells from a patient's blood sample or for the purification and characterization of fetal cells from a maternal blood sample. .
- CTCs circulating tumor cells
- the ability to purify and characterize circulating tumor cells is of major interest in diagnosing and monitoring the evolution of breast cancer, but also other types of cancer such as prostate, kidney, bladder cancer. , liver, colon, lung.
- WO 03/091730 discloses a device for controlling the migration of leukocytes, this device comprising two chambers interconnected by a channel. The goal is to study the mechanisms of leukocyte binding to the walls of blood vessels. Compounds mediating the migration of leukocytes composed of endothelial cells can be placed in the channel. Compounds to be tested can also be placed in this device.
- US-2003/0044766 discloses a method and apparatus for detecting an interaction between two types of cells, one being fixed in a passage subjected to a laminar flow of a dispersion comprising the second type of cell. This device is intended to study the interactions between leukocyte populations and endothelial cells.
- A. Pierres et al, Faraday Discuss., 1998, 111, 321-330 discloses the use of a laminar flow chamber to study the formation of bonds between streptavidin coated spheres and the graft-coated chamber walls. biotin.
- the shear force, the strength of the ligand-receptor binding, the density of receptors on the surface are important parameters.
- WO 00/23802 discloses a diagnostic method for determining the cell binding capacity in a biological sample of passing this sample over a covered surface of a substrate binding to these cells.
- This document is more particularly directed towards the diagnosis of platelet anomalies and thromboses.
- the shear conditions that are applied are intended to mimic the conditions that these cells experience in vivo in the blood vessels.
- the documents of the prior art essentially relate to devices and methods for studying interactions between two cell populations: leukocytes and endothelial cells, to study platelet adhesion or cellular models (liposome spheres) carrying receptors. .
- the cells to which the present invention is more particularly concerned have the particularity of being present in very low concentration in the biological samples studied, in the middle of other predominantly large cell populations.
- the specific receptors of these cells are expressed in low density on their surface. It is these two particularities that make their capture and identification within a biological sample particularly difficult.
- the use of a prior art laminar flow device whose wall is conventionally grafted with a CTC ligand does not make it possible to capture CTC within a blood sample because these are present in excess.
- a small amount and the antigen-antibody interaction concerned by this capture is of the order of 50 to 150 piconewtons, which is extremely low compared to the interactions of the adhesion molecules between two cell populations.
- the invention more particularly relates to the capture of a population of cells whose specific surface markers interact with their receptors with an intensity ranging from 1OpN to InN, advantageously from 20pN to 50OpN, preferably from 30 to 30OpN, more specifically from 50 to 15OpN.
- the problems to be solved by the present invention are the capture of a CTC minority cell population within a biological sample, using a reduced amount of biological sample, and having as high a capture yield as possible. .
- the grafting endowed with the device of the invention has the particularity of presenting a homogeneous regular surface onto which a population of ligands specific for the sub-population of cells which it is sought to capture can be grafted.
- the surface homogeneity favors weak ligand-receptor or antigen-antibody interactions which under other material conditions would not allow this capture.
- a fluid flow chamber comprises a cavity, preferably a parallelepiped-type cavity, having two orifices placed at two ends of the cavity, the fluid being injected through a first orifice and collected by a second orifice.
- the dimensions of the fluid circulation chamber are advantageously chosen so that it is a laminar flow chamber. It can also be a cavity of a microfluidic device such as that described in FIG. US-6,408,878.
- the internal cavity of the fluid circulation chamber has a volume less than or equal to 30 .mu.l, more preferably still less than or equal to 12 .mu.l, which makes it possible to treat biological samples of reduced volume.
- the device is provided with a pump for injecting the sample fluid into the fluid circulation chamber.
- the device comprises a circuit for circulating the fluid in a loop connected to a pump, so that the sample to be treated passes more than once in the fluid flow chamber before being collected.
- At least one wall of the fluid circulation chamber is provided with a particular grafting.
- it is a wall parallel to the flow that passes through the fluid flow chamber.
- the fluid flow chamber is a parallelepiped and one of its internal faces comprises a particular grafting.
- the wall of the specially grafted fluid flow chamber consists of a solid support, which may be a glass or silicon slide or any metallic solid surface with -OH surface features, which is placed in the fluid circulation chamber.
- the solid support surface, glass slide, Si or other is functionalized by a homogeneous, monomolecular, self-assembled layer of chlorosilane chains.
