title: Cryopreservation of Human Mucosal Leukocytes.
abstract: 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.
  
  
  RESULTS
  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.
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Sean M
authors.identifier: http://orcid.org/0000-0002-9409-9405
authors.initials: SM
authors.lastname: Hughes
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.|School of Mechanical and Materials Engineering, Washington State University, Everett, Washington, United States of America.
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Shu
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Claire N
authors.initials: CN
authors.lastname: Levy
authors.affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: April L
authors.initials: AL
authors.lastname: Ferre
authors.affiliation: Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Heather
authors.initials: H
authors.lastname: Hartig
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Cifeng
authors.initials: C
authors.lastname: Fang
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Gretchen
authors.initials: G
authors.lastname: Lentz
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Fialkow
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Anna C
authors.initials: AC
authors.lastname: Kirby
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Adams Waldorf
authors.affiliation: Division of Comparative Pathology, Tulane National Primate Research Center, Tulane University School of Medicine, Covington, Louisiana, United States of America.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Veazey
authors.affiliation: Division of Medical Biotechnology, Fraunhofer Institute for Biomedical Engineering IBMT, Sulzbach/Saar, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Germann
authors.affiliation: Division of Medical Biotechnology, Fraunhofer Institute for Biomedical Engineering IBMT, Sulzbach/Saar, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: von Briesen
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.|Department of Medicine, University of Washington, Seattle, Washington, United States of America.|Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.|Department of Global Health, University of Washington, Seattle, Washington, United States of America.
authors.forename: M Juliana
authors.initials: MJ
authors.lastname: McElrath
authors.affiliation: School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.|Magee-Womens Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Charlene S
authors.initials: CS
authors.lastname: Dezzutti
authors.affiliation: Division of Experimental Medicine, Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sinclair
authors.affiliation: Division of Experimental Medicine, Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America.|Core Immunology Lab, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Chris A R
authors.initials: CA
authors.lastname: Baker
authors.affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California Davis, Davis, California, United States of America.|Department of Medicine, Division of Infectious Diseases, School of Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Shacklett
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.|Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Dayong
authors.initials: D
authors.lastname: Gao
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.|Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.|Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Florian
authors.initials: F
authors.lastname: Hladik
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D005260:Female; D006801:Humans; D007962:Leukocytes / Q000166:cytology*; D009092:Mucous Membrane / Q000166:cytology*; D014621:Vagina / Q000166:cytology