Anais
Brasileiros
de
Dermatologia
www.anaisdedermatologia.org.br
INVESTIGATION
Standardization
of
organoid
culture
for
evaluation
of
melanogenesis
induced
by
UVB,
UVA
and
visible
light
夽,夽夽
Thainá
Oliveira
Felicio
Olivatti
a,
Giovana
Piteri
Alcantara
b,
Ana
Cláudia
Cavalcante
Espósito
Lemos
c,
Márcia
Guimarães
da
Silva
d,
Hélio
Amante
Miot
b,∗aFaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,SP,Brazil
bDepartmentofDermatologyandRadiotherapy,FaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,
SP,Brazil
cGraduatePrograminPathology,DepartmentofDermatologyandRadiotherapy,FaculdadedeMedicinadeBotucatu,
UniversidadeEstadualPaulista,Botucatu,SP,Brazil
dDepartmentofPathology,FaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,SP,Brazil
Received9April2019;accepted13June2019 Availableonline6December2019
KEYWORDS
Melanosis; Organoids; Photobiology
Abstract
Background: Organoidculturesareprimaryculturesthatmaintainarchitecturalcharacteristics andtherelationshipsbetweencells,aswellastheextracellularmatrix.Theyarealternatives forpathophysiologicalortherapeuticinvestigationratherthananimalandinvitrotests. Objective: Development of a cutaneous organoid culture model, aiming at the study of radiation-inducedmelanogenesis.
Method: Avalidationstudy,whichinvolvedbiopsiesoftheskinofthebackoftheadultear.One samplewasirradiatedwithdifferentdosesofUVB,UVA,orvisiblelight(VL);theotherwas main-tainedinthedarkfor72h.Theviabilityofthetissueswasevaluatedfromthemorphological andarchitectural parameters ofthehistology, andthe expressionofthe glyceraldehyde-3-phosphatedehydrogenase(GAPDH)gene,byreal-timepolymerasechainreaction(PCR).The radiation-inducedmelaninpigmentationwasstandardizedaccordingtothedosesofeach radi-ationandevaluatedbydigitalimageanalysis(Fontana-Masson).
夽 Howtocitethisarticle:OlivattiTOF,AlcantaraGP,LemosACCE,SilvaMG,MiotHA.Standardizationoforganoidcultureforevaluation
ofmelanogenesisinducedbyUVB,UVAandvisiblelight.AnBrasDermatol.2020;95:46---51.
夽夽StudyconductedattheDepartmentofDermatologyand Radiotherapy,FaculdadedeMedicinadeBotucatu, UniversidadeEstadual
Paulista,Botucatu,SP,Brazil.
∗Correspondingauthor.
E-mail:heliomiot@fmb.unesp.br(H.A.Miot).
https://doi.org/10.1016/j.abd.2019.06.005
0365-0596/©2019SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC BYlicense(http://creativecommons.org/licenses/by/4.0/).
Results: TheprimaryskinculturewasstandardizedatroomtemperatureusingDMEMmedium. The doses ofUVB,UVA, andVL (blue light) thatinduced differential melanogenesiswere: 166mJ/cm2,1.524J/cm2,and40J/cm2.TheexpressionoftheGAPHDconstitutionalgenedid
notdifferbetweenthesampleofskinprocessedimmediatelyaftertissuecollectionandthe sampleculturedfor72hinthestandardizedprotocol.
Studylimitations: Thiswasapreliminarystudythatevaluatedonlytheviabilityandintegrity ofthemelanogenicsystem,andtheeffectoftheradiationalone.
Conclusions: Thestandardizedmodelmaintainedviablemelanocyticfunctionfor72hatroom temperature,allowingtheinvestigationofmelanogenesisinducedbydifferentformsof radia-tion.
©2019SociedadeBrasileira deDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Introduction
The physiopathological investigation and therapeutic
responses of certain dermatoses, in anima nobile, may
belimited by elements relatedtotreatment toxicity, the
need fordifferentcomparativegroups, adherenceaspects
in therapeutic trials, and the uncertainty regarding the
biologicalresponseofanintervention(e.g.,carcinogenesis).
