1. ISMETT
(Istituto Mediterraneo per i Trapianti
e Terapie ad Alta Specializzazione)
Palermo
1
Fondazione RiMED
Palermo – Italy

2. 2
Experimental
Laboratory
GMP Facility
• Cell Production Laboratory
• QC Laboratoy
Preclinical Laboratory
(large animals)
q Regenerative Medicine
q Immunobiology
q Molecular Medicine
q Biomedical Technologies
Advanced therapies
Department of Laboratory Medicine and Advanced Biotechnologies
Laboratory of Clinical Pathology,
Microbiology and Virology
Adoptive Immunotherapies
Transplantation of fetal hepatocytes
Transplantation of pancreatic islets

3. 3
ü Immunotherapeutic approaches (CTLs, NK, dendritic
cells) for treating viral infections and virus-related
tumors
ü Development of regenerative therapies for treating
chronic liver diseases
ü Characterization of stem/progenitors cells in fetal
and adult tissues/organs
ü Characterization of soluble factors produced by stem
cells
Competence

4. GMP Facility - ISMETT
4
Unit of Regenerative Medicine and Biomedical Technologies

5. Unit of Regenerative Medicine and Biomedical Technologies
Organization and Personnel
5
Laboratory of experimental research
n.12 Researchers, n.2 Technicians
(supported by RiMED Foundation)
GMP Facility
(QP, QA, QC, Validation Manager, Production Head)
ISMETT Labs
Laboratory of molecular biology
Laboratory of microbiology
Laboratory of immunology
Cytofluorimetry – Mass Spectrometry
Pre-Clinical Research Lab
Animal Facility
n. 1 Veterinarian
n. 1 Bioengineer

6. Technical setting
6
Unit of Regenerative Medicine and Biomedical Technologies
§ Facility for experimental cell biology (primary cell cultures, cell immortalization, phenotypic &
genotypic analysis , 3D cultures, etc.)
§ Facility for cell production (infected/non-infected cells)
§ Facility for molecular biology (NA amplification, gene expression, sequencing, gene vector
production)
§ Facility for experimental and clinical immunology (lymphoid cells purification, activation and
characterization; immunopheno yping; cell sorting)
§ Facility for microbiology/virology (virus production and analysis; bacterial/yeast cultures for
genetic engineering; control of microbial contamination)
§ Mass spectrometry (proteomics, metabolomics)
§ Biobanking (lymphoid cells for therapy, stem/progenitor cells, primary cell cultures, clinical samples
etc.)
§ Clinical setting for phase I/III studies (cohorts of patients affected by end-stage diseases and
solid organ transplants, highly-specializedsurgical and interventional procedures)

7. Services currently offered
Ø Development of adoptive immunotherapies:
Ø Ag-specific T Cell therapy – Innate immunity–mediated therapy
Ø Production of stem cells in GMP Facility
Ø Mass spectrometry (qualitative/quantitative) analysis of stem cells
secreted factors
Ø Biobanking of primary cultures of adult/fetal human tissues and
pathologic samples of patients affected by end-stage organ
diseases and serious viral/microbial infections
Ø QA assistance for GMP- compliant Standard Operative Procedures
Ø Clinical trials (phase I/II studies)
7

8. Ex vivo production and in vivo infusion of
autologous (or heterologous) cytotoxic/helper
T lymphocyte clones specific to
EBV
CMV
Adoptive cell immunotherapy for prevention and treatment of
post-transplantation herpesvirus infections
Prevention and
treatment of PTLD
Treatment of CMV infections
caused by drug-resistant viral
variants
Trapianti – Infettivologia - Immunoterapia
8

9. ITA 005 colon - treatment with anti-EBV-CTLs
before treatment after treatment
CD20+ B cellsCD20+ B cells
EBEREBER
In vitro production of EBV-specific T cells
Immunoterapia adottiva
CD20+ B cells
EBER
60 Gy
irradiation
9

