Journal of ophthalmic and Vision research 2014;Vol.9,No.4 407

INTRODUCTION

Apterygiumisatriangularorwing‑shapedfibrovascularovergrowthofabnormalconjunctivaontothecornea.The

TransplantationofAutologousEx VivoExpandedHumanConjunctivalEpithelialCellsforTreatmentofPterygia:AProspectiveOpen‑labelSingleArm

MulticentricClinicalTrialViraf Sam Vasania1, PhD; Aarya Hari1, MSc; Radhika Tandon2, FRCOphth; Sanjay Shah3, MS

Suhas Haldipurkar4, DOMS; Smitesh Shah5, DOMS; Shailendra Sachan1, MBBS Chandra Viswanathan1, MD, PhD

1Regenerative Medicine Group, Reliance Life Sciences Pvt. Ltd., Navi Mumbai, Maharashtra, India2Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

3Department of Ophthalmology, King Edward Memorial Hospital, Pune, Maharashtra, India4Laxmi Eye Institute, Panvel, Mumbai, Maharashtra, India

5Dr. Shah’s Laser Eye Institute, Kalyan West, Thane, Maharashtra, India

Original Article

Correspondence to: ChandraViswanathan,MD,PhD.RegenerativeMedicineGroup,RelianceLifeSciencesPvt.Ltd.,R‑282, T.T.CAreaofMIDC,ThaneBelapurRoad,Rabale, NaviMumbai‑400701,India. E‑mail:chandra.viswanathan@ril.com

Received:12‑10‑2012 Accepted:12‑10‑2013

etiologyofpterygiaremainslargelyunknown;[1]butitisbelievedtobecausedbyexposuretoultravioletradiation,dust, anddry climates.[2] In severe cases, visual loss

AbstractPurpose:Toestablishtheefficacyandsafetyofex vivoculturedautologoushumanconjunctivalepithelialcell(hCjEC)transplantationfortreatmentofpterygia.Methods:Twenty‑fivepatientswithpterygiawererecruitedatdifferentcentersacrossthecountry.AutologoushCjECgraftswerepreparedfromconjunctivalbiopsyspecimensexcisedfromthehealthyeyeandculturedex vivoonhumanamnioticmembranemountedoninsertsusingauniquemountingdevice.ThehCjECgraftswerethentransportedinanin‑housedesignedtransportcontainerfortransplantation.Post‑surgery,thepatientswerefollowedupondays1,7,14,30,90,and180aspertheapprovedstudyprotocol.Clinicaloutcomeswereassessedbyslitlampexamination,visualacuity,imprintcytology,fluorescein/rosebengalstaining,Schirmer’stest,andphotographicevaluationthreeand6monthspost‑transplantation.Results: Twopatientswere lost to follow‑upandfinal analysis included23 cases.No recurrenceofpterygiumwasobservedin18(78.3%)patients;alloftheseeyesshowedasmoothconjunctivalsurfacewithoutepithelialdefects.Recurrencewasobservedin5(21.7%)patientsat3monthspost‑treatment.Noconjunctivalinflammation,secondaryinfectionsorothercomplicationswerereported.Adequategobletcellswerepresentin19(82.6%)patientsatthesiteoftransplantation.Conclusion:Wehave,forthe1sttime,standardizedaprotocolforpreparingautologoushCjECgraftsthatcanbesafelytransportedtomultiplecentersacrossthecountryfortransplantation.Theclinicaloutcomewassatisfactoryfortreatingpterygia.

Keywords: Autologous;HumanAmnioticMembrane;ConjunctivalEpithelialCells;GobletCells;Pterygium;MulticenterStudy

J Ophthalmic Vis Res2014;9(4):407‑416.

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DOI: 10.4103/2008‑322X.150800

Ex vivo Expansion of Autologous Human Conjunctival Epithelial Cells; Vasania et al

408 Journal of ophthalmic and Vision research 2014; Vol. 9, No. 4

mayarisefrominducedirregularcornealastigmatism,cornealstromalscarringandobscurationofthevisualaxis.[3,4]Severalsurgicalmethodshavebeenemployedformanagementofpterygium.Simpleexcisionusingthebarescleratechniquehasbeenshowntobeassociatedwitharecurrencerateofmorethan70%.[5‑8]Thishighrecurrence rate has led to the search for adjunctive treatmentoptionssuchaschemotherapyorradiotherapy.Differentsurgicaltechniquescombinedwithadjuvantssuch asmitomycin‑C,[9] β-irradiation,[10] thiothepa,[11] anti‑VEGF(vascularendothelialgrowthfactor)agents,[12] andmorerecently,alcohol[13]havebeentriedinvariouscombinationsforthetreatmentofpterygia.Theuseofamnioticmembranegrafts(AMG)hasbeen

showntobeefficaciousintreatingprimarypterygia.[14] AMGservesasanalternative toconjunctival tissue ineyeswithlargeconjunctivaldefectsandinadequatetissueto cover thebare sclera, commonly seen in recurrentpterygia.[15]However,intermsofpreventingrecurrence,most studieshave shown this treatmentmodality tobe less effective thanconjunctivalautografting.[16] The conjunctivalautograftingtechniquewas introducedbyKenyonetalin1985andhasbecomeapopulartreatmentforrecurrentandadvancedpterygia.[17]Althoughmoretimeconsuming,conjunctivalautograftingisamuchsaferandeffectiveapproachthanchemotherapyorradiotherapy.This technique, targeted to reduce recurrence, hasresulted invaryingdegreesof success.[18] Recurrence ratesashighas39%afterconjunctivalautograftinghasbeen reported.[19] Limbal‑conjunctival autografts afterpterygiaexcisionhavealsoshownpromisefortreatmentofrecurrentpterygia.[20,21]

