Advances in induced anhydrobiosis for cell and gamete storage

Trends in Biotechnology OPEN ACCESS Review Advances in induced anhydrobiosis for cell and gamete storage Pasqualino Loi 1 ,7, *, 1 Department of Veterinary Medicine, University of Teramo, Teramo 64100, Italy Luca Palazzese 1,7 , Margherita Moncada 1 , Martina Lo Sterzo 1 , Domenico Iuso 1 , Marta Czernik 1,2 , 2 Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzebiec, 05-552, Warsaw, Poland Teruhiko Wakayama 3 ,4 , and 3 Advanced Biotechnology Center, University of Yamanashi, Yamanashi 400-8510, Japan 4 Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan Takahiro Kikawada 5 ,6 5 Division of Biomaterial Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8634, Japan 6 Department of Integrated Biosciences, Graduate School of Frontier Science, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan It will appear strange to most readers, but lyophilization of spermatozoa was described by Chris Polge’s first article that demonstrated the viability of spermatozoa stored in liquid nitrogen (LN 2) . Since then, cryopreservation has become the only biobanking choice. The demonstration that lyophilized spermatozoa remain fertile has r ekindled interest in noncryogenic biobanking. This perspective has recently been embraced by engineers, physicists, and anhy drob iotic physiologists, and their synergy has remarkably advanced the field. Progress in water subtraction procedures and dehydration medium formulated by biomimicking anhydrobiotic organisms has brought spermatozoa drying close to practical application for biodiversity preservation and human-assisted reproduction, allowing at the same time easy a nd safe transport of genetic resources on Earth and eventually space travel. Trends in Biotechnology, July 2025, Vol. 43, No. 7 https://doi.org/10.1016/j.tibtech.2025.02.010 1599 © 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biobanking is a primary societal need Biobanks (see Glossary) that store a plethora of biol ogical samples includ e internationa l and national banks, large and small universit y banks, and private for and not-for-profit banks [1]. Most of their users gravitate around life sciences, clinical research, and medicine. In 2009, TIME named biobanking as one of the ten ideas that will change to world [2], and the prediction was ultimately correct. Since then, population and di sease-based biobanks have grown steadily until novel therapeutic perspectives drifted away from the one-size-fits-all strategy. For obvious reasons, “individual sample storage” was exclusively linked to banking for reproductive purposes [3] to counteract human fertility decline [4]. More recently however, precision medicine drifted toward patient-derived s ample s torage [5], exponentially e xpanding the size, number, and cost of biobanks. The costs represent the need for specialized facilities, equipment, and personnel and, above all, the continuous liquid nitrogen (LN 2 ) supply required for cryogenic storage, the universal gold standard storage approach in biobanks. The 2023 global LN 2 market size has been of US $19.4 billion and is set to expand further, with a foreseen growth rate of 5.2%, leading to reaching US$30.9 billion by 2032 (https://www.imarcgroup.com/liquid-nitrogen-market). The growing importance of biobanks and the consequent spike in the associated costs nurtured initiatives in search for alternative storage options, noting that few options are available. Natur e has only two strategies to reversibly suspend l ife, both leveraging intrac ellular water: keep it still, as in freezing, or remove it, as in anhydrobiotic organisms. Curiously, both strategies were attempted in the paper published by Chris Polge and colleagues in 1949, in which they demonstrated that rooster spermatozoa maintained fertilizing ability after cryopreserving with glycerol in LN 2 [6]. However, for reasons deta iled elsewhere [7], cryopreservation became the method of choice. The birth of mice from dry spermatozoa in 1998 reopened the quest for alternative storage options to conventional freezing [8], including other reports aiming at repeating the mouse’s work. Some of these studies reported the production of live offspring. This undertaking was only Highlights Cryogenic storage is effective but comes with negative side effects, like elevated costs and a high carbon footpri nt. Alternative storage options based of the induction of anhydrobiosis followed by dry storage are highly desirable. A biomimicry approach borrowed from anhydrobiotic organisms resulted in successful induction of reversible drying in spermatozoa and cells. The current state of the art of spermatozoa dry storage is close to practical application, but cell and oocyte drying is still out of reach. Dry storage would enormously simplify the establishment and maintenance of working biobanks for human infertility treatment, biodiversity preservation, and easy transport of samples through ordinary mail and in space missions. 