Development of a Neuronal Microscope for Cell Characterization and Manipulation (Neuronal micRoscopy for cEll Behavioral Examination and mAnipuLation)
Phase: NA
Conditions: Hepatic Carcinoma
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico



Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
FLOWCHART 
 
 
Screening 
Intervention 
Period 
(-t1) 
(t1) 
Enlistment 
informedconsent 
- 
inclusion/exclusioncriteria 
- 
Administrationoftheintervention 
Biologicalsamplecollection 
- 
 
 
 
 
LISTOFABBREVIATIONS 
 
ALS:Automated 
Lab 
Solutions 
GmbH 
THEREIS:EthicsCommittee 
CEA:Commissariatàl’énergieatomiqueetauxénergiesalternatives 
CI:InformedConsent 
CRF:CaseReportForm,datacollectionformEC: 
EuropeanCommunity 
ENS:EcoleNormaleSupérieuredeLyonGCP:GoodClinicalPractic
e, good clinical practice HCC:HepatocellularCarcinoma, 
hepatocellularcarcinomaLMU:LudwigMaximilian 
University 
of Munich WUT:PolitechnikaWarszawska 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
TITLEPAGE 
FLOWCHART 
LISTOFABBREVIATIONSINDEX1.INTRO
DUCTION 
1.1Backgroundandrationale 
2. OBJECTIVEOFTHETESTING 
2.1 Primaryobjective 
2.2 Secondaryobjective(s). 
3. STUDYDESIGN 
3.1 Studydesign 
3.2 Inclusioncriteria 
3.3 Exclusioncriteria 
4. PROCEDURES RELATINGTOTHESTUDY 
4.1 Intervention 
4.2 Randomization 
4.3 Blindness 
5. ENDPOINTS 
5.1 Primaryendpoint 
5.2 Secondaryendpoint 
6. DURATION/TIMELINEOFTHESTUDY 
7. STATISTICALANALYSIS 
7.1 Samplesizeanddataanalysis 
8. ADVERSEEVENTS 
9. RISK/BENEFITASSESSMENT 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
10. PRACTICEMANAGEMENT 
10.1 Datacollectionandmanagement 
10.2 Regulatoryaspectsandethicalconsiderations 
10.2.1 Approvalofthecompetentauthority 
10.2.2 ApprovaloftheEthicsCommittee 
10.2.3 Informedconsent 
10.3 Dutiesoftheinvestigator 
10.4 Studymonitoring 
10.5 Qualityassuranceofthestudy 
10.6 Closureofthestudy 
10.7 Archivingofdocuments 
10.8 Disclosureofinformationregardingscientificdiscovery 
10.8.1 Confidentiality 
10.8.2 Publications 
10.9 Intellectualpropertyrightsonthestudyresults 
11. Indemnityandcompensationincaseofdamages 
 
12. FinancialArrangements 
 
13. ConflictsofInterestDisclosure 
14. References 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
1. INTRODUCTION 
1.1 Backgroundandrationale 
 
Hepatocellularcarcinoma(HCC)isthemostcommontypeofprimarylivercancerandisaleadingcause 
of 
cancer-related 
death 
worldwide. 
The 
prognosis 
of 
HCC 
remains 
poor, 
with 
a 
5-
yearsurvivalrateof18%(1).RiskfactorsforHCCincludeviralinfection,autoimmunehepatitis,chronicalcoh
ol 
use 
or 
metabolic 
fatty 
liver 
disease, 
obesity, 
and 
diabetes 
mellitus 
(2). 
Furthermore,alterationsandchronicinflammationofthemicroenvironmentcanfacilitatethetransformati
onofnormalliverstemcellsintoprecanceroustumorstemcells(3).Alltheseunderlyingpathogenicstimulic
aninduceaspectrumofgeneticandepigeneticmodifications,whichareinvolvedinthecellcycle,cellgrowth
andregulationofadhesion.Therefore,heterogeneityandtumorprimingpotentialarisefromacombinatio
nofbothendogenousandexogenousfactors(4).Inparticular,hepatocellularcarcinomaisanextraordinari
lyheterogeneousdiseaseduetothemorphologicalandhistologicaldiversityoflivercelltypes,andsuchintr
atumoralheterogeneityrepresentsamajorchallengefortumorcharacterizationandtherapeuticmanage
mentofpatientswithHCC. 
 
Currentinvitromodels,basedonconventionalhepatomaandhepatocarcinomacelllines,failtorecapitulate
keycharacteristicsoftumortissuesuchasthree-dimensionaltissuearchitecture,cellular 
heterogeneity, 
and 
cell-cell 
interactions. 
Organoids, 
which 
are 
3D 
cellular 
structuresgeneratedfrombothinducedpluripotentstemcellsandadulttissue-
residentstemcells,haverecently been exploited to overcome the limitations of 2D cell culture 
systems, emerging aspowerful tools for studying human diseases (5, 6). This is possible as they are 
able 
to 
stablypreservethegeneticinformationoftheautologoustissueandmimicthepathologicalstateofthetissu
eitself. 
These structures will be the basis for developing a new and promisingmethodology of microscopy, 
called "neuronal", which is strongly based on neural networks capable of perception, interpretation, 
inference, prediction, decision, and action. 
 
2. OBJECTIVE/HYPOTHESISOFTHEEXPERIMENT 
 
Theaimoftheproject,withinwhichthisclinicalstudyispart,istovalidatetheabilityofa"neuronal"microscopetod
ecipherthemolecular 
mechanismsattheoriginofcancer,especiallybyaddressingtheproblemofbiologicalheterogeneity. 
Molecular characterization of tumor cell subtypes at the single-cell level has the potential 
toresolvethetemporalorderofeventsgoverningthedevelopmentaltrajectoriesoflivercancer.Thankstot
heneuronalmicroscopeable 
toselectindividualcells,weintendtoidentifyandisolatecellsatdifferentpointsofthetrajectoriesthatleadfr

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
omthenormalstatetothe 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
tumor(“past,presentandfuture”). 
Morespecifically,thescientificprojectaimstodevelopa"cell-
harvestingneuronalmicroscope"thatwillallowincombinationwithbiologicalanalyses(proteomics,genet
ics,etc.)tocharacterizethecellularheterogeneityknowntocauselivercancer.Thechallengeistodevelopth
isnewmolecularimagingtechniquewhichcanpotentiallyrevealnewcellularmechanismsattheoriginofad
isease. 
Todemonstratethat,thisneuronal 
microscopy 
canleadtounderstandingthecellularoriginofadisease,itwillbeusedtodecipher,identifyandpredictthesta
teofcellsoriginatingfromlivertumorsderivedfrompatientsandmousemodelsofthisdisease. 
 
2.1 Primaryobjective 
 
TheprimaryobjectiveofthestudyconductedattheFondazione 
IRCCS Ca’ 
Granda 
Ospedale Maggiore 
Policlinicoistogenerateandcharacterizethree-dimensionalcell 
culture 
models,calledorganoids,ofthemainlivercellpopulations,inordertoimitatetheearlystages 
of 
human 
carcinogenesisand 
then 
analyze 
the 
onsetof 
HCC, 
to 
identifythe 
specific 
mechanismsinvolvedinhepatocellularcarcinoma. 
 
2.2 Secondaryobjectives 
 
1. Liverorganoids(normal,pre-tumorortumor) are used to“train”theneuronalmicroscope in order 
todistinguishvariouscellsubtypeswithinheterogeneouscultures 
and 
recognizepre-neoplastic 
cellsable to initiatethe transition froma normalstatetoatumorstate. 
 
2. On 
thisbasis, 
a“singlecell 
sampling”system 
willbedeveloped 
thatwillallow 
usto 
detectandcollectdifferentsubtypesoflivercellsthatareexpectedtobecomecancerousandtoanalyzethem
beforethetumormanifestsitself. 
 
3. STUDYDESIGN 
3.1 Studydesign 
Interventional,withthecollectionofbiological,non-pharmacological,monocentricmaterial. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
RESPONSIBILITY(roleofthepromoterandcollaborators) 
 
Operationalunit 
Participantname 
Roleandfunctionsinthefirm 
Department 
of 
TransfusionMedicineandHematolo
gy, 
Fondazione 
IRCCSCa' 
Granda 
Ospedale 
MaggiorePoliclinico 
 
Dr.DanielePrati 
Prof.LucaValenti(principalinvestigato
r) 
Samplerecruitmentandcharacterization; 
Isolation 
and 
generation 
oforganoids,spheroidsandtissues;Ch
aracterizationoforganoidsbygeneand
proteinexpression 
 
Dataanalysis 
 
Internalcollaborations: 
 
Operationalunit 
Participantname 
Roleandfunctionsinthefirm 
UOCGeneralSurgeryandLi
verTransplants, 
Fondazione IRCCS Ca' Granda 
OspedaleMaggiorePoliclinico 
 
Prof.GiorgioRossi 
Reportingofeligiblepatientsandsampl
echaracterization 
UOCScientificDirectionFondazio
ne 
IRCCSCa' 
Granda 
OspedaleMaggiorePoliclinico 
 
Dr.StefanoGatti 
Reporting 
of 
eligible 
patients,sample 
characterization 
andsupportincell isolationprotocols 
 
 
Externalcollaborations: 
 
 
Collaboratingbody 
Roleandfunctionsintheproject 
Commissariatàl’énergieatomiqueetauxénergiesalternatives,Fra
nce(CEA) 
Development of the 
2D neuronal 
scanning lens-free 
microscopeandprototypingofthe3Dneuronalmicroscope;valida
tionofthemicroscopeprototypesthroughtheuseofhumanandmu
rinesamplesgeneratedrespectivelyattheFoundationandENSLy
on 
Iprasense,France 
Technicaldevelopmentandimplementationofthelens-free 
neuronalmicroscope 
EcoleNormaleSupérieuredeLyon,France(ENS) 
Generation 
of 
mouse 
models 
of 
liver 
disease;processingandisolationofsamplesgeneratedattheF
oundation;sequencingandtranscriptomicanalysisofsinglec
ellstogeneratecelltrajectories 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
 
LudwigMaximilianUniversityofMunich,Germany(LMU) 
Proteomicandmulti-omicanalyzesofcellandorganoidclusters 
AutomatedLabSolutionsGmbH,Germany(ALS) 
Developmentandvalidationofthelens-
freeneuronalmicroscope;creationoftheprototypefortheis
olationofsinglecells 
PolitechnikaWarszawska,Poland(WUT) 
Metrological evaluation and validation of the developed 
2Dand 3D microscopes, through the design and production 
ofcalibrationsamplesusing3Dprintingtechnology 
 
 
3.2 Inclusioncriteria 
 
Adultpatientsundergoingsurgicalcholecystectomy,liverresectionforhepatocellularcarcinomaor whole 
liver explants, who have given consent to participate in the study, will be included. 
 
3.3 Exclusioncriteria 
 
Patientswhoarepositiveforchronicviralhepatitis(HCV-RNAandHBsAg)willbeexcluded. 
 
4. PROCEDURESRELATINGTOTHESTUDY 
4.1 Intervention 
 
IncollaborationwiththeLiverTransplantUnit of Fondazione IRCCS Ca’ Granda Ospedale Maggiore 
Policlinico,wastetissuesampleswillbecollectedfrompatientsundergoingcholecystectomy,liverresecti
onforhepatocellularcarcinoma(bothintra-tumoralandextra-
tumoraltissues)ortakenfromexplantedwholelivers. 
Thiswillallowustoisolatecellsandgenerateorganoidsstartingfromthethreeconditionsofinteresttous: 
1) normallivertissues(cholecystectomy); 
2) diseasedliver(extra-tumoralparenchymaandliverexplants); 
3) hepatocellularcarcinoma. 
Weaimtocollectupto10independentsamplespertissuetype. 
To validate the heterogeneity obtained in vitro using organoids, mono- and multi-lineage 
liverspheroidswillbegeneratedbyexploitingpreviouslyestablishedhepatomacelllines(11),namely:phenoty
pically 
differenthepatomacelllines 
(HepG2andSNU398cells) 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
andprimarycelllinesrepresentativeoftumorheterogeneity(HCC-1,HCC-2,HCC-3,generatedwithin 
thestudy“ImpactoftheI148MvariantofthePNPLA3geneontheregulationofretinolmetabolismand activation 
of 
human 
hepatic 
stellate 
cells”, 
Opinion 
No. 
29_2016 
and 
29_2016bis, 
proceedings300/2016),culturedin96-wellplates. 
Tostudytheroleofspecificgeneticpathwaysinregulatingtumorheterogeneity,wewillknockdowngenesof
interestforHepG2hepatomacells.Toregulatetheexpressionofspecificproteinsincellularmodels,wewillu
sespecificantisenseoligonucleotides(morpholinos:MPO,GeneTools)thatblockproteintranslation,orMP
OthatdoesnotrecognizeanymRNAsequenceasacontrol. 
 
ThebiopsyfragmentsorfragmentsobtainedfromsurgicalresectionswillbetransportedinCelsiorperfusio
nsolutionat4°CandwillbeprocessedattheTranslationalMedicinelaboratoryintheshortestpossibletimet
oisolateliverorganoids.Thetissueswillbemechanicallyfragmentedintosmallpieces,avoidingreducingth
emtosinglecells;thiswillincreasetheformationefficiencyoftheorganoids.Thenthefragmentswillbefurth
ersubjectedtoenzymaticdigestionwithasolutioncontainingCollagenaseandDNaseat37°C.Thecellclust
ersobtainedfromenzymaticdigestionwillbeincludedinreducedgrowthfactorMatrigel.OncetheMatrigel
haspolymerized,thecompleteculturemediumto 
growliverorganoidswillbeadded.Isolationsobtainedfromnormaltissueswillbeculturedinmediumcontai
ning:AdvancedDMEM/F12supplementedwith1%N2and1%B27(bothfromGIBCO),1.25mMN-
Acetylcysteine(Sigma),10nMLeu-
Gastrin(Sigma),50ng/mlEGF(Peprotech),1g/mlRSPO1(Peprotech),100ng/mlFGF10(Peprotech),25 
ng/ml HGF (Peprotech), 10 mM Nicotinamide (Sigma), 5 M A83.01 (Peprotech), and 10 M 
Forskolin(Peprotech).Inordertoincreasetheisolationefficiencyoftheorganoidsforthefirstfewdays,25ng
/ml Noggin (Peprotech), 100 ng/ml Wnt3a (peprotech) and 10 M Rock inhibitor Y27632 
(Peprotech) 
willbeaddedtothemedium.Isolationsobtainedfromtumortissueswillbedivided 
and 
culturedinclassicalmediumor 
intumoroidmediumcontaining:AdvancedDMEM/F12supplementedwith1%N2and1%B27(both 
from 
GIBCO), 
1.25 
mM 
N-Acetylcysteine(Sigma), 
10 
nM 
Leu-Gastrin 
(Sigma), 
50 
ng/ml 
EGF(Peprotech),100ng/mlFGF10(Peprotech),25ng/mlHGF(Peprotech),10mMNicotinamide (Sigma), 
5 M A83.01(Peprotech), 10 M Forskolin (Peprotech),and 3 nM dexamethasone(Sigma)(8). 
 
The samples isolated at the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico will be 
sent 
to 
the 
ENS 
in 
Lyon, 
that 
incollaborationwithCEA 
will 
developthetranscriptomicanalysisonbothwholehumanorganoidsandisolatedcells, 
usingtheneuronalmicroscope. 
CEA,throughtheuseofhumanandmurinecellsamplesisolatedattheFondazione 
IRCCS 
Ca’ 
Granda 
Ospedale Maggiore PoliclinicoandENSLyonrespectively,willdevelopthe2Dand3Dimagingplatforms. 
LMUincollaborationwithCEAwilldevelopproteomicanalysisbothonwholehumanorganoidsandonisolat
edcellsusingtheneuronalmicroscope. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
5. ENDPOINTS 
 
5.1 PrimaryEndpoint 
 
Isolation of epithelial cells in order to generate models of major hepatocellular 
carcinoma cellpopulations in a three-dimensional culture, called liver organoids, and 
their molecularcharacterizationofthegeneratedmodels. 
 
5.2 SecondaryEndpoints 
 
• Knowledgeofthebehavioralandmorphologicaldifferencesbetweenphysiologicalandtumorhepatocytes; 
 
• Knowledgeofthemolecularmechanismsunderlyingcellularlevelobservationsexploiting“o
mics”technologiesandCRISPR-Cas9geneticengineering. 
 
6. DURATION / TIMELINE OF THE STUDY 
Month and year of study start: 01/2021  
Monthand year of enrollment closure: 01/2024  
Month and yearofstudyend:01/2025 
The study will have a duration of 48 months. In the first three years we expect to enroll 
approximately 30 subjects inaccordancewiththeinclusioncriteria. 
 
PROJECTGANTT 
 
Intervention 
Period(months) 
1-12 
13-24 
25-36 
37-48 
Enlistment 
- 
- 
-- 
Informedconsent 
- 
- 
- 
Inclusion/exclusion criteria 
- 
- 
- 
Generationoforganoids 
- 
- 
- 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
Evaluationofgeneexpressioninliveror
ganoids 
-- 
-- 
-- 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
Evaluationofspecificliver 
proteinsinliverorganoids 
-- 
-- 
-- 
 
Datacorrelationanalysis 
- 
- 
- 
 
 
7. STATISTICANALYSIS 
 
7.1 Samplesizeanddataanalysis 
 
Previouslypublishedworks(7,8,9,10)havehighlightedthatthegenerationefficiencyofliver
 
organoidsis approximately 90%starting from surgicalresections and 33%from biopsies (7,8). 
Consideringtheseestimates,inordertoisolateatleast6independentorganoidculturesofeachtypeof 
conditionof interestinour study,itis estimatedto recruitatleast 30donors(10 
foreachconditionstudied).Theintentistoobtainreplicatesforanalyseswithasufficientrepresentationofthedif
ferentstagesofliverdamage(normalliver,inflammation/fibrosisandhepatocarcinoma)andtobeabletomapth
eimpactofthemostcommonhereditarygeneticvariants(presentinatleast20%ofindividuals)inthepopulation.
 
 
Themainanalyseswillbeconductedfromcellculturesisolatedfromasingleindividual.Giventhedescriptive
natureofmappingcellularphenotypesandgeneexpression,andthelargenumberoforganoids(>103̂)andc
ells(>109̂)fromasingleindividual,wewillhavesufficientstatisticalpower(>80%)toidentifyandanalyzespe
cificcellularsubsetsrepresenting>1%ofthepopulationoverall. 
 
 
8. ADVERSEEVENTS 
 
Theprojectdoesnotinvolvetheadministrationofdrugsorothersubstancesoradhocinvasiveintervention
soutsideofnormalclinicalpractices.Therefore,noadverseeventsareexpected. 
 
 
9. RISK/BENEFITASSESSMENT 
The 
study 
does 
not 
predict 
an 
immediate 
benefit 
for 
the 
donors,buttheresultsofthistrialwillhavethepotentialtoleadtoadecodingofthemechanismsattheoriginof
tumordevelopment,resolvethetemporalorderoftheeventsthatregulatetheevolutionarytrajectory,under
standtheprogression,characterizationandimprovethetherapeuticmanagementofpatients. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
10. PRACTICEMANAGEMENT 
10.1 Datacollectionandmanagement 
 
For each participant will be assigned a unique code upon enrollment. The file associating 
theparticipant'scodewiththerelevantidentificationdatawillbestoredseparatelyonacomputer protected 
with 
a 
password.Thestudydatabasewillbeprotectedwith 
a 
different 
passwordandaccessibleonlytostaff designatedbytheprincipalinvestigator.Theunique code will be 
used to prevent that unauthorized individual will be able to discover the identity of the enrolled 
patient.Only designed investigators will be able to know the identity of the enrolled subjects. 
 
Regardingdatastorage:datathat 
can 
be 
made 
publicly 
accessible,to 
all 
partnerswillberequiredtouseanopenaccessrepository,linkedtothetoolsproposedbytheEuropeanCommun
ity(e.g.OpenAIRE)toensureaccesstopublicationsandabibliographicmetadataarchiveinastandardformat,i
ncludingtheinformationrequiredbytheEC. 
 
10.2 
Regulatoryaspectsandethicalconsiderations 
 
10.2.1 ApprovaloftheCompetentAuthority 
Inaccordancewithcurrentregulations,theprincipalinvestigatormustobtainapprovalfromtheappropriateC
ompetentAuthoritybeforestartingtheclinicalstudy. 
ThisstudywillbeconductedinaccordancewiththerulesoftheICH/GCP(InternationalConferenceofHarmoniz
ation/GoodClinicalPractice)andallapplicablelaws,includingtheDeclarationofHelsinkiofJune1964,asamen
dedbythelatestWorldMedicalAssociationGeneralAssemblyinSeoul,2008. 
 
10.2.2 ApprovaloftheEthicsCommittee 
Theinvestigatormustensurethattheprotocolhasbeenseenandapprovedbythelocalindependent 
Ethics Committee (EC) beforestartingthestudy. 
TheEC 
mustalsoreviewandapprovetheinformedconsent(IC)formandallwritteninformationreceivedfromthep
atientpriortoenrollmentinthestudy. 
Ifitisnecessarytomodifytheprotocoland/ortheICduringthestudy,theinvestigatorwillbetheguarant
orandthereforethepersoninchargeofensuringthereviewandapprovalofthismodifieddocumentacc
ordingtotheEC'srequest. 
ThecontentofthesechangeswillbeimplementedonlyaftertheEChasapprovedthem.Untilthattime,itwillb
enecessarytorefertothepreviousversionofthealreadyapproveddocument. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
10.2.3 InformedConsent(IC) 
Theinvestigatororotherstaffdesignedbythe 
investigatorshouldinformpeopleaboutallaspectsandproceduresofthestudy. 
Theprocessforobtaininginformedconsentmustcomplywithcurrentregulatoryprocedures.Theinvestiga
tor(ordesignatedcollaborator)andthesubjectmustdateandsigntheinformedconsentformbeforethat 
thepatientinitiatesanystudy-relatedprocedures.Thesubjectwillreceiveacopy of the CI dated and 
signed 
by 
both 
parties; 
the 
original 
copy 
will 
be 
kept 
in 
the 
archivesdesignatedforstudy.Neithertheinvestigatornordesignatedpersonnelshouldinanywayexertany 
coercion or influence on a subject to participate or continue to participate in the study. 
Asubject'sdecisiontoparticipateinthestudymustbecompletelyvoluntary.Theinvestigatoranddesignate
dpersonnelshouldemphasizetothesubjectthathemaywithdrawconsentatanytimewithoutpenaltyorlos
sofanybenefitstowhichhemaybeentitled. 
Writtenororalinformationrelatingtothestudy,includingthewrittenconsentform,mustnotcontainanyling
uisticexpressionthatforcesthesubjectto(evenapparently)waivehisorherlegalrights,orthatwouldexoner
atetheinvestigator,theinstitutionorthesponsorfromliabilityfornegligence. 
 
10.3 Dutiesoftheexperimenter 
Inaccordancewithapplicablelocalregulations,theinvestigatormustsendperiodicreportsregardingthepr
ogressofthestudyinhiscentertotheECandnotifytheECoftheclosureofthe study. Periodic reporting and 
closure notification are part of the investigator'sresponsibilities. 
 
10.4 Studymonitoring 
In accordance with applicable regulations and good clinical practice (GCP), the clinical monitor 
must visit or contactthe center periodically. The duration, nature and frequency of such 
visits/contacts depend on the frequencyof recruitment,the qualityof thedocuments heldby 
thecenter andtheir adherenceto theprotocol. 
 
 
Throughthesecontacts,themonitormust: 
• monitorandevaluatetheprogressofthestudy 
• examinethedatacollected 
• conductverificationofthesourcedocument 
• identifyeachproblemanditssolutions 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
 
Thepurposesofthemonitoringactivityaretoverifythat: 
• the rights and welfare of the subject are respected 
• The study data are accurate, complete, and can be verified from the original 
documents 
• thestudyisconductedinaccordancewiththeprotocolandanyapprovedamendments,GCPan
dapplicableregulations 
Theinvestigatormust: 
• giveto the clinical monitordirectaccesstoallrelevantdocumentation 
• dedicatepartofhisandhisstaff'stimetotheclinical 
monitortodiscussthemonitoringresultsandanyotherpossibleaspects. 
 
Theclinical 
monitorshouldalsocontactthecenterpriortothestartofthestudytodiscusstheprotocolanddatacollectionpr
ocedureswiththestaff. 
 
10.5 Qualityassuranceofthestudy 
AsPromoter,FondazioneIRCCSCa'Granda,OspedaleMaggiorePoliclinicocancarryoutaqualitycontrolche
ckonthestudyatitsdiscretion.Inthiscase,theinvestigatormustallowtheclinical 
monitordirectaccesstoallrelevantdocumentationanddedicatesomeofhisorhertimeandstafftothereview
ertodiscussthemonitoringresultsandanyotheraspectsofthestudy. 
 
Furthermore,RegulatoryAuthoritiescancarryoutinspections.Inthiscase,theinvestigatormustgrantthein
spectordirectaccesstoallrelevantdocumentation,anddedicatepartofhistimeandstafftotheinspectortod
iscussthemonitoringresultsandanyotheraspectsofthestudy. 
 
10.6 Closureofthestudy 
Atthetimeofstudyclosure,themonitorandtheinvestigatormustactivateaseriesofprocedures: 
• reviewallstudydocumentation 
• reconcilestudydata 
• reconcileallclarificationreports. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
10.7 Archivingofdocuments 
Inaccordancewithcurrentnationalregulations,theinvestigatormustkeepacopyofalldocumentationa
ndstoreitinadryandsafeplaceafterthestudyisclosed. 
 
10.8 Disclosureofinformationregardingscientificdiscovery 
10.8.1 Confidentiality 
Theinvestigatorandotherpersonnelinvolvedinthestudymustprocessallinformationrelatingtothestudy(including
theprotocol,dataobtainedandalldocumentationproducedduringthestudy)andmustnot use suchinformation, 
dataor reportsfor purposesother than thosedescribed inthe protocol. 
Theserestrictionsdonotapplyto: 
1) informationthatbecomespubliclyavailable,notduetonegligenceonthepartoftheinvestiga
tororhisstaff; 
2) informationrequiringconfidentialdisclosuretoCEforthesolepurposeofevaluatingthestudy; 
3) informationthatmustbedisclosedinordertoobtainappropriatemedicalcareforastudysubject. 
 
10.8.2 Publications 
 
TheIRCCSCa'GrandaFoundation,OspedaleMaggiorePoliclinicoisthesoleownerofthedata.Awebsitededicated
totheprojectwillbebuiltwithprivatesectionsthatwillservetheprojectpartnersas 
update 
and 
exchange 
channels, as well as to build a data archive to be used in the post-projectexploitation phase. The public 
section 
will 
contain 
the 
project 
description 
(e.g. 
objectives, 
partners,fundingsource,etc.),publishedarticlesandongoingwork.Thescientificdirectorofthestudywillwritea 
final report and make the results public at the end of the study. The data will be made 
publicanonymouslyandpresentedasrequiredinaggregatemode.InaccordancewiththeprovisionsoftheGrant
Agreement,thedisseminationoftheresultswilltakeplaceassoonaspossible,byappropriatemeans,includings
cientificpublications. 
 
10.9 Intellectualpropertyrightsonthestudyresults 
 
Most data will be associated with results that may have commercial or industrial 
protectionpotential and therefore cannot be made accessible for verification and re-use 
generally duetointellectualpropertyprotectionmeasures. 
However, relevant data necessary for verifying results published in scientific journals can be 
madeaccessibleonacase-by-casebasis. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
AConsortiumAgreementwillbenegotiatedandsignedbyallpartiesinordertospecify,amongotherthings,thetermsa
ndconditionsrelatingtoownership,accessrights,exploitationofthebackgroundandresultsaswellasthedisseminat
ionoftheresults,inaccordancewiththegrantagreementandRegulationn°1290/2013of11December2013. 
TheconsortiumagreementwillbebasedontheDESCAHorizon2020ModelConsortiumAgreementwiththenecessaryadaptatio
nsconsideringthespecificcontextandpartiesinvolvedintheprojectand 
willbebasedonthefollowingprinciples: 
 
• Thepartieswillidentifytheknowledge,information,data(ofanyformornature),know-how,technical material 
and/or 
assets 
protected 
by 
industrial 
and/or 
intellectual 
property 
rights 
orsusceptibletoprotection,(Background)heldand/ordevelopedinanycapacityindependentlybyeachof 
the 
Parties at a time prior to the start of the Project which they will make available for 
theimplementationoftheprojectandwillevaluatetheiravailabilityforaccessrightswithregardtotherightsofpot
entialthirdpartiesonsuchbackgroundtotheextentthatthisinformationisknowntotherespectivepartyattheti
meoftheassessment; 
• Ownershipofresults,includingjointresultsgeneratedbytwoormoreparties,willgotothepartyorpartiesthat
generatedsuchresults; 
• Theproprietarypartieswilltakeallappropriatemeasurestoprotecttheresultssusceptibletocommerci
alorindustrialexploitation,inparticularthroughintellectualpropertyrights,whereappropriate; 
• Thepartieswillusereasonableeffortstoexploitanddisseminatetheresults,directlyorindirectl
y,forexamplebylicensingsuchresults; 
• Each party will grant the other parties access rights (through licenses) to its Background and the 
resultsgenerated during the Project, only if strictly necessary for the implementation of the project itself 
and/orfortheexploitationoftheresultsofsuchotherparties(onfairandreasonableterms). 
 
11. INDEMNITYANDCOMPENSATIONINCASEOFDAMAGES 
 
In the event of unwanted events or any damage that may arise from participation 
inresearch,ourInstitute'sInsurancePolicyisalsoextendedtocoverindividualsparticipatinginresearc
hprojects. 
 
12. FINANCIALAGREEMENTS 
Thecostsofstudyproceduresexceedingnormalclinicalpracticewillbeentirelycoveredbythefundsderivingfromt
heHorizon2020–EuropeEUfinancing. 

Department of Services and Preventive Medicine 
UOCTransfusion Center – Director: Dr. Daniele Prati 
Tel. 0255036595–email:luca.valenti@unimi.it 
 
 
 
13. DISCLOSURESONCONFLICTSOFINTEREST 
Theinvestigatorsdeclarenoconflictsofinterest. 
 
14. REFERENCES 
 
1- YarchoanM.etal.'Recentdevelopmentsandtherapeuticstrategiesagainsthepatocellular 
carcinoma'.CancerRes,2019 
2- YangJDetal.'Hepatocellularcarcinoma:Aglobalview'.NatRevGastroenterolHepatol,2010 
3- PlaksV.etal.'Thecancerstemcellniche:howessentialisthenicheinregulatingstemnessoftumor 
cells?'.CellStemCell,2015 
4- LawsonDAetal.'Tumorheterogeneityandmetastasisatsingle-cellresolution'.NatCellBiol,2018 
5- CleversH.'ModelingDevelopmentandDiseasewithOrganoids'.Cell,2016 
6- FatehullahA.etal.'Organoidsasaninvitromodelofhumandevelopmentanddisease'.NatCellBiol,20
16 
7- HuchM,GehartH,vanBoxtelR,HamerK,BlokzijlF,VerstegenMM,EllisE,vanWenumM,Fuchs 
SA, 
de 
LigtJ, 
vande 
WeteringM, 
SasakiN, 
Boers 
SJ,Kemperman 
H,de 
JongeJ 
,Ijzermans 
JN,NieuwenhuisEE,HoekstraR,StromS,VriesRR,vanderLaanLJ,CuppenE,CleversH.Long-
termcultureofgenome-stablebipotentstemcellsfromadulthumanliver.Cell2015Jan 
15;160(1-2):299-312.doi:10.1016/j.cell.2014.11.050. 
8- Broutier,L.,Mastrogiovanni,G.,Verstegen,M.etal.Humanprimarylivercancer–
derivedorganoidculturesfordiseasemodelinganddrugscreening.NatMed23,1424–
1435(2017).https://doi.org/10.1038/nm.4438 
9- NuciforoS,FofanaI,MatterMS,BlumerT,CalabreseD,BoldanovaT,PiscuoglioS,WielandS, 
RingnaldaF,SchwankG,TerraccianoLM,NgCKY,HeimMH.OrganoidModelsofHumanLiverCancersDerivedfr
omTumorNeedleBiopsies.CellRep.2018Jul31;24(5):1363-1376.doi:10.1016/j.celrep.2018.07.001. 
10- Sorrentino,G.,Rezakhani,S.,Yildiz,E.etal.Mechano-modulatorysyntheticnichesforliver 
organoidderivation.NatCommun11,3416(2020). 
11- PingitoreP,SasidharanK,EkstrandM,PrillS,LindénD,RomeoS.HumanMultilineage3DSpheroidsasaM
odelofLiverSteatosisandFibrosis.IntJMolSci.2019;20(7):1629.Published2019Apr2.doi:10.3390/ijms
20071629