Cryonics, the low-temperature preservation of tissues and organs, offers the possibility of ‘suspended animation’ and long term storage for future use. Although the idea seems promising, there are several challenges to achieving successful cryopreservation, especially for large volumes. A key issue is recrystallization - the growth of ice crystals upon rewarming that can damage cells and tissues. Controlled rewarming at high rates is critical to overcoming this problem, but difficult to achieve uniformly in large samples.
A New Approach: Focused Ultrasound
A new proof-of-concept study has demonstrated the use of high intensity focused ultrasound (HIFU) waves to achieve the rapid rewarming needed to avoid recrystallization during cryopreservation. HIFU uses acoustic waves to generate targeted heat at a focus point. The study, published in Scientific Reports, used HIFU to successfully cryopreserve and rewarm the nematode worm Caenorhabditis elegans.
HIFU has several advantages for cryopreservation. It does not create stationary heating patterns and avoids thermal runaway, allowing precise control of rewarming rates. It can also be used with conventional cryoprotectants and monitored in real-time using magnetic resonance imaging (MRI). Importantly, HIFU is scalable to larger sample volumes using transducer arrays. These properties make HIFU a promising tool for achieving the rapid and uniform rewarming needed for successful cryopreservation of tissues and organs.
Proof of Principle: Cryopreserving C. elegans
To demonstrate the feasibility of HIFU for cryopreservation, the researchers used C. elegans as an animal model system. C. elegans is a well-studied model organism used in many laboratories, with a complete set of organs. Populations of about 200 worms at all life stages were cryopreserved using a standard slow freezing method in 15% glycerol and cooled to -80°C for storage.
After 48 hours, the worms were rewarmed either using HIFU or conventional methods (air or water bath). Conventional rewarming led to only 35% survival for early larval stages and 0% for adults, showing the need for more rapid rewarming.
Optimizing the HIFU Parameters
A series of 53 experiments were performed to optimize the HIFU parameters, varying the rewarming rate and exposure time. The rewarming rate was measured from -60 to -40°C, where recrystallization is most likely to occur. They found that higher rewarming rates of 157.8°C/min and 217.8°C/min led to the highest survival rates when applied for 60-70 seconds.
Insufficient exposure to HIFU (less than 50 seconds) did not achieve rapid enough rewarming and resulted in no survival. Excessively long exposure (over 70 seconds) also reduced survival, likely due to overheating. At the optimal parameters of 217.8°C/min for 70 seconds, survival rates reached 90% across all life stages, compared to only 35% for early larval stages and 0% for adults with conventional rewarming.
The researchers also simulated the experimental setup using finite element modeling to help determine the focal region dimensions and verify the rewarming rates. The highest temperature achieved was within 10°C between points in the samples, demonstrating the precision achievable with HIFU.
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Scalability and Potential Applications
A key advantage of HIFU is that it is scalable to larger sample volumes. The researchers used a single transducer in this study, but scaling up the number of transducers in an array could allow targeted rewarming of tissues and whole organs. HIFU rewarming could also be monitored in real-time using magnetic resonance imaging (MRI), allowing precise control of heating.
The successful cryopreservation and recovery of C. elegans at all life stages demonstrates the potential of HIFU rewarming to enable long-term biobanking of many sample types. While further research is needed, HIFU may help solve the longstanding challenge of recrystallization during rewarming and enable the cryopreservation of larger tissues and whole organs for regenerative medicine. With its scalability, controllability, and routine use in other applications, HIFU is a promising new tool for advancing the field of cryonics.
