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Background Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. Materials and methods The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. Results MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. Conclusions MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Background PBSC collection using a blood cell separator in very low weight patients can be frequently complicated by severe adverse effects and technical difficulties. Material and methods From March 2013 to January 2017, 14 PBSC collections were performed in 12 children weighing less than 10 kg, affected by different solid tumours. PBSC collection was performed with a "homemade" aseptically assembled circuit. The circuit is composed by a 150 mL collection bag connected with a 4 stopcock ramp, perfused with ACD. This circuit allows collection of a specific total blood amount from CVC, depending on CD34+ /kg target. Results Mean CD34+ cell performance per collection was 9.3 × 106 /kg. Tolerance to the procedure was very good as none of the patients experienced complications, with the exception of a patient who showed mild cyanosis and pallor after collection. Moreover, no bleeding or thrombotic complications have been observed. To date, 16 PBSC reinfusions have been performed in 7 children with a mean CD34+ cells viability of 98.1% ± 2.7 and mean WBC viability of 57% ± 10. Cell recovery after thawing was 87% ± 10.8. A rapid graft intake for both neutrophils and platelets, between day 7 and 20 after reinfusion was observed. Discussion The procedure of total blood collection without the use of a cell separator is feasible and allows a good PBSC collection without significant side effects in very low-weight children. Moreover, this method could represent a valid and safe alternative to leukapheresis in patients where classic procedure could be difficult to apply.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Background The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. Methods As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. Conclusions PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production. Electronic supplementary material The online version of this article (10.1186/s13287-018-0863-8) contains supplementary material, which is available to authorized users.

Expression of the solute carrier (SLC) transporter SLC22A3 gene is associated with overall survival of pancreatic cancer patients. This study tested whether genetic variability in SLC22A3 associates with pancreatic cancer risk and prognosis. Twenty four single nucleotide polymorphisms (SNPs) tagging the SLC22A3 gene sequence and regulatory elements were selected for analysis. Of these, 22 were successfully evaluated in the discovery phase while six significant or suggestive variants entered the validation phase, comprising a total study number of 1,518 cases and 3,908 controls. In the discovery phase, rs2504938, rs9364554, and rs2457571 SNPs were significantly associated with pancreatic cancer risk. Moreover, rs7758229 associated with the presence of distant metastases, while rs512077 and rs2504956 correlated with overall survival of patients. Although replicated, the association for rs9364554 did not pass multiple testing corrections in the validation phase. Contrary to the discovery stage, rs2504938 associated with survival in the validation cohort, which was more pronounced in stage IV patients. In conclusion, common variation in the SLC22A3 gene is unlikely to significantly contribute to pancreatic cancer risk. The rs2504938 SNP in SLC22A3 significantly associates with an unfavorable prognosis of pancreatic cancer patients. Further investigation of this SNP effect on the molecular and clinical phenotype is warranted. National Institute of Public Health Ministry of Education Youth and Sports of the Czech Republic Italian Cancer Genome Project Ministero Salute Pancobank Heidelberger Stiftung Chirurgie EU ERA-Net TRANSCAN Italian Ministry of Health Division of Gastroenterology, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy

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Tattoo is going to be a very common practice especially among young people and we are witnessing a gradual increase of numerous potential complications to tattoo placement which are often seen by physicians, but generally unknown to the public. The most common skin reactions to tattoo include a transient acute inflammatory reaction due to trauma of the skin with needles and medical complications such as superficial and deep local infections, systemic infections, allergic contact dermatitis, photodermatitis, granulomatous and lichenoid reactions, and skin diseases localized on tattooed area (eczema, psoriasis, lichen, and morphea). Next to these inflammatory skin reactions we have to consider also the possibility of the development of cutaneous conditions such as pseudolymphomatous reactions and pseudoepitheliomatous hyperplasia. The aim of this study is to underline the importance of an early diagnosis by performing a histological examination especially when we are in front of suspected papulonodular lesions arising from a tattoo, followed by a proper treatment, since cutaneous neoplastic evolution is known to be a rare but possible complication.