- a protein entity for recognizing the population of target cells, in particular circulating tumor cells is grafted directly or via a suitable coupling agent onto the layer of chlorosilanes.
- the selectivity of the recognition biomolecule is critical for the capture of the target cell population.
- the solid support (blade) prepared is then mounted in the fluid circulation device.
- the functionalization of the solid support for cellular capture comprises 3 steps: the grafting of cholorosilanes on the glass slide, the grafting of a coupling agent (which may be a chemical coupling agent or a protein A or G protein agent) and finally grafting a recognition protein entity (antibody, or the ligand of a membrane receptor of a cell).
- FIG. 1 illustrates a glass slide of this type grafted with an antibody: the solid support is grafted by an organized self-assembled silane layer, to which is attached by a covalent bond a layer of coupling agent (linker).
- a biomolecule layer of specific recognition of the target cell population is covalently or non-covalently attached to the coupling agent.
- the presence of the coupling agent layer is optional: according to a variant of the invention, it is possible for the recognition biomolecules to be directly attached to the organized self-assembled silane layer.
- To carry out the covalent immobilization of organic molecules on a mineral surface it is first necessary to graft thereon coupling molecules that will ensure the attachment of organic molecules on the mineral substrate.
- Organosilicon coupling molecules have been proposed for this purpose by LA Chrisey et al. ⁇ Nucletic Acids Research, 1996, 24, 15, 3031-3039) and U. Maskos et al. (Nucleic Acids Research, 1992, 20, 7, 1679-1684).
- the molecules used namely 3-glycidoxypropyltrimethoxysilane and various aminosilanes, however, have the disadvantage of being deposited randomly and not reproducibly on the surface. They form an inhomogeneous film whose thickness can not be controlled, a film which is also not very robust with respect to subsequent chemical treatments, the inhomogeneity of the film indicating indeed poor protection of the siloxane bonds. It is therefore very difficult to obtain a reproducible grafting of these molecules.
- Prior to binding or synthesizing oligonucleotides on the substrate additional surface reactions are required to decrease the steric gene at the surface (e.g.
- organosilicon compounds have been described in WO 01/53303. These compounds can be used as coupling molecules to deposit on the surface of a solid support, an organized self-assembled monolayer. Such molecules have been used in particular for the immobilization of biomolecules such as nucleic acids.
- the method described in WO 01/53303 makes it possible to obtain a solid support grafted by immobilized biomolecules on an organized self-assembled monolayer. This characteristic results in the presentation of a homogeneous surface of biomolecules that promotes the capture of a minority population of cells, with a low density of specific sensors in a biological sample flowing on this surface.
- the grafts of the prior art did not make it possible to obtain a satisfactory surface homogeneity and this defect resulted in a low rate of capture of minority target cells within a biological sample.
- the solid support is modified by an organized self-assembled monolayer comprising at least one organosilicon compound corresponding to formula (I):
- n is between 15 and 35, preferably between 20 and 25, m is equal to 0 or 1,
- X 1 , X 2 and X 3 which may be identical or different from each other, are selected from the group consisting of linear or branched, saturated C 1 to C 6 alkyl groups, and the hydrolysable groups, at least one X 1 , X 2 or X 3 representing a hydrolysable group,
- A represents a group -O- (CH 2 -CH 2 -O) k - (CH 2 ) j - in which k represents an integer between 0 and 100, preferably between 0 and 5, and i represents a number an integer greater than or equal to 0, preferably equal to 0 or 1, - B represents a group chosen from -OCOR, -OR,
- R, Ri, R 2 being chosen from: a hydrogen atom, a hydrocarbon chain optionally substituted by one or more halogen atoms, saturated or unsaturated, linear or branched, comprising 1 to 24 carbon atoms, preferably 1 to 6 carbon atoms, or an aromatic group optionally substituted by one or more halogen atoms.
- aromatic is meant any group which has one or more aryl rings, for example a phenyl ring.
- organized self-assembled monolayer means an assembly of molecules in which the molecules are organized, an organization due to interactions and a strong cohesion between the chains of the molecules, giving rise to a stable and ordered anisotropic film (A. Ulman, Chem Rev., 1996, 96, 1533-1554).
- An organized self-assembled monolayer formed on a solid support makes it possible to obtain a dense, homogeneous organic surface and well defined parameters both chemically and structurally.
