Transfusion Service and Cell Therapy Laboratory
via Luca Giordano 13, 50132 - Florence
Tel: 055 5662991/448 Fax: 055 587010
Principal Investigator: Franco Bambi (director Blood bank, qualified personnel - GMP unit)
Staff
- Mariani Maria Pia (haematologist)
- Muscarella Elisa (biologist)
- Spitaleri Irene (quality control personnel- GMP unit)
- Taddei Alessandro (biotechnologist)
- Bindi Barbara (laboratory technician)
- Bisin Silvia (laboratory technician Production personnel – GMP unit)
- Ceccantini Riccardo (laboratory technician)
- Cunial Vanessa (laboratory technician)
- Pavan Paola (laboratory technician)
Cellular therapies are being increasingly explored for both regenerative (stem cells) and immunotherapy (T, NK cells; mesenchymal stem cells (MSC) ) purposes. The Cellular Therapy Laboratory was specifically developed to provide access to cellular therapies for the tertiary care pediatric referral center in Tuscany. This program was designed to comply with international regulations of good laboratory and manufacturing practices (GLP, GMP).
Research activity is currently focusing on the use of partially matched family donors for the cure of malignancy and standardizing of procedures for in vitro MSC expansion and for their phenotypical evaluation.
Concerns regarding the use of MSC
In recent years, there has been increasing interest in a population of adult stem cells found in the bone marrow, commonly called mesenchymal stem cells (MSC), due to their potential use in regenerative medicine. MSC have been extensively studied in research facilities and have rapidly reached the clinical trial phase worldwide.
Under appropriate conditions MSC may differentiate in vitro into one of many cell phenotypes. The ability of MSC to differentiate in several tissues after engraftment has been confirmed in vivo following systemic injection. The feasibility of systemic infusions of MSC has been demonstrated in patients with solid, hematological malignancies and congenital diseases. The results confirmed pre-clinical data regarding safety, proving that infused cells engraft in the recipient's tissues, sometimes contributing to amelioration of the disease. It is necessary to expand and characterize human MSC in animal-serum-free media to improve the compliance of the MSC infusions in patients to Good Clinical Practices (GCP).
The use of cellular products for therapy has been generally challenged by the necessity of bovine-derived sera and/or serum-derived products in the culture media. These factors increase the risk of well known infectious diseases of xenogeneic origin or the chance of host immune reactions.
Serum-free media for expansion of hematopoietic stem cells and dendritic cells have already been established. Dealing specifically with MSC, the only published clinical trial reporting the infusion of allogeneic MSC infusion showed that in one of the patients the possible benefit of the MSC infusion has been compromised by a lack of engraftment. In this particular patient, an immune reaction against a bovine derived protein was found, suggesting that the use of bovine serum from the earliest phase of cell isolation might be responsible for the immune reaction against MSC. In our project, efforts will be made to develop an animal serum deprived procedure for isolation and expansion of MSC.
In our laboratory the MSC will be ex-vivo manipulated in the GMP environment testing new serum deprived media for their isolation.
Isolation and expansion of MSC with serum-free media and according to GMP criteria
It is clear that to move from the pre-clinical findings of any study dealing with MSC to a clinical setting it is mandatory to produce the cells in the appropriate manner.
In order to reach this target, at the laboratory of the Transfusion Center it is realized a controlled contamination area consistent with the standards provided by European GMP; furthermore the ministerial authorization that will lead to the production is already started. The facility is characterized by an A/B grade area for sterile processing and by a C grade area for less crucial procedures, with PCL2 biohazard level. A continuous particle monitoring system is already present, as established by the recent revision of EU GMP.
In this environment we will test several media for the MSC isolation. A major problem is the strict dependence of human MSC on bovine calf serum, which is obsolete and dangerous in human transplant settings. It is as yet unclear which individual factors are supporting the growth of MSC.
Our objective is the identification of the optimal in vitro requirement for MSC growth without serum derived additives, in the comparative analysis of the serum dependent (SD) MSC (MSCsd) versus the serum free (SF) MSC (MSCsf), to detect possible differences in the in vitro and in vivo behavior. Once able to get the cells survive without serum, tests dealing with cell cycle analysis and phenotypical characterization will be performed. The proliferation analysis will be performed by FACS with BrDU and Propidium staining protocols. The phenotype analysis will reproduce the one previously described for the MCSsd, using the same panel of monoclonal antibodies.
The further step will be the longevity and telomere length. The MSCsf and MSCsd will be tested analyzing the telomere length and telomerase activity in cultures by real time PCR. These parameters are well established markers for long-lived cells and generally ensure the plasticity of MSC. This is of utmost importance as several cytokines and growth factors that will be tested have the potential to induce cell differentiation under certain circumstances. In addition, in vitro plasticity of MSCsf will be tested with standard protocol for bone cartilage and fat differentiation, similarly in vivo ability of a multi-tissues engraftment will be tested in the SCID model. The end-point is to compare the published data dealing with conventional MSC isolation with the ones obtained from MSCsf cultures.
