Method for real-time measurement of the individual secretions of a cell

Abstract

The present invention relates to a method for real-time measurement of the secretion of at least one compound by at least one individual cell, comprising: the culturing, in a liquid medium, of at least one cell in a culture chamber, at least one wall of which comprises at least one sensitive area, a sensitive area comprising a plurality of ligands, attached to a solid support, each ligand being able to bind specifically to the compound, and an element for real-time transduction of a signal produced by the binding of the compound to one of the ligands; the identification, in a sensitive area, of at least one spot producing a signal; the real-time measurement of the signal produced by the spot identified, representing the amount of compound secreted by an individual cell.

Claims

1. A method for measuring real-time secretion of a compound by an individual cell, the method comprising: (a) culturing the cell in a culture chamber comprising a wall with an area sensitive to the secreted compound, wherein the sensitive area comprises: (i) a ligand attached to a solid support and is capable of binding specifically to the compound and (ii) an optical reading system for measuring in real-time a signal produced during, from, or during and from, the binding of the compound to the ligand, wherein the signal is not mediated by molecules added to, or present in, the culture chamber other than the compound and the ligand; (b) identifying, in the sensitive area, a spot emitting the signal produced in (a), the spot being defined by a continuous and discrete portion of the sensitive area in contact with the secretions of the individual cell, wherein the whole of the portion emits signal; and (c) measuring in real-time the signal produced by the spot, wherein the signal represents the amount of the compound secreted by the individual cell.

2. The method of claim 1, wherein the signal is measured using a near-field optical technique.

3. The method of claim 1, wherein the cell is bacterial, fungal, algal, protozoan, or metazoan.

4. The method of claim 1, wherein the cell originates from a biological sample, a food sample, a water sample, a soil sample, a sludge sample, or an air sample.

5. The method claim 4, wherein the biological sample is blood, cerebrospinal fluid, an oral secretion, sperm, a vaginal secretion, urine, feces, synovial fluid, a biopsy, a specimen originating from a lavage of an organ or of an anatomical cavity, a sample from drainage of a biological fluid, a specimen of a cutaneous or conjunctival serous fluid, or combinations thereof.

6. The method of claim 1, wherein the cell is an immune cell, a nerve cell, an endocrine cell, a stem cell, an epithelial cell, a cell infected with an infectious agent, or a cancer cell.

7. The method of claim 1, wherein the compound is a protein, a polypeptide, a peptide, a lipid, a glycoprotein, a glycolipid, a lipoprotein, an inorganic ion, a small organic molecule, a vesicle, a microbial particle, a viral particle, or combinations thereof.

8. The method of claim 1, wherein the compound is an intercellular signaling compound, an extracellular matrix protein, an immunoglobulin, an infectious agent or a subunit of an infectious agent, or combinations thereof.

9. The method of claim 1, wherein the ligand is a peptide, a polypeptide, a protein, a glycoprotein, an oligosaccharide, a polysaccharide, a nucleic acid, a lipid, a polymer, or an organic compound comprising from 1 to 100 carbon atoms.

10. The method of claim 1, wherein the ligand is an antibody, an antibody fragment, a scFv, an antigen, a hapten, an aptamer, a lectin, or a chelating agent.

11. The method of claim 1, wherein the solid support is a material comprising glass, silicon, an organic polymer, a metal material, or a carbon-based conductor.

12. The method of claim 1, wherein the wall of the culture chamber comprising the sensitive area is a basal wall in which cells can sediment, and the amount of cells in the culture chamber is in a range of about 1 to about 10.sup.6 cells/mm.sup.2 of the surface area of the sensitive area.

13. The method of claim 1, wherein the density of the ligand in the sensitive area is in a range of about 10.sup.8 to 10.sup.12 ligands/mm.sup.2.

14. The method of claim 1, wherein the signal is measured by surface plasmon resonance.

15. The method of claim 1, wherein the solid support is glass, silicon, an organic polymer, metal, a carbon based conductor, or combinations thereof.

