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Towards storage of cells and gametes in dry form
Review
Towards storage of cells and gametes in dry form P. Loi, D. Iuso, M. Czernik, F. Zacchini, and G. Ptak Department of Comparative Biomedical Sciences, University of Teramo, Piazza Aldo Moro 45, 64100, Teramo, Italy
We review published data on cell/gamete lyophilization. Most studies have utilized the same established protocols for cryopreservation (storage in liquid nitrogen) as for cell lyophilization (dehydration of frozen samples by water sublimation). Surveying natural lyoprotectants, we suggest trehalose and late embryogenesis abundant (LEA) proteins as ideal candidates for the reversible desiccation of mammalian cells/gametes. We find that despite the numerous water subtraction techniques, scientists have relied almost exclusively on lyophilization. There is thus room for improvement in both medium formulation and water subtraction strategies for dry cell/gamete storage. We believe the development of dry processing protocols for use in biobanks of cells/ gametes, at reduced cost and with minimal carbon footprint, is within our grasp. Introduction The demand for long-term eukaryotic cell storage methods emerged in the first half of the 20th century once experimentalists began successfully exploiting spermatozoa and cells for medicine, research, and animal breeding. Initially, the options for storage were freezing, vitrification, and freeze drying (i.e., lyophilization) [1–3]. For the difficulties to maintain structural integrity in dry cells, lyophilization was partially dropped, whereas freezing was developed further; helped also by technological advances in mechanical freezer development, coupled with the availability of liquid nitrogen (LN). LN was first liquefied by two Polish physicists, Wroblewski and Olszewski in 1883 [4], and was widely used after World War II, becoming a key element in early cryobiology. The route to successful cryopreservation was far from straight and owes a great deal to serendipity. The first successful cryopreservation of spermatozoa [5] was accomplished with a bottle of sucrose – the common cryoprotectant in vogue at that time – contaminated with glycerol. The accidental discovery of glycerol and its analogs as the most powerful cryoprotectants paved the way for deep-freezing cells in LN. The science of cryobiology started in 1940 and laid down all physical/chemical Corresponding authors: Loi, P. ([email protected]); Ptak, G. ([email protected]). Keywords: lyophilization; liquid nitrogen; anydrobiosis; gametes; long-term storage. 0167-7799/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2013.09.004
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requirements enabling a cell to withstand reversibly the long-term exposure at subzero temperatures. A common feature of all cryopreservation protocols is the addition of a suitable permeable cryoprotectants, for example, glycerol or ethylene glycol, to the freezing medium. The cryoprotectant binds to intracellular water, thus reducing intracellular damage. Then the sample undergoes controlled cooling in order to induce progressive dehydration prior to being plunged into LN. The dehydration is essential, because it avoids/reduces intracellular ice injury. The search for avoiding intracellular ice formation resulted in the evolution of the freezing paradigms, which is now named vitrification. Vitrification relies on the ability of highly concentrated solutions of cryoprotectants to supercool to low temperatures, forming a glassy matrix without the formation of ice [6]. Since the first report, there has been massive investment in vitrification research, which has now become the first cryostorage choice for human oocytes and embryos [7,8]. Overall, the current freezing protocols are straightforward and efficient, with a good recovery rate at thawing [9], but they are not devoid of problems. Liquid nitrogen storage is expensive, requiring continuous monitoring of its levels and supply [9]; it is inconvenient, necessitating dedicated facilities and equipment, especially for many developing countries; it is potentially dangerous for the operator and can become contaminated by viruses or other pathogens [10]; and finally, it makes the shipping of samples difficult (Table 1). Aside from these technical and practical inconveniences, LN storage also poses an environmental concern, given that its industrial production and the maintenance of its storage centers have a high carbon footprint. Given the increasing demand of biobanks for various purposes, including medicine, research, pharmaceutical endeavors, and biodiversity preservation, there is a pressing need to develop alternative storage options. In this review we critically analyze the data published on dry preservation of mammalian cells and gametes; surveying all organisms undergoing desiccation in their life cycle; listing the naturally available lyoprotectant molecules; and finally, we speculate about their potential use for dry storage of cells and gametes. Freeze-dried spermatozoa Following the successful lyophilization of spermatozoa [5], in the 1950s, some attempts were made to lyophilize red blood cells and platelets [11,12]. Although drying red blood cells proved unsuccessful, the lyophilized platelets nevertheless preserved the morphology and function of the