There iswide variation in the extent of surgicalexcisionofpterygiaandsubconjunctivalfibrovasculartissuebyvarious investigators.[22,23]Excisionof largerconjunctival grafts from thebulbar conjunctivamayhelp reduce the recurrence rate ofpterygia butmayresult in complications such as scarring,fibrosis andinflammationatthedonorsite.[24]Sincethepreferredsiteforautograftexcisionisthesuperiorbulbarconjunctiva,patientsrequiringsubsequentglaucomasurgerycouldfacefurtherproblems.In1997,Pellegrinietalreportedthefirstsuccessful

use of ex vivoexpansionforautologoustransplantationwithoutinducingiatrogenicinjurynormallyassociatedwith autograft excision.[25] Since then, this techniqueis beingused extensively for treatingvariousoculardisorderswithlong‑termpositiveclinicaloutcome.[26,27] The use of cultivated and ex vivoexpandedconjunctivalepithelialcellsheetsfortreatmentofpterygiahasbeeninpracticeforafewyearsnow.Theadvantagesofusingcultivated conjunctival epithelium include reductionin inflammationandearlyepithelialization leading tofasterrecovery.[28‑29]

Wehavestandardizedamethod for ex vivo culture ofautologousconjunctivalepithelialcellswhichwould

benefitpatientswhoaregeographicallydistantfromthecellculturefacility.Duringthedevelopmentstage,themajorchallengewaspreparationofhumanconjunctivalepithelialcell(hCjEC)graftswhichcouldbetransportedtohospitalsacrossthecountry.Toovercomethisissue,wedevelopedanoveldeviceformountinghumanamnioticmembrane (HAM)whichwould serve as a substratefor culturing the cells.[30] Further,wealsodesignedatransportcontainerwhichwouldensuregraftintegrityduringshipment.[31]Tothebestofourknowledge,thisisthefirstmulticentricclinicalstudytoassessthesafetyandefficacyofautologoushCjECgraftstransportedacrossthecountryandusedfortreatmentofpterygia.

METHODS

ThisstudyadheredtothetenetsoftheDeclarationofHelsinki andwasapprovedby the ethics committeesofthestudysites.Informedconsentwasobtainedfromallparticipants.ThestudywasconductedatfoursitesacrossthecountryfromJanuary2008toDecember2009.Twenty‑fivepatientswereenrolledinthestudyaspertheinclusioncriteria[Table1].

Human Amniotic Membrane ProcessingPlacentaswereobtained,afterdueconsentingprocess,frommothersundergoingCaesareansectionandwereused toprepareHAM.Screening tests for infectiousdiseasewere done for human immunodeficiencyviruses1and2(HIV‑1,HIV‑2),hepatitisBandCviruses

Table 1. Criteria for selection of patients for the study

InclusioncriteriaPatientswithunilateralconjunctivaldisordersMaleorfemalepatientsof18yearsandabovePatientwillingtocomplywithprotocoldescribedproceduresWritteninformedconsentfrompatient/patient’slegalrepresentative

ExclusioncriteriaPreviousconjunctivaltransplantsurgeryPregnantorlactatingwomenPatientwithdocumentedHIVinfection/AIDSHistoryofsignificanthematological,hepatic,renal,cardiovascular,respiratory,neurological,endocrinalorallergicdiseasePatientswithhistoryofdiabetesCoexistingconditionslimitingthesuccessfuloutcomeofthetransplantsurgerysuchasdryeye(Schirmer’s<6mm),lidmargindisorderoractivelyinflamedeyeAnydebilitatingdisease/disorderorpsychiatricconditionthatinthejudgmentoftheinvestigatorwouldinterferewiththeadherencetothestudyprotocolorabilitytogiveinformedconsent

HIV,humanimmunodeficiencyviruses;AIDS,acquiredimmunedeficiencysyndrome

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(HBV,HCV) by polymerase chain reaction (PCR)and for cytomegalovirus (CMV‑IgM,CMV‑IgG), andSyphilis IgM/IgGby serology.Amnioticmembranewasprocessedaccording to themethodproposedbyKimet al.[32] Briefly, theplacentawas cleanedunderaseptic conditions and the amnionwas separatedfrom the chorionbybluntdissection.Themembranewas cut into pieces admeasuring 4 cm× 4 cm andplacedonseparatepiecesofnitrocellulosepaper.Eachmembranewasplacedinthesterilespecimencryogenicvial(ThermoFisherScientific‑Nunc,DK‑4000Roskilde,Denmark) containingDulbecco’smodified Eagle’smedium (DMEM) (Invitrogen‑Gibco,Carlsbad,CA,USA) and glycerol (SigmaAldrich, St. Louis,MO,USA)[1:1]andcryopreservedat−80°C.Sterilitychecksandendotoxintestswereperformedbeforereleasingthemembranesforclinicaluse.

