一种改进的小鼠原代肝细胞分离时间和劳动效率方案
October 25th, 2021
原代肝细胞是 研究体外肝脏反应和新陈代谢的宝贵工具。利用市售试剂，开发了一种改进的小鼠原代肝细胞分离时间和劳动效率方案。
小鼠原代肝细胞在肝脏研究中很重要。特别适用于葡萄糖代谢和肝脏药物检测。从时间上讲，隔离过程可能非常耗时且能源昂贵。
该协议可以提供一种有效的方法来分离小鼠原代肝细胞。这是时间和劳动友好的。对于所有区域，只需很少的步骤即可实现。
首先使用蠕动泵开始以每分钟4 mL的速度泵送预热的双蒸馏水，持续5分钟。然后将泵送管从水改为预热的灌注介质。监测水和灌注介质之间的气泡。
将有助于跟踪灌注介质通过系统的流动。接下来用70%乙醇对麻醉的八周龄C57黑6J雌性小鼠的腹部进行消毒。并用剪刀切开腹部，露出肝脏，门静脉和下腔静脉。
然后停止蠕动泵。在确保泵送管中含有灌注介质而不是水之后。将 24 号导管插入下腔静脉。
重新打开泵，切开门静脉。在泵送灌注介质时，每分钟按压门静脉，让液体到达肝脏的每个角落。继续泵送约三到五分钟，或直到冲洗出的液体变清。
接下来将泵送管从灌注培养基改为胶原酶分散酶培养基。并每分钟按压门静脉，让液体到达肝脏的每个角落。继续泵送，直到所有 25 mL 胶原酶分散酶培养基耗尽。
接下来分离整个肝脏，没有胆囊。并将其置于30 mL洗涤培养基中，置于冰上的培养皿中。使用镊子将肝脏撕成碎片，将原代肝细胞释放到溶液中。
在这个阶段，洗涤培养基变成含有释放的原代肝细胞和小肝碎片的浑浊溶液。通过70微米的细胞过滤器过滤这种浑浊的溶液，在50 mL的冰管中加入20 mL，每次调用1X HBSS。并通过倒置管子20次来混合溶液。
接下来离心溶液。然后在组织培养罩中，吸出上清液。并用30mL冷洗培养基洗涤沉淀。
再次用离心沉淀细胞。除去上清液，并将沉淀重新悬浮在25mL培养基中。计数细胞并将其接种在所需的培养板上。
根据实验设计。接种后，分离的原代肝细胞牢固附着1小时，12小时后完全扩增。在分离的肝细胞中，与整个肝脏相比，肝细胞标志物（如转甲状腺素，CD95，ASGR1和ASGR2）的mRNA水平显着增加。
相比之下，免疫细胞，星状细胞和内皮细胞的存在较低。CD45、胶原蛋白 1 型 α 1 和内皮细胞 2 激酶 2 mRNA 水平急剧下降。这表明该方案可以大大减少来自其他肝细胞的干扰。
电镀后，ASGR1和ASGR2水平随时间下降，但直到12小时时间点仍与整个肝脏相当。特别是对于ASGR1。膜结合CD81的表达水平在长达48小时内保持一致。
相比之下，Toll样受体4的表达通常在48小时后增加并变得高于体内水平。胰岛素敏感性测定表明，胰岛素在丝氨酸473处显着促进两种AKT的磷酸化。叉头盒01在丝氨酸256处，在肝细胞中。
表明原发性肝细胞对胰岛素的敏感性。磷酸烯醇丙酮酸羧激酶蛋白水平在胰高血糖素处理后显著升高。表明葡萄糖产生途径被激活。
这种激活通过增加葡萄糖产生得到进一步证实。还有其他葡萄糖生产刺激剂，如毛喉素+ IBMX。该方案可以节省使用小鼠原代肝细胞研究的时间和精力。
按照该协议，可以进行相关实验，例如假设的葡萄糖代谢和药物生物标志物测定。
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概览
本研究提出了一种分离小鼠原代肝细胞的高效、省时协议，这对于肝脏功能和代谢的研究至关重要。该方法利用市售试剂来提高产量并最大限度地减少非实质细胞的干扰。
关键研究组成部分
研究领域
- 肝脏代谢
- 葡萄糖代谢
- 药物测试
背景
- 小鼠原代肝细胞对于研究肝脏生物学至关重要。
- 传统的分离方法通常耗时且费力。
- 缩短分离时间可以促进更高效的生物实验。
使用的方法
- 使用蠕动泵和胶原酶/弹性酶培养基的分离协议。
- 小鼠模型：C57BL/6J雌性小鼠。
- 细胞培养和基因表达测量的技术。
主要结果
- 与整个肝脏相比，肝细胞标记物的mRNA水平增加。
- 分离后，免疫和内皮细胞的存在减少。
- 展示了胰岛素敏感性和激活的葡萄糖生产途径。
结论
- 该协议显著提高了原代肝细胞分离的效率。
- 增强了对肝脏研究中葡萄糖代谢和药物反应的理解。
常见问题解答
Executive Industry Relevance
Efficient isolation of primary hepatocytes is critical for liver metabolism and drug testing studies, where time and labor constraints impact throughput in discovery workflows. This protocol reduces isolation time to ~35 minutes while maintaining hepatocyte yield and purity, supporting rapid hypothesis testing in preclinical models. The method enables scalable preparation of disease-relevant systems for mechanistic de-risking in target validation and lead identification pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Provides purified hepatocytes for interrogating therapeutic hypotheses in glucose metabolism and hepatic drug response pathways.
- Operational Value: Reduces time and labor burden, enabling higher throughput in target validation assays.
- Predictive Value: Yields hepatocytes with demonstrated insulin sensitivity and gluconeogenesis activation, supporting mechanistic de-risking of liver targets.
Screening & Assay Development
