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.
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*Tel: 612-625-5513. E-mail: email@example.com. ORCID Kanav Khosla: 0000-0001-9075-8445 John Bischof: 0000-0001-6726-7111 Author Contributions K.K., L.Z., M.H., and J.B. conceived the experiments. K.K., A.B., and A.C.-C. performed the experiments and analyzed the data. L.Z. developed and analyzed the FEM model. All authors contributed to writing of the article. Funding This work was supported by NIH R41 OD024430-01, Kuhrmeyer Chair, Institute for Engineering in Medicine at the University of Minnesota (funding provided to J.B.). This work was also supported by the Anela Kolohe Foundation, the Cedarhill Foundation, the Skippy Frank Translational Medicine Fund, the Roddenberry Foundation, the Paul M. Angell Family Foundation, the Hawaii Institute of Marine Biology, and the Smithsonian Institution (funding provided to M.H.). Notes The authors declare no competing financial interest.