Thyroid‐on‐a‐Chip: An Organoid Platform for In Vitro Assessment of Endocrine Disruption

Abstract

Thyroid is a glandular tissue in the human body in which the function can be severely affected by endocrine disrupting chemicals (EDCs). Current in vitro assays to test endocrine disruption by chemical compounds are largely based on 2D thyroid cell cultures, which often fail to precisely evaluate the safety of these compounds. New and more advanced 3D cell culture systems are urgently needed to better recapitulate the thyroid follicular architecture and functions and help to improve the predictive power of such assays. Herein, the development of a thyroid organoid‐on‐a‐chip (OoC) device using polymeric membranous carriers is described. Mouse embryonic stem cell derived thyroid follicles are incorporated in a microfluidic chip for a 4 day experiment at a flow rate of 12 µL min−1. A reversible seal provides a leak‐tight sealing while enabling quick and easy loading/unloading of thyroid follicles. The OoC model shows a high degree of functionality, where organoids retain expression of key thyroid genes and a typical follicular structure. Finally, transcriptional changes following benzo[k]fluoranthene exposure in the OoC device demonstrate activation of the xenobiotic aryl hydrocarbon receptor pathway. Altogether, this OoC system is a physiologically relevant thyroid model, which will represent a valuable tool to test potential EDCs.

A Modular Microfluidic Organoid Platform Using LEGO‐Like Bricks

“…In a previous work, we have demonstrated that perfusion promotes the functionality of mESC‐derived thyroid organoids. [ 30 ] However, on‐chip culture of organoids was difficult to execute, in part due to the high complexity of the microfluidic apparatus and time‐consuming cell culture protocols. In the present work, Matrigel‐embedded thyroid organoids (30–100 µm), were pre‐formed and cultured in microthermoformed carriers for three days under static conditions.…”

“…The FCBs occupied a total volume of 1.3 L (excluding pump) and allowed a minimal used tubing length of 34 cm per FCB, which corresponded to a 64% drop as compared to our previously used OoC platform. [ 30 ] This decrease in tubing and connectors yielded 15× shorter assembly times of microfluidic peripherals (Figure S8, Supporting Information), facilitating the assembly of the total fluidic system by two operators within 2 h.…”

“…Organoid Culture: Thyroid organoids were differentiated from a recombinant murine embryonic stem cell (ESC) line (A2LoxNkx2-1-Pax8) as previously described. [30,34,35] In brief, ESCs were cultured on 𝛾-ray irradi-ated mouse embryonic fibroblasts (MEF) feeders in DMEM supplemented with 15% ES Cell qualified FBS (Sigma Aldrich, St. Louis, MO), IK0701 LIF (1000 U mL −1 ) (ORF Genetics, Kopavogur, Iceland), non-essential amino acids (0.1 × 10 −3 m), sodium pyruvate (1 × 10 −3 m), penicillin and streptomycin (50 U mL −1 ), and 2-mercaptoethanol (0.1 × 10 −3 m). ESCs were cultured in hanging drops (1000 cells per droplet) in differentiation medium containing DMEM supplemented with 15% FBS, vitamin C (50 μg mL −1 ), non-essential amino acids (0.1 × 10 −3 m), sodium pyruvate (1 × 10 −3 m), penicillin and streptomycin (50 U mL −1 ), and 2-mercaptoethanol (0.1 × 10 −3 m).…”

“…The polymeric carrier is a PC membrane (50 μm in thickness) that can be microthermoformed to include different patterns, such as trays, pockets, or microcavities. [30,31] Moreover, multiple cell types can be directly cultured onto the PC carrier [32] and on both sides. [33] For example, this offers the opportunity to create a vascular barrier beneath the tissue model off-chip, which once matured, can be transferred inside the OoC device.…”

In vitro construction of liver organoids with biomimetic lobule structure by a multicellular 3D bioprinting strategy

“…[3][4][5] Organoid models have gradually matured and become prominent in recent years. [6][7][8][9] Compared with traditional monolayer culture and animal models, organoids are able to partially reproduce key physiological features of the native human organs, including cell heterogeneity, spatial structure and microenvironment, and provide important cell-cell and extracellular matrix (ECM) interactions. 10,11 These can promote hepatocyte proliferation, differentiation, expression of specific genes and proteins, and response to exogenous stimuli.…”