- Scientific Value: Delivers hepatocytes with low non-parenchymal cell interference, improving assay specificity for compound screening.
- Operational Value: Uses commercially available reagents and standardized steps, enhancing reproducibility across screening campaigns.
- Scalability: Enables preparation of validated biological systems suitable for high-content or phenotypic screening platforms.
Translational & Preclinical Research
- Translational Continuity: Supports disease-relevant systems for preclinical validation of liver-targeted compounds.
- Risk-Adjusted Advancement: Generates hepatocytes with validated glucose production and insulin response, informing go/no-go decisions.
- Biomarker Alignment: Maintains hepatocyte marker expression (ASGR1, ASGR2, CD81) for up to 12 hours, enabling biomarker-assay compatibility.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation through lead identification to preclinical studies, providing a reliable source of primary hepatocytes for liver metabolism assays.
- Discovery Biology: Enables hypothesis testing of hepatic pathways by delivering hepatocytes with confirmed marker expression and functional responses.
- Screening: Produces hepatocytes suitable for compound evaluation due to reduced stromal cell contamination and consistent insulin sensitivity.
- Analytics: Supports quantitative readouts such as glucose production, phosphorylation assays, and mRNA expression of hepatocyte markers.
- Translational Research: Connects discovery to preclinical work by providing hepatocytes that maintain key metabolic functions for up to 48 hours.
- Enterprise Reuse: Establishes a reusable isolation capability that standardizes hepatocyte preparation across projects and teams.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in liver metabolism studies through reduced cellular interference and functional validation.
- Operational Value: Streamlines workflow with minimal steps and commercially available reagents, improving lab efficiency.
- Strategic Value: Accelerates discovery timelines by reducing isolation time from hours to ~35 minutes, increasing assay frequency.
- Portfolio Impact: Enables risk-adjusted prioritization of liver targets by providing reliable preclinical models for mechanism validation.
Implementation Considerations
- Requires expertise in mouse surgery and perfusion techniques.
- Depends on access to a peristaltic pump and sterile tissue culture environment.
- Necessitates standardized reagent preparation (perfusion medium, collagenase dispase, wash and culture media).
- Involves adaptation considerations for different mouse strains or ages beyond the C57BL/6J female model used.
- Limited by the need for immediate use of isolated hepatocytes to maintain functional integrity, as marker expression declines after 12 hours.
