The Protocol Of Cryoprotectant Agent For Rabbit Intervertebral Disc Vitrification

Objectives: This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents(CPA),and to select the optimal cryoprotectant agents for the vitrification of intact intervertebral disc.Methods: We chose five most commonly used cryoprotectant agents including dimethyl sulphoxide,formamide,ethylene glycol,propylene glycol and glycerol.Using these agents,five single-CPA aqueous solutions,ten two-CPA solutions,and ten three-CPA solutions were formulated.Cell viability of nucleus pulposus cells was determined using Cell Counting Kit-8(CCK-8)and Fluorescein Diacetate/Propidium Iodide(FDA/PI).Then investigating the effect of preservation of the rabbit intervertebral disc by vitrification.Results: The results of CCK-8 test was consistent with the results of FDA/PI test,the order of the toxicity of these five CPA from low to high is Ethylene Glycol,Glycerol,Formamide,DMSO,Propylene Glycol.The toxicity of 2-CPA and 3-CPA was lower than any of the CPA which formulated them.The toxicity of the mixed CPA that contain Ethylene Glycol or Glycerol was lower than any other mixed CPA.The survival rate and relative activity of the rabbit lumbar disc nucleus pulposus cells in the vitrification preservation group were better than those in the cryopreservation group.Conclusions: We can choose the mixed CPA that contain Ethylene Glycol and(or)Glycerol for the vitrification of intervertebral disc.