METRICS FOR DEFINING CELL 3DNESS: A GUIDE FOR THE DEVELOPMENT OF SCAFFOLDS FOR 3D CELL CULTURE
Tanya M. Farooque, Charles H. Camp, Christopher K. Tison, Girish Kumar, Sapun H. Parekh, Carl G. Simon, Jr.
Three-dimensional (3D) cell culture is the preferred method for cell study in the fields of tissue regeneration, drug delivery, cell migration, and cell signaling. Cells cultured in 3D behave more physiologically than cells cultured on two-dimensional (2D) flat surfaces. However, it is not clear which 3D culture systems provide a 3D niche for cell culture. Presented is a methodology that can be utilized to assess the degree of 3Dness of cells on scaffolds of differing architecture in order to define scaffolds systems that drive cells into a 3D morphology. Geometrically, 3D is defined as an object with four points in space that occupy more than one plane. The morphology of primary human bone marrow stromal cells (hBMSCs) cultured on a variety of scaffold systems was captured via confocal microscopy z-stacks. Z-stacks were assessed from multiple perspectives (x, y, and z) to assess 3Dness of cell morphology and new metrics based on cell area and principal moments of the gyration tensor were developed and used to score cell 3Dness. Cells on flat surfaces had morphologies similar to flat disks, cells in fibers were mostly rod shape with branches in multiple planes, and cells in non-degradable hydrogels were spherical. These results provide a new method for measuring cell 3Dness that can be used to determine if a scaffold system provides a 3D niche for cells.