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Chieregato K.,Advanced Cellular Therapy Laboratory | Albiero E.,Advanced Cellular Therapy Laboratory | Castegnaro S.,Advanced Cellular Therapy Laboratory | Bernardi M.,Advanced Cellular Therapy Laboratory | And 5 more authors.
Blood Cells, Molecules, and Diseases | Year: 2012

Recently a number of cellular therapy based-clinical trials have been carried out using mesenchymal stromal cells (MSC) or cytokine-induced-killer (CIK) cells aiming to improve outcome of allogeneic hematopoietic stem cell transplantation.We have isolated MSC from umbilical cord (UC) exploring the interaction between CIK cells and UC-MSC. We found that UC-MSC could suppress CIK cells activity, when co-cultured in a cell-to-cell system. In addition, CIK cells could potentially lyse UC-MSC in a time and ratio dependent manner that could have implications for their in vivo use.Here we provide experimental data on the mutual interaction of CIK cells and UC-MSC, suggesting a negative interference when the two cell types are used in combination. In the light of our observations, when CIK and UC-MSC will be used in clinical trials, timing and sequencing of their infusion should be considered. © 2012 Elsevier Inc.. Source

Bernardi M.,Hematology Project Foundation Research Laboratories | Bernardi M.,Advanced Cellular Therapy Laboratory | Adami V.,University of Trento | Albiero E.,Hematology Project Foundation Research Laboratories | And 5 more authors.
Experimental and Toxicologic Pathology | Year: 2014

Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line.PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective.Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. © 2013 Elsevier GmbH. Source

Izeta A.,Hospital Universitario Donostia | Herrera C.,University of Cordoba, Spain | Mata R.,Andalusian Initiative for Advanced Therapies | Astori G.,Advanced Cellular Therapy Laboratory | And 8 more authors.
Cytotherapy | Year: 2016

In June 2015, European Medicines Agency/Committee for Advanced Therapies (CAT) released the new version of the reflection paper on classification of advanced therapy medicinal products (ATMPs) established to address questions of borderline cases in which classification of a product based on genes, cells or tissues is unclear. The paper shows CAT's understanding of substantial manipulation and essential function(s) criteria that define the legal scope of cell-based medicinal products. This article aims to define the authors' viewpoint on the reflection paper. ATMP classification has intrinsic weaknesses derived from the lack of clarity of the evolving concepts of substantial manipulation and essential function(s) as stated in the EU Regulation, leading to the risk of differing interpretations and misclassification. This might result in the broadening of ATMP scope at the expense of other products such as cell/tissue transplants and blood products, or even putting some present and future clinical practice at risk of being classified as ATMP. Because of the major organizational, economic and regulatory implications of product classification, we advocate for increased interaction between CAT and competent authorities (CAs) for medicines, blood and blood components and tissues and cells or for the creation of working groups including representatives of all parties as recently suggested by several CAs. © 2016 International Society for Cellular Therapy. Source

Zanon C.,Advanced Cellular Therapy Laboratory | Stocchero M.,S IN Soluzioni Informatiche | Albiero E.,Advanced Cellular Therapy Laboratory | Castegnaro S.,Advanced Cellular Therapy Laboratory | And 4 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2014

Background Cytokine-induced killer (CIK) cells, obtained after mononucleated cell stimulation with interferon-γ, interleukin-2, and anti-CD3 antibody, are constituted by CD3+CD56+ (CIK) cells and a minority of natural killer (NK; CD3-CD56+) cells and T-lymphocytes (CD3+CD56-) with antitumor effect against hematological malignancies, thus representing a promising immunotherapy strategy. To ensure in vivo antitumor activity it is mandatory to maximize the percentage of CD3+56+ effector cells, which is highly variable depending on the starting sample and the harvesting day. Based on cytofluorimetric data, we have retrospectively applied multivariate statistical data analysis (MVDA) to 30 expansions building mathematical models able to predict the expansion fate and the optimal CIK harvesting day. Methods Cell phenotype was monitored during culture; multivariate batch statistical process control was applied to monitor cell expansion and orthogonal projections to latent structures to predict CIK percentage. Results Ten expansions had CD3 +CD56+ cells ≥40% (good batches) and 20 had CD3 +CD56+ cells ≤40%. In 36.7%, CD3+CD56 + cells reached the highest concentration at day 17 and the others at day 21. We built a highly predictive regression model for estimating CD3 +CD56+ cells during culture. Three variables resulted highly informative: NK % at day 0, cytotoxic T-lymphocytes % (CTLs, CD3 +CD8+) at day 4, and CIK % at day 7. "Good batches" are characterized by a high percentage of CTLs and CD3 +CD56+ cells at day 4 and day 7, respectively. Conclusion By applying MVDA it is possible to optimize CIK expansion, deciding the optimal cell harvesting day. A predictive role for CTL and CIK was evidenced. © 2013 International Clinical Cytometry Society © 2013 Clinical Cytometry Society. Source

Bernardi M.,Advanced Cellular Therapy Laboratory | Bernardi M.,Hematology Project Foundation Research Laboratories | Albiero E.,Advanced Cellular Therapy Laboratory | Albiero E.,Hematology Project Foundation Research Laboratories | And 7 more authors.
Cytotherapy | Year: 2013

Background aims: A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Methods: Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. Results: After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P< 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. Conclusions: The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. © 2013 International Society for Cellular Therapy. Source

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