U-Net Deep-Learning-Based 3D Cell Counter for the Quality Control of 3D Cell-Based Assays through Seed Cell Measurement

Conventional cell-counting software uses contour or watershed segmentations and focuses on identifying two-dimensional (2D) cells attached on the bottom of plastic plates. Recently developed software has been useful tools for the quality control of 2D cell-based assays by measuring initial seed cell numbers. These algorithms do not, however, quantitatively test in three-dimensional (3D) cell-based assays using extracellular matrix (ECM), because cells are aggregated and overlapped in the 3D structure of the ECM such as Matrigel, collagen, and alginate. Such overlapped and aggregated cells make it difficult to segment cells and to count the number of cells accurately. It is important, however, to determine the number of cells to standardize experiments and ensure the reproducibility of 3D cell-based assays. In this study, we apply a 3D cell-counting method using U-net deep learning to high-density aggregated cells in ECM to identify initial seed cell numbers. The proposed method showed a 10% counting error in high-density aggregated cells, while the contour and watershed segmentations showed 30% and 40% counting errors, respectively. Thus, the proposed method can reduce the seed cell-counting error in 3D cell-based assays by providing the exact number of cells to researchers, thereby enabling the acquisition of quality control in 3D cell-based assays.