Method for obtaining cells from corneas

Abstract

The present invention relates to a method for obtaining corneal cells with an increased yield and kits for carrying out said method.

Description

[0001]METHOD FOR OBTAINING CELLS FROM CORNEASFIELD OF INVENTIONThe present invention relates to the field of medicine, in particular to a method to samplecorneal cells.BACKGROUND ARTThe impression cytology (IC) is a minimally invasive method widely used to obtainepithelial cells from the ocular surface, mostly from the conjunctiva, from normal individuals,human and animal, and subjects with a number of ocular surface pathologies and whichrequires the use of anesthetic. IC is used to evaluate dry eye disease (DED), blepharitis andmeibomian gland dysfunction (MGD), allergic conjunctivitis, ocular burns, and squamousmetaplasia, amongst others. IC is based on the use of filter membranes which are pressedagainst the surface of the ocular surface for a set time and peeled off. This leads to the peelingof the first layers of the surface cells which stay attached to the filter membrane. Despite itsadvantages of being a quick procedure, minimally invasive and requiring minimal equipmentand expertise, IC yields a relative low number of cells and the cells obtaining are of relativelypoor quality, especially when collecting corneal samples.Therefore, a new method of collecting cells from the cornea dealing with the existentproblem of IC is necessary.SUMMARY OF THE INVENTIONThe inventors have developed a new method for obtaining cells from corneas, preferablyepithelial cells, which improves the yield of the number of cells from one single samplecollection, opening up the possibility of complex analysis of the cells obtained as well asreducing the number of samples required to diagnose or detect corneal diseases. In addition,the inventors improved the quality of the cells obtained leading to physiological more relevantanalysis. In a first aspect, the present invention relates to a method for obtaining corneal cellscomprising the steps of:(i) applying an anesthetic solution to the ocular surface to be sampled, and(ii) scraping the corneal surface with a polyethersulfone (PES) membrane.A further aspect of the present invention relates to a kit for carrying out the methodaccording to the invention, comprising an anesthetic solution and a PES membrane.A further aspect of the present invention relates to the use of the kit according to theinvention for obtaining corneal cells.BRIEF DESCRIPTION OF THE FIGURESFigure 1. Relative cell viability (%) of A-431 cells treated with different concentrations ofthe Barraquer in-house anesthetic, and different incubation times, in comparison withuntreated cells, n=6, * p < 0.001. SDS, sodium dodecyl sulfate.Figure 2. Relative cell viability (%) of A-431 cells treated with different concentrations ofthe “Colirofta” or “Colirofta doble” anesthetics, and different incubation times, in comparisonwith untreated cells, n=6, * p < 0.001. SDS, sodium dodecyl sulfate.Figure 3. Comparison of the relative cell viability (%) of cells treated with the Barraquerin-house, the “Colirofta” or the “Colirofta Doble” anesthetics in comparison with untreated cells,n=6, * p < 0.001.Figure 4. Comparison of the cell count with the 12 tested conditions. (A) Cell count ofpositive (hybridoma cells) and negative (PBS) controls with the 6 tested membranes. (B) Cellcount for each of the 12 impression cytology conditions, n=3, *p < 0.05. CelAc, celluloseacetate; NiCel, nitrocellulose; PES, polyethersulfone; PI, patient interface; s, seconds.Figure 5. Comparison of the number of cells collected from the cornea of pig eye witheach membrane type, both impression cytology and membrane scraping cytology included.CelAc, cellulose acetate; PES, polyethersulfone; IC, impression cytology; MSC, membranescraping cytology.Figure 6. Comparison of the number of cells collected from the cornea of pig eye byforceps or scraping using three different membranes, cellulose acetate (CelAc) 0.45 µm, PES0.45 µm and PES 0.20 µm, and including or not lubrication, n=3, * p < 0.05.Figure 7. Comparison of the number of cells collected according to eye lubrication in themembrane scraping cytology method with PES 0.20 µm membrane performed on humancornea. Figure 8. Total amount of protein quantified by BCAs assays in pig corneas collectedwith membrane scraping cytology method and lysed with 5 different protein extraction buffers.Mean and SD are shown.Figure 9. Proteins identified in human live corneas collected with membrane scrapingcytology method. Mean and SD are shownFigure 10. A. Schematic cell with all subcellular compartments analyzed inSubcelullaRVis. B. Schematic cell showing the presence of identified proteins in thosesubcellular compartments that are colored. Plasma membrane is shown with a differentintensity for improving visualization.DETAILED DESCRIPTION OF THE INVENTIONThe method according to the invention obtains a higher number of cells from the corneain comparison with previous methods, allowing the realization of complex analysis in a highernumber of cells, and reducing the number of samples required from individuals. This greatlyimproves biomolecular analysis of corneal cells as well as biomarker analysis for cornealdiseases such as dry eye syndrome, keratoconus and squamous metaplasia.Therefore, in a first aspect, the present invention relates to a method for obtainingcorneal cells, from here onwards the method of the invention, comprising the steps of:(i) applying an anesthetic solution to the ocular surface to be sampled, and(ii) scraping the corneal surface with a polyethersulfone (PES) membrane.The term “cornea”, as used herein, refers to the transparent front part of the eye thatcovers the iris, pupil, and anterior chamber. The cornea is composed of five layers: theepithelium, Bowman’s layer, stroma, Descemet’s membrane, and the endothelium, and eachof those layers exerts an essential role in maintaining corneal transparency and stable visualfunction. Alternatively, the invention relates to a polyethersulfone (PES) membrane for use in amethod for obtaining corneal cells comprising the steps of:(i) applying an anesthetic solution to the ocular surface to be sampled, and(ii) scraping the corneal surface with a polyethersulfone (PES) membrane.In a particular embodiment of the method of the invention, the corneal cells are epithelialcorneal cells.The “corneal epithelium” is composed fairly uniformly of 5–7 layers of cells. It is about50 micrometers (μm) in thickness. The epithelium is uniform to provide a smooth regularsurface and is made up of nonkeratinized stratified squamous epithelium. The term “epithelialcorneal cells”, as used herein, refers to the cells present in the 5–6 layers epithelial structure,namely: superficial cells, wing cells, and the basal cells. In a preferred embodiment of themethod of the invention, the epithelial corneal cells obtained are superficial cells, wing cellsand / or basal cells.The corneal cells obtained from the method of the invention can be used for severalapplications, such as, and without limitation, diagnostic of diseases, analysis of biomarkers,biomolecular omics analysis such as transcriptomics and / or proteomics analyses. In aparticular embodiment of the method of the invention, the corneal cells obtained are used fordiagnostic analysis, biomarker analysis, transcriptomics analysis and / or proteomics analysis.Step (i) of the method of the inventionThe first step of the method of the invention comprises the application of an anestheticsolution to the ocular surface to be sampled. The term "anesthetic" or "anesthesia", as usedherein, denotes a compound / formulation for the management of acute physical pain, suitablefor short-term, temporary use, which has an effect that produces numbing or decreasedsensitivity in the body part / organ to which the compound / formulation is administered (e.g.,decreased corneal sensitivity of the eye). The term “solution”, as in “anesthetic solution”, asused herein, denotes that the anesthetic is of aqueous or liquid composition.In some particular embodiments, the anesthetic solution comprises a tear substitutecomprising an active ingredient, which may include, without limitation: a polyol, a dextran, awater-soluble protein, a carbomer, a gum, a cellulose derivative, or combinations thereof.Other suitable tear substitute components known in the art may be used in the anestheticsolution. Suitable cellulose derivatives for use in anesthetic solutions include, withoutlimitation, hydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC) sodium,hydroxypropyl cellulose, hydroxyethyl cellulose, methyl cellulose, or combinations thereof. Ina particular embodiment of the method of the invention, the anesthetic solution comprises acellulose derivative, preferably hydroxypropylmethyl cellulose.As the expert will be aware, the anesthetic solution can be combined with apharmaceutically suitable or acceptable carrier (e.g., a pharmaceutically suitable (oracceptable) excipient, physiologically suitable (or acceptable) excipient, pharmaceuticallysuitable (or acceptable) vehicle, or physiologically suitable (or acceptable) carrier). As usedherein, the term "pharmaceutically acceptable vehicle" refers to any formulation that isacceptable, i.e., is safe and provides the appropriate delivery of an effective anesthetic for thedesired route of administration. In a particular embodiment of the method of the invention, theanesthetic comprises a pharmaceutically acceptable vehicle, wherein the pharmaceuticallyacceptable vehicle is to an extent of more than 50%, preferably more than 75%, morepreferably more than 95% by weight water.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises an aminoamide anesthetic. In another particular embodiment of the method of theinvention, the anesthetic solution comprises an aminoesther anesthetic. In a particularembodiment of the method of the invention, the anesthetic solution comprises a combinationof an aminoamide anesthetic and an aminoesther anesthetic. In yet another particularembodiment of the method of the invention, the anesthetic solution comprises one, two, three,or more different anesthetics.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises an aminoester anesthetic selected from the group consisting of: benzocaine,chloroprocaine, cocaine, cyclomethycaine, oxybuprocaine, dimethocaine, larocaine,piperocaine, propoxycaine, procaine, novocaine, proparacaine, tetracaine, and amethocaine.In another particular embodiment of the method of the invention, the anesthetic solutioncomprises an aminoamide anesthetic selected from the group consisting of: articaine,bupivacaine, cinchocaine, dibucaine, etidocaine, levobupivacaine, lidocaine, lignocaine,mepivacaine, prilocaine, ropivacaine, and trimecaine.In a more particular embodiment of the method of the invention, the anesthetic solutioncomprises an anesthetic selected from the group consisting of lidocaine, tetracaine,oxybuprocaine, a salt thereof and a combination thereof.The person skilled in the art will understand that the salt of the anesthetic to be used isa pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt thereof”, asused herein, refers to derivatives of the anesthetic wherein the parent compound is modifiedby making acid or base salts thereof. Examples of pharmaceutically acceptable salts include,but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali ororganic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parentcompound formed, e.g., from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 1,2-ethanedisulfonic, 2-acetoxybenzoic, 2-hydroxyethanesulfonic,acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodide,hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulfamic,sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic. The pharmaceutically acceptable salts of the anesthetics can be synthesized from theparent compound that contains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are useful. Lists of suitable salts are found inRemington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.,1990, p. 1445.The person skilled in the art will also understand that the salt to be used mustbe an ophtalmically acceptable salt. In a preferred embodiment, the salt is an inorganic salt, acitrate salt or an acetate salt. In a more preferred embodiment, the salt is a hydrochloride salt.In a particular embodiment of the method of the invention, the anesthetic solution isselected from the group consisting of:- an anesthetic solution comprising hydroxypropylmethylcellulose and lidocaine;- an anesthetic solution comprising tetracaine hydrochloride and naphazoline; and- an anesthetic solution comprising tetracaine hydrochloride and oxybuprocainehydrochloride. In a particular embodiment of the method of the invention, the anesthetic solutioncomprises between about 0.02 mg / mL to about 0.12 mg / mL lidocaine and between about 5mg / mL to about 35 mg / mL HPMC, preferably comprises at least about 0.04 mg / mL, at leastabout 0.06 mg / mL, at least about 0.08 mg / mL, at least about 0.1 mg / mL lidocaine and at leastabout 10 mg / mL, at least about 11 mg / mL, at least about 12 mg / mL, at least about 13 mg / mL,at least about 14 mg / mL, at least about 15 mg / mL, at least about 16 mg / mL, at least about 17mg / mL, at least about 18 mg / mL, at least about 19 mg / mL, at least about 20 mg / mL, at leastabout 21 mg / mL, at least about 22 mg / mL, at least about 23 mg / mL, at least about 24 mg / mL,at least about 25 mg / mL, at least about 26 mg / mL, at least about 27 mg / mL, at least about 28mg / mL, at least about 29 mg / mL, at least about 30 mg / mL, at least about 35 mg / mL HPMC.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises between about 1 mg / mL to about 10 mg / mL tetracaine hydrochloride and betweenabout 0.1 mg / mL to about 1 mg / mL naphazoline hydrochloride, preferably at least about 2mg / mL, at least about 3 mg / mL, at least about 4 mg / mL, at least about 5 mg / mL, at least about6 mg / mL, at least about 7 mg / mL, at least about 8 mg / mL, at least about 9 mg / mL tetracainehydrochloride and at least about 0.2 mg / mL, at least about 0.3 mg / mL, at least about 0.4mg / mL, at least about 0.5 mg / mL, at least about 0.6 mg / mL, at least about 0.7 mg / mL, at leastabout 0.8 mg / mL, at least about 0.9 mg / mL naphazoline hydrochloride.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises between about 0.1 mg / mL to about 2 mg / mL tetracaine hydrochloride and betweenabout 1 mg / mL to about 8 mg / mL oxybuprocaine hydrochloride, preferably at least about 0.2mg / mL, at least about 0.3 mg / mL, at least about 0.5 mg / mL, at least about 0.7 mg / mL, at leastabout 0.9 mg / mL, at least about 1.0 mg / mL, at least about 1.1 mg / mL, at least about 1.3mg / mL, at least about 1.5 mg / mL, at least about 1.7 mg / mL, at least about 1.9 mg / mLtetracaine hydrochloride and at least about 2 mg / mL, at least about 3 mg / mL, at least about 4mg / mL, at least about 5 mg / mL, at least about 6 mg / mL, at least about 7 mg / mL oxybuprocainehydrochloride. The concentration of the anesthetic in the anesthetic solution can also be defined bystating the weight to volume percentage (w / v) of the specific components. Therefore, in aparticular embodiment of the method of the invention, the anesthetic solution comprisesbetween about 0.002 % w / v to about 0.012 % w / v lidocaine and between about 0.5 % w / v toabout 3.5 % w / v HPMC, preferably comprises at least about 0.004 % w / v, at least about 0.006% w / v, at least about 0.008 % w / v, at least about 0.01 % w / v lidocaine and at least about 0.5% w / v, at least about 1 % w / v, at least about 1.5 % w / v, at least about 2 % w / v, at least about2.5 % w / v, at least about 3 % w / v, at least about 3.5 % w / v HPMC.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises between about 0.1 % w / v to about 1 mg / mL tetracaine hydrochloride and betweenabout 0.01 % w / v to about 0.1 % w / v naphazoline hydrochloride, preferably at least about 0.2% w / v, at least about 0.3 % w / v, at least about 0.4 % w / v, at least about 0.5 % w / v, at leastabout 0.6 % w / v, at least about 0.7 % w / v, at least about 0.8 % w / v, at least about 0.9 % w / vtetracaine hydrochloride and at least about 0.02 % w / v, at least about 0.03 % w / v, at leastabout 0.04 % w / v, at least about 0.05 % w / v, at least about 0.06 % w / v, at least about 0.07 %w / v, at least about 0.08 % w / v, at least about 0.09 % w / v naphazoline hydrochloride.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises between about 0.01 % w / v to about 0.2 % w / v tetracaine hydrochloride andbetween about 0.1 % w / v to about 0.8 % w / v oxybuprocaine hydrochloride, preferably at leastabout 0.02 % w / v, at least about 0.03 % w / v, at least about 0.05 % w / v, at least about 0.07 %w / v, at least about 0.09 % w / v, at least about 0.1%, at least about 0.11 % w / v, at least about0.13 % w / v, at least about 0.15 % w / v, at least about 0.17 % w / v, at least about 0.19 % w / vtetracaine hydrochloride and at least about 0.2 % w / v, at least about 0.3 % w / v, at least about0.4 % w / v, at least about 0.5 % w / v, at least about 0.6 % w / v, at least about 0.7 % w / voxybuprocaine hydrochloride.In a more particular embodiment of the method of the invention, the anesthetic solutionis selected from the group consisting of:- an anesthetic solution comprising 0.06 mg / mL lidocaine hydrochloride and 20mg / mL hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 5 mg / mL tetracaine hydrochloride and 0.5mg / mL naphazoline hydrochloride; and- an anesthetic solution comprising 1 mg / mL tetracaine hydrochloride and 4mg / mL oxybuprocaine hydrochloride.In a more particular embodiment of the method of the invention, the anesthetic solutionis selected from the group consisting of:- an anesthetic solution comprising 0.006 % w / v lidocaine hydrochloride and 2 %w / v hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 0.5 % w / v tetracaine hydrochloride and 0.05% w / v naphazoline hydrochloride; and- an anesthetic solution comprising 0.1 % w / v tetracaine hydrochloride and 0.4 %w / v oxybuprocaine hydrochloride.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises 0.06 mg / mL lidocaine hydrochloride and 20 mg / mL hydroxypropyl methylcellulosegel. In another particular embodiment of the method of the invention, the anesthetic solutioncomprises 0.006 % w / v lidocaine hydrochloride and 2 % w / v hydroxypropyl methylcellulosegel. In a particular embodiment of the method of the invention, the anesthetic solutioncomprises 1 mg / mL tetracaine hydrochloride and 4 mg / mL oxybuprocaine hydrochloride. Inanother particular embodiment of the method of the invention, the anesthetic solutioncomprises 0.1 % w / v tetracaine hydrochloride and 0.4 % w / v oxybuprocaine hydrochloride.In a particular embodiment of the method of the invention, the anesthetic solutioncomprises 5 mg / mL tetracaine hydrochloride and 0.5 mg / mL naphazoline hydrochloride. Inanother particular embodiment of the method of the invention, the anesthetic solutioncomprises 0.5 % w / v tetracaine hydrochloride and 0.05 % w / v naphazoline hydrochloride.In a particular embodiment of the method of the invention, the anesthetic solution furthercomprises buffering agents such as sodium dihydrogen phosphate, sodium hydrogenphosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, boric acid,sodium borate, citric acid, tartaric acid, sodium tartrate, and any combination thereof.In another particular embodiment of the method of the invention, the anesthetic solutionfurther comprises pH adjusters to produce a final solution having a pH suitable for use.Examples of pH adjusters may include hydrochloric acid, acetic acid, sodium hydroxide,potassium hydroxide, and the like. Conventional acids and bases are well-known pH adjusterswithin the art.In another particular embodiment of the method of the invention, the anesthetic solutionfurther comprises one or more non-steroidal anti-inflammatory agents (NSAIDS), preferablyselected from the group consisting of: naproxen, diclofenac, celecoxib (Celebrex®, Pfizer),sulindac, diflunisal, piroxicam, indomethacin, etodolac, meloxicam, ibuprofen, ketoprofen,r-flurbiprofen (Myriad