An organoid is a 3D organization of cells that can recapitulate some of the structure and function of native tissue. Recent work has seen organoids gain prominence as a valuable model for studying tissue development, drug discovery, and potential clinical applications. The requirements for the successful culture of organoids in vitro differ significantly from those of traditional monolayer cell cultures. The generation and maturation of high-fidelity organoids entails developing and optimizing environmental conditions to provide the optimal cues for growth and 3D maturation, such as oxygenation, mechanical and fluidic activation, nutrition gradients, etc. To this end, we discuss the four main categories of bioreactors used for organoid culture: stirred bioreactors (SBR), microfluidic bioreactors (MFB), rotating wall vessels (RWV), and electrically stimulating (ES) bioreactors. We aim to lay out the state-of-the-art of both commercial and in-house developed bioreactor systems, their benefits to the culture of organoids derived from various cells and tissues, and the limitations of bioreactor technology, including sterilization, accessibility, and suitability and ease of use for long-term culture. Finally, we discuss future directions for improvements to existing bioreactor technology and how they may be used to enhance organoid culture for specific applications.

Mitochondrial respiratory chain (RC) disease therapies directed at intra-mitochondrial pathology are largely ineffective. Recognizing that RC dysfunction invokes pronounced extra-mitochondrial transcriptional adaptations, particularly involving dysregulated translation, we hypothesized that translational dysregulation is itself contributing to the pathophysiology of RC disease. Here, we investigated the activities, and effects from direct inhibition, of a central translational regulator (mTORC1) and its downstream biological processes in diverse genetic and pharmacological models of RC disease. Our data identify novel mechanisms underlying the cellular pathogenesis of RC dysfunction, including the combined induction of proteotoxic stress, the ER stress response and autophagy. mTORC1 inhibition with rapamycin partially ameliorated renal disease in B6.Pdss2^kd/kd mice with complexes I–III/II–III deficiencies, improved viability and mitochondrial physiology in gas-1(fc21) nematodes with complex I deficiency, and rescued viability across a variety of RC-inhibited human cells. Even more effective was probucol, a PPAR-activating anti-lipid drug that we show also inhibits mTORC1. However, directly inhibiting mTORC1-regulated downstream activities yielded the most pronounced and sustained benefit. Partial inhibition of translation by cycloheximide, or of autophagy by lithium chloride, rescued viability, preserved cellular respiratory capacity and induced mitochondrial translation and biogenesis. Cycloheximide also ameliorated proteotoxic stress via a uniquely selective reduction of cytosolic protein translation. RNAseq-based transcriptome profiling of treatment effects in gas-1(fc21) mutants provide further evidence that these therapies effectively restored altered translation and autophagy pathways toward that of wild-type animals. Overall, partially inhibiting cytosolic translation and autophagy offer novel treatment strategies to improve health across the diverse array of human diseases whose pathogenesis involves RC dysfunction.

Biomarkers for α‐synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD). Endogenous auto‐antibodies to α‐synuclein could serve as biomarkers for underlying synucleinopathy, but previous assessments of auto‐antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto‐antibodies to α‐synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme‐linked immunosorbent assay for measuring α‐synuclein auto‐antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto‐antibody levels than females in both fluids. CSF auto‐antibody levels were significantly higher in PD patients as compared with HC, whereas serum levels were not significantly different. CSF auto‐antibody levels did not associate with amyloid‐β_1–42, total tau, or phosphorylated tau. CSF auto‐antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ_1–42. CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α‐synuclein with N‐ and C‐terminal truncations, we found that CSF auto‐antibodies target amino acids 100 through 120 of α‐synuclein. We conclude that endogenous CSF auto‐antibodies are significantly higher in PD patients as compared with HC, suggesting that they could indicate the presence of underlying synucleinopathy. These auto‐antibodies associate with poor cognition, independently of CSF amyloidβ_1–42, and target a select C‐terminal region of α‐synuclein. image Read the Editorial Highlight for this article on page 433.

Introduction: The successful implantation of laboratory-grown cardiac tissue requires phenotypically mature cardiomyocytes capable of electrophysiological integration with native heart tissue. Pulsed electrical stimulation (ES) has been identified as a promising strategy for enhancing cardiomyocyte maturation. However, there are discrepancies in the literature as to best practices for promoting cardiac differentiation using ES.Methods: This study presents a novel, 3D printed bioreactor that delivers in vitro ES to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), promoting cell maturity and functional readiness for implantation. Finite element analysis and mathematical modeling were used to model the fluid dynamics and to characterize in detail the delivery of pulsatile electrical signals, providing precise control over stimulation parameters such as voltage, current, and charge.Results: The bioreactor developed here provides an easy-to-use, inexpensive platform for culturing hiPSC-CMs under the influence of ES and low-shear fluid flow for enhanced nutrient availability, while its “drop-in” design facilitates real-time observation of cultured cells. The electrical stimulation provided is controlled, modeled, and predictable, enabling reproducible experimental conditions and promoting comparability across future studies. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) grown in the bioreactor with ES showed improved differentiation and an enhanced ability to respond to external electrical pacing signals.Discussion: By offering a standardized platform for ES-based cardiomyocyte maturation, this bioreactor aims to accelerate advancements in cardiac tissue engineering. Future research will explore how variations in ES parameters influence cardiomyocyte phenotype and maturation, contributing to a deeper understanding of cardiac cell development and optimization for therapeutic applications.

The differentiation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into specific subtypes, including ventricular, atrial, conduction, and nodal, remains a significant challenge for in vitro disease modeling and regenerative medicine. While chemical approaches have been explored for subtype specification, these protocols often result in heterogeneous CM populations. In this study, we tested the hypothesis that differential electrical stimulation (ES) can guide/modulate the differentiation of subtype-specific CMs from hiPSCs. By varying stimulation parameters, such as frequency and onset of ES at different developmental time points, we demonstrate that ES alone promotes the differentiation of hiPSC into either ventricular or atrial CMs, without changing any chemical cues. Our results show that lower frequency stimulation earlier in development promotes atrial gene expression, while higher frequency ES later in development promotes ventricular differentiation. These findings were validated by gene expression analysis, immunostaining, and measurement of calcium signaling. This study highlights the potential of ES as a tunable tool for directing CM subtype specification, offering a promising strategy for the generation of pure populations of CM subtypes for use in precision medicine, disease modeling, and regenerative therapies.