Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

International Journal of Molecular Sciences

on behalf of the ENDIA Study Group¹  Minh Bui²  Ace Y. L. Choo³  Sabrina Binkowski³  Christopher M. Hope⁴  Trung Nguyen⁴  Timothy Sadlon⁴  Simon C. Barry⁴  Dao Huynh⁴  Jennifer J. Couper⁴  Ying Ying Wong⁴  Griffith Boord Perkins⁴  Helena Oakey⁴  Megan A. S. Penno⁴  Ki Wook Kim⁵  Emily Gibson⁵  William D. Rawlinson⁵  Dexing Huang⁶  Katrina Ngui⁶ 

[1] ;Child Health Research Unit, Barwon Health, Geelong, VIC 3220, Australia;Children’s Diabetes Centre, Telethon Kids Institute, The University of Western Australia, Perth, WA 6009, Australia;Robinson Research Institute, Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia;School of Women’s and Children’s Health, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia;Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia;

关键词: PBMC;    blood handling;    delay in processing;    cryopreservation;    cell concentration;   

DOI  :  10.3390/ijms22179129

来源: DOAJ

【 摘 要 】

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10⁶ cells/mL to 1.67 × 10⁶ cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10⁶ cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

【 授权许可】

Unknown