Large-Scale Production of Wholly-Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.
Advanced healthcare materials
Combining the sustainable culture of billions of human cells and the bioprinting of wholly-cellular bioinks offers a pathway towards organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, we optimize the suspension culture of human induced pluripotent stem cell-derived aggregates using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, we demonstrate that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. Our 4-day culture resulted in a 16.6- to 20.4-fold expansion of cells, we generate approximately 4 billion cells per vessel, while maintaining > 94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly-cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adhm.202201138
View details for PubMedID 36314397
Biomanufacturing human tissues via organ building blocks.
Cell stem cell
2022; 29 (5): 667-677
The construction of human organs on demand remains a tantalizing vision to solve the organ donor shortage. Yet, engineering tissues that recapitulate the cellular and architectural complexity of native organs is a grand challenge. The use of organ building blocks (OBBs) composed of multicellular spheroids, organoids, and assembloids offers an important pathway for creating organ-specific tissues with the desired cellular-to-tissue-level organization. Here, we review the differentiation, maturation, and 3D assembly of OBBs into functional human tissues and, ultimately, organs for therapeutic repair and replacement. We also highlight future challenges and areas of opportunity for this nascent field.
View details for DOI 10.1016/j.stem.2022.04.012
View details for PubMedID 35523137
A Microwell Cell Capture Device Reveals Variable Response to Dobutamine in Isolated Cardiomyocytes
2019; 117 (7): 1258-1268
Isolated ventricular cardiomyocytes exhibit substantial cell-to-cell variability, even when obtained from the same small volume of myocardium. In this study, we investigated the possibility that cardiomyocyte responses to β-adrenergic stimulus are also highly heterogeneous. To achieve the throughput and measurement duration desired for these experiments, we designed and validated a novel microwell system that immobilizes and uniformly orients isolated adult cardiomyocytes. In this configuration, detailed drug responses of dozens of cells can be followed for intervals exceeding 1 h. At the conclusion of an experiment, specific cells can also be harvested via a precision aspirator for single-cell gene expression profiling. Using this system, we followed changes in Ca2+ signaling and contractility of individual cells under sustained application of either dobutamine or omecamtiv mecarbil. Both compounds increased average cardiomyocyte contractility over the course of an hour, but responses of individual cells to dobutamine were significantly more variable. Surprisingly, some dobutamine-treated cardiomyocytes augmented Ca2+ release without increasing contractility. Other cells responded with increased contractility despite unchanged Ca2+ release. Single-cell gene expression analysis revealed significant co-expression of β-adrenergic pathway genes PKA regulatory subunit type I, PKA regulatory subunit type II, and Ca2+/calmodulin-dependent protein kinase II across cardiomyocytes. Other data supported a connection between the effects of dobutamine on relaxation rate and the expression of protein phosphatase 2. These findings suggest that variable drug responses among cells are not merely experimental artifacts. By enabling direct comparison of the functional behavior of an individual cell and the genes it expresses, this new system constitutes a unique tool for interrogating cardiomyocyte drug responses and discovering the genes that modulate them.
View details for DOI 10.1016/j.bpj.2019.08.024
View details for Web of Science ID 000488457600009
View details for PubMedID 31537313
View details for PubMedCentralID PMC6818147