Abstract
BackgroundCryopreservation 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 findingsTo 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.ConclusionsCryopreservation 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.
Highlights
The availability of mucosal tissue specimens is crucial for the study of mucosal biology and immunity
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
We have recently reported methods for the cryopreservation of leukocytes isolated from mucosal tissues [1], but limited and conflicting data are available for intact tissues [2,3,4,5]
Summary
The availability of mucosal tissue specimens is crucial for the study of mucosal biology and immunity. Working with mucosal specimens is complicated by the difficulty of storing tissues long-term while maintaining good viability. A protocol for preserving tissue specimens with good viability and function would enable their collection and analysis in clinical trials involving STIs such as human immunodeficiency virus (HIV) or herpes simplex virus. These trials are conducted at sites around the world and samples must be transported to analysis laboratories. The ability to preserve tissues with good viability would allow functional assays to be performed, and might allow samples to be stored without pre-determining the type of assay to be performed on them. Storing viable samples could thereby give the investigator more options, as the viable samples could be thawed and preserved according to the needs of the assay later on
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