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Langmuir. 2019 Jun 11;35(23):7354-7363. doi: 10.1021/acs.langmuir.8b02831. Epub 2019 Jan 9.

Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome.

de Vries RJ1,2,3,4, Banik PD1,2,4, Nagpal S1,2,4, Weng L1,2, Ozer S1,2,4, van Gulik TM3, Toner M1,2,4, Tessier SN1,2,4, Uygun K1,2,4.

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Department of Surgery , Massachusetts General Hospital , Boston , Massachusetts 02114 , United States.
Center for Engineering in Medicine , Harvard Medical School , Boston Massachusetts 02114 , United States.
Department of Surgery, Academic Medical Center , University of Amsterdam , 1105 AZ Amsterdam , The Netherlands.
Shriners Hospital for Children , Boston , Massachusetts 02114 , United States.


Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3-5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960-1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.

[Available on 2020-06-11]

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