16. The method of claim 15, wherein the solid support is glass.

17. The method of claim 16, wherein the glass is a glass prism coated with a 50 nm film of gold.

18. The method of claim 1, wherein the ligand is an antibody or an antigen binding fragment thereof, an antigen, a hapten, an aptamer, a lectin, or a chelating agent.

19. The method of claim 18, wherein the antibody is a monoclonal antibody or an antigen binding fragment thereof.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a diagrammatic representation of the experimental device used in the example. The sensitive area of the device (gold prism) is chemically modified by the attachment of antibodies specific for a compound secreted by cells. Two cell samples were cultured together on the device: in the culture chamber on the left, cells secreting the compound of interest, and in the culture chamber on the right, cells not secreting this molecule. This configuration comprising two culture chambers makes it possible to measure and quantify the nonspecific parasitic signals. The secreted compound is detected in real time, and without labeling, by SPRi, using a LED, a polarizing filter and a CCD camera.

(2) FIG. 2 represents an SPR image of the surface of the gold prism of the device used in the example. This surface is divided up into a sensitive area (at the top) functionalized with the antibody, and a control area (at the bottom) which is not functionalized. The cells were incubated on these areas, in two distinct culture chambers, one in which the cells are not stimulated (on the right), the other in which the cells are stimulated with ConA (on the left). Light spots corresponding to the secreted compounds attached to the sensitive surface appeared on the part of the sensitive area on which the ConA-stimulated cells were incubated (top left). The part of the sensitive area which is boxed-in in white is magnified in FIG. 3.

(3) FIG. 3 is a magnification of the part of the sensitive area boxed-in in white in FIG. 2. Masks in the shape of a circle are represented; they delimit portions of interest of the sensitive area corresponding to a spot (circles c, d, e and f). Control portions (no spot) were also selected (circles a and b).

(4) FIG. 4 represents the variation in relative reflectivity (y-axis, as %) of the portions a, b, c, d, e and f of the sensitive area that are represented in FIG. 3, as a function of time (x-axis, in minutes).

EXAMPLE

(5) In this example, the interferon gamma (IFNγ) secreted by mouse splenocytes was detected in real time, on the individual-cell scale, by surface plasmon resonance imaging (SPRi), following cell stimulation using concanavalin A (conA). The stimulated cells were studied, as were cells that were not stimulated, and therefore not secreting IFNγ, that were used as a negative control, on the same device.

(6) 1. Experimental Device and Reagents

(7) In this example, the device (see FIG. 1) comprises: two culture and analysis chambers containing AIMV medium (Gibco) making it possible to maintain in culture (viability and secretory activity) two cell samples in parallel; a rat monoclonal antibody directed against mouse interferon gamma (anti-IFNγ, BD biosciences) used as specific ligand for the secreted product; a glass prism coated with a 50 nm film of gold (GenOptics) as solid support for the anti-IFNγ antibody; an SPRi-lab+ surface plasmon resonance imager (GenOptics) as signal transduction element enabling real-time optical reading of the interactions between the antibody (anti-INFγ IgG) and the secreted compound (mouse INFγ); an incubator (Memmert) for maintaining the whole of the device at 37° C., a temperature favorable to cell secretion.

