Development of oxidative stress-associated disease models using feto-maternal interface organ-on-a-chip.

Oxidative stress (OS) and inflammation arising from cellular derangements at the fetal membrane-decidual interface (feto-maternal interface [FMi]) is a major antecedent to preterm birth (PTB). However, it is impractical to study OS-associated FMi disease state during human pregnancy, and thus it is difficult to develop strategies to reduce the incidences of PTB. A microfluidic organ-on-chip model (FMi-OOC) that mimics the in vivo structure and functions of FMi in vitro was developed to address this challenge. The FMi-OOC contained fetal (amnion epithelial, mesenchymal, and chorion) and maternal (decidua) cells cultured in four compartments interconnected by arrays of microchannels to allow independent but interconnected co-cultivation. Using this model, we tested the effects of OS and inflammation on both fetal (fetal → maternal) and maternal (maternal → fetal) sides of the FMi and determined their differential impact on PTB-associated pathways. OS was induced using cigarette smoke extract (CSE) exposure. The impacts of OS were assessed by measuring cell viability, disruption of immune homeostasis, epithelial-to-mesenchymal transition (EMT), development of senescence, and inflammation. CSE propagated (LC/MS-MS analysis for nicotine) over a 72-hour period from the maternal to fetal side, or vice versa. However, they caused two distinct pathological effects on the maternal and fetal cells. Specifically, fetal OS induced cellular pathologies and inflammation, whereas maternal OS caused immune intolerance. The pronounced impact produced by the fetus supports the hypothesis that fetal inflammatory response is a mechanistic trigger for parturition. The FMi disease-associated changes identified in the FMi-OOC suggest the unique capability of this in vitro model in testing in utero conditions.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

A Dynamic Flow Fetal Membrane Organ-on-a-Chip System for Modeling the Effects of Amniotic Fluid Motion.

Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. An amnion membrane (AM) organ-on-chip (OOC) was utilized to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 hours flow culture to mimic fluid motion. Static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control for pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to CK-18 ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a flow culture environment is not necessary to mimic any in utero physiologic cellular conditions of fetal membrane-derived cells.

A multi-organ, feto-maternal interface organ-on-chip, models pregnancy pathology and is a useful preclinical extracellular vesicle drug trial platform.

Pregnant women and their fetuses are often excluded from clinical trials due to missing drug-related pre-clinical trial information at the human feto-maternal interface (FMi). The two interfaces-placenta/decidua and fetal membranes/decidua are gatekeepers of drug transport; however, testing their functions is impractical during pregnancy. Limitations of current in-vivo/in-vitro models have hampered drug development and testing during pregnancy. Hence, major complications like preterm births and maternal and neonatal mortalities remain high. Advancements in organ-on-chip (OOC) platforms to test drug kinetics and efficacy and novel extracellular vesicle-based fetal drug delivery are expected to accelerate preclinical trials related to pregnancy complications. Here we report the development and testing of a humanized multi-organ fetal membrane/placenta (fetal)-decidua (maternal) interface OOC (FMi-PLA-OOC) that contains seven cell types interconnected through microchannels to maintain intercellular interactions as seen in-utero. Cytotoxicity, propagation, mechanism of action, and efficacy of engineered extracellular vesicles containing anti-inflammatory interleukin (IL)-10 (eIL-10) were evaluated to reduce FMi inflammation associated with preterm birth. A healthy and disease model (lipopolysaccharide-infectious inflammation) of the FMi-PLA-OOC was created and co-treated with eIL-10. eIL-10 propagated from the maternal to fetal side within 72-hours, localized in all cell types, showed no cytotoxicity, activated IL-10 signaling pathways, and reduced lipopolysaccharide-induced inflammation (minimized NF-kB activation and proinflammatory cytokine production). These data recapitulated eIL-10s' ability to reduce inflammation and delay infection-associated preterm birth in mouse models, suggesting FMi-PLA-OOC as an alternative approach to using animal models. Additionally, we report the utility of eIL-10 that can traverse through FMis to reduce inflammation-associated pregnancy complications.

Endocrine-disrupting compounds and their impact on human placental function: evidence from placenta organ-on-chip studies.

The effects of endocrine-disrupting compounds (EDCs) on the placenta, a critical gestational organ for xenobiotic protection, are well reported; however, models to determine the role of EDCs in placental disruption are limited. An advanced 2nd-trimester human placenta organ-on-chip model (2TPLA-OOC) was developed and validated, with six representative cells of the maternal and the fetal interface interconnected with microchannels. Various EDCs (150 ng mL-1 each of bisphenol A, bisphenol S, and polybrominated diphenyl ethers-47 and -99) were gradually propagated across the chip for 72 hours, and their various effects were determined. Cigarette smoke extract (CSE), an environmental risk factor, was used as a positive control. EDCs produced overall oxidative stress in the placental/decidual cells, induced cell-specific endocrine effects, caused limited (<10%) apoptosis/necrosis in trophoblasts and mesenchymal cells, induced localized inflammation but an overall anti-inflammatory shift, did not change immune cell migration from stroma to decidua, and did not affect placental nutrient transport. Overall, (1) the humanized 2TPLA-OOC recreated the placental organ and generated data distinct from the trophoblast and other cells studied in isolation, and (2) at doses associated with adverse pregnancies, EDCs produced limited and localized insults, and the whole organ compensated for the exposure.