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用于肝毒性检测的柱形/灌注板中的再生人肝类器官(HLOs)。

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays.

作者信息

Shrestha Sunil, Acharya Prabha, Kang Soo-Yeon, Vanga Manav Goud, Lekkala Vinod Kumar Reddy, Liu Jiafeng, Yang Yong, Joshi Pranav, Lee Moo-Yeal

机构信息

Department of Biomedical Engineering, University of North Texas, Denton, Texas, USA.

Bioprinting Laboratories Inc., Dallas, Texas, USA.

出版信息

bioRxiv. 2024 Nov 3:2024.03.25.586638. doi: 10.1101/2024.03.25.586638.

Abstract

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate the structure and function of human fetal liver tissues, thus being considered as a promising tissue model for liver diseases and predictive compound screening. However, the adoption of HLOs in drug discovery faces several technical challenges, which include the lengthy differentiation process with multiple culture media leading to batch-to-batch variation, short-term maintenance of hepatic functions post-maturation, low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity levels compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for organoid culture and analysis. The dynamically cultured Diff-HLOs exhibited greater maturity and reproducibility than those cultured statically, especially after a 10-day differentiation period. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate liver recovery from DILI. The assessment of liver regeneration post-injury is critical to understanding the mechanism of recovery and determining the threshold drug concentration beyond which there will be a sharp decrease in the liver's regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to the short-term differentiation of passage-able Exp-HLOs, stable hepatic function post-maturation, high reproducibility, and high throughput with capability of organoid culture, testing, staining, imaging, and analysis.

摘要

从胚胎干细胞(ESC)、诱导多能干细胞(iPSC)和成体干细胞(ASC)分化而来的人肝脏类器官(HLO)可以重现人胎儿肝脏组织的结构和功能,因此被认为是一种有前景的肝脏疾病组织模型和预测性化合物筛选模型。然而,在药物发现中采用HLO面临一些技术挑战,其中包括分化过程漫长,需使用多种培养基,导致批次间差异;成熟后肝功能的短期维持;由于基质胶解离和将HLO转移到微孔板中,检测通量较低;与原代肝细胞相比,成熟水平不足。为了解决这些问题,通过优化分化方案,从人iPSC中生成了可扩增的HLO(Exp-HLO),将其快速打印在带有侧壁和狭缝的144柱板(144PillarPlate)上,并在带有灌注孔和储液器的144灌注板(144PerfusionPlate)中动态培养长达20天,使其分化为分化型HLO(Diff-HLO),用于类器官培养和分析。动态培养的Diff-HLO比静态培养的表现出更高的成熟度和可重复性,尤其是在10天的分化期之后。此外,对柱/灌注板中的Diff-HLO用对乙酰氨基酚和曲格列酮进行3天测试,以评估药物性肝损伤(DILI),然后在扩增培养基中孵育10天,以评估肝从DILI中的恢复情况。损伤后肝脏再生的评估对于理解恢复机制和确定药物浓度阈值至关重要,超过该阈值肝脏再生能力将急剧下降。我们设想,由于可传代的Exp-HLO短期分化、成熟后肝功能稳定、可重复性高以及具有类器官培养、测试、染色、成像和分析能力的高通量,柱/灌注板中的生物打印Diff-HLO可用于肝毒性化合物的高通量筛选(HTS)。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f3a1/11541619/08b49abc4696/nihpp-2024.03.25.586638v3-f0002.jpg

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