An adaptable soft-mold embossing process for fabricating optically-accessible, microfeature-based culture systems and application toward liver stage antimalarial compound testing.

Steven P Maher,Chiara Andolina,Chris Palmiotti,Jetsumon Sattabongkot,Amy J Conway,Jessica Turgeon,Alison Roth,Raahil Patel,Samantha J Barnes,Virginia Van Grod,Phillip Cualing,Dennis E Kyle,Rays H Y Jiang,Brice Campo,Naresh Singh,Myles Johnson,Victor Chaumeau,François Nosten,James C Hsiao ,Swamy Rakesh Adapa ,Wajeeh Saadi ,H-C Steve Sun ,John Adams ,Caroline Robert

doi:10.1039/c9lc00921c

Abstract

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.

Highlights

In vitro organ models are gaining popularity as tools for drug discovery, due to their potential to serve as more predictive models of human diseases.[1]
These and other long-term primary hepatocyte culture methods demonstrate the key to long-term culture is to maintain hepatocyte membrane polarization and cuboidal morphology characteristically found in vivo, thereby preventing dedifferentiation and stabilizing function in vitro.[12,13,14]
To fully optimize our μHEP system for liver stage (LS) parasite formation we hypothesized LS parasite formation rate could be dependent on the specific microfeature design, and the identification of an ideal design could be predicted by how each design supports hepatic differentiation, measured by individual hepatic phenotypes such as production of albumin and factor IX, or induction of CYP3A4

Summary

Introduction

In vitro organ models are gaining popularity as tools for drug discovery, due to their potential to serve as more predictive models of human diseases.[1]. We hypothesized we could engineer microfeatures onto the culture area of a microtiter plate, maintain optical accessibility for HC/RI, and fabricate the plate in a one-step process, thereby making microfeaturebased hepatocyte culture more congruent with existing laboratory equipment and in a throughput amendable to many applications, including liver stage antimalarial drug development

Methods

Results

Conclusion