1. 3D Model of Cells on Plate with Growth and Division Sudin Bhattacharya 1 Wan-Yun Cheng Christopher Eklund Rory Conolly

2. Motivation Computational modeling of in vitro data is sensitive to the surface area that cells present to the culture medium. Surface area varies with degree of confluency of the culture and among cells at a given degree of confluency. Agent-based computational modeling can provide spatially realistic simulations of cells growing in a culture dish, and of the surface area that each cell presents to the medium. Expect to help with characterization of how degree of confluency contributes to variability of in vitro data. Directly relevant to IVIVE. 2

3. Agent-based modeling as a means for testing biological hypotheses Agent-based modeling (ABM) is a style of modeling in which individuals (cells) and their interaction with each other and their environment are explicitly represented. - Jeff Schank http://www.agent-based-models.com/blog/category/agent-based-modeling/ Virtual tissues Cells in a culture dish We use Monte Carlo (MC) simulations of a 3D multicellular ABM developed in CompuCell3D (free tool, Glazier et al., Indiana U.) 3

4. Assumptions used for model development Cells grow on a culture dish. Cells strongly adhere to dish. Cells adhere to each other less strongly. Periodic Boundary Conditions – models growth and flux of cells in a square section of the dish. There is a distribution of initial cell sizes (cells are initially in different stages of cycle). Each cell grows gradually – volume incremented by 1 unit every 5 th MC step, and symmetrically divides into two when volume is roughly doubled (normal distribution about a mean doubling volume). Orientation of cell division is random. Cells unattached to dish die with an 80% probability – monolayer of cells. 4

5. Cell Growth 5

6. Cell Growth – top view 6

7. Distribution of Initial Cell Volumes (125 – 245 units) and Normal Distribution of Doubling Volume (225 – 275 units) 7

8. 8 Surface Area Vs. Time

9. Surface Area Vs. Confluence 9

10. Confluence Vs. Time 10

11. Surface Area Distribution among Cells t(MCS) = 0 t(MCS) = 500t(MCS) = 1000 t(MCS) = 1500 t(MCS) = 2000 t(MCS) = 2500 t(MCS) = 2750 t(MCS) = 3000 11

12. 12 Data Needs Time-course of confluence? Imaging of cell growth? Distribution of cellular surface areas at various levels of confluence?

13. Laboratory measurements Cells under different confluence Aiming for different levels of confluence: low, medium, high, and over confluence Low (10%-25% confluence) Imaging for 5 consecutive days Image acquisition 2D image DIC or fluorescence image 10 images at each level of confluence 3D image (Z-stack) 5 Z-stack images at each level of confluence Image post processing 2D image: cell surface area (NIS Elements) 3D image: cell thickness (NIS Elements) and cell volume (MatLab) 13