Department of Dermatology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany.
Children's Hospital, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany.
Int J Mol Sci. 2022 Jun 11;23(12):6543. doi: 10.3390/ijms23126543.
Hepatocytes comprise the majority of the liver and largely exert metabolic functions, whereas non-parenchymal cells (NPCs)-comprising Kupffer cells, dendritic cells and liver sinusoidal endothelial cells-control the immunological state within this organ. Here, we compared the suitability of two isolation methods for murine liver NPCs. Liver perfusion (LP) with collagenase/DNase I applied via the portal vein leads to efficient liver digestion, whereas the modified liver dissociation (LD) method combines mechanical dissociation of the retrieved organ with enzymatic degradation of the extracellular matrix. In cases of both LP and LD, NPCs were enriched by subsequent gradient density centrifugation. Our results indicate that LP and LD are largely comparable with regards to the yield, purity, and composition of liver NPCs. However, LD-enriched liver NPCs displayed a higher degree of activation after overnight cultivation, and accordingly were less responsive towards stimulation with toll-like receptor ligands that are frequently used as adjuvants, e.g., in nano-vaccines. We conclude that LP is more suitable for obtaining liver NPCs for subsequent in vitro studies, whereas LD as the less laborious method, is more convenient for parallel isolation of larger numbers of samples for ex vivo analysis.
肝细胞构成了肝脏的大部分，主要发挥代谢功能，而非实质细胞（包括库普弗细胞、树突状细胞和肝窦内皮细胞）控制着该器官内的免疫状态。在这里，我们比较了两种分离小鼠肝脏非实质细胞（NPCs）的方法的适用性。门静脉内应用胶原酶/DNase I 进行肝脏灌流（LP）可有效进行肝脏消化，而改良的肝脏解离（LD）方法则将回收器官的机械解离与细胞外基质的酶降解相结合。在 LP 和 LD 的情况下，通过后续的梯度密度离心来富集 NPCs。我们的结果表明，LP 和 LD 在肝脏 NPCs 的产量、纯度和组成方面基本相当。然而，LD 富集的肝脏 NPCs 在过夜培养后表现出更高程度的激活，因此对经常用作佐剂的 Toll 样受体配体的刺激反应性较低，例如在纳米疫苗中。我们得出结论，LP 更适合用于获得用于后续体外研究的肝脏 NPCs，而 LD 作为一种不那么繁琐的方法，更适合于并行分离大量样本进行体外分析。