Genetics, Inc.), mefenamic, nabumetone, tolmetin, sodium salts of eachof the foregoing: ketorolac bromethamine, ketorolac tromethamine (Acular®, Allergan, Inc.),choline magnesium trisalicylate, rofecoxib, valdecoxib, lumiracoxib, etoricoxib, aspirin,salicylic acid and its sodium salt, salicylate esters of α,β,γ-tocopherols and tocotrienols (andall their d, l, and racemic isomers); methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl,esters of acetylsalicylic acid, tenoxicam, aceclofenac, nimesulide, nepafenac, amfenac,bromfenac, flufenamate, and phenylbutazone.In a particular embodiment of the method of the invention, the anesthetic solution furthercomprises one or more angiostatic and / or anti-inflammatory steroids, preferably selected froma group consisting of: anecortive acetate (Retaane®, Alcon, Inc., Fort Worth, Tex.);tetrahydrocortisol; 4,9(11)-pregnadien-17α,21-diol-3,20-dione (Anecortave) and its -21-acetate salt, 11-epicortisol, 17α-hydroxyprogesterone, tetrahydrocortexolone, cortisone,cortisone acetate, hydrocortisone, hydrocortisone acetate, fludrocortisone, fludrocortisoneacetate, fludrocortisone phosphate, prednisone, prednisolone, prednisolone sodiumphosphate, methylprednisolone, methylprednisolone acetate, methylprednisolone, sodiumsuccinate, triamcinolone, triamcinolone-16,21-diacetate, triamcinolone acetonide and its -21-acetate, -21-disodium phosphate, and -21-hemisuccinate forms, triamcinolone benetonide,triamcinolone hexacetonide, fluocinolone, fluocinolone acetate, dexamethasone and its 21-acetate, -21-(3,3-dimethylbutyrate), -21-phosphate disodium salt, -21-diethylaminoacetate, -21-isonicotinate, -21-dipropionate, and -21-palmitate forms; betamethasone and its -21-acetate, -21-adamantoate, -17-benzoate, -17,21-dipropionate, -17-valerate, and -21-phosphate disodium salts, beclomethasone, beclomethasone dipropionate, diflorasone,diflorasone diacetate, mometasone furoate, and acetazolamide (Diamox®, LederleParenterals, Inc., Carolina, Puerto Rico; several other manufacturers).In a particular embodiment of the method of the invention, the anesthetic solution furthercomprises anti-neovascularization steroids selected from the group consisting of: 21-nor-5β-pregnan-3α,17α,20-triol-3-acetate, 21-nor-5α-pregnan-3α,17α,20-triol-3-phosphate, 21-nor-5β-pregn-17(20)en-3α,16-diol, 21-nor-5β-pregnan-3α,17β,20-triol, 20-acetamide-21-nor-5α-pregnan-3α,17α-diol-3-acetate, 313 acetamido-5β-pregnan-11β,17α,21-triol-20-one-21-a-cetate, 21-nor-5α-pregnan-3α,17β,20-triol, 21α-methyl-5β-pregnan-3α,11β,17α,21-tetrol-20-1-one-21-methyl ether, 20-azido-21-nor-5β-pregnan-3α,17α-diol, 20(carbethoxymethyl)thio-21-nor-5β-pregnan-3α,17α-diol, 20-(4-fluorophenyl)thio-21-nor-5β-pregnan-3α,17α-diol, 16α-(2-hydroxyethyl)-17β-methyl-5β-androstan-3α,17α-diol, 20-cyano-21-nor-5β-pregnan-3α,17α-diol, 17α-methyl-51-androstan-3α,17β-diol, 21-nor-5β-pregn-17(20)en-3α-ol, 21- or -5β-pregn-17(20)en-3α-ol-3-acetate, 21-nor-5-pregn-17(20)-en-3α-ol-16-acetic acid 3-acetate,3β-azido-5β-pregnan-11β,17α,21-triol-20-one-21-acetate, and 5β-pregnan-11β,17α,21-triol-20-one, 4-androsten-3-one-17β-carboxylic acid, 17α-ethynyl-5(10)-estren-17β-ol-3-one, and17α-ethynyl-1,3,5(10)-estratrien-3,17β-diol. In another particular embodiment of the method of the invention, the anesthetic solutionfurther comprises a vasoconstrictor selected from naphazoline, phenylephrine, oxymetazolineor tetrahydrozoline; preferably naphazoline.In another particular embodiment of the method of the invention, the anesthetic solutiondoes not contain an active ingredient different from an anesthetic; preferably the anestheticsolution does not contain a non-steroidal anti-inflammatory agent, an angiostatic, an anti-inflammatory steroid, an anti-neovascularization steroid, and a vasoconstrictor; preferablydoes not contain a non-steroidal anti-inflammatory agent, an angiostatic, an anti-inflammatorysteroid and an anti-neovascularization steroid.The expert in the field will know how to administer the anesthetic solution to ocularsurface to be sampled. It is preferable that the administration is made by drops into the ocularsurface either by the subject to be sampled or by another person.The term “ocular surface”, as used herein, refers to the interface between the functioningeye and the environment. The ocular surface comprises both the cornea and the conjunctiva,and the anesthetic solution, or any other solution applied during the method of the invention,can be applied to any part of the ocular surface, as long as it acts on the corneal surface, thesurface from which the sample will be obtained.Step (ii) of the method of the inventionStep (ii) comprises the sampling process of the method of the invention, wherein thecorneal surface is scraped with a PES membrane.The term “scraping”, as used herein, refers to the action of rubbing or dragging the PESmembrane across part of the whole surface of the cornea as to obtain corneal cells, preferablyepithelial corneal cells, while keeping the membrane in contact with said surface. It will beunderstood that the action of scraping the PES membrane does not involve keeping themembrane still in any one position of the corneal surface and then peeling off said membraneof the corneal surface, but instead, while the membrane is in contact with the corneal surface,the membrane is being dragged across the corneal surface area.In a particular embodiment of the method of the invention, at least one quarter, at leasthalf, at least three quarters or the whole corneal surface is sampled in step (ii).During the process of scraping the corneal surface the person performing the scrapingmay use only one side of the PES membrane or use both sides of the PES membrane, byflipping the membrane to the opposite side during the scraping process. In a particularembodiment of step (ii) of the method of the invention, the scraping uses one side of the PESmembrane. In another particular embodiment of step (ii) of the method of the invention, thescraping uses both sides of the PES membrane.In another particular embodiment of step (ii) of the method of the invention, the cornealsurface is scraped with PES membrane, once, twice, three or more times preferably twice;more preferably three times.The scraping of the corneal surface is carried out with a polyethersulfone (PES)membrane. The term “polyethersulfone”, also known as “PES”, as used herein, refers to anamorphous, transparent, and pale amber high-performance thermoplastic. PES is stable athigh temperatures in both air and water and has a relatively high water absorption.“Polyethersulfone (PES) membrane”, as used herein, refers to a membrane made of PES, i.e.,a polymeric membrane which is characterized by having oxidative, thermal and hydrolyticstability as well as good membrane properties. PES membranes are prepared by a phaseinversion method. The membrane structure is influenced by the composition such asconcentration, solvent and additives, temperature of PES solution, the non-solvent or themixture of non-solvents, and the coagulation bath or the environment, etc. This allows fordifferent structure membranes to be developed. One important structural characteristic of thePES membranes is the pore size. Membranes can also be divided into four types dependingon their application: microfiltration, ultra-filtration, nano-filtration and osmosis. Theclassification corresponds to their average pore sizes which are in the range of 50–500 nm,1–50 nm, ≤1 nm and 0.3–0.6 nm, respectively. The pore size of the membrane can bedetermined by direct measurement of the diameter of the pores, using for example, scanningelectron microscopy (SEM) which allows to visualize the PES membrane pores and measuretheir diameters. In a particular embodiment of the method of the invention, the PES membranehas a pore size of between about 0.2 micrometers (µm) and about 0.45 µm, preferably a poresize of about 0.25 µm, about 0.3 µm, about 0.35 µm, about 0.4 µm, about 0.45 µm. In aparticular embodiment of the method of the invention, the PES membrane has a pore size of0.20 µm. In another particular embodiment of the method of the invention, the PES membranehas a pore size of 0.45 µm. In another embodiment, the PES membrane has a pore size of0.22 µm.In order to improve the yield of viable cells sampled by the method of the invention, thetime between step (i) and step (ii) should not be more than two minutes. Therefore, in aparticular embodiment of the method of the invention, the time elapsed between step (i) andstep (ii) is less or equal to 2 minutes. In another particular embodiment of the method of theinvention, the time elapsed between step (i) and step (ii) is about 0.2 min, about 0.4 min, about0.6 min, about 0.8 min, about 1 min, about 1.2 min, about 1.4 min, about 1.6 min, about 1.8min, about 2 min.Further steps of the method of the inventionIn addition to steps (i) and (ii) of the method of the invention, the method optionally mayfurther comprise an additional step which is performed after step (i) and preceding step (ii) ofthe method of the invention. In a particular embodiment of the method of the invention, themethod comprises a step of lubricating the ocular surface after step (i) and preceding step (ii),being that step (ii) is carried out immediately after the lubrication.The term “lubricating” or “lubrication”, as used herein, refers to the process of applyinga substance such as saline solution to the ocular surface as to minimize friction and allowsmooth movement of the scraping action of the PES membrane. If performed, this stepimmediately precedes the action of scraping the ocular surface with the PES membrane.In a particular embodiment of the method of the invention, the step of lubricating iscarried out with a saline solution. The term “saline solution”, as used herein, refers to a mixtureof sodium chloride and water. Saline solution has a number of uses in medicine includingcleaning wounds, removal and storage of contact lenses, and help with dry eyes. In a particularembodiment, the saline solution is selected from physiological saline solution and balancedsalt solution.In addition to the saline solution salts, other components may be combined with thesaline solution such as steroids, antihistamines, sympathomimetics, beta receptor blockers,parasympathomimetics, parasympatholytics, prostaglandins, non-steroidal anti-inflammatorydrugs (NSAIDs) or antibiotics. In a particular embodiment, the saline solution does not containany other component different from water, inorganic salts, citrate salts and acetate salts;preferably does not contain steroids, antihistamines, sympathomimetics, beta receptorblockers, parasympathomimetics, parasympatholytics, prostaglandins, NSAIDs or antibiotics.In a particular embodiment of the method of the invention, the saline solution is sodiumchloride (NaCl) 9 mg / mL in purified water.In another particular embodiment, the saline solution is a solution comprising, preferablyconsisting of, sodium chloride, potassium chloride, calcium chloride, magnesium chloride,sodium acetate, sodium citrate and water; more particularly is a saline solution comprising,preferably consisting of, sodium chloride 6.4 mg / mL, potassium chloride (KCl) 0.75 mg / mL,calcium chloride dihydrate (CaCl2·2H2O) 0.48 mg / mL, magnesium chloride hexahydrate(MgCl2•6H2O) 0.3 mg / mL, sodium acetate trihydrate (C2H3NaO2·3H2O) 3.9 mg / mL, sodiumcitrate dihydrate (C6H5Na3O7·2H2O) 1.7 mg / mL, and water at a pH of 7.5. Preferably, saidsaline solution has an osmolality of 300 mOsm / kg.Kit of the inventionAnother aspect of the present invention relates to a kit for carrying out the methodaccording to the invention, comprising an anesthetic solution and a PES membrane, from hereonwards the kit of the invention.All terms and definitions previously described are equally applicable to the presentaspects. All embodiments previously described in relation to previous aspects are equally andvalidly applied to the current aspect.In the context of the present invention, “kit” is understood as a product of the differentreagents for performing the methods described in the present invention, both in those casesin which the only steps (i) and (ii) are performed or wherein further steps are also performed.In the kit of the invention the different reagents may be packaged together to allow for transportand storage or separately in which case the transport and storage may be performedseparately also. Nevertheless, if the kits defined in the present invention do not comprise thereagents necessary for putting the methods of the invention into practice, such reagents arecommercially available and can be found as part of a kit. Suitable materials for packaging thecomponents of the kit include, without being limited to, glass, plastic (polyethylene,polypropylene, polycarbonate and the like), bottles, vials, paper, sachets and the like. Kits canadditionally contain instructions for using the different components in the kit. Said instructionscan be in printed format or in an electronic device capable of storing instructions such thatthey can be read by a person, such as electronic storage media (magnetic discs, tapes andthe like), optical means (CD-ROM, DVD, USB) and the like. The media can additionally oralternatively contain Internet addresses where said instructions are provided.In a particular embodiment of the kit of the invention, the PES membrane has a poresize of between 0.20 µm and 0.45 µm.In another particular embodiment of the kit of the invention, the PES membrane has apore size of 0.20 µm. In another particular embodiment of the kit of the invention, the PESmembrane has a pore size of 0.45 µm. In another embodiment, the PES membrane has apore size of 0.22 µm.In yet another particular embodiment of the kit of the invention, the anesthetic solutioncomprises an anesthetic selected from the group consisting of lidocaine, tetracaine,oxybuprocaine, a salt thereof and a combination thereof.In a further particular embodiment of the kit of the invention, the anesthetic solution isselected from the group consisting of:- an anesthetic solution comprising hydroxypropylmethylcellulose and lidocaine;- an anesthetic solution comprising tetracaine hydrochloride and naphazoline; and- an anesthetic solution comprising tetracaine hydrochloride and oxybuprocainehydrochloride. In a further particular embodiment of the kit of the invention, the anesthetic solution isselected from the group consisting of:- an anesthetic solution comprising 0.06 mg / mL lidocaine hydrochloride and 20mg / mL hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 5 mg / mL tetracaine hydrochloride and 0.5mg / mL naphazoline hydrochloride; and- an anesthetic solution comprising 1 mg / mL tetracaine hydrochloride and 4mg / mL oxybuprocaine hydrochloride.In a particular embodiment of the kit of the invention, the anesthetic solution comprises1 mg / mL tetracaine hydrochloride and 4 mg / mL oxybuprocaine hydrochloride.In a particular embodiment of the kit of the invention, the anesthetic solution comprises5 mg / mL tetracaine hydrochloride and 0.5 mg / mL naphazoline hydrochloride.In a particular embodiment of the kit of the invention, the kit further comprises alubricating saline solution. In a more particular embodiment, the saline solution is sodiumchloride (NaCl) 9 mg / mL in purified water.In another particular embodiment, the saline solution comprises, preferably consists of,sodium chloride 6.4 mg / mL, potassium chloride (KCl) 0.75 mg / mL, calcium chloride dihydrate(CaCl2·2H2O) 0.48 mg / mL, magnesium chloride hexahydrate (MgCl2•6H2O) 0.3 mg / mL,sodium acetate trihydrate (C2H3NaO2·3H2O) 3.9 mg / mL, sodium citrate dihydrate(C6H5Na3O7·2H2O) 1.7 mg / mL, and water at a pH of 7.5.Uses of the inventionAnother aspect of the invention relates to the use of the kit of the invention for obtainingcorneal cells, from here onwards the use of the invention.In a preferred embodiment, the corneal cells obtained are epithelial corneal cells.All terms and definitions previously described are equally applicable to the presentaspects. All embodiments previously described in relation to previous aspects are equally andvalidly applied to the current aspect.In a particular embodiment the use of the invention is to carry out the method of theinvention. The present invention can be further defined by the following aspects:1. Method for obtaining corneal cells comprising the steps of:(i) applying an anesthetic solution to the ocular surface to be sampled, and(ii) scraping the corneal surface with a polyethersulfone (PES) membrane.2. The method according to aspect 1, wherein the PES membrane has a pore size ofbetween 0.20 µm and 0.45 µm.3. The method according to aspect 1 or 2, wherein the PES membrane has a pore sizeof 0.20 µm.4. The method according to any one of aspects 1 to 3, wherein the anesthetic solutioncomprises an anesthetic selected from the group consisting of lidocaine, tetracaine,oxybuprocaine, a salt thereof and a combination thereof.5. The method according to aspect 4, wherein the anesthetic solution is selected fromthe group consisting of:- an anesthetic solution comprising hydroxypropylmethylcellulose and lidocaine;- an anesthetic solution comprising tetracaine hydrochloride and naphazoline; and- an anesthetic solution comprising tetracaine hydrochloride and oxybuprocainehydrochloride.6. The method according to aspect 5, wherein the anesthetic solution is selected fromthe group consisting of:- an anesthetic solution comprising 0.06 mg / mL lidocaine hydrochloride and 20mg / mL hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 5 mg / mL tetracaine hydrochloride and 0.5mg / mL naphazoline hydrochloride; and- an anesthetic solution comprising 1 mg / mL tetracaine hydrochloride and 4mg / mL oxybuprocaine hydrochloride.7. The method according to aspect 6, wherein the anesthetic solution comprises 1 mg / mLtetracaine hydrochloride and 4 mg / mL oxybuprocaine hydrochloride.8. The method according to aspect 6, wherein the anesthetic solution comprises 5 mg / mLtetracaine hydrochloride and 0.5 mg / mL naphazoline hydrochloride.9. The method according to any one of aspects 1 to 8, wherein the time elapsed betweenstep (i) and step (ii) is less than 2 minutes.10. The method according to any one of aspects 1 to 9, wherein the method comprises astep of lubricating the ocular surface after step (i) and preceding step (ii), being thatstep (ii) is carried out immediately after the lubrication.11. The method according to aspect 10, wherein the step of lubricating is carried out witha saline solution.12. The method according to aspect 11, wherein the saline solution is selected from agroup consisting of:- Sodium chloride (NaCl) 9 mg / mL in purified water; and- A solution comprising sodium chloride 6.4 mg / mL, potassium chloride (KCl)0.75 mg / mL, calcium chloride dihydrate (CaCl2·2H2O) 0.48 mg / mL, magnesiumchloride hexahydrate (MgCl2•6H2O) 0.3 mg / mL, sodium acetate trihydrate(C2H3NaO2·3H2O) 3.9 mg / mL, sodium citrate dihydrate (C6H5Na3O7·2H2O) 1.7mg / mL, and water at a pH of 7.513. The method according to any of the aspects 1 to 12, wherein the corneal cells areepithelial corneal cells.14. A kit for carrying out the method according to any one of claims 1 to 13, comprising ananesthetic solution and a PES membrane.15. The kit according to aspect 14, wherein the PES membrane has a pore size of between0.20 µm and 0.45 µm.16. The kit according to aspect 14 or 15, wherein the PES membrane has a pore size of0.20 µm.17. The kit according to any one of aspects 14 to 16, wherein the anesthetic solutioncomprises an anesthetic selected from the group consisting of lidocaine, tetracaine,oxybuprocaine, a salt thereof and a combination thereof.18. The kit according to aspect 17, wherein the anesthetic solution is selected from thegroup consisting of:- an anesthetic solution comprising hydroxypropylmethylcellulose and lidocaine;- an anesthetic solution comprising tetracaine hydrochloride and naphazoline; and- an anesthetic solution comprising tetracaine hydrochloride and oxybuprocainehydrochloride.19. The kit according to aspect 18, wherein the anesthetic solution is selected from thegroup consisting of:- an anesthetic solution comprising 0.06 mg / mL lidocaine hydrochloride and 20mg / mL hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 5 mg / mL tetracaine hydrochloride and 0.5mg / mL naphazoline hydrochloride; and- an anesthetic solution comprising 1 mg / mL tetracaine hydrochloride and 4mg / mL oxybuprocaine hydrochloride.20. The kit according to aspect 19, wherein the anesthetic solution comprises 1 mg / mLtetracaine hydrochloride and 4 mg / mL oxybuprocaine hydrochloride.21. The kit according to aspect 19, wherein the anesthetic solution comprises 5 mg / mLtetracaine hydrochloride and 0.5 mg / mL naphazoline hydrochloride.22. The kit according to any one of aspects 14 to 21, further comprising a lubricating salinesolution. 