Irmi ismett


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Irmi ismett

  1. 1. ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione) Palermo 1 Fondazione RiMED Palermo – Italy
  2. 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. 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. 4. GMP Facility - ISMETT 4 Unit of Regenerative Medicine and Biomedical Technologies
  5. 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. 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. 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. 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. 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. 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. 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. 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. 13 Isolamento Isole di Langerhans Programma di trapianto insule pancreatiche - trapianto eterologo, autotrapianto –
  14. 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. 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. 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. 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. 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. 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