7 These authors contributed equally to this work *Correspondence: [email protected] (P. Loi). marginally accomplished elsewhere, with three laboratory [9–11] and one farm [12] animal species being produced so far. Interestingly, scientists raised the stakes by inducing genome viability of dried somatic cells through nuclear transfer into nucleated oocytes [13–15]. Since then, an increasing number of researchers have cautiously engaged in the reversible drying of cells. Given that cells are extremely vulnerable to water loss and are devoid of genes endowing them with desiccation tolerance, the only possible approach to making them tolerant to water deprivation is to load them with gene products discovered in anhydrobiotic organisms, starting with disaccharides such as tre halose [16]. Trehalose-expressing fibroblasts were reported to withstand water removal [ 17], formalizing the entry of somatic cells as cand idates for dry storage. Notably, the first somatic cells to be lyophilized were erythrocytes, enucleated cells, platelets, and cellular fragments, indicating that early drying biologists feared that the nuclear structure was vulnerable to dehydration [18,19]. Trends in Biotechnology OPEN ACCESS Besides trehalose, late embryogenesis abundant (LEA) proteins are the most acquainted and interesting xeroprotectants expressed in anhydrobiotic organisms, as well as in seeds [20]. LEA proteins are a large family of highly di sordered proteins, that is, they lac k organized secondary and tertiary structures in the presence of water but acquire their final ordered structure upon dehydration (for a review on the properties of LEA proteins, please see [21]). The first review article on cells and gamete lyophilization, published a decade ago [7], marked the beginning of what was then a niche research topic, with limited publications primarily focused on spermatozoa lyophilization. However, in the years since, the landscape has dramatically shifted. As biobanks have proliferated, significant advancements have been made in the field of cell and gamete-controlled dehydration. This review highlights key developments in the technology, sho wcas ing progre ss in techniques for controlled water removal, which are now being refined to allow for the long-term storage of spermatozoa through lyophilization. These advancements are particularly noteworthy as they open up practical applications in reproductive medicine and animal husbandry. Additionally, this review e mphasizes that while spermatozoa storage has made great st rides, other areas of gamete a nd cell preservation (such as embryo and somatic cell drying) remain more challenging. We see the most promise in the near term for establishing anhydrobiosis for the short-term storage of sperm in dry working banks. This paper not only surveys the work that has been done so far but also points to future directions for tackling these more complex issues, particularly for cell drying, where the only suitable path cannot be simpl y turning the m into ‘temporary anhydrobiotes’ by expressing or loading into them the most effective kit of xeroprotectants identified in model organisms, selected with AI, and validated through empirical trial s. Induction of reversible drying in cells and spermatozoa: state of the art An analysis of published data over the past 10 years provides two main insights: (i) A robust leap in the reversible drying of spermatozoa and a proof of concept of reversible drying in cells have been achieved. (ii) Several approaches for cells and spermatozoa dehydration are now available. As for point (i), advancing the state of the art in sperm drying has been relatively easy, taking advantage of the peculiar nuclear organization of spermatozoa. The simple solutions put into practice revealed that every minor technical change that reduced DNA damage resulted in a significant and immediate improvement in the fertilizing capacity of the rehydrated spermatozoa. For instance, it has been established that freezing spermatozoon at mild subzero temperatures, in the 1600 Trends in Biotechnology, July 2025, Vol. 43, No. 7 range of –20 to –30°C before water sublimation, significantly reduced singleand doublestrand DNA breaks. Consequently, their fertilization potential was markedly increased, and the n eed for LN 2 , even in the early steps of t he process, was eliminated [22]. The benefits of this simplification are likely because it eliminates the osmotic and mechanical damage to the genome caused by deep freezing. Trends in Biotechnology OPEN ACCESS Less struc tured and exhausti ve are the available data on somatic cell drying, which remain s a niche endeavor for just a few laboratories. The paucity of available data does not allow us to have a complete picture, nor can it be sa id tha t true progress has been achieved. However, the available data let us indulge in some optimism. Our group explored the behavior of fibroblasts expressing three LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydr in-1dhn, expressed in the nucl eus–cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, which h as a h igh affinity for cellular membranes; and pTag-LEA -BFP, Artemia franciscana LEA protein that targets the mitochondria. Fibroblasts expressin g one or all three LEA proteins were air dried for 4 hours, and cell viability was monitored immediately after the stress and after a long recovery. Almost all untransfected cells were nonviable (1% live cells), while over 30% of LEA protein-expressing cells were viable and recovered rapidly after rehydration [23]. These findings confirmed and expanded previous data [24] and are truly remarkable first because proteins normally expressed predominantly in the plant kingdom are tolerated by mammalian cells and second because they protect them from the same physical stress. In vivo experiments with LEA proteins in a model organism (Caenorhabditis elegans) confirmed that LEA proteins can induce desiccation tolerance in multicellular organisms and that their protection resides in specific motifs of the protein [25], adding to our optimism that a medium formulated with the most effective peptides of LEA proteins (easily loadable) will confer full desiccation tolerance to cells and, perhaps, to oocytes and embryos. Clearly, the path for cell reversible drying is far more complicated than spermatozoa drying. The transformation of cells into ‘transient anydrobiontes’, as suggested here, might help, but it hardly confers full protection; therefore, industrial processing expertise is required. The shielding of the cells with external membranes before drying, as is carried out in microencapsulation, m ight result in their effective protection against dehydration stress. Yeast cells are the best example. Yeast lyophilization is routine in the food industry, and a pivotal role in their desiccation tolerance, among other factors, is provided by the c ell wall [26]. Yeast cell wall components are increasingly used for encapsulation of p robiotics [27]. Microencapsulation of cells with cell wall components be fore drying might be key in conferring desiccation tolerance. As for point 2, a retrospective review of dehydration attempts, all directed toward spermatozoa, indicates that the few researchers who dared to venture into gamete drying resorted to sticking to the path indicated by the original article: canonical lyophilization. However, recent data have clearly indicated a drift from lyophilization toward gentler water subtraction techniques, like spin or vacuum drying (Figure 1). Spin drying is a soft dehydration procedure originally adapted to store nucleic acids [28]. The technique is based on the dehydration of the sample under a vacuum while spinning it in a dedicated centrifuge. The procedure is very rapid, in the order of a few minutes, and requires a mild vacuum. Adopting spin drying has considerably reduced DNA damage and increased the developmental performance of embryos derived from spin-dried ram spermatozoa [29]. These latest water subtraction methods are highly interesting. If we really want to stick to a biomimicry approach in our attempts to dry cells, we have to remember that anhydrobiotic organisms do not freeze before drying. Noncryogenic approaches are also emerging in oocyte drying. Microwave-assisted drying in a medium enriched with trehalose and choline acetate facilitated preserving DNA integrity in the nuclei of dried Glossary Artemia franciscana: species of brine shrimp endemic to the Americas but now widely introduced throughout the tropics and temperate zones worldwide. Biobanks: nonprofit storage units dedicated to the collection, processing, conservation, and distribution of human and nonhuman biological samples (e.g., various cell types, gametes, and embryos) and related data for research, diagnosis, toxicology testing, and cell replacement therapy. Officially recognized by health authorities, they implement a quality system and ensure the rights of the subjects involved. Caenorhabditis elegans: phasmidary nematode worm, about 1 mm long, that lives in the soil in temperate regions; largely used as an experimental model. ES: embryonic stem cell; pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo. LEA: late embryogenesis abundant proteins (LEA proteins) are found in plant seeds as well as in some bacteria and invertebrates. LEA proteins protect against protein aggregation caused by desiccation or osmotic stresses, including those related to low temperatures. LEA proteins were first identified in cotton seeds, where they accumulate during the late stages of embryogenesis, hence their name. Liquid nitrogen (LN 2 ): liquid nitrogen, a cryogenic fluid used for the conservation of biological samples (as in biobanks), with a temperature between −210°C and −196° C. Polypedilum vanderplanki: or the sleeping chironomid, is a dipteran in the family Chironomidae. It is found in semiarid regions of the African continent. Pv11 cells: a cell line derived from the anhydrobiotic insect Polypedilum vanderplanki. Singleand double-strand DNA breaks: single-strand breaks occur when the nucleotide sequence of only one strand is altered (damaged or break). Double-strand