Study on thermophysical properties and effect of lyoprotectants in freezing human hepatoma Hep-G<sub>2</sub> cells_Journal of Biomedical Engineering

Authors：

LI Weijie , SONG Ping ,  LIU Baolin

School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, P.R.China;

Corresponding author：

LIU Baolin, Email: blliuk@163.com

Keywords：

freeze-drying; lyoprotectants; freezing; differential scanning calorimeter

DOI：

10.7507/1001-5515.201711004

Video：

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Cell freeze-drying can be divided into the freezing and drying processes. Mechanical damage caused by ice crystals and damage from solute during freezing shall not be ignored and lyoprotectants are commonly used to reduce those damages on cells. In order to study the mechanism of lyoprotectants to protect cells and determine an optimal lyoprotectant formula, the thermophysical properties and percentage of unfrozen water of different lyoprotectants in freezing were investigated with differential scanning calorimeter (DSC). The survival rate indicated by trypan blue exclusion test and cell-attachment rate after 24 h using different lyoprotectants to freeze hepatoma Hep-G₂ cells were measured after cell cryopreservation. The results show that 40% (W/V) PVP + 10% (V/V) glycerol + 15% (V/V) fetal bovine serum + 20% (W/V) trehalose formula of lyoprotectant demonstrate the best effect in protecting cells during freezing, for cell-attachment rate after 24 h is 44.56% ± 2.73%. In conclusion, the formula of lyoprotectant mentioned above can effectively protect cells.

Citation： LI Weijie, SONG Ping, LIU Baolin. Study on thermophysical properties and effect of lyoprotectants in freezing human hepatoma Hep-G₂ cells. Journal of Biomedical Engineering, 2019, 36(5): 803-809. doi: 10.7507/1001-5515.201711004 Copy

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4.	Akhoondi M, Oldenhof H, Stoll C, et al. Membrane hydraulic permeability changes during cooling of mammalian cells. Biochim Biophys Acta, 2011, 1808(3): 642-648.
5.	Sieme H, Oldenhof H, Wolkers W F. Mode of action of cryoprotectants for sperm preservation. Anim Reprod Sci, 2016, 169: 2-5.
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10.	周新丽, 陈光明, 张绍志. 血液细胞冷冻干燥保存的研究进展. 国际输血及血液学杂志, 2005, 28(5): 428-431.
11.	Han B, Bischof J C. Direct cell injury associated with eutectic crystallization during freezing. Cryobiology, 2004, 48(1): 8-21.
12.	Oldenhof H, Friedel K, Sieme H, et al. Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy. Cryobiology, 2010, 61(1): 115-122.
13.	Wolkers W F, Balasubramanian S K, Ongstad E L, et al. Effects of freezing on membranes and proteins in LNCaP prostate tumor cells. Biochim Biophys Acta, 2007, 1768(3): 728-736.
14.	Fahy G M, Levy D I, Ali S E. Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology, 1987, 24(3): 196-213.
15.	Wang Shangping, Oldenhof H, Dai Xiaolei, et al. Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules. Biochim Biophys Acta, 2014, 1844(2): 430-438.
16.	何晖, 刘宝林, 华泽钊, 等. 甘油保护剂对冷冻干燥保存红细胞作用的实验研究. 上海理工大学学报, 2006, 28(5): 413-417.
17.	Wowk B, Darwin M, Harris S B, et al. Effects of solute methoxylation on glass-forming ability and stability of vitrification solutions. Cryobiology, 1999, 39(3): 215-227.
18.	Shamblin S L, Zografi G. The effects of absorbed water on the properties of amorphous mixtures containing sucrose. Pharm Res, 1999, 16(7): 1119-1124.
19.	Tomczak M M, Hincha D K, Estrada S D, et al. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein. Biophys J, 2002, 82(2): 874-881.
20.	Imamura K, Asano Y, Maruyama Y, et al. Characteristics of Hydrogen bond formation between sugar and polymer in freeze-dried mixtures under different rehumidification conditions and its impact on the glass transition temperature. J Pharm Sci, 2008, 97(3): 1301-1312.

1. 华泽钊. 冷冻干燥新技术. 北京: 科学出版社, 2006.
2. Nash T. Chemical constitution and physical properties of compounds able to protect living cells against damage due to freezing and thawing. Cryobiology, 1966: 179-211.
3. Wu Zhuangyuan, Zheng Xinbiao, Luo Yongming, et al. Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. Anim Reprod Sci, 2015, 163: 75-81.
4. Akhoondi M, Oldenhof H, Stoll C, et al. Membrane hydraulic permeability changes during cooling of mammalian cells. Biochim Biophys Acta, 2011, 1808(3): 642-648.
5. Sieme H, Oldenhof H, Wolkers W F. Mode of action of cryoprotectants for sperm preservation. Anim Reprod Sci, 2016, 169: 2-5.
6. Crowe J H, Oliver A E, Hoekstra F A, et al. Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification. Cryobiology, 1997, 35(1): 20-30.
7. 司徒镇强, 吴军正. 细胞培养. 西安: 世界图书出版公司, 1996.
8. Jabrane S, Létoffé J M, Claudy P. Vitrification and crystallization in the R(−)1,2-propanediol-S(+)1,2-propanediol system. Thermochim Acta, 1995, 258(7): 33-47.
9. 高才, 周国燕, 胥义, 等. 乙二醇和丙三醇水溶液冻结特性的研究. 物理化学学报, 2004, 20(2): 123-128.
10. 周新丽, 陈光明, 张绍志. 血液细胞冷冻干燥保存的研究进展. 国际输血及血液学杂志, 2005, 28(5): 428-431.
11. Han B, Bischof J C. Direct cell injury associated with eutectic crystallization during freezing. Cryobiology, 2004, 48(1): 8-21.
12. Oldenhof H, Friedel K, Sieme H, et al. Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy. Cryobiology, 2010, 61(1): 115-122.
13. Wolkers W F, Balasubramanian S K, Ongstad E L, et al. Effects of freezing on membranes and proteins in LNCaP prostate tumor cells. Biochim Biophys Acta, 2007, 1768(3): 728-736.
14. Fahy G M, Levy D I, Ali S E. Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology, 1987, 24(3): 196-213.
15. Wang Shangping, Oldenhof H, Dai Xiaolei, et al. Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules. Biochim Biophys Acta, 2014, 1844(2): 430-438.
16. 何晖, 刘宝林, 华泽钊, 等. 甘油保护剂对冷冻干燥保存红细胞作用的实验研究. 上海理工大学学报, 2006, 28(5): 413-417.
17. Wowk B, Darwin M, Harris S B, et al. Effects of solute methoxylation on glass-forming ability and stability of vitrification solutions. Cryobiology, 1999, 39(3): 215-227.
18. Shamblin S L, Zografi G. The effects of absorbed water on the properties of amorphous mixtures containing sucrose. Pharm Res, 1999, 16(7): 1119-1124.
19. Tomczak M M, Hincha D K, Estrada S D, et al. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein. Biophys J, 2002, 82(2): 874-881.
20. Imamura K, Asano Y, Maruyama Y, et al. Characteristics of Hydrogen bond formation between sugar and polymer in freeze-dried mixtures under different rehumidification conditions and its impact on the glass transition temperature. J Pharm Sci, 2008, 97(3): 1301-1312.

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Study on thermophysical properties and effect of lyoprotectants in freezing human hepatoma Hep-G₂ cells