- the formation of this monolayer obtained thanks to the self-assembling properties of the compounds of formula (I) for well-defined values of n, m, k and i, it is perfectly reproducible for each organosilicon compound.
- the formation of an organized, very dense, self-assembled monolayer protects the siloxane bonds against chemical treatments (acidic or basic), which allows to realize various chemical reactions on this surface.
- organosilicon compounds of formula (I) used in the present invention advantageously have very varied functionalities and a high reactivity, having regard to the nature of the group A and the diversity of the terminal groups B usable, these group B may of course be modified and functionalized at will according to the organic chemistry reactions well known to those skilled in the art.
- the compounds described above make it particularly advantageous for the selected organosilicon compounds of formula (I) to immobilize biomolecules on a support in a reliable and reproducible manner, with regard to the homogeneity and stability of the self-assembled organized monolayer formed on the support.
- the biomolecules are immobilized on the modified support by strong covalent bonds, without degradation of the siloxane bonds developed between the organosilicon compounds and the solid support.
- Suitable solid supports are, in general, those whose surface is hydrated and / or those whose surface has hydroxyl groups.
- said support is selected from the group consisting of glasses, ceramics (for example oxide type), metals (for example aluminum or gold) and metalloids (such as silicon).
- hydrolyzable means any group capable of reacting with an acid in an aqueous medium so as to give the compounds XiH, X 2 H or X 3 H, X 1 , X 2 and X 3 being such that defined in formula (I).
- said hydrolysable group is selected from the group consisting of halogen atoms, the group -N (R ') 2 and the groups -OR', R 'being a saturated alkyl group in C 1 to C 6 , linear or branched.
- suitable halogen atoms are fluorine as well as chlorine, bromine or iodine.
- X 1 , X 2 and X 3 represent chlorine atoms.
- n is greater than or equal to 22.
- solid supports modified according to the present invention is particularly advantageous for the preparation of supports on which are covalently attached protein entities (antibodies, ligand of a cellular receptor, protein ...) capable of selectively binding cells representing a subpopulation within a biological sample.
- protein entities antibodies, ligand of a cellular receptor, protein
- the preparation of the grafted solid support comprises the following steps: a) preparation of a modified solid support by an organized self-assembled monolayer comprising at least one organosilicon compound corresponding to formula (I) as defined above, wherein said organosilicon compounds have at their end a carboxylic acid function hydroxyl or amine protected; b) optionally, deprotection of the hydroxyl or amine carboxylic acid function; c) optionally grafting a coupling agent onto the modified solid support; d) grafting the protein entity.
- Step a) is advantageously carried out by the following steps: i) removal of the contaminants from the solid support and hydration and / or hydroxylation of its surface; j) introduction into a mixture of at least two solvents comprising at least one non-hydrocarbon solvent; polar, under an inert atmosphere, of an organilicon compound of formula (I) as defined above, said compound having at one end a hydroxyl or protected amino carboxylic acid function, k) silanization of the support obtained in step i) by immersion in the solution prepared in step j),
- step k) 1) optionally, annealing the silanized support obtained in step k), carrying the self-assembled monolayer, at a temperature of between 50 and 120 ° C., for a duration of 5 minutes to one night, and m) rinsing the modified support obtained in step k) or 1) to the acid of a solvent, preferably polar.
- contaminants of the solid support is meant any compound such as grease, dust or other, present on the surface of the support and which is not part of the chemical structure of the support itself.
- step i) can be carried out using one or more solvents and / or oxidizing and / or hydroxylating (for example a sulfochromic mixture), a detergent solution (for example Hellmanex®), photochemical ozone treatment or any other appropriate treatment.
- solvents and / or oxidizing and / or hydroxylating for example a sulfochromic mixture
- a detergent solution for example Hellmanex®
- photochemical ozone treatment or any other appropriate treatment.
- Step j) may advantageously be carried out in a mixture of at least one non-polar hydrocarbon solvent and at least one polar solvent.
- the volume proportions of non-polar solvent and polar solvent are preferably between 70/30 and 95/5.
- a usable non-polar hydrocarbon solvent is cyclohexane and a polar solvent that can be used is chloroform.
- the concentration of the organosilicon compound in the solvent mixture is preferably between 1 ⁇ 10 -5 and 10 -2 mol / liter.
