8 research outputs found
Gold Nanorod Induced Warming of Embryos from the Cryogenic State Enhances Viability
Zebrafish
embryos can attain a stable cryogenic state by microinjection
of cryoprotectants followed by rapid cooling, but the massive size
of the embryo has consistently led to failure during the convective
warming process. Here we address this zebrafish cryopreservation problem
by using gold nanorods (GNRs) to assist in the warming process. Specifically,
we microinjected the cryoprotectant propylene glycol into zebrafish
embryos along with GNRs, and the samples were cooled at a rate of
90âŻ000 °C/min in liquid nitrogen. We demonstrated the
ability to unfreeze the zebrafish rapidly (1.4 à 10<sup>7</sup> °C/min) by irradiating the sample with a 1064 nm laser pulse
for 1 ms due to the excitation of GNRs. This rapid warming process
led to the outrunning of ice formation, which can damage the embryos.
The results from 14 trials (<i>n</i> = 223) demonstrated
viable embryos with consistent structure at 1 h (31%) and continuing
development at 3 h (17%) and movement at 24 h (10%) postwarming. This
compares starkly with 0% viability, structure, or movement at all
time points in convectively warmed controls (<i>n</i> =
50, <i>p</i> < 0.001, ANOVA). Our nanoparticle-based
warming process could be applied to the storage of fish, and with
proper modification, can potentially be used for other vertebrate
embryos
Conduction Cooling and Plasmonic Heating Dramatically Increase Droplet Vitrification Volumes for Cell Cryopreservation
Abstract Droplet vitrification has emerged as a promising iceâfree cryopreservation approach to provide a supply chain for offâtheâshelf cell products in cell therapy and regenerative medicine applications. Translation of this approach requires the use of low concentration (i.e., low toxicity) permeable cryoprotectant agents (CPA) and high post cryopreservation viability (>90%), thereby demanding fast cooling and warming rates. Unfortunately, with traditional approaches using convective heat transfer, the droplet volumes that can be successfully vitrified and rewarmed are impractically small (i.e., 180 picoliter) for 400âfold improvement in warming rates over traditional convective approach. High viability cryopreservation is then demonstrated in a model cell line (human dermal fibroblasts) and an important regenerative medicine cell line (human umbilical cord blood stem cells). This approach opens a new paradigm for cryopreservation and rewarming of dramatically larger volume droplets at lower CPA concentration for cell therapy and other regenerative medicine applications
Successful cryopreservation of coral larvae using vitrification and laser warming
Abstract Climate change has increased the incidence of coral bleaching events, resulting in the loss of ecosystem function and biodiversity on reefs around the world. As reef degradation accelerates, the need for innovative restoration tools has become acute. Despite past successes with ultra-low temperature storage of coral sperm to conserve genetic diversity, cryopreservation of larvae has remained elusive due to their large volume, membrane complexity, and sensitivity to chilling injury. Here we show for the first time that coral larvae can survive cryopreservation and resume swimming after warming. Vitrification in a 3.5âM cryoprotectant solution (10% v/v propylene glycol, 5% v/v dimethyl sulfoxide, and 1âM trehalose in phosphate buffered saline) followed by warming at a rate of approximately 4,500,000â°C/min with an infrared laser resulted in up to 43% survival of Fungia scutaria larvae on day 2 post-fertilization. Surviving larvae swam and continued to develop for at least 12âhours after laser-warming. This technology will enable biobanking of coral larvae to secure biodiversity, and, if managed in a high-throughput manner where millions of larvae in a species are frozen at one time, could become an invaluable research and conservation tool to help restore and diversify wild reef habitats