Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction

Abstract

There is a significant need for in vitro methods to study drug-induced liver injury that are rapid, reproducible, and scalable for existing high-throughput systems. However, traditional monolayer and suspension cultures of hepatocytes are difficult to handle and risk the loss of phenotype. Generally, three-dimensional (3D) cell culture platforms help recapitulate native liver tissue phenotype, but suffer from technical limitations for high-throughput screening, including scalability, speed, and handling. Here, we developed a novel assay for cytochrome P450 (CYP450) induction/inhibition using magnetic 3D cell culture that overcomes the limitations of other platforms by aggregating magnetized cells with magnetic forces. With this platform, spheroids can be rapidly assembled and easily handled, while replicating native liver function. We assembled spheroids of primary human hepatocytes in a 384-well format and maintained this culture over five days, including a 72 h induction period with known CYP450 inducers/inhibitors. CYP450 activity and viability in the spheroids were assessed and compared in parallel with monolayers. CYP450 activity was induced/inhibited in spheroids as expected, separate from any toxic response. Spheroids showed a significantly higher baseline level of CYP450 activity and induction over monolayers. Positive staining in spheroids for albumin and multidrug resistance-associated protein (MRP2) indicates the preservation of hepatocyte function within spheroids. The study presents a proof-of-concept for the use of magnetic 3D cell culture for the assembly and handling of novel hepatic tissue models.

Engineered Liver-On-A-Chip Platform to Mimic Liver Functions and Its Biomedical Applications: A Review

“…Hepatic cell lines, such as HepG2 (derived from human hepatocellular carcinoma of a 15-year-old male) and HepaRG (terminally differentiated hepatic cells derived from a hepatic progenitor cell line of a female hepatocarcinoma), have been widely used in toxicological investigations, because they have a stable phenotype, are essential for drug metabolism and toxicity responses, and are easily manipulated with unlimited proliferation [59,60]. For example, HepG2 cells have been used for toxicological and pharmacological research since the 1970s.…”

“…The cell-repellent plate method is commonly used to self-assemble liver cell spheroids [59][60][61]69]. Desai et al used magnetic nanoparticles to modify PHHs and then applied magnetic force to rapidly assemble and easily handle the spheroids [59].…”

“…The cell-repellent plate method is commonly used to self-assemble liver cell spheroids [59][60][61]69]. Desai et al used magnetic nanoparticles to modify PHHs and then applied magnetic force to rapidly assemble and easily handle the spheroids [59]. Moreover, in toxicological applications, by integrating microsensors with a liver-on-a-chip, the metabolic parameters and cell viability of a single spheroid could be monitored without microscopy [60].…”

Cell Aggregation Culture Induces Functional Differentiation of Induced Hepatocyte-like Cells through Activation of Hippo Signaling

“…Thus, in vitro analyses of hepatocyte-specific properties are important for biomedical studies and drug development. However, because it is difficult to expand and maintain differentiated hepatocytes in culture (Desai et al, 2017;Guo et al, 2017;Khetani and Bhatia, 2008), alternative cell sources for hepatocytes are desirable for analyses of hepatic functions in vitro. As an alternative to hepatocytes, hepatocyte-like cells can be induced from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Sancho-Bru et al, 2011;Si-Tayeb et al, 2010b).…”

A magnetic three‐dimensional levitated primary cell culture system for the development of secretory salivary gland‐like organoids

“…Moreover, the nanoparticles used to render these cells magnetic are non‐toxic as they do not affect normal cellular proliferation and homeostasis of in vivo organs, and neither induce an inflammatory response (Daquinag et al, ; Jaganathan et al, ; Lin et al, ; Souza et al, ; Tseng et al, ; Tseng et al, ). So far, M3DL‐based organoids have been developed for many applications such as drug toxicity screening; regeneration of tissues in vitro including liver, adipose, and lung; and to recapitulate 3D breast tumours (Daquinag et al, ; Desai, Tseng, & Souza, ; Jaganathan et al, ; Tseng et al, ). Yet the ability of this M3DL system in forming functional SG tissue organoids or mini‐glands remains unknown.…”