Search

Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio-maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. J. Cell. Physiol. 232: 548-555, 2017. © 2016 Wiley Periodicals, Inc.

Autologous graft is considered the gold standard of graft materials; however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro-grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro-grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy, and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro-grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential.

INTRODUCTION: Extracorporeal photoapheresis (ECP) is an immunomodulatory therapy for patients with acute or chronic graft-versus-host disease (aGVHD/cGVHD). So far, few studies have explored the molecular regulation of GVHD, and to date no studies have addressed how specific is miRNA expression change during ECP, and whether selected miRNA profiles might be predictive of clinical responses to ECP. OBJECTIVE: The aim of the study was to analyze the expression profile of miRNAs in plasma of patients with GVHD candidates for ECP, and their changes in responding and non-responding patients to this therapy. PATIENTS AND METHODS: Patients with GVHD underwent ECP therapy by off-line methods according to internal protocols. Peripheral blood samples were drawn pre-ECP and after 6 months of treatment. Data on patient characteristics, medical therapies and responses were obtained from medical records. We included the following study cohorts: 1) Initial cohort of 10 GVHD patients (7 cGVHD, 3 aGVHD) and 3 controls; 2) Internal validation cohort with 21 GVHD patients (14 cGVHD, 7 aGVHD) and 10 controls; and 3) External validation cohort (Policlinico S. Matteo, Pavia) composed of 24 GVHD patients (17 cGVHD, 7 aGVHD) and 12 controls. Additionally, samples from 12 patients undergoing ECP due to lung transplantation were also included. Plasma miRNAs were purified with NucleoSpin miRNA Plasma (Macherey-Nagel). In the initial cohort, we analyzed 178 miRNAs, using the Plasma focus miRNAs PCR array (Exiqon). In the validation cohorts we quantified, by qRT-PCR, candidate miRNAs using miRcury LNA RT miRNA PCR (Exiqon) and specific Exiqon primers. RESULTS: In the initial cohort, 4 miRNAs (miR-22-5p, miR-34a-5p, miR-148a-3p, and miR-505-3p) showed higher expression in patients with GVHD compared to controls (p CONCLUSION: This study identifies miR-34a-5p and miR-148a-3p as potential biomarkers to predict responses to ECP in patients with GVHD. The combinatorial function of these miRNAs can provide a more informative outlook on the pathophysiology of the disease by identifying potential target genes being inhibited and the pathways involved, both in the development of GVHD and following responses to ECP. Overall, this information would enable the implementation of more personalized patient treatment strategies, and likely lead to significant advances in the management of GVHD patients. Disclosures Lozano: Amgen: Consultancy; Terumo S.A.: Consultancy; Grifols S.A.: Consultancy; Novartis: Consultancy; Macopharma: Consultancy.

Critical limb ischemia (CLI) is burdened by a 40% major amputation rate, and a 5-year life expectancy

The aim of the present work was the development of heart patches based on gelatin (G) and chondroitin sulfate (CS) to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD). Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL) as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system's general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses) onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

Cell-based therapies have been employed to promote neovascularization mainly through the release of paracrine factors inhibiting apoptosis and supporting migration and proliferation of resident differentiated cells. We tested in vitro pro-angiogenic effects of apoptotic cord blood-derived mononuclear cells (CB-MNCs) and their conditioned medium (CM) on mature endothelial cells (HUVECs) and peripheral blood-derived endothelial progenitor cells (ECFCs). CB-MNCs were γ-irradiated to induce apoptosis and cultured for 72 h to obtain the release of CM. MNCs viability, evaluated by flow cytometry, decreased progressively after γ-irradiation reaching 41% at 72 h. γ-Irradiated MNCs (γMNCs) released increasing amounts of EGF, PDGF-AB and VEGF in their CM over time, as assessed by ELISA. γ-MNCs and their CM enhanced capillary-like network formation (in a dose-dependent and time-persistent manner), proliferation and migration of HUVECs in vitro, while they primed capillary-like network formation (dose-independent and not time-persistent) and induced migration but did not support proliferation of ECFCs. Our data support the hypothesis of paracrine mechanism as prevalent in regenerative medicine and demonstrate the efficacy of MNCs secretome in inducing neovascularization. To our knowledge, this is the first paper highlighting differential pro-angiogenic effects of CM on mature and progenitor endothelial cells, adding a tile in the understanding of mechanisms involved in neovascularization.

Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34+ cells together with efficient T and B cell depletion are required to minimize the risks of graft versus host disease (GVHD) and Epstein-Barr virus (EBV)-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, we compared 73 ICS performed following the manufacturer's recommended platelet depletion versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34+ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34+ cell recovery by LKF platelet contamination.

