Rewarming strategies for cryopreservation: Technological challenges and opportunities in energy conversion

Ruidong Ma; Ziyuan Wang; Ren Shen; Zhiquan Shu; Chen Ming; Dayong Gao

doi:10.1515/fzm-2025-0010

Rewarming strategies for cryopreservation: Technological challenges and opportunities in energy conversion

doi: 10.1515/fzm-2025-0010

Ruidong Ma¹,
Ziyuan Wang¹,
Ren Shen^{1, 2},
Zhiquan Shu^{1, 3},
Chen Ming¹,
Dayong Gao^{1
, ,}

1.
Department of Mechanical Engineering, University of Washington, Seattle 98195, USA
2.
Department of Mechanical Engineering, Seattle University, Seattle 98122, USA
3.
School of Engineering and Technology, University of Washington Tacoma, Tacoma 98402, USA

Funds:

National Natural Science Foundation of China 2346842

More Information

Corresponding author: Dayong Gao, E-mail: dayong@uw.edu

摘要

Abstract: Cryopreservation of living cells and tissues plays a vital role in biomedical research, clinical applications, biotechnology innovation, the development of new vaccines and drugs, and the conservation of endangered species. While significant technological breakthroughs have been achieved in cooling methods—particularly through vitrification for large tissue and organs—the lack of optimal rewarming technology remains a key obstacle to successful cryopreservation, especially for larger samples such as tissues and organs. The primary challenges during the warming process include non-uniformity heating and insufficient rewarming rates, which can lead to thermal stress-induced structural damage and lethal ice recrystallization, ultimately compromising the integrity and functionality of biological materials. In recent years, various advanced warming techniques have emerged, employing different energy conversion approaches to achieve volumetric heating while minimizing the risk of overheating. These techniques involve thermal, mechanical-thermal, and electromagnetic-thermal energy conversions. However, each method presents its own limitation. This review aims to summarize recent advancements in rewarming technologies for cryopreservation, with a focus on their mechanisms, applications, and the key challenges that must be addressed to enable broader adoption in medical and commercial contexts.
- cryopreservation /
- rewarming technology /
- energy conservation /
- electromagnetic

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Figure 1. Cryopreservation standard procedures and Peter Mazur's "two-factor" hypothesis

(A) Major steps of cryopreservation. The sample temperature is initially lowered to 0 ℃ to allow for the addition of cryoprotective agents (CPAs), followed by further cooling to reach the target cryogenic temperature. Storage requires proper moisture control and maintenance of a stable low temperature. Rewarming then returns the sample from ultra-low temperatures to 0 ℃, after which CPAs are removed. Finally, sample functionality and metabolic activity are assessed before future application. (B) Peter Mazur's "Two-factor" Hypothesis. During slow cooling, cells undergo dehydration due to the solution effect. Conversely, rapid cooling can lead to intracellular ice formation, which is lethal to cells. Therefore, an optimal, cell-type-specific cooling rate is required to avoid both intracellular ice formation and excessive dehydration.

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Figure 2. Energy conversion sources and methods used in cryopreservation rewarming technologies

Three primary categories of energy sources-thermal, mechanical, and electromagnetic-are employed to achieve rapid and uniform heating. Each category involves distinct heating mechanisms and has specific applications in cryopreservation rewarming.

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Figure 3. Illustration of the conventional heating method in cryopreservation

(A) Dry thawing utilizing a conduction heat transfer mechanism, where heated plates warm the sample via direct contact. (B) Water bath rewarming, where the sample is immersed in warm water to enable heat transfer through conduction and convection. (C) Hot moving fluid rewarming (typically heated air), demonstrating a convection heat transfer mechanism from left to right.

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Figure 4. Heating mechanism of laser rewarming and its setup

(A) The mechanism of laser rewarming with nanorods or nanoparticles infused into the cryopreserved sample. The laser is directed onto the cryopreserved sample, such as cells, and dispersed by the nanorods across the sample volume. The scattered wave uniformly heats the cells. (B) The general setup of the laser rewarming application. NIR, near infrared.

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Figure 5. Relationship between dielectric properties of materials and frequency

As frequency increases, the corresponding wavelength decreases, and the type of oscillating mass shifts from larger entities such as ions to smaller ones such as electrons.

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Figure 6. Schematic diagram of the Joule heating setup

The left panel illustrates a configuration in which the sample is directly placed on a flat sheet electrical conductor. Upon application of power, Joule heating is initiated, with the bottom layer of the sample being heated first due to direct contact with the conductor. The right panel depicts a sandwich structure designed for tissue slices, enabling more uniform heating by enclosing the sample between conductive layers.

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Figure 7. Molecular structure of water (left), DMSO (middle), and glycerol (right)

The red regions indicate areas of negative charge, while the blue regions represent positive charge. The black arrow denotes the direction of the electric field, illustrating the alignment behavior of dipolar molecules under an applied alternating electric field.

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Figure 8. Configuration of capacitor heating system

(A) Concentric cylindrical electrode configuration; (B) Parallel plate electrode configuration; (C) Multiple electrode configuration. Each setup offers distinct electric field distributions and is suited for different sample sizes and heating uniformity requirements.

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Figure 9. Schematic drawing of the antenna heating systems

The left panel illustrates a horn antenna system equipped with a microwave absorber. The microwave absorber is employed to prevent reflected waves from disrupting the electric field, and should be integrated within the horn antenna. The right panel depicts the hybrid helical antenna heating method combined with a water bath. The water bath is placed outside the sample holder to enhance heating efficiency.

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Figure 10. SMER technology setup and results

(A)-(C) A schematic diagram of the SMER cavity with automatic loading and unloading system. (D) & (E) The simulation of the electric and magnetic field distribution. (F) The rewarming rate of the 25 mL DPVP solution from vitrified state to room temperature.

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Figure 11. Eddy current illustration

The left figure shows a 3D representation of the eddy current generated by an alternating current-induced magnetic field. As the direction of the current changes, a magnetic flux is created and is located at the center of the coil. The right figure displays the top cross-section view. Eddy currents occur when the magnetic field is tangential to the surface of the conductive material.

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Figure 12. Magnetic hysteresis effect and nanowarming results

(A) Illustration of the magnetic hysteresis curve (B-H). (B) Relationship between magnetic field strength and the specific absorption rate (SAR) at frequencies of 360 and 190 kHz, with power levels of 120 kW and 15 kW, respectively. SAR represents the rate at which energy is absorbed by biological tissue exposed to an electromagnetic field. (C) SAR of magnetic nanoparticles at various temperatures and magnetic field strengths.

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Table 1. Water Bath Rewarming Experiments of Cryopreserved Samples

Sample Name	Total Volume (mL)	Water Bath Temperature(℃)	Shaking	Rewarming Rate (C/min)	Recovery Rate (%)
Rat Islet slice1^[61]	0.1	37	-	> 1000	79
Bovine embryo^[25]	0.25	35	×	> 1000	42.1
Testicular interstitial cell^[62]	1	37	√	20	65.3±2.1
Rabbit Carotid Artery^[63]	10	37	×	130	71
DPVP Solution^[48]	25	37	√	48.2	-
DPVP is a vitrification solution contains 41% (v/v) dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO) and 6% (w/v) polyvinylpyrrolidone (Sigma-Aldrich) in phosphate buffered saline solution (Sigma Aldrich). - means information is not mentioned in cited references.

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