10. Immunoterapia adottiva per il trattamento di patologie
indotte da herpesvirus in pazienti immunocompromessi
§ Autorizzazione dell’AIFA per il trattamento delle patologie EBV e CMV-correlate nei
pazienti trapiantati
QP/QA: QC: Controllo impianti:
§ Uso di CTL eterologhe anti-EBV e anti-CMV
§ Biomarkers diagnostici di trasformazione EBV- indotta su linfociti B
§ Linee guida di diagnostica molecolare, immunologica e immunoistochimica per
trattamenti terapeutici virus-specifici in fase sintomatica e pre-emptive
(Gruppo di Lavoro su Infezioni in Trapianto: AMCLI-SIV)
§ Partecipazione di ISMETT (socio fondatore) a EATRIS
10
Workshop: L’Infrastruttura di Ricerca EATRIS e il Nodo Italiano:
Stato di avanzamento e prospettive
26-27 Febbraio 2013 - Istituto Superiore di Sanità
The EATRIS consortium of academic institutes provides cutting edge
infrastructure and expertise along the entire translational value chain.
With top quality facilities for basic research, manufacture, non-clinical and
clinical development, our EATRIS institutes offer a truly multidisciplinary
environment.
Prfoducts:
Advanced Therapy Medicinal Products – Biomarkers - Imaging and
Tracing - Small Molecules - Vaccines

11. Isolation of Buffy coat
from liver perfusate
Leukapheresis
Hepatic Lymph nodes
PBMC
from HCV+ patients and
healthy donors
B cell
B cell
HCV-specific
Neutralizing Antibodies
B cell
B cellB cell
EBV- Immortalized
HCV-specific B cells
IV infusion
activated NK
IL2/IFNα/IL12
Low viremic Recipient
(anhepatic phase)
Adapted from: Ohira M et al JCI 2009,119:11 3226-3235
Depletion of CD3+ cells
Isolation of CD56+ NK cells
CliniMACS
Adoptive immunotherapy plan against HCV recurrence
after liver tranplantation

12. Liver perfusate (whole hepatic blood) processing
Infusion in Liver
Recipient
Transfer in 500ml conical
Centrifugation
In vitro NK cells
Activation/expansion
3 X ~3Lt
Reservoirs
Volume reduction
7-10 Lt → 0.5-1.5 Lt
buffy coat
Isolation
Apheresis COM.Tec
Transfer in double
blood bag
Purification of
CD3-CD56+ NK cells
CliniMACS
Clinical grade
MagMACS
research grade
GMP-conforming
12

13. 13
Isolamento Isole di Langerhans
Programma di trapianto insule pancreatiche
- trapianto eterologo, autotrapianto –

14. Insule pancreatiche – diabete
14
Nano Engineering for Cross Tolerance: New approaches for bioengineered, vascularized,
chimeric islet transplantation in non-immunosuppressed hosts (NEXT)
Progetto presentato per FP7 HEALTH.2013.1.3-2: Innovative approaches to address
adverse immune reactions to biomedical devices, implants and transplant tissues -
Coordinator dr. Severino – Explora srl
Studio di piccole molecole cito-protettive con duplice applicabilità nella demenza di Alzheimer
e nel trattamento del diabete mediante trapianto di insule pancreatiche
Progetto finanziato PON 02- 00697 Potenziamento laboratori pubblico-privati
In collaborazione con CNR IBB di Catania, RiMED, Myrmex SpA (ccordinatore CNR –IBB)l

15. Transplantion of human fetal liver cells in patients
with end-stage liver cirrhosis
Fetal liver cell
suspension
Collagenase digestion
Infusion into patient
splenic arteryHuman fetal
liver
Phase I-II Control Study of Human Fetal Liver Cell Transplantation for
Treatment of Chronic Liver Disease
(Submitted manuscript)
Efficient human fetal liver cell isolation protocol based on
vascular perfusion for liver cell-based therapy and case report on
cell transplantation
(Liver Transpl. 18:226-237, 2012)
15