Introduction Ischemic cardiovascular diseases are a leading cause of mortality in the industrialized world. Cell-based therapies have been tested to promote revascularization, mainly through the release and actions of paracrine factors that may prevent apoptosis and support migration and proliferation of resident endothelial cells (EC) and circulating endothelial progenitors (EPC). The mononuclear cell fraction of umbilical cord blood (CB-MNC) is rich in pluripotent progenitors and represents an attractive cell source for therapeutic angiogenesis. In order to demonstrate the paracrine action of CB-MNC we tested the angiogenic potential of conditioned medium (CM) derived from apoptotic CB-MNC evaluating the effects exerted over mature EC and peripheral blood (PB)-derived EPC in terms of proliferation, migration and capillary-network formation in vitro. Methods Human Umbilical Vein Endothelial Cells (HUVEC, Lonza) were cultured in Endothelial Basal Medium (EBM2) supplemented with SingleQuotes (EGM2). EPCs were obtained from PB as “Endothelial Colony Forming Cells” (ECFC). Both HUVEC and ECFC were used for subsequent experiments from passage 2 to passage 6. MNC were isolated from CB by density gradient centrifugation, gamma-irradiated (70Gy) to induce apoptosis and incubated in EBM2 for 72h to obtain the release of CM. CM was recovered from culture, centrifuged (4000 rpm 10 min) to remove cell debris and stored at -80°C. MNC viability after gamma-irradiation was evaluated by flow cytometry (7-AAd staining). RayBio Human Angiogenesis Array and ELISA assays (to quantify PDGF-AB, VEGF165, EGF, IGF-I) were used to assess CM soluble factors content. The angiogenic potential of soluble factors released by gamma-irradiated MNC was assessed by Matrigel capillary-like network formation assay using the Transwell Permeable Supports and confirmed using the sole CM in the Matrigel assay. CM capacity to support HUVEC and ECFC viability was assessed by the MTT assay and the capacity to induce HUVEC and ECFC migration by the Chemicon Cell Migration Assay. EBM2 was used as control medium. All experiments were repeated at least 4 times. Results Gamma-irradiation induced a significant time-dependent reduction in MNC viability (p CM contained various cytokines and growth factors involved in the regulation of angiogenesis, wound healing, extracellular matrix remodeling and inflammation as showed by Angiogenesis Array. ELISA assays showed that irradiated MNC released an increasing amount of PDGF-AB, VEGF165 and EGF over time, while IGF-I was not detected in the CM. Soluble factors released from irradiated MNC in the Transwell system showed a significant pro-angiogenic effect over HUVEC (p Results obtained with the Matrigel Transwell system using irradiated MNC were confirmed employing CM from irradiated MNC cultured 72 hours: CM induced capillary-like network formation in both HUVEC (p CM compared to control medium significantly increased HUVEC viability (p Conclusion Our experiments show that soluble factors released by lethally irradiated CB-MNC have an important angiogenic effect, inducing HUVEC but not ECFC proliferation, HUVEC and ECFC migration and capillary network formation in vitro. These results outline an interesting difference between mature and progenitor EC in their response to CM, which may reflect the different physiological roles that mature and progenitor EC exert during angiogenesis. This study confirms the paracrine effect of factors released from stem cells contained in the MNC fraction, which could be exploited in regenerative medicine to induce therapeutic revascularization using CB, an easy available stem cells source. Disclosures: No relevant conflicts of interest to declare.

Recent evidence suggests that leukemia is not solely a cancer autonomous process, but rather a disease in which the bone marrow microenvironment, the niche, plays a crucial role too (Raaijmakers, 2011). MSCs are key component of the niche. Thus, several studies have tested whether these cells from haematological patients contain chromosomal defects identical or different from those present in leukemic cells. Based on these findings the principal aim of the present study was to evaluate whether leukemic and MSC from six AML patients shared the same cytogenetic defects after examination with three different technologies, conventional cytogenetics (CC), FISH and aCGH/SNPa. At the onset of the disease and after informed consent all the six patients were submitted to bone marrow (BM) aspiration. BM cells were submitted to CC and FISH analyses. In addition, MSC were isolated from BM cell suspension (10-15 ml) as previously described. Briefly, mononucleated cells were isolated from BM by density gradient centrifugation using Lympholyte®-H and seeded in 75 cm2 cell culture flasks at a cell density of 106 cells/cm2. Cells were cultured at 37°C, 5% CO2 in MEM-alpha medium containing 1% Penicillin/Streptomycin, 1% L-Glutamine and 10% fetal bovine serum. After 48-h adhesion, non-adherent cells were removed and culture medium replaced (Achille et al, 2011). Growth medium was changed every three days. MSCs were examined after the first passage and their phenotype was evaluated by flow cytometry. Cells were detached from culture using Tripsin-EDTA, washed twice with PBS and stained for ten minutes with the following fluorochrome-conjugated antibodies: anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD73-PE, anti-CD34-FITC, anti-CD80-PE, anti-CD133-APC, anti-CD31-PE and anti-CD45-APC-Alexa750. Stained cells were acquired with a Beckman Coulter Navios instrument and data analyzed with Kalooza software. The commercial FISH probes used were LSI D7S486/CEP7, LSI AMLETO from Abbot Molecular Inc. (Chicago, Il, USA) and ON c-Myc/SE8, SE10(D10Z1) from Kreatech (Amsterdam, NL). These probes were applied according to manufactures guidelines and cut-off values determined by applying a one-sided 95% confidence interval using a binomial distribution. aCGH/SNPa was carried out with the SureScan Microarray Scanner G4900DA (Agilent Technologies Inc. Santa Clara, CA). CC revealed a monosomy 7 in two patients, a del(7)(q31) in one, a trisomy 8 and a trisomy 10 in one patient each, a t(8;21)(q24,q22) translocation in the last patient. All these defects were confirmed by FISH. In order to establish whether leukemic cells and MSCs shared these same abnormalities, MSCs cultures were tested with FISH. MSC purity assessed by flow-cytometry was 50-87%. FISH revealed a normal pattern in all the cultures examined. In contrast, aCGH/SNPa revealed neither gains/losses nor LOH in four patients, a trisomy 5 in one and the LOH of a 3.8 Mb sized region located on 13q31.1 in one patient. This study, the first one that applied aCGH/SNPa to investigate the MSC chromosomal pattern, suggests that i) MSCs from chromosomally abnormal AML patients may show a normal FISH pattern, but may be either normal or contain chromosomal aberrations different from those present in leukemic cells on aCGH/SNPa analysis; ii) these defects are uncommonly seen in AML; iii) MSCs defects may flag that the leukemogenic event targets not only the hematopoietic tissue but also the stromal cell compartment, i.e. the niche; iii) aCGH/SNPa provides an in-depth view of MSC chromosomal pattern allowing the identification of potential clonal markers. Disclosures: No relevant conflicts of interest to declare.

