Magnetic induction heating of superparamagnetic nanoparticles during rewarming augments the recovery of hUCM-MSCs cryopreserved by vitrification

Abstract

Cryopreservation by vitrification has been recognized as a promising strategy for long-term banking of living cells. However, the difficulty to generate a fast enough heating rate to minimize devitrification and recrystallization-induced intracellular ice formation during rewarming is one of the major obstacles to successful vitrification. We propose to overcome this hurdle by utilizing magnetic induction heating (MIH) of magnetic nanoparticles to enhance rewarming. In this study, superparamagnetic (SPM) Fe₃O₄ nanoparticles were synthesized by a chemical coprecipitation method. We successfully applied the MIH of Fe₃O₄ nanoparticles for rewarming human umbilical cord matrix mesenchymal stem cells (hUCM-MSCs) cryopreserved by vitrification. Our results show that extracellular Fe₃O₄ nanoparticles with MIH may efficiently suppress devitrification and/or recrystallization during rewarming and significantly improve the survival of vitrified cells. We further optimized the concentration of Fe₃O₄ nanoparticles and the current of an alternating current (AC) magnetic field for generating the MIH to maximize cell viability. Our results indicate that MIH in an AC magnetic field with 0.05% (w/v) Fe₃O₄ nanoparticles significantly facilitates rewarming and improves the cryopreservation outcome of hUCM-MSCs by vitrification. The application of MIH of SPM nanoparticles to achieve rapid and spatially homogeneous heating is a promising strategy for enhanced cryopreservation of stem cells by vitrification.

Statement of Significance

Here we report the successful synthesis and application of Fe₃O₄ nanoparticles for magnetic induction heating (MIH) to enhance rewarming of vitrification-cryopreserved human umbilical cord matrix mesenchymal stem cells (hUCM-MSCs). We found that MIH-enhanced rewarming greatly improves the survival of vitrification-cryopreserved hUCM-MSCs. Moreover, the hUCM-MSCs retain their intact stemness and multilineage potential of differentiation post cryopreservation by vitrification with the MIH-enhanced rewarming. Therefore, the novel MIH-enhanced cell vitrification is valuable to facilitate the long-term storage of hUCM-MSCs and possibly many other important cells to meet their ever-increasing demand by the burgeoning cell-based medicine.

Introduction

Mesenchymal stem cells (MSCs) are capable of self-renewal and multi-lineage differentiation and are important for cell-based therapies to treat a variety of disorders [1], [2], [3], [4]. The umbilical cord matrix (UCM), which is routinely discarded after childbirth, is a valuable resource for non-invasive collection of MSCs without any ethical issues [5]. MSCs from human UCM (hUCM-MSCs) are an abundant source of adult stem cells and are an alternative to embryonic stem cells [6], [7]. UCM-MSCs show low immunogenicity and elicit a lower incidence of graft rejection and post-transplant infections compared with other sources of adult stem cells [8]. Therefore, hUCM-MSCs are an extremely valuable candidate for cell-based regenerative medicine [9], [10]. Moreover, banking hUCM-MSCs is essential to meet the ever increasing demand of the cells for clinical applications and research [11].

Cell cryopreservation is a technology for long-term storage of cells by cooling the cells to cryogenic temperatures with minimal metabolic activities [12], [13], [14], [15]. Although it has been commonly used for cell cryopreservation, the conventional slow-freezing approach is time-consuming and usually requires an expensive programmable freezer to achieve the desired cooling rates that are different for different types of cells [16], [17], [18]. Vitrification is attracting more and more attention in recent years as a fast and economic alternative to slow freezing, particularly for the cryopreservation of stem cells and reproductive cells [19], [20], [21], [22], [23]. Vitrification is a process by which the disordered liquid state of water is brought to a standstill as a solid without crystallization during cooling [24]. High cooling rates and high concentrations (4–8 M) of cryoprotectants have been conventionally used to achieve vitrification. To improve the recovery of cells post vitrification, most research effort has been focused on achieving an ultra-rapid cooling rate and lowering the concentrations of cryoprotectants required for vitrification [18], [19], [25], [26], [27]. However, due to the low thermal conductivity of biological samples, the conventional approach of rewarming large-volume cryopreserved samples in a water bath is associated with non-uniform distribution of temperature, and this non-uniformity can induce thermal stress that can crack the brittle cryopreserved sample [28], [29], [30]. Moreover, a high heating rate for rewarming is crucial, because devitrification and recrystallization will occur if the temperature cannot be rapidly increased above the melting points of the aqueous sample. Therefore, both the heating rate and uniformity of heating during rewarming are important to cryopreservation by vitrification.

Electromagnetic (EM) heating, which produces a higher warming rate and more uniform heating than the conventional water bath rewarming method, is considered to be an effective approach for rewarming cryopreserved samples [31], [32]. Previous studies have been focused on microwave and high frequency RF rewarming (hundreds of MHz or even GHz in frequency). Ketterer et al. thawed cryopreserved canine kidney using a microwave oven [33], [34]. However, because the microwave penetration depth is limited and a uniform heating may not be achieved, the rewarming outcome was not favorable [35]. Ruggera et al. proposed rapid EM heating using a resonant helical coil applicator to prevent the formation of ice crystals during rewarming for the cryopreservation of cells and organs [32]. Robinson et al. further developed this EM heating system with a cylindrical resonance cavity [31], [36]. Gao et al. developed a single-mode resonant cavity to achieve rapid and uniform warming of cryopreserved biomaterials [37]. In addition, Jin et al. used infrared laser pulses to achieve ultra-rapid warming rates (10,000,000 °C/min) for rewarming frozen or vitrified oocytes [38], [39], [40]. However, these approaches need complicated instruments and are time consuming.

Magnetic nanoparticles, which possess several unique characteristics including biocompatibility and SPM properties, are widely applied in medicine [41], [42], [43], [44]. They can be rapidly heated upon exposure to an AC magnetic field and are uniformly spread throughout the biomaterial [30], [45]. Heating magnetic nanoparticles can be conveniently realized with an induction apparatus over a medium frequency range (several hundreds of kHz), and has been investigated to treat tumors [41], [42], [45], [46], [47], [48], [49]. Magnetic nanoparticles have also been shown to improve the efficiency of the microwave rewarming process, and augment cancer treatment with cryosurgery [50], [51]. However, the effect of magnetic induction heating (MIH) of SPM nanoparticles in an AC magnetic field on cryopreservation by vitrification has not been reported. In this study, we synthesize and characterize SPM nanoparticles and apply them, as part of the vitrification solution, for hUCM-MSCs cryopreservation by vitrification. We found that SPM nanoparticles improve the rewarming of hUCM-MSCs cryopreserved by vitrification under an alternating magnetic field.