Animalexperimentationincreasestherangeofresearch
possibilities at lower cost, with greater accessibility to
subjects and greater exposure control and environmental
influence.1However,thephysiologicalresponsesofanimals
maynotbesimilartohumans(e.g.,melanogenesis),which
mayleadtomisleadingconclusions.2Inaddition,anumber
ofskindiseaseslackanimalmodels(e.g.,melasma),leading
totheneedforotherinvestigativeprocesses.
Computational simulationsof biologicalphenomenaare
an option to experiment with living beings, and provide
verycredibleresultswhensimulatinginteractionsbetween
variableswidelyknownfromanexperimentalpointofview.
Unfortunately,thevariables thatinterferewithcutaneous
processes (e.g., melanogenesis) are still not fully
eluci-dated,northeirinteractionwithotherexternalelements.3
Human cell cultures of primary or commercial strains
(e.g.,HaCaT)arewidelyusedinpathophysiologicalresearch
orfor responsetospecificstimuli.Despitetheirhomology
withhuman tissues,celllineculturesdonotreplicate the
interactionbetween differentcelltypes in theskin (e.g.,
theepidermo-melanicunit).4
Commercial systems with multiple cell cultures (e.g.,
3Dskin: keratinocytes,melanocytes, andfibroblasts)have
been propagated asa solutiontoperceivetheinteraction
between skin cells and are more likely to be related to
the tissue structure of the host.5---7 However, the strains
areusuallyfromdifferentdonors,withreplicationpatterns
somewhatdifferentfromwhatisfoundinthetissue;also,
theydonotpresentendothelium,dendriticcells,orMerkel
cells,which,althoughtheyarenotverynumerousinthe
tis-sue,mayproducesomerelevantinteractionsdependingon
thestudiedphenomenon.Inaddition,todate,3D
commer-cial culturesand commercial skin cell co-cultures do not
yetcharacterizecertaindiseases.7---9 Organoidculturesare
primarytissueculturesthatmaintainthearchitectural
char-acteristics and the main relationships between different
celltypes,inadditiontotheextracellularmatrix.10---12They
have the additional advantage that they can be sampled
fromhealthyskinandskinwithdiseasefromthesamehost,
favoringtheexvivocomparisonbetweenthesites.13,14The
skinis arelativelyviable tissuefor organoidculture, with
reportsoffunctionalmaintenanceformorethansevendays
atroomtemperature.12,15---19Melanogenesisisacomplex
pro-cess, which depends mainly on the action of ultraviolet
radiation(UVAandUVB)ontheepidermo-melanicunit,with
importantparacrineregulation,butwhichalsosuffers
inter-ferencefromelementsofthedermis.20,21 Visiblelight(VL)
hasbeendescribedasapromoterofmelanogenesisinhigh
phototypes,andthemostbiologicallyactivefractionofVL
intheskincomprisestheblue-violetrange(400---500nm).22
This study aimed at the development of an organoid
culturemodelfromaskinfragment,in ordertostudythe
melanogenesisinducedbydifferentformsofradiation.
Methods
The project was approved by the institution’s research
ethics committee (No. 2,700,889), and all participants
signedtheinformedconsentbeforeinclusioninthestudy.
Atotaloftenadultvolunteers(>18years)attendedtoat
theHospitaldasClínicasdaFaculdadedeMedicinade
Botu-catu (Brazil),whounderwent biopsy ofthe retroauricular
regionwitha3-mmpunchunderlocalanesthesia(lidocaine
2%),afterantisepsiswithalcoholicchlorhexidine(2%)anda
sterilefield.The fragmentsweresectionedatthelevelof
thedeepdermistoavoidlargenumbersofadipocytesthat
interferewithcompletesubmergenceofthefragmentinthe
culturemedium.
