Cryopreservation of human mucosal tissues

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.

研究背景
从宫颈阴道黏膜与结直肠黏膜中分离得到的白细胞的冷冻保存，可用于细胞免疫相关研究（详见Hughes SM等，《PLOS ONE》2016年发表的研究）。然而，针对黏膜生物学与性传播感染的部分研究问题，使用完整黏膜组织开展实验能获得更优的研究结果，但目前尚无针对完整黏膜组织的标准冷冻保存方案。

研究方法与结果
为筛选适配黏膜组织的最优保存方案，我们分别测试了两种冷冻保存策略：一是以10%二甲基亚砜（dimethylsulfoxide）为冷冻保护剂的慢速降温法（降温速率1℃/分钟，以下简称“冷冻保存法”）；二是以20%二甲基亚砜与20%乙二醇（ethylene glycol）为冷冻保护剂的快速降温法（直接投入液氮，以下简称“玻璃化（vitrification）法”）。
我们通过一系列实验手段，对比了新鲜与人源宫颈阴道、结直肠保存组织的多项指标，包括代谢活性、人类免疫缺陷病毒（HIV）感染性、细胞表型、经苏木精-伊红染色（hematoxylin-and-eosin staining）观察的组织结构、细胞数量与活力、细胞因子分泌水平以及杀微生物剂药物浓度。
在阴道组织样本中，冷冻保存法与玻璃化法处理后的组织在代谢活性、HIV感染性以及组织结构方面表现相近。但结直肠黏膜组织经玻璃化法处理后，细胞回收率极低：从玻璃化处理的结直肠组织中分离得到的细胞数量，较冷冻保存法处理的样本减少90%。
新鲜与冷冻保存的宫颈外上皮组织的HIV感染率相近，但冷冻保存的结直肠组织较新鲜组织更难被感染（风险比0.7，95%置信区间0.4~1.2）。最后，我们对比了冷冻保存前后的细胞分离效果：先冷冻保存再解冻后分离得到的细胞回收率（71%，95%置信区间59%~84%）高于先分离细胞再进行冷冻保存的组别（50%，95%置信区间38%~62%）。两种方式处理后的细胞功能均与新鲜组织相近。此外，冷冻保存的组织外植体中的杀微生物剂药物浓度低于新鲜组织样本。

研究结论
采用二甲基亚砜对完整宫颈阴道与结直肠组织进行冷冻保存，可适配多数实验检测场景，而玻璃化法的适用范围相对有限。从完整组织块经冷冻保存后回收的细胞产量，高于先分离细胞再冷冻保存后解冻得到的单细胞悬液，但二者的T细胞功能无显著差异。