Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use

Abstract

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 120 hours 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 Me2SO 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 2 × 106 cells/mL. It was further established that DPSC can be stored at −85°C or −196°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.

Tooth Organ Engineering: Biological Constraints Specifying Experimental Approaches

“…The biological constraints, which become better characterized as research progresses, will necessarily restrict the number of protocols and strategies. Potential human cell sources have been reported (Gronthos et al, 2000;Miura et al, 2003), while banks for autologous cell sources are being developed (Arora et al, 2009;Tamaoki et al, 2010;Woods et al, 2009). The biological potentialities of the different cells types, which recently were identified from human sources, will have to be tested both in vitro and in vivo.…”

Clinical grade adult stem cell banking

“…13 Moreover, MSCs are robust and can survive freezing temperatures without any significant loss in viability, thus allowing for future "off-the-shelf" therapy approaches. 5,28,29 In a clinical transplant or transfusion application, a large number of frozen stored MSCs are usually required and therefore the development of stem cell banks is necessary. These banks must assure the quality and safety of these cell products especially when the stored MSCs are intended for clinical use in cell therapy and regenerative medicine.…”

Multipotent Dental Stem Cells: An Alternative Adult Derived Stem Cell Source for Regenerative Medicine

“…The potential use of dental stem cells has become even more viable for tissue regeneration and other therapies with the recent advances in cryopreservation. These advances allow proliferation and long term storage of these cells for future cell therapy treatments while maintaining their differentiation potential (Ding et al, 2010;Papaccio et al, 2006;Seo et al, 2005;Woods et al, 2009;Zhang et al, 2006).…”