23. The kit according to aspect 22, wherein the saline solution is selected from a groupconsisting of:- Sodium chloride (NaCl) 9mg / mL in purified water; and- A solution comprising sodium chloride 6.4 mg / mL, potassium chloride (KCl)0.75 mg / mL, calcium chloride dihydrate (CaCl2·2H2O) 0.48 mg / mL, magnesiumchloride hexahydrate (MgCl2•6H2O) 0.3 mg / mL, sodium acetate trihydrate(C2H3NaO2·3H2O) 3.9 mg / mL, sodium citrate dihydrate (C6H5Na3O7·2H2O) 1.7mg / mL, and water at a pH of 7.5.24. Use of the kit according to any one of aspects 14 to 23 for obtaining corneal cells.25. Use of the kit according to aspect 24 to carry out the method according to any one ofaspects 1 to 13.*** The invention will be described by way of the following examples, which are to beconsidered as merely illustrative and not limitative of the scope of the invention.EXAMPLESExample 1Membrane Scraping Cytology (MSC) procedureMaterials and methods1.1. Instruments and materials- Filters Supor® 200, (VWR, Sterile Polyethersulphone filter 0.2 µm 47 mmgridded individual; #514-4201)- Barron Vacuum Donor cornea punch® (Katena Products, 9.00 mm, #K20-2112) 1.5 mL- Adson forceps (NOPA, AB 100 / 12)- Self-sealing pouches (VWR, #129-1407)- Eppendorf (VWR, sterile conical Polypropylene Tube with cap, #525-1126)- Eppendorf (VWR, Protein LoBind Tube 1.5 mL Safe-Lock Tubes, #525-0133) -Eppendorf (Thermo Fisher (VWR), RNase-free tubes 1.5 mL, #AM12400)- Plastic Eppendorf storage box (VWR, 155x140x55, #KOEHA-1192)- Cryo Eppendorf storage box (VWR, #5160035)- Nitrile gloves (VMR, Ref.112-2371, 112-2379)- Pre-printed self-adhesive labels to indicate the patient code, the eye fromwhich the sample has been taken.- Colirofta anesthetic, 5 mg / mL Tetracaine hydrochloride + 0.5 mg / mLNaphazoline hydrochloride (Alcon Laboratories)- 70 % ethanol- Sterile Saline solution (for example: Saline Solution Senti2#8436538552506 or BSS Serag-Wiessner #012956)- RPMI 1640 Medium (GIBCO, #21875-034)- Fetal Bovine Serum (FBS) (BIOWEST, # S181B)1.2. Material preparationMost of the materials used were previously cleaned / disinfected and / or prepared understerile conditions except the 1.5 mL “protein LoBind safe-lock tube” and “RNase-free tubes“which were prepared under cleanroom conditions.^ Adson surgical forceps were disinfected and sterilized.^ 1.5 mL Eppendorf tubes were sterilized (in pouches of 1 or 2 units).Preparation of RPMI / 10 % FBS aliquots^ All materials were sprayed with ethanol 70% and wiped with a clean-room paper towelbefore placing them inside the hood.^ 1 mL RPMI / 10 % FBS was aliquoted in sterile 1.5 mL polypropylene tubes using amicropipette with sterile tips.^ The cap of all the polypropylene tubes was closed.^ The plastic Eppendorf box was stored with aliquots at -20° C for 6 months or in thefridge at +4° C for 4 weeks.Membrane Cutting^ Aliquots were thawed at room temperature before use.^ All materials were sprayed with ethanol 70 % and wiped with a clean-room paper towelbefore placing them inside the hood.^ A sterile PES membrane bag was opened and the membrane was placed on top ofthe Vacuum Donor Cornea Punch®.^ The PES membrane was cut to 9 mm. If necessary, tweezers were used to removeany rough edge.^ One, two or four membranes were placed inside a sterile self-sealing pouch with thetweezer. ^The pouch was sealed and stored at room temperature until use.1.3. Sample collectionThe person who was going to manipulate the sample had to wear clean nitrile gloves.^ If the sample was collected inside the operating theatre, the patient was placed lyingface up.^ If the sample was collected inside the eye exam room, the patient was asked to resttheir chin and forehead on the slit lamp.^ 1 drop of Colirofta 0.5 % anesthetic was instilled to the eye to be sampled. The timebetween dosing the anesthesia and the sample collection should be less or equal to 2minutes. ^If the sample was collected inside the operating theatre, the patient eye was openedwith a blepharostat. If the sample was collected inside the eye exam room, the personcollecting the sample used their index finger and thumb to open the patient’s eye.^ The patient’s eye was well lubricated with a sterile saline solution and rested 5 secondsto avoid tear meniscus.^ One of the sterile pre-cut membranes was taken from the pouch with sterile Adsonforceps. If the sample was collected for flow cytometry or other cell analysis method, only one sideof the membrane was used (stripe marks facing up – no contact with the eye) for scrapingall the cornea, passing twice / three times per each part of the cornea. If the sample wascollected for protein or mRNA analysis, both sides of the membrane were used for scrapingall the cornea, passing it twice on each side.^ If the sample was collected for flow cytometry analysis, the membrane was placed inone of the RPMI / 10 % FBS aliquots ensuring it was completely immersed in the liquid.If the sample was collected for mRNA analysis, it was placed in an empty sterileEppendorf RNAse free tube.If the sample was collected for protein analysis, it was placed in an empty proteinLoBind tube.^ The tubes were labelled with pre-printed self-adhesive labels indicating the patientcode, the eye from which the sample had been taken.^ The tubes were placed with the samples in a storage box.If samples were going to be analysed fresh (flow cytometry), they were kept inside thefridge after their collection and were analysed on the same day.If the samples were going to be analysed by molecular biology methods, they werefrozen at -80º C (within 3h from collection) until the analysis.Evaluation of the advantage of the Membrane Scraping Cytology (MSC) procedureMaterials and methods2.1. Experiment 1: Toxicity studyTo optimize the yield and quality of cells collected by the method of the invention, thetoxicity of the following three different anesthetics used in the collaborating clinical centerswas tested in the human epithelial cell line A-431.^ Barraquer in-house: 0.06 mg / mL lidocaine hydrochloride (0.006 % w / v) + 20 mg / mLhydroxypropyl methylcellulose gel (2 % w / v).^ Colirofta (Alcon Laboratories): 5 mg / mL tetracaine hydrochloride (0.5 % w / v) + 0.5mg / mL naphazoline hydrochloride (0.05 % w / v).^ Colirofta doble (Alcon Laboratories): 1 mg / mL tetracaine hydrochloride (0.1 % w / v) +4 mg / mL oxybuprocaine hydrochloride (0.4 % w / v).A-431 cells were seeded in 96 wells plates (2 x104 cells / well), incubated at 37º C for 48hours and then treated with different dilutions of anesthetics for different periods of incubation(Table 1). Six replicates were tested for each condition. One or two dilutions of anestheticswere tested to simulate the dilution of these solutions with tears when used in real samples.Table 1. Experimental design of the toxicity study.Dilution Anesthetic (µL) Medium (µL)1 / 3 35 701 / 5 35 140Condition Anesthetic Dilution Time1 Barraquer in-house 1 / 3, 1 / 5 5, 15, 30 and 60min 2Colirofta 1 / 3 2, 5 and 15 min3 Colirofta Doble 1 / 3 2, 5 and 15 minCells incubated with culture medium only or with 2 % SDS for the longest incubationtimes were used as controls.Cell viability was then assessed using the MTT assay according to the following method.At the end of the incubation, the cells were washed with Hank’s Balanced Salt Solution(HBSS) 1X and incubated with 100 μL of the MTT labelling reagent (Calbiochem, 475989) for2 hours. The MTT solution was then removed and 100 μL of dimethyl sulfoxide (DMSO) (VWR,23500.260) were added for 10 minutes in the dark. The purple formazan product formed wasmeasured at 570 nm with a plate reader.In each plate the mean of the absorbance of the 6 untreated replicates (cells incubatedwith medium culture only) was used as the relative absorbance value for the calculation of thecell viability:(^^^^^^^^^^ ^^^^ − ^^^^ ^^^^^^ )% ^^^^ ^^^^^^^^^ = ^^^^ ^^^^^^^^^ × 100^^^^ ^^^^^^^^^^ ^^^^^^^^^2.2. Experiment 2: Optimization of the method of impression cytologyTo optimize the impression cytology process, a series of experiments were performedto evaluate the effect of:1) The membrane material (PES, Cellulose acetate, Nitrocellulose, hydrophilic PTFE)2) The membrane pore size (0.20 μm, 0.45 μm)3) The duration of the membrane application4) The method of the membrane applicationUsing fresh (less than 8 hours postmortem) pig eyes, the following parameters weretested: -Membrane typeo PES Supor® Membrane, 0.20 µm, Gridded (Product ID: 66234, Pall)o PES Supor® Membrane, 0.45 µm, Gridded (Product ID: 60043, Pall)o Cellulose acetate circles (OE 66), 0.20 μm (Product ID: WHA10404106, Merck)o Cellulose acetate circles, 0.45 μm (Product ID: WHA70000002, Merck)o Whatman® nitrocellulose membrane filters, NC 45, pore size 0.45 μm (ProductID: WHA10401114, Merck)o Biopore Membrane Filter roll, Hydrophilic PTFE, 0.4 μm (Product ID:BGCM00010, Merck)- Duration of the membrane application on the corneao 4 secondso 8 seconds- Method of the membrane applicationo Goldmann coneo Patient interface (PI)o Adson ForcepsConsidering all the parameters to be tested, a total of 36 conditions would have beenrequired to test all the possible conditions, and 108 pig eyes would have been used to performeach combination in triplicate. To simplify the protocol and make the experiment doable, areduced experimental design (with the minimum number of conditions to be able to implementan analysis model) was generated using the JMP software (Table 2). The 12 conditionscalculated were tested in triplicate and the results were implemented in the scientific model tobe able to determine the best impression cytology (IC) method.Table 2. Experimental design of the optimization of the impression cytology.Condition Membrane Application Time Application Method1 CelAc 0.20 8 seconds Goldmann Cone2 NiCel 0.45 8 seconds Goldmann Cone3 Biopore 8 seconds Patient Interphase4 PES 0.20 4 seconds Forceps5 CelAc 0.20 4 seconds Forceps6 PES 0.45 4 seconds Goldmann Cone7 NiCel 0.45 4 seconds Patient Interphase8 PES 0.20 8 seconds Patient Interphase9 Biopore 4 seconds Goldmann Cone10 PES 0.45 8 seconds ForcepsCondition Membrane Application Time Application Method11 CelAc 0.45 4 seconds Patient Interphase12 CelAc 0.45 8 seconds ForcepsIn addition, 6 positive controls and 6 negative controls were added: one positive controland one negative control per membrane type.For the positive control, 200 µL of a suspension of hybridoma cells (1 x106 cells / mL inPBS) were added onto a membrane placed in a well of a 12-well plate. The membrane wasincubated with the cell solution for 2 minutes to allow the cells to adhere.For the negative control, the membrane was incubated with PBS alone for 2 minutesbefore performing the Hoechst staining.The corneal cell samples were collected from fresh (less than 8 hours post-mortem) pigeyes. Excess extraocular tissue was removed from the pig eyes which were then rinsed withnon-preserved sterile saline solution. The eyes were left to dry at room temperature for acouple of minutes before performing the IC according to each of the 12 conditions. Right afterperforming the IC, the membranes were treated with a cell fixing solution and stained with theHoechst stain. The Hoechst is a fluorescent stain which labels DNA. It therefore allows thevisualization of each fixed cell attached to the membrane used for IC.Hoechst staining was performed according to the following protocol:• The membrane was transferred to a well of a 12-well plate, cells facing upward.• 1 mL of fixation buffer (70 % Ethanol Absolute, 25 % Paraformaldehyde-4 %,5 % Glacial Acetic) prepared on the day of the experiment was added carefullyand incubated for 15 minutes.• The fixation buffer was removed, 2 mL of H2O were added and incubated for 5minutes. •The H2O was removed, 1 mL of Hoechst solution (1:1000 in PBS) was addedand incubated in the dark for 15 minutes.• The membrane was transferred to a well of a 6-well plate with the cells facingdownward and covered with a coverslip.• The Cytation (Automated Imaging Microplate Reader, BioTek Cytation 5,Agilent) was used to visualize the membranes.To quantify the number of cells collected, a photo of the entire membrane was takenwith the Cytation microscope for each sample and was then analyzed with the ImageJsoftware. Positive and negative controls for each membrane type were used to subtractmembrane autofluorescence and establish the cell quantification parameters.2.3. Experiment 3: Test of the method of membrane scraping cytologyTo further optimize the collection of cells by cytology, fresh (less than 8 hourspostmortem) pig eyes were used to compare the IC method investigated in experiment 2 withthe membrane scraping cytology (MSC) method. In this comparative study, to simulate theconditions of sample collection in clinical centers, anesthetic was added on the cornea beforethe application of the membrane. IC was performed by applying the membrane on the corneafor 4 seconds with Adson forceps by applying gentle pressure over the whole surface of themembrane while MSC was performed by rubbing the membrane 4 times over the cornea.Moreover, the advantage observed with the use of the PES membrane (experiment 2)was also evaluated with the MSC method. Finally, the effect of the lubrication of the eye witha non-preserved sterile saline solution was assessed (Table 3). After adding 1 drop ofanesthetic on the cornea and letting the eye dry for 2 minutes, 1 drop of sterile saline solutionwas added on the cornea and immediately the membrane was applied or scrapped on thecornea. Table 3. Experimental design of the validation of the membrane scraping cytology.Membrane Membrane applicationLubrication NPES 0.20 1PES 0.20 1PES 0.20 IC No 3PES 0.20 IC Yes 3PES 0.20 MSC No 3PES 0.20 MSC Yes 3PES 0.45 1PES 0.45 1PES 0.45 IC No 3PES 0.45 IC Yes 3PES 0.45 MSC No 3PES 0.45 MSC Yes 3Cel Ac 0.45 1Cel Ac 0.45 1 Membrane Membrane applicationLubrication NCel Ac 0.45 IC No 3Cel Ac 0.45 IC Yes 3Cel Ac 0.45 MSC No 3Cel Ac 0.45 MSC Yes 3Twelve sample collection conditions were assessed in triplicate with the addition of 3positive and 3 negative controls (one positive and one negative control per membrane type).For the positive control, 200 µL of a suspension of hybridoma cells (1 x106 cells / mL inPBS) were added onto a membrane placed in a well of a 12-well plate. The membrane wasincubated with the cell solution for 2 minutes to allow the cells to adhere.For the negative control, the membrane was directly incubated in the fixing solution.The corneal cell samples were collected from fresh pig eyes. After removing the excessextraocular tissue, one drop of “Colirofta doble” anesthetic was added and the eye wasallowed to dry at room temperature for 2 minutes. IC or MSC was then performed accordingto the 12 conditions described in Table 3.Right after performing the IC or MSC, the membranes were fixed, stained with Hoechstand visualized with the Cytation according to the protocol described in Experiment 2.3. Results3.1 Experiment 1: Toxicity studyA cell viability higher than 80 % was observed with the anesthetic Barraquer in-houseat the shorter incubation times (5 min and 15 min). The viability decreased after 30 min and60 min incubation, especially at the higher concentration (dilution 1 / 3) (Figure 1).A cell viability similar to the one observed with the Barraquer in-house anesthetic wasobtained with the “Colirofta Doble” after 2 min or 5 min incubation, ~80 % cell viability. A lowercell viability was observed with the “Colirofta”, ~65 % viability. The cell viability decreasedsignificantly with a longer incubation for both anesthetics (Figure 2).At a comparable dilution (1 / 3), there was no significant difference between the Barraquerin-house, the “Colirofta” and the “Colirofta Doble” anesthetics after 5 min of incubation.However, after 2 min of incubation, a higher cell viability was observed with the Colirofta Doblein comparison with the Colirofta, and after 15 min incubation the anesthetic Barraquer in-housedemonstrated the highest cell viability of all three anesthetics (Figure 3).3.2. Experiment 2: Optimization of the method of impression cytologyThe membranes allowing the adhesion of more cells, in both the positive controls andthe samples of impression cytology, were the PES membranes (0.20 and 0.45 µm) and thenitrocellulose membrane. The Biopore membrane also demonstrated a good cell adhesion inthe positive control, but it was more difficult to use in impression cytology with the membranebecoming transparent and sticking excessively to the cornea.More cells were collected with the Goldmann cone, but the application time did notinfluence the efficiency of collection (Figure 4).The results obtained for each of the 12 predefined conditions were introduced into TheJMP Functional DOE Analysis option, which uses a generalized regression model to identifythe optimum condition. According to our results and the calculated model, the best method ofimpression cytology should be with a PES membrane 0.20 µm applied on the cornea with aGoldmann cone for 4 seconds.3.3. Experiment 3: Test of the method of membrane scraping cytologyThe results obtained in experiment 2 showed the advantage of using the PES andNitrocellulose membranes over the other membrane types. However, the number of cellscollected with the different IC methods tested remained low. To improve the yield of cellcollection MSC was compared with IC.Very little background was observed in the negative controls while all the positivecontrols presented adhering cells, especially the PES 0.20 µm and PES 0.45 µm membranes(Figure 5).Less than 500 cells were collected using the 0.45 µm cellulose acetate membrane withany of the four tested conditions and no condition-specific advantage was observed with thismembrane. In comparison, both PES membrane (0.20 µm and 0.45 µm) allowed the collectionof more than 500 cells with any of the four conditions tested (Figure 6 and Table 4).The two PES membranes (0.20 and 0.45 µm) allowed the collection of more cells thanthe cellulose acetate membrane using both IC and MSC methods.Using the cellulose acetate membrane, a mean of 260 cells per membrane was collectedby impression cytology (on a dry or lubricated eye) and a mean of 201 cells per membranewas collected by membrane scraping cytology (on a dry or lubricated eye). Significantly morecells were collected with the PES 0.20 µm membrane: a mean of 836 cells per membrane byIC (on a dry or lubricated eye) and a mean of 4907 cells per membrane by MSC (on a dry orlubricated eye) (Figure 6 and Table 4).A significant improvement in terms of cell collection was noted with the PES 0.20 µmmembrane using MSC, 5.8 times more cells were collected with MSC compared to IC. Inaddition, the lubrication of the eye with a non-preserved sterile saline solution improved thecell collection by MSC by 84.4 % (Figure 6 and Table 4).Similar results were observed with the PES 0.20 µm and PES 0.45 µm membranes witha better reproducibility for the PES 0.20 µm membrane (Figure 6 and Table 4).Table 4: Comparison of the number of cells collected by IC (forceps) or MSC (scraping)using three different membranes.Cell countMembrane type Collection Mean SD % CVForceps 333 191 57.3Cellulose Acetate Forceps + lubrication 187 173 92.70.45 µm Scraping 231 204 88.2Scraping + lubrication 172 134 78.0Forceps 628 381 60.7Forceps + lubrication 719 287 39.9PES 0.45 µmScraping 4790 4879 101.8Scraping + lubrication 5763 2613 45.3Forceps 755 551 73.0Forceps + lubrication 917 820 89.4PES 0.20 µmScraping 3450 1006 29.2Scraping + lubrication 6365 2392 37.64. ConclusionsDifferent steps of the corneal cytology process were assessed to improve the qualityand efficiency of the collection of corneal cells.Corneal cytology is a minimally invasive method that requires the use of anesthetic.Therefore, the