Development of protocols for the in vitro expansion of hematopoietic potential under good medical practice (GMP) conditions
Once the tissue-engineered construct is developed it will be validate in a clinical setting.
The use of human Hematopoietic Stem Cells (HSC) isolated from bone marrow or mobilized peripheral blood as a source of cells for reconstitution following high-dose chemotherapy is now a common therapeutic modality for the treatment of malignancy. To obtain numbers of cells sufficient for such applications in children or adults, ex vivo expansion has been utilized, resulting in successful engrafment and accelerated recovery from neutropenia and thrombocytopenia.
Enhancement of stem cell self-renewal is the natural issue of such a procedure, as an increased number of stem cells is a key factor in the overall numerical expansion of the explanted cell population. In a number of studies carried out over the last 15 years, it was demonstrated that very low oxygen tensions in vitro (severe hypoxia) help to preserve the stem cell potential within hematopoietic cell populations obtained ex vivo (Blood 82:2031-2037, 1993; Leukemia 14:735-739,2000). Severe hypoxia was shown to influence the balance between stem cell differentiation commitment and self-renewal in favor of the latter and to restrain the effects of cytokines boosting commitment and clonal expansion (Exp. Hematolo. 30:67 – 73,2002 ).
Main Research Themes
- Development of-GMP compliant culture conditions for mesenchymal stem cell culture and expansion for therapeutic application
There are several nutritional supplements, such as fetal bovine serum (FBS) that can be used to expand MSC in ex-vivo cultures. For safety reasons, medical practitioners are compelled to avoid the use of animal serum to expand the cells for therapeutic purposes in humans, while serum-free media or human serum and plasma can be used.
Main achievements
Platelets play a fundamental role in hemostasis and tissue repair. Platelets are a natural source of growth factors that are released upon activation. Therefore platelets are a safe and readily available source of growth factors for cell culture. In collaboration with Rizzoli Institute (Bologna) a protocol for MSC ex-vivo expansion with autologous platelet-rich plasma (PRP) has been established.
Current work
Good Manufacturing Practices (GMPs) standards are necessary for the manufacture of cell-based therapeutic agents that are administered to patients enrolled in clinical trials in Europe.
Currently a collaborative effort is underway to establish a protocol for the culture and expansion of MSC according to GMP standards for clinical use.
Future plans
Once a protocol for the ex-vivo expansion of MSC has been established with PRP, according to GMP standards for clinical use, it will be validated in a clinical setting.
fig.1
Fig. 1, 2 - Mesenchymal stem cells (MSC)
fig.2 - Development of a tissue-engineered construct to improve allograft integration for osteosarcoma patients
Osteosarcoma is a primary malignant tumor of the skeleton that occurs mainly in young patients.
Before chemotherapy was introduced, all patients with osteosarcoma were treated by amputation and almost all patients died within a year from diagnosis. Today the treatment is pre- and postoperative chemotherapy associated with surgery and the percentage of patients cured varies between 60% and 70%. Surgery is conservative (limb salvage) in more than 90% of patients and most of them require reconstruction.
Massive bone allografts are used with increased frequency in reconstructive surgery to replace missing bone parts, such as critical size defects. The effectiveness of the procedure depends on healing time and type of graft host integration. Although most of the massive allografts have long-term success, 25% of reconstructions fail. To improve the integration of the graft a tissue-engineered construct can be used. A tissue-engineered construct can be composed of stem cells, growth factors and biodegradable biomaterial.
Main achievements
MSC can differentiate into various cells of mesenchymal origin, among them bone. MSC have been shown to be effective in preclinical studies to regenerate bone in massive defects.
Current work
The composition and the safety of a tissue-engineered construct are currently under study. It is important that the biomaterial utilized is biodegradable and compatible with the differentiation of the MSC toward the osteogenic lineage. - Severe hypoxia enhances ex vivo hematopoietic expansion under "clinical grade" conditions
Main achievements
The target of this study was the development of protocols for the in vitro expansion of hematopoietic potential under good medical practice (GMP) conditions, suitable for clinical use. Serum-free incubation media were therefore used, supplemented with a combination of stem cell-active cytokines, to incubate explanted hematopoietic populations in order to compare their in vitro expansion under severe hypoxia with the one in normoxia.