16. Human fetal hepatocytes cultured in high density
demonstrate mature hepatic functionality
*
0
50
100
150
200
250Conc(ng/ml)/1.8x10
6
cells
Albumin secretion
Fetal Adult Liver
MSCs
0
2
4
6
8
10
12
14
16
18
Conc(mg/dL)/1.8x10
6
cells
Urea production
Fetal Adult Liver
MSCs
0
50000
100000
150000
200000
250000
300000
RLU/1.8x106
cells
CYP3A4 activity
Fetal Adult
Liver
MSCs
G6Pase activity Glycogen storage ICG uptake
Primary culture of
fetal hepatocytes
High density culture Low density culture Flat cell culture
1:3 split
0
50
100
150
200
250
Conc(ng/ml)/1.5x10
6
cells
Down-regulation albumin
secretion in flat cell cultures
Fetal hep. Flat
cells
Liver
MSCs
*
Cryopreservation up to 1 year storage is well tolerated as did not significantly affect viability (A), and
functions (B, C) of fetal hepatocytes
60
65
70
75
80
85
90
FRESH CRYO
Percentageofviability
Fresh Cryo
10
30
50
70
90
110
130
150
170
190
F R ES H C R Y O
Conc.(ng/ml)/1.8x10
6
cells
Fresh
Cryo
A B
C
Cryo
16

17. 17
Medicina rigenerativa: cellule staminali dermiche
Ø Multipotent fetal dermal cells are easy to isolate, with a high yield
Ø Cultured fetal dermal stem cells possess a mesenchymal phenotype
Ø Cells with a regenerative potential can be successful isolated from a
small fetal skin biopsy and maintained in culture for long periods without
losing their potential, thus generating large quantities for clinical
applications.

18. 18
In vitro Expansion and Characterization of Skin
Precursor Cells Isolated from Human Fetal Dermis
CD71
CD105
dermal
papilla dermal
papilla
A B C
CD105
CD90 CD73 CD105
Fetal Adult
SSEA4SSEA4
90% 10.5%
Pluripotency marker SSEA-4 in fetal
and adult dermal cells
Approx. 90% positive for the classical
mesenchymal markers CD90, CD73 and CD105
Localized in close proximity of skin
dermal papillae
Low immunogenicity. (A) Fetal dermal
cells co-cultured with allogenic PBMCs
do not evoke T cell proliferation. (B)
Positive control MLR
A B
A
0
5
10
15
20
25
30
P1
P3
P5
P7
P9
P11
P13
P15
P17
P19
P21
P23
P25
P27
P29
Passage number
Numberofdaystothenext
passage
Adult Fetal
Day 0
CB
Day 6
Retention of a stable phenotype
after 12 subcultivations
Expansion potential of fetal vs. adult dermal cells
(A) In vitro angiogenic potential. (B, C) Epithelial-like
potential DMSO-induced
A B
AD P10Fet P25
Higher retention of differentiation
potential in fetal than adult cells
0
20
40
60
80
100
120
CD90
CD105
CD73
HLAclassI
CD44
CD166
CD71
Vimentin
CD29
Nestin
CD49b
CD49e
CD106
CD117
CD45
CD34
CD14
HLADR
%positivecells
P3
P12
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Cellnumber(106
)
(C) Non enzymatic isolation technique of “cell outgrowth”. (D)
cell yield after isolation: fetal cells can be isolated with a 3-fold
higher efficiency than adult cells
Adult
Fetal

19. Tissue Stem/Progenitor Cells & Microenvironment Modulation
TISSUE OF ORIGIN:
≠ types of tissues ; ≠ age (e.g. fetal and adult tissues)
DEVELOPMENT & OPTIMIZATION of CULTURE SYSTEMS:
2D and 3D ; use of scaffolds ; growth factors + ; hypoxia
Mesenchymal Stem Cells
“Stemnessdegree”
Tuning&Evaluation
Models of assessment of therapeutic and regenerative potentials in vivo
SILAC
Microvescicles
cell-cell contacts
Secreted Factors: Qualitative & Quantitative ProteomicsSecret-OMICS
miRNA
Light AA Heavy AA
Conditioned Media Processing
Quantitative identification of
full differential secretome
LC-MS/MS
Systems Biology
Bioinformatics
Data Functional Analysis
Integration of
Transcriptomics Data
Secreted Factors Mix &
Systems Biology
In Gel
Digestion
Protein identification
Protein quantification