Mounting on Human Amniotic MembraneOnthedayofbiopsyprocessing,twomembraneswerethawed andwashed thricewith sterileDulbecco’sphosphate‑buffered saline (DPBS) (Invitrogen‑Gibco,Carlsbad,CA,USA)for5mineachtime.Thebasementmembrane side of HAMwas then treated withtrypsin‑EDTA(Invitrogen‑Gibco,Carlsbad,CA,USA)for15minat37°C.Theamnioticepitheliumwasgentlyremovedusinga cell scrapperandwashed thrice for5mininDPBS(1X)toremovecellulardebris.TheHAMwasoriented correctly, asdescribedby

Zakaria et al,with its basementmembrane facingupwards.[33]Thenitrocellulosemembraneonthemillicellinsert(MilliporeCorporation,Billerica,MA,USA)wasgentlypeeledoff.Theinsertwasthenplacedontopofthemembrane and the edgesof themembranewerepulledovertherimoftheinsert.Thisisnowreferredtoas“HAMconstruct”.TheHAMconstructwasthenflippedoverandplacedon thebaseof themountingdevice[Figure1a].AsiliconeringwasslippedontotheHAMconstructusingtheplungerofthemountingdeviceinordertoprovideawrinkle‑freesubstrate[Figure1b].TheHAMconstructwasthenflippedbackandplacedinthe6‑wellplate(ThermoFisherScientific‑Nunc,DK‑4000Roskilde,Denmark)containingsupplementalhormonalepithelialmedium(SHEM).

Preparation of Human Conjunctival Epithelial Cell GraftsConjunctival biopsies, approximately 2mm× 4mmin size,withunderlying stromawereobtainedunderstrictasepticconditionsfromthesuperiorfornixofthecontralateralhealthyeyeofpatients.Biopsiescollectedattherespectivesitesbytrainedinvestigatorsweresentto the currentGoodManufacturingPractices (cGMP)facilityforprocessing.Theyweretransportedat2°C–8°CinSHEM[Table2].Thebiopsywascutintopiecesand

seededontwoHAMconstructsinSHEM.Thecultureswereincubatedat37ºCinanatmosphereof5%CO2 and 95%air.Growthofcellsfromtheexplantswasmonitoredwithaninvertedphase‑contrastmicroscope(OlympusCorporation,Tokyo,Japan)foraperiodof14–21days.Theculturemediumwasreplenishedonalternatedays.

Uniqueness of the Transport ContainerThe transport containerwas designed to hold thegraft assembly in place and protect the graft from

Table 2. Composition of supplemental hormonal epithe‑lial medium

Medium components

Final concentration

Manufactured by

DMEM/F‑12(1:1) 1X Invitrogen‑Gibco,Carlsbad,CA,USA

Fetalbovineserum(EScelltested)

10% HyCloneLaboratoriesInc.,Logan,Utah,USA

Dimethylsulphoxide

0.1% Sigma‑Aldrich,St.Louis,MO,USA

Epidermalgrowthfactor

10ng/ml Sigma‑Aldrich,St.Louis,MO,USA

Insulin‑transferrin ‑selenium

1X Invitrogen‑Gibco,Carlsbad,CA,USA

Hydrocortisone 0.5µg/ml Sigma‑Aldrich,St.Louis,MO,USA

Basicfibroblastgrowthfactor

4ng/ml R&Dsystems,Minneapolis,MN,USA

Penicillin‑ Streptomycin

1X Invitrogen‑Gibco,Carlsbad,CA,USA

Gentamicin 50µg/ml Sigma‑Aldrich,St.Louis,MO,USA

DMEM,dulbecco’smodifiedeagle’smedium;ES,embryonicstem

Figure 1. (a and b) Indigenously designed andpatentedmountingdevice formountinghumanamnioticmembraneonto amillicell insert, (c) In‑housedesigned andpatentedstainless steel transport container for transportationof thegrafttohospitalsite.

c

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410 Journal of ophthalmic and Vision research 2014; Vol. 9, No. 4

damageduring transport. The transport container iscylindricalinshape,madeofSS316Landhasascrewcaplid[Figure1c].Aspeciallydesignedsiliconegasketholdsthegraftfirmlyinplaceandpreventsleakageofmediumduringtransport.

Packaging of the GraftUponattainingconfluence(approximately14–21days),thegraftswereindividuallypackagedinthein‑housedesignedtransportcontainers.Thegraftassemblywasplacedgentlyinsidethecontainerandthemediumwasaddedslowlyalongthesides.Thesiliconegasketwasplacedontopandthecontainerwasclosedwiththelid.Thepreparedgraftsweretransportedtothehospitalsitefortransplantationwithin24hofpackaging.Onlyonegraftwasneeded for the transplantation.The secondgraftwas sent as a standby in theunlikely event ofdamagetothefirstgraft.

