Improving the uniformity of cell seeding.

(A) Uniform spacing between each cell is the targeted distribution of cells in a dish when seeding to form a monolayer. The reality is that the same number of cells aggregate around the well edge and well centre resulting in a heterogeneous density distribution. Dashed outline shows example of a surface such as a coverslip in the well, which would often not be centred and suffer from further uneven distribution. Movement of the culture vessel to the incubator causes further redistribution of cells due to fluid flow, ultimately resulting in variation in cell phenotype depending on local changes in density. (B) The uniform cell seeder (UCS) device can be used to control the cell suspension, and therefore cell density, across a surface. A capping lid (blue) above the culture surface (grey) holds the cell suspension (pink) at a uniform height across the area (arrows), which evenly distributes the cell population. (C) The UCS device is filled by pipetting the cell suspension through an inlet. In this case, a 22mm coverslip in a 24 well plate is seeded with a uniform distribution of cells. Through capillary force, the cell suspension is held between the UCS and culture surface. (D) Nuclear staining of cells seeded in a 24-well plate without (left) and with (right) the UCS device after 2 weeks culture. Some aggregation around the edges remains, but the uniformity across the well is significantly improved.