Year: 2018
Type: article
Abstract: BACKGROUND: Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol. METHODS AND FINDINGS: To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones. CONCLUSIONS: Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.
Source: PLoS ONE
Authors: Sean M. Hughes, April L. Ferre, Sarah E. Yandura, Cory Shetler, Chris A. R. Baker, Fernanda Calienes, Claire N. Levy, Rena D. Astronomo, Zhiquan Shu, Gretchen M. Lentz, Michael Fialkow, Anna C. Kirby, M. Juliana McElrath, Elizabeth Sinclair, Lisa C. Rohan, Peter L. Anderson, Barbara L. Shacklett, Charlene S. Dezzutti, Dayong Gao, Florian Hladik
Language: English
Topic: HIV Research and Treatment
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,386
Funders: Bill and Melinda Gates Foundation, Fred Hutchinson Cancer Research Center, University of Washington, University of Pittsburgh, National Institutes of Health, Medical Center, University of Pittsburgh, Center for AIDS Research, University of Washington, National Cancer Institute, National Institute of Allergy and Infectious Diseases
Awards: G1676129921, G2173125444, G2219438539, Film Antiretroviral Microbicide Evaluation, G3085519557, G3985073149, G4160532866, Nanoparticle microbicides for delivery of combination antiretroviral drugs, G4393346530, University of Washington - to develop the optimal techniques for cryopreservation of human mucosal tissues for use in assessing HIV vaccines and microbicides., G465034039, G5012080466, G5769972797, HIV-Specific T Cell Responses in Rectal Mucosa, G6966134840, G7391576593, Laboratory Center (LC): Microbicide Trials Network, G7791962275, G9000115589, G9000186388, Cancer Center Support Grant
OpenAlex ID: W2950956622