Characterization of Laser Gold Nanowarming: A Platform for Millimeter-Scale Cryopreservation

Preventing ice formation during cryopreservation by vitrification has led to the successful storage and banking of numerous cellular- and tissue-based biomaterials. In their breakthrough work, Peter Mazur's group achieved over 90% survival by using a laser warming technique for 100 μm mice oocytes that were cooled in 0.1 μL droplets with 2.3 M CPA and extracellularly loaded India ink (laser absorber). Laser warming can provide rapid and uniform warming rates to "outrun" damaging ice crystal growth. Here we generalize Mazur's technique for microliter-sized droplets using laser nanowarming to rewarm millimeter-scale biomaterials when loaded extracellularly and/or intracellularly with biocompatible 1064 nm resonant gold nanoparticles. First, we show that droplets containing low-concentration cryoprotectants (such as 2 M propylene glycol ± 1 M trehalose) can be rapidly cooled at rates up to 90 000 °C/min by plunging into liquid nitrogen to achieve either a visually transparent state (i.e., vitrified) or a cloudy with ice (i.e., nonvitrified) state. Both modeling and experiments were then used to characterize the laser nanowarming process for different laser energy (2-6 J), pulse length (1-20 ms), droplet volume (0.2-1.8 μL), cryoprotectant (2-3 M), and gold concentration (0.77 × 10¹⁷-4.8 × 10¹⁷ nps/m³) values to assess physical and biological success. Physical success was achieved by finding conditions that minimize cloudiness and white spots within the droplets during cooling and warming as signs of damaging ice formation and ice crystallization, respectively. Biological success was achieved using human dermal fibroblasts to find conditions that achieve ≥90% cell viability normalized to controls postwarming. Thus, physical and biological success can be achieved using this platform cryopreservation approach of rapid cooling and laser gold nanowarming in millimeter-scale systems.