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Table 1. Pros and cons of cryoconservation versus lyophilization storage Method Ease/practicality Specialized equipment Cost for long-term storage Hazard Pathogen contamination
Cryoconservation + ++++ +++++ +++++ +++++
Lyophilization +++++ +++++ + + +
cells [13]. Why were nucleated cells or cell particles such as platelets not the first targets for lyophilization trials? We speculate that there were at least two reasons; one practical and one biological. In practical terms, freeze-dried blood would simplify the storage and transport of blood banks enormously. Biologically, red blood cells and platelets are devoid of nuclei, and it is likely that early ‘lyobiologists’ feared that the nuclear structure would be incompatible with dry storage. At the end of 1998, the cell and gamete storage paradigm shifted, when Wakayama and Yanagimachi demonstrated that lyophilized mouse spermatozoa, stored at room temperature for 3 months, were able to generate normal offspring [14]. The spermatozoa were nonviable and motionless at rehydration, but the handicap of the lost motility was easily overcome by their direct injection into the oocytes. These findings demonstrated for the first time that nuclear and cellular viability are not equivalent. The lyophilized spermatozoa were essentially dead, yet were still able to interact with the programming machinery of the oocytes, giving rise to normal pups. This discovery was an important milestone; the fertilizing ability of lyophilized spermatozoa has since been confirmed in other species including horses [15], dogs [16], rats [17], cattle [18], pigs [19], rabbits [20], marsupials [21], and humans [22]. Other experiments, performed after the publication of Wakayama and Yanagimachi’s data, demonstrated that somatic cells subjected to thermal stress (558C and 758C) for 30 min, and obviously killed by the treatment, were capable of generating normal lambs upon transfer into enucleated sheep oocytes [23]. Hence, it was established that cell viability and nuclear viability are likewise not equivalent in somatic cells. Soon after, sheep somatic cells – granulosa cells and lymphocytes – lyophilized with the addition of trehalose in the freezing medium in Israel, were shipped by ordinary mail to Italy. The lyophilized cells were kept on a shelf in a cardboard box and used 5 years later for nuclear transfer. Surprisingly, the reconstructed embryos developed to the blastocyst stage [24]. Subsequent work reproduced these results in mice [25] and pigs [26]. The current list of successful nuclear transfers of lyophilized cells includes sheep, pigs, mice, and cattle (Japanese Brown Cattle; Matzukawa, personal communication). Thus, multiple research teams have shown that lyophilized cells can be successfully reprogrammed after nuclear transfer and can develop until the blastocyst stage. The next step would be to demonstrate that these cloned embryos can develop into normal offspring, and experiments to that end are currently ongoing. The above results prompted scientists to look beyond LN for the storage of cells. Two independent research
groups reported the successful dry storage of human mesenchymal stem cells and cord blood cells. In both cases, the cells maintained the capacity to form colonies under appropriated conditions in vitro [27,28]. The findings are significant in showing that dry storage of cells and gametes is a viable option. We are clearly in the early stages of dry storage of cells and gametes, and there is a large margin for improvement. For one, if we exclude the addition of trehalose, the freezing protocol we used in our original study [24], but also in those leading to successful dry storage of spermatozoa [14], is even more primitive that those used for drying yeast. The cells and spermatozoa were suspended in small aliquots (200 ml) of standard buffered medium, snap-frozen in LN, and water-extracted with a conventional lyophilizer. Yeast cells are instead lyophilized efficiently using a combination of sugars and yeast extracts, which probably exert a bulk effect analogous to the egg yolk in sperm freezing medium. None of these ingredients have ever been used for cell lyophilization. It appears likely that improvements in the composition of lyophilization media will improve the dry storage of cells and gametes overall, but there is also room for improvement in the drying procedures. Is lyophilization the only way, or are alternative options also worthy of exploration? Selection of ideal lyoprotectants The discovery of the protective action of glycerol ensured the success of deep-freezing