USE OF FLUORESCEIN DICARBONATE DERIVATIVES AS A CELL MARKER

Abstract

The invention relates to fluorescein derivative compounds and the use thereof as fluorescent markers. Said markers can be used in particular in biology and in medicine, particularly in the field of ophthalmology. The non-fluorescent fluorescein derivatives are activated in the cell, thereby allowing the detection of living cells as well as the quantification of cell viability by fluorescence, without being toxic to cells.

Claims

1. A method of cellular marking of cells comprising the steps of contracting the cells with a compound of general formula (I), ##STR00019## wherein: R.sup.1 is selected from a C.sub.3-C.sub.20 alkyl, a polyethylene glycol or a polypropylene glycol, malic acid derivatives and corresponding esters, sugars, and natural polysaccharides; X.sup.1 is selected from H, Cl, F, Br and NO.sub.2; X.sup.2 is selected from H, Cl, Br and N(CH.sub.2CO.sub.2H).sub.2; R.sup.2 is selected from H, NCS and CO.sub.2R.sup.3; and R.sup.3 is selected from H or a C.sub.1-C.sub.3 alkyl, and its pharmaceutically acceptable salts, assessing the presence of stain or fluorescence in said cells.

2. The method according to claim 1, wherein R.sup.1 is a C.sub.5-C.sub.10 alkyl.

3. The method according to claim 1, wherein R.sup.2 is hydrogen.

4. The method according to claim 1, wherein X.sup.2 is hydrogen.

5. The method according to claim 1, wherein X.sup.2 is hydrogen.

6. The method according to claim 1, wherein the cell marking is carried out in vitro.

7. The method according to claim 1, wherein the cell marking is carried out in vivo or ex vivo.

8. The method according to claim 1 to 7, wherein the cells are endothelial cells.

9. The method according to claim 8, wherein the cells are corneal endothelial cells.

10. A pharmaceutical composition comprising a compound of formula (I) defined in claim 1 and an excipient and/or a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, wherein the composition is an aqueous composition.

12. The pharmaceutical composition according to claim 10, wherein the composition is in the form of a gel or a film.

13. The pharmaceutical composition according to claim 11, wherein the excipient and/or the carrier is suitable for ophthalmic use.

14. A method for preparing a compound of formula (I) ##STR00020## wherein R.sup.1 is selected from a C.sub.3-C.sub.20 alkyl, a polyethylene glycol or a polypropylene glycol, malic acid derivatives and corresponding esters, sugars, and natural polysaccharides; X.sup.1 is selected from H, Cl, F, Br and NO.sub.2; X.sup.2 is selected from H, Cl, Br and N(CH.sub.2CO.sub.2H).sub.2; R.sup.2 is selected from H, NCS and CO.sub.2R.sup.3; and R.sup.3 is selected from H or a C.sub.1-C.sub.3 alkyl and its pharmaceutically acceptable salts, wherein the method comprises the steps of: a) Reaction between a diphenol (II), ##STR00021## with phosgene or a derivative thereof in the presence of base leading to the formation of the synthesis intermediate of formula (III) ##STR00022## Reaction of the intermediate (III), ##STR00023## with an alcohol of formula (IV), R.sup.1OH, in the presence of base to obtain the compound of formula (I).

15. A method for assessing the cellular viability of cells which comprises the steps of contacting the cells with a compound of general formula (I) ##STR00024## wherein R.sup.1 is selected from a C.sub.3-C.sub.20 alkyl, a polyethylene glycol or a polypropylene glycol, malic acid derivatives and corresponding esters, sugars, and natural polysaccharides; X.sup.1 is selected from H, Cl, F, Br and NO.sub.2; X.sup.2 is selected from H, Cl, Br and N(CH.sub.2CO.sub.2H).sub.2; R.sup.2 is selected from H, NCS and CO.sub.2R.sup.3; and R.sup.3 is selected from H or a C.sub.1-C.sub.3 alkyl and its pharmaceutically acceptable salts, assessing the presence of staining or fluorescence of the cells, the staining or fluorescence being indicative of the presence of living cells.

16. The method according to claim 15, wherein R.sup.1 is a C.sub.5-C.sub.10 alkyl, and/or R.sup.2 is hydrogen and/or X.sup.1 is hydrogen and/or X.sup.2 is hydrogen.