- Step (k) of silanization of the support may be carried out for a time of between 1 minute and 3 days and at a temperature of between -10 ° C. and 120 ° C., depending on the solvents used.
- Step c) grafting a coupling agent may be implemented differently depending on the nature of the terminal function of the group of formula (I).
- a homo-bifunctional or hetero-bifunctional coupling agent may be chosen.
- the functional group for reacting with the protein is either carbonyl at sulfosuccinimidyl (BS3) or sulfo NHS ester (Sulfo-SMCC). It can be:
- BS3 (Sulfosuccinimidyl) suberate (Staros JV (1982) N-Hydroxysulfosuccinimide active esters: Bis (7H-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linker, Biochemistry 21: 3950. ), which is implemented according to scheme 1:
- Sulfo-SMCC 1-cyclohexane-4- (N-maleimidomethyl) sulfosuccinimidylcarboxylate (Samoszuk MK et al., (1989) Antibody, Immunocojugates, Radiopharm 2:37), which is implemented according to Scheme 2 :
- PA or PG can be coupled directly to the chlorosilane chain terminated in NH 2 in the presence of EDC [1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride] (Grabarek Z and Gergely J (1990) Zero- length crosslinking procedure with the use of active esters, Anal Biochem 185: 131).
- EDC 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride
- a hetero-bifunctional coupling agent is:
- the hetero-bifunctional coupling agent is:
- KMUH N-hydrazide of k-maleimidoundecanoic acid (Trail PA, et al (1993) Science 261: 212), the implementation of which is as illustrated by Scheme 5:
- step d) the protein entity is chosen according to the use that is desired of the device:
- the nature of the cell subpopulation that is to be captured is critical to the choice of this protein entity. The latter must have a specific affinity for the targeted subpopulation.
- the CTC capture strategy is based on the recognition properties of cell surface molecules. These molecules are cell adhesion molecules, or membrane receptors. Cellular capture is mediated through the interaction of specific ligands with its receptors or antibodies directed against an epitope of an adhesion molecule or receptor. Whatever the protein entity used for cellular capture
- the strategy for attachment of the recognition protein entity may be in the use of the (5-Succinimidyl-S-acetyl) SATA Acetate Reagent by a two-step process (Durican RJS, Weston PD et al (1983) A It can be used to provide sulfhydryl groups in proteins, and its use in the preparation of conjugates for immunoassays, Anal Biochem 132: 68) as shown in Schemes 6 and 7, or TRAUT reagent (2-immothiolane * HCl).
- Ep-CAM 5 an intercellular adhesion molecule (Gastl G Spizzo G Obrist P et al. (2000) Ep-CAM overexpression in breast cancer as a predictor of survival. The Lancet 356: 1981).
- Ep-CAM is also referred to as 17-1A, ESA, EGP40. It is a 40 kDa transmembrane epithelial glycoprotein encoded by the GA 733-2 gene (Linnenbach AJ,
- Ep-CAM a human antigen is a homophilic cell. cell adhesion molecule J Cell Biol 125: 437). Ep-CAM is suggested as a therapeutic target, particularly in immunotherapy.
- Ber-EP4, or MOC31 or HA-125 may be linked either directly to protein A or protein G, or to the NH 2 , OH or COOH-terminated chlorosilane chain, via a suitable coupling agent, b) Over-expression of HER-2
- the human growth factor receptor 2 (HER-2) / new (c-erbB-2) gene is located on chromosome 17q and encodes a transmembrane protein receptor with tyrosine kinase activity, from the family of factor receptors. epidermal growth (EGFR) or the HER family.
- EGFR epidermal growth
- Anti-HER-2 antibody (trastuzumab) is used in antitumor therapy.
- the anti-HER-2 (anti-human ErB2) antibody is used as a specific marker to capture breast cancer CTCs. Combined with detection by the Ber-EP4 antibody, it will act as a potential CTC marker.
- MUC1 Mucins are expressed by various types of normal epithelial cells in a rough environmental setting such as the air / water interface of the respiratory system, the acidic environment of the stomach, and the complex environment of the tract. intestinal and secretory epithelial surfaces of specialized organs such as the liver, pancreas, gallbladder, kidneys, salivary glands, lacrimal glands and the eye.
- the mucin family comprises at least 17 secreted and uncreated molecules and plays a central role in the maintenance of homeostasis (Hollingsworth MA and Swanson BJ (2004) Mucins in cancer: protection and control of the cell surface. : 45).
