RT Journal Article
SR Electronic
T1 Cryopreservation of human mucosal leukocytes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 039578
DO 10.1101/039578
A1 Sean M. Hughes
A1 Zhiquan Shu
A1 Claire N. Levy
A1 April L. Ferre
A1 Heather Hartig
A1 Cifeng Fang
A1 Gretchen Lentz
A1 Michael Fialkow
A1 Anna C. Kirby
A1 Kristina M. Adams Waldorf
A1 Ronald S Veazey
A1 Anja Germann
A1 Hagen von Briesen
A1 M. Juliana McElrath
A1 Charlene S. Dezzutti
A1 Elizabeth Sinclair
A1 Chris A. R. Baker
A1 Barbara L Shacklett
A1 Dayong Gao
A1 Florian Hladik
YR 2016
UL http://biorxiv.org/content/early/2016/03/24/039578.abstract
AB Background Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.Methods and findings To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes.Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.Conclusions Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.