17. The method according to claim 15, wherein the cell marking is carried out in vitro.

18. The method according to claim 15, wherein the cell marking is carried out in vivo or ex vivo.

19. The method according to claim 15, wherein the cells are endothelial cells.

20. The method according to claim 15, wherein the cells are corneal endothelial cells.

Description

DESCRIPTION OF FIGURES

[0118] FIG. 1: LDH assay by enzymatic reaction

[0119] FIG. 2: Cytotoxicity of calcein-AM (4 ?M) and compound 1 in solution in Cremophor? EL [CAS 61791-12-6] (molar ratio 1/1) designated by the reference F4267 (40 ?M and 1000 ?M) on HCE-2 cells, expressed in % as a function of time after single contact (0=45 min, 24 h and 48 h).

[0120] FIG. 3: Images of HEL-299 cells obtained using the MVX10 macroscope under the FITC filter after staining: A: Calcein-AM (4 ?M); B: F4267 (4 ?M); C: F4267 (40 ?M) (?2.5 and ?6.3 objectives for the first and second line respectively).

[0121] FIG. 4: Endothelial cell loss of human corneas preserved in organoculture and contacted with the product F4267 (40 ?M) either repeatedly every day for 7 days or uniquely on DO, before each being exposed daily to the light under the FITC filter

[0122] FIG. 5: Viability measured in % by fluorescence analysis on human corneas, one of which is incubated daily with the product F4267 (images A and A) and the other uniquely (images B and B) on D0, but both exposed daily to light under the FITC filter. Images A and B represent the fluorescence after incubation with the product F4267 on D0 and images A and B represent the fluorescence after incubation with Calcein-AM at the end of the experiment on D7 to measure cell viability.

[0123] FIG. 6: Images of B4G12 cells obtained using an epifluorescence microscope under the FITC channel and after marking with calcein-AM (4 ?M) and the product F4267 (40 ?M) with and without rinsing with PBS according to example 5.

[0124] FIG. 7: Images of B4G12 cells obtained using an epifluorescence microscope under the FITC channel after marking for 5 days with calcein-AM (4 ?M) and the product F4267 (40 ?M), with and without rinsing with PBS, according to example 5.

[0125] FIG. 8: Images of B4G12 cells obtained using an epifluorescence microscope under the DAPI channel (405 nm) after marking for 5 days with calcein-AM (4 ?M) and the product F4267 (40 ?M), with and without rinsing with PBS, according to example 5.

EXAMPLES

[0126] The examples given in the present application are illustrative and do not limit the scope of the invention.

Example 1: Synthesis of Compounds

1. Abbreviations

[0127] AcOEt Ethyl acetate [0128] DCE 1,2-dichloroethane [0129] DCM Dichloromethane [0130] DMAP 4-Dimethylaminopyridine [0131] DMSO Dimethyl sulfoxide [0132] PE Petroleum ether [0133] HRMS High resolution mass spectrometry [0134] NMR Nuclear magnetic resonance

2. Operating Procedures

[0135] The general synthesis of fluorescein dicarbonate compounds is carried out from fluorescein or corresponding derivatives. The synthesis is carried out in 2 steps: a) fluorescein or the corresponding derivative reacts with a reagent generating phosgene (phosgene, diphosgene or triphosgene) in the presence of base and the solvent is evaporated, b) the synthesis intermediate obtained reacts with an alcohol in the presence of a base to obtain the final compound of general formula (I).

a) Synthesis Protocol in the Presence of Primary Alcohols

[0136] Diphosgene (6 eq.) then triethylamine (1 eq.) are successively added drop by drop to a suspension of fluorescein (1 eq.) in DCE. The reaction mixture is heated at 60? C. for 2 hours. The solvent is evaporated with a Schlenk line and the product is dried for several hours in the dark.

[0137] In another Schlenk tube, the primary alcohol (2 eq.) is dissolved in DCE and triethylamine (2 eq.) is added drop by drop. The fluorescein residue, previously obtained, is dissolved in DCE and the alcohol solution is added drop by drop over 10 minutes. The reaction mixture is heated for 3 hours at 70? C.

[0138] The reaction medium is poured into water and extracted with DCM. The organic extracts are combined, washed with water, then with a saturated aqueous NaCl solution, dried on MgSO.sub.4, and the solvent is evaporated by rotary evaporation. The crude product is finally purified by column chromatography on silica gel (AcOEt/EP) to obtain the desired product.

b) Synthesis Protocol in the Presence of Secondary Alcohols

[0139] Diphosgene (6 eq) then diisopropylethylamine (7 eq) are successively added drop by drop to a suspension of fluorescein (1 eq.) in DCE. The reaction mixture is heated at 60? C. for 2 hours. The solvent is evaporated with a Schlenk line and the product is dried for several hours in the dark.

