Physical problems with the vitrification of large biological systems

Area of Science:

• Cryobiology
• Biotechnology
• Materials Science

Background:

• Vitrification offers a promising method for cryopreserving mammalian organs.
• Current research primarily focuses on small biological samples (microns to millimeters).
• Scaling vitrification to larger volumes (milliliters to liters) presents unique challenges.

Purpose of the Study:

• To investigate the effects of sample size on vitrification outcomes for large biological systems.
• To provide data on nucleation, ice crystal growth, and fracture in larger samples.
• To evaluate the influence of cooling rate and thermal gradients on vitrification success.

Main Methods:

• Qualitative and quantitative analysis of thermal gradients, cooling rates, and fracture temperatures.
• Experiments conducted on propylene glycol solutions across a range of sample sizes (10 ml to 1.5 L).
• Assessment of nucleation probability, ice crystal size, and fracture occurrence at or below the glass transition temperature (Tg).

Main Results:

• Cooling rates significantly impact nucleation and crystal growth in vitrification solutions.
• Slow, uniform cooling can postpone fracturing in large samples (e.g., 482 ml) to approximately 25°C below Tg.
• Larger sample sizes ( > 10 ml) require higher cryoprotectant concentrations for vitrification, even with carrier solutions.

Conclusions:

• Sample size is a critical factor influencing the success of organ vitrification.
• Optimized cooling protocols and understanding thermal gradients are essential for large-volume cryopreservation.
• Increased cryoprotectant concentration is necessary for larger samples, posing a challenge for clinical application.