Colangioids to Define the Genetic Factors Involved in Atypical Primary Sclerosing Cholangitis
Phase: NA
Conditions: PSC
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

Department of Services Area
SC Transfusion Medicine 3 Director: Daniele Prati 
Transfusion Service
Tel. 02 5503.4687
<Cholangioids to define the genetic factors involved
in atypical primary sclerosing cholangitis=
(English title: <Cholangioids to dissect the genetic drivers of atypical Primary
Sclerosing Cholangitis=)
Acronym: CILIA
FRRB Project #4976687
Protocol version number: v1.0
Date: 25/09/2024
Promoter:IRCCS Foundation Ca' Granda Maggiore Hospital Polyclinic,
Via Sforza 28, 20122 Milan, Italy
Coordinating center:SC Transfusion Medicine
IRCCS Ca' Granda Foundation Maggiore Hospital Polyclinic, 
Via Francesco Sforza 35, 20122 Milan, Italy
Principal Investigator:Prof. Luca Valenti
______________________________
PRIVACY STATEMENT
All information presented in this document will be considered confidential and will remain the exclusive property 
of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. The use of such confidential information must be 
limited to the recipient for the agreed purpose and must not be disclosed, published or otherwise communicated 
to unauthorized persons, for any reason, in any form without the prior written consent of Fondazione IRCCS Ca' 
Granda Ospedale Maggiore Policlinico.
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Translated from Italian to English - www.onlinedoctranslator.com

FLOWCHARTS
Screening
Intervention
Period
(-t1)
(t1)
Enlistment
informed consent
inclusion/exclusion criteria
Administration of the intervention
Biological sample collection: blood sampling and/or liver biopsy/resection
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LIST OF ABBREVIATIONS
aPSC:Atypical primary sclerosing cholangitis THERE 
IS:Ethics Committee
THERE:Informed Consent
CRF:Case Report Form, data collection form 
GCP:Good Clinica Practice, good clinical practice 
GWAS:Genome-wide association studies HLA:
Human leukocyte antigen IBD:Inflammatory 
bowel disease iPSC:Induced Pluripotent Stem 
Cells PBMC:peripheral blood mononuclear cells
RESPONSIBILITY (role of the promoter and collaborators)
Promoter: IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico 
Coordinating Center: SC Transfusion Medicine
PI of the study: Prof. Luca Valenti
Scientific Director of the FRRB Project: Dr. Alessandro Cherubini
Structure
Participant Name
Role and functions in the study
SC Transfusion Medicine, 
IRCCS Foundation Ca' Granda 
Maggiore Hospital Polyclinic
Dr. Alessandro Cherubini
Recruitment (LV) and
Sample characterization; 
Isolation and generation of 
organoids, spheroids and 
tissues; Characterization of 
organoids by gene and protein 
expression
Data Analysis
Prof. Luca Valenti
Dr. Daniele Prati
Internal collaborations
Structure
Participant Name
Role and functions in the study
SC General Surgery and 
Liver Transplantation,
IRCCS Foundation Ca' Granda 
Hospital Maggiore Polyclinic
Dr. Daniele Dondossola
Reporting of eligible patients 
and characterization of 
samples
SC Scientific Direction IRCCS 
Foundation Ca' Granda 
Maggiore Hospital Polyclinic
Dr. Stefano Gatti
Support in cell isolation 
protocols
External collaborations (biological sample analysis, data analysis, diagnostic procedures, etc.)