Abstract 2141 Poster Board II-118 Background: CD133+ (Prominin-1 positive) is a 5-transmembrane glycoprotein that identifies immature progenitor stem cells. Immature hematopoietic stem cells retain the possibility to give origin to tissues different from hematopoietic cell lines (transdifferentiation). Material and methods: In this preliminary study we investigated the possibility to mobilize, collect, immunoselect and reinfuse autologous CD133+ immature stem cells in liver cancer patients. This approach was adopted to obtain, in a short time, an adequate volume increase of the disease free liver thus extending the resectability criteria of the liver, with the final goal to prolong survival. We enrolled 4 patients with a large liver cancer and no chance of resection. The mobilization protocol consisted in: G-CSF administration (10 μg/kg/day ) for 3-5 days, peripheral blood stem cell (PBSC) monitoring starting from the 3rd day, leukapheresis (LKF) collection processing 3 blood volumes when CD133+ cells>15 μL. Patients were monitored during mobilization, collection and post collection phase for clinical status, blood pressure and bleeding. Positive CD133+ immunoselection (Miltenyi Clinimacs) was performed on LKF product after overnight storage. Quality controls on positive fraction consisted in viability and purity of CD133+ cells by cytofluorimetric analysis and clonogenic assays. Microbial tests were performed on the negative fraction. After LKF, patients underwent right portal embolization and infusion of CD133+ cells into the opposite portal vein by a percutaneous access. Evaluation of liver regeneration was performed 30 days after stem cell infusion by spiral CT and galactose clearance. Liver resection was carried out if liver regeneration reached 30-40%. Results: Stem cell mobilization, LKF content and immunoselected cells are detailed in tab 1. No relevant side effects were observed. We obtained an efficient stem cell mobilization in all patients enrolled. No bacterial or fungal contamination was observed in cells infused. Results about liver regeneration and patients' follow up are detailed in table 2. Conclusions: Our approach to liver regeneration was feasible and safe with no relevant side effects. We observed an efficient stem cell mobilization comparable to healthy donors also in liver cancer patients. The infusion of CD133+ cells allowed a significant hepatic tissue regeneration in all patients. Controlled clinical trials are needed to confirm our preliminary results. Disclosures: No relevant conflicts of interest to declare.

The presence of mesenchymal progenitors in full-term umbilical cord blood (UCB) has been object of discussion in recent years, as attempts to obtain these cells have either failed or yielded low frequency of mesenchymal stromal cells (MSCs). MSCs have been so far mainly expanded in vitro in the presence of fetal calf serum (FCS), which potentially carries the risk of both transmitting zoonoses and causing immune reactions against animal proteins. For these reasons, alternative culture supplements, devoid of animal components, such as platelet lysate (PL), have been tested, allowing efficient MSC isolation and expansion from bone marrow (BM). In this study, we tested the ability of PL-additioned medium to isolate and expand ex vivo MSCs from full-term UCB (UCB-MSCs) and we characterized these cells in terms of clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential and biosafety profile, comparing these characteristics with those of PL-expanded BM-MSCs. Moreover, we focused our attention on immunoregulatory properties of UCB-MSCs on alloantigen-specific immune responses and on the mechanisms by which these cells exert their effect. Ten UCB units (median volume 45 ml, range 40–60), from full-term deliveries, were selected according to the following criteria: total nucleated cell (TNC) count ranging from 500 to 750 ×106; isolation performed within 24 hours after delivery; overall cell viability > 75%, investigated by 7-amino-actinomycin D (7-AAD) and Aldeflour (ALDH). Two of the 10 UCB units (20%, UCB3 and UCB6) gave rise to MSC-like clones, which were expanded ex vivo and characterized. UCB-MSCs displayed the typical morphology, immunephenotype and differentiation capacity into osteoblasts and adypocytes reported in the literature. Although displaying a rather low clonogenic efficiency, UCB-MSCs showed to have a higher proliferative potential compared to BM-MSCs, as demonstrated by the calculated cumulative cell counts from P0 to P5. Thereafter, UCB3- and UCB6-MSCs displayed a progressive decrease in proliferative capacity, until they reached senescence after 83 (P10) and 90 (P11) days of culture. The cells progressively died during the senescence period, without showing any alteration in morphology or proliferative rate. The lack of spontaneous transformation into tumor cells was demonstrated by both the absence of telomerase activity and hTERT transcripts and by molecular karyotyping through array-Comparative Genomic Hybridization (array-CGH) assay. The immune-regulatory effect of UCB-MSCs on alloantigen-specific immune response in mixed lymphocyte culture (MLC) was investigated, together with some of the mechanisms potentially responsible for this effect, including PGE2 production and IDO activity. We found that, similarly to BM-MSCs, UCB-MSCs expanded in PL are able to: strongly inhibit alloantigen-induced lymphocyte subset (CD3+, CD4+, CD8, CD3negCD56+ NK lymphocytes) proliferation; decrease alloantigen-induced cytotoxic activity; increase secretion of IL-6 and IL-10 in MLC supernatant. While the addition of BM-MSCs to MLC increased the percentage of CD4+CD25+FoxP3+T cells, the addition of UCB-MSCs did not result in any increase of this cell subset. Moreover, we found that the immune modulation of UCB-MSCs is apparently due to PGE2 production, while the addition of IDO-specific inhibitor was not able to reverse the suppressive effect exerted by MSCs. Altogether, these data indicate that relevant differences exist between UCB- and BM-MSCs, ex vivo cultured in the presence of PL, in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties. These aspects may be relevant for the clinical application of UCB-MSCs.

