C. Perotti

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell‐therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet‐lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell‐therapy approaches. We confirm previously published data showing 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. We further extended these data by investigating the immune‐regulatory effect of MSCs on the alloantigen‐specific immune response in mixed lymphocyte culture (MLC). We found that MSCs‐PL are comparable to MSCs‐FCS in their capacity to: (i) decrease alloantigen‐induced cytotoxic activity; (ii) favor differentiation of CD4+ T‐cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL‐10 in MLC supernatant, as well as 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 reducing early IFNγ‐secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs‐PL can be used as an alternative to MSCs‐FCS, although these latter cells might be more suitable for preventing/treating alloreactivity‐related immune complications. J. Cell. Physiol. 211: 121–130, 2007.

A new automated cell washer device for thawed cord blood units.

BACKGROUND:  The current available techniques to wash out DMSO from thawed umbilical cord blood (UCB) units are based essentially on standard centrifugation in an open system with various degrees of cell loss.

Sponge-like dressings based on biopolymers for the delivery of platelet lysate to skin chronic wounds

The aim of the present work was the development of sponge-like dressings, obtained by freeze-drying, based on chitosan glutamate and sodium hyaluronate for platelet lysate (PL) delivery to chronic skin wounds. A first phase of the research focused on the choice of the best dressing composition to obtain formulations endowed with the desired mechanical and hydration properties. In particular glycine amount (cryoprotectant agent), and water content were considered as formulation variables. The addition of glycerophosphate, used to solubilize chitosan at pH close to neutrality, was also investigated. In the second phase of the research, dressings were loaded with different amounts of PL. The influence of freeze-drying process and of excipients on the biological activity of platelet growth factors was investigated by means of a cell proliferation test using human fibroblasts. PDGF AB (platelet derived growth factor) content was assayed by means of ELISA test. Depending on composition, dressings showed different mechanical and hydration properties that make them suitable to wounds with different exudate amounts. Both freeze-drying process and excipients employed did not disturb the activity of platelet growth factors. The dressings loaded with platelet lysate were characterized by % proliferation values on fibroblast cell comparable to those observed for the fresh hemoderivate. The PDGF AB assay confirmed the results obtained from cell proliferation test.

Autologous platelet lysate for treatment of refractory ocular GVHD

Current treatment of ocular GVHD (oGVHD), represented by systemic immunosuppressive regimens and local therapies (mainly artificial tears and corticosteroids), gives unsatisfactory results. We investigated the safety and efficacy of autologous plasma rich in PDGFs to treat oGVHD unresponsive to standard medications. A total of 23 patients with refractory oGVHD (grade II–IV) unresponsive to standard therapy were treated with autologous plasma rich in PDGFs eye drops (PRGD) four times/day for 6 months. Symptoms and signs (best visual acuity, Schirmer’s test and tear break up time (TBUT), evaluation of the anterior segment and fluorescein and lissamine staining) were always assessed by the same ophthalmologist. Patients were defined as ‘responders’ when showing improvement for total complaints and at least one sign. At 30 days of treatment, 17 patients (73.9%) were classified as responders. The symptom that improved most was photophobia (improved in 19 patients, 82.6%). TBUT improved in 20 patients (86.9%) and anterior segment score in 19 patients (82.6%). Response was maintained over time. No serious adverse events occurred. PRGD proved to be safe and effective in treating oGVHD and may be a valid treatment option from the early stages of the disease to avoid irreversible ocular damage.

Extracorporeal photopheresis as a new supportive therapy for bronchiolitis obliterans syndrome after allogeneic stem cell transplantation.

Extracorporeal photopheresis as a new supportive therapy for bronchiolitis obliterans syndrome after allogeneic stem cell transplantation

A cross-sectional study on vision-related quality of life in patients with ocular GvHD

Ocular GvHD affects about 40–60% of patients receiving bone marrow transplantation. Ocular complaints worsen quality of life (QoL), which, besides survival time, is a primary end point in a patients follow-up. The aim of our study was to assess the ocular surface status and vision-related QoL (VRQoL) and explore the potential determinants in VRQoL in patients with chronic GvHD with ocular involvement. In this cross-sectional study, we investigated 40 patients with ocular GvHD after allogeneic hematopoietic stem cell transplantation assessing ocular symptoms and signs, VRQoL and ophthalmologic parameters. The median age was 52.1 years; 32.5% were females. Most of them presented a multiple organ involvement. Ophthalmological parameter examinations were on average abnormal. Corneal staining was severe/very severe in 25%; conjunctival staining in 10% of subjects. The worse QoL scores were on ‘general vision’, ‘ocular pain’, ‘vision-specific mental health’ and ‘vision-specific role difficulties’. Both symptoms and sign scores indicate poor VRQoL. A lower VRQoL was related to schooling level, job position, underlying disease and extracorporeal photopheresis. Corneal staining, Schirmer and tear film breakup time were negatively associated to visual function-related subscales. An accurate ophthalmological and VRQoL assessment should be mandatory for a long time to promptly recognize early signs of ocular suffering, and to prevent irreversible ocular complications.

Quality control on mononuclear cells collected for extracorporeal photochemotherapy: comparison between two UV-A irradiation devices.

Recently, MacoPharma released a new UV‐A cell irradiator device (Macogenic G2) for extracorporeal photopheresis (ECP), smaller and lighter than the Macogenic G1 but with no integrated cooling system. We compared the two devices at different working temperatures (G1 at standard irradiation temperature – 21°C – and G2 set by purpose at 34°C) in patients affected with chronic graft‐versus‐host disease and chronic lung allograft dysfunction treated by ECP. We demonstrate that both G1 and G2 devices are efficient in inducing the inhibition of lymphocytic proliferation and mononuclear cells apoptosis after 48 h even when G2 is set at higher‐than‐standard temperature.

Luminex® xMAP® technology is an effective strategy for high-definition human leukocyte antigen typing of cord blood units prior to listing

Introduction: Allele-level donor–recipient match at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci impacts the outcome after cord blood transplantation for hematologic malignancies and modifies the strategy of donor selection. High definition of both class I and II HLA loci at time of listing is a way to improve the attractiveness of cord blood bank inventories, reducing the time for donor search and procurement and simplifying donor choice, in particular, for patients of non-European heritage. Methods: In 2014, Luminex® xMAP® technology was introduced in our laboratory practice and was applied to cord blood units typing. In this study, we evaluated the impact of this strategy in comparison with the platform in use until 2013, relying on LiPA reverse polymerase chain reaction–sequence-specific oligonucleotide (revPCR-SSO) plus polymerase chain reaction–sequence-specific primer (PCR-SSP). Results: In 2014, the time for testing was shorter (141 vs 181 days on average), the number of test repetitions was lower (in particular for HLA-A locus, p = 0.026), and the cost reduced (240.7 vs 395.6 euros per unit on average) compared to 2013, demonstrating that Luminex xMAP technology is superior to the previous approach. Conclusion: Luminex xMAP platform has useful application in cord blood banking programs, to achieve high-definition HLA typing of cord blood units at the time of banking in a quick, accurate, and cost-effective manner.

HLA-A, -B, -DRB1 allele and haplotype frequencies of 674 cord blood donors from North Italy

http://dx.doi.org/10.1016/j.humimm.2017.04.009 0198-8859/ 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. ⇑ Corresponding author at: IRCCS Policlinico San Matteo Foundation, viale Golgi 19, 27100 Pavia, Italy. E-mail address: cristina.capittini@libero.it (C. Capittini). C. Capittini a,⇑, A. De Silvestri , M. Guarene , A. Pasi , C. Tinelli , C. Perotti b