五种玻璃化冻存试剂单独或组合应用对髓核细胞的毒性分析

Five kinds of vitrified cryoprotectants:toxicity of their alone or combination to nucleus pulposus cells

背景:冻存椎间盘可为同种异体椎间盘移植创造条件,传统的低温冷冻法常导致细胞内外冰晶形成从而造成细胞损伤,玻璃化冻存法可避免冻存过程中细胞内外形成冰晶而得到广泛应用,然而玻璃化法冻存椎间盘在内外文献中却少见报道,冻存试剂对髓核细胞的毒性研究也比较少见。目的:分析5种玻璃化冻存试剂单独或组合应用对于兔髓核细胞的毒性作用,从而为玻璃化冻存椎间盘选择合适的冻存试剂。方法:选取目前最常用的5种玻璃化冻存试剂:二甲基亚砜、甲酰胺、乙二醇、丙二醇、丙三醇,采用单一5种、任意2种及任意3种玻璃化冻存试剂互相组合成各10种冻存试剂组合共25种。用CCK-8和FDA/PI两种方法检测髓核细胞的细胞存活率。用统计学方法对所得数据进行统计分析,选出对髓核细胞毒性较小的玻璃化冻存试剂组合方案。结果与结论:1两种检测方法检测结果:5种玻璃化试剂对髓核细胞的毒性由小到大依次为:乙二醇、丙三醇、甲酰胺、二甲基亚砜、丙二醇。22种冻存试剂组合和3种冻存试剂组合的毒性均比组成该组合溶液的任意一种单一玻璃化冻存试剂小。3组合试剂中,含有乙二醇或丙三醇的2种冻存试剂组合或3种冻存试剂组合毒性均较小。故玻璃化冻存椎间盘可选用包含乙二醇和(或)丙三醇的玻璃化组合试剂。

BACKGROUND： Transplantation of allogeneic intervertebral disc can be facilitated by the cryopreservation of the intervertebral disc. But the traditional cryopreservation methods always lead to the appearing of ice crystals inside and outside the cells which can cause cellular injury. The vitrification method that can avoid the formation of ice crystals have been widely applied in the cryopreservation field. However, only a few reports have assessed the vitrified cryopreservation of the intervertebral disc, and the toxicity of cryoprotectants to the nucleus pulposus cells have not been fully explored. OBJECTIVE： To determine the order of toxicity of five commonly used cryoprotectants that are used alone or in combination to rabbit nucleus pulposus cells, and to select the optimal cryoprotectant for the vitrification of the intervertebral disc.METHODS： We chose five most commonly used cryoprotectants including dimethyl sulphoxide, formamide, ethylene glycol, propylene glycol and glycerol. Then, 5 single commonly used cryoprotectants, 10 mixed agents containing any 2 commonly used cryoprotectants, and 10 mixed agents containing any 3 commonly used cryoprotectants were formulated. Cell viability of nucleus pulposus cells was determined using cell counting kit-8 and fluorescein diacetate/propidium iodide method. All data obtained were analyzed statistically to choose the appropriate combining scheme with less toxicity. RESULTS AND CONCLUSION： The order of the toxicity of these five commonly used cryoprotectants from low to high was ethylene glycol, glycerol, formamide, dimethyl sulphoxide, and propylene glycol. The toxicity of the combined agents containing two or three commonly used cryoprotectants was lower than that of any commonly used cryoprotectants that were used to formulate them. The toxicity of the mixed agents that contained ethylene glycol or glycerol was lower than that of any other mixed agents. So we can choose the mixed cryoprotectants that contain ethylene glycol and（or） glycerol for the vitrification of the intervertebral disc.