[0140] In another Schlenk tube, the secondary alcohol (2 eq.) is dissolved in DCE. Diisopropylethylamine (2 eq.) and DMAP (1 eq.) are added. The fluorescein residue, previously obtained, is dissolved in DCE and the alcohol solution is added drop by drop over 10 minutes. The reaction mixture is heated for 3 hours at 70? C.

[0141] The reaction medium is poured into water and extracted with DCM. The organic extracts are combined, washed with water, then with a saturated aqueous NaCl solution, dried on MgSO.sub.4, and the solvent is evaporated by rotary evaporation. The crude product is finally purified by column chromatography on silica gel (AcOEt/cyclohexane, then DCM/AcOEt) to obtain the desired product.

3. Synthesized Compounds

Compound 1: Fluorescein Pentyl Dicarbonate

[0142] ##STR00015##

[0143] Fluorescein pentyl dicarbonate is purified on silica gel (AcOEt/EP 15:80) and isolated in the form of a transparent oil (36%). .sup.1H NMR (500 MHz, CDCl.sub.3) ? (ppm): 8.04 (dt, .sup.3J=7.5 Hz, .sup.4J=1.0 Hz, 1H), 7.69 (td, .sup.3J=7.4 Hz, .sup.4J=1.1 Hz, 1H), 7.64 (td, .sup.3J=7.4 Hz, .sup.4J=1.1 Hz, 1H), 7.22-7.14 (m, 3H), 6.91 (dd, .sup.3J=8.7 Hz, .sup.4J=2.3 Hz, 2H), 6.84 (d, .sup.3J=8.7 Hz, 2H), 4.26 (t, .sup.3J=6.7 Hz, 4H), 1.82-1.70 (m, 4H), 1.46-1.31 (m, 8H), 0.94 (t, .sup.3J=6.7 Hz 6H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? (ppm): 169.11, 153.08, 152.93, 152.44, 151.56, 135.34, 130.12, 129.07, 126.10, 125.31, 124.05, 117.26, 116.61, 109.93, 81.54, 69.41, 28.26, 27.81, 22.30, 13.95.

Compound 2: Fluorescein Decyl Dicarbonate

[0144] ##STR00016##

[0145] Fluorescein decyl dicarbonate is purified on silica gel (AcOEt/EP 20:80) and isolated in the form of a transparent oil (77%). H NMR (500 MHz, DMSO-d.sub.6) ? (ppm): 8.07 (dt, 3J=7.6 Hz, .sup.4J=1.0 Hz, 1H), 7.83 (td, .sup.3J=7.4 Hz, .sup.4J=1.1 Hz, 1H), 7.77 (td, .sup.3J=7.4 Hz, .sup.4J=1.1 Hz, 1H), 7.46-7.36 (m, 3H), 7.06 (dd, .sup.3J=8.7 Hz, .sup.4J=2.4 Hz, 2H), 6.91 (d, .sup.3J=8.7 Hz, 2H), 4.22 (t, .sup.3J=6.6 Hz, 4H), 1.77-1.59 (m, 4H), 1.38-1.21 (m, 28H), 0.85 (t, .sup.3J=6.6 Hz, 6H). .sup.13C NMR (126 MHz, DMSO) ? (ppm): 168.85, 152.93, 152.60, 151.23, 136.52, 131.09, 129.75, 125.84, 125.55, 124.57, 118.52, 116.90, 110.48, 81.28, 69.39, 31.75, 29.38, 29.15, 29.04, 28.39, 25.59, 22.56, 14.42. HRMS (ESI-TOF) C.sub.42H.sub.52O.sub.9 found: 701.3644 [M+H].sup.+, calculated: 701.3684.

