Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me(2)SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2x10(6) cells/mL. It was further established that DPSC can be stored at -85 degrees C or -196 degrees C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.