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Optimization of the Cultivation of Donor Mesenchymal Stromal Cells for Clinical Use in Cellular Therapy

CellBio 2014年第1期3卷 25-33页

作者： Vanessa Valim bruna amorin Annelise Pezzi Maria Aparecida Lima da Silva Ana Paula Alegretti Lucia Silla Cellular Therapy Center Hospital de Clinicas de Porto Alegre Porto Alegre Brazil Post-Graduation in Medicine: Medical Sciences Federal University of Rio Grande do Sul Porto Alegre Brazil Cellular Therapy Center Hospital de Clinicas de Porto Alegre Porto Alegre Brazil Post-Graduation in Medicine: Medical Sciences Federal University of Rio Grande do Sul Porto Alegre Brazil Hematology and Bone Marrow Transplantation Hospital de Clinicas de Porto Alegre Porto Alegre Brazil

Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction;2) The number of cells indicated for therapy that is relatively high, with the need to optimize the expansion, number and time wise;3) The utilization of third party donors;4) Culture passage number(P);and 5) Source of the cells. This study was designed to determine the superiority of the Platelet Lysates(PL)over FBS on the expansion of MSC, the optimal cell’ plating density and days between each pass, and to investigate if donor total nucleated cells(TNC)obtained from the washouts of discharged bags and filters of hematopoietic stem cell transplantation(HSCT)can be expanded to be used at clinical grade. TNC were removed, plated and after the first passage were cultivated in different concentrations with FBS or PL, and the number of days to reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or with FBS and after seven days counted to analyze how much they had grown in that period. The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC and to allow the proliferation of enough number of MSC at P2 for clinical use.

关键词： Platelet Lysate MSC Optimization Cell Therapy

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Adipocyte Derived Mesenchymal Stromal Cell and Platelet Lysate: Ideal Cell and Supplement for the Treatment of Immune-Inflammatory Diseases?

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CellBio 2016年第2期5卷 15-25页

作者： Felipe Rodrigues Annelise Pezzi Álvaro Laureano Vanessa Valim bruna Zambonato Alice Dahmer Letícia Baggio Filipe Sehn Ianaê Wilke Maria Aparecida L. da Silva bruna amorin Cellular Therapy Center Hospital de Clínicas de Porto Alegre Porto Alegre Brazil Laboratory of Cell Culture and Molecular Analysis of Human Cells Hospital de Clínicas de Porto Alegre Porto Alegre Brazil Laboratory of Cell Culture and Molecular Analysis of Human Cells Hospital de Clínicas de Porto Alegre Porto Alegre Brazil Post-graduation in Medicine: Medical Sciences Federal University of Rio Grande do Sul Porto Alegre Brazil

Background: Mesenchymal stromal cells (MSC) are being tested for the treatment of immune diseases. MSC are present in several adult tissues which milieu may influence MSC behavior particularly under inflammatory conditions. Additionally, culture conditions also can modify cell function or state of activation. Methods: To address the influence of the MSC source on its characteristics, we studied a xenofree, platelet lysate supplemented MSC from dental pulp, adipose tissue and bone marrow, co-cultured with isolated T cells and PBMC subset, and studied the effect of culture animal or human supplements immunomodulatory effect. Results: All three sources were efficient in inhibiting T cells. Among all MSC sources, as also described by others, adipose MSC was capable to significantly induce Treg phenotype and decrease T CD8+. Furthermore, comparing fetal bovine serum and platelet lysate, results demonstrate that platelet lysate alone is capable to induce immunomodulatory phenotype. Additional studies have to be made to elucidate the PL immunomodulatory effect.

关键词： Mesenchymal Stromal Cells Dental Pulp Adipose Tissue Bone Marrow T Cells Immunomodulatory Effect

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Characteristics of Mesenchymal Stem Cells under Hypoxia

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CellBio 2013年第1期2卷 11-19页

作者： bruna amorin Ana Paula Alegretti Vanessa de Souza Valim Annelise Martins Pezzi da Silva Maria Aparecida Lima da Silva Felipe Sehn Lucia Silla Cell Technology Center Experimental Research Center Hospital de Clínicas de Porto Alegre-Porto AlegreRio Grande do Sul Brazil

Mesenchymal stem cells (MSC) are considered non-hematopoietic multipotent stem cells with self-renewal properties and the ability to differentiate into a variety of mesenchymal tissues. Optimal conditions for the culture of these cells have been the subject of investigation for several years. In particular, ideal oxygen tension levels have not been established in the literature. In physiological environments, oxygen tension may vary from 12% in peripheral blood to 1% in the deep zone of cartilage regions. In any case, oxygen tension is considerably lower in vivo when compared with the normal atmosphere of standard cell culture conditions (21%). The objective of this study was to review the literature available on MSC characteristics (cell cycle, survival, proliferation, differentiation, morphology, immunophenotype, cytogenetics) when cultured under hypoxic conditions. Our focus on optimal culture conditions is justified by the key role currently played by these cells in regenerative medicine.

关键词： Mesenchymal Stem Cells Culture Hypoxia

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β-Globin Gene Cluster Haplotypes and Clinical Severity in Sickle Cell Anemia Patients in Southern Brazil

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Open Journal of Blood Diseases 2014年第2期4卷 16-23页

作者： Maria ALda Silva Joao RFriedrisch Christina MBittar Meide Urnau Joice Merzoni Vanessa SValim bruna amorin Annelise Pezzi Jose Artur BChies Lucia Mda Rocha Silla Laboratory of Hematopoietic Cell Culture and Molecular Analysis Center for Experimental ResearchPorto AlegreBrazil Hematology and Bone Marrow Transplantation of Hospital de Clinicas de Porto Alegre Porto AlegreBrazil Post-Graduation in Medicine:Medical Sciences Federal University of Rio Grande do SulPorto AlegreBrazil Laboratory of Immunogenetics Department of GeneticsUFRGSPorto AlegreBrazil

Hematopoietic stem cell transplantation(HSCT)has emerged as a curative strategy for sickle cell anemia(SCA);it is necessary to find markers of SCA clinical severity to spare those SCA patients whose clinical course is mild from the morbidity and mortality associated with HSCT. Haplotypes have been correlated with the severity of clinical manifestations in SCA patients, and fetal hemoglobin(HbF)and socioeconomic status(SeS)have also been described as negative factors. We studied these factors and their impact on clinical manifestations in a population of Southern Brazilian patients attending the Center for Sickle Cell Anemia at Hospital de Clínicas de Porto Alegre/RS, Brazil. Clinical severity was defined as two or more veno-occlusive episodes per year. The βS haplotypes were determined by PCR in 75 SCA patients. Among the 150 βS chromosomes analyzed, 99(66%)were identified as Bantu(Ban), 41(27%)asBenin(Ben), and 10(7%)as other haplotypes. Most patients in our sample(62.7%)belonged to lower SeS groups, precluding meaningful statistical analysis of SeS impact on clinical severity. There was no correlation between haplotypes or HbF level and SCA clinical severity. Gene polymorphisms and environmental issues have to be taken into consideration.

关键词： Sickle Cell Anemia β-Globin Fetal Hemoglobin and Clinical Severity

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