Institution
Operational unit
Participant Name
Role and functions in the 
study
IRCCS Foundation
SC Gastroenterology
Prof. Pietro Invernizzi
Scientific support in
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Saint Gerard of the
Dyers - Monza
how much expert center
in rare liver 
diseases such as
sclerosing cholangitis
primitive
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Department of Services Area
SC Transfusion Medicine 3 Director: Daniele Prati 
Transfusion Service
Tel. 02 5503.4687
INDEX
1. INTRODUCTION
1.1 Background and rationale
2. OBJECTIVE OF THE EXPERIMENTATION
2.1 Primary objective
2.2 Secondary objective(s)
3. STUDY DESIGN
3.1 Study design
3.2 Inclusion criteria
3.3 Exclusion criteria
4. PROCEDURES RELATING TO THE STUDY
4.1 Intervention
4.2 Randomization
4.3 Blindness
5. ENDPOINT
5.1 Primary Endpoint
5.2 Secondary Endpoint
6. DURATION/TIMELINE OF THE STUDY
7. STATISTICAL ANALYSIS
7.1 Sample size
7.2 Data analysis
8. ADVERSE EVENTS
9. RISK/BENEFIT ASSESSMENT
10. STUDIO MANAGEMENT
10.1 Data collection and management
10.2 Regulatory aspects and ethical considerations
10.2.1 Approval by the competent authority
10.2.2 Ethics Committee Approval
10.2.3 Informed consent
10.3 Duties of the experimenter
10.4 Study monitoring
10.5 Study quality assurance
10.6 Closing of the study
10.7 Document storage
10.8 Disclosure of information regarding scientific discovery
10.8.1 Confidentiality
10.8.2 Publications and Intellectual Property Rights on the Results of the Study
11. Indemnity and compensation in case of damages 
12 Amendments to the protocol
13 Financial agreements
14 Disclosure on conflicts of interest
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15 References
16 Appendix
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1. INTRODUCTION
1.1 Background and rationale
Primary sclerosing cholangitis (PSC) is a rare, progressive and often fatal disease of the intrahepatic 
or extrahepatic bile ducts, with an estimated prevalence in Western countries of 1/10,000.
1,2. Biliary disease in PSC is represented by cholestasis, chronic inflammation of the bile ducts, the 
small tubes through which bile passes, progressive concentric fibrosis around the bile ducts2. 
This results in an obstruction to the passage of bile, which can lead to the development of 
cirrhosis with complications related to portal hypertension, cholangitis and often progress to bile 
duct cancer (cholangiocarcinoma). The only curative therapy in patients with PSC is liver 
transplantation, since no drug has been shown to be effective in preventing disease progression. 
The etiology is most likely multifactorial immune-mediated, where the onset of PSC is triggered 
by environmental factors in a genetically susceptible host2Genome-wide association studies 
(GWAS) have identified variations at the human leukocyte antigen (HLA) complex on 
chromosome 6 and several other loci, but these explain only a small part of the heritability of 
PSC.3.4In most cases, PSC occurs in men in their 30s and 40s who have inflammatory bowel 
disease (IBD).5, suggesting a key role of altered intestinal permeability and inflammation. 
However, approximately 30% of patients do not present colonic inflammation, which is 
consistent with the heterogeneity of the disease. Preliminary data obtained in our laboratory 
analyzing a cohort of Italian individuals with atypical PSC (aPSC), identified a suggestive 
enrichment of rare variants in genes involved in cilia morphogenesis (e.g. CEP120 and AHI1). 
These data are consistent with previous findings, showing the correlation between gene variants 
involved in ciliopathies, including the DCDC26 gene, and chronic cholestatic disorders that can 
mimic PSC.
Primary cilia are organelles present on the outer membrane of ductal cells, called cholangiocytes. 
These organelles function as antennas that detect stimuli from bile and transmit information to 
cells by regulating various signaling pathways involved in secretion, proliferation and apoptosis.6
. Therefore, the alteration of primary cilia plays an important role in the de-differentiation of 
cholangiocytes and therefore in the development of cholangiopathies, in the invasion of 
inflammatory cells and in the fibrotic process. However, to date little is known about the 
contribution of genetic variants to the severity and progression of PSC, perhaps also due to the 
lack of a reliable model of bile duct.
Recently, three-dimensional cell cultures, called organoids, have been proposed as a 
revolutionary tool in the field of cell biology, as they are able to mimic the corresponding 
organ in vivo.7Organoids can be derived from either induced pluripotent stem cells (iPSCs) or 
tissue-resident adult stem cells.8. Compared to conventional 2D cultures and animal models, 
organoids allow to reproduce the genetic background of the patient in the model, 
recapitulating in vitro structures and functions similar to in vivo tissues. For this reason, 
organoids have been exploited in different applications, including drug discovery and 
testing, precision medicine and cell therapy9311. However, organoids still show several 
limitations to model liver diseases. Indeed, they are only able to recapitulate the hepatic 
epithelial component, cholangiocytes and/or hepatocytes and above all they lack the 3D 
hepatic microenvironment, such as stromal and immune cells, which play an important role 
in the pathogenesis of several liver diseases.