gametes and cells [4]. Since then, other cryoprotectants have been identified and optimal cell type–cryoprotectant combinations have been established. We must do the same for dry cell and gamete storage, starting with the individuation and selection of the best lyoprotectants. This is particularly important for eukaryotic cells and gametes. Mammals are sensitive to water loss, and dehydration can be life-threatening; therefore, a coordinated physiological pathway keeps water loss to a minimum. Eukaryotic cells sense desiccation and respond by increasing stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein kinases (MAPKs [29]); a general protective pathway activated by a wide range of stimuli. However, even if the cells sense water loss, the cellular response protects cells from an increase in ionic concentration and osmotic stress, and not water loss [29]. The sugar trehalose was the first lyoprotectant; its effectiveness was shown by Crowe et al. in platelet-drying studies, following the observation that many anhydrobiotes accumulate the sugar when drying [13]. The protective effects of trehalose and the underlying mechanisms have been described in many authoritative reviews [30]. The induced expression of trehalose in cultured cells confers a partial desiccation tolerance in fibroblasts [31,32]. However, whether the cells can resume growing following rehydration is unclear. Trehalose has also been used as a cryoprotectant, for oocyte freezing [33], but it is during drying that trehalose is most helpful. In a recent study, Caenorhabditis elegans was engineered to express the sugar. Only trehalose-expressing dauer larvae survived dehydration, whereas controls did not [34]. Even in our 689
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Table 2. Anhydrobiotic organisms; adapted from [63] Anhydrobiotic organism Rotifers
Tardigrades
Nematodes
Bacteria
Fungi Seeds of several plants
Eggs of certain crustaecans
Larvae of certain insects
Large animal
Species Philodina roseola Mniobia sp., Mniobia magna, Mniobia russeola, Macrotrachela musculosa Macrobiotus hufelandi Macrobiotus dispar Ramazzottius oberhaeuseri Scottnema lindsayae Aphelenchus avenea Caenorhabditis elegans, Steinernema feltiae Tylenchus polyhypnus Anguina tritici Heterodera avenae Clostridium sporogenes Deinococcus radiodurans Nostoc commune Chroococcidiopsis Escherichia coli Pseudomonas putida Thermoplasma acidophilum Schizophyllum commune Saccharomyces cerevisiae Matricaria chamomilla Sinapis arvensis Nelumbo nucifera Gossypium hirsutum Artemia salina Artemia francescana Branchinecta packardi Streptocephalus sealii Branchinecta mackini Thamnocephalus platyurus Streptocephalus proboscideus Macrobiotus sp. Branchinecta lindahli Polypedilum vanderplanki Margarodes vitium Eburia quadrigeminata, Buprestis aurulenta Drosophila melanogaster Placobdella parasitica Margarodes vitium Eburia quodrigeminata Buprestis aurulenta Allolobophora chlorotica
first paper on somatic cell lyophilization, trehalose was a crucial element for maintaining nuclear viability [24]. However, trehalose alone does not allow somatic cells or oocytes to undergo lyophilization followed by rehydration and remain viable [24]; hence, other lyoprotectants are needed to fulfill the task. Fortunately, we can look to nature for some clues: a plethora of model organisms (Table 2) have developed elegant mechanisms for coping with water loss, and their physiologies may provide us with worthwhile lyoprotectant ideas. Table 3 lists the major lyoprotectants expressed by anhydrobiots, classified on the basis of their molecular structures and functions. Most of these exert bulk effects, establishing hydrogen bonds with structural and functional proteins and lipids, or increasing the viscosity of the cytosol, thus facilitating its transition to an amorphous, biologically inertial ‘glassy’ state. 690
Family Philodinidae Philodinidae Philodinidae Philodinidae Philodinidae Macrobiotidae Macrobiotidae Hypsibiidae Cephalobidae Aphelenchoididae Rhabditidae Steinernematidae Tylenchinae Anguinidae Heteroderidae Clostridiaceae Deinococcaleae Nostocaceae Xenococcaceae Enterobacteriaceae Pseudomonadaceae Thermoplasmataceae Schizophyllaceae Saccharomycetaceae Asteraceae Brassicaceae Nelumbonaceae Malvaceae Artemiidae Artemiidae Branchinectidae Streptocephalidae Branchinectidae Thamnocephalidae Streptocephalidea Macrobiotidae Branchinectidae Chironomidae Margarodidae Cerambycidea Buprestidae Drosophilidae Glossiphoniidae Margarodidae Cerambycidae Buprestidae Lumbricidae
Anhydrobiotic longivity n/a 9 years (eggs) 2.5 months (adult) 2.5 months 2–3 months (adult)
9 years (eggs) n/a 2.2 years n/a n/a 39 years (adult) 32 years (adult) 5.5 years 35 years till 100 years
15 yeras 35 years n/a from 50 to about 1000 years
n/a n/a 15 years 16 years 15 years 14 years 13 years 6 years 7 years 4 years 17 years n/a n/a n/a n/a n/a 17 years 40 years (larvae) 26 years n/a