Compound 3: Fluorescein Dicarbonate Diethyl (L-)Malate

[0146] ##STR00017##

[0147] Fluorescein dicarbonate diethyl (L-) malate is purified on silica gel (AcOEt/cyclohexane 35:65, then DCM/AcOEt 95:5).sup.1H NMR (400 MHz, CDCl.sub.3) ? (ppm): 8.06 (d, .sup.3J=7.1 Hz, 1H), 7.78-7.60 (m, 2H), 7.27-7.12 (m, 3H), 6.97 (dd, .sup.3J=8.7 Hz, .sup.4J=2.4 Hz, 2H), 6.87 (d, .sup.3J=8.8 Hz, 2H), 5.50 (t, .sup.3J=6.1 Hz, 2H), 4.40-4.13 (m, 8H), 3.10-2.92 (m, 4H), 1.43-1.16 (m, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) ? (ppm): 169.02, 168.74, 168.08, 152.87, 152.18, 152.13, 151.48, 151.48, 135.37, 130.14, 129.12, 125.98, 125.32, 123.98, 117.09, 116.89, 109.82, 77.33, 77.02, 76.70, 72.27, 62.30, 61.43, 36.02, 14.13, 14.08.

Compound 4: Fluorescein (L)-Malic Acid Dicarbonate

[0148] ##STR00018##

[0149] Fluorescein (L)-malic acid dicarbonate is purified on silica gel (AcOEt/EP 75:25, then DCM/AcOEt 97:3).sup.1H NMR (400 MHz, CDCl.sub.3) ? (ppm): 8.08-8.01 (d, .sup.3J=7.1 Hz 1H), 7.73-7.58 (m, 2H), 7.17 (d, .sup.3J=7.1 Hz, 1H), 7.13 (d, .sup.4J=2.2 Hz, 2H), 6.86 (dd, .sup.3J=8.7 Hz, .sup.4J=2.2 Hz, 2H), 6.81 (d, .sup.3J=8.6 Hz, 2H), 4.36 (hept, .sup.3J=6.7 Hz, 2H), 3.35 (d, .sup.3J=7.4 Hz, 4H).

Example 2: In Vitro Study on Adherent Cells of the Research Laboratory Test Type

1. Material and Methods

Cytotoxicity

[0150] The cytotoxicity of a solution of compound 1 in Cremophor? EL [CAS 61791-12-6](molar ratio 1/1), designated under the reference F4267, compared to that of calcein-AM, was evaluated on the human corneal epithelium cell line immortalized by SV40, HCE-2 (ATCC CRL-11135) using two solutions of F4267 of concentrations [40 ?M] and [1000 ?M] or calcein-AM (FP-F19820, Interchim) [4 ?M] diluted in optiMEM (11058021, Gibco, ThermoFisher) and deposited for 45 minutes on the cells (100 ?L per well seeded 24 hours previously at 1.10.sup.5 cells/mL [3788, Corning, surface: 0.32 cm.sup.2]. After 45 minutes of contact with compound 1 or with calcein-AM, the cells were rinsed with their culture medium, then returned to culture in an oven with 5% CO.sub.2, at 37? C. Cytotoxicity was measured by dosing lactate dehydrogenases (LDH) released into the supernatant using the CyQUANT LDH Cytotoxicity Assay kit (C20301, ThermoFisher, see cytotoxicity test below) and expressed as % of dead cells using an internal standard. Cytotoxicity was evaluated 45 minutes (before rinsing), 24 h and 48 h after the start of incubation (3 wells per condition, n=1 experiments).

Cytotoxicity Test

[0151] Lactate DeHydrogenases (LDH) are a family of ubiquitous cytosolic enzymes present in almost all cell types. Membrane damage causes a release of LDH into the external environment. There is a direct correlation between cellular cytotoxicity which causes membrane damage and the amount of LDH present in the extracellular environment. In the cytotoxicity test, LDH is measured by the enzymatic reaction shown in FIG. 1. LDH transforms pyruvate into lactate at the same time as there is a reduction of NAD+ into NADH. The NADH thus produced reacts with fluorescent or luminescent molecules.

[0152] The cytotoxicity test was carried out with tetrazolium salt (INT) which is converted into formazan. The staining was then studied using a spectrophotometer. The concentration of formazan being proportional to the amount of LDH present in the medium, this was quantified by an absorbance reading at 555 nm, against a blank measurement at 650 nm to eliminate the parasitic absorbance of the medium. The percentage of dead cells is then calculated from the formula below, with the maximum LDH activity being that of a well from which all cells have been lysed, and the basal LDH activity, that of a non-treated well.