impact on sample viability of 3 anesthetics used in collaborative centers wasevaluated. Every anesthetic tested showed a toxic effect after a long exposition (> 15 min).Moreover, the anesthetic Barraquer in-house showed an increase of toxicity at a higherconcentration. The ideal protocol would be to use the minimum anesthetic volume needed forthe shorter time possible. As the three anesthetics showed a similar toxicity at a similar dilutionand after a similar incubation time (dilution 1 / 3 and 5 minutes incubation).Out of the 6 tested membranes, the PES membranes showed a higher cell collectionyield. More specifically, the 0.45 µm cellulose acetate membrane allowed very few cells to becollected compared to the PES membranes (0.20 µm and 0.45 µm).With the PES membranes (0.20 µm and 0.45 µm) significantly more cells were collectedwith the membrane scraping cytology compared to the impression cytology and the lubricationof the eye allowed to further improve the collection. The advantage of the lubrication of theeye observed in pig eyes was confirmed with membrane scraping cytology in human cornea(Figure 7) following the procedure on Materials and methods.Example 21. Materials and methodsAll samples used for the following experiments were collected under the methodaccording to the first aspect herein described. Briefly, an anesthetic (Colirofta 5 mg / mLTetracaine hydrochloride + 0.5 mg / mL Naphazoline hydrochloride (CN 672095, AlconLaboratories)) solution was applied to the eye surface and, within 2 min, a moisturizer solution(saline or BSS) was applied before scraping the corneal surface with a polyethersulfone (PES)membrane. Each side of the membrane was scraped 2-3 times over the cornea.For each experiment, PES membrane negative controls were also obtained followingthe same preparation procedure but without contacting the eye surface or any other solution.In the case of human specimens, the local ethical committee approved the collection ofthese samples for molecular analysis and written consent was obtained from all participantsin agreement with the Declaration of Helsinki.1.1. Total protein quantificationEpithelia corneal samples were collected from the surface of 20 cadaveric pig eyes andimmediately transferred to an empty and clean Protein LoBind Eppendorf tube and stored at-80º C until further use.These pig cornea samples were processed as follows to extract total protein content.Five different cell lysis buffers were tested (Table 5); thus, diverse downstream processes canbe performed without interferences. Briefly, samples were lysed with 140 µL cold buffer byintermittent vortexing for 10 min.Afterwards, samples were centrifuged at 1500 g (4º C) for 1 min and supernatant lysateswere recovered in a clean Protein LoBind tube and stored at -20º C until further analysis.Table 5: Lysis buffers tested for protein extraction.Commercial Buffer Preparation reference Addition of Extraction Enhancer Buffer 50X(Ab193971, Abcam) (1:50Ab193970, PTRdilution),1 mM PMSF (78830, Merck), 1 mM SodiumAbcam Orthovanadate (56740, Merck), 2.5 mM NaF (201154, Merck)Cell FNN0011, Extraction ThermoFisher Buffer 89900,Addition of Halt™ Protease Inhibitor Cocktail (87785,RIPA ThermoFisher ThermoFisher) CelLytic™ C2978,(1:100 dilution).M Sigma-Aldrich 78503, M-PER™ ThermoFisher Total protein content of the lysates was determined by means of the bicinchoninic acid(BCA) assay. Concretely, two different assays were used: Pierce™ BCA Protein Assay(23227, ThermoFisher) and Pierce™ Dilution-Free™ Rapid Gold BCA Protein Assay (A55861,ThermoFisher). In short, standard BSA curves ranging from 150 to 10 µg / mL (BCA) and 250to 15 µg / mL (Rapid Gold BCA) were prepared using each of the tested buffers as diluents.Slightly modifying manufacturer’s instructions, BCA assay was conducted at 37º C for 2 h andvolumes were adjusted to perform the analysis in a 384-well plate. Rapid Gold BCA assaywas conducted following the recommended procedure, at room temperature for 5 min. Finally,absorbance was measured at 562 nm (BCA) or 480 nm (Rapid Gold BCA) in a Cytation 5 CellImaging Multimode reader. Blank OD (0 µg / mL) was subtracted from each sample andstandard read and standard curves were adjusted with 4PL in GraphPad Prism software inorder to extrapolate sample protein concentration. Both standards and samples were testedin duplicate and the average protein concentration was used for further analyses. Thosesamples which CV between duplicates was higher than 10% were excluded from analyses.Statistical analysis was performed with JMP 16 software: due to normal distribution ofthe data, ANOVA with Tukey-Kramer HSD posthoc tests were applied to compare among lysisbuffers for each of the protein quantification methods. P-values < 0.05 were consideredsignificant.1.2. LC-MS untargeted proteomics analysisSurface epithelial cell samples were collected from 63 live human corneas andimmediately transferred to an empty and clean Protein LoBind Eppendorf tube and stored at-80º C until further use.These human epithelia corneal samples were processed for proteomics analysisfollowing CORNEA_Project internal SOP. Briefly, samples were mechanically homogenizedwith a paramagnetic particle (BeatBox tissue kit & equipment, PreOmics) using 120 µL lysisbuffer for 10 min. Commercial sample preparation kits were employed (SP3 add-on & iST kits,PreOmics) following manufacturer’s instructions. In short, cell lysates were denaturalized at95º C for 10 min before their protein content was enzymatically digested for 2 h using amagnetic bead-assisted method. Tryptic peptides obtained were cleaned up in C18 cartridgesby elution with a basic organic solution. Finally, these peptides were evaporated to drynessand dissolved in an acidic solution to be run in LC-MS.All samples were randomly analyzed by LC-MS / MS using DIA-PASEF mode on anEvosepOne-TIMSTOF-Flex platform. Peptide identification and bottom-up proteincharacterization was performed by Spectronaut v18 software; the FASTA human referenceproteome data from Uniprot (UP000005640) was used for protein inference.The list of identified proteins was submitted to SubcellulaRVis web-based tool(http: / / phenome.manchester.ac.uk / subcellular / ) in order to locate those proteins in thedifferent subcellular compartments of the cell. Representation of groups of proteins in eachcell location was highlighted by coloring that area in a schematic picture; no Gene Ontologyenrichment analysis was conducted at this point.1.3. RT-qPCR transcriptomics analysisSurface epithelial cell samples were collected from 25 live human corneas andimmediately transferred to an empty and clean Rnase-free Eppendorf tube and stored at -80ºC until further use.Total RNA was isolated from these human epithelia corneal samples with RNeasy PlusMicro Kit (74034, Qiagen), following manufacturer’s instructions, using 150 µL RLT Plus bufferfor cells lysis and eluting RNA with prewarmed water (60º C). The integrity and amount of theextracted RNA was determined using the 4200 TapeStation system (Agilent) before beingretrotranscribed to cDNA by NZY First-Strand cDNA Synthesis Kit, following manufacturer’sinstructions. RT-qPCR was run in triplicates in a 384-well plate format for GAPDH and B2M usingcustomized forward and reverse primers (IDT) and PerfeCta SYBR Green FastMix ROX(95073, Quantabio) in a CFX Opus 384 Real-Time PCR System (12011452, Bio-Rad).Threshold cycle (Ct) values were recorded from each sample and both mean and standarderror were calculated. Only samples showing a standard error lower than 30 % at least induplicates were considered for further analysis.2. Results2.1. Total protein quantificationEpithelia corneal cells from cadaveric pig eyes collected by means of the membranescraping method under the first aspect of the invention, were lysed and total protein contentwas extracted and quantified. Figure 8 shows the amount of protein quantified by 2 differentassays (BCA and Rapid Gold BCA) for 5 different buffers tested.The average yield of protein extraction from membrane scraped epithelia corneal cellsfrom pig eyes was 41.96 ± 28 µg / mL.None of the buffers tested showed statistically significant increase or decrease in proteinyield, showing that different buffers are equally efficient to extract protein content from cellscollected by the membrane scraping method.2.2. LC-MS untargeted proteomics analysisProtein extracts from epithelial cells of human live corneas collected by means of themethod herein described were processed to conduct protein identification by means of LC-MS. On average, more than 4000 proteins were identified (4371 ± 580 proteins), with a CVof 13 % among all samples tested (Figure 9).The list of identified proteins showed full representation over all subcellularcompartments, demonstrating a complete protein extraction and an unbiased proteomicsanalysis (Figure 10).2.3. RT-qPCR transcriptomics analysisRNA was extracted from epithelia cells of human live corneas collected by means of themembrane scraping method of the first aspect of the invention. The average concentration ofRNA extracted was 0.83 ± 1.49 ng / µL and RNA integrity number ranged between 5 and 9.5.Previous results from the inventors with complete epithelia corneal samples obtained byde-epithelization procedure, showed an average RNA concentration of 466.04 ± 418.61 ng / µLand RNA integrity number ranged between 6.1 and 9.9. Running 500 ng of retrotranscribedRNA (N = 29 de-epithelization samples), mean Ct values were 20.80 ± 0.88 for GAPDH and22.27 ± 1.16 for B2M. The average Ct in epithelial corneal cells samples collected bymembrane scraping of GAPDH and B2M was 27.56 ± 1.40 for GAPDH and 24.07 ± 2.12 forB2M.Thus, similar qPCR results were obtained with epithelial cells collected by the membranescraping collection method, even with much less starting RNA.3. ConclusionsThe method of the invention allows obtaining enough surface epithelia cells even fromlive corneas to conduct molecular analysis. With this method, it is possible to extract andquantify protein and RNA molecules, which showed quality enough to be used for omicsanalysis, both at protein and gene transcript levels.

