Isolation Methods of Peripheral Blood Mononuclear Cells in Spanish Biobanks: An Overview

Consuegra, Irene; Rodríguez-Aierbe, Clara; Santiuste, Inés; Bosch, Anna; Martínez-Marín, Rosario; Fortuto, Ma Antonia; Díaz, Tatiana; Martí, S.; Muñoz‐Fernández, María Ángeles

doi:10.1089/bio.2016.0105

Cited by 13 publications

(7 citation statements)

References 33 publications

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“…Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from anonymized healthy blood donors coming from the transfusion Center of Madrid following national guidelines. PBMCs were isolated by a standard Ficoll - Hypaque density gradient (Rafer, Madrid, Spain) and cultured following the procedures of Spanish HIV HGM BioBank [42–45]. PBMCs were kept with 60 U/mL of interleukin 2 (IL-2; Bachem AG, Bubendorf, Switzerland) and stimulated with 60 U/mL of interleukin 2 (IL-2; Bachem AG, Bubendorf, Switzerland) and 2 μg/mL of phytohemaglutinin (PHA; Remel, Santa Fe, NM, USA) for 48 h before the experiments for T cell analysis.…”

Section: Methodsmentioning

confidence: 99%

G2-S16 dendrimer microbicide does not interfere with the vaginal immune system

et al. 2019

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It is essential that prophylactic drugs do not interfere with the normal function of the immune system. The use of nanoparticles as vaginal microbicides is a promising prevention strategy against sexually transmitted infections. With that aim, our group is working with the G2-S16, a second generation carbosilane dendrimer with sulfonate groups in the periphery, which has been previously shown to be effective against HIV-1 and HSV-2 infection, and it is now on the road to clinical trials. Our objective in this new study is to assess the effects of G2-S16 on the immune barrier of the female reproductive tract. The expression of differentiation, maturation and activation markers was measured in epithelial cells, dendritic cells, M and GM macrophages, and T cells using RT-qPCR and flow cytometry. The results demonstrate that G2-S16 does not alter the natural immunity of the vagina, strongly supporting the biosafety of this dendrimer for clinical use.

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Section: Methodsmentioning

confidence: 99%

G2-S16 dendrimer microbicide does not interfere with the vaginal immune system

et al. 2019

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“…PBMCs were isolated from buffy coats obtained from anonymous healthy blood donors donated by the Transfusion Center of Madrid following national guidelines. PBMCs were isolated by a standard Ficoll-Hypaque density gradient (Rafer, Madrid, Spain) and cultured following the procedures of Spanish HIV HGM BioBank [ 21 , 22 , 23 ]. PBMCs were cultured in RPMI-1640 medium (Biochrom AG, Berlin, Germany) with 10% fetal bovine serum (FBS; Gibco, London, UK), 1% l -glutamine (Lonza, Walkersville, MD, USA) and antibiotic cocktail (125 mg/mL ampicillin,125 mg/mL cloxacillin and 40 mg/mL gentamicin; all from Normon, Madrid, Spain).…”

Section: Methodsmentioning

confidence: 99%

Nanoparticle-Delivered HIV Peptides to Dendritic Cells a Promising Approach to Generate a Therapeutic Vaccine

et al. 2020

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Finding a functional cure for HIV-1 infection will markedly decrease the social and economic burden of this disease. In this work, we have taken advantage of the antigen presenting cell role of human dendritic cells (DCs) to try to induce an immune response to HIV-derived peptide delivered to DCs using two different polycationic nanoparticles: a G4 PAMAM dendrimer modified to a 70/30 ratio of hydroxyl groups/amines and a cyclodextrin derivative. We have studied peptide delivery using a fluorescence peptide and have studied the immune response generation by cytokine determination and flow cytometry. We have found a robust delivery of the antigenic peptide to DCs and activated dendritic cell-mediated peripheral blood mononuclear cells (PBMCs) proliferation using the mixed lymphocyte reaction. However, no expression of markers indicating activation of either B or T lymphocytes was observed. Moreover, the release of the pro-inflammatory cytokine TNF-α or IL-2 was only observed when DCs treated with either the dendrimer or the dendriplex containing the peptide. Antigenic peptide delivery to DCs is a promising approach to generate a vaccine against HIV-1 infection. However, more studies, including the simultaneous delivery of several antigenic peptides from different viral proteins, can markedly improve the immune response.

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“…PBMC were then immediately processed for flow cytometry or frozen in fetal bovine serum/7.5% dimethyl sulfoxide (DMSO) (Sigma Aldrich) (Consuegra et al, 2017;Mitrus et al, 2013) and conserved in liquid nitrogen for 3 months. For whole blood freezing, samples were precooled 15 min on ice.…”

Section: Sample Preparationmentioning

confidence: 99%

An easy and reliable whole blood freezing method for flow cytometry immuno‐phenotyping and functional analyses

Braudeau

Salabert

Chevreuil

et al. 2021

Cytometry Part B Clinical

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Background: Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. Methods:In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor ® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.Results: Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly,we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). Conclusions:In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

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Isolation Methods of Peripheral Blood Mononuclear Cells in Spanish Biobanks: An Overview

Cited by 13 publications

References 33 publications

G2-S16 dendrimer microbicide does not interfere with the vaginal immune system

G2-S16 dendrimer microbicide does not interfere with the vaginal immune system

Nanoparticle-Delivered HIV Peptides to Dendritic Cells a Promising Approach to Generate a Therapeutic Vaccine

An easy and reliable whole blood freezing method for flow cytometry immuno‐phenotyping and functional analyses

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