[00001]$\%\mathrm{dead}\mathrm{cells}=\left(\frac{L\mathrm{DH}\mathrm{activity}\mathrm{of}\mathrm{the}\mathrm{treated}\mathrm{well}-\mathrm{basal}L\mathrm{DH}\mathrm{activity}}{\mathrm{maximum}L\mathrm{DH}\mathrm{activity}-\mathrm{basal}L\mathrm{DH}\mathrm{activity}}\right)*100$

Quality of Marking

[0153] The quality of cytoplasmic marking by compound 1 on cells was evaluated on the adherent line of normal human fetal lung fibroblasts HEL299 [ATCC CCL-137], because they form a cellular monolayer (a single thickness facilitating the evaluation of cellular morphology and enumeration). Cells were seeded at 1.10.sup.5 cells/mL 72 h before marking, then marked with the solution of compound 1 designated by the reference F4267 at [4 ?M] and [40 ?M] or with a solution of Calcein-AM [4 ?M], then observed in a sterile Petri dish under the FITC filter of a macroscope (macro-zoom microscope, MVX10, Olympus, Tokyo, Japan) (2 wells per condition, n=1 experiments).

2. Results

Cytotoxicity

[0154] At 45 min, the cytotoxicity (%) induced on HCE-2 was very low: 0.6?0.5% for calcein-AM, 0.8?0.5% for F4267 at 40 ?M and 2.2?0.6% for F4267 1000 ?M.

[0155] At 24 h, the cytotoxicities were 26.4?14.1% for calcein-AM, 7.3?3.4% for F4267 at 40 ?M and 21.6?2.7% for F4267 at 1000 ?M.

[0156] At 48 h, the cytotoxicities were 18.8?11.1% for calcein-AM, 6.1?0.9% for F4267 at 40 ?M and 22.3?3.8% for F4267 at 1000 ?M (FIG. 2).

Quality of Marking

[0157] The marking of HEL299 cells allowed to visualize the cell cytoplasms in a similar way with the Calcein-AM solution [4 ?M] and with the F4267 solution at [4 ?M] and [40 ?M] respectively (FIG. 3).

Example 3: Ex Vivo Study on Endothelium of Whole Corneas, of Cornea Bank Test Type

1. Material and Methods

Cytotoxicity

[0158] The cytotoxicity of compound 1 (solution F4267) was evaluated on the endothelium of whole corneas, using pairs of human corneas, preserved in organoculture and rejected for transplantation by a cornea bank. The 2 corneas from the same donor have the same biological features and constitute the best comparator of each other. Endothelial Cell Density (ECD) was first evaluated before experimentation using cell counting by image analysis [Cell Tissue Bank, 2017, 18 (2), 185-191, Jumelle and al.], then the endothelium of both corneas was contacted with a solution of F4267 [40 ?M] for 45 minutes at room temperature, under sterile conditions [Invest Ophthalmol Vis Sci. 2011, 52 (8), 6018-6025, Pipparelli and al.]. The corneas were then observed using the MVX10 macroscope with the ?0.8 objective under the FITC filter, then returned to organoculture. In order to evaluate the toxicity by repeated incubation, the cornea of the right eye (RE) was contacted daily with a solution of F4267 [40 ?M in OptiMEM, 45 minutes, room temperature] for 7 days while the cornea of the left eye (LE) was only incubated with optiMEM (solvent alone). The corneas were exposed daily to blue light from the FITC filter (same intensity of XCite lamp at 12/100) under the ?0.8 objective of the MVX10 macroscope. The DCE was measured again on D7 using the Hoechst-Ethidium-Calcein-AM staining (Invest Ophthalmol Vis Sci. 2011, 52 (8), 6018-6025, Pipparelli and al) (1 pair of corneas, n=1).

Quality of Marking

[0159] The quality of cytoplasmic marking by compound 1 (solution F4267) on corneal endothelium was compared to that of Calcein-AM by staining paired corneas. The endothelial surface of the corneas was incubated for 45 minutes either with the F4267 solution (40 ?M on the left eye, LE) or with Calcein-AM (4 ?M on the right eye, RE) following a validated staining protocol. (Invest Ophthalmol Vis Sci. 2011, 52 (8), 6018-6025, Pipparelli and al.) (n=2 pairs of corneas).

2. Results

Cytotoxicity

[0160] The DCE in viable cells on D7, after daily contact of the cornea of the RE with the product F4267 followed by observation at low magnification for 7 days, was 1832 cells/mm.sup.2. This DCE was 1612 cells/mm.sup.2 for a single contact of the cornea of the LE but also with daily exposure to light. Cell loss is therefore comparable, indicating that repeated incubation is well tolerated. (FIG. 4).