In‑Process TestingDuringgraftpreparation,spentmediumwascheckedforsterilitybydirectinoculationmethodasprescribedin the current IndianPharmacopoeia and endotoxintest using the Limulus Amebocyte Lysate (LAL)gel‑clotmethod (WakoChemicals, Richmond,VA,USA)atdifferent stagesduring theprocess [Table3].Mycoplasmatestingofthespentmediumwascarriedoutasperthemanufacturer’sinstructions(MinervaBiolabsGmbH,Berlin,Germany).

Characterization of the Human Conjunctival Epithelial Cell GraftAfter successful transplantation, the second graftwassentbacktothefacility.Theexpandedcellswerecheckedforviabilityandexpressionofkeymarkersbysemi‑quantitativeRT‑PCR.

Gene Expression Profiling by Semi‑ Quantitative Reverse Transcription‑ polymerase Chain ReactionRNA extraction was performed using RNeasyMini Kit (QIAGEN,Hilden, Germany) as per themanufacturer’sinstructions.OnemicrogramofRNAwasconvertedintocomplementaryDNA(cDNA)utilizingthefirststrandsynthesiskit(Invitrogen,Carlsbad,CA,USA) as per themanufacturer’s protocol. PCRwasperformedasreportedpreviously.[34]TheprimerdetailsareasgiveninTable4.

Cellular CharacterizationDuring the standardizationprocess for preparationof hCjEC grafts, immunofluorescence stainingwasperformed to confirm the identity of conjunctival

epithelial cells. Formalin‑fixed paraffin‑embeddedsectionsofhCjECgraftweredeparaffinizedwithxyleneandgradedalcoholtreatment.Thesectionsweredippedin 10mM sodium citrate buffer, pH 6.0 for antigenretrieval.Thesectionswereheatedinamicrowaveovenfor30 s andpermeabilizedwith0.2%TritonX‑100 inphosphate‑bufferedsaline(PBS).Thenon‑specificbindingsiteswereblockedwith3%bovineserumalbumin(BSA)inPBSfor60minutesat4°C.Sectionswereincubatedwithmouseanti‑cytokeratinepithelialcloneAE1(Millipore,Billerica,MA,USA),mouseanti‑cytokeratin epithelialcloneAE3(Millipore,Billerica,MA,USA),goatanti‑mucinMUC4(SantaCruzBiotechnology,SantaCruz,CA,USA),andmouseanti‑mucinMUC5AC (Millipore,Billerica,MA,USA)overnightat4°CfollowedbyincubationwiththeirrespectiveFITC‑conjugatedsecondaryantibodiesfor30minutesat4°C.Excessantibodieswerewashedoffandsamplesweremountedinimmunofluorescencemountingmedium(Sigma‑Aldrich,St.Louis,MO,USA)andobservedunderafluorescencemicroscope(NikonE600)forthepresenceofimmunoreactivecells.

Surgical ProcedureThe surgicalprocedurewas carriedout asdescribedpreviously.[18]Underlocalanesthesiawith2%lignocaine,theheadofthepterygiumwascompletelyremovedbybluntdissection.ThehCjECgraftwaswashed4timeswithDPBS (1X) containingD‑glucose and sodiumpyruvate (Invitrogen‑Gibco,Carlsbad,CA,USA)andplacedoverthediseasedconjunctivalsurfacewiththeepithelialcellsfacingupwards.Thegraftwastrimmedtofittheentireconjunctivaldefect,includingthebulbarsurface of the fornix and the deeper portion of thepalpebralaspectofthefornix, ifrequired.Itwasthensecured to the recessed conjunctival edgewitha fewinterrupted or running 8.0 vicryl sutures so that itsmarginremainedplacedundertheconjunctivalmargins.Sutureswere tied carefully ensuring that the trailing

Table 3. Schedule for in‑process testing

Stage In‑process quality checks

Sterility testing

Endotoxin testing

Mycoplasma detection

Conjunctival culture medium

√* √ ‑†

Spentconjunctivalbiopsycollectionmedium

√ √ ‑

Spentconjunctivalculturemedium(day10)

√ √ ‑

Spentconjunctivalculturemedium(day12)

‑ ‑ √

Spentconjunctivalculturemedium (atthetimeofpackaging)

√ √ ‑

*Testwasdone;†Testwasnotdone

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suturedidnotdragalongthesurfaceofthemembrane.Ifclinicallyrequired(basedoninvestigator’sdiscretion),the conjunctival graftwas then tucked to the deepfornixbyamusclehookanchoredtothepalpebralsideofthefornixbypassingtwoorthreedouble‑armed6.0vicrylsuturesthroughthefullthicknessofthelidandsecuredtotheskinwithsiliconebolsters.Therestoftheconjunctivalgraftwasthensecuredandflattenedtothebulbar aspectby interrupted10.0nylon sutureswithsuperficialscleralbites.Atopicalbroad‑spectrumantibioticwasusedandthe

eyewaspatched.Thepatchwasnotdisturbedforthenext24h.Post‑operatively,prednisoloneeyedropswereinstilled4timesadayfor4weeks,followedbytopicalfluorometholoneandtobramycin4timesadayforthenext4weeks.Lubricatingeyedropswereadministeredat2‑4hintervalsforthefirst1‑monthandthentapereddownto3timesadayforthenext2months.