Oral Mucositis (OM) frequently occurs in patients (pts) who develop acute or chronic Graft versus Host Disease (GvHD) after an allogeneic stem cell transplant. Severity and extension of the disease vary, ranging from mild atrophy to severe ulceration. The consequences of OM often include pain, inadequate nutrition and potentially life-threatening infections. Conventional treatments (topical anaesthetics, oral hygiene, protective coative agents, analgesics) often fail in curing OM. Recently, platelet derived growth factors (PDGF), in liquid or gel form have shown efficacy in repairing different tissues (bone, cartilage, skin) damaged by trauma, vascular or metabolic dysfunction. Basing on these evidences, we decided to test PDGFs combined with a mucoadhesive, biocompatible vehicle for : safety and tolerability when applied on oral mucosa, efficacy in controlling the damaged mucosa of GvHD patients. Patients and Methods 6 male pts were enrolled in the study. OM was assessed according to the WHO scale. Platelet lysate derived either from allogeneic platelet apheresis or autologous peripheral blood was added to a mucoadhesive biocompatible vehicle under sterile conditions and stored at 4°C for a maximum of 15 days. A sample for microbial and fungal detection was taken from each preparation. Platelet Lysate in mucoadhesive Vehicle (PLV) was applied on the oral mucosa 3 times/day for a maximum of 30 days. At the time of application there was no evidence of local infection. Use of analgesics, local infection and percentage of body weight increase (as index of trophism) were evaluated every 2 days. Pts characteristics after 30 days of treatment are shown in table 1. The response to treatment was defined as following: no response: no improvement or worsening of OM. 25% response: reduction of extension in one oral lesion, slight reduction of pain, unchanged dosage of analgesic. 50% response: reduction of extension in two or more lesions, important improvement of pain, nutrition with solid food, reduction of analgesic. 100% response: disappearance of oral lesions and pain. No use of analgesics, normal nutrition. Results: Results are shown in table 2. No evidence of bacterial or fungal contamination in PLV emerged. No local infections during application were observed. Conclusions Our preliminary experience demonstrate that oral application of PLV is safe, well tolerated and may be effective in treating OM (grade II–IV) in GvHD patients. Confirmation of our data in a larger randomized study could open a simple and economic possibility to cure lesions otherwise very difficult to treat. Table 1. Characteristics of pts treated with PLV. Pts n. Age GvHD Mucositis (grade) Plt lysate 1 13 Acute (grade III) IV allogeneic 2 51 chronic extensive III autologous 3 34 Chronic extensive III allogeneic 4 33 Chronic extensive III autologous 5 17 Chronic extensive III allogeneic 6 45 Chronic extensive II autologous Table 2. Results after 30 days of treatment of OM with PLV. Pts n. Weight (% increase) Response Use of analgesics Oral infection 1 0 a unchanged no 2 10 d no no 3 7 c reduction no 4 2 b unchanged no 5 10 d no no 6 3 b unchanged no

Introduction. Platelets contain a mixture of growth factors (GF). These can be released from platelet lysate (PL) whose application to wounds is supposed to favour the healing process. The application of PL in a suitable formulation can improve therapeutic efficacy and patient compliance. A formulation of PL with a selected biocompatible vehicle has been developed for application in buccal mucosal damages (mucositis), that are the most common complications of the nonsurgical therapy of cancer. The same vehicle can be proposed also for ophthalmic formulations as several studies have shown that topical application of the growth factors to the injured cornea facilitates wound repair by rapid regrowth of the epithelial cells. Aim of the present work was to develop in vitro tests for a fast evaluation of PL activity in the formulation to evidence excipient compatibility, and the effect of formulative parameters on PL stability. The proliferative effect on two different models, fibroblasts and a corneal epithelial cell line, was evaluated. The stability of PL in the formulation has been assessed after two weeks at 4–8 °C storage. Methods. PL was mixed in a 1:1 ratio to the selected vehicle and stored at 4–8 °C. Immediately after preparation (time zero, T0) and after 2, 7, 10 and 15 days (T2–T15), aliquots were used to assess proliferative effect on cell cultures. Both fibroblast (primary human cell lines) and RCE (Rabbit corneal epithelial cell lines, ECACC) were seeded in 96-well plates with area of 0.34 cm2 at the concentrations of either 10000 or 5000 cells/well. After 24 hours the cells were added with either complete growth medium (Reference), minimal medium not supplemented with foetal calf serum and for REC cells without also EGF (Control), PL formulation diluted 1/20 (containing PL at 5%) and PL formulation diluted 1/40 (containing PL at 2.5%). After 24 hours, neutral red (NR) test was performed (Tox Kit 4, Sigma-Aldrich, Milano I). The NR solution absorbance was determined by means of a plate reader (Perkin Elmer, Milan, I) at wavelength of 490 nm. The absorbance read for each sample was compared with that of Reference, whose proliferation was assumed as 100%. Results. At time zero, the percentage of proliferation showed no significant differences with respect to the cells in complete growth medium (Reference) in the case of fibroblasts, both at 5000 and 10000 seeding level. The two cell models gave comparable results although slightly higher values of proliferation were obtained in fibroblasts. This can be attributed to the epithelial nature of the RCE and to the possible different percentage in the platelet derived mixture of FGF and EGF. In both cases it can be argued that the vehicle is compatible with cell growth. Both for fibroblasts and for RCE no decrease of activity with time was observed, and no statistical differences with respect to the control have been obtained until 15 days storage. (See figure 1). The results obtained suggest that the two cell culture models evaluated can be suitable to assess the activity of PL in easy and fast way. This will be useful to develop formulations intended for the treatment of mucositis and of damages of corneal epithelia. In particular, the formulation tested seems to be stable and to allow the release of GF from PL until 15 days of storage. Figure 1. Proliferation (5000 cells/well) induced by the PL formulation as a function of storage time. The dotted line indicates the proliferation of the Reference (assumed as 100%). Figure 1. Proliferation (5000 cells/well) induced by the PL formulation as a function of storage time. The dotted line indicates the proliferation of the Reference (assumed as 100%).

