Chinese Journal of Tissue Engineering Research ›› 2016, Vol. 20 ›› Issue (11): 1570-1576.doi: 10.3969/j.issn.2095-4344.2016.11.008

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

Li Jian-guo1, Li Pan2, 3, 4, Yin He-yong3, 5, Liu Xi2, Zhou Yu-jun2, Li Hai-tao2, Li Yan-fei2, Wang Fei2, Hu Cheng-dong2
  

  1. 1Emergency Department, Shexian County Hospital, Handan 056400, Hebei Province, China; 2Second Department of Orthopedics, Handan Central Hospital, Handan 056001, Hebei Province, China; 3Institute of Orthopedics, Chinese PLA General Hospital, Beijing 100039, China; 4Hebei Medical University, Shijiazhuang 050011, Hebei Province, China; 5School of Medicine, Nankai University, Tianjin 300073, China
  • Received:2015-12-28 Online:2016-03-11 Published:2016-03-11
  • Contact: Hu Cheng-dong, M.D., Chief physician, Master’s supervisor, Second Department of Orthopedics, Handan Central Hospital, Handan 056001, Hebei Province, China
  • About author:Li Jian-guo, Attending physician, Emergency Department, Shexian County Hospital, Handan 056400, Hebei Province, China
  • Supported by:

    Handan Science and Technology Research and Development Project, No. 0928108052

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Abstract:

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.