ABSTRACT
Conventional frozen cryopreservation (CFC) is currently the gold standard for cardiovascular allograft preservation. However, inflammation and structural deterioration limit transplant durability. Ice-free cryopreservation (IFC) already demonstrated matrix structure preservation combined with attenuated immune responses. In this study, we aim to explore the mechanisms of this diminished immunogenicity in vitro. First, we characterized factors released by human aortic tissue after CFC and IFC. Secondly, we analyzed co-cultures with human peripheral blood mononuclear cells, purified monocytes, T cells and monocyte-derived macrophages to examine functional immune effects triggered by the tissue or released cues. IFC tissue exhibited significantly lower metabolic activity and release of pro-inflammatory cytokines than CFC tissue, but surprisingly, more active transforming growth factor ß. Due to reduced cytokine release by IFC tissue, less monocyte and T cell migration was detected in a chemotaxis system. Moreover, only cues from CFC tissue but not from IFC tissue amplified αCD3 triggered T cell proliferation. In a specifically designed macrophage-tissue assay, we could show that macrophages did not upregulate M1 polarization markers (CD80 or HLA-DR) on either tissue type. In conclusion, IFC selectively modulates tissue characteristics and thereby attenuates immune cell attraction and activation. Therefore, IFC treatment creates improved opportunities for cardiovascular graft preservation.
Subject(s)
Aorta/immunology , Cryopreservation/methods , Immunity, Cellular/immunology , Leukocytes, Mononuclear/immunology , Macrophages/immunology , Monocytes/immunology , T-Lymphocytes/immunology , Aorta/metabolism , Biomarkers/metabolism , Cell Movement , Cell Proliferation , Cells, Cultured , Coculture Techniques , Cryopreservation/classification , Cytokines/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Macrophages/metabolism , Monocytes/metabolism , T-Lymphocytes/metabolismABSTRACT
Flow cytometric analysis is important for the investigation and clinical preparation of lymphocytes from children. However, the strict requirement of cell freshness and interassay variability limits the application of this methodology for pediatric investigations. Therefore, it is necessary to identify a reliable cryopreservative method capable of maintaining high cell viability and proper cell function in lymphocytes from children. In the present study, eight commonlyused cell cyropreservative methods were used, and their effects on cell viability, surface marker expression and cell function were examined. In addition, how these methods affect the distribution of Tcell receptor Vß subfamilies were also determined. The results of the present study provided valuable experimental evidence, based on which the optimal method for the cryopreservation of lymphocytes from children in pediatric investigations and clinical applications can be selected.
Subject(s)
Cryopreservation/methods , Flow Cytometry , Lymphocytes/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Antigens, Surface , Cell Survival/genetics , Child , Child, Preschool , Cryopreservation/classification , Female , Humans , Immunophenotyping , Infant , Lymphocytes/immunology , Male , Receptors, Antigen, T-Cell, alpha-beta/immunologyABSTRACT
BACKGROUND: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still requires improvement. METHODS: Frozen/thawed sheep ovarian cortical biopsies were embedded in collagen matrix with or without isoform 165 of vascular endothelial growth factor (VEGF165) and transplanted into ovaries of immunodeficient mice. Ovaries were chosen as transplantation sites to more closely resemble clinical conditions in which orthotopic transplantation has previously allowed several spontaneous pregnancies. RESULTS: We found that VEGF165 significantly increased the number of Dextran-FITC positive functional vessels 3 days after grafting. Dextran- fluorescein isothiocyanate (FITC) positive vessels were detectable in 53% and 29% of the mice in the VEGF-treated and control groups, respectively. Among these positive fragments, 50% in the treated group displayed mature smooth-muscle-actin-alpha (alpha-SMA) positive functional vessels compared with 0% in the control group. CD31 positive murine blood vessels were observed in 40% of the VEGF165 transplants compared with 21% of the controls. After 3 weeks, the density of murine vessels was significantly higher in the VEGF165 group. CONCLUSION: The encapsulation of ovarian tissue in collagen matrix in the presence of VEGF165 before grafting has a positive effect on functional blood vessel recruitment. It can be considered as a useful technique to be improved and further developed before human clinical applications in female cancer patients in the context of fertility preservation.
Subject(s)
Fertility Preservation/methods , Ovary/transplantation , Vascular Endothelial Growth Factor A/metabolism , Animals , Collagen , Cryopreservation/classification , Cryopreservation/methods , Female , Mice , Mice, SCID , Neovascularization, Physiologic , Ovary/blood supply , Ovary/metabolism , Protein Isoforms/metabolism , Sheep , Tissue Culture Techniques , Transplantation, HeterologousABSTRACT
The Food and Drug Administration (FDA) is classifying a cord blood processing system and storage container into class II (special controls). The special control that will apply to this device is the guidance document entitled "Class II Special Controls Guidance Document: Cord Blood Processing System and Storage Container." FDA is classifying this device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of this device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance document that will serve as the special control for this device.
Subject(s)
Cell Separation/classification , Cryopreservation/classification , Fetal Blood , Specimen Handling/instrumentation , Cell Separation/instrumentation , Cryopreservation/instrumentation , Device Approval/legislation & jurisprudence , Equipment Safety , Hematology/instrumentation , Hematology/legislation & jurisprudence , Humans , Pathology/instrumentation , Pathology/legislation & jurisprudence , United States , United States Food and Drug AdministrationABSTRACT
OBJETIVO: O objetivo deste trabalho foi avaliar, retrospectivamente, os primeiros oito anos de funcionamento do Banco de Valvas Cardíacas Humanas do Hospital de Caridade da Irmandade da Santa Casa de Misericórdia de Curitiba (BVCHSC), analisando aspectos relacionados às atividades de captacão, processamento, armazenamento e distribuicão de enxertos homólogos cardiovasculares. MÉTODO: A selecão inicial dos doadores seguiu as diretrizes nacionais para captacão de órgãos humanos, além de critérios específicos do BVCHSC. Os coracões foram obtidos de doadores de múltiplos órgãos, doadores em parada cardíaca e receptores de transplante cardíaco, com somatória dos tempos de isquemia inferior a 48 horas. A idade dos doadores variou desde recém-nascidos até 60 anos para as valvas aórticas e 65 para as pulmonares. Os enxertos dissecados tiveram suas dimensões mensuradas e sua morfologia avaliada, sendo classificados como categoria 0 (descartados), 1 (alteracões morfológicas mínimas) ou 2 (perfeitos). Foram determinados a incidência e os germes responsáveis pela contaminacão nos enxertos recebidos, assim como a eficiência da solucão de descontaminacão. Foram também avaliados aspectos relacionados à distribuicão dos enxertos. RESULTADOS: De setembro de 1996 a fevereiro de 2005, 1059 coracões provenientes de 19 Estados foram recebidos no BVCHSC. Destes, 977 (92,3 por cento) eram de doadores de morte encefálica. Foram processados 2105 enxertos, e dentre os aórticos e pulmonares analisados, 783 eram da categoria 2, 697 pertenciam à categoria 1 e 186 foram descartados por alteracões morfológicas. No total, 433 enxertos recebidos estavam contaminados, sendo a solucão de antibióticos eficiente na esterilizacão de 330 destes casos. Quinhentos e setenta e um (27,1 por cento) enxertos foram rejeitados em alguma fase do processo, sendo a contaminacão e as alteracões morfológicas as causas mais freqüentes. Foram distribuídos 1338 enxertos para 74 instituicões de saúde do país, sendo mais comumente empregados na substituicão da valva aórtica (529), na correcão de cardiopatias congênitas (478) e durante operacões de Ross (272). CONCLUSÕES: As atividades do BVCHSC durante estes oito anos de funcionamento foram satisfatórias, atingindo os objetivos propostos.
