Microalgae cryopreservation techniques: choosing cryoprotectants and cooling regimens
Keywords:
microalgae, cryoprotectants, cryopreservation regimes, plant vitrification solutions, low temperature storageAbstract
This review was aimed to summarize and array current methods to cryopreserve microalgae, particularly, the approaches to selecting cryoprotectants and optimal cooling parameters, aimed at preserving their viability and functional activity after thawing. The paper analyses the key aspects of cryopreservation of microalgae, including the efficiency comparison of different classes of cryoprotectants as well as their impact on cells; mechanisms of cryodamage and strategies for minimizing it; optimal cooling and warming regimens for various types of microalgae. Special attention has been paid to the dependency of cryopreservation efficiency on the concentration and composition of cryoprotective solutions, which vary depending on the specific species and cultures. Some microalgae has been noted to maintain high viability using elevated concentrations of non-penetrating cryoprotectants. The necessity for further research using new and combined cryoprotective solutions has been emphasized to expand the possibilities of long-term storage of microalgae.
Probl Cryobiol Cryomed. 2026; 36(1): 5—15
References
Ali P, Fucich D, Shah AA, et al. Cryopreservation of cyanobacteria and eukaryotic microalgae using exopolysaccharide extracted from a glacier bacterium. Microorganisms [Internet]. 2021 Feb 15 [cited 2025 Jun 27]; 9: 395. Available from: https://www.mdpi.com/2076-2607/9/2/395 CrossRef
Ananina GE, Stepanyuk LV, Vysekantsev IP, Petrov IV. Impact of hypothermal and low temperature storage conditions on viability of immobilized probiotics Bifidobacterium bifidum Actual Problems of Modern Medicine: Bulletin of the Ukrainian Medical Stomatological Academy. 2020; 20(3): 185-91. CrossRef
Arav, A. Cryopreservation by directional freezing and vitrification focusing on large tissues and organs. Cells [Internet]. 2022 Mar 22 [cited 2025 Jul 23]; 11(7): 1072. Available from: https://www.mdpi.com/2073-4409/11/7/1072 CrossRef
Aray-Andrade MM, Uyaguari-Diaz MI, Bermudez JR. Short-term deleterious effects of standard isolation and cultivation methods on new tropical freshwater microalgae strains. PeerJ [Internet]. 2018 July 19 [cited 2025 Jun 27]; 6: e5143. Available from: https://peerj.com/articles/5143/ CrossRef
Ben-Amotz A, Gilboa A. Cryopreservation of marine unicellular algae. II. Induction of freezing tolerance. Mar Ecol Prog Ser. 1980; 2: 221-4. CrossRef
Benelli C. Plant cryopreservation: A look at the present and the future. Plants [Internet]. 2021 Dec 13 [cited 2025 Jul 23]; 10(12): 2744. Available from: https://www.mdpi.com/2223-7747/10/12/2744 CrossRef
Brand JJ, Diller KR. Application and theory of algal cryopreservation. Nova Hedwig. 2004; 79(1): 175-89. CrossRef
Bui TV, Ross IL, Jakob G, Hankamer B. Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae. PLoS ONE [Internet]. 2013 Nov 11 [cited 2025 Jul 23]; 8: e78668. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078668 CrossRef
Cañavate JP, Lubian LM. Effects of slow and rapid warming on the cryopreservation of marine microalgae. Cryobiology. 1997; 35(2): 143-9. CrossRef
Chernobai NA, Kadnikova NG, Vozovyk KD, et al. Temperature-salt stress increases yield of valuable metabolites and shelf life of microalgae. Biophysical Bulletin. 2022;(48):7-17. CrossRef
Chernobai NA, Vozovik KD, Kadnikova NG. Comparative analysis of methods for assessing the safety of Dunaliella salina Teodoresco and Chlorococcum dissectum Korshikov (Chlorophyta) microalgae cultures after exposure to stress factors. Algologia. 2021; 31(4): 353-64. CrossRef
Chong G, Tsai S, Wang LH, et al. Cryopreservation of the gorgonian endosymbiont Symbiodinium. Sci Rep [Internet]. 2016 Jan 12 [cited 2025 Jul 23]; 6: 18816. Available from: https://www.nature.com/articles/srep18816 CrossRef
da Costa BB, Lassen PG, Streit DP Jr. Cryopreservation-induced morphological changes in freshwater fish sperm: a systematic review. Biopreserv Biobank. 2024; 22(5): 416-27. CrossRef
Day JG, Childs KH, Stacey GN. Implications of a catastrophic refrigeration failure on the viability of cryogenically stored samples. Protist [Internet]. 2022 Oct 23 [cited 2025 Jul 23]; 173(6): 125915. Available from: https://www.sciencedirect.com/science/article/abs/pii/S1434461022000608 CrossRef
Day JG, Fleck RA. Cryo-injury in algae and the implications this has to the conservation of micro-algae. Microalgae Biotechnol. 2015; 1(1): 1-11. CrossRef
Day JG, Tytor S, Egardt J, et al. Challenges for the maintenance and cryopreservation of multiple isolates of model microorganisms: an example using the marine diatom Skeletonema marinoi. Biopreserv Biobank. 2017; 15(3): 191-202. CrossRef
Day JG. Cyanobacteria: An Economic Perspective. In: Sharma NK, Rai AK, Stal LJ, editors. Hoboken, NJ, USA: John Wiley & Sons; 2013. p. 319-27.
Esteves-Ferreira AA, Corrêa DM, Carneiro AP, et al. Comparative evaluation of different preservation methods for cyanobacterial strains. J Appl Phycol. 2013; 25: 919-29. CrossRef
Deniz I, Demirel Z, Imamoglu E, Conk-Dalay M. Long-term storage of microalgae: determination of optimum cryopreservation conditions. JMBA. 2022; 102(3-4): 276-84. CrossRef
Engels JM, Ebert AW. A critical review of the current global ex situ conservation system for plant agrobiodiversity. I. History of the development of the global system in the context of the political/legal framework and its major conservation components. Plants [Internet]. 2021 July 29 [cited 2025 Jul 23]; 10(8): 1557. Available from: https://www.mdpi.com/2223-7747/10/8/1557 CrossRef
Fernandes MS, Calsing LC, Nascimento RC, et al. Customized cryopreservation protocols for chlorophytes based on cell morphology. Algal Res [Internet]. 2019 Mar 01 [cited 2025 Jun 21]; 38: 101402. Available from: https://www.sciencedirect.com/science/article/abs/pii/S2211926418301760 CrossRef
Fleck RA, Benson EE, Bremner DH, Day JG. Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: a novel approach for developing protistan cryopreservation strategies. Free Radic Res. 2000; 32(2): 157-70. CrossRef
Foo SC, Mok CY, Ho SY, Khong NM. Microalgal culture preservation: progress, trends and future developments. Algal Res [Internet]. 2023 Feb 21 [cited 2025 Jun 21]; 71(1): 103007. Available from: https://www.sciencedirect.com/science/article/pii/S2211926423000401 CrossRef
Gaget V, Chiu YT, Lau M, Humpage AR. From an environmental sample to a long-lasting culture: the steps to better isolate and preserve cyanobacterial strains. J Appl Phycol. 2017; 29: 309-21. CrossRef
Garrido-Cardenas JA, Han X, Alonso DL, García-Maroto F. Evaluation and optimization of a methodology for the long-term cryogenic storage of Tetradesmus obliquus at –80 °C. Appl Microbiol Biotechnol. 2019; 103(5): 2381-90. CrossRef
Guermazi W, Sellami-Kammoun A, Elloumi J, et al. Microalgal cryo-preservation using dimethyl sulfoxide (Me2SO) coupled with two freezing protocols: influence on the fatty acid profile. J Therm Biol. 2010; 35(4): 175-81. CrossRef
Harding K. Plant and algal cryopreservation: issues in genetic integrity, concepts in cryobionomics and current applications in cryobiology. Asia–Pacific J Mol Biol Biotechnol. 2010; 18(1): 151-4.
