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

ABSTRACT: 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 ?m mice oocytes that were cooled in 0.1 ?L 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 ?L), 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.

SUBMITTER: Khosla K

PROVIDER: S-EPMC6536355 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Khosla Kanav K Zhan Li L Bhati Aditya A Carley-Clopton Aiden A Hagedorn Mary M Bischof John J

Langmuir : the ACS journal of surfaces and colloids 20181025 23

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 grow ...[more]

PMID: 30299961

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Project description:BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.

Dataset Information

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

Publications

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

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