PDF(Pubmed)
Abstract：
Glycerol and dimethyl sulfoxide (DMSO) are widely used cryoprotectants for freezing human cell cultures. During the manufacturing process of ocular stem cell-based autographs, ex vivo cultivated ocular cells are cryopreserved for quality control purposes in accordance with regulatory requirements. The efficiency of the cryopreservation methods is limited by their effect on cell survival and quality. We compared two cryopreservation reagents, glycerol and DMSO, for their influence on the survival and quality of human primary conjunctival cultures. We found increased cell viability after cryopreservation in DMSO compared to cryopreservation in glycerol. The clonogenic and proliferative capacity was unaffected by the cryopreservation reagents, as shown by the colony forming efficiency and cumulative cell doubling. Importantly, the percentage of p63α- and keratin 19 (K19)-positive cells following cryopreservation in DMSO or glycerol was comparable. Taken together, our results demonstrate that cryopreservation in DMSO improves cell survival compared to cryopreservation in glycerol, with no subsequent effect on cell proliferative-, clonogenic-, or differentiation capacity. Therefore, we advise the use of a 10% DMSO-based cryopreservation medium for the cryopreservation of human primary conjunctival cells, as it will improve the number of cells available for the manufacturing of conjunctival stem cell-based autografts for clinical use.
摘要：
甘油和二甲基亚砜(DMSO)是广泛用于冷冻人细胞培养物的冷冻保护剂。在基于眼干细胞的亲笔签名的制造过程中，根据监管要求,将离体培养的眼细胞冷冻保存用于质量控制目的。冷冻保存方法的效率受限于它们对细胞存活和质量的影响。我们比较了两种冷冻保存试剂,甘油和DMSO，它们对人类原发性结膜培养物的生存和质量的影响。我们发现与在甘油中冷冻保存相比,在DMSO中冷冻保存后细胞活力增加。克隆形成和增殖能力不受冷冻保存试剂的影响，如集落形成效率和累积细胞倍增所示。重要的是,在DMSO或甘油中冷冻保存后，p63α-和角蛋白19（K19）-阳性细胞的百分比相当。一起来看,我们的结果表明，与甘油中的冷冻保存相比，DMSO中的冷冻保存提高了细胞存活率，对细胞增殖没有后续影响-，克隆-，或分化能力。因此,我们建议使用10％基于DMSO的冷冻保存培养基冷冻保存人原代结膜细胞，因为它将提高可用于制造临床使用的基于结膜干细胞的自体移植物的细胞数量。