The most interesting lyoprotectant candidates are the LEA proteins, normally expressed during the final maturation of seeds [35]. First, LEA proteins are expressed not only in seeds,but also in other organisms (i.e., Drosophila melanogaster and Artemisia franciscana) and surprisingly in eukaryotes, suggesting that mammalian cells may be protected by the same mechanisms, although the path to lyophilization of larger organisms is not so straightforward [36,37]. Second, LEA proteins are unfolded and randomly coiled in the presence of water but acquire functional structures in the form of a helices and b sheets, upon dehydration [38]. Third, LEA proteins target subcellular organelles such as mitochondria [37], nucleus [39], cytosol [37], and membranes [40], including the endoplasmic reticulum. The protective action of LEA proteins varies, depending on membrane binding, DNA protection, and
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Table 3. List of lyoprotectant molecules and their action Lyoprotectant molecules SUGAR
PROTEIN
Name Trehalose Sucrose Raffinose Fructans LEA
Heat shock protein (e.g., p26, p27, and p70) Anhydrin
OTHER
Extracellular polysaccharides (EPSs)
Lipids Hydroxyethyl starch (HES) Proline
Yeast extract Skimmed milk
Action Sugars confer protection of cellular and macromolecular structure by replacing water and formation of a glassy matrix in the cytoplasm. In particular, sugars can reduce lipid membranes melting temperature (Tm), inhibit fusion between membranes and stabilize them Highly hydrophilic and unstructured proteins that acquire secondary structures during desiccation. They protect cells from desiccation by - reducing aggregation of denatured proteins, - stabilization of target proteins via chaperon-like activity, - protection of cell membranes - stabilization of vitrified sugar glasses by increasing glass transition temperature (Tg) - sequestration of divalent ions - synergic interaction with other lyoprotectants, as trehalose Chaperon activity by preventing protein aggregation
Refs [57,58]
Highly hydrophilic and unstructured proteins that acquire secondary structures during desiccation. They act as - chaperon-like molecular shields by creating electrostatic and/or steric obstacle that avoid aggregation of denatured proteins in nucleus - endonuclease that might be involved in repair process of desiccation-induced DNA damage incurred during anhydrobiosis EPS can - help to maintain the integrity, due to their ability to retain water layer around the cells, - stabilize cellular membranes when in combination with nonreducing sugars, such as trehalose and sucrose Lipids, as phosphatidylinositol, contribute to protein and membrane stabilization thanks to hydroxyl groups HES inhibits fusion between membranes. It has been observed that a combination of glucose and HES can increase desiccation tolerance Proline can act as osmoprotectant amino acid. It has been suggested that proline can be involved in membrane and protein stabilization, in reduction of DNA Tm and response to oxidative stress Bulk action Bulk action
[61]
ion chelation (Table 3). Finally, a synergistic effect between trehalose and LEA proteins has been described [37]. Several recent studies have considered that LEA proteins confer desiccation tolerance in somatic cells. In one study, a cell line (human HepG2) stably expressing two LEA proteins – one localized to the cytoplasm and nucleus (AfrLEA2), and one targeted to mitochondria (AfrLEA3m) – plus a trehalose transporter, was deprived of water with using spin drying [36]. The results were encouraging, with 98% of the cells expressing LEA plus trehalose alive after rehydration. The resistance to desiccation was primarily attributable to the LEA proteins. The same group repeated the work on cells of another model organism, D. melanogaster, with similar findings [41]. The preliminary conclusion is that LEA proteins, by themselves but preferably in association with a sugar like trehalose, can contribute to a successful lyophilization medium. These papers [36,41] provide a proof of principle that LEA proteins can confer desiccation tolerance in somatic cells. However, the approach taken in these studies, involving genetic transformation of the cells, is not broadly applicable. Hence, simpler and more practical solutions are urgently needed. Further molecular studies exploring which part of the LEA proteins confers the desiccation
[41–59]
[60]
[62] [57]
tolerance, may help the development of simpler protocols. An ideal approach may involve short peptides that can be easily introduced into cells using electroporation or another membrane permeabilization technique. Alternatives to lyophilization Lyophilization is an established industrial process for foodstuff, pharmaceutical, and biological specimens preservation, involving two steps: freezing and vacuum drying. When cells or gametes are frozen, the experimentalist typically defaults to using LN. LN freezing requires a high concentration of permeable cryoprotectants. However, the most common cryoprotectants, such as glycerol or ethