Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn's disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. By review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared with human and rodent MSCs, and limited lifespan is an obstacle for preclinical investigation and therapeutic use. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories are problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average >90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.

中文翻译：

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脐带来源的犬间质基质细胞的分离，培养和冷冻保存的协议：细胞附着在长期维持中的作用。

间充质基质细胞（MSCs）在再生医学领域具有广阔的前景，因为它们能够在诸如克罗恩氏病和骨关节炎之类的损伤中或通过掺入组织工程构建体中产生可变的局部抗炎作用。当前，MSC文献将啮齿动物用于临床前疾病模型。在大型动物中使用自然发生的疾病来模拟人类疾病的兴趣日益浓厚。通过对犬MSC文献的回顾，看来犬MSC可能难以在培养物中维持较长的传代时间，并且与人和啮齿类MSC相比，在组织来源之间存在很大差异，并且使用寿命有限是临床前研究和治疗用途的障碍。使用犬类MSC的研究一直集中在源自骨髓或脂肪组织的细胞上，由于缺乏标准化，实验室之间制造MSC的差异是有问题的。为了解决这些问题，在这里，采用分步过程来优化犬MSC的分离，扩增和利用犬脐带来源的MSC进行冷冻保存。培养方案利用组织培养表面的涂层，该涂层增加细胞粘附，增加集落形成单位-成纤维细胞效率并减少群体倍增时间。用我们的方案分离的犬MSC的维护时间比已发表的犬MSC的检测方法更长。我们改良的冷冻保存方案可在融化时平均产生90％以上的存活MSC。