Post‑operative Evaluation of Clinical OutcomesThroughout the study, operated eyewas observedforsignsofinflammationandinfection.Patientsweremonitoredon atpost‑operativedays 1, 7, 14, 30, 90,and180forgraftedema,lacrimation,graftretraction/necrosisandirritation.Physiologicalandanatomicalassessmentofconjunctival

epitheliumwasdoneonday90andday180usinglocal

andslitlampexamination,imprintcytology,fluorescein/rose‑bengalstaining,Schirmer’stest,andvisualacuity.Thegraftsuccesswasassessedbasedonthesmoothness,vascularity, epithelial integrity, adequatehydration,tear formationand thepresenceofgoblet cellson theconjunctivalsurface.Anyevidenceofinflammationoftheconjunctivalsurface,necrosisatthesiteoftransplant,drycorneawithblurredvisionandmucopurulentdischargefromtherecipienteyewithin1‑weekoftransplantationwereconsideredassignsofgraftfailure.

Statistical AnalysisDataprocessing, tabulation of descriptive statistics,andcalculationof inferentialstatisticswasperformedprimarilyusingSASsoftware(release9.0orhigher)forWindows.Statisticalanalysiswasperformedusing95%confidenceintervalalongwithcountsandproportions.

RESULTS

ThedemographicandclinicaldetailsaresummarizedinTable 5.A total of 25patients from fourdifferentcenterswere recruited. These patients underwentpterygium excision followed by transplantation ofautologoushumanconjunctivalepithelialcellgraft.Thepatientsincluded12(48.0%)maleand13(52.0%)femalesubjectswithmeanageof43.9(range,25–67)years.Allpatientswerefollowedfor6months.Two(8.0%)patientswerelosttofollow‑up.

Table 4. Primers used for semi‑quantitative RT‑PCR

Gene Primer sequence Annealing temperature (°C)

PCR product (bp)

Gene bank accession number

p63 F‑AGCAGCAAGTTTCGGACAGTR‑GCTGCTGAGGGTTGATAAGC

60 378 NM_001114978

Oct4 F‑AGTGAGAGGCAACCTGGAGAR‑CAAAAACCCTGGCACAAACT

60 447 NM_002701

CK4 F‑CCAGGAGCTCATGAGTGTGAR‑CCAAACTCCAAGAGGCAGAG

60 491 NM_002272

CK7 F‑CAGGAACTCATGAGCGTGAAR‑GGGTGGGAATCTTCTTGTGA

60 346 NM_005556

CK12 F‑AGGACTGGGTGCTGGTTATGR‑CAGGGCCAGTTCATTCTCAT

58 436 NM_000223

CK19 F‑AGCAGGTCCGAGGTTACTGAR‑CCTCCAAAGGACAGCAGAAG

60 367 NM_002276

ABCG2 F‑TTATCCGTGGTGTGTCTGGAR‑CCTGCTTGGAAGGCTCTATG

58 429 NM_004827

Integrinβ1 F‑AATGAAGGGCGTGTTGGTAGR‑CCTCGTTGTTCCCATTCACT

63 664 NM_002211

Connexin43 F‑GGACATGCACTTGAAGCAGAR‑GGTCGCTCTTTCCCTTAACC

60 368 NM_000165

MUC4 F‑AAAACAGCCCACTGATGTCCR‑CCAGCCTTCACGAAACTCTC

60 353 NM_004532

GAPDH F‑TTCACCACCATGGAGAAGGR‑CATGTGGGCCATGAGGTC

60 690 NM_002046

RT‑PCR,reversetranscription‑polymerasechainreaction;CK,cytokeratin;MUC,mucin;GAPDH,glyceraldehyde‑3‑phosphatedehydrogenase

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All conjunctival biopsies even from the farthestsite (approximately 2,000 km away)were receivedat the facilitywithin24hofexcision.Theywerewelltransported at 2°C–8°C in a validated shipper. ThebiopsieswerecutintosmallpiecesandseededasexplantsontwodifferentHAMinparallel.Initialmigrationoftheconjunctivalepithelialcellsfromtheexplantswasseenbyday2[Figure2a].Thecellsstartedexpandinginaconcentricmanner and showed typical cobble‑stonemorphology [Figure 2b]. On day 12,mycoplasmatestingbyPCRwasdoneonthespentmedium.Allthecultures (n=24)werefoundtobenegative[Figure3].Aconfluentsheetofepithelialcellswasobtainedwithin14‑21days.ThesehCjECgraftswerethenpackagedandtransportedinspeciallydesignedstainlesssteeltransportcontainersthatmaintainedgraftintegrityduringtransit.All the cultivated conjunctival grafts, at process

development stage, showed high expression ofcytokeratin epithelial clones AE1 and AE3; andgoblet‑cell richmucins,MUC4 andMUC5AC byimmunofluorescencestaining,thusconfirmingthattheywereconjunctivalcells[Figure4].Muchasitwouldhavebeendesirabletocharacterize

thecultivatedconjunctivalgraftspriortotransplantation,

thesizeoftheexcisedtissuefromthedonoreyedidnotprovide adequate cells for characterization.Hence, itwasdecidedtoconfirmtheidentityoftheculturedcellspost‑operatively,fromthesecondgraftsentbackbytheinvestigator.The secondhCjECgraftwas transported back at

