PT  - JOURNAL ARTICLE
AU  - Sean M. Hughes
AU  - Zhiquan Shu
AU  - Claire N. Levy
AU  - April L. Ferre
AU  - Heather Hartig
AU  - Cifeng Fang
AU  - Gretchen Lentz
AU  - Michael Fialkow
AU  - Anna C. Kirby
AU  - Kristina M. Adams Waldorf
AU  - Ronald S Veazey
AU  - Anja Germann
AU  - Hagen von Briesen
AU  - M. Juliana McElrath
AU  - Charlene S. Dezzutti
AU  - Elizabeth Sinclair
AU  - Chris A. R. Baker
AU  - Barbara L Shacklett
AU  - Dayong Gao
AU  - Florian Hladik
TI  - Cryopreservation of human mucosal leukocytes
AID  - 10.1101/039578
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 039578
4099  - http://biorxiv.org/content/early/2016/03/25/039578.short
4100  - http://biorxiv.org/content/early/2016/03/25/039578.full
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.