breaks occur when both strands of the DNA are broken. Trehalose: disaccharide that presents a 1α-1′α glycosidic bond by condensation between two glucose molecules; it is widespread in yeasts, fungi, and inse cts. Tricum aestivum: soft wheat, an herbaceous plant of the Poaceae family. Trends in Biotechnology, July 2025, Vol. 43, No. 7 1601 immature cat oocytes stored for 7 weeks at room temperature [30]. The method is based on nonionizing microwave radiation that results in rapid water loss from samples kept in a medium with disaccharides such as trehalose and sucrose. The relative increase in disaccharide concentration consequent to water loss resulted in isothermal vitrification during drying [31]. WGA: whole-genome analysis, also known as whole-genome sequencing, is the process of determining the entire DNA sequence of an organism’s genome. Working biobanks: different from master biobanks (large facilities that store countless numbers of a large variety of biological samples, often on an international scale), working biobanks are small-sized biobanks that are typically established by research laboratories or infertility clinics. Xeroprotectant: xero means ‘dry’ in Greek. A xeroprotectant is a molecule or protein that protects an organism from dry damage . Zea mays: Corn (Zea mays L., 1753), also called maize. Trends in Biotechnology OPEN ACCESS Toward the establishment of dry working banks At the time that our previous review article was published [7], the only available certainty was that lyophilized mouse spermatozoa maintain fertility, and a couple of sporadic reports were published on somatic cell lyophilization revived by nuclear transfer [13,15]. These could hardly be defined as state of the art. Since then, inducing reversible drying has stopped being a task of single groups; rather, it has evolved into a truly multidisciplinary effort (Figure 2). In other w ords, the ‘tria l and error’ approach previously adopted has been replaced by the collaborative efforts of biophysical and chemical theoretical modelers that predicted the molecular interaction between xeroprotectants and cellular subcompartments, DNA included, under various water concentrations, negative pressures, and temperatures. These theoretical models encouraged the exploration of alternative drying methods, lik e spin dry ing, or dry spermatozoa after exposure to mild subzero temperatures. In addition to the basic theoretical m odelers, substantial contributions are continuously being made by molecular biologists exploring the basic mechanisms conferring reversible drying in model anhydrobiotic organisms, including Polypedilum vanderplanki. Pv11 cell-derived P. vanderplanki embryos can show their anhydrobiosis ability when pretreated for 48 h in high concentrations of tr ehalose, which induces the expression of genes involved in desiccation tolerance [32]. Trehalose is known as one of the xeroprotectants that fills the space previously occupied by water in dehydrating cells, eventually forming an organic glass to protect biomolecules from desiccation damage [33]. It is interesting to note that in Pv11 cells, in addition to this protective function, trehalose is also invo lved in the gene induction mechanism [34]. The trehalose-induced genes include some intrinsically disordered proteins called LEA proteins [35]. In Pv11 cells, LEA proteins are likely to function to prevent irreversible aggregation and denaturation of proteins and cell membranes during the desiccation process, and the coexistence of trehalose greatly enhances this a ntiaggregation activity [36]. Of course, other trehalose-induced genes may also act as xeroprotectants in Pv11 cells [37]. In addition to canonical xeroprotectants such as trehalose and LEA proteins , xeroprotectants should also include molecules that repair biomolecules damaged by desiccation. For example, repair mechanisms for genomic DNA damage caused by reactive oxygen species generation during rehydration are obviously important. Several DNA repair factors are known to function during the rehydration process in P. vanderplanki [38]. Recently, molecular research using a genome editing technique revealed that the sodium-dependent trehalose transporter STRT1 effectively releases unwanted intracellular trehalose from desiccated Pv11 cells immediately after rehydration [16]. STRT1 activity is important for the effective regulation of intracellular osmolarity during the rehydration process, allowing desiccate d Pv11 cells to resume vital activity without bursting. Although it is very important for cells to take up a large amount of xeroprotectants, it is also important to implement an effective extracellular efflux mechanism of protective factors to avoid cell bursting. Hence, streamlini ng the gene expression path trigger ed by water removal in Pv11 cells has proved to be an astonishing tool to mine powerful xeroprotectants, identified through gene expression analysis and validated by reverse genetics and biochemistry. The number of candidate xeroprotect ant molecules t o be validated on sper matozoa or soma tic cells is increasing rap idly. Clearly, these new, high-performance xeroprotectors, preferably selected through in sili co modelling and even bett er by arti ficial intelligence (AI), sh ould be incorporated into the drying medium and validated though empirical trials. Put simply, thanks to collaborative efforts, we now have a robust state-of-theart of reversible somatic cell and sp ermatozoa drying, schematically presented in Table 1 . 1602 Trends in Biotechnology, July 2025, Vol. 43, No. 7 Trends in Biotechnology OPEN ACCESS TrendsTrends inin BiotechnologyBiotechnology Figure 1. Water subtraction techniques, alternative to the standard lyophilization, being explored. Table 1 provides an overview and establishes pri orities. The ove rview underlin es the inc redible progress achieved over the last 10 years, progress that contemplates mostly the male gamete but also goes through somatic cells and touches ultimately the female gamete. Some of the outcomes achieved are truly remarkable, especially if we consider that they were realized by borrowing drying devices developed for other purposes, like drying large amounts of pharmaceutical or food products rather than tiny volumes of cells. Ad hoc devises for gamete and cell drying, discussed in the next section, will unquestionably accelerate progress. As for priorities, while it appears realistic to assume that spermatozoa, including human spermatozoa, might be successfully dried while maintaining fertility in the short term, that is, in the next 5–10 years, reversible drying in somatic cells, and of course oocytes, remains daring, even in the long term. It is also clear that dry storage will never replace the major cryobanks (for a list of the ten largest cryobanks in the word, see https://www.biobanking.com/10-largest-biobanks-in-the-world/); however, and limited to spermatozoa, working banks temporarily storing these gametes for a short time, like those in human fertility treatment centers, might opt to store spermatozoa in dry form for intuitive economic reasons, in addition to the advantages presented elsewhere [39]. G iven that a limi ted number of samples are stored during infertility treatment cycles and that the storage is usually limited to t he c ycle’s length, there will be no need to scale up the Trends in Biotechnology, July 2025, Vol. 43, No. 7 1603 storage process. The same consideration applies to genetic samples of endangered species, especially in low-income countries, which also might start as dry working banks. It is worth remembering that 75 % of the world’s endangered species live in low-income countries, like Asia and Africa (together accounting for 9500 of over 18 500 total endangered species; IUCN red list (International Union for Conservation of Nature). Dry working biobanks of any size have enormous potential in biodiversity preservation, even with the current state of the art that does not guarantee viability after rehydration. However, this is not necessarily an issue. Dry mouse somatic cells have been converted into embryonic stem (ES) cells by nuclear transfer, followed by blastocyst production. It was demonstrated on several occasions that ES cells could be programmed toward primordial germ cells and then instructed to become mature gametes, capable of generating normal offspring [40]. However, bona fide ES cells are presently available in laboratory animal, mouse and rat, and human embryos but not in other species. This inconvenience might be overcome by generating embryonic-like cells from nuclear transfer embryos. Furthermore, both gametes can be generated from somatic cells by converting them into induced pluripotent stem cells, which can be directed to develop into primordial germ cell-like cells and then into mature gametes [41,42]. It is certainly challenging at the moment and a rather radical shortcut on a path full of uncertainties, but we think it is the only one currently available [43] because many species on the brink of extinction will disappea r before we can adequately know how to control their reproductive biology. Trends in Biotechnology OPEN ACCESS TrendsTrends inin BiotechnologyBiotechnology Figure 2. Multidisciplinary approach for inducing reversible drying of cells and gametes. While backup working banks at fertility clinics do not require long-term storage, storage of genetic backup material from endangered species is a fundamental issue, requiring long-t erm storage, 1604 Trends in Biotechnology, July 2025, Vol. 43, No. 7 ideally at the lowest possible cost, like room temperature storage. Unfortunately, despite its importance , this is an unexplored field. Going back in time, the first group that explored the issue started off with bad news; they stated that dry mouse spermatozoa could by no means be stored at room temperature and retain fertilization potential [44]. The more positive news was provided later by two independent reports in mice, one with conventionally lyophilized spermatozoa stored under vacuum in vials and the other with evaporation-dried spermatozoa stored