Extending the shelf life of HLM chips through freeze-drying of human liver microsomes immobilized onto thiol–ene micropillar arrays†

IF 6.1 2区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS
Lab on a Chip Pub Date : 2024-07-31 DOI:10.1039/D4LC00429A
Iiro Rautsola, Markus Haapala, Leo Huttunen, Ossi Korhonen and Tiina Sikanen
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Abstract

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic in vitro organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping. The shelf life of the HLM chips in the wetted state is ca. 2–3 weeks only and requires cold storage at 4 °C. In this study, we developed a freeze-drying method for lyophilization of HLMs that are readily immobilized inside microfluidic pillar arrays made from off-stoichiometric thiol–ene polymer. The success of lyophilization was evaluated by monitoring the cytochrome P450 and UDP-glucuronosyltransferase enzyme activities of rehydrated HLMs for several months post-freeze-drying. By adapting the freeze-drying protocol, the HLM chips could be stored at room temperature (protected from light and moisture) for at least 9 months (n = 2 independent batches) and up to 16 months at best, with recovered enzyme activities within 60–120% of the non-freeze-dried control chips. This is a major improvement over the cold-storage requirement and the limited shelf life of the non-freeze-dried HLM chips, which can significantly ease the design of experiments, decrease energy consumption during storage, and reduce the shipping costs with a view to commercial adaptation.

Abstract Image

通过冷冻干燥固定在硫醇烯微柱阵列上的人肝脏微粒体来延长 HLM 芯片的保质期
用固定化人肝微粒体(HLM 芯片)功能化的微流体流动反应器是药物发现和开发的有力工具,可在流动条件下进行基于机理的酶抑制研究。此外,HLM 芯片还可用于简化人类药物代谢物的生产,以用于后续的微流控体外器官模型,或作为代谢物标准用于药物安全性评估。然而,生物功能化微反应器的保质期有限,这通常会在储存和运输方面对其商业化应用构成重大障碍。湿润状态下的 HLM 芯片保质期大约只有 2-3 周,并且需要在 4°C 下冷藏。在本研究中,我们开发了一种冻干方法,用于将 HLM 冻干,使其易于固定在由非计量硫醇烯聚合物制成的微流控柱阵中。通过监测冻干后几个月内重新水合的 HLMs 的细胞色素 P450 和 UDP-葡萄糖醛酸转移酶的酶活性,评估了冻干的成功率。通过调整冻干方案,HLM 切片可在室温下(避光和防潮)保存至少 9 个月(2 个独立批次),最长可达 16 个月,恢复的酶活性为未冻干对照组的 60-120%。这与非冻干 HLM 芯片的低温储存要求和有限的货架期相比是一个重大改进,可大大简化实验设计,降低储存过程中的能耗,并减少运输成本,从而实现商业化应用。
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来源期刊
Lab on a Chip
Lab on a Chip 工程技术-化学综合
CiteScore
11.10
自引率
8.20%
发文量
434
审稿时长
2.6 months
期刊介绍: Lab on a Chip is the premiere journal that publishes cutting-edge research in the field of miniaturization. By their very nature, microfluidic/nanofluidic/miniaturized systems are at the intersection of disciplines, spanning fundamental research to high-end application, which is reflected by the broad readership of the journal. Lab on a Chip publishes two types of papers on original research: full-length research papers and communications. Papers should demonstrate innovations, which can come from technical advancements or applications addressing pressing needs in globally important areas. The journal also publishes Comments, Reviews, and Perspectives.
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