Harding K, Day JG, Lorenz M, et al. Introducing the concept and application of vitrification for the cryo-conservation of algae - a mini-review. Nova Hedwig. 2004; 79(1): 207-26. CrossRef
Harding K, Muller J, Lorenz M, et al. Deployment of the encapsulation-dehydration protocol to cryopreserve microalgae held at the Sammlung von Algenkulturen, Universitat Gottingen, Germany. CryoLetters. 2008; 29(1): 15-20. PubMed
Holm-Hansen O. Viability of blue-green and green algae after freezing. Physiol Plant. 1963; 16(3): 530-40. CrossRef
Hong SS, Lee SY, Kim YN, et al. A modified cryopreservation method of psychrophilic chlorophyta Pyramimonas sp. from Antarctica. Ocean Polar Res. 2011; 33(3): 303-8. CrossRef
Hu Y, Liu X, Liu F, et al. Trehalose in biomedical cryopreservation - properties, mechanisms, delivery methods, applications, benefits, and problems. ACS Biomat Sci Eng. 2023; 9(3): 1190-204. CrossRef
Iwamoto K, Fukuyo S, Okuda M, et al. Cryopreservation of the chlorophyll d-containing cyanobacterium Acaryochloris marina. Procedia Environ Sci. 2012; 15: 118-25. CrossRef
Jia B, Allai L, Li C, et al. A review on the functional roles of trehalose during cryopreservation of small ruminant semen. Front Vet Sci [Internet]. 2024 Nov 19 [cited 2025 Aug 23]; 11: 1467242. Available from: https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2024.1467242/full CrossRef
Joseph I, Panigrahi A, Chandra PK. Tolerance of three marine microalgae to cryoprotectant dimethyl sulfoxide, methanol and glycerol. Indian J Geo Mar Sci. 2000; 29(3): 243-7.
Kapoore RV, Huete-Ortega M, Day JG, et al. Effects of cryopreservation on viability and functional stability of an industrially relevant alga. Sci Rep [Internet]. 2019 Feb 14 [cited 2025 Aug 23]; 9: 2093. Available from: https://www.nature.com/articles/s41598-019-38588-6 CrossRef
Kihika JK, Wood SA, Rhodes L, et al. Cryoprotectant treatment tests on three morphologically diverse marine dinoflagellates and the cryopreservation of Breviolum sp. (Symbiodiniaceae). Sci Rep [internet]. 2022 Jan 13 [cited 2025 Aug 23]; 12: 646. Available from: https://www.nature.com/articles/s41598-021-04227-2 CrossRef
Kim HJ, Koo BW, Kim D, Seo YS, Nam YK. Effect of marine-derived ice-binding proteins on the cryopreservation of marine microalgae. Mar Drugs [Internet]. 2017 Dec 1 [cited 2025 Aug 23]; 15(12): 372. Available from: https://www.mdpi.com/1660-3397/15/12/372. CrossRef
Kim HH, Popova E. Unifying principles of cryopreservation protocols for new plant materials based on alternative cryoprotective agents (CPAs) and a systematic approach. CryoLetters. 2023; 44(1): 1-12. CrossRef
Kim T, Pradhan B, Ki JS. Staining to machine learning: An emerging technology for determination of microalgal cell viability. J Appl Phycol. 2024; 36(5): 2573-92. CrossRef
Kugler A, Kumari P, Kokabi K, et al. Resilience to freezing in the vegetative cells of the microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta). J Phycol. 2020; 56(2): 334-45. CrossRef
Kumari N, Gupta MK, Singh RK. Open encapsulation-vitrification for cryopreservation of algae. Cryobiology. 2016; 73(2): 232-9. CrossRef
Landecker H. Cell freezing and the biology of inexorability: on cryoprotectants and chemical time. BioSocieties. 2024; 19(4): 635-55. CrossRef
Lee E, Baiz CR. How cryoprotectants work: hydrogen-bonding in low-temperature vitrified solutions. Chem Sci. 2022; 13(34): 9980-4. CrossRef
Lomba L, García CB, Benito L, et al. (2023). Advances in cryopreservatives: exploring safer alternatives. ACS Biomat Sci Eng. 2024; 10(1): 178-90. CrossRef
Mafaldo ÍM, de Medeiros VPB, da Costa WKA, et al. Survival during long-term storage, membrane integrity, and ultrastructural aspects of Lactobacillus acidophilus 05 and Lacticaseibacillus casei 01 freeze-dried with freshwater microalgae biomasses. Food Res Int [Internet]. 2022 July 7 [cited 2025 Aug 23]; 159: 111620. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0963996922006780 CrossRef
Malajczuk CJ, Stachura SS, Hendry JO, Mancera RL. Redefining the molecular interplay between dimethyl sulfoxide, lipid bilayers, and dehydration. The J Phys Chem B. 2022; 126(13): 2513-29. CrossRef
Matsuo S, Yamazaki K, Yasui M, et al. Cooling-rate dependence of the cryopreservation of aquaporin-overexpressing cells with a non-permeable cryoprotectant. Cryobiology [Internet]. 2025 Mar 28 [Cited 2025 Aug 23]; 119: 105237. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0011224025000434 CrossRef
Mokoena K, Sym S, Mycock D. Cryopreservation of Pyramimonas mucifera. CryoLetters. 2022; 43(1): 18-24. CrossRef
Morris GJ. The cryopreservation of Chlorella. 1. Interactions of rate of cooling, protective additive and warming rate. Arch Microbiol. 1976; 107(1): 57-62. CrossRef
Morris GJ. Cryopreservation of 250 strains of Chlorococcales by the method of two-step cooling. Br Phycol J. 1978; 13(1): 15-24. CrossRef
Morschett H, Reich S, Wiechert W, Oldiges M. Simplified cryopreservation of the microalga Chlorella vulgaris integrating a novel concept for cell viability estimation. Eng Life Sci. 2015; 16(1): 36-44. CrossRef
Mortain-Bertrand A, Etchart F, Boucaud MT. A method for the cryoconservation of D. salina (Chlorophyceae): effect of glycerol and cold adaptation. J Phycol. 1996; 32(2): 346-52. CrossRef
Nagel M, Pence V, Ballesteros D, et al. Plant cryopreservation: Principles, applications, and challenges of banking plant diversity at ultralow temperatures. Annu Rev Plant Biol. 2024; 75: 797-824. CrossRef
Nakanishi K, Deuchi K, Kuwano K. Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15 years of cryostorage. J Appl Phycol. 2012; 24(6): 1381-5. CrossRef
Nausch H, Buyel JF. Cryopreservation of plant cell cultures - diverse practices and protocols. N Biotechnol. 2021; 62: 86-95. CrossRef
Nozaki H, Mori F, Tanaka Y, et al. Cryopreservation of vegetative cells and zygotes of the multicellular volvocine green alga Gonium pectorale. BMC Microbiol [Internet]. 2022 Apr 14 [cited 2025 Aug 23]; 22(1): 103. Available from: https://link.springer.com/article/10.1186/s12866-022-02519-9 CrossRef
Paredes E, Ward A, Probert I, et al. Cryopreservation of algae. Methods Mol Biol. 2021; 2180: 607-21. CrossRef
Pence VC, Ballesteros D, Walters C, et al. Cryobiotechnologies: Tools for expanding long-term ex situ conservation to all plant species. Biol Conserv [Internet]. 2020 Sep 20 [cited 2025 Jun 26]; 250: 108736. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0006320720307941 CrossRef
Poncet JM, Véron B. Cryopreservation of the unicellular marine alga, Nannochloropsis oculata. Biotechnol Lett. 2003; 25(23): 2017-22. CrossRef
Prieto-Guevara M, Alarcón-Furnieles J, Jiménez-Velásquez C, et al. Cryopreservation of the microalgae Scenedesmus sp. Cells [Internet]. 2023 Feb 5 [cited 2025 Jun 26]; 12(4): 562. Available from: https://www.mdpi.com/2073-4409/12/4/562 CrossRef
Probert I, Gouhier L, Campbell CN. Cryopreservation of algae. Methods Mol Biol. 2021; 2180: 607-21. CrossRef
Rana S, Giordano M, Tsai S, Lin C. Cryopreservation of the green microalga Tetraselmis suecica with a controlled, slow-cooling technique. Platax. 2021; 18: 7-16. CrossRef
Rhodes L, Smith J, Tervit R, et al. Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology. 2006; 52(1): 152-6. CrossRef
Saadaoui I, Al Emadi M, Bounnit T, et al. Cryopreservation of microalgae from desert environments of Qatar. J Appl Phycol. 2015; 28(4): 2233-40. CrossRef
Salas Leiva JS, Dupre E. Cryopreservation of the microalgae Chaetoceros calcitrans (Paulsen): analysis of the effect of DMSO temperature and light regime during different equilibrium periods. Lat Am J Aquat Res. 2011; 39(2): 271-9. CrossRef
Santarius KA. Freezing of isolated thylakoid membranes in complex media. X. Interactions among various low molecular weight cryoprotectants. Cryobiology. 1996; 33: 118-26. CrossRef
Shah MR, Morrison EN, Noble AJ, Farrow SC. A simple and effective cryopreservation protocol for the industrially important and model organism, Euglena gracilis. STAR Protoc [Internet]. 2022 Mar 18 [cited 2025 Jun 26]; 3(1): 101043. Available from: https://www.sciencedirect.com/science/article/pii/S2666166721007498 CrossRef
Sharma Y, Sharma M. Biophysics of cryopreservation. (IjoT) Int J Thermodyn. 2022; 25(1): 17-27. CrossRef
Shevchenko N, Mozgovska A, Bobrova O, et al. Post-thaw survival of meristems from in vitro sweet potato (Ipomoea batatas (L.) Lam.) plants. Biol Life Sci Forum [Internet]. 2020 Dec 1 [cited 2025 Jun 26]; 4(1): 43-9. Available from: https://www.mdpi.com/2673-9976/4/1/43 CrossRef
Shin A, Choi SR, Yim JH, et al. Synergistic effect of polyglycerol and dmso for long-term cryopreservation of Stichococcus species. Biomacromolecules. 2024; 26(1): 635-43. CrossRef
Smolyaninova YI, Shigimaga VO, Kolesnikova AO, Popivnenko LI, Todrin AF. Electric conductivity and resistance of mouse oocyte membranes to effect of pulsed electric field in cryoprotectant solutions. Probl Cryobiol Cryomed. 2019; 28(4): 311-21. CrossRef
Stock W, Pinseel E, De Decker S, et al. Expanding the toolbox for cryopreservation of marine and freshwater diatoms. Sci Rep [Internet]. 2018 Mar 09 [cited 2025 Jun 26]; 8(1): 4279. Available from: https://www.nature.com/articles/s41598-018-22460-0 CrossRef
Tzovenis I, Triantaphyllidis G, Naihong X, et al. Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain. Aquaculture. 2004; 230(1-4): 457-73. CrossRef
Vozovyk K, Chernobai N, Kadnikova N, Shevchenko N. Effect of cryoprotective solutions on metabolic activity of Chlorococcum dissectum and Dunaliella salina cell cultures. Probl Cryobiol Cryomed. 2023; 33(1): 14-24. CrossRef
Vozovyk K, Shevchenko N. Effect of low temperature storage conditions on the viability of microalgae Chlorococcum dissectum. The Journal of V.N.Karazin Kharkiv National University. Series "Biology". 2022; 39, 12-9. CrossRef
Wang M-R, Bi W, Shukla MR, et al. Epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants. Plants [Internet]. 2021 Sep 13 [cited 2025 Jun 26]; 10: 1889. Available from: https://www.mdpi.com/2223-7747/10/9/1889 CrossRef
Yim JH, Seo YB, Kim SM, Jeon YJ. [Recent research trends of cryopreservation technology based on microalgae chlorophyta]. J Life Sci. 2021; 31(10): 960-8. Korean. CrossRef
Yee JC, Yang H. Cryopreservation of Tetraselmis striata through systematic evaluation of multiple parameters in the cooling process. Aquaculture [Internet]. 2023 Mar 15 [cited 2025 Jun 26]; 566: 739172. Available from: https://www.sciencedirect.com/science/article/abs/pii/S004484862201290X CrossRef
Zamecnik J, Faltus M, Bilavcik A. Vitrification solutions for plant cryopreservation: modification and properties. Plants [Internet]. 2021 Nov 29 [cited 2025 Jun 26]; 10(12): 2623. Available from: https://www.mdpi.com/2223-7747/10/12/2623 CrossRef
Zhang E, Zhang L, Wang B, et al. Cryopreservation of marine diatom algae by encapsulation-vitrification. CryoLetters. 2009; 30(3): 224-31. CrossRef
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