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

Area of Science:

• Cellular biology
• Biotechnology
• Cryopreservation science

Background:

• Hepatocyte viability and function loss after cryopreservation remains a significant challenge.
• Vitrification shows promise but is limited for large cell volumes needed clinically.

Purpose of the Study:

• To develop and validate a novel bulk droplet vitrification method for large hepatocyte volumes.
• To improve post-preservation viability and metabolic function compared to cryopreservation.

Main Methods:

• Utilized rapid osmotic dehydration to concentrate intracellular cryoprotective agents (CPAs) before vitrification.
• Employed a low preincubated CPA concentration (15% v/v) to minimize toxicity.
• Compared bulk droplet vitrified, cryopreserved, and fresh hepatocytes for viability, morphology, and metabolic function.

Main Results:

• Bulk droplet vitrified hepatocytes exhibited significantly higher viability post-preservation and after 1 day in culture compared to cryopreserved cells.
• Vitrified hepatocytes demonstrated improved morphology and significantly higher metabolic activity in long-term cultures.
• Simulations and measurements confirmed adequate intracellular CPA concentration and high cooling rates for successful vitrification.

Conclusions:

• A novel bulk droplet vitrification method for large cell volumes was developed and validated.
• This method significantly enhances hepatocyte viability and metabolic function over cryopreservation.
• The technique offers a viable alternative for clinical applications requiring preserved hepatocytes.