Current work
Cells have been obtained from mobilized peripheral blood donors. CD34+ cells have been isolated from buffy coat by the Miltenyi Biotec "indirect" immunomagnetic technique, using for the first passage the "midi" column and for the second the "mini" column. Cells have then been analyzed by flow cytometry using anti-CD34,-CD133, -CD61, -CD3, -CD19 antibodies. The purity of the isolated CD34+ cells was 35-75%. Cells have been cultured in the HP01 medium (MacoPharma), in the presence of the FKT6 cytokine combination, including Flt3-ligand, Stem Cell Factor/Kit-ligand, ThromboPoietin and Interleukin-6. Incubation under severe hypoxia (0.1% O2) has been carried out in a water-saturated Ruskinn Concept 400 anaerobic incubator, flushed with a preformed gas mixture (0.1% O2, 5% CO2, 95% N2); incubation in normoxia (21% O2) has been carried out in a 5% CO2, 95% air, water-saturated atmosphere. Culture expansion has been measured after 14 days of incubation, with respect to either the overall number of viable cells or the number of colony-forming cells (CFC), as determined by following cell transfer to secondary FKT6-supplemented MethoCult (StemCell Technologies Inc.) semisolid cultures and a further 7-day incubation therein, in any case in normoxia.
Hematopoietic expansion in hypoxia was 2.8-fold higher than in normoxia as determined by counting the overall number of viable cells, and 3.5-fold higher as determined by counting the number of CFC.
In the presence of the FKT6 cytokine combination, incubation in severe hypoxia resulted an efficient method to markedly improve the expansion of hematopoietic cell populations under "clinical grade" conditions.
Research Grants
- Associazione Noi per Voi, Genitori contro le leucemie e i tumori solidi del bambino, Florence
- Fondazione Tommasino Bacciotti, Florence
Publications
- Bambi F, Fontanazza S, Messeri A, Lippi A, Tucci F, Tamburini A, Tintori V, Casini T, Lacitignola L, Tondo A, Veltroni M, Bernini G, Faulkner LB. Use of percutaneous radial artery catheter for peripheral blood progenitor cell collection in pediatric patients. Transfusion 2003;43:254-258.
- Pagani A, Macri L, Faulkner LB, Tintori V, Paoli A, Garaventa A, Bussolati G. Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology. Diagn.Mol.Pathol. 2002;11:98-106
- Sardi I, Tintori V, Marchi C, Veltroni M, Lippi A, Tucci F, Tamburini A, Bernini G, Faulkner L. Molecular profiling of high-risk neuroblastoma by cDNA array. Int.J Mol.Med 2002;9:541-545.
- Faulkner, L., Lacitignola, L., Tintori, V., Bambi, F., Mariani, M., Marchi, C., Muscarella, E., Tamburini, A., Tucci, F., Lippi, A., and Bernini, G. Graft vs. neuroblastoma effect after partially matched related hematopoietic transplantation. Cytotherapy 6(4), 407. 2004
- Luceri C., De Filippo C., Giovannelli L., Blangiardo M., Cavalieri D., Aglietti F., Pampaloni, M., Bambi F., Biggeri A. and Piero Dolora - Extremely Low-Frequency Electromagnetic Fields do not Affect DNA Damage and Gene Expression Profiles of Yeast and Human Lymphocytes Radiation Research 164, 277–285 (2005)
- Lodovici M, Caldini S, Luceri C, Bambi F, Boddi V, Dolara P. Active and passive smoking and lifestyle determinants of 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human leukocyte DNA. Cancer Epidemiol Biomarkers Prev. 2005 Dec;14(12):2975
- Iannalfi A, Bambi F, Tintori V, Lacitignola L, Bernini G, Mariani MP, Sanvito MC, Pagliai F, Brandigi F, Muscarella E, Tapinassi F, Faulkner L. Peripheral blood progenitor uncontrolled-rate freezing: a single pediatric center experience. Transfusion. 2007 Aug 21
- Astori G, Vignati F, Bardelli S, Tubio M, Gola M, Albertini V, Bambi F, Scali G, Castelli D, Rasini V, Soldati G, Moccetti T. "In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells. J Transl Med. 2007 Oct 31;5(1):55
Collaborations
- Divisione di Chirurgia Oncologica Ortopedica (direttore dott. Rodolfo Capanna) Azienda O.U. Careggi, Firenze
- Dipartimento di Patologia e Oncologia Sperimentali, Università degli Studi di Firenze, Firenze;
- Istituto Mediterraneo di Ematologia, Policlinico di Torvergata, Roma
- Laboratorio di Biologia cellulare e Terapie avanzate - Università di Modena e Reggio Emilia (Mo)
- Cardiocentro Ticino Lugano, (CH)
- Laboratorio di Rigenerazione Tessutale Ossea, Istituti Ortopedici Rizzoli, Bologna (BO)
- Etablissement Français du Sang, Bordeaux (Fr)
