不同组织冻存体系对牙周膜干细胞体外扩增的影响

背景：冻存是保证干细胞移植治疗的关键步骤之一。传统的冻存是将细胞直接置于冻存液中进行保存,然而冻存液中二甲基亚砜虽能减少细胞复苏过程中冰晶对细胞膜产生的机械性损伤,但同时又对细胞具有毒性作用,直接影响细胞生存状态,不利于临床移植治疗。目的：寻找适宜牙周膜干细胞体外扩增的牙周周组织冻存的最佳方案。方法：收集健康人牙,刮取牙周组织后将其平均分为3等份,随机分为新鲜组、5%二甲基亚砜组和10%二甲基亚砜组,后2组分别以体积分数5%和10%二甲基亚砜添加冻存1个月后提取牙周膜干细胞。新鲜组直接提取牙周膜干细胞。结果与结论：5%二甲基亚砜组原代细胞游出组织团块所需时间和细胞收获量虽不及新鲜培养组,但却明显优于10%二甲基亚砜组（P〈0.05）。新鲜组、5%二甲基亚砜组和10%二甲基亚砜组第1代牙周膜干细胞克隆形成率、活细胞比率、第3代牙周膜干细胞BrdU细胞增殖能力、MTT细胞生长曲线和牙周膜干细胞表面标志物表达差异没有显著性意义（P〉0.05）。提示5%二甲基亚砜添加冻存体系不仅能比10%二甲基亚砜添加的普通冻存体系明显缩短牙周膜干细胞体外扩增所需时间,增加细胞收获量同时还能保持细胞基本生物学特性,降低二甲基亚砜的总体用量和其在反复冻存复苏细胞过程中对细胞造成的直接损伤,为未来更加安全的实施临床移植治疗提供了保障,是供体组织储存新的选择。

Periodontal ligament stem cells expansion in vitro under different cryopreservation systems

BACKGROUND： The cryopreservation is the key step to ensure the successful stem cell transplantation treatment. The traditional cryopreservation is to place the cells directly into the cryopreservation solution, but dimethyl sulfoxide in the cryopreservation liquid likes a double-edged sword during the repeated cryopreservation and recovery process, that is, although dimethyl sulfoxide can reduce the cell membrane mechanical injury by ice crystals in the recovery process, at the same time, it can enhance the toxic effects on cells directly and affect cell survival situation and is not conducive to clinical transplantation therapy. In recent years, extracted stem cells from frozen tissue had become another research direction. OBJECTIVE： To look for the best solution of periodontal ligament stem cells expansion in vitro by the appropriate cryopreservation periodontal tissue. METHODS： Periodontal tissue was scraped from healthy human teeth and divided them into three equal parts： fresh group, harvesting periodontal ligament stem cells from fresh tissue; 5% dimethyl sulfoxide group, 5% dimethyl sulfoxide added into the cryopreservation system; 10% dimethyl sulfoxide group, 10% dimethyl sulfoxide added into the cryopreservation system. One month later, periodontal ligament stem cells were extracted from the cryopreserved periodontal ligament from the latter two groups. RESULTS AND CONCLUSION： In the time that cells swam out of tissue mass and cell harvest amount, the 5% dimethyl sulfoxide group was inferior to the fresh group but better than the 10% dimethyl sulfoxide group （P〈0.05）. No differences were found among the three groups in the following aspects （P〈0.05）： colony formation rate of passage 1 periodontal ligament stem cells, cell survival rate, proliferation ability of passage 3 periodontal ligament stem cells, cell growth curve and surface marker expression of periodontal ligament stem cells. The results suggest that the 5% dimethyl sulfoxide added cryopreserved system for periodontal ligament tissue cannot only shorten the time of periodontal ligament stem cells amplification in vitro, ensure cell harvest and maintain basic cellular biological characteristics, but also reduce the total amount of the dimethyl sulfoxide and the direct damage to the cells caused by repeatedly frozen cells, thereby providing a more secure guarantee for the future implementation of clinical transplantation therapy. So, the 5% dimethyl sulfoxide added cryopreserved system may be the new selection for donor tissue storage.