Introduction Dimethilsulfoxyde (DMSO) is a cryoprotectant routinely used for stem cell cryopreservation. At reinfusion, DMSO is toxic for the recipient and the risk of adverse effects (chills, flushing, nausea, vomiting, abdominal pain, chest tightness, blood pressure changes) are proportional to the DMSO amount. DMSO may also induce lifethreatening complications (cardiac and pulmonary arrest, acute renal failure) in low body weight patients or in subjects with a pre-existing cardiac disease. DMSO related complications are emphasized in “bad mobilizer” patients (CD34+/mL Herein we present our experience in washing out DMSO from thawed PBSC with an alternative technique. Material and Methods 48 PBSC bags, with a mean volume of 110 ml (range: 107–112) containing 10% DMSO were thawed for autotransplant in 8 patients affected with NHL-B (3), HL (3) and AML (2). 2 patients suffered from cardiac arrhythmia and 6 patients weighted less than 40 kgs (range 31–39.5). Each bag was thawed in a water bath at 37°C and immediately after was made a sterile connection with a bag containing a solution of the same volume (saline and 20% ACD-A) previously refrigerated at 4°C to minimize the DMSO cell damage. Afterward, the diluted PBSC bag was centrifuged at 1200g× 5mins at 4°C and the excess supernatant was removed using a plasma extractor. Finally, the cells were resuspended in an equal volume of refrigerated saline solution and human serum albumin (2%) and promptly reinfused to the patient. Pre-freezing, post thawing and post washing samples were collected to determine TNC, MNC, CD34+ cell content, viability and microbial contamination. Results Pre-freezing, post thawing and post washing TNC, MNC, CD34+ cell content, viability and pre-freezing versus post washing cell recovery are shown in table 1. Data are expressed as mean and range (min-max). No microbial contamination was detected in all samples analyzed. No infusion related toxicity was recorded. No clotting was observed. No delay in WBC and PLT engrafment was documented. Conclusions Our DMSO wash out technique is feasible, simple and safe and permits a satisfying recovery of MNC and CD34+ cells, preserving cell viability. PBSC manipulation at 4°C employing refrigerated solutions during every step mantains the quality of the thawed graft, minimizing DMSO stem cell damage with no delay in engraftment time. Finally, our method may be considered expecially when DMSO infusion could be lifetreathening in patients at high risk for a pre-existing cardiac, pulmonary or renal disease or in low body weight patients. Table 1. Pre-freezing Post-thawing Post-washing Recovery (%) TNC (109) 46.2 (41.2–53.1) 40.4 (37.6–46.3) 31.7 (26.2–35.8) 67.3 (64–71.2) MNC (109) 24.5 (20.3–28.6) 22.4 (17.1–26.8) 20.3 (14.7–26.4) 83.3 (81–85.2) CD34+ (106) 35.7 (29–38.6) 31.3 (25–35.4) 27.2 (21.1–30.5) 77.1 (73.1–80) Viability (%) 98.9 (97.5–99.9) 87 (86.2–89.4) 79 (76.7–81.9)

There is large interest in the use of mesenchymal stromal cells (MSCs) in approaches of cell-therapy and tissue engineering. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns in the case of clinical grade cellular preparations because of the theoretical risk of transmission of zoonoses and triggering immune reactions in the host. Therefore, the identification of a serum-free medium appropriate for both the extensive expansion necessary to reach the large numbers of MSCs required for clinical application, and the exclusion of risks connected with the use of animal products, is warranted. Aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL) are endowed with biological properties appropriate for cell-therapy approaches. MSCs were generated from bone-marrow of 8 healthy hematopoietic stem cell donor; 4 different culture conditions were tested: 10 % FCS; 5% PL; 2,5% PL; 1% PL. MSCs were harvested when reaching ≥ 80% confluence and replated for expansion at 4.000 cells/cm² until passage 5. CFU-F frequency, proliferative capacity, morphology, surface phenotype and differentiation capacity were evaluated. In particular, the immune regulatory effect on alloantigen-specific immune response, the kinetics of cytokine production and the resistance to spontaneous transformation into tumor cells of MSC expanded in the presence of either PL or FCS were investigated. Our results demonstrate that MSCs expanded in either FCS or PL display comparable morphology, phenotype and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. Immune-regulatory effect of MSCs was investigated on alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: decrease alloantigen-induced cytotoxic activity; favor differentiation of CD4+ T-cell subsets expressing Treg phenotype; increase early secretion of IL-10 in MLC supernatant, as well as to induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and in reducing early IFNg-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by both molecular karyotyping (array-comparative genomic hybridization) and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the hypothesis of a remarkable immune functional plasticity of human MSCs and suggest that the use of MSCs-PL, which seem to be endowed with a relatively low immune suppressive activity, could be more appropriate in approaches of reparative/regenerative cell-therapy or in strategies aimed at improving hematopoietic/immune recovery after hematopoietic stem cell transplantation (HSCT). On the contrary, as MSCs-FCS seem to display a more pronounced immune suppressive function, they might be more suitable for preventing or treating alloreactive-related immune complications, such as severe Graft-versus-host disease (GvHD) in HSCT and graft rejection in HSCT and solid organ transplantation.

Besides large-scale banking of unrelated units, CB banks (CBBs) collect and store dedicated cord blood (CB) for sibling patients. In this setting the recipient is already designated, therefore special care is applied to all critical steps of CB management, from collection to infusion, to ensure the highest quality of the graft and the successful outcome of transplant. CB collection procedures must also protect mother and infant donors. According to FACT Netcord standards, delivery practice cannot be modified in attempt to increase CB volume. In case of in utero collection, additional safeguards are requested and in multiple gestation pregnancies, all infants shall be delivered before any CB collection begins. The mother must sign a written informed consent and CB can be drawn from infants after at least 34 week gestation. Moreover, the CBB shall maintain all details of clinical outcome for related allotramsplants. In particular, data on adverse events associated with the infusion of CB shall be carefully recorded. We report our experience of banking CB for sibling recipients to be transplanted at the onchohematologic-paediatric division of our hospital. From 1996 we have collected and banked 104 dedicated CBs (64 for hematological and 40 for inherited disorders). Collections were carried out mostly in the obstetric ward of our hospital (77%); 40 (39%) and 64 (61%) units by vaginal and caesarean delivery, respectively. Eligibility to caesarean delivery was strictly dictated by obstetric factors and never due to CB collection requirements. Mean maternal age was 32 years (18–42). Infant donors were 61 (59%) males and 43 (41%) females; mean neonatal weight was 3178 g (1270–4720) and gestation week 38 (35–41). 6/104 (5.7%) collections were obtained during twin pregnancies. No adverse reaction on both newborn and mother was reported by the obstetric or neonatology staff. The characteristics of CB donations at collection were: volume 102 ml (25–199), nucleated cells (NC) 924 ×106 (59.6–2193), CD34+ cells 269 ×104 (8.9–1226.4), viability 96.5 % (54.7–99.9), CFU-GM 343.1 ×104 (8.5–2112.5). Bacterial contamination occurred in 2 cases (1.9%). Prenatal HLA typing was available for only 21/104 (20%) donors and most of the units were typed after collection. 39/104 units (37%) matched their recipient and 28/104 (27%) were successfully transplanted. The characteristics of CBs at infusion were: collected NC dose 6.3 ×107 (1.8–15.6), infused NC dose 4.6 ×107 (1.4–14) and collected CD34+ cells dose 1.9 ×106 (0.3–10.5), all shown per kg recipient weight. The outcome data were available for 24/28 (86%) CBs, in particular no adverse event associated to CB infusion or DMSO toxicity was reported. Preserving the uniqueness of the graft for sibling transplantation imposes a rigorous organizational network to prevent any technical or organisational setback. In our hands, this strategy was effective in ensuring the best quality and safety of the stem cell product guaranteeing at the meantime the protection of mother and infant donors.

