Formulation and characterization of self-assembling cardiac spheroids as a model for drug-screening

Anticancer drug development needs to be shifted to a patient-specific platform that can be performed in high volume. Here, we report the generation and characterization of self-assembled cardiac spheroids formed from human induced pluripotent stem cells to be used for drug-screening applications. In doing so, we describe our employed cardiomyocyte differentiation and spheroid formation protocols to generate self-assembled spheroids whose sizes increased as a function of seeding density. We found that cardiac cells seeded at numbers of 2.5 x 10³, 10 x 10³, 15 x 10³, 20 x 10³ and 20 x 10³ resulted in the generation of spheroids with mean effective diameters of 264 ± 7 µm, 386 ± 4 µm, 445 ± 9 µm, 561 ± 10 µm, and 733 ± 8 µm, respectively. Using spheroid seeding with 10 x 10³ enabled the formation of spheroids with an approximate 386 ± 4 µm diameter, our desired size range emulative of the average in vivo intercapillary distance for downstream drug-treatment studies. We observed that implementing a centrifugation step during spheroid seeding greatly reduced any size variation without impacting the spheroid’s ability to form in-tact structures and retention of spontaneous beating properties. To quantify cardiomyocyte efficiency in our 2D differentiation protocols, we examined immunomarkers for cardiomyocyte and fibroblasts and observed an average cardiomyocyte prevalence of 4.86 ± 2.81 %, within our cell population. Lastly, we simulated acute drug-induced cardiotoxicity on the generated cardiac spheroids by examining their viability and spontaneous beating rate changes in response to doxorubicin-hydrochloride treatment. From the tested drug concentrations and our subsequent live fraction area curve fitting, we tabulated a half maximal inhibitory concentration of 2.11 µM, agreeable with reported concentrations in the clinic. Lastly, we observed that after 24 hours of drug exposure, statistically significant changes to the beating rates of spheroids exposed to concentrations ≥ 5 µM. Taken together, these data indicate that our stem cell-derived cardiac spheroids show promise as a potential patient-specific candidate model for assessing cardiotoxicity introduced by anticancer therapies.Mechanical Engineerin

OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. The most efficient way of studying drug toxicity and efficacy requires high-resolution imaging of a large number of organoids acquired in the least amount of time. Currently missing are suitable platforms capable of fast-paced high-content imaging of organoids. To address this knowledge gap, we present the OrganoidChip, a microfluidic imaging platform that incorporates a unique design to immobilize organoids for endpoint, fast imaging. The chip contains six parallel trapping areas, each having a staging and immobilization chamber, that receives organoids transferred from their native culture plates and anchors them, respectively. We first demonstrate that the OrganoidChip can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. Next, we show the capability of our device in assessing the dose-dependent responses of organoids’ viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-chip experiments. Importantly, the chip enables organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevents the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the OrganoidChip is a promising microfluidic platform that can serve as a building block for a multiwell plate format that can provide high-throughput and high-resolution imaging of organoids in the future.This article is published as Moshksayan, K., Harihara, A., Mondal, S. et al. OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids. Sci Rep 13, 11268 (2023). https://doi.org/10.1038/s41598-023-38212-8. Posted with permission

ApoE Receptor 2 mediates trophoblast dysfunction and pregnancy complications induced by antiphospholipid antibodies in mice

OBJECTIVE: Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine if the LDL receptor family member apoE receptor 2 (apoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. METHODS: Placental and trophoblast apoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG (NHIgG) and aPL were purified from healthy individuals and APS patients, respectively. The role of apoER2 in aPL-induced changes in trophoblast proliferation, migration and kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of apoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant apoER2(+/+) and apoER2(-/-) mice injected with aPL or NHIgG. RESULTS: We found that apoER2 is abundant in human and mouse placental trophoblasts, and in multiple trophoblast-derived cell lines including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2-glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by EGF in trophoblasts was mediated by apoER2. Furthermore, in a murine passive transfer model of pregnancy complications of APS, apoER2(-/-) mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. CONCLUSION: ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS. This article is protected by copyright. All rights reserved