High-Throughput Microfluidic Platform for 3D Cultures of Mesenchymal Stem Cells, Towards Engineering Developmental Processes

Supplementary Information Correlation between microaggregates diameter and cell number A previously implemented algorithm [1] for determining the number of chondrocytes in an aggregate starting from its two-dimensional macroscopic profile was applied to our model. Briefly, the algorithm correlates the diameter of an aggregate with the corresponding cell number by means of a sphere packing theory. Fig.SI1 shows the theoretical relationship between diameter and cell number, having upper and lower bounds. Experimental results obtained through our microfluidic approach were compared to the algorithm in order to determine the degree of matching. In details, hBM-MSC microaggregates were formed within the platform and after 3 hours live phase contrast images were acquired by means of a Olympus BX-61 microscope. The diameter of each microaggregate was calculated as the average of two measurements. Microaggregates were subsequently fixed in 4% PFA and immunofluorescence stained for Dapi as described in the Materials and Methods section. Confocal Z-stack images of the immunofluorescence microaggregates were acquired by means of a Nikon A1R Nala Confocal microscope. The number of cells was then calculated through the ImageJ software (NIH). The experimental results obtained with hBM-MSCs microaggregates were shown to match with the theoretical model (Fig.SI1) with a high degree of fidelity, namely R 2 =0.9565. The model was thus employed during the experiments for estimating the number of cells starting from the measurement of microaggregates diameter.