Introduction: Primitive hematopoietic progenitor cells may be characterized by the detection of the intracellular enzymatic activity of aldehyde dehydrogenase (ALDH ). The use of a fluorescent substrate for ALDH (Aldefluor) permits the cytofluorimetric study of different stem cell populations in the functional way rather then as markers of cell surface. Aim of this study was to evaluate the ALDH activity of the stem cells in pre-freezing (fresh) leukapheresis (LKF) colllections and in cord blood (CB) units, to verify the feasibility of detecting the ALDH activity in the post-thawing units and finally to compare the results between the fresh and post thawed stem cells to be transplanted. Materials and methods: Samples containing 2 x106 total nucleated cells obtained from 5 LKF and 5 CB units immediately after collection and from the same units after thawing, were incubated with Aldeflour (StemCo Biomedical, Durham, NC, USA) at 37°C for 45 min. and successively for 15 min. with anti CD34 PE, (Beckman Coulter, Fullerton, CA, USA) and anti CD45 PerCP (BD Biosciences, San Jose, CA, USA). Viability of CD34+ cells was evaluated using 7AAD, (Beckman Coulter, Fullerton, CA, USA). The setting of thawed samples was carried out immediately after thawing, taking care to make the sample dilution very quickly, in order to avoid the detrimental action on the cells exerted by DMSO at 37°C and at room temperature. All samples were analized using a BD Biosciences FACSCalibur flow cytometer device (San Jose, CA, USA). Results: The results of the ALDH and CD34+ cell analysis and stem cell viability on fresh and thawed samples are detailed in table 1. Conclusions: Our results demonstrate the feasibility of ALDH determination even in post thawed samples on condition that the test is conducted with accuracy with particular attention at the dilution step that must be completed in a very short time. The ALDH activity tends to decline in thawed stem cells demonstrating the detrimental action on stem cell functionality of the entire cryopreservation and thawing processes. In particular the contact of the stem cell with cryoprotectant mixture (DMSO) at room temperature has a negative, time dependent, impact on stem cell quality, confirming that the reinfusion phase of the graft must be carried out as soon as possible. The introduction of a functional test to evaluate the graft quality may be helpful in transplant clinical practice. Table 1: Results of ALDH assayes on pre-freezing and post-thawing leukapheresis and cord blood collections. CD34+ (%) ALDH+ (%) CD34+/ALDH+ (%) ALDH+/CD34+ (%) Viability (%) Fresh LKF n=5 0.56 0.12 64.6 97.8 99.2 Fresh CB n=5 0.28 0.23 76.7 99.8 98.1 Thawed LKF n=5 0.48 0.09 65.5 94.4 81.2 Thawed CB n=5 0.24 0.19 71.5 96.8 77.3

Introduction: At moment PBSC collections can be performed using semiautomated or automated cell separator devices. The collection with semiautomated methods implies an augmented working load for the dedicated personnel and is strongly influenced by the operator. On the contrary, the automated methods offer the advantages of a diminuished working load for the dedicated personnel and an high standardization of the collection procedure. Herein we report our experience on 60 PBSC collections employing the new automated COM.TEC Fresenius autoMNC program that provides the possibility to predict the total number of CD34+ cells collected basing on the CD34+ cell count (x μL) pre-leukapheresis (LKF) collection in peripheral blood. Materials and Methods: 39 patients affected with various onchohematological diseases and10 healty donors were mobilized with chemotherapy + G-CSF or G-CSF alone, respectively, and subsequently underwent LKF collection for auto or allotransplant. According to our internal protocol 60 LKF collections were performed starting with a CD34+ cell count in peripheral blood at least of 20/μL. Net weight of the final LKF product and its CD34+ cell content were evaluated at the end of each PBSC collection procedure and then compared to the expected data calculated by the cell separator device. Moreover a post collection peripheral blood Plt count was evaluated for each patient/donor. Results: The mean starting WBC count was 25.86x103/μL (range: 4–82.3), Plt count was 151.38x103/μL (20–395), CD34+ cells was 96.63/μL (20–332). The mean WBC and CD34+ cells in the LKF collection were 224.78x103/μL (20.71–425.3) and 565.45x106 (59.3–1609.3), respectively. The mean volume of the LKF collection was 237.28 ml (120–503). The mean estimated CD34+ cell content was 498.37x106 while the real mean CD34+ LKF cell content was 623.32x106. The mean CD34+ cell collection efficiency was 91% (66–126). Finally, the mean post procedure Plt count in patient/donor was 77.91x103/μL (12–164). Conclusions: The automatized PBSC collection with the new program COM.TEC Fresenius autoMNC demonstrated a very high CD34+ cell collection efficiency. Moreover the possibility to predict the CD34+ cell yield permits an optimal management of the LKF collection, reducing the number of procedures per patient/donor. The difference observed between the mean estimated CD34+ cells and the real CD34+ cell content may be due to the intra-procedure stem cell mobilization phenomenon. Finally, this new automatized collection system demonstrated to limit the collection related thrombocytopenia either in patient or in donor.

Introduction: Platelet concentrates (PC) collected by apheresis are effective in supporting deeply thrombocytopenic patients. The reduced risk of multiple allogeneic exposure and transmissible infectious diseases together with the high WBC depletion and diminished transfusion reactions are the main advantages offered by PC transfusion. At moment, the availability of several synthetic solutions for platelets storage permits to prepare hyperconcentrate(dry) apheresis platelets with the advantage of reducing febrile non-hemolytic transfusion reactions and, in low body weight patients the citrate toxicity, without the necessity of further manipulations. The aim of this study was to test the quality of 20 dry platelets (DP) in comparison to 20 standard plateletpheresis (SP) concentrates. Materials and methods: A total of 40 apheresis procedures were performed by the single-needle Cobe Trima separation device (Gambro BCT, Lakewood, CO, USA) collecting either DP or SP concentrates. Within 1h after collection, the bag containing DP was added with the appropriate amount (70% of DP final volume) of synthetic solution for platelets storage (SSP, MacoPharma). Both DP and SP concentrates were stored at room temperature with gentle agitation for 4 days. For both concentrates, platelet yield was calculated and in vitro studies of membrane glycoproteins expression and aggregation at day +1 and day +4 were carried out. Results: The comparison between 20 DP and 20 SP concentrates in terms of ability to aggregate in vitro and membrane glycoproteins expression at day +1 and day +4 of storage is reported in table A and B respectively. Conclusions: The in vitro tests documented a major activation of dry platelets. In particular, the ability to aggregate was reduced in the 20 DP concentrates analised and this phenomenon was more evident at day +4 of storage. The alteration of membrane glycoproteins expression (markers of storage lesion) confirms the lower in vitro quality of DP concentrates. The effectiveness of this new blood component in vivo should be evaluated in a controlled clinical trial. Table A. At collection Day +1 Day +4 SP DP SP DP SP DP Collagen μg/ml 4 93 88 97 82 80 53 ADP 10 μM 32 11 24 16 16 5 Ristocetin 1.5 mg/ml 91 97 77 81 71 52 Collagen 10 μg/ml + Adrenaline 10 μM 98 95 93 94 93 80 ADP 10 μM + Adrenaline 10 μM 87 72 76 61 57 30 Table B. At collection Day +1 Day +4 SP DP SP DP SP DP GPIb alfa (MFI) 5.06 5.75 6.31 6.13 5.26 4.54 GPIIb-IIIa (MFI) 35.47 36.71 34.61 37.7 31.76 40.9 GP IV (MFI) 11.49 11.4 11.67 10.28 11.65 11.38 GP 53 24.23 27.11 21.74 25.91 21.46 31.84 GMP 140 21.79 29.29 22.65 30.38 20.58 34.89

Introduction: Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in auto and allotransplant settings. In particular, when an haploidentical transplant is performed an high dose of purified CD34+ cells together with an efficient T and B cells depletion are requested to minimize the risks of GVHD and EBV related lymphoma. In order to ameliorate the performances of the ICS device, we compared 73 ICS performed following the customer’s recommendations vs 20 ICS performed after 3 centrifugations in order to increase the platelet depletion of the starting leukapheresis (LKF) product. Matherials and Methods: 93 ICS (from single or fractioned LKF product) were carried out using the Miltenyi Clinimacs device on 76 LKF collected from 37 healty donors mobilized with G-CSF alone (12μg/kg). In tab 1 are summarized the characteristics of the starting 93 pre-immunoselection products in terms of TNC, CD34+ cells, plt and T and B lymphocyte content. 73 LKF were prepared following the standard manufacturer’s protocol and the remaining 20 with an adjunctive plt depletion obtained by 3 low speed centrifugations (900 rpm). Results: a statistical significance in terms of CD34+ cells recovery, final TNC (x106) and CD34+ cell (x106) absolute count was found in favour of the modified immunoselection procedure (see tab.1). Conclusions: ICS demonstrated to be an highly efficient procedure when the Miltenyi Clinimacs device is employed. Moreover, the modification of the standard manufacturer’s technique simply by adjunctive centrifugations on the starting LKF product can further improve the recovery of stem cells maintaining, at the same time, a good purity and not influencing the T and B cells log of depletion, thus demonstrating the negative influence of plt contamination on ICS. These results are more remarkable in the context of haploidentical transplant where the plt contamination of LKF is particularly high. Table 1 Standard procedure(n=73) Modified procedure (n=20) LKF content Pre-ICS Post-ICS Pre-ICS Post-ICS Mann Withney test TNC(x10e6) 58150 (24150–146700) 264 (73.1–784.5) 54250 (32790–87570) 330.41 (126.84–537.9) p 0.02 CD34+(x10e6) 354.25 (115.67–941.22) 245.55 (59.39–768.81) 377.52 (149.84–604.23) 310.38 (115.87–489.49) p 0.04 PLT(x10e9/L) 3220 (555–9320) - - 3522 (744–6600) - - n.s. LymphocytesT(x10e6) 12880 (1200–36050) 0.13 (0.01–0.6) 11820 (1690–25050) 0.38 (0.08–2.34) n.s. LymphocytesB(x10e6) 3180 (70–15000) 1.06 (0.02–8.42) 2810 (1120–6100) 0.82 (0.19–2.98) n.s. CD34+(%)recovery 70.98 (9.5–115.32) 82.34 (66.37–99.94) p 0.04 Purity(%) 92 (69–99) 94 (89–98) n.s. LogT-Depletion 5.1 (3.74–7.91) 4.6 (3.4–5.2) n.s. LogB-Depletion 3.65 (2.45–6.08) 3.55 (2.58–4.44) n.s.

Extracorporeal photochemotherapy (ECP) is an effective, relatively new technique, FDA approved for cutaneous T-cell lymphoma, employed as second-third line treatment in patients affected with GVHD not or poorely responsive to standard immunosuppressive drugs. Both aGVHD and cGVHD are the major complications after stem cell transplantation (till to 50% and 80% of the patients, respectively). Corticosteroids, cyclosporine, micofenolate, tacrolimus and various experimental monoclonal antibodies (anti CD40 ligand, anti TNFα etc) are the drugs employed to control GVHD and are burdened with important short and long term side effects. Recently we revised our data about 102 pts (children and adults) treated by ECP in our Institution. The overall response was 75%, permitting to taper or suspend the immunosuppressive therapy (IST) in 67.6% of the pts. The economical and social impact (cost analysis and quality of life) of ECP vs standard IST was calculated basing on the National Price List and on Short Form Quality of Life (QoL) Scoring System. The cost of ECP comprehensive of the leukapheresis procedure, waste matherials and dedicated personnel was estimated as 684.51 Euro/procedure. On the other side, when the solely costs of the most common IST drugs (corticosteroids, cyclosporine) were considered for 5 months of treatment, an evident and obvious economical advantage emerged (120,3 E). On the contrary, when the costs of hospitalisation and day hospital regimen derived from the most common side effects related to the solely use of IST were included in our cost analysis studies, an economical advantage for long term ECP treatment (calculated on 16 procedures) was demonstrated (14952.16 E vs 17553 E). Moreover, when the “real life implication” calculated on the QoL parameters were considered, the advantages were more evident. In conclusion, the tie of respecting strictly an imposed program of budget calculated on the short period may exert an inhibitory effect in introducing new diagnostic or therapeutic procedures ignoring that the improvement or the cure of the patient has always a positive economical counterpart, expecially when the impact of a new technology is considered in a long term view.

Introduction: Nowadays UCB represents an established source of hematopoietic stem cells for unrelated transplants in children and the employ in adults is quickly growing up. Nucleated cells (NCs) content is one of the main predictors to evaluate UCB for clinical use; however, other indicators of the hematopoietic potential, such as CD34+ cell and colony-forming cells (CFUs), have recently showed similar correlations. According to Netcord-FACT standards, it is recommended to test all the above mentioned parameters before releasing UCB to the transplant center; a segmented tubing of the UCB bag should be used but a satellite cryotube is more often available. We preliminarily report the results of quality controls performed on thawed cryovials corresponding to each of 15 units delivered by our UCB Bank. Material and methods: in our policy, all UCBs are stored accompanied by 3 satellite cryovials, treated under the same conditions of the unit. For each of the 15 UCBs released for transplantation, one cryotube was thawed in a 37°C water bath with a gentle agitation, without washing out DMSO. NCs and mononucleated cells (MNCs) were estimated with an automated cell counter. Viability and CD34+ cell count were evaluated by flow cytometry, with a no-wash, single-platform technique and 7-aminoactinomycin D. CFU assay was performed using commercial reagents (Methocult GF H4434, StemCell Technologies) and colonies were classified after a 14 days incubation. The same parameters referring to fresh UCBs (before cryopreservation) were always available. Results: the UCB characteristics measured after thawing a cryovial were compared with those of the fresh cord and are detailed in the table below. fresh UCB before cryopreservation UCB cryovial after thawing Yield (%) NC (x106) 1491.3 (148–2262) 1354.2 (167.4–2119.9) 92 (83–113) MNC (x106) 662.7 (96.5–900.3) 638.8 (295–1238.7) 95 (63–159) CD 34+ cells (x106) 3.47 (0.38–10.87) 3.19 (0.4–9.27) 85 (37–118) Viability (%) 96 (88–100) 62 (39–77) Viability of CD34+ cells (%) 92 (71–99) CFU-GM (x104) 838.5 (57.2–3581.2) 424.11 (65.29–917.64) 69 (16–184) BFU-E (x104) 1709.61 (159.84–5116) 473.34 (39.69–1204.14) 41 (5–139) total CFU (x104) 2565.17 (217.04–8953) 911.68 (164.43–2075.22) 57 (9–160) Excellent yields were found for NCs, MNCs and CD34+ cells. Despite of the decrease in the overall viability, the viable CD34+ cells as percentage was always highly satisfactory. The colonies growth was found lower in the thawed sample in comparison with fresh UCB before cryopreservation. Conclusion: in our experience, highly satisfactory evaluations of UCB content could be obtained using thawed cryotubes with regard to NC, MNC and also CD34+ cell. However, concerning the latter, the different methods employed on fresh UCBs, such as CD34+ cells detection without beads, may be advocated to explain some discrepancies in the yield range. The results of CFU assay confirmed to be poorly useful, essentially because affected by a subjective interpretation even if the reduced cell growth may be also related to the presence of DMSO as inhibiting factor.

Stem cell transplantation from unrelated cord blood (CB) donors is nowadays a standard practice for treatment of both malignant and non-malignant haematological disorders. The degree of HLA disparities, the cell dose and the prompt availability of the CB unit strongly influence the donor choice. According to FACT Netcord standards, HLA A, B and DRB1 shall be determined on a pre-cryopreservation sample from each CB. Once a CB unit is identified for potential use, a sample is tested to verify HLA type and confirmation of maternal haplotype is provided. The recipient’s HLA typing and its matching to the donor are confirmed as well prior to CB release for transplant. Quality of the stem cell product represents a basic assumption for the final success of transplant and an essential requirement for CB Banks (CBB). Each CBB shall establish a program of quality assurance that includes identification and traceability. The protection of confidentiality of mother and infant donors and the maintenance of linkage of a particular CB to its mother/neonate pair is of the highest priority. For this purpose each CB is assigned a unique numeric identifier that accompanies the unit in all steps of management and by which it is related to its maternal/infant data and reference samples. At the Pavia CBB, all CB donations are routinely typed for both HLA class I and class II by molecular techniques (low resolution for A and B loci and high resolution for DRB1). The mother haplotype is also assessed by molecular methods at time of banking. Therefore each CB/mother pair is checked before inclusion in the inventory. We retrospectively evaluate the impact of such a strategy on ensuring both identification and linkage of our CB repository. We review the data referring to the CBs stored in our Bank, all typed by LR-DNA for class I loci and by LR/HR-DNA for class II loci. Our inventory consists of 1987 units available for donor selection. In 2 pairs (0.1%) the mother’s typing was found fully disparate as respect to the supposed corresponding CB/neonate. The identification cannot be confirmed leading to discard of the unit. We also review the data pertinent to the 45 units released for transplant by our CBB: in all cases the cord’s confirmatory typing was coherent to the previous typing thus confirming the identity of the product ready for shipment. In our hands, the practice of confirming maternal haplotype at time of banking provides the final evidence of correct assignment and identification before inclusion in the inventory, ensuring the highest quality of the product supplied. This strategy also optimizes the time for the search requests management contributing to prompt availability of the graft.