Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized 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, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating 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.

Biomanufacturing human tissues via organ building blocks.

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.

A Microwell Cell Capture Device Reveals Variable Response to Dobutamine in Isolated Cardiomyocytes.

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 ß-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 ß-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.

Using fiber optics to detect moisture intrusion into a landfill cap consisting of a vegetative soil barrier.

The intrusion of moisture into landfills can pose a health hazard because of the possibility that the moisture will carry harmful substances into the groundwater. Early detection of moisture anywhere within these landfills is essential if corrective action is to be taken well before an occurrence of this kind. This paper presents the results of a field-scale simulation test of the use of fiber optics to detect the presence of moisture within landfill covers, using a detection method based on the thermal response of soils as a function of their moisture content. By sending electrical current through an embedded stainless-steel tube, soils of varying moisture content were heated and time-dependent temperature measurements were obtained with a fiber-optic distributed temperature sensor system. The optical fiber itself lay within the tube, but its temperature was a function of how rapidly heat was conducted into the surrounding medium. The results of this experiment, which are in agreement with those obtained using more traditional "point" sampling and laboratory analysis, are presented along with the strengths and limitations of the thermal-response method of detecting moisture.