Ultra-Rapid Laser Calorimetry for the Assessment of Crystallization in Low-Concentration Cryoprotectants

Abstract

Cryoprotective agents (CPAs) are routinely used to vitrify, attain an amorphous glass state void of crystallization, and thereby cryopreserve biomaterials. Two vital characteristics of a CPA loaded system are the critical cooling and warming rates (CCR and CWR), the temperature rates needed to achieve and return from a vitrified state respectively. Due to the toxicity associated with CPAs, it is often desirable to use the lowest concentrations possible, driving up CWR and making it increasingly difficult to measure. This paper describes a novel method for assessing CWR between the 0.4×105-107 °C/min in microliter CPA loaded droplet systems with a new ultra-rapid laser calorimetric approach. Cooling was achieved by direct quenching in liquid nitrogen, while warming was achieved by the irradiation of plasmonic gold nanoparticle-loaded vitrified droplets by a high-power 1064 nm millisecond pulsed laser. We assume "apparent" vitrification is achieved provided ice is not visually apparent (i.e. opacity) upon imaging with a camera during cooling or highspeed camera during warming. Using this approach, we were able to investigate CWR in single CPA systems such as glycerol, PG, and Trehalose in water, and mixtures of glycerol-trehalose-water and propylene glycol-trehalose-water CPA at low concentrations (20-40 wt%). Further, an phenomenological model for determining the CCRs and CWRs of CPA was developed which allowed for predictions of CCR or CWR of single component CPA and mixtures, providing an avenue for optimizing CCR and CWR and perhaps future CPA cocktail discovery.

The first proof of concept demonstration of nanowarming in coral tissue

“…The copyright holder for this preprint this version posted March 19, 2023. ; https://doi.org/10.1101/2023.03. 16.533048 doi: bioRxiv preprint regression and skeletal exposure at the edges (Fig. 3A-B), but some vitrification solution exposures caused more pronounced tissue regression and expulsion of symbionts (Fig.…”

“…The copyright holder for this preprint this version posted March 19, 2023. ; https://doi.org/10.1101/2023.03. 16.533048 doi: bioRxiv preprint incubation step, although it was likely less than the 50% of external GNR concentration used for modelling. To increase the concentration of GNR within the tissue it may be necessary to devise methods for microinjections of GNR and CPA into the polyp mouth or increase the duration of incubation of SPMFs in GNR, to improve post-warming recovery.…”

“…The copyright holder for this preprint this version posted March 19, 2023. ; https://doi.org/10.1101/2023.03. 16.533048 doi: bioRxiv preprint titanium heaters (finnex.net) per tank controlled by an external controller (Inkbird Technical Corporation Limited, Shenzen, China). Controllers were programmed such that temperature did not fluctuate by more than 1.3 °C in a day, confirmed using a bluetooth temperature logger that recorded temperature every 30 seconds (Inkbird Technical Corporation Limited, Shenzen, China).…”

“…; https://doi.org/10.1101/2023.03. 16.533048 doi: bioRxiv preprint 3 For cryopreservation and biobanking, adult coral tissues have the advantage of being available all year round; however, they have the disadvantage of being highly complex, containing algal symbionts and a calcium carbonate skeleton, and of being sensitive to chilling injury 13 . One of the most effective tools available for the cryopreservation and recovery of complex tissues is vitrification and laser warming with the aid of plasmonic gold nanorods, referred to as 'nanowarming'.…”

Photothermal heating of titanium nitride nanomaterials for fast and uniform laser warming of cryopreserved biomaterials

“…This study extrapolated data from higher CPA measurements. Recently Kangas et al (2022) , measured the CWR via laser calorimetry in microliter (μL) droplets of low CPA concentrations, 20 to 40 wt%. This study found that the CWR and CCR range from 0.4 × 10 5 –10 7 °C/min and ∼10 4 –10 5 °C/min, respectively.…”