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Automated, Image-Based Analysis of a Hanging Drop Micro-Hole Plate Model to Create and Differentiate 3D Mesenchymal Stem Cell Spheroids for Downstream Tissue FormationTélécharger
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February 13, 2017
Human mesenchymal stem cells (hMSCs) have recently emerged as a leading candidate in cellular therapies due to their ability to differentiate and mature into multiple lineages including adipocytes, chondrocytes, and osteocytes, their immunesuppressive properties, and distinct migratory and trophic effects during tissue repair and regeneration. Early work with hMSCs incorporated two-dimensional (2D) cell culture, leading to a realization that this culture method alters the native phenotype of hMSCs. This has been shown by tests with self-assembled 500-10,000 cell hMSC aggregates revealing the ability to create an “in vivo-like microenvironment and better preserve MSC phenotype”, in addition to preclinical studies where intramyocardial transplantation of three-dimensional (3D) cultured MSCs into porcine models improved cell survival and integration. This has increased the desire to fully understand the impact that aggregation has on hMSCs within spheroidal structures.
One such therapeutic area is the creation of complex tissues from preformed 3D hMSC spheroids. Here, large numbers of spheroids are typically required. Current methods commonly incorporate single or low spheroidal numbers per well. Therefore, high numbers of plates are used to generate the tissue density required, which can be expensive and labor intensive. Others methods that do create higher spheroid numbers allow spheroids with great variation in size, and also suffer from undesired “preaggregation”. Multiple pore hanging drop microplates can eliminate these complications by offering a method to create large numbers of spheroids of consistent size from a single well of a 6-well plate. Using an insert design, cell suspension is added across the top of the insert where cells are free to fall into the 650 micro-holes in the insert. Spheroids form and remain in the plate while undergoing differentiation without further manual manipulation. Upon completion of the desired process, spheroids are easily dropped from the insert by touching off the insert to media filled wells below.
Here we demonstrate the ability to automate the steps necessary to place hMSCs into the microplate inserts, track spheroid formation, and monitor spheroids during differentiation into chondrocyte lineages. By incorporating automation, the simplicity and repeatability of cell dispensing and spheroid formation monitoring procedures can be improved compared to manual processing. Use of a non-contact liquid dispenser ensures that consistent cell numbers are dispensed evenly across each insert. Automated brightfield imaging allows cells and spheroids across the entire area of each insert to be visualized without manual intervention. Expression of normal mesenchymal cell protein markers in undifferentiated hMSC spheroids, as well as chondrocyte differentiation of spheroidal cells upon induction was confirmed through immunofluorescence. The combination of an appropriate high density plate model and automated processing offers an easy-to-use, robust method to create the high density numbers of hMSC spheroids needed for important therapeutic applications.