in LiCl desiccation jars. The lengths of room temperature storage in these studies were 1 and 2 years, respectively [45,46]. Our data on ram spermatozoa confirm these latest r eports, showing that spermatozoa retained the capacity to generate normal embryos through intracytoplasm ic sperm injection after 2 years of storage at room temperature [29]. Clearly, in vitro development needs to be completed with suitable in vivo trials and the production of live off spring. We and others have demonstrated that room temperature storage was a realistic option, even for a relatively long time, 2 years, in our published report. Managing the long-term storage of dried genetic material in biobanks is an indispensable task that has just started to be touched. The starting condition of the sample (namely its water content) and the packaging atmosphere composition (the rehyd ration modalities) are the elements to maneuver around. Normally, dry spermatozoa are packed under vacuum in glass vials. The integrity of the packaging is crucial for long-term preservation, as any leakage allowing atmospheric air to enter the vials could cause irreversible DNA damage [29,45]. Ideally, dehydration should leave a minimal amount of water in the samples, kn own as ‘bound water’, to prevent structural collapse, particularly of membranes [47]. Once this crucial parameter has been established, the next step is to determine the composition of the packaging atmosphere. Our data, borrowed from dry storage of n uclei acids [29], have shown that dried spermatozoa, stored in a saturated atmosphere of a mix of inert gases like helium and argon, show preserved fertilizing ability after 2 years of storage at room temperature. However, the field is far from being complete, and there is much room fo r improvement. Aging Trends in Biotechnology OPEN ACCESS Table 1. Recent milestones in reversible dry of cells and gametes Milestones Species Drying technique Storage Year Refs I Spermatozoa drying under mild cryostorage Sheep Freeze-dry 4°C, up to 18 months 2020 [22] II Spermatozoa fertility preservation after 2 years of storage at RT Sheep Vacuum-dry encapsulation RT, 2 years 2023 [29] III Lyophilized spermatozoa mailed across countries using ordinary mail Mouse Freeze-dry on postcards RT, 3 days or −30°C, up to 3 months 2021 [49] IV Lyophilized spermatozoa retained full fertility (offspring production) following cosmic ray exposure at the space station Mouse Freeze-dry −95°C International Space Station, 9 months 2017 [48] V Dehydration of oocyte nuclei by microwave-assisted drying Cat Microwave-assisted dehydration (T max , 40°C) 4°C, 24 hours 2019 [31] VI Somatic cells expressing LEA proteins survived water stress Sheep Air-dry 16°C, No storage 2020 [23] VII Lyophilized cells maintain genome stability and cell viability following nuclea r transfer and embryonic stem cell isolation Mouse Freeze-dry −30°C, up to 9 months 2022 [40] Abbreviations: LEA, Late Embryogenesis Abundant proteins; RT, room temperature; T max , maximum temperature. Trends in Biotechnology, July 2025, Vol. 43, No. 7 1605 models that could supply information before the scientist became too old to check, like accelerated aging simulations by thermal treatment [44], would greatly benefit these studies. Trends in Biotechnology OPEN ACCESS Concluding remarks and future perspectives Even though the progress achieved is truly remarkable, we need to attain two main milestones to provide r eversible drying full steam: (i) the development of dedicated dryers for small-volume samples, in the order of 100 μL or less, and (2) the production of large animals with rehydrated spermatozoa, followed by long-term studies on their well-being, behavior, and health. (i) All the data achieved thus far on sperm drying have been accomplished using devices designed to process very large volumes of products that require long processing times and extreme force magnitudes, like negative pressure. While these processing paths do not h arm unstructured pharmaceutical or food products, they might cause mechanical damage to the fragile structures of the cell. Therefore, ad hoc device s designed to process small-volume samples are key to speeding up the progress in reversible drying of spermatozoa, with p ossible positive benefits for somatic cells and oocytes. (2) Proof-of-concept demonstration of the normalcy of offspring derived from embryos produced with dried s permatozoa . This is a fundamenta l requirement before applying dry spermatozoa storage for human fertilit y treat ments. A genome-wide screening of DNA from mice produced from dry spermatozoa has been conducted [48]. The screening detected minor genomic dif ferences, probably ascribed to indels resulting from DNA damage and repair after 9 months of exposure to cosmic radiation. Yet, no problems were found in the offspring that grew i nto normal and fertile adults [40]. The m ouse data are encouraging, TrendsTrends inin BiotechnologyBiotechnology Figure 3. Overview of xeroprotectant actions, modalities of administration, recovery of dry