# More Sustainable Cryopreservation **URL:** \ **Category:** Methods & Reagents\ **Created:** [June 25, 2024, 12:04am UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712 "2024-06-25T00:04:42Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![MQGoulding](https://community.alliancegenome.org/letter_avatar_proxy/v4/letter/m/90ced4/32.png) [@MQGoulding](https://community.alliancegenome.org/u/MQGoulding)\ **Post date:** [June 25, 2024, 12:04am UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712/1 "2024-06-25T00:04:42Z")
Dear Worm Folks: What if we could keep worm stocks in long-term suspended animation using a regular old Minus Twenty (-20C) chest freezer? Would this be a great thing, for your lab and/or for the World? (Saving Energy+Carbon+Real Estate+Money+Hassle?) If Yes, please Like! Or if Meh, can you Downvote with a quick reply to that effect? Feedback is appreciated. I have a surprising experimental result, and a hypothesis that might lead to a breakthrough - thinking of sharing via MicroPub. I want to challenge others to follow up and vie for the potential prize, since I don’t have a lab at the moment. Thanks! This is an answer to an existing challenge: www [dot] freezerchallenge [dot] org/the-challenge [dot] html. ---
**Author:** ![Janet\_Duerr](https://community.alliancegenome.org/letter_avatar_proxy/v4/letter/j/5fc32e/32.png) [@Janet\_Duerr](https://community.alliancegenome.org/u/Janet_Duerr)\ **Post date:** [July 8, 2024, 9:26pm UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712/2 "2024-07-08T21:26:10Z")
I have not been able to keep my worms alive very long (not years) in a -30C freezer. I suspect you are using something other than the standard worm freezing media? If so, please share (perhaps by publishing a micropub). Janet ---
**Author:** ![MQGoulding](https://community.alliancegenome.org/letter_avatar_proxy/v4/letter/m/90ced4/32.png) [@MQGoulding](https://community.alliancegenome.org/u/MQGoulding)\ **Post date:** [October 15, 2024, 10:21pm UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712/3 "2024-10-15T22:21:42Z")
Yeah, I was using Kevin O’Connell’s trehalose medium - it works WAY better than the traditional freezing solution (at -80). ---
**Author:** ![c.w](https://community.alliancegenome.org/letter_avatar_proxy/v4/letter/c/bbe5ce/32.png) [@c.w](https://community.alliancegenome.org/u/c.w)\ **Post date:** [October 27, 2026, 12:49pm UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712/4 "2026-10-27T12:49:23Z")
Hijacking this old thread about sustainable cryopreservation… Has anyone compared how well the trehalose medium cryopreservation methods works at -80C vs -70C? My institution is pushing labs to switch the temperature of deep freezers to -70C (and I suspect many other places are doing this as well). I’m wondering whether to push back on this, or if there is any reassuring evidence out there that frozen nematodes happily tolerate -70C. Thanks in advance for any thoughts on this! ---
**Author:** ![MQGoulding](https://community.alliancegenome.org/letter_avatar_proxy/v4/letter/m/90ced4/32.png) [@MQGoulding](https://community.alliancegenome.org/u/MQGoulding)\ **Post date:** [October 31, 2026, 12:27am UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712/5 "2026-10-31T00:27:38Z")
OK, my MS was declined by microPub - not enuf replicates, and I’ve lost my lab so no hope. Wheresoever, here it is, spilling the beans in what is left of the Worm Breeder’s Gazette. Enjoy! (Is there a word limit on these posts?) A new perfusion medium for ultra-low temperature (-80°C) cryopreservation of nematodes was reported to yield “a significant improvement in survival” (O’Connell, 2022). On a whim, I tried using this new formulation for freezing worms in an ordinary chest freezer, where temperatures had been observed to hover around -22°C. Populations of _C. elegans_ strain CB1467 from three freshly-starved 60mm plates were frozen in three 2ml screw-cap cryovials, using standard methodology (see Methods and Reagents). After 24 hours one tube (manifestly frozen solid) was thawed at room temperature and dumped on a 60mm plate. Quick examination five minutes later, and again on the following day when the puddle had dried, showed that nearly every young adult and adolescent (L3-L4) worm had survived and resumed normal behavior. After 30 days, a second tube was removed from the freezer and examined in the same way. Every animal on the plate was dead. The third frozen population was immediately thawed and examined, with the same result observed. The thawed worms did not languish into gradual demise: they all just never woke up, indicating fatal damage inflicted pre-thawing. This set of results prompts the question of what kind of lethal physiological insult might be incurred over a multiday timespan in a completely solid medium, i.e., one where molecules as large as phospholipids, amino acids, etc., presumably experience no translational movement. A plausible answer is that much smaller molecules continue to diffuse through interstices of the otherwise fixed matrix, colliding with frozen membranes, proteins, etc., and doing damage at a rate directly related to temperature. Prime candidates are of course the reactive oxygen species (ROS) perennially present in all aerobic life, including many particles composed of three atoms or fewer (Juan et al., 2021). Alternatively (or additionally), peroxidation chain reactions between juxtaposed lipid molecules might wreak widespread damage across the planes of gellated membrane leaflets, without any need for ROS diffusion (Porter, 2013). This is not a new idea; ROS are indeed recognized as a threat to cryopreservation. Further, they may be an especial occupational hazard of cryobiosis, as their concentrations have been shown to rise by over five-fold during slow cooling of mammalian sperm (Evangelista-Vargas & Santiani, 2017). Testing the hypothesis of oxidative damage in cryonic trauma to nematodes at -20°C should be straightforward, thanks to two facts. First is the well-established fact that both small-molecule ROS production and lipid peroxidation are mostly metabolically downstream of molecular oxygen (O2). Second is the remarkable fact that _C. elegans_ can survive complete anoxia for hours (Van Voorhies & Ward, 2000); such pretreatment is predicted to boost cryopreserved lifespan. (Conversely, the same study found that worms can live for days in a pure O2 atmosphere; this would be predicted to ruin cryopreservation, perhaps even at -80°C.) Another test would be to pre-treat worms with cocktails of small-molecule antioxidants, ideally a maximal tolerable dose of every kind available (Charlton et al., 2023). If an affordable cocktail confers adequate cryoprotection at -20°C, then worm breeders could save a lot on ultracold freezer space, electrical bills, and liquid nitrogen refills. If the time-dependent lethal factor operating at -20°C be identified and cured, then this far cheaper refrigeration regimen may indeed turn out to be optimal, given that -80°C (with no cryoprotectant) has been observed to cause protein denaturation to a much greater extent (Bischof et al., 2002). Methods and Reagents: Worms were cultured on lawns of E. coli OP50 on ‘poor people’s Nematode Growth Medium’ (ppNGM), consisting of agar (1.7% w/v) supplemented with high-salt LB nutrient mix (0.6% w/v, 10% of the concentration used for optimal bacterial growth) and plant sterols (13μg/ml). The nutrient agar was dissolved and sterilized by tyndallization (see below), and sterols added from a warmed aliquot of 1000x ethanol stock solution immediately before pouring plates. Worm handling preparatory to freezing was done as described by O’Connell (2022); worm suspensions (1.0ml volumes) were finally transferred into 2ml screw-cap cryo-tubes, which were placed right-side-up in a polystyrene foam rack designed to fit 15ml conical tubes; a second such rack was inverted over the first to make a thermoprotective sandwich; the two racks were tightly rubberbanded together and placed at low altitude in a 5 cubic foot chest freezer pre-loaded with plenty of frozen aqueous material. The following method was routinely used for tyndallization. Each 500ml of nutrient medium in a 1L Erlenmeyer flask (capped with an inverted beaker) was simmered for two hours in an oven (150°C), then cooled to room temperature; the following day, flasks were heated to a boil by microwave oven, then simmered another hour in the 150°C oven. References Porter NA. A perspective on free radical autoxidation: the physical organic chemistry of polyunsaturated fatty acid and sterol peroxidation. J Org Chem. 2013 Apr 19;78(8):3511-24. doi: 10.1021/jo4001433. Epub 2013 Apr 9. PMID: 23445181; PMCID: PMC3674788. O’Connell KF. Cryopreservation of _C. elegans_ and Other Nematodes with Dimethyl Sulfoxide and Trehalose. Methods Mol Biol. 2022;2468:43-49. doi: 10.1007/978-1-0716-2181-3\_3. PMID: 35320559. Van Voorhies WA, Ward S. Broad oxygen tolerance in the nematode _Caenorhabditis elegans._ J Exp Biol. 2000 Aug;203(Pt 16):2467-78. doi: 10.1242/jeb.203.16.2467. PMID: 10903161. Charlton NC, Mastyugin M, Török B, Török M. Structural Features of Small Molecule Antioxidants and Strategic Modifications to Improve Potential Bioactivity. Molecules. 2023 Jan 20;28(3):1057. doi: 10.3390/molecules28031057. PMID: 36770724; PMCID: PMC9920158. Bischof JC, Wolkers WF, Tsvetkova NM, Oliver AE, Crowe JH. Lipid and protein changes due to freezing in Dunning AT-1 cells. Cryobiology. 2002 Aug;45(1):22-32. doi: 10.1016/s0011-2240(02)00103-7. PMID: 12445547. Evangelista-Vargas S, Santiani A. Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa. Reprod Domest Anim. 2017 Oct;52(5):819-824. doi: 10.1111/rda.12984. Epub 2017 Apr 29. PMID: 28455949. Fleming SD, Thomson LK. The Oxidative Stress of Human Sperm Cryopreservation. Antioxidants (Basel). 2025 Mar 28;14(4):402. doi: 10.3390/antiox14040402. PMID: 40298642; PMCID: PMC12024095. Juan CA, Pérez de la Lastra JM, Plou FJ, Pérez-Lebeña E. The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies. Int J Mol Sci. 2021 Apr 28;22(9):4642. doi: 10.3390/ijms22094642. PMID: 33924958; PMCID: PMC8125527. ---
**Author:** ![c.w](https://community.alliancegenome.org/letter_avatar_proxy/v4/letter/c/bbe5ce/32.png) [@c.w](https://community.alliancegenome.org/u/c.w)\ **Post date:** [November 3, 2026, 2:30pm UTC](https://community.alliancegenome.org/t/more-sustainable-cryopreservation/7712/6 "2026-11-03T14:30:43Z")
Thanks for sharing your info and ideas about this