Solid tumor cells grow in a stiff microenvironment with dense extracellular matrix (ECM) and condensed packing of adjacent cells. Tumor cells are capable of migrating through constricted pores formed by ECM or surrounded by other cells, and the nuclear envelope can break with repair factor mislocalization, further leading to DNA damage and genetic changes, or even accumulated to be genomic variations. Cell division, likewise, is confined by a stiff niche of adjacent cells and extracellular matrix, and such confinement has been reported to cause chromosome mis-segregation. The chromosome-loss live cell reporter system was developed to prove that cells undergoing specific types of chromosome missegregation can survive and maintain heritability, resulting in permanent genomic variations. Mitotic cells under in vitro confinement and in vivo conditions exhibit more abnormal division and more fluorescence-null reporter-negative cells, for both cancer and normal types. Confinement and SAC inhibition both lead to chromosome mis-segregation but do not superimpose, and Topoisomerase IIa plays an essential role in cells to survive after confined mitosis. Myosin II was found to lead to increased nuclear envelope rupture and, therefore, more DNA damage, while it protects mitotic cell rounding within 3D confined environments, since the increase of reporter-negative cells was observed after Myosin II knockdown.