- Mucins are high molecular weight glycoproteins with tandem repeats of serine, threonine and proline-rich sequences attached to oligosaccharides by O-glycosidic linkages.
- MUCl is an integral membrane protein. In breast tumors, MUC1 is over-expressed and aberrantly glycosylated (Rahn JJ, Dabbagh L, Pasdar M et al (2001) The importance of MUCl cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients Cancer 91: 1973).
- the anti-MUC1 antibody (CT2 Mab) is used as a specific marker to capture breast cancer CTCs. Combined with detection by the Ber-EP4 antibody, it will act as a potential CTC marker.
- the grafting of the anti-MUC1 antibody can be done like that of the Ber-EP4 antibody.
- Selected membrane antigens comprising an Ep-CAM membrane glycoprotein, the MUC1 glycoprotein, and a HER-2 / neu growth factor receptor are over-expressed tumor markers in CTCs.
- the tumor cells can reveal optimized cell surface molecule density, thereby promoting specific cell uptake.
- the density of CTC in the peripheral blood is a totally unknown parameter. This parameter can vary according to the degree of the pathology and revolves around 1 CTC for 10 3 to 2 ⁇ 10 4 leukocytes.
- the antibodies directed against these molecules are specific and sufficiently selective to allow CTC capture under the best possible conditions in terms of conservation, cell morphology and viability.
- the length of the X 3 -Si (CH 2 ) 22- antibody-coupling agent complex varies between 40 to 50 ⁇ thus producing great flexibility in a favorable presentation of the antibody to the cellular antigen.
- a rapid evaluation gives 8 receptors in a square area of 10 ⁇ .
- the evaluation by atomic force microscopy (AFM) the graft density of organosilicon chains being 3 to 5 molecules per nm, this configuration is very favorable to obtain a good bonding agent grafting and antibody.
- Figure 2 illustrates cellular capture by means of a laminar flow chamber.
- the present method of cellular capture uses a laminar flow chamber (1), made of Plexiglas ®, comprising a cavity (2) of size 20 ⁇ 6 ⁇ 0.2 mm 3 (L ⁇ 1 ⁇ h), and connected to the external through inlet (3) and outlet (4) openings for the circulation of cells and fluids.
- a laminar flow chamber (1) made of Plexiglas ®, comprising a cavity (2) of size 20 ⁇ 6 ⁇ 0.2 mm 3 (L ⁇ 1 ⁇ h), and connected to the external through inlet (3) and outlet (4) openings for the circulation of cells and fluids.
- the wall (5) constituting the floor of the chamber is a glass slide or crystalline Si removable and chemically functionalized.
- This blade is applied in a notch (6) against the cavity (2) of the chamber (1) by means of an O-ring (7) for sealing. It can be provided that the ends of the cavity (2) are rounded so as to promote the positioning of the O-ring (7).
- It is screwed (8) onto the Plexiglas® base by a metal plate (9).
- a Teflon® gasket (9a) separates the glass slide from the metal plate. Any other means known to those skilled in the art and allowing the attachment of the solid support in the cavity can be used. In the case illustrated in FIG.
- the metal plate (9) comprises in its central part a transparent zone (not hatched) which makes it possible to observe the interior of the cavity (2 ) using a microscope (not shown).
- Teflon® joint (9a) has a transparent central area (not hatched). The proportion of opaque and transparent areas may vary.
- the inflow and evacuation of the fluids through the openings (3, 4) are placed under the control of a peristaltic pump (10) imposing the flow rate and the flow velocity.
- the peristaltic pump (10) takes the biological sample (11) in a receptacle (12), provided for this purpose, closed so as to be kept sterile, and injected into the circulation tube (13). It can take reagents (14a, 14b) in the receptacles (14), also closed so as to ensure the proper preservation of the products they contain, and inject them into the circulation tubes (15).
- a valve (16) at the intersection of the circulation tubes (15), (13) and (17) regulates the connection between the circulation tubes (15) and (13) and the introduction tube (17). in the laminar flow chamber (1). The fluids pass through the laminar flow chamber (1) under control of the peristaltic pump (10) and exit through the outlet tube (18).
- a valve (19) allows the fluid of the outlet tube (18) to be directed either towards an evacuation tube (20) or to a recycling tube (21) connected to the cavity (2), which makes it possible to pass again the fluid in the laminar flow chamber (1).
- the recycling tube (21) is connected to the cavity (2) via the receptacle (12), but it is possible to provide a direct connection between the recycling tube (21) and the cavity ( 2).
- the recycling of the biological sample by this circuit allows a better capture efficiency of the target cells.
- the flow direction of the fluids in the device is indicated by arrows.
- the device described above is an example of implementation of the invention. Other variations are included within the scope of the invention.
- the essential characteristic lies in the circulation of the biological sample in a fluid circulation chamber allowing a flow laminar flow whose inner wall is provided with a particular grafting described above.
- the fluid circulation circuit outside the fluid circulation chamber can be arranged according to the reagents that one wishes or not to use.
- the device may comprise a plurality of fluid circulation chambers placed in series or in parallel.
- This fluid flow chamber may be included in any microfluidic device of configuration suitable for cell capture, such as for example a microfluidic device such as that described in US-6,408,878.
- the biological sample is passed through the fluid circulation chamber one or more times, depending on the experimental protocol chosen, it is possible to recover the cells which have been fixed on the grafted wall according to the invention by injecting into the fluid circulation chamber a reagent allowing the stalling of these cells. They are then recovered for analysis and counting.
- the device of FIG. 2 can be constructed as follows: A solid support grafted with hydroxyl-terminated, amino-protected or acid-protected chlorosilane functions is fixed in the cavity (2) as described above, so as to close the cavity (2). The other mechanical elements of the device are placed as in FIG. 2. A deprotection reagent for the terminal functions is injected into the cavity (2) from one of the receptacles (14), then a coupling agent is injected into the cavity (2) and finally the biomolecule of recognition. It is indeed possible, from the mechanical device described in FIG. 2, to functionalize the wall (solid support) grafted with chlorosilanes by an appropriate sequence of injections of reagents, rinsing steps that can be provided intermediately.
- a volume of 3-5 ml of peripheral blood of patients developing breast cancer and / or cancer-free controls is harvested in a conventional manner in Hanks medium (In VitroGen) containing 5mM EDTA (ethylene diamine tetra acid). acetic).
- the nucleated cells (leukocytes and tumor cells) are then separated from red blood cells and platelets on a gradient containing ficoll (Amersham) in pre-adapted tubes (Leucosep).
- Cell fractions containing leukocytes and CTCs are then diluted in Hanks medium containing 0.1% HSA (human serum albumin) at 1 x 10 6 cells per ml before subjecting them to isolation. average of the laminar flow chamber.
- the flow rate of the peristaltic pump is set around 50 ⁇ l / min corresponding to a shear rate of 15 sec -1 and a shear stress of 0.15 dyne / cm 2 .
- IHC immunohistochemistry
- the present method can also be used in obstetrics in prenatal diagnosis for early genetic analyzes of fetal cells in maternal blood (Bianchi, D., 1999) Fetal cells in maternal circulation: feasibility for prenatal diagnosis Br J Maematol 105: 574 Fisk N. (1998) Maternal-fetal medicine and prenatal diagnosis, Curr Opin Obstet Gynecol 10:81).
- the target fetal cell population in our process is trophoblastic, epithelial, and short-lived cells during the first trimester of pregnancy (Shulman LP., 2003) Fetal cells in maternal blood. ).
- the density of this cell population is very low: ⁇ 1 fetal trophoblast for 10 6 maternal cells.
- Cellular uptake can be by means of antibodies directed against membrane epithelial antigens, or an antibody against the human placental lactogen (Latham SE, Suskin HA, Petropoulos A et al (1996)
- a monoclonal antibody to human placental lactogen hormone facilitates isolation of fetal cells from maternal blood in a model system Prenat Diagn 16: 813).
- the present method has a certain advantage over existing methods such as flow cytometry or magnetic beads.
- Isolated fetal cells undergo molecular genetic characterization for the detection of genetic abnormalities (PCR, FISH).
- PCR PCR, FISH.
- Another application of the present method is the detection of CTC in bone marrow and peripheral blood (Chapter 15: Tumor Cell Contamination (2001) Autologous blood and Marrow Transplantation: Proceedings of the Tenth International Symposium, edited by Karel A. Dicke and A Keating).