(8) Reagents required: Phosphate buffered saline (PBS, Sigma-Aldrich); Roswell Park Memorial Institute culture medium (RPMI, Sigma-Aldrich); 11-Mercaptoundecanoic acid (Sigma-Aldrich); N,N′-Dicyclohexylcarbodiimide (DCC, Sigma-Aldrich); N-hydroxysuccinimide (NHS, Sigma-Aldrich); dimethylformamide (DMF, Sigma-Aldrich); Antibiotic solution: 5000 U/ml penicillin—5 mg/ml streptomycin (Sigma-Aldrich); Concanavalin A (Sigma-Aldrich).
2. Production of the Sensitive Area: Attachment of the Antibodies to the Gold Surface

(9) The antibodies were attached to the gold surface by complexation of thiolated products. Briefly, the antibodies were coupled to a molecule of 11-mercaptoundecanoyl-1-N-hydroxysuccinimide ester (Thiol-NHS), synthesized using 11-mercaptoundecanoic acid, N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in dimethylformamide (DMF). The coupling with the antibody was carried out in PBS equilibrated at pH8, overnight at 4° C., with a Thiol-NHS/antibody molar ratio of 10:1. The antibodies thus coupled were subsequently purified by ultracentrifugation on a membrane having a cut-off threshold of 30 kDa, then concentrated to 2 μM in PBS containing 10% glycerol. The coupled antibodies were deposited and spread out on the gold surface and thus grafted via sulfur-gold bonding. A nonfunctionalized control area and a sensitive area, functionalized with the antibodies, were delimited on the same chip.

(10) 3. Cell Culture

(11) Murine splenocytes were taken from a C57B1/6 mouse. After removal of the spleen, the cells were dissociated on a sieve grille and suspended in a RPMI medium. After centrifugation for 5 min at 300 g, the cell suspension was incubated for 5 min in the presence of a red blood cell lysis buffer (8.3 g/l of NH.sub.4Cl, 0.8 g/l of NaHCO.sub.3, 0.04 g/l of EDTA) in order to remove this cell type from the sample. After washing in PBS, the cells were again centrifuged (5 min, 300 g) and suspended at a density of 10.sup.6 cells/ml in RPMI medium containing 10% of fetal calf serum (FCS) and 1% of antibiotic solution. The cell viability was verified by counting after staining with trypan blue. Just before the experiment, the cells were centrifuged for 5 min at 300 g and resuspended at a density of 200 000 cells/ml in a buffered AIMV medium containing or not containing concanavalin A at 2 μg/ml.

(12) 4. Detection of Cell Secretions

(13) Before the analysis, the sensitive area was treated by incubation in AIMV medium for 30 min. The prism was then inserted into the SPR imager, under the culture chambers, in order to complete the device. The whole device was placed in an incubator at 37° C. 500 μl of cell suspension prepared as described above were deposited in each of the culture chambers and incubated for the time of the experiment. Thus, cells stimulated with ConA were cultured in one chamber, while nonstimulated control cells were incubated in the other chamber. The binding of the secreted compounds by the antibodies was detected by SPRi. A 12-bit CCD camera made it possible to quantify the signal consisting of the variations in reflectivity caused by the IFNγ-antibody interaction in the sensitive area. Images of the variations in reflectivity at the surface of the sensitive area were recorded in real time.

(14) Spots appeared on the surface of the sensitive area (FIG. 2), formed by the compounds secreted by each individual cell that were bound by the antibodies of the sensitive area. “Masks” were then drawn on the images (represented by the circles denoted by a, b, c, d, e, and f in FIG. 3) in order to delimit portions of the sensitive area corresponding, a priori, to antibodies binding the IFNγ produced by one and the same cell (circles c, d, e and f). Portions with no spot were also selected as controls (circles a and b) in the sensitive area in the presence of stimulated cells.

(15) The mean of the variation in reflectivity per pixel contained in each of the portions selected was plotted over time for each portion c, d, e and f (likewise for the control portions a and b). The variation in “relative reflectivity” expressed as percentage reflectivity, of the portions of the sensitive area containing the stimulated cells, was calculated by subtraction of the background noise, i.e. of the mean variation in reflectivity recorded in the part of the sensitive area containing the nonstimulated and therefore nonsecretory control cells (FIG. 4).

(16) The curves c, d, e and f of FIG. 4 show variations in reflectivity with kinetics specific to each spot, thus demonstrating individual behaviors specific to each cell.