2°C–8°C and received at the cGMP facilitywithin48h of transplantation.Upon receipt, theseunusedgrafts were assessed for viability andmolecularcharacterization.Theviabilityofthecellsasdeterminedbytrypanbluedyeexclusionassaywasbetween78%and90%[Figure5a]inallspecimens.Semi‑quantitativeRT‑PCRanalysiswasdoneonconjunctivalcells.HighlevelsofmRNAexpressionofepithelialstemcellmarker,p63;conjunctivalepithelialcellmarkers,CK4andCK7;epithelial basalmarker, CK19; integrinβ1 (CD29);connexin 43 (CX43); and goblet‑cellmarker,MUC4were observed in these cells.We also observed thatthesecellsweaklyexpressedCK12andABCG2.Theseresultsclearlyindicatethatthecellsretaintheirnormalcharacteristics[Figure5b].The focus of the studywas also todetermine the

clinicalacceptanceandsuccessofthegraftandtherateofpterygiumrecurrence.Figure6a‑fshowsacasewithpterygiumextendinguptothecentralcorneafromthenasal region in therighteyeprior toandafterhCjECgraftingondays1,14,30,90,and180.One of the evaluated parameterswas reduction

in ocular surface inflammation.We observed thatconjunctival inflammation subsided tonormal levelsin16(69.6%)patientswithin7daysandin18(78.3%)

Table 5. Demographic and clinical details of the study

Numberofpatients 25(100.0)Left eye affected 15(60.0)Righteyeaffected 10(40.0)Meanage(years)±SD 43.9±11.09Rangeofage(years) 25‑67Gender 12(48.0)male

13(52.0)femaleRace Indian/AsianTreatmentmodality hCjECgrafttransplantationGraftrejection NonePterygiumrecurrence 5.0(21.7)Patientslosttofollow‑up 2.0(8.0)SD,standarddeviation;hCjEC,humanconjunctivalepithelialcell

Figure 3.Detection ofMycoplasmaDNA. Spentmedium(day12)wastestedbypolymerasechainreaction(PCR)usingprimersetsspecifictothehighlyconserved16SrRNAcodingregioninthemycoplasmagenome.Theexpectedproductsizesforpositiveandnegativecontrolsare270and191basepairs,respectively.Spentmedium(day12)wasfoundnegative.A100bpDNAladderwasusedasthemolecularsizemarker.PCRdatapresentedherearerepresentativeof24independentclinicalsamples.

Figure 2. Phase‑contrastmicroscopy of ex vivo expandedhCjECsfromconjunctivalbiopsy.(a)Primaryexplantculturefromconjunctival explants seededonHAMshowbuddingofcellsbyday2. (b)Confluentmonolayerofcells showingcuboidalmorphologytypicallyofepithelialcellsbyday14.Totalmagnificationofthephase‑contrastimage:×100.Arrowshowsbuddingof cells from the explants. hCjECs,humanconjunctivalepithelialcells;HAM,humanamnioticmembrane.

ba

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patientswithin14daysoftransplantation.Onday90,noconjunctivalinflammationwasseenintheoperatedeyeof22(95.7%)patientsbutin1(4.3%)patientitpersisteduntil theendof studyperiod.Otherparameters suchas overall luster, discharge, andbleedingwere alsomonitoredinthetreatedeyeinallpatients.Lusteranddischargefromtherecipienteyewasnormalfromday14onwardsuntiltheendofthestudy.Minorbleedingwasobservedin2patientsonday7buttherewasnoconjunctivalbleedinginanypatientfromday14uptotheendofthestudyasshowninTable6.Therewereno

secondary infectionsorother complicationsobservedwithrespecttothetransplant.Visualacuityoftherecipienteyewascheckedonday

1andpost‑operativedays90and180.Thenumberofpatientswithvisualacuityof6/6increasedfrom7(28%)pre‑operativelyto12(48%)atfinalvisit.Noworseningof

Figure 4.CellularcharacterizationofhCjECgraft.Representativeimagesofimmunofluorescencestainingfor(a)acidic(typeI)cytokeratin (AE1), (b)basic (type II) cytokeratin (AE3), andgoblet‑cellrichmucins,(c)MUC4and(d)MUC5AConparaffinsectionsoftheconjunctivalgraft.hCjEC,humanconjunctivalepithelialcell.