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The present study is part of a project funded by the Regional Foundation for Biomedical 
Research (FRRB) whose general objective is to generate three-dimensional models of 
primary sclerosing cholangitis (PSC), called assemblyloids, and to study the cellular and 
molecular mechanisms through which genetic variants associated with genes involved in 
ciliopathies accelerate the progression of PSC. Our hypothesis is that the loss of function of 
cilia in cholangiocytes may represent a link between cellular senescence, development of 
inflammation, fibrosis and finally liver cancer. The variants related to ciliopathies could lead 
to an incomplete maturation of cholangiocytes with consequent malfunction that can 
therefore lead to a chronic inflammation of ductal cells and therefore to a persistent and 
uncontrolled activation of stromal cells and infiltration of immune cells.
Furthermore, the generation of assemblyloids capable of reproducing native tissue as 
faithfully as possible will provide a new in vitro model for testing new pharmacological 
approaches aimed at correcting genetic mutations for improved precision medicine.
2. OBJECTIVE/S/I HYPOTHESIS OF THE EXPERIMENTATION
2.1 Primary objective
The primary objective of the study is to explore the behavior of genetic variants involved 
in ciliopathies in the onset and progression of aPSC by analyzing in three-dimensional 
models called assemblyloids:
-
-
-
-
Morphology of primary cilia 
Differentiation of cholangiocytes 
Activation of fibrogenesis 
Infiltration of immune cells
2.2 Secondary objective
Using assemblyloids as a templatein vitroto test new pharmacological approaches, such as 
extracellular vesicles containing the CRISPR/Cas9 genome editing system. This model more 
faithfully reproduces the counterpartin vivowill allow to have a more faithful answer than 
using the conventional 2D model, organoids or animal model.
3. STUDY DESIGN
3.1 Study design
This is a non-pharmacological interventional study, with collection of biological material, single-
center, non-profit.
3.2 Inclusion criteria
Patients will be included:
- Aged between 18 and 90 years old
- Of both sexes
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- willingness to sign informed consent for the study;
Additional criteria group 1
- patients with a confirmed diagnosis of aPSC
Additional criteria group 2
- patients with presumed aPSC who are candidates for liver biopsy
Additional criteria group 3
- non-aPSC patients scheduled for the following procedures:
- liver resection for hepatocellular carcinoma or other liver lesions (including secondary lesions from 
other neoplasms and benign focal lesions, which will allow obtaining healthy starting liver tissue);
- post-transplant healthy liver biopsies;
- cholecystectomies.
Additional criteria group 4
- patients previously genotyped in the study <Impact of whole exome sequencing on the 
clinical management of patients with non-alcoholic fatty liver disease and cryptogenic 
liver disease 3 Ricerca Finalizzata 2016, project code RF-2016-02364358= found to be 
carriers of gene variants associated with ciliopathies.
3.3 Exclusion criteria
Patients who do not meet the above study criteria and with the following criteria will be 
excluded:
- positivity for chronic viral hepatitis (HCV-RNA and/or HBsAg);
- positivity to other liver diseases such as autoimmune and viral hepatitis (hepatitis B and C), 
hereditary hemochromatosis, alpha-1-antitrypsin deficiency, Wilson's disease.
4. PROCEDURES RELATING TO THE STUDY
4.1 Enrollment and collection of biological samples
Patient enrollment in the study will be coordinated by the Transfusion Medicine 
Unit at the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
Specifically, patients with a confirmed diagnosis of aPSC (group 1) in the context of 
scheduled clinical visits will be subjected to a blood sample of approximately 7 cc.
From patients with presumed aPSC who are candidates for liver biopsy to confirm the diagnosis of 
aPSC (group 2), any liver biopsy waste tissue will be collected. In addition, these patients will be 
required to have an additional peripheral blood sample of 7 cc.