Claims

CLAIMS1. Method for obtaining corneal cells comprising the steps of:(i) applying an anesthetic solution to the ocular surface to be sampled, and(ii) scraping the corneal surface with a polyethersulfone (PES) membrane.

2. The method according to claim 1, wherein the PES membrane has a pore size ofbetween 0.20 µm and 0.45 µm.

3. The method according to claim 1 or 2, wherein the PES membrane has a pore size of0.20 µm.

4. The method according to any one of claims 1 to 3, wherein the anesthetic solutioncomprises an anesthetic selected from the group consisting of lidocaine, tetracaine,oxybuprocaine, a salt thereof and a combination thereof.

5. The method according to claim 4, wherein the anesthetic solution is selected from thegroup consisting of:- an anesthetic solution comprising 0.06 mg / mL lidocaine hydrochloride and 20mg / mL hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 5 mg / mL tetracaine hydrochloride and 0.5mg / mL naphazoline hydrochloride; and- an anesthetic solution comprising 1 mg / mL tetracaine hydrochloride and 4mg / mL oxybuprocaine hydrochloride.

6. The method according to any one of claims 1 to 5, wherein the time elapsed betweenstep (i) and step (ii) is less than 2 minutes.

7. The method according to any one of claims 1 to 6, wherein the method comprises astep of lubricating the ocular surface after step (i) and preceding step (ii), being thatstep (ii) is carried out immediately after the lubrication.

8. The method according to claim 7, wherein the step of lubricating is carried out with asaline solution.

9. The method according to claim 8, wherein the saline solution is selected from a groupconsisting of:- Sodium chloride (NaCl) 9 mg / mL in purified water; and- A solution comprising sodium chloride 6.4 mg / mL, potassium chloride (KCl)0.75 mg / mL, calcium chloride dihydrate (CaCl2·2H2O) 0.48 mg / mL, magnesiumchloride hexahydrate (MgCl2•6H2O) 0.3 mg / mL, sodium acetate trihydrate(C2H3NaO2·3H2O) 3.9 mg / mL, sodium citrate dihydrate (C6H5Na3O7·2H2O) 1.7mg / mL, and water at a pH of 7.5.

10. The method according to any one of claims 1 to 9, wherein the corneal cells areepithelial corneal cells.

11. A kit for carrying out the method according to any one of claims 1 to 10, comprising ananesthetic solution and a PES membrane.

12. The kit according to claim 11, wherein the PES membrane has a pore size of 0.20 µm.

13. The kit according to claim 11 or 12, wherein the anesthetic solution is selected fromthe group consisting of:- an anesthetic solution comprising 0.06 mg / mL lidocaine hydrochloride and 20mg / mL hydroxypropyl methylcellulose gel;- an anesthetic solution comprising 5 mg / mL tetracaine hydrochloride and 0.5mg / mL naphazoline hydrochloride; and- an anesthetic solution comprising 1 mg / mL tetracaine hydrochloride and 4mg / mL oxybuprocaine hydrochloride.

14. The kit according to any one of claims 11 to 13, further comprising a lubricating salinesolution.

15. Use of the kit according to any one of claims 11 to 14 for obtaining corneal cells or tocarry out the method according to any one of claims 1 to 10.

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