Clinical hepatocyte transplantation short-term efficacy has been demonstrated; however, some major limitations, mainly due to the shortage of organs, the lack of quality of isolated cells and the low cell engraftment after transplantation, should be solved for increasing its efficacy in clinical applications. Cellular stress during isolation causes an unpredictable loss of attachment ability of the cells, which can be aggravated by cryopreservation and thawing. In this work, we focused on the use of a Good Manufacturing Practice (GMP) solution compared with the standard cryopreservation medium, the University of Wisconsin medium, for the purpose of improving the functional quality of cells and their ability to engraft in vivo, with the idea of establishing a biobank of cryopreserved human hepatocytes available for their clinical use. We evaluated not only cell viability but also specific hepatic function indicators of the functional performance of the cells such as attachment efficiency, ureogenic capability, phase I and II enzymes activities and the expression of specific adhesion molecules in vitro. Additionally, we also assessed and compared the in vivo efficacy of human hepatocytes cryopreserved in different media in an animal model of acute liver failure. Human hepatocytes cryopreserved in the new GMP solution offered better in vitro and in vivo functionality compared with those cryopreserved in the standard medium. Overall, the results indicate that the new tested GMP solution maintains better hepatic functions and, most importantly, shows better results in vivo, which could imply an increase in long-term efficacy when used in patients.

中文翻译：

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临床级冷冻人肝细胞对急性肝衰竭小鼠体内疗效的改善

临床肝细胞移植的短期疗效已得到证实；然而，一些主要的局限性，主要是由于器官短缺、分离细胞质量不足和移植后细胞植入率低，需要解决一些主要限制，以提高其在临床应用中的疗效。分离过程中的细胞压力会导致细胞附着能力的不可预测的丧失，冷冻保存和解冻可能会加剧这种情况。在这项工作中，我们专注于使用良好生产规范 (GMP) 解决方案与标准冷冻保存培养基威斯康星大学培养基相比，目的是提高细胞的功能质量及其在体内移植的能力，建立一个可供临床使用的冷冻人肝细胞生物库的想法。我们不仅评估了细胞活力，还评估了细胞功能性能的特定肝功能指标，如附着效率、产尿能力、I 期和 II 期酶活性以及体外特定粘附分子的表达。此外，我们还评估和比较了在急性肝功能衰竭动物模型中不同培养基中冷冻保存的人肝细胞的体内功效。与在标准培养基中冷冻保存的人肝细胞相比，在新 GMP 溶液中冷冻保存的人肝细胞具有更好的体外和体内功能。总体而言，结果表明新测试的 GMP 解决方案保持了更好的肝功能，最重要的是，在体内显示出更好的结果，这可能意味着在患者中​​使用时长期疗效会增加。