Maher Steven P, Conway Amy J, Roth Alison, Adapa Swamy R, Cualing Phillip, Andolina Chiara, Hsiao James, Turgeon Jessica, Chaumeau Victor, Johnson Myles, Palmiotti Chris, Singh Naresh, Barnes Samantha J, Patel Raahil, Van Grod Virginia, Carter Robert, Sun H-C Steve, Sattabongkot Jetsumon, Campo Brice, Nosten François, Saadi Wajeeh M, Adams John H, Jiang Rays H Y, Kyle Dennis E
Center for Global Health and Infectious Diseases Research, Department of Global Health, College of Public Health, University of South Florida, Tampa, Florida, USA.
Shoklo Malaria Research Unit, Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand & Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Lab Chip. 2020 Mar 17;20(6):1124-1139. doi: 10.1039/c9lc00921c.
Advanced cell culture methods for modeling organ-level structure have been demonstrated to replicate in vivo conditions more accurately than traditional in vitro cell culture. Given that the liver is particularly important to human health, several advanced culture methods have been developed to experiment with liver disease states, including infection with Plasmodium parasites, the causative agent of malaria. These models have demonstrated that intrahepatic parasites require functionally stable hepatocytes to thrive and robust characterization of the parasite populations' response to investigational therapies is dependent on high-content and high-resolution imaging (HC/RI). We previously reported abiotic confinement extends the functional longevity of primary hepatocytes in a microfluidic platform and set out to instill confinement in a microtiter plate platform while maintaining optical accessibility for HC/RI; with an end-goal of producing an improved P. vivax liver stage culture model. We developed a novel fabrication process in which a PDMS soft mold embosses hepatocyte-confining microfeatures into polystyrene, resulting in microfeature-based hepatocyte confinement (μHEP) slides and plates. Our process was optimized to form both microfeatures and culture wells in a single embossing step, resulting in a 100 μm-thick bottom ideal for HC/RI, and was found inexpensively amendable to microfeature design changes. Microfeatures improved intrahepatic parasite infection rates and μHEP systems were used to reconfirm the activity of reference antimalarials in phenotypic dose-response assays. RNAseq of hepatocytes in μHEP systems demonstrated microfeatures sustain hepatic differentiation and function, suggesting broader utility for preclinical hepatic assays; while our tailorable embossing process could be repurposed for developing additional organ models.
用于模拟器官水平结构的先进细胞培养方法已被证明比传统的体外细胞培养更能准确地复制体内条件。鉴于肝脏对人类健康尤为重要，已经开发了几种先进的培养方法来实验肝脏疾病状态，包括感染疟原虫，即疟疾的病原体。这些模型表明，肝内寄生虫需要功能稳定的肝细胞才能茁壮成长，而对寄生虫群体对研究性疗法反应的强有力表征依赖于高内涵和高分辨率成像（HC/RI）。我们之前报道过非生物限制可延长微流控平台中原代肝细胞的功能寿命，并着手在微量滴定板平台中引入限制，同时保持HC/RI的光学可达性；最终目标是建立一个改进的间日疟原虫肝期培养模型。我们开发了一种新颖的制造工艺，其中PDMS软模具将肝细胞限制微特征压印到聚苯乙烯中，从而得到基于微特征的肝细胞限制（μHEP）载玻片和平板。我们的工艺经过优化，可在单个压印步骤中形成微特征和培养孔，从而得到100μm厚的底部，非常适合HC/RI，并且发现可以低成本地对微特征设计进行更改。微特征提高了肝内寄生虫感染率，并且μHEP系统被用于在表型剂量反应试验中重新确认参考抗疟药的活性。μHEP系统中肝细胞的RNA测序表明微特征维持肝分化和功能，这表明在临床前肝脏检测中具有更广泛的用途；而我们可定制的压印工艺可重新用于开发其他器官模型。
