Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of ⁵⁴Mn import and export in mutant cells. While impaired ⁵⁴Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired ⁵⁴Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated ⁵⁴Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.

锰 (Mn) 是一种必需金属，在高浓度时可能有毒。2012 年，在 SLC30A10（一种 Mn 流出转运蛋白）突变的患者中报告了 Mn 过量的第一个遗传原因。为了在体外探索 SLC30A10 的功能，本研究使用 CRISPR/Cas9 基因编辑开发了稳定的 SLC30A10 突变 Hep3B 肝癌细胞系和活小鼠中的胶原酶灌注，以分离 Slc30a10 缺陷的原代肝细胞。我们还比较了原代和非原代细胞系的表型，以确定它们是否都可以作为 SLC30A10 已知生理作用的可靠体外模型。突变体 SLC30A10 Hep3B 细胞在暴露于过量 Mn 时增加了 Mn 水平并降低了活力。运输研究表明，突变细胞的进口和出口减少了^54百万。受损时鉴于 SLC30A10 在细胞锰出口中的重要作用，假设^{54百万出口， 54}百万进口受损是出乎意料的。全基因组测序未发现突变 Hep3B 突变细胞系中已知 Mn 转运蛋白的任何其他突变。然后，我们评估了从具有不同肝锰水平的转基因小鼠中分离的原代肝细胞培养物中^{54锰的转运。}基于这些实验的结果，我们建议可以在体外研究 SLC30A10 缺乏对 Mn 稳态的影响，但仅限于特定类型的细胞系。