The cylindrical fragments were divided longitudinally
into twoequivalent half-cylinders and stored in the
spe-cific DMEMmedium (Dulbecco’s Modified Eagle Medium
---SigmaInc.,UnitedKingdom),23inasteriletransparent
plas-ticvial,according totheprotocolestablishedbyAyresfor
organoidculture(standardizationofviability)atroom
tem-perature(20◦C),forposteriordosimetryoftheradiationfor
melanogenesis.24Onefragmentwasirradiated(immediately
aftercollection),andtheother,keptinthedarkfor72h.
The fragments were irradiated with increasing
doses of UVA, UVB, and VL from artificial sources
of UVB (230W/cm2; source FS72T12/UVB/HO), UVA
(1270W/cm2, source Phillips TL 100W/10R) and LED
light (110mW/cm2 in the blue-violet range, source
GBR-LUX 200W) standardized at 10cm. Radiation levels were
initiated at 166mJ/cm2, 1.270J/cm2, and 40J/cm2,
A
B
50 µm 50 µm
Figure1 Histologicalsectionoforganoidculture(72h)ofretroauricularskin(Fontana-Masson,×400).(A)Culturenotirradiated; (B)cultureirradiatedwithUVB,166mJ/cm2.
consecutive sample until differential pigmentation was
identified.Theabsorbanceofthewallsoftheplasticbottles
wasdeterminedforeachformofradiation.A10mLvolume
oftheculturemediumwasusedtoensuresubmergenceof
thefragment.
Tissue viability was assessed from histological and
morphological parameters (H&E), and the quantitative
expressionoftheconstitutiveGAPDHgene
(glyceraldehyde-3-phosphatedehydrogenase)in freshlyharvested skinsvs.
thoseculturedfor 72h wasdeterminedby real-time
poly-merasechainreaction(PCR).TheGAPHDgeneencodesan
elementalproteintoperformbasiccellularfunctions(e.g.,
energymetabolism),commonlyusedasapositivecontrolin
real-timePCRreactions,andrepresentsthemaintenanceof
thebasalmetabolicfunctionsofthecell.
The quantificationofthereal-timePCR wasperformed
fromtheamplificationoftheGAPDHtargetgenebyprimer
andavalidatedTaqManprobe.AliquotsofthecDNA(3L)
extracted fromthe cutaneoussamples weresubmitted to
the PCR reaction using the TaqMan system. Each
reac-tion used 15L of the TaqMan Gene Expression Master
Mix(Life Technologies--- Carlsbad,CA,United States)and
1.0---1.5Loftheprimerset.Amplificationwasperformed
onABI7300equipment(AppliedBiosystems---Carlsbad,CA,
UnitedStates)usingparametersstandardized bythe
man-ufacturer. The data were processed by the SDS Software
System 7300 andthe results expressedas thresholdcycle
(Ct)todeterminetheexpressionoftheGAPHDcDNA from
thenumberofcyclesrequiredtoreachthedetectionlimit
ofthefluorescentsignal,whichwaspredefinedbetween0.2
and0.3.
The radiation-inducedmelanin pigmentation was
stan-dardizedasafunctionofthedosesofeachformofradiation
asafunctionofanincreaseofmorethan10%ofthe
pigmen-tationofthebasallayer,evaluatedbydigitalimageanalysis
oftheslidesstainedbyFontana-Masson.25 Standardization
wascompletedwhentheabovecriteriaweremetand
repro-duced.
Results
Theprimaryskinculturewasstandardizedatroom
temper-atureusingtheDMEMmedium.ThedosesofUVB,UVA,and
LVthatinduceddifferentialmelanogenesiswereasfollows:
166mJ/cm2,1.524J/cm2,and 40J/cm2,respectively. The
absorbanceoftheplasticbottleswasabout50%forallforms
ofradiation.
Themelanogenesisofthebasallayercouldbeperceived
byFontana-Massonstainingafter72hofculture(Figs.1---3).
In the validated protocol, epidermal detachments,
pyknosis, and/or necrosis of keratinocytes were not
observed.Oneoftheculturesshowedcontaminationafter
72h andwasdiscarded,thus totallingninepatients inthe
standardization.
The GAPHDconstitutional geneexpression was
equiva-lentbetweentheskinsampleprocessedimmediatelyafter
tissue collection and the sample cultured for 72h in the
standardizedprotocol(Fig.4).
Discussion
Thepresent studyvalidatesareliablelowcostalternative
toorganoidcultureoftheskinforthestudyof
melanogene-sis.Thestandardizedmodelmaintainedviablemelanocytic
functionfor72hatroomtemperature,allowingthe
inves-tigation of melanogenesis induced by different forms of
radiation.Tissueabundance,easyaccessibilityformaterial
collection, and architectural and functional maintenance
encourage the use of cutaneous organoidcultures in
der-matologicalresearch.6,15,18,25,26
Melanocyteculturesareusedfor thestudyofinhibitors
of melanogenesis. However, in monocultures, irradiated
melanocytessynthesizemorepheomelaninthaneumelanin,
which dependsontheinteractionwithkeratinocytes.The
investigation ofphenomenarelatedtoeumelanogenesis is
favored, therefore,by organoid modelsthatmaintain the
influenceofdifferentcellulargroups.24,27
Cutaneousorganculturesusingair---liquidmediaaremore
commonly used in the study of keratinization, while
sus-pendedcultures(asusedinthisstudy)allowbetterperfusion
andviability.12
Smaller fragments (e.g., 2mm2) tend to have greater
viability and architectural preservation due to the more
homogeneous imbibition by the culture medium and
oxy-gen distribution to the tissue.19,26 In the present study,
3mm fragmentsweresectionedinthemedium,with
ade-quatepreservationoffunction.Oneofthefragmentsthat
was inadvertently grown without the longitudinal section
showedevidentnuclearpyknosisinthecenterofthepiece
A
B
50 µm 50 µm
Figure2 Histologicalsectionoforganoidculture(72h)ofretroauricularskin(Fontana-Masson,×400).(A)Culturenotirradiated; (B)cultureirradiatedwithUVA,1.524J/cm2.
A
B
50 µm 50 µm
Figure3 Histologicalsectionoforganoidculture(72h)ofretroauricularskin(Fontana-Masson,×400).(A)Culturenotirradiated; (B)cultureirradiatedwithvisiblelight,40J/cm2(400---500nmblue-violet).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1.0e+002 1.0e+001 1.0e+000 1.0e-001 1.0e-002 1.0e-003 1.0e-004 17 18 19 Cycle number Delta Rn vs cycle Delta Rn 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Figure4 Geneexpressionsoforganoidcultures(72h)ofretroauricularskin.Curvesobtainedfromsamplesamplifiedbyreal-time polymerasechainreaction(PCR)fortheendogenousglyceraldehyde-3-phosphatedehydrogenase(GAPDH)gene,whosethreshold amplificationcyclenumbers(Ct)werecalculated.
Modernmeansofpreservingandtransportingtissues
pro-vide supportfor theformation of skin banks andalso the
developmentoforganoidculturesforthestandardizationof
experiments.28---31Theuseofbovineserumincreasesthe
via-bilitypotentialofthecultures;however,itcompromisesthe
tissuecultureasafunctionoftime.12 Havingthecultureat
bodytemperature(37◦C)alsoincreasestheviabilityofthe
tissue,aswell asitsmetabolicrate,enhancingthe
explo-rationofoxidativeandmitogenicphenomenasuchasthose
involvedincarcinogenesis.
Organoid cultures are kept out of interaction with
the organism, without thermal, neurological, hormonal,
immune,andhypoxicstimuli,whichhindertheirtissue
prop-ertiesasafunction oftime.12,32 Challengesandstimuli to
fragmentsmust beimposedearly inordertopreservethe
credibilityofresults.
The melanogenesis of the basal layer from the early
stimuluswasevidentin theproposed model.Digital
anal-ysisofbasalmelanogenesisincreasesthesensitivityofthe
each type of radiation (alone or in combination) should
beexploredlater,aswell asthemelanogenic responsein
melasma.
An additional limitationto theuse of whole skin
frag-mentsforgenomicstudiesistheheterogeneityofthecells
presentinthesample,andincreasedexpression ofagene
cannotbeattributedtoasinglecelltype.Forthispurpose,
primarycell cultures,single-cell, or laser microdissection
techniquesmaybeused.33
Cutaneous organoid cultures are more available and
lower-cost than 3D cultures, presenting greater
architec-turalcomplexityby contemplatingthetotalityofresident
cells (e.g., endothelium, nerves, mast cells) and the
extracellular matrix, andthey may moreadequately
rep-resenttopographicdifferences,allowing bothgeneticand
metabolicmanipulation, aswellascell isolation;
further-more,theymaybederivedprimarilyfromdermatosis,with
the possibility of comparison with normal skin from the
sameindividual.However,theyhavelesspredictable
kinet-ics and cannot be constituted from genetic engineering
(e.g.,CRISPR,knock-outgenes).Bothformsofculturehave
anexperimental limitationovertimeanddonotmaintain
thecutaneous microbiome.12 These characteristics should
beconsideredinthechoiceofexperimentalmodels,which
shouldbeappropriatelystandardizedandvalidatedforeach
trial.
The irradiance of sunlight at noon in the interior of
Brazil(latitude: 22◦5309 S; longitude:48◦2642 W;
alti-tude: 804m; 9th of December, 2018) was as follows:
5mW/cm2(UVB);1.7mW/cm2(UVA),and0.2W/cm2
(blue-violet light). This leads to the hypothesis that less than
15min of unprotected sun exposure could potentially
induce pigmentation of the basal layer, maintaining the
limitationsof comparisonwithindependentformsof
radi-ation, while the effect of combined radiation or with
different photoprotection regimes may present different
behaviors.
In vivo experimental studies have resulted in
effec-tive skin pigmentation from 40J/cm2 in melanodermic
individuals (Fitzpatrick IV and V), but not in fair-skinned
phototypes.PigmentationduetoUVA1(340---400nm) could
already be perceived from 5J/cm2 in participants with
darkerphototypes.34
Solar radiation representsa continuum of
electromag-netic frequenciesthat interactwithbiological tissuesand
promotediversestimuli.35 Forreasonsof systematization,
thebiological effects of radiation arestudied from
sepa-rate wavelengths, but there is some interactionbetween
them.36Inthecaseofcutaneouspigmentation,VLpromotes
greaterintensityandpersistenceofUVA-induced
pigmenta-tion1.22,37
The UVA, UVB, and VL sourcesof thisstudy promoted
independentirradiations,sincetheydidnotpresent
signif-icantreadingsoftheother wavelengths(datanotshown).
Thismayberelevantbecauseinclassicalpigmentation
stud-ies,suchasthatbyMahmoudetal.,usedVLsourcesthat
emitted0.19%UVAand1.5%infrared;aswellassourcesof
UVA1thathad0.12%UVA2and0.0001%UVB.34Theeffectof
theheterogeneityoftheradiationtypesinthegroupsmakes
itdifficulttocomparethemwithresultsfromhomogeneous
sources.
Conclusion
Anorganoidculturemodelwasstandardizedtoevaluatethe
melanogenesisinducedbyUVB,UVA,andVL.
Financial
support
FundodeApoioaDermatologiadeSãoPaulo---FUNADERSP.
Authors’
contribution
ThaináOliveiraFelicioOlivatti:Approvalofthefinalversion
ofthemanuscript;collection,analysis,andinterpretationof
data;intellectualparticipationinthepropaedeuticand/or
therapeuticconductofthestudiedcases.
GiovanaPiteriAlcantara:Approvalofthefinalversionof
themanuscript;conceptionandplanningofthestudy;
col-lection,analysis,andinterpretation ofdata;participation
inthedesignofthestudy;criticalreviewoftheliterature.
AnaCláudiaCavalcanteEspósitoLemos:Approvalofthe
finalversionofthemanuscript;conceptionandplanningof
thestudy; compositionof themanuscript;participationin
the design of the study; intellectual participation in the
propaedeutic and/or therapeutic conduct of the studied
cases; critical review of the literature; criticalreview of
themanuscript.
Márcia Guimarães da Silva: Composition of the
manuscript; collection, analysis, and interpretation of
data;participationinthedesignofthestudy.
Hélio Amante Miot: Approval of the final version of
the manuscript; conception and planning of the study;
composition of the manuscript; collection, analysis, and
interpretation of data; participation in the design of the
study;intellectualparticipationinthepropaedeuticand/or
therapeuticconductofthestudiedcases;criticalreviewof
theliterature;criticalreviewofthemanuscript.
Conflicts
of
interest
Nonedeclared.
References
1.OlsonH,BettonG,RobinsonD,ThomasK,MonroA,KolajaG, etal.Concordanceofthetoxicityofpharmaceuticalsinhumans andinanimals.RegulToxicolPharmacol.2000;32:56---67.
2.BastoniniE,KovacsD,PicardoM.Skinpigmentationand pig-mentarydisorders:focusonepidermal/dermalcross-talk.Ann Dermatol.2016;28:279---89.
3.HandelAC,MiotLD,MiotHA.Melasma:aclinicaland epidemi-ologicalreview.AnBrasDermatol.2014;89:771---82.
4.MinuthWW,SittingerM,KlothS.Tissueengineering:generation ofdifferentiatedartificialtissuesforbiomedicalapplications. CellTissueRes.1998;291:1---11.
5.RaviM,ParameshV,KaviyaSR,AnuradhaE,SolomonFD.3Dcell culturesystems: advantagesandapplications. JCellPhysiol. 2015;230:16---26.
6.YoshidaGJ, SayaH,ZouboulisCC.Three-dimensionalculture ofsebaceousglandcellsrevealingtheroleofprostaglandin
E2-inducedactivationofcanonicalWntsignaling.BiochemBiophys ResCommun.2013;438:640---6.
7.HaycockJW.3Dcell culture:areviewofcurrentapproaches andtechniques.MethodsMolBiol.2011;695:1---15.
8.KimY, ParkN, RimYA, NamY, JungH, LeeK, et al. Estab-lishmentofacomplexskinstructurevialayeredco-cultureof keratinocytesandfibroblastsderivedfrominducedpluripotent stemcells.StemCellResTher.2018;9:217.
9.Sriram G, Bigliardi PL, Bigliardi-Qi M. Full-thickness human skinequivalentmodelsofatopicdermatitis.MethodsMolBiol. 2019;1879:367---83.
10.BredenoordAL,CleversH,KnoblichJA.Humantissuesinadish: theresearchandethicalimplicationsoforganoidtechnology. Science.2017;355.
11.Mebarki M, Bennaceur A, Bonhomme-Faivre L. Human-cell-derivedorganoidsas anewexvivomodelfordrug assaysin oncology.DrugDiscovToday.2018;23:857---63.
12.Zhou L, Zhang X, Paus R, Lu Z. The renaissance of human skin organ culture: a critical reappraisal. Differentiation. 2018;104:22---35.
13.Boisnic S, Branchet-Gumila MC,Le CharpentierY, Segard C. Repairof UVA-induced elastic fiber and collagen damageby 0.05%retinaldehyde cream inanexvivohumanskinmodel. Dermatology.1999;199Suppl.1:43---8.
14.DuongHS,ZhangQ,KobiA, LeA,MessadiDV.Assessmentof morphologicalandimmunohistologicalalterationsinlong-term keloidskinexplants.CellsTissuesOrgans.2005;181:89---102.
15.SarkanyI,GaylardePM.Ultrastructuralchangesinhumanskin maintainedinorganculture.BrJDermatol.1970;83:572---81.
16.HentzerB,KobayasiT.Adulthumanskinmaintainedinorgan cultureII.Theultrastructureofthecellularcompartment of connectivetissue.ActaDermVenereol.1980;60:465---76.
17.BoisnicS,Branchet-GumilaMC,NizriD,BenSlamaL. Histochem-ical and biochemical modifications induced byexperimental irradiationofhumanskinmaintainedinsurvivalconditionsand modulationbyapplicationofanemulsioncontainingtrolamine. IntJTissueReact.2003;25:9---18.
18.deJesus RibeiroC,OharaMT,GamaP.Alternativemodelto humanskinorganculture:a preliminarystudywithLeibovitz L15medium.MicroscResTech.2005;66:139---44.
19.VaraniJ.Preservationofhumanskinstructureandfunctionin organculture.HistolHistopathol.1998;13:775---83.
20.VideiraIF,MouraDF,MaginaS.Mechanismsregulating melano-genesis.AnBrasDermatol.2013;88:76---83.
21.ImokawaG.Autocrineandparacrineregulationofmelanocytes inhumanskinandinpigmentarydisorders.PigmentCellRes. 2004;17:96---110.
22.RamasubramaniamR, Roy A, Sharma B, Nagalakshmi S. Are theremechanisticdifferencesbetweenultravioletandvisible
radiationinducedskinpigmentation?PhotochemPhotobiolSci. 2011;10:1887---93.
23.Zeltinger J, Holbrook KA. A model system for long-term serum-free suspension organculture ofhuman fetal tissues: experimentsondigitsandskinfrommultiplebodyregions.Cell TissueRes.1997;290:51---60.
24.AyresE,CostaA,EberlinS,ClericS.Exvivostudyforevaluating thewhiteningactivityofPycnogenol®afterex-posureto
ultra-violetandinfraredradia-tions,andvisiblelight.SurgCosmet Dermatol.2015;7:303---7.
25.MiotHA,BrianeziG,TamegaAdeA,MiotLD.Techniquesof digi-talimageanalysisforhistologicalquantificationofmelanin.An BrasDermatol.2012;87:608---11.
26.HentzerB,KobayasiT.Adulthumanskinmaintainedinorgan culture:I.Theultrastructureoftheacellularcompartmentof connectivetissue.ActaDermVenereol.1979;59:389---400.
27.DuvalC,SmitNP,KolbAM,RégnierM,PavelS,SchmidtR. Ker-atinocytescontrolthepheo/eumelaninratioinculturednormal humanmelanocytes.PigmentCellRes.2002;15:440---6.
28.AhmedAK,HahnDE,HageJJ,BleikerEM,WoerdemanLA. Tem-porarybankingofthenipple-areolacomplexin97skin-sparing mastectomies.PlastReconstrSurg.2011;127:531---9.
29.KaganRJ,RobbEC,PlessingerRT.Humanskinbanking.ClinLab Med.2005;25:587---605.
30.SheridanR,MaheJ,WaltersP.Autologousskinbanking.Burns. 1998;24:46---8.
31.Arora M. Cell culture media: a review. Mater Methods. 2013;3:175.
32.Danso MO,BerkersT, MieremetA,Hausil F,BouwstraJA. An exvivohumanskinmodelforstudyingskinbarrierrepair.Exp Dermatol.2015;24:48---54.
33.SantoroS,LopezID,LombardiR,ZauliA,OsiceanuAM,Sorosina M,etal.Lasercapturemicrodissectionfortranscriptomic pro-filesinhumanskinbiopsies.BMCMolBiol.2018;19:7.
34.Mahmoud BH, Ruvolo E, Hexsel CL, Liu Y, Owen MR, Kol-lias N, et al. Impact of long-wavelength UVA and visible lightonmelanocompetentskin.JInvestDermatol.2010;130: 2092---7.
35.Sklar LR, Almutawa F,Lim HW, Hamzavi I. Effects of ultra-violet radiation, visible light, and infrared radiation on erythemaandpigmentation:areview.PhotochemPhotobiolSci. 2013;12:54---64.
36.SchalkaS,SteinerD,RavelliFN,SteinerT,TerenaAC,Marc¸on CR,etal.BrazilianSocietyofDermatologyBrazilianconsensus onphotoprotection.AnBrasDermatol.2014;89:1---74.
37.Kohli I, Chaowattanapanit S, Mohammad TF, Nicholson CL, Fatima S, Jacobsen G, et al. Synergistic effects of long-wavelengthultravioletA1andvisiblelightonpigmentationand erythema.BrJDermatol.2018;178:1173---80.
