Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73

Purinergic signalling

article

Ross J. Turner¹  Nicholas J. Geraghty¹  Jonathan G. Williams¹  Diane Ly¹  Daniel Brungs¹  Martin G. Carolan¹  Thomas V. Guy¹  Debbie Watson¹  Jeremiah F. de Leon⁴  Ronald Sluyter¹ 

[1]Illawarra Health and Medical Research Institute

[2]Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong

[3]CONCERT - Centre for Oncology Education and Research Translation

[4]Illawarra Cancer Care Centre, Wollongong Hospital

[5]St Vincent’s Clinic

关键词: Ecto-enzymes;    ENTPD1;    NT5E;    Purinergic signalling;    Purinergic receptor;    Liquid biopsy;   

DOI  :  10.1007/s11302-020-09714-1

学科分类：分子生物学，细胞生物学和基因

来源: Springer

【 摘 要 】

CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.

【 授权许可】

CC BY