cells and gametes, and applications. Xeroprotectants are compounds that protect cells and biological structures from damage during desiccation by stabilization of genetic material and cellular membranes, maintenance of an osmotic balance, reduction of oxidative stress, induction of a dormancy-like state to reduce metabolic activity and conserve energy, and preservation of the mitochondrial membrane and function, ensuring cellular energy metabolism upon rehydration. They have been applied to spermatozoa, germinal vesicle (GV) oocytes, somatic cells, or embryonic stem cells (ESCs). After rehydration, cells functionally recover almost exclusively genetic material, allowing spermatozoa to be used for intracytoplasmic sperm injection (ICSI), GV oocytes for GV nuclear transfer (GVNT), and somatic or embryonic stem cells for somatic cell nuclear transfer (SCNT). Such xeroprotectant-assisted cell drying holds potential use in human and veterinary reproductive technologies (spermatozoa and GV oocytes) and long-term preservation of spermatozoa and somatic or embryonic stem cells as a strategy for safeguarding biodiversity and support endangered species. (See [40,43]). Abbreviation: ROS, reactive oxygen species. Outstanding questions The production of offspring from dry spermatozoa of a large animal model is of pivotal importance before application of dry storage in h uman infertility clinics. The development of bench-top dryers specifically designed for small aliquots of cells or gametes is of great importance to speed up the progress and to customize drying condition according to cell type. Selection and validation of the newest and most effective xeroprotectant for cell drying is important. The definition of optimal packaging conditions for long-term storage of gametes first and then cells is compulsory. 1606 Trends in Biotechnology, July 2025, Vol. 43, No. 7 but similar or even more compelling data from large animals are compulsory before applying dry storage to human spermatozoa. Whole-genome analysis ( WGA), including DNA sequenc ing and trans criptome and methylome analyses, are increasingly adopted as proof to define normalcy or absence of negative side effects of a certain procedure. We believe that it is a reductive approach, if we plan t o generate humans with dry-stored spermatozoa. Suitable large models, like sheep or pigs, should be generated and carefully followed up to exclude negative metabolic, c linical, fertility, a nd behavioral effects later in life arising from subtle g enomic damage caused by water subtraction in the sp ermatozoa, probably not detected by the available WGA. Trends in Biotechnology OPEN ACCESS To summarize, the induction of reversible drying has made a remarkable leap in the last 10 years, taking advantage of a truly interdisciplinary approach, where insect biologists, embryologists, and chemical engineers joined forces to implement physiological insights gained from anhydrobiotic organisms and various technicalities to achieve harmless water extraction from somatic cells and gametes (Figure 3 and Table 2). While it is unrealistic to replace cryostorage with dry storage in master biobanks, dry storage of spermatozoa and somatic cells in working banks, ideally at room temperature, is now within our grasp. Table 2. Application of working dry banks, risk and feasibility Cell type Application Application risk and feasibility Spermatozoa Human infertility preservation Medium Farm animal breeding Low–medium Space travel and exoplanet colonization Low Oocyte (MII) Human infertility treatment Medium Farm animal breeding Medium–high Space travel and exoplanet colonization High Somatic cells and tissue Cloning for diversity preservation Medium–high Space travel and exoplanet colonization Medium–high Embryonic stem cells Cloning for diversity preservation Medium Space travel and exoplanet colonization Medium Abbreviation: MII, metaphase II. Many benefits could be gained from this green storage opt ion, and all are important (see Outstanding questions). These include spermatozoa storage in human fertility clinics and for farm animal breeding, biobanking for biodiversity conservation, easy sample transport by ordinary mail [49], and, one day, possible transport of dried cells and germplasm for space travel and Mars colonization [50]. Acknowledgments This work has been funded by the European Union–Next Generation EU under the Italian Ministry of University and Research (MUR) N ational Innovation Ecosystem grant ECS00000041VITALITY-CUP C43C 2200038 0007. Moreover, this work was supported by funding from the Europea n Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodow ska-Cur ie a ction, projec t ‘WhyNotDry’ (GA-101131087), MIUR, PRIN 2022CSHPAS, and PNRR P2022FA79R to P.L. M.C. acknowledges s upport from the Na tional Science Centre, Poland, through grant number 2019/35/B/NZ3/02856 (Opus). Part of this work was funded by JSPS KANENHI, Grant Number JP22H00372, to T .K. Declaration of interests No interests are declared. Trends in Biotechnology, July 2025, Vol. 43, No. 7 1607