- CTC CTC in bone marrow and peripheral blood
- the present method relates to a two-step evaluation on the same patient: 1) on the presence and percentage of CTC in the peripheral blood before ablative chemotherapy of the cord, and 2) the absence of CTC on the blood sample processed and purged to obtain a hematopoietic stem cell population for autologous transplantation.
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Abstract
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JP2007519842A JP2008505630A (ja) | 2004-07-09 | 2005-07-07 | 新規のマイクロ流体システム及び細胞捕獲方法 |
CA002573044A CA2573044A1 (fr) | 2004-07-09 | 2005-07-07 | Nouveau systeme microfluidique et procede de capture de cellules |
US11/571,808 US20080318203A1 (en) | 2004-07-09 | 2005-07-07 | Novel Microfluidic System and Method for Capturing Cells |
EP05788488A EP1766404A1 (fr) | 2004-07-09 | 2005-07-07 | Nouveau systeme microfluidique et procede de capture de cellules. |
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FR0407722A FR2872912B1 (fr) | 2004-07-09 | 2004-07-09 | Nouveau systeme microfluidique et procede de capture de cellules |
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US (1) | US20080318203A1 (fr) |
EP (1) | EP1766404A1 (fr) |
JP (1) | JP2008505630A (fr) |
CA (1) | CA2573044A1 (fr) |
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JP2007315955A (ja) * | 2006-05-26 | 2007-12-06 | Fujitsu Ltd | 検出素子、その製造方法、および標的検出装置 |
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GB201003886D0 (en) * | 2010-03-09 | 2010-04-21 | Univ Leeds | Biosensor apparatus and use thereof |
WO2012120266A1 (fr) * | 2011-03-10 | 2012-09-13 | University Of Leeds | Appareil capteur et utilisation de celui-ci |
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US20130330711A1 (en) * | 2012-06-06 | 2013-12-12 | National Taiwan University | Sensor for detection of a target of interest |
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CN103937658B (zh) * | 2014-03-28 | 2015-11-04 | 武汉介观生物科技有限责任公司 | 一种稀有细胞检测芯片及其应用 |
IL284235B (en) | 2015-04-22 | 2022-07-01 | Berkeley Lights Inc | Kits and methods for preparing a microfluidic device for cell culture |
US10799865B2 (en) | 2015-10-27 | 2020-10-13 | Berkeley Lights, Inc. | Microfluidic apparatus having an optimized electrowetting surface and related systems and methods |
SG11201809539RA (en) | 2016-05-26 | 2018-12-28 | Berkeley Lights Inc | Covalently modified surfaces, kits, and methods of preparation and use |
HUE064189T2 (hu) * | 2016-07-28 | 2024-02-28 | Captec Medical Kft | Áramlásból befogó eszköz sejtek vérbõl történõ eltávolítására |
CN107338184A (zh) * | 2017-08-02 | 2017-11-10 | 苏州博福生物医药科技有限公司 | 一种用于捕获细胞或溶液中生物分子的捕获筛及装置 |
EP3444034A1 (fr) * | 2017-08-18 | 2019-02-20 | XanTec bioanalytics GmbH | Cellule d'écoulement pour enrichissement sélectif de particules ou de cellules cibles |
JP2021520847A (ja) * | 2018-04-07 | 2021-08-26 | ルマサイト リミティド ライアビリティ カンパニー | 流体オートサンプラ及び培養器 |
KR20210063362A (ko) | 2018-09-21 | 2021-06-01 | 버클리 라잇츠, 인크. | 작용화된 웰 플레이트, 이의 제조 및 사용 방법 |
CN113000079B (zh) * | 2020-06-02 | 2023-09-22 | 山东大学 | 一种重金属离子检测电化学微流控传感芯片及其制备方法 |
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- 2005-07-07 EP EP05788488A patent/EP1766404A1/fr not_active Withdrawn
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Cited By (1)
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JP2007315955A (ja) * | 2006-05-26 | 2007-12-06 | Fujitsu Ltd | 検出素子、その製造方法、および標的検出装置 |
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US20080318203A1 (en) | 2008-12-25 |
EP1766404A1 (fr) | 2007-03-28 |
CA2573044A1 (fr) | 2006-02-16 |
JP2008505630A (ja) | 2008-02-28 |
FR2872912A1 (fr) | 2006-01-13 |
FR2872912B1 (fr) | 2007-03-02 |
WO2006016044B1 (fr) | 2006-04-06 |
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