[0161] The images in FIG. 5 illustrate the viability measured in % after marking with Calcein-AM on D7 by fluorescence image analysis: cornea A (RE) repeatedly contacted every day for 7 days with the product F4267, cornea B (LE) contacted only on DO but both exposed daily to light under the FITC filter.

Quality of Marking

[0162] The product F4267 [40 ?M] allows a marking differentiating dead cells from living cells (FIGS. 5A and 5B, on DO). In addition, it has been shown that the product F4267 [40 ?M] allows a marking differentiating dead cells from living cells in a manner similar to Calcein-AM [4 ?M] on the entire cornea.

Example 4: Marking Different Cell Types

[0163] Corneal endothelial cells (B4G12), fibroblasts (Hel-299), and corneal epithelial cells (HCE-2) were cultured on an 8-well culture slide (LabTek, 177445) for 5 days in corresponding culture media described below.

B4G12 culture medium: Gentamycin, OptiMEM, SVFD, CaICl.sub.2, Ascorbic acid, EGF, Chondroitin sulfate, SB203580, Y27632.
HCE-2 culture medium: DMEM GlutaMax, F12-HAM, SVFD, Antibiotic/mycotic, EGF.
Hel-299 culture medium: DMEM GlutaMax, SVFD, ATB/M

[0164] Upon confluence, cell marking is carried out for 45 minutes with solutions comprising Calcein-AM or the product F4267 at different concentrations (solutions A, B, C and D), before rinsing with PBS. Then the culture wells are removed, and the slide is mounted with a coverslip and Vectashield.

Solution A: calcein-AM (4 ?M), Hoescht ( 1/200), ethydium ( 1/500)
Solution B: F4267 (40 ?M), Hoescht ( 1/200), ethydium ( 1/500)
Solution C: F4267 (4 ?M), Hoescht ( 1/200), ethydium ( 1/500)
Solution D: F4267 (400 ?M), Hoescht ( 1/200), ethydium ( 1/500)

[0165] The slides are then observed under a macroscope with ?0.8 and ?6.3 magnification and with an epifluorescence microscope with ?10 and ?60 magnification. The different wells are observed under the same conditions at each magnification to allow a comparison of the intensities and the quality of the marking with identical image processing.

[0166] The cytoplasms of living cells, regardless of the cell type, are clearly visible with compound F4267.

Example 5: Evaluation of the Impact of Cell Marking on Cell Viability

[0167] The experiment is carried out with HCEC-B4G12 corneal endothelial cells (CVCL_2065). The cells are thawed and cultured in their culture medium. The cells are trypsinized once a week before being seeded in wells of 24-well slides at a concentration of 1.10.sup.5 cells/mL. The cells are then incubated for 5 days at 37? C., 5% CO.sub.2 until 90% confluence.

[0168] The different wells are stained daily with Calcein-AM (4 ?M) or product F4267 (40 ?M), according to the following conditions: marking for 45 minutes, then rinsing with PBS or marking for 45 minutes without rinsing. The marking and possibly rinsing step is repeated every day for 4 successive days, then after 2 days without marking, the marking is repeated again for 2 consecutive days.

[0169] The marking steps are carried out in duplicate. Afterwards, each marking, a well for each of the conditions is observed by epifluorescence microscopy (Olympus IX81 Microscope, FITC channel, ?4 objective, L: 25, Texp: 142.7 ms), while the second well (control) is kept in the darkness in order to evaluate the impact of fluorescence observation at the end of the study. On the last day of the study, the marking is carried out with calecin-AM (4 ?M) in the presence of Hoescht ( 1/500) or the product F4267 (40 ?M) in the presence of Hoescht ( 1/500), and the wells are imaged in MIA?4 (reconstruction of several fields to obtain a complete image of the well).

[0170] The marking steps are carried out in a clean room under PSM to ensure sterility, and the culture dishes are parafilmed during observation to limit possible contamination.

[0171] FIGS. 6 and 7 represent the cell layers observed on DO, therefore the first day of marking, and on D5, that is to say the last day of marking. It appears that the cell layers of cells marked with Calcein-AM are less dense whether for the experiments in the presence or absence of rinsing, unlike the cell layers whose cells were marked with F4267.

[0172] FIG. 8 corresponding to the images of the cells under the DAPI channel confirms the absence of cell layer for the cells marked with calcein-AM.

[0173] These different observations conclude that the product F4267 appears to be non-toxic, or at least less toxic than calcein-AM for cells, since no negative influence of the marker F4267 on the cell layer is demonstrated.