dc

ba

Figure 5.Viabilityandgeneexpressionanalysisof returnedhCjECgrafts. (a)Cell viabilitywasdeterminedby trypanbluedye‑exclusionassayonconjunctivalcellsrecoveredfromthereturnedgrafts.(b)RNAwasisolatedfromthegraftandexpressionofp63,Oct4,CK4,CK7,CK12,CK19,ABCG2,integrinβ1(Intβ1),connexin43(CX43),andMUC4wereanalyzedbysemi‑quantitativeRT‑PCR.GAPDHwasusedasan internalstandard.TheexpectedproductsizesforGAPDH,p63,Oct4,CK4,CK7,CK12,CK19,ABCG2,integrinβ1(Intβ1),connexin43(CX43),andMUC4are690,378,447,491,346,436,367,429,664,368,and353basepairs,respectively.A100bpDNAladderwasusedas themolecular sizemarker.Datapresentedherearerepresentativeof24independentclinicalsamples.RT‑PCR,reversetranscriptase‑polymerasechainreaction.

b

a

Figure 6. (a)Pre‑operativephotographof the eyeof apatientwith recurrentpterygiumextending from thenasal region.Post‑operativephotographs,afterhCjECgrafttransplantation,takenon(b)day1,(c)day14,(d)day30,(e)day90,and(f)day180respectively,showsnorecurrenceofpterygium.hCjEC,humanconjunctivalepithelialcell.

d

cb

f

a

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visionwasobservedinanypatientduringthefollow‑upperiod.Imprintcytologyshowedthatgobletcellsatthesiteof

transplantationwereseenin17(73.9%)patientsonday30andin19(82.6%)patientsbyday180.Nogobletcellwasseenin3(13%)patientsattheendofthestudy.Inaddition,Schirmer’stestconfirmedadequatehydrationwithpresenceoftearsinall23(100%)patientsatdays90and180.No recurrence of pterygiumwas observed in

18 (78.3%) patients until day 180 and conjunctivalsurfaceappearedclinicallynormalinthesepatientsfrom3monthspost‑operatively.Intheremaining5(21.7%)patients, pterygium recurrencewasobservedwithin6months.

DISCUSSION

Pterygia are complex fibrovascular conjunctivalovergrowthsonthecorneaandsurgicalexcisionisthemostprevalenttreatmentoption.Recurrenceiscommonand therefore, newer approaches to treat pterygiaarebeing contemplatedand triedby researchersandophthalmic surgeons.[15]More recently, autologousconjunctival epithelial cellgraftinghasbeen reportedforreconstructingconjunctivaldefects.[29] The results of ourclinicaltrialtoassessthesafetyandefficacyofsuchautologousgrafts to stabilize the conjunctival surfacehavebeenpromising.ThisclinicalstudywasconductedwithformalapprovalfromtheDrugControllerGeneralofIndia,thehighestapprovingauthority,intheIndiancontext.Allproceduresforgraftpreparationincludingcell characterization and transport conditionswereestablishedpriortocommencementofthestudy.Conjunctivalepithelialcell transplantationrequires

a carrier tissue, as it is notpossible to transfer thesecells alone.Anatural basementmembrane,HAM isalreadyinclinicaluseandisbelievedtominimizeocularinflammation,reducepainandaidinepithelialization.[35] HAMhasbeenusedascarriertissueforconjunctivalstemcells.[29]Inourstudy,weusedHAMasthesubstratumforculturingconjunctivalepithelialcells.ThehCjECgraftswerepreparedfromtheipsilateral

conjunctivaltissue.Regardlessofage,thebiopsysampleswere all 2mm×4mm in size andall of themwereculturedonHAMaspertheproceduredescribedinthemethods section.Thequalityof thepreparedgraft isdirectlyrelatedtotheconjunctivalbiopsytissueexcisedfromthedonoreye.Despitethisinherentvariation,ourresultsshowedconsistencyinthequalityofthegraftspreparedusingthisstandardizedmethod.Transporting the grafts todistant sites across the

countrywas a challenge.Wedidvalidation runs toestablish the logistics and transport conditions. Thein‑house designed transport containermaintainedproper orientation and integrity of the graft during

transit.Our validation study showed that the graftcan be transported at 2°C–8°Cwithout affecting itsviabilityand integrity [Table7].No instanceofgraftdamagewasnotedduringtransport.Also,cellviabilityofallreturnedgraftswasfoundtobegreaterthan80%whenassessed48haftersurgery.Atthehospitalsite,the doctors and the paramedical staffwere trainedtohandlethegrafttoensurethatthereisnodamageduringtransplantation.Mostofourpatientshadprimarypterygiawhilefive

caseshad recurrent lesions.Theprimaryobjectiveofthestudywastoassessfeasibilityofthegraftthroughmanifestations such as inflammation, graftmelting,mucopurulentdischarge,necrosis,andrednessontherecipienteye.Theentirestudywasuneventfulwithnocaseshavingpost‑operativeinfectionsorredness.Minoradverseeventslikeeyepainandlocalirritationsubsidedwithmedications.

