Analysis of protective mechanism of silk protein based cryoprotectants_Journal of Biomedical Engineering

Authors：

 ZHOU Xinli , DU Yukun , TENG Yun , ZHANG Xiaomin

Institute of Biothermal Technology, University of Shanghai for Science and Technology, Shanghai 200093, P.R.China;

Corresponding?author：

ZHOU Xinli, Email: zjulily@163.com

Keywords：

silk fibroin; cryoprotectant; mechanism analysis

DOI：

10.7507/1001-5515.201807061

Video：

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Dimethyl sulfoxide (Me₂SO) supplemented with fetal bovine serum (FBS) is a widely used cryoprotectant combination. However, high concentration of Me₂SO is toxic to cells, and FBS presents problems related to diseases such as bovine spongiform encephalopathy and viral infections. Silk protein is a kind of natural macromolecule fiber protein with good biocompatibility and hydrophilicity. The aim of this paper is to analyze the cryoprotective mechanism of silk protein as cryoprotectant. Firstly, differential scanning calorimetry (DSC) was used to measure the thermal hysteresis activity (THA) of silk protein. The THA of 10 mg/mL sericin protein was 0.96°C, and the THA of 10% (V/V) fibroin protein was 1.15°C. Then the ice recrystallization inhibition (IRI) of silk protein-PBS solution was observed with cryomicroscope. The cold stage was set at ? 7°C, after 40 minutes’ incubation, the mean grain size rate (MGSR) of sericin protein and fibroin protein were 28.99% and 3.18%, respectively, which were calculated relative to phosphate buffer saline (PBS) control. It is indicated that sericin and silk fibroin have certain effects of inhibiting recrystallization of ice crystals. Finally, the structure and physicochemical properties of silk protein were analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that the content of the random coil was 75.62% and the β-sheet structure was 24.38% in the secondary of sericin protein. The content of the β-sheet structure was 56.68%, followed by random coil structure 22.38%, and α-helix 16.84% in the secondary of fibroin protein. The above analysis demonstrates the feasibility of silk fibroin as a cryoprotectant, and provides a new idea for the selection of cryoprotectants in the future.

Citation： ZHOU Xinli, DU Yukun, TENG Yun, ZHANG Xiaomin. Analysis of protective mechanism of silk protein based cryoprotectants. Journal of Biomedical Engineering, 2019, 36(6): 986-993. doi: 10.7507/1001-5515.201807061 Copy

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2.	Lovelock J E, Bishop M W. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature, 1959, 183(4672): 1394-1395.
3.	Guillouzo A, Rialland L, Fautrel A, et al. Survival and function of isolated hepatocytes after cryopreservation. Chem Biol Interact, 1999, 121(1): 7-16.
4.	Nagahara Y, Sekine H, Otaki M, et al. Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices. Cryobiology, 2016, 72(1): 53-59.
5.	李佳佳, 程騰, 賀小英, 等. 二甲基亞砜和胎牛血清對293T細胞冷凍效果的影響. 信陽師范學院學報:自然科學版, 2013, 26(2): 217-220.
6.	Hunter N, Foster J, Chong A, et al. Transmission of prion diseases by blood transfusion. J Gener Virol, 2002, 83(Pt 11): 2897-2905.
7.	睢曉潔, 潘超, 楊靜, 等. 以氧化三甲胺作為細胞凍存保護劑的研究. 中國科技論文, 2017, 12(12): 1341-1345.
8.	滕蕓. 基于蠶絲蛋白的低溫保護劑的研究. 上海: 上海理工大學, 2018.
9.	Halwani D O, Brockbank K G, Duman J G, et al. Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions. Cryobiology, 2014, 68(3): 411-418.
10.	Sun K H, Jung K E, Won Y H, et al. Improvement in ovarian tissue quality with supplementation of antifreeze protein during warming of vitrified mouse ovarian tissue. Yonsei Med J, 2018, 59(2): 331-336.
11.	Rubinsky B, Arav A, Mattioli M, et al. The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Commun, 1990, 173(3): 1369-1374.
12.	Arav A, Rubinsky B, Fletcher G, et al. Cryogenic protection of oocytes with antifreeze proteins. Mol Reproduct Dev, 1993, 36(4): 488-493.
13.	Carpenter J F, Hansent T N. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci U S A, 1992, 89(19): 8953-8957.
14.	Amir G, Horowitz L, Rubinsky B, et al. Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III. Cryobiology, 2004, 48(3): 273-282.
15.	馬慶保, 劉志東. 南極磷蝦抗凍蛋白熱滯活性的差示掃描量熱法評價. 食品科學, 2018, 39(11): 1-7.
16.	紀瑞慶, 劉愛國, 陳龍, 等. 魚類抗凍蛋白結構與抗凍活性的關系. 食品科學, 2015, 36(5): 274-282.
17.	Mao Xinfang, Liu Zhongyuan, Li Honglei, et al. Calorimetric studies on an insect antifreeze protein ApAFP752 from Anatolica polita. J Therm Anal Calorim, 2011, 104(1): 343-349.
18.	張換成, 胥義. 深低溫保存生物材料快速復溫方法的研究進展. 中國醫學物理學雜志, 2015, 32(1): 144-148.
19.	Tomczak M M, Marshall C B, Gilbert J A, et al. A facile method for determining ice recrystallization inhibition by antifreeze proteins. Biochem Biophys Res Commun, 2003, 311(4): 1041-1046.
20.	Cao Tingting, Zhang Yuqing. Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines. Mater Sci Eng C Mater Biol Appl, 2016, 61: 940-952.
21.	Rockwood D N, Preda R C, Yücel T, et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc, 2011, 6(10): 1612-1631.
22.	尉姍姍, 尹林克, 牟書勇, 等. 新疆沙冬青抗凍蛋白的提取分離及其熱滯活性測定. 云南植物研究, 2007, 29(2): 251-255.
23.	Stubbs C, Lipecki J, Gibson M I. Regioregular alternating polyampholytes have enhanced biomimetic ice recrystallization activity compared to random copolymers and the role of side chain versus main chain hydrophobicity. Biomacromolecules, 2017, 18(1): 295-302.
24.	Jia Chunli, Huang Weining, Wu Chao, et al. Characterization and yeast cryoprotective performance for thermostable icestructuring proteins from Chinese Privet (Ligustrum Vulgare) leaves. Food Research International, 2012, 49(1): 280-284.
25.	Wu Jinhong, Zhou Yanfu, Wang Shaoyun, et al. Laboratory-scale extraction and characterization of ice-binding sericin peptides. Eur Food Res Technol, 2013, 236(4): 637-646.
26.	任禾盛, 許娜飛, 華澤釗. 抗凍蛋白活性的差示掃描量熱測定及其吸附-抑制機制. 細胞生物學雜志, 2004, 26(4): 413-416.
27.	Du N, Liu X Y, Hew C L. Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein. J Biol Chem, 2003, 278(38): 36000-36004.
28.	Raymond J A, DeVries A L. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci U S A, 1977, 74(6): 2589-2593.
29.	Li Ling, Wu Jinhong, Zhang Li, et al. Investigation of the physiochemical properties, cryoprotective activity and possible action mechanisms of sericin peptides derived frommembrane separation. LWT, 2017, 77: 532-541.
30.	Gupta D, Agrawal A, Chaudhary H, et al. Cleaner process for extraction of sericin using infrared. J Clean Prod, 2013, 52(4): 488-494.
31.	周小進, 董雪. 不同脫膠方法對蠶絲性能的影響分析. 針織工業, 2013(4): 44-48.
32.	Zhang D Q, Liu B, Feng D R, et al. Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein. Biochem J, 2004, 377(3): 589-595.
33.	Wierzbicki A, Madura J D, Salmon C, et al. Modeling studies of binding of sea raven type II antifreeze protein to ice. J Chem Inf Comput Sci, 1997, 37(6): 1006-1010.

1. Baust J G, Gao Dayong, Baust J M. Cryopreservation: An emerging paradigm change. Organogenesis, 2009, 5(3): 90-96.
2. Lovelock J E, Bishop M W. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature, 1959, 183(4672): 1394-1395.
3. Guillouzo A, Rialland L, Fautrel A, et al. Survival and function of isolated hepatocytes after cryopreservation. Chem Biol Interact, 1999, 121(1): 7-16.
4. Nagahara Y, Sekine H, Otaki M, et al. Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices. Cryobiology, 2016, 72(1): 53-59.
5. 李佳佳, 程騰, 賀小英, 等. 二甲基亞砜和胎牛血清對293T細胞冷凍效果的影響. 信陽師范學院學報:自然科學版, 2013, 26(2): 217-220.
6. Hunter N, Foster J, Chong A, et al. Transmission of prion diseases by blood transfusion. J Gener Virol, 2002, 83(Pt 11): 2897-2905.
7. 睢曉潔, 潘超, 楊靜, 等. 以氧化三甲胺作為細胞凍存保護劑的研究. 中國科技論文, 2017, 12(12): 1341-1345.
8. 滕蕓. 基于蠶絲蛋白的低溫保護劑的研究. 上海: 上海理工大學, 2018.
9. Halwani D O, Brockbank K G, Duman J G, et al. Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions. Cryobiology, 2014, 68(3): 411-418.
10. Sun K H, Jung K E, Won Y H, et al. Improvement in ovarian tissue quality with supplementation of antifreeze protein during warming of vitrified mouse ovarian tissue. Yonsei Med J, 2018, 59(2): 331-336.
11. Rubinsky B, Arav A, Mattioli M, et al. The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Commun, 1990, 173(3): 1369-1374.
12. Arav A, Rubinsky B, Fletcher G, et al. Cryogenic protection of oocytes with antifreeze proteins. Mol Reproduct Dev, 1993, 36(4): 488-493.
13. Carpenter J F, Hansent T N. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci U S A, 1992, 89(19): 8953-8957.
14. Amir G, Horowitz L, Rubinsky B, et al. Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III. Cryobiology, 2004, 48(3): 273-282.
15. 馬慶保, 劉志東. 南極磷蝦抗凍蛋白熱滯活性的差示掃描量熱法評價. 食品科學, 2018, 39(11): 1-7.
16. 紀瑞慶, 劉愛國, 陳龍, 等. 魚類抗凍蛋白結構與抗凍活性的關系. 食品科學, 2015, 36(5): 274-282.
17. Mao Xinfang, Liu Zhongyuan, Li Honglei, et al. Calorimetric studies on an insect antifreeze protein ApAFP752 from Anatolica polita. J Therm Anal Calorim, 2011, 104(1): 343-349.
18. 張換成, 胥義. 深低溫保存生物材料快速復溫方法的研究進展. 中國醫學物理學雜志, 2015, 32(1): 144-148.
19. Tomczak M M, Marshall C B, Gilbert J A, et al. A facile method for determining ice recrystallization inhibition by antifreeze proteins. Biochem Biophys Res Commun, 2003, 311(4): 1041-1046.
20. Cao Tingting, Zhang Yuqing. Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines. Mater Sci Eng C Mater Biol Appl, 2016, 61: 940-952.
21. Rockwood D N, Preda R C, Yücel T, et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc, 2011, 6(10): 1612-1631.
22. 尉姍姍, 尹林克, 牟書勇, 等. 新疆沙冬青抗凍蛋白的提取分離及其熱滯活性測定. 云南植物研究, 2007, 29(2): 251-255.
23. Stubbs C, Lipecki J, Gibson M I. Regioregular alternating polyampholytes have enhanced biomimetic ice recrystallization activity compared to random copolymers and the role of side chain versus main chain hydrophobicity. Biomacromolecules, 2017, 18(1): 295-302.
24. Jia Chunli, Huang Weining, Wu Chao, et al. Characterization and yeast cryoprotective performance for thermostable icestructuring proteins from Chinese Privet (Ligustrum Vulgare) leaves. Food Research International, 2012, 49(1): 280-284.
25. Wu Jinhong, Zhou Yanfu, Wang Shaoyun, et al. Laboratory-scale extraction and characterization of ice-binding sericin peptides. Eur Food Res Technol, 2013, 236(4): 637-646.
26. 任禾盛, 許娜飛, 華澤釗. 抗凍蛋白活性的差示掃描量熱測定及其吸附-抑制機制. 細胞生物學雜志, 2004, 26(4): 413-416.
27. Du N, Liu X Y, Hew C L. Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein. J Biol Chem, 2003, 278(38): 36000-36004.
28. Raymond J A, DeVries A L. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci U S A, 1977, 74(6): 2589-2593.
29. Li Ling, Wu Jinhong, Zhang Li, et al. Investigation of the physiochemical properties, cryoprotective activity and possible action mechanisms of sericin peptides derived frommembrane separation. LWT, 2017, 77: 532-541.
30. Gupta D, Agrawal A, Chaudhary H, et al. Cleaner process for extraction of sericin using infrared. J Clean Prod, 2013, 52(4): 488-494.
31. 周小進, 董雪. 不同脫膠方法對蠶絲性能的影響分析. 針織工業, 2013(4): 44-48.
32. Zhang D Q, Liu B, Feng D R, et al. Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein. Biochem J, 2004, 377(3): 589-595.
33. Wierzbicki A, Madura J D, Salmon C, et al. Modeling studies of binding of sea raven type II antifreeze protein to ice. J Chem Inf Comput Sci, 1997, 37(6): 1006-1010.

Journal of Biomedical Engineering

Analysis of protective mechanism of silk protein based cryoprotectants

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