Cellular microarrays are powerful experimental tools for high-throughput screening of large numbers of test samples. Miniaturization increases assay throughput while reducing reagent consumption and the number of cells required, making these systems attractive for a wide range of assays in drug discovery, toxicology, stem cell research and potentially therapy. Here, we provide an overview of the emerging technologies that can be used to generate cellular microarrays, and we highlight recent significant advances in the field. This emerging and multidisciplinary approach offers new opportunities for the design and control of stem cells in tissue engineering and cellular therapies and promises to expedite drug discovery in the biotechnology and pharmaceutical industries.

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Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT + cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-β/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the α subunit of the hypoxia-inducible factor (HIF-1α) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1α, 2-methoxyestradiol (2ME2), on the levels of HIF-1α using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1α levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.

Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron’s migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.