Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells

The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs) in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and highthroughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code) to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology.