Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells

Abstract

The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate-buffered saline (PBS) was employed, with a slow-ramp freezing rate. We observed > 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive-cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze-thawing process. Real-time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis- and transcription-related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long-term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright © 2014 John Wiley & Sons, Ltd.

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo

“…The cell survival rate of cryopreserved dental pulp tissue-derived MSCs was found to be 79.5 ± 3.0% as evaluated by propidium iodine (PI) and Hoechst 33342 staining, similar to our previous studies [21,23]. Fibroblast-like spindle colonies were observed within the first week of culture, which became homogeneous at passage 3 upon sub-culturing (Figure 1A).…”

“…Dental follicles from extracted wisdom teeth were previously cryopreserved using a newly developed tissue cryopreservation protocol [21]. MSCs from these cryopreserved dental follicles were subsequently shown to have immunomodulatory properties that further enhanced osteogenesis under in vivo conditions [23].…”

“…Dental pulp tissues harvested from wisdom teeth extracted ( n = 8, four for tissue cryopreservation, and four for fresh dental pulp harvesting; aged 14–19 years, average: 18.5 years old) at the Department of Oral and Maxillofacial Surgery at Changwon Gyeongsang National University Hospital were cryopreserved as previously described [21,23]. Briefly, after being collected from the extracted wisdom tooth using a sterile scalpel (Figure 1A), the dental pulp tissue from a single donor was minced into 1–3-mm 2 explants and tissue segments, and was placed into a 1.8 mL cryovial (Thermo scientific, Roskilde, Denmark) containing 1 mL of cryoprotectant, consisting of 0.05 M glucose, 0.05 M sucrose, and 1.5 M ethylene glycol in phosphate-buffered saline (PBS) (osmolality: 1900 mOsm/kg).…”

“…Interestingly, in our previous studies, a long-term tissue cryopreservation method was developed using slow-programmed cryopreservation protocols for human dental tissues and Wharton’s jelly for subsequent use as an autologous stem-cell resource [21,22]. More specifically, dental follicle tissues from extracted wisdom teeth were cryopreserved for more than a year, and stem cells were successfully isolated from the cryopreserved tissues thereafter [21,23].…”

“…More specifically, dental follicle tissues from extracted wisdom teeth were cryopreserved for more than a year, and stem cells were successfully isolated from the cryopreserved tissues thereafter [21,23]. Stem cells from cryopreserved dental follicles (hDFCs-cryo) displayed the same MSC characteristics as stem cells isolated from fresh dental follicles (hDFCs-fresh) [21]. Moreover, hDFCs-cyro and hDFCs-fresh revealed the same in vivo bone regeneration potential and immunomodulatory effects following in vivo transplantation [23].…”