An official website of the United States government
Introduction:The current liver organ shortage has pushed the field of transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at high subzero storage temperatures (−10°C to −15°C), while simultaneously maintaining a sufficient unfrozen fraction to limit ice-mediated injury. Methods and results:Using glycerol as the main permeating cryoprotectant agent, this research first demonstrated that partially frozen rat livers showed similar outcomes after thawing from either −10°C or −15°C with respect to subnormothermic machine perfusion metrics. Next, we assessed the effect of adding ice modulators, including antifreeze glycoprotein (AFGP) or a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Discussion:Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, including testing additional ice modulators, can help improve the viability of these partially frozen organs.
more »
« less
Partial freezing of rat livers extends preservation time by 5-fold
Abstract The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between −4 and −6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (−10 to −15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.
more »
« less
- Award ID(s):
- 1941543
- PAR ID:
- 10369034
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Additives that help cells survive the stresses of freezing and thawing are known as cryoprotective agents (CPAs). Two different types of CPAs have been identified: penetrating and non-penetrating. Common penetrating CPAs include dimethylsulfoxide (DMSO) and glycerol. The location of a CPA (intracelluar or extracellular) is important for understanding the molecular mechanisms of action for the agent. Low-temperature Raman spectroscopy is a label-free method of detecting the location of CPAs at low temperature with high spatial resolution and chemical specificity. To this end, cells cryopreserved in DMSO using a variety of cooling rates and DMSO concentrations and imaged using Raman spectroscopy were analyzed using automated image analysis to determine the partitioning ratio (concentration of DMSO outside/concentration of DMSO inside the cell). The partitioning ratio was roughly 1 for Jurkat cells frozen at 1°C/min in varying concentrations of DMSO with the exception of 1% DMSO which had a partitioning ratio of 0.2. The partitioning ratio increased from 1 to 1.3 as the cooling rate increased from 1°C to 5°C/min. Different cell types, specifically sensory neurons cells and human induced pluripotent stem cells, exhibited differences in partitioning ratio when frozen in 10% DMSO and 1°C/min suggesting that differences in freezing response may result from differences in solute partitioning. The presence of intracellular ice changed the distribution of DMSO inside the cell and also the partitioning ratio.more » « less
-
Temperature-controlled 3D cryoprinting (TCC) is an emerging tissue engineering technology aimed at overcoming limitations of conventional 3D printing for large organs: (a) size constraints due to low print rigidity and (b) the preservation of living cells during printing and subsequent tissue storage. TCC addresses these challenges by freezing each printed voxel with controlled cooling rates during deposition. This generates a rigid structure upon printing and ensures cell cryopreservation as an integral part of the process. Previous studies used alginate-based ink, which has limitations: (a) low diffusivity of the CaCl2 crosslinker during TCC’s crosslinking process and (b) typical loss of print fidelity with alginate ink. This study explores the use of an ink made of agar and alginate to overcome TCC protocol limitations. When an agar/alginate voxel is deposited, agar first gels at above-freezing temperatures, capturing the desired structure without compromising fidelity, while alginate remains uncrosslinked. During subsequent freezing, both frozen agar and alginate maintain the structure. However, agar gel loses its gel form and water-retaining ability. In TCC, alginate crosslinking occurs by immersing the frozen structure in a warm crosslinking bath. This enables CaCl2 diffusion into the crosslinked alginate congruent with the melting process. Melted agar domains, with reduced water-binding ability, enhance crosslinker diffusivity, reducing TCC procedure duration. Additionally, agar overcomes the typical fidelity loss associated with alginate ink printing.more » « less
-
null (Ed.)Abstract. Ice-nucleating particles (INPs) are efficiently removed fromclouds through precipitation, a convenience of nature for the study of thesevery rare particles that influence multiple climate-relevant cloudproperties including ice crystal concentrations, size distributions andphase-partitioning processes. INPs suspended in precipitation can be used toestimate in-cloud INP concentrations and to infer their originalcomposition. Offline droplet assays are commonly used to measure INPconcentrations in precipitation samples. Heat and filtration treatmentsare also used to probe INP composition and size ranges. Many previousstudies report storing samples prior to INP analyses, but little is knownabout the effects of storage on INP concentration or their sensitivity totreatments. Here, through a study of 15 precipitation samples collected at acoastal location in La Jolla, CA, USA, we found INP concentration changes upto > 1 order of magnitude caused by storage to concentrations ofINPs with warm to moderate freezing temperatures (−7 to−19 ∘C). We compared four conditions: (1) storage at roomtemperature (+21–23 ∘C), (2) storage at +4 ∘C, (3) storage at −20 ∘C and (4) flash-freezing samples with liquid nitrogen prior to storage at −20 ∘C. Results demonstrate that storage can lead to bothenhancements and losses of greater than 1 order of magnitude, withnon-heat-labile INPs being generally less sensitive to storage regime, butsignificant losses of INPs smaller than 0.45 µm in all tested storageprotocols. Correlations between total storage time (1–166 d) and changesin INP concentrations were weak across sampling protocols, with theexception of INPs with freezing temperatures ≥ −9 ∘C in samples stored at room temperature. We provide thefollowing recommendations for preservation of precipitation samples fromcoastal or marine environments intended for INP analysis: that samples bestored at −20 ∘C to minimize storage artifacts, thatchanges due to storage are likely an additional uncertainty in INPconcentrations, and that filtration treatments be applied only to freshsamples. At the freezing temperature −11 ∘C, average INPconcentration losses of 51 %, 74 %, 16 % and 41 % were observed foruntreated samples stored using the room temperature, +4, −20 ∘C, and flash-frozen protocols, respectively.Finally, the estimated uncertainties associated with the four storage protocolsare provided for untreated, heat-treated and filtered samples for INPsbetween −9 and −17 ∘C.more » « less
-
Abstract All cloud and climate models assume ice crystals grow as if they were formed from pure water, even though cloud and haze droplets are solutions. The freezing process of a solution droplet is different than that of a pure water droplet, as shown in prior work. This difference can potentially affect the particle’s subsequent growth as an ice crystal. We present measurements of ice crystal growth from frozen sodium chloride (NaCl) solution droplets in the button electrode levitation diffusion chamber at temperatures between −61° and −40°C. Measured scattering patterns show that concentrated solution droplets remain unfrozen with classical scattering fringes until the droplets freeze. Upon freezing, the scattering patterns become complex within 0.1 s, which is in contrast with frozen pure water particles that retain liquid-like scattering patterns for about a minute. We show that after freezing, solution particles initially grow as spherical-like crystals and then transition to faster growth indicative of a morphological transformation. The measurements indicate that ice formed from solution droplets grows differently and has higher growth rates than ice formed from pure water droplets. We use these results to develop a power-law-based parameterization that captures the supersaturation and mass dependencies.more » « less
