Mesenchymal stem cells (MSCs) are primary candidates in cell therapy and tissue engineering and are being tested in clinical trials for a wide range of diseases. Originally isolated and expanded as plastic adherent cells, MSCs have intriguing properties of in vitro self-assembly into three-dimensional (3D) aggregates reminiscent of skeletal condensation in vivo. Recent studies have shown that MSC 3D aggregation improved a range of biological properties, including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition. Hence, the formation of 3D MSC aggregates has been explored as a novel strategy to improve cell delivery, functional activation, and in vivo retention to enhance therapeutic outcomes. This article summarizes recent reports of MSC aggregate self-assembly, characterization of biological properties, and their applications in preclinical models. The cellular and molecular mechanisms underlying MSC aggregate formation and functional activation are discussed, and the areas that warrant further investigation are highlighted. These analyses are combined to provide perspectives for identifying the controlling mechanisms and refining the methods of aggregate fabrication and expansion for clinical applications.
Three-dimensional cell culture is emerging as a more relevant alternative to the traditional two-dimensional format. Yet the ability to perform cytometry at the single cell level on intact three-dimensional spheroids or together with temporal regulation of the cell microenvironment remains limited. Here we describe a microfluidic platform to perform high-density three-dimensional culture, controlled stimulation, and observation in a single chip. The method extends the capabilities of droplet microfluidics for performing long-term culture of adherent cells. Using arrays of 500 spheroids per chip, in situ immunocytochemistry and image analysis provide multiscale cytometry that we demonstrate at the population scale, on 104 single spheroids, and over 105 single cells, correlating functionality with cellular location within the spheroids. Also, an individual spheroid can be extracted for further analysis or culturing. This will enable a shift towards quantitative studies on three-dimensional cultures, under dynamic conditions, with implications for stem cells, organs-on-chips, or cancer research.
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