Damage mechanisms for osteoblast cells (OBs) attached to hydroxyapatite (HA)discs and glass coverslips were comprehensively investigated. Cell-cell, cell-matrix interaction altered the cryobiological properties of cells. Attached cells were subject to more severe mechanical damage than isolated cells because attached cells had larger contacting area with ice and the three dimensional movements of isolated cells made them more flexible than attached cells that could only deform in one dimension. Results showed that the viability of attached OB cells decreased significantly compared with the viability of isolated OB cells under the same cryopreservation procedure. Extracellular ice, differential thermal contraction, and mechanical stresses were the major damaging factors for OB cells attached to HA discs and glass coverslips.