Table 6. Parameters monitored in the recipient eye post‑transplantation

Parameter Follow‑up (days)

Number of patients (n=23) (%)

Normal Abnormal

Inflammation 7 16(69.6) 7(30.4)14 18(78.3) 5(21.7)30 18(78.3) 5(21.7)90 22(95.7) 1(4.3)180 22(95.7) 1(4.3)

Luster 7 23(100) 0(0)14 23(100) 0(0)30 23(100) 0(0)90 23(100) 0(0)180 23(100) 0(0)

Discharge 7 22(95.7) 1(4.3)14 23(100) 0(0)30 23(100) 0(0)90 23(100) 0(0)180 23(100) 0(0)

Bleeding 7 21(91.3) 2(8.7)14 23(100) 0(0)30 23(100) 0(0)90 23(100) 0(0)180 23(100) 0(0)

Table 7. Stability study of the hCjEC* grafts (n=10)

Parameter analyzed

Storage time at 2°C‑8°C

6 h 12 h 24 h 48 h

Attachmentofcells Good Good Good GoodMorphologyofcells Cuboidal Cuboidal Cuboidal CuboidalCellviability% 95±2 90±2 88±3 82±3pHofthetransportmedium

7.2 7.3 7.5 8.3

*hCjEC,humanconjunctivalepithelialcell

Journal of ophthalmic and Vision research 2014;Vol.9,No.4 415

Ex vivo Expansion of Autologous Human Conjunctival Epithelial Cells; Vasania et al

Surgicalexcisionremainstheconventionaltreatmentfor pterygium and recurrence is themost commonundesirableoutcome.Thetwoimportantconcernsinpterygiumsurgeryaretoreducepterygiumrecurrenceandminimize complications arising from surgery.Thereisnostandarddefinitionofrecurrencebutwhenafibrovascular outgrowth is observed at the site ofpreviouslyexcisedpterygium,itisgenerallyacceptedasrecurrence.[15]Ithasbeenreportedthatatleast97%of all recurrencesmanifestwithin the 1st year after excision.[36]Muchas itwouldhavebeendesirabletoextend follow‑up to 1‑year, our approved protocolwaslimitedto6months.However,wedidfollowthepatientsforupto1‑yearthroughtelephoneinteractionswith the investigator, purely out of interest. In ourstudy, 18 (78.3%) patients had no recurrence ofthe pterygia at 6months. Informal communicationconfirmed that therewas no recurrence in thesepatientsevenafter1‑year.Ocular surface inflammation plays a significant

roleinincreasingtheriskofpterygiumrecurrence.Hence, control of inflammation before and aftersurgeryisessentialforreducingrecurrencerate.Inour study, conjunctival inflammation subsided in18(78.3%)patientswithin14dayspost‑operativelyand no conjunctival inflammationwas present in22 (95.7%) patients by day 90. This observationcontinued until the end of the study. No otherseriouscomplicationswerereportedduringthestudyindicatingthathCjECgraftwaswelltoleratedatthesiteoftransplantation.Integrity of the conjunctiva is a very important

endpoint. The conjunctival surface became smoothgraduallybytheendof3monthsandcontinueduptotheendofthestudy.Further,attheendof1‑year,wehadinformalcommunicationthattherewasnochangeinthesmoothnessatthetransplantsite.Presence of adequate goblet cells is a critical

parameterthatreflectstheoverallhealthoftheocularsurface.[37]Atleast19(82.6%)patientsshowedsufficientgoblet cells at day 180. This is another importantindicator of normal conjunctival regenerationpost‑transplant.In other studies, transplantationof an autologous

cultivatedconjunctivalepithelialsheetfacilitatedearlypost‑operativeepithelializationandrecovery.However,true recurrencewas observed in 22.7% cases.[18] Inourclinical study,5 (21.7%)patients showedsignsofrecurrentpterygia 3monthspost‑transplantwhich isconsistentwithotherreports.Inconclusion,wedemonstratedforthe1sttime,the

feasibilityoftransportingex vivoexpandedautologoushumanconjunctivalepithelialcells(hCjECs)todistantlocationswithoutcompromisingviabilityandintegrityofthegraft.Thismethodoftransportingculturedgraftsinthepresentstudyseemssatisfactory.Largerstudies

with longer follow‑upwill beneededbeforeone canconsider thismethodas thepreferred alternative fortreatmentofpterygia.

ACKNOWLEDGEMENTS

Theauthorswould like to thankRelianceLifeSciencesPvt.Ltd. (www.rellife.com) forproviding the opportunity andfinancial support to carryout the clinical study.TheyalsothankAllIndiaInstituteofMedicalSciences,NewDelhi;KingEdwardMemorialHospital,Pune;LaxmiEyeInstitute,Panvel;andDr.Shah’sLaserEyeInstitute,Kalyanforconductingthisstudy.

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How to cite this article: Vasania VS, Hari A, Tandon R, Shah S, Haldipurkar S, Shah S, et al. Transplantation of autologous ex vivo expanded human conjunctival epithelial cells for treatment of pterygia: A prospective open-label single arm multicentric clinical trial. J Ophthalmic Vis Res 2014;9:407-16.

Source of Support: Nil. Conflict of Interest: None declared.