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Finally, after informed consent, in collaboration with the SC General Surgery and Liver Transplantation of the 
IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, samples of waste tissue will be collected 
(approx. 1 cm3) starting from patients not affected by aPSC (group 3) who for clinical needs will undergo liver 
resection for hepatocellular carcinoma or other liver lesions, cholecystectomy or post-transplant whole liver 
biopsies. This procedure will not entail any additional risk for the patient compared to the usual routine, nor 
will it reduce the availability of material for standard pathological analyses. Finally, a blood sample 
(approximately 7 cc) will be required, in addition to those already foreseen during regular clinical checks, in 
order to isolate PBMC cells from which macrophages will be generated.
In general:
- The liver samples collected in the study will allow us to isolate cells (ovalocytes 3 liver 
progenitor cells) to generate organoids and stromal cells.
- The blood samples will allow us to 1) isolate PBMC cells from which macrophages and iPSCs 
will be generated; 2) isolate DNA from PBMC in order to identify any genetic variants 
related to ciliopathies.
Given the rarity of the pathology under study, in order to reach the number of samples planned in 
the study, during visits already scheduled for routine, patients affected by aPSC previously 
genotyped in the context of the study promoted by the Foundation (group 4) may be enrolled for 
a blood sample equal to 7 cc in order to isolate peripheral blood mononuclear cells to be 
reprogrammed to iPSC.
Following gene sequencing, a group of patients carrying the gene variants related to 
ciliopathies and a group not carrying these variants will be identified.
All cell types and genetic material obtained will be biobanked at the biobank of the Fondazione 
IRCCS Ca' Granda Ospedale Maggiore Policlinico at the end of the study, following the signing of 
the informed consent obtained at the time of patient enrollment.
4.2 Isolation and culture of organoids
Biopsy fragments or fragments obtained from surgical resections will be transported in Celsior 
perfusion solution at 4°C and will be processed at the Translational Medicine laboratory, SC 
Transfusion Center of the IRCCS Ca' Granda Foundation Ospedale Maggiore Policlinico within 24 
hours of collection to isolate liver organoids. The tissues will be mechanically fragmented into 
small pieces avoiding reducing them to single cells; this will increase the efficiency of organoid 
formation. Then the fragments will be further subjected to enzymatic digestion with a solution 
containing Collagenase and DNase at 37°C. The cell clusters obtained from enzymatic digestion will 
be included in reduced growth factor Matrigel. Once the Matrigel has polymerized, the complete 
culture medium for liver organoids will be added: Advanced DMEM/F12 supplemented with 1% N2 
and 1% B27 (both from GIBCO), 1.25 mM N-
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Acetylcysteine   (Sigma), 10 nM Leu-Gastrin (Sigma), 50 ng/ml EGF (Peprotech), 1 -g/ml RSPO1 
(Peprotech), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 10 mMNicotinamide (Sigma), 
5 -M A83.01 (Peprotech) and 10 -M Forskolin (Peprotech). In order to increase the efficiency of 
organoid isolation, 25 ng/ml Noggin (Peprotech), 100 ng/ml Wnt3a (peprotech) and 10 -M Rock 
inhibitor Y27632 (Peprotech) will be added to the medium for the first few days. The organoids will 
then be differentiated towards a hepatocyte phenotype.
4.3 Isolation and culture of stromal cells
A part of the biopsy fragments or those obtained from surgical resections, once digested to 
obtain the epithelial cells that will form the organoids, will be processed to obtain the 
stromal cells.
The liver sample will first be perfused with perfusion buffer (0.5 -M EGTA, 2 mM HCl2, 20 
mM HEPES, 1 mg/ml pronase, 0.25 mg/ml collagenase type IV). Stromal cells will then be 
isolated by separation on a density gradient generated with OptiPrep solution (StemCell 
Technologies). Stromal cells will be cultured on plastic in DMEM medium containing 10% 
FBS and we will characterize their activation status and the purity of the cell population, 
by flow cytometry and quantitative Real Time PCR (RT-qPCR), evaluating the presence of 
specific cellular markers such as CD90, CD29 and CD7312.
4.4 Isolation and culture of peripheral blood mononuclear cells (PBMC)
PBMC cells will be isolated from blood samples by density gradient using SepMate tubes 
(StemCell Technologies). The isolated cells will be cultured in RPMI 1640 containing 10% FBS and 
then differentiated into macrophages for 6 days by adding 50 ng/mL M-CSF to the culture 
medium.
4.5 Generation of induced pluripotent stem cells (iPSCs)
A portion of the PBMCs will be cultured in medium composed of StemPro#-34 SFM 
(ThermoFisher Scientific) containing 100 ng/mL SCF, 100 ng/mL FLT-3, 20 ng/mL IL-3 and 20 
ng/mL IL-6 and then reprogrammed to iPSCs using the CytoTune#-iPS 2.0 Sendai 
Reprogramming kit (ThermoFisher Scientific). The obtained iPSCs will be maintained in 
Essential 8# medium (ThermoFisher Scientific) on vitronectin-coated plates and their ability 
to differentiate to all three germ layers meso-, endo- and ectoderm will be assessed by RT-
qPCR and immunofluorescence.
4.6 Generation of assemblyloids
In order to obtain the assemblyloids, both organoids and stromal cells and macrophages 
isolated directly from liver tissue or differentiated from iPSCs, will be dissociated into single 
cells with enzymatic digestion using trypsin. The cells will then be included in reduced 
growth factorMatrigel in different ratios in order to mimic the physiological conditions 
present in the liver tissue. Once the Matrigel has polymerized, the culture medium 
appropriately formulated to allow the survival of both ductal cells, stromal cells and immune 
cells will be added: Advanced DMEM/F12 supplemented with 1% N2 and 1% B27 (both from 
GIBCO), 1.25 mM N-Acetylcysteine   (Sigma), 10 nM Leu-Gastrin (Sigma), 50 ng/ml EGF
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(Peprotech), 25 ng/ml HGF (Peprotech) and 2% FBS (GIBCO). The assemblyloids will be grown 
in suspension using a shaker in order to increase the diffusion of nutrients and oxygen and 
promote the correct organization of the cells inside the matrix drop similar to that which 
would be found in the organ.in vivo.
4.7 Characterization of assemblyloids
The assemblyloids will be characterized in order to confirm their ability to mimic the complexity 
of the organ in vivo, both at the molecular level by single cell RNA-sequencing using the 
Chromium technology (10x Genomics) present at the Fondazione IRCCS Ca' Granda Ospedale 
Maggiore Policlinico, and at the morphological level by immunohistochemical analysis.
4.8 Genetic analysis
DNA will be extracted by phenol-chloroform from peripheral blood and genetic variants of 
interest will be determined by TaqMan assay on an ABI 7500 Fast apparatus (Life 
Technologies, Carlsbad, CA, USA).
4.9 RNA isolation
RNA will be extracted from cultured organoids, stromal cells, macrophages and assemloids using 
Trizol reagent (Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. 1 
µg of total RNA will be reverse transcribed using the VILO kitrandom hexamers synthesis system 
(Life Technologies, Carlsbad, CA). Gene expression will be assessed by quantitative real-time PCR 
(RT-qPCR) using an ABI 7500 Fast thermal cycler (Life Technologies, Carlsbad, CA) and SYBR Green 
chemistry (Fast SYBR Green Master Mix; Life Technologies, CA). All reactions will be performed in 
triplicate.
4.10 Characterization of the role of cilia in the progression of sclerosing cholangitis
primitive
In order to define the role of genetic variants involved in ciliopathies in the progression of aPSC, 
assemblyloids will be characterized at the molecular level by cutting edge technologies including 
single cell RNA-sequencing (scRNA-seq), using the Chromium 10x instrument present in the 
Foundation, which will allow us to understand how the variants influence the different cell 
populations at the molecular level. The morphology of primary cilia will be studied by electronic 
and confocal microscopy with instruments present in the Foundation. Finally, the role of genetic 
variants on the progression of the disease will be studied, looking at collagen deposition and 
fibrosis formation, by confocal microscopy and macrophage activation and infiltration, with 
functional assays and confocal microscopy.
4.11 Use of liver assembloids as a model to test new pharmacological approaches
Considering the ability of the assembloids to morphologically and physiologically reproduce the 
native tissue, they will be used as an in vitro model to test new pharmacological approaches such 
as engineered extracellular vesicles to carry the CRISPR/Cas9 genome editing system. Once 
generated, the assembloids will be treated with isolated extracellular vesicles at the Translational 
Medicine laboratory of the SC Transfusion Center of the IRCCS Foundation Ca' Granda Ospedale 
Maggiore Policlinico for 72h. The genetic editing will be evaluated
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by TaqMan assay on ABI 7500 Fast apparatus as described in point 4.8.
5. ENDPOINT
5.1 Primary Endpoint
Exploration and description of the behavior of genetic variants associated with ciliopathies on 
the aPSC phenotype within the variant carrier group compared to the non-carrier group.
5.2 Secondary Endpoints
Testing of new pharmacological approaches based on extracellular vesicles containing the 
CRISPR/Cas9 genome editing system to correct mutations involved in ciliopathies using 
assemblyloids as a modelin vitro.
6. DURATION / TIMELINE OF THE STUDY
Study start month and year: 12/2024 
Enrollment close month and year: 12/2026 
Study end month and year: 12/2027
PROJECT GANTT
Intervention/months
1-6
7-12
13- 18 19-24
25-30
31-36
Enlistment
Informed consent
Inclusion/exclusion criteria
Isolation of primary cells
iPS Generation
Assembloid Generation
Gene expression analysis
Morphological analysis by mycoscopy
Functional analyses of the role of cilia in primary sclerosing cholangitis
Using Assemblloids to Test EVs as a Novel Pharmacological Approach
7. STATISTICAL ANALYSIS
7.1 Sample size
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Since the disease is rare and to 
the inferential, but it is only 
descriptive sample size. Those 
patients carrying the variant to 
achieve this goal we set 
ourselves in point 4.1, given that 
as generation of organoids is
the inside of the primary endpoint are not
and exploratory, for this reason it is not 
that we will be able to collect however
and 6 samples of non-carrier patients we are 
going to recruit at least 20 patients for each
indicated by previously published works
make explicit the differences
essario to decline a
at least 6 samples from
of the variant. For
each group indicated
ncethe efficiency of
or
or
And
c
to
And
d
And
30% starting from biopsies
13.14.
7.2 Data analysis
Statistical analyses will be ef f
scRNA-seq analyses will be performed using the Seurat package of the R software (https://www.r-
, with a pipeline already standardized within our laboratory. The results
will be represented by descriptive statistics: categorical variables will be reported as frequency 
and percentage, while continuous variables will be reported as mean ± SD or median 
(interquartile range), depending on the distribution. If deemed appropriate, graphs will be used 
to integrate the descriptive analyses.
carried out using the JMP software
(SA, Cary, NC) and the
(project.org)
8. ADVERSE EVENTS
The project does not involve the administration of drugs or other substances or ad hoc invasive 
interventions outside of normal clinical practices. Therefore, no adverse events are expected.
9. RISK/BENEFIT ASSESSMENT
The study does not foresee an immediate benefit for patients, but the results of this trial will 
have the potential to lead to a decoding of the mechanisms underlying the development of 
PSC, resolve the temporal order of events that regulate its evolutionary trajectory, 
understand its progression, characterization and improve the therapeutic management of 
patients.
10. STUDIO MANAGEMENT
10.1 Data collection and management
Each participant, at the time of enrollment, will be assigned a unique code. The de-identification of 
the data will be done in such a way that the people who access the database will not be able to 
trace the identity of the subjects in any way. Only local experimenters will be able to trace the 
identity of the enrolled subjects.
The data needed for the study will be recorded in a specific eCRF in a Data Management System 
validated according to national regulations, provided by the Scientific Direction of the Foundation. 
The platform used will be RedCap (Research Electronic Data Capture).
The REDCap Consortium is composed of >1000 institutional partners worldwide (research institutions, 
universities, ministries etc). The consortium supports a secure web application (REDCap) designed 
exclusively to support data acquisition for research studies. The REDCap application allows users to 
create and manage online databases quickly and securely, and is currently in use for more than 
110,000 projects with approximately 150,000 users covering numerous areas of research interest 
across the consortium.
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Through REDCap, for this study the following will be implemented: a) user-level 
identification, with specific restrictions based on the role in the study b) real-time data 
integrity validation and control c) patient de-identification before data export d) 
centralized data storage with daily backup, a secure server within the Foundation's IT 
structure.
10.2 Regulatory aspects and ethical considerations
10.2.1 Approval by the Competent Authority
In accordance with applicable regulations, the principal investigator must obtain approval from the 
appropriate Competent Authority prior to initiating the clinical study.
This study will be conducted in accordance with the ICH/GCP (International Conference of 
Harmonization/Good Clinical Practice) rules and all applicable laws, including the Helsinki 
Declaration of June 1964, as amended by the last World Medical Association General 
Assembly in Seoul, 2008.
10.2.2 Ethics Committee Approval
The investigator must ensure that the protocol has been reviewed and approved by the local 
independent Ethics Committee (EC) before starting the study.
The CE must also review and approve the informed consent (IC) form and all written 
information received from the patient prior to enrollment in the study.
Should it be necessary to modify the protocol and/or the IC during the study, the investigator will be 
the guarantor and therefore the person responsible for ensuring the review and approval of such 
modified document as requested by the CE.
The content of these changes will be implemented only after the CE has approved them. Until 
then, it will be necessary to refer to the previous version of the document already approved.
10.2.3 Informed consent (IC)
The investigator or other personnel designated by him have the task of informing the subjects about all 
aspects and procedures of the study.
The process for obtaining informed consent must comply with applicable regulatory 
procedures. The investigator (or designated staff member) and the subject must date and 
sign the informed consent form prior to the patient initiating any study-related procedures. 
The subject will receive a copy of the IC dated and signed by both parties; the original copy 
will be retained in designated study archives. Neither the investigator nor designated staff 
member should in any way coerce or influence a subject to participate or continue to 
participate in the study. A subject's decision to participate in the study must be completely 
voluntary. The investigator and designated staff member should emphasize to the subject 
that he or she may withdraw consent at any time without penalty or loss of any benefits to 
which the subject may be entitled.
Written or oral information relating to the study, including the written consent form, 
must not contain any language that forces the subject to waive (even apparently) his or 
her legal rights, or that would exonerate the investigator, the institution or the
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sponsor from liability for negligence.
10.3 Duties of the experimenter
In accordance with applicable local regulations, the investigator must submit periodic 
reports regarding the progress of the study at his/her site to the CE and notify the CE 
of study closure. Periodic reports and closure notification are part of the investigator's 
responsibilities.
10.4 Study monitoring
In accordance with applicable regulations and good clinical practice (GCP), the monitor must visit or 
contact the center periodically. The duration, nature and frequency of such visits / contacts depend 
on the recruitment frequency, the quality of the documents held by the center and their adherence 
to the protocol.
Through these contacts, the monitor must:
" monitor and evaluate the progress of the study
" examine the collected data
" conduct source document verification
" identify each problem and related solutions
The purposes of the monitoring activity are to verify that:
" the rights and well-being of the subject are respected
" the study data are accurate, complete and verifiable from the original documents
" the study is conducted in accordance with the protocol and any approved amendments, GCP and 
applicable regulations
The experimenter must:
" give the monitor direct access to all relevant documentation
" dedicate part of his time and his staff to the monitor to discuss the monitoring results and 
any other possible aspects.
The monitor must also contact the center prior to the start of the study to discuss the protocol 
and data collection procedures with the staff.
10.5 Study quality assurance
As the sponsor, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico may, at 
its discretion, carry out a quality control of the study. In this case, the investigator must 
allow the monitor direct access to all relevant documentation and dedicate part of his 
or her time and personnel to the reviewer to discuss the results of the monitoring and 
any other aspects of the study.
In addition, Regulatory Authorities may conduct inspections. In this case, the 
investigator must allow the inspector direct access to all relevant documentation, and 
dedicate part of his time and personnel to the inspector to discuss the monitoring 
results and any other aspects of the study.
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10.6 Closing of the study
At the time of study closure, the monitor and the experimenter must activate a series
of procedures:
" review all study documentation
" reconcile study data
" reconcile all clarifying reports.
10.7 Document storage
In accordance with current national regulations, the investigator must keep a copy of all 
documentation and store it in a dry and safe place after the study has been closed.
10.8 Disclosure of information regarding scientific discovery
10.8.1 Confidentiality
The investigator and other personnel involved in the study must handle all information relating to 
the study (including the protocol, data obtained and all documentation generated during the study) 
and must not use such information, data or reports for purposes other than those described in the 
protocol. These restrictions do not apply to:
1) information that becomes publicly available, not due to negligence on the part of 
the investigator or his staff;
2) information that requires confidential disclosure to CE for the sole purpose of evaluating the study;
3) information that must be disclosed in order to obtain appropriate medical care for a 
study subject.
10.8.2 Publications and Intellectual Property Rights on the Results of the Study
SINGLE-CENTER STUDY that includes collaborations:
Publications:
Foundation as a Prom studio, even in 
the case of results visibility 
proportional to the actual 
contribution provided in the form in 
aggregate or common the 
identification of the subject involved. 
Each magazine or publication has the 
role and the participation of the Ce
ortore will guarantee the diffusion and publication of 
negatives, without any constraint and guaranteeing 
their participation.
notification containing the results and data of the 
collaborating institution and the Foundation, under 
study and the role covered. The data may therefore 
be anonymized, so as not to permit the identification 
of the person to whom the data refer.
one of the results of the 
collaborating center
t
And
uGod, will have to indicate the 
proportional number to the
or be published in any 
manner whatsoever
the
n
And
n
Intellectual Property Rights 
The Parties acknowledge that 
for the to be used and shared 
by each party that right of 
access and use,
And
:
management of collaboration in the field of ti, 
information,know-how,inventions (br and remains 
the exclusive owner even if n non-exclusive and 
free, for the sole purposes of the real
of the study will be able to
(whether or not it is 
possible) and grant the other 
a ization of the activities
And
n
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object of the study and limited use 
does not include the ownership of 
the Promoter, except in compliance 
with the legislation.
m
the
g
And
entity for the duration of the same. It remains 
interrupted to sublicense to third parties.
entity, data and results generated within the framework of specific 
agreements between the Promoter and the centre/centre
I know that this right of
The Studio will be staffed by 
our collaborators.
And
11. INDEMNITY AND COMPENSATION IN CASE OF DAMAGES
In case of unwanted events or any damages that may arise from participation in 
research, our Institute's Insurance Policy also extends to cover subjects participating in 
research projects.
12. AMENDMENTS TO THE PROTOCOL
In case of changes in the study protocol, the amendment procedure will be activated in 
advance at the local independent CE. The CE will have to verify and approve again the CI 
form and all written information received from the patient before enrollment in the study.
13. FINANCIAL AGREEMENTS
The costs of study procedures exceeding normal clinical practice will be entirely covered by funds 
deriving from the EarlyCareerAward II Edition 3 Regional Foundation for Biomedical Research 
funding.
14. DISCLOSURE ON CONFLICTS OF INTEREST
The experimenters declare no conflicts of interest.
15. REFERENCES
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2. Karlsen, T.H., Folseraas, T., Thorburn, D. & Vesterhus, M. Primary sclerosing cholangitis - a
comprehensive review.J Hepatol67,129831323 (2017).
3. Karlsen, T.H.et al.Genome-wide association analysis in primary sclerosing cholangitis.
gastroenterology138,110231111 (2010).
4. Alberts, R.et al.Genetic association analysis identifies variants associated with disease
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5. Kim, Y.S., Hurley, E.H., Park, Y. & Ko, S. Primary sclerosing cholangitis (PSC) and
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Exp Mol Med55,138031387 (2023).
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Acta Mol Basis Dis1864,124531253 (2018).
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medicine.Nat Rev Mol Cell Biol21,5713584 (2020).
8. Hofer, M. & Lutolf, M. P. Engineering organoids.Nat Rev Mater6,4023420 (2021).
9. Clevers, H. Modeling Development and Disease with Organoids.Cell165,158631597 (2016).
10. Kim, M.et al.Patient-derived lung cancer organoids as in vitro cancer models for therapeutics
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11. Zhou, Z., Cong, L. & Cong, X. Patient-Derived Organoids in Precision Medicine: Drug
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