Department of Dental Basic Sciences, Faculty of Odonto-Stomatology, University of Medicine and Pharmacy, Ho Chi Minh City, Viet Nam.
Department of Physiology and Animal Biotechnology, Laboratory of Tissue Engineering and Biomedical Materials, Faculty of Biology - Biotechnology, University of Science, Vietnam National University, Ho Chi Minh City, Viet Nam.
Arch Oral Biol. 2017 Dec;84:74-81. doi: 10.1016/j.archoralbio.2017.09.002. Epub 2017 Sep 7.
This study aimed to simplify the collection, isolation and cryopreservation procedure of human dental pulp stem cells (DPSCs) to ease the establishment of dental stem cell banking.
Extracted third molars were collected and stored either in growth medium or in gentamicin-saline (480μg/ml) for 6, 9 or 12h. DPSCs were isolated and subjected to cryopreservation by a controlled-rate or rapid freezing method in 5 or 10% DMSO. Flow cytometry and growth pattern of DPSCs before and after cryopreservation were conducted.
Rate of contamination by which the extracted teeth were stored in control and gentamicin-saline were 9.1% (N=33) and 2.3% (N=43), respectively. Successful cell isolation rate of teeth preserved in gentamicin-saline at 6h (92.9%) was comparable to those of growth media group (90.3%). At 9 and 12h, the rates dropped significantly to 75% and 54%, respectively. Cryopreservation by controlled-rate freezing either in 5 or 10% DMSO resulted in a significantly higher percentage of viable cells than by rapid freezing. Cells conserved by controlled-rate freezing in 5% DMSO showed a pattern of growth similar to control unfrozen cells; 10% DMSO significantly deteriorated the growth pattern of the cells. After thawing, DPSCs conserved by controlled-rate freezing still expressed stemness characteristics, although hematopoietic stem cell markers were slightly increased.
Gentamicin-saline was effective in preserving human teeth for DPSC isolation. Controlled-rate freezing in 5% DMSO gave the highest rate of cell viability. This study simplifies the storage conditions and proposes a simple method for cryopreservation of DPSCs.
本研究旨在简化人牙髓干细胞（DPSCs）的采集、分离和冻存程序，以方便建立牙髓干细胞库。
采集第三磨牙并分别储存在生长培养基或庆大霉素-生理盐水（480μg/ml）中 6、9 或 12 小时。分离 DPSCs 并采用控速或快速冷冻法在 5%或 10%DMSO 中进行冻存。比较冻存前后 DPSCs 的流式细胞术和生长模式。
保存在对照组和庆大霉素-生理盐水中的牙齿污染率分别为 9.1%（N=33）和 2.3%（N=43）。保存在庆大霉素-生理盐水 6 小时的牙齿（92.9%）成功分离细胞率与生长培养基组（90.3%）相当。9 小时和 12 小时时，分离率显著下降至 75%和 54%。5%或 10%DMSO 控速冷冻冻存的细胞存活率显著高于快速冷冻。5%DMSO 控速冷冻保存的细胞保持与未冷冻对照细胞相似的生长模式；10%DMSO 显著恶化了细胞的生长模式。冻存后，通过控速冷冻保存的 DPSCs 仍表达干细胞特性，尽管造血干细胞标志物略有增加。
庆大霉素-生理盐水可有效保存人牙用于 DPSCs 分离。5% DMSO 控速冷冻可获得最高的细胞活力率。本研究简化了储存条件，并提出了一种简单的 DPSCs 冻存方法。
