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Cryopreservation of human mucosal leukocytes

Hughes, SM and Shu, Z and Levy, CN and Ferre, AL and Hartig, H and Fang, C and Lentz, G and Fialkow, M and Kirby, AC and Waldorf, KMA and Veazey, RS and Germann, A and Von Briesen, H and McElrath, MJ and Dezzutti, CS and Sinclair, E and Baker, CAR and Shacklett, BL and Gao, D and Hladik, F (2016) Cryopreservation of human mucosal leukocytes. PLoS ONE, 11 (5).

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Abstract

© 2016 Hughes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Hughes, SM
Shu, Z
Levy, CN
Ferre, AL
Hartig, H
Fang, C
Lentz, G
Fialkow, M
Kirby, AC
Waldorf, KMA
Veazey, RS
Germann, A
Von Briesen, H
McElrath, MJ
Dezzutti, CScsd13@pitt.eduCSD13
Sinclair, E
Baker, CAR
Shacklett, BL
Gao, D
Hladik, F
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorReeves, R. KeithUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, or Units > Magee-Women's Research Institute
Date: 1 May 2016
Date Type: Publication
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Journal or Publication Title: PLoS ONE
Volume: 11
Number: 5
DOI or Unique Handle: 10.1371/journal.pone.0156293
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Medicine
Refereed: Yes
Date Deposited: 22 Dec 2016 16:02
Last Modified: 26 Jan 2019 19:55
URI: http://d-scholarship.pitt.edu/id/eprint/28226

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