Isolation and freezing of human peripheral blood mononuclear cells from pregnant patients

Efthymiou, Athina; Mureanu, Nicoleta; Pemberton, Rebecca; Tai-MacArthur, Sarah; Mastronicola, Daniela; Scottà, Cristiano; Lombardi, Giovanna; Nicolaides, Kypros H.; Shangaris, Panicos

doi:10.1016/j.xpro.2022.101204

Cited by 5 publications

(2 citation statements)

References 5 publications

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“…Blood was captured in BD Vacutainer® EDTA Tubes with a volume of 10 mL, and the samples were kept at room temperature (18-22°C) while being agitated using an orbital shaker at 90-100 rpm. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described 53 . Brie y, Ficoll-Paque PLUS medium was placed into a SepMate™-50 tube, and the collected blood was transferred to a separate 50 mL Falcon tube where it was diluted 1:1 with PBS-EDTA containing 2% FBS.…”

Section: Pbmc Isolation and Facsmentioning

confidence: 99%

Single-cell transcriptomics reveals markers of regulatory T cell dysfunction in Gestational Diabetes Mellitus

Shangaris,

Mensah,

Efthimiou

et al. 2024

Preprint

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Gestational diabetes mellitus (GDM) is a common pregnancy complication associated with hyperglycaemia, chronic inflammation and adverse health outcomes. Regulatory T cells (Tregs) are thought to contribute to GDM due to their role in suppressing inflammation. However, it remains unclear whether specific Treg subsets are impaired in patients with GDM. To investigate transcriptional variation in GDM Tregs, we applied single-cell RNA sequencing to Tregs isolated from the blood of 13 healthy pregnant women and 10 patients with GDM. We identified naive and effector Treg subsets, none of which significantly differ in the proportion of cells captured from GDM and controls. We report a naive Treg subset with reduced expression of AP-1 transcription factor subunits in GDM, including JUN, FOS, and EGR1, and an effector Treg subset with increased signalling of angiogenesis marker genes. Genes dysregulated in GDM Tregs independently predicted GDM status in pseudobulk and whole blood mRNA from independent cohorts. Remarkably, TXNIP, which regulates glucose levels, emerged as the most reliable standalone predictor in bulk mRNA (minimum AUC 0.7) equivalent to using body mass index (AUC 0.72) in our cohort. This study uncovers a disrupted molecular pathway in Treg cell subsets from GDM patients and proposes a panel of genes with translational potential as early disease biomarkers.

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Section: Pbmc Isolation and Facsmentioning

confidence: 99%

Single-cell transcriptomics reveals markers of regulatory T cell dysfunction in Gestational Diabetes Mellitus

Shangaris,

Mensah,

Efthimiou

et al. 2024

Preprint

Self Cite

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“…Density gradient centrifugation is perhaps the most common traditional method to isolate subpopulations of WBCs from blood. Cells sediment through layers of liquids of different densities formed by gradient media, such as Ficoll-Paque 9 , 10 or Histopaque 11 , 12 , until reaching their points of neutral buoyancy. Drawbacks with this method are that ( i ) the accuracy is limited by the difficulties of accurately collecting the thin sheaths of layered cells from the test tube for cells of contiguous densities, ( ii ) it cannot easily be implemented for applications of handling small sample volumes, and ( iii ) it is challenging to implement in a flow-through format for in-line integration in compact automated analytical instrumentation to facilitate diagnosis or treatment of a patient at the point-of-care.…”

Section: Introductionmentioning

confidence: 99%

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing

Alsved,

Rezayati Charan,

Ohlsson

et al. 2024

Sci Rep

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Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

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Recent advances in the production, reprogramming, and application of CAR-T cells for treating hematological malignancies

et al. 2022

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Isolation and freezing of human peripheral blood mononuclear cells from pregnant patients

Cited by 5 publications

References 5 publications

Single-cell transcriptomics reveals markers of regulatory T cell dysfunction in Gestational Diabetes Mellitus

Single-cell transcriptomics reveals markers of regulatory T cell dysfunction in Gestational Diabetes Mellitus

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing

Recent advances in the production, reprogramming, and application of CAR-T cells for treating hematological malignancies

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