Dynamic42 GmbH, Jena, Germany.
upcyte technologies GmbH, Hamburg, Germany.
Toxicology. 2023 Jan 1;483:153374. doi: 10.1016/j.tox.2022.153374. Epub 2022 Nov 14.
Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells forming the hepatic sinusoidal wall. Besides their high endocytic potential, LSECs have been demonstrated to markedly contribute to liver homeostasis and immunity, and may partially explain unexpected hepatotoxicity of drug candidates. However, their use for in vitro investigations is compromised by poor cell yields and a limited proliferation capacity. Here, we report the transient expansion of primary human LSECs from three donors by lentiviral transduction. Transduced ("upcyte®") LSECs were able to undergo at least 25 additional population doublings (PDs) before growth arrest due to senescence. Expanded upcyte® LSECs maintained several characteristics of primary LSECs, including expression of surface markers such as MMR and LYVE-1 as well as rapid uptake of acetylated LDL and ovalbumin. We further investigated the suitability of expanded upcyte® LSECs and proliferating upcyte® hepatocytes for detecting acetaminophen toxicity at millimolar concentrations (0, 0.5, 1, 2, 5, 10 mM) in static 2D cultures and a microphysiological 3D model. upcyte® LSECs exhibited a higher sensitivity to acetaminophen-induced toxicity compared to upcyte® hepatocytes in 2D culture, however, culturing upcyte® LSECs together with upcyte® hepatocytes in a co-culture reduced APAP-induced toxicity compared to 2D monocultures. A perfused Dynamic42 3D model was more sensitive to acetaminophen than the 2D co-culture model. Cytotoxicity in the 3D model was evident by decreased cellular viability, elevated LDH release, reduced nuclei counts and impaired cell morphology. Taken together, our data demonstrate that transient expansion of LSECs represents a suitable method for generation of large quantities of cells while maintaining many characteristics of primary cells and responsiveness to acetaminophen.
肝窦内皮细胞（LSEC）是高度特化的内皮细胞，构成肝窦壁。除了高内吞能力外，LSEC 还显著促进肝脏稳态和免疫，并且可能部分解释候选药物的意外肝毒性。然而，由于细胞产量低和增殖能力有限，它们在体外研究中的应用受到限制。在这里，我们报告了通过慢病毒转导从三个供体短暂扩增原代人 LSEC。转导的（“upcyte®”）LSEC 能够在衰老导致生长停滞之前至少进行 25 次额外的群体倍增（PD）。扩增的 upcyte® LSEC 保持了原代 LSEC 的几个特征，包括表达表面标志物，如 MMR 和 LYVE-1 以及快速摄取乙酰化 LDL 和卵清蛋白。我们进一步研究了扩增的 upcyte® LSEC 和增殖的 upcyte® 肝细胞在静态 2D 培养物和微生理 3D 模型中检测毫米浓度（0、0.5、1、2、5、10 mM）对乙酰氨基酚毒性的适用性。在 2D 培养物中，与 upcyte® 肝细胞相比，upcyte® LSEC 对乙酰氨基酚诱导的毒性更敏感，然而，在共培养物中培养 upcyte® LSEC 与 upcyte® 肝细胞可降低与 2D 单培养物相比的 APAP 诱导的毒性。灌注的 Dynamic42 3D 模型对乙酰氨基酚的敏感性高于 2D 共培养模型。3D 模型中的细胞毒性通过降低细胞活力、升高 LDH 释放、减少核计数和损害细胞形态来证明。总之，我们的数据表明，LSEC 的瞬时扩增代表了一种生成大量细胞的合适方法，同时保持原代细胞的许多特征和对乙酰氨基酚的反应性。
