Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Abstract

Many immunological experiments would be greatly facilitated if peripheral blood mononuclear cells (PBMC) preserved important functional properties over longer periods of time. Regarding adhesive interactions with endothelial cells, a crucial step of inflammatory processes, this had not been investigated yet. We demonstrate that PBMC subsets subjected to controlled cryopreservation retain their phenotypic traits inasmuch as the proportion of viable T cells, monocytes/macrophages and B cells was comparable with their freshly isolated counterparts. More importantly, we demonstrate for the first time that the procedure does not impede crucial adhesion-mediated dynamic interactions with endothelial cells. Using a flow chamber system, freshly isolated and cryopreserved PBMC showed similar rolling and firm adhesion on TNF-a-activated endothelial cells under shear flow as compared to freshly isolated PBMC from the same donors. Thus, our observation is an important prerequisite for functional studies of leucocyte recruitment when sequential investigations with material from the same patients are required.Key words: endothelial cell -leukocyte rolling -method for preserving PBMC functions Accepted for publication 20 February 2013Peripheral blood mononuclear cells (PBMC) are indispensable for many immunological analyses, both in basic and clinical research. Long-term or repeated studies require PBMC storage while reliably preserving functional traits. Since the first successful preservation of mouse lymphocytes (1), different freezing protocols have been developed. Phenotypic traits of lymphocytes remained stable after freezing at À80°C, while monocytes, macrophages and granulocytes were more prone to damage (2). Programmed freezing and mechanical freezing methods appeared to be comparable regarding recovery and viability of PBMC (3). Concerning apoptosis, storage of PBMC at À150°C was superior compared to storage at À30°C or to storage of whole-blood samples at either temperature (4,5).Notwithstanding a fair number of approaches to store PBMC, we were surprised to find that virtually all of these earlier studies had a peculiar view on a limited array of cellular functions or mere phenotypic characterization (5-7). Thus, the capability of cryopreserved PMBC to properly interact with endothelial cells, a crucial early step in inflammatory reactions, still had to be established. Using a dynamic flow chamber system, we provide here the first experimental evidence for intact functional traits of PBMC after cryopreservation regarding rolling on and adhesion to cultured endothelial cells.Towards this end and with ethical approval, venous blood (20-40 ml) was collected from a total of six healthy volunteers (five female non-smokers, one male smoker; age range 22-28 years). PBMC were isolated using Ficoll-Paque PLUS density gradient centrifugation and washed with Hank's buffered salt solution (HBSS). PBMC were labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, 2 lM in HBSS) for flow chamber analysis dir...

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

“…The fitness-for-purpose of PBMC cryopreservation has been demonstrated in the scope of preservation of lymphocyte immunophenotypes and proliferative responses [48,49], pyrogenic responses [50,51], establishment of LCLs [52], Th1/Th2 cytokine ratio after stimulation [53], Tcell culture and subsequent polyfunctional antigen-specific CD4 and CD8 T cell responses [16,54], host cell reactivation (HCR) assay [55], and mutagen sensitivity assay for assessment of DNA repair capacity [56]. Cryopreservation does not induce any significant difference in the proportions of T cells expressing CD3 and monocytes/ macrophages expressing CD14 [57]. The impact of cryopreservation on B cells expressing CD19, if it exists, is small [48].…”

“…Contradictory conclusions have been published concerning the impact of cryopreservation on the adhesion-mediated interactions between mononuclear and endothelial cells. Cryopreservation does not alter integrins on the mononuclear cells and hence does not affect rolling and adhesion on TNF-α-activated endothelial cells [57]. Another study found a change in the adhesive phenotype and a modulation of the ability of monocytes to migrate across endothelial monolayers when cells were tested immediately post-thaw, but not if they were allowed to recover before testing [58].…”

Protective effect of curcumin against ultraviolet A irradiation‑induced photoaging in human dermal fibroblasts

“…Furthermore, increasing scientific evidence has demonstrated that Cur is able to inhibit chemical-induced carcinogenesis/tumor promotion and radiation-induced mammary tumorigenesis ( 12 – 15 ). In addition, a molecular biology-based study demonstrated that Cur may exert inhibitory effects on the UVB-induced production of reactive oxygen species (ROS) and expression of matrix metalloproteinase (MMP) in vitro by blocking the activation of the UVB-induced mitogen-activated protein kinase, nuclear factor-κB (NF-κB) and AP-1 transcription factor signal pathways ( 16 ). Tsai et al ( 17 ) demonstrated that Cur provided protection against UVB radiation-induced skin cancer growth in a mouse model.…”

Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry

“…We tested 100% FBS, 20% FBS mixed with 80% RPMI, and 100% RPMI, and found the highest cell recovery using 20% FBS 80% RPMI. Others have used 100% RPMI [ 19 , 20 ], 10% FBS [ 3 , 7 , 9 , 13 , 21 ], 20% FBS [ 5 ], 30% FBS [ 22 ] or 50% FBS [ 23 ] mixed with RPMI. And some studies simply fail to mention the FBS concentration in the washing medium [ 24 ].…”