Cell seeding simulation on micropatterned islands shows cell density depends on area to perimeter ratio, not on island size or shape.

Protein micropatterned substrates have been used to control cell size, shape, and cell-cell contacts, characteristics that influence a range of cell behaviors from early cell differentiation to late stages of maturation. Knowing the initial island cell seeding density is important to interpreting results and understanding downstream cell behavior. While studies routinely report the intended or target cell seeding density, they do not report the actual cell seeding density on the islands. As cells proliferate, differences in initial cell seeding density could compound and may lead to misinterpretation of results. In this work, we present a cell seeding simulation and apply it to 100s of islands with a range of geometries (sizes and shapes) to explore how island cell seeding density relates to the target or unpatterned cell seeding density. We first experimentally validate the simulation and then show that normalized island cell seeding density depends on island size, shape, and spacing, but can be predicted solely from island area to perimeter ratio, A2P, via a power law relationship for a wide range of island geometries. Interestingly, normalized island cell seeding density is the same as the normalized unpatterned cell seeding density for A2P ≥ 17 µm. This simulation will help to design micropatterned substrates and to have more accurate representation of the island cell seeding density at the start of experiments. By knowing the island cell seeding density, we can more easily reproduce results across research groups to understand the roles of cell-cell contact and cell size and shape on cell behavior. STATEMENT OF SIGNIFICANCE: We present a cell seeding simulation on protein-micropatterned substrates and use it to simulate seeding across 100s of island geometries (size, shape, and spacing) covering two orders of magnitude in size. The simulation shows that island cell density varies significantly with island geometry compared to the target seeding density. However, island cell density can be predicted from one geometric parameter - the island's area to perimeter ratio. Results will help direct researchers on how to achieve uniform cell density across all island geometries. Since cell density and island shape both influence cell behaviors, such as differentiation, this simulation may help to isolate these factors, facilitate micropatterned substrate design, and provide a mechanism for more reproduceable results across studies.