Droplet freeze-thawing system based on solid surface vitrification and laser rewarming_Journal of Biomedical Engineering

Authors：

ZHU Wenxin ¹ , PAN Ping’an ¹ ,  HUANG Yonghua ¹ , CHEN Wei ² , HAN Sha ² , LI Zheng ² , CHENG Jinsheng ³

1. Institute of Refrigeration and Cryogenics, Shanghai Jiao Tong University, Shanghai 200240, P. R. China;
2. Department of Andrology, Center for Men’s Health, Institute of Urology, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, P. R. China;
3. Shanghai Tofflon Medical Equipment Co., Ltd, Shanghai 201108, P. R. China;

Corresponding author：

HUANG Yonghua, Email: huangyh@sjtu.edu.cn

Keywords：

Cell cryopreservation; Vitrification; Solid surface vitrification; Laser warming

DOI：

10.7507/1001-5515.202305004

Video：

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Abstract Full text Figures/Tables Video References Cited by

Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10³ °C/min. The volume range of 1–8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400–1 200 W laser power, and the rewarming rate was up to the order of 10⁶ °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400–1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.

Citation： ZHU Wenxin, PAN Ping’an, HUANG Yonghua, CHEN Wei, HAN Sha, LI Zheng, CHENG Jinsheng. Droplet freeze-thawing system based on solid surface vitrification and laser rewarming. Journal of Biomedical Engineering, 2023, 40(5): 973-981. doi: 10.7507/1001-5515.202305004 Copy

1.	喻梓瑄, 张宵敏, 周新丽. 微滴喷射高通量玻璃化细胞保存的实验研究. 生物医学工程学杂志, 2019, 36(5): 850-855, 861.
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10.	Akiyama Y, Shinose M, Watanabe H, et al. Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing. Proc Natl Acad Sci, 2019, 116(16): 7738-7743.
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12.	Jin B, Kleinhans F W, Mazur P. Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse. Cryobiology, 2014, 68(3): 419-430.
13.	Bhattacharya M S. A review on cryoprotectant and its modern implication in cryonics. Asian J Pharmac, 2016, 10(3): 154-159.
14.	Zhan L, Guo S Z, Kangas J, et al. Conduction cooling and plasmonic heating dramatically increase droplet vitrification volumes for cell cryopreservation. Adv Sci, 2021, 8(11): 2004605.
15.	Khosla K, Zhan L, Bhati A, et al. Characterization of laser gold nanowarming: a platform for millimeter-scale cryopreservation. Langmuir, 2018, 35(23): 7364-7375.
16.	Hopkins J B, Badeau R, Warkentin M, et al. Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions. Cryobiology, 2012, 65: 169-178.
17.	Elliott G D, Wang S, Fuller B J. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology, 2017, 76: 74-91.
18.	Han Z, Bischof J C. Critical cooling and warming rates as a function of CPA concentration. CryoLetters, 2020, 41: 185-193.

1. 喻梓瑄, 张宵敏, 周新丽. 微滴喷射高通量玻璃化细胞保存的实验研究. 生物医学工程学杂志, 2019, 36(5): 850-855, 861.
2. Best B P. Cryoprotectant toxicity: Facts, issues, and questions. Rejuv Res, 2015, 18(5): 422-436.
3. Marx V. Cell-line authentication demystified. Nat Methods, 2014, 11: 483-488.
4. Gonzales F, Luyet B. Resumption of heart-beat in chick embryo frozen in liquid nitrogen. Biodynamica, 1950, 7(126-128): 1-5.
5. Brockbank K G M, Chen Z Z, Song Y C. Vitrification of porcine articular cartilage. Cryobiology, 2010, 60(2): 217-221.
6. Campbell L D, Betsou F, Garcia D L, et al. Development of the ISBER best practices for repositories: Collection, storage, retrieval and distribution of biological materials for research. Biopreserv Biobank, 2012, 10(2): 232-233.
7. Fischbach M A, Bluestone J A, Lim W A. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med, 2013, 5(179): 1-6.
8. Singh V K, Kalsan M, Kumar N, et al. Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev Biol, 2015, 3(2): 1-18.
9. Shi M, Ling K, Yong K W, et al. High-throughput non-contact vitrification of cell-laden droplets based on cell printing. Sci Rep, 2015, 5(1): 1-10.
10. Akiyama Y, Shinose M, Watanabe H, et al. Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing. Proc Natl Acad Sci, 2019, 116(16): 7738-7743.
11. Jae R Y, Young S S. Vitrification and devitrification of micro-droplets. J Micromech Microeng, 2012, 22: 115018.
12. Jin B, Kleinhans F W, Mazur P. Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse. Cryobiology, 2014, 68(3): 419-430.
13. Bhattacharya M S. A review on cryoprotectant and its modern implication in cryonics. Asian J Pharmac, 2016, 10(3): 154-159.
14. Zhan L, Guo S Z, Kangas J, et al. Conduction cooling and plasmonic heating dramatically increase droplet vitrification volumes for cell cryopreservation. Adv Sci, 2021, 8(11): 2004605.
15. Khosla K, Zhan L, Bhati A, et al. Characterization of laser gold nanowarming: a platform for millimeter-scale cryopreservation. Langmuir, 2018, 35(23): 7364-7375.
16. Hopkins J B, Badeau R, Warkentin M, et al. Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions. Cryobiology, 2012, 65: 169-178.
17. Elliott G D, Wang S, Fuller B J. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology, 2017, 76: 74-91.
18. Han Z, Bischof J C. Critical cooling and warming rates as a function of CPA concentration. CryoLetters, 2020, 41: 185-193.

Journal of Biomedical Engineering

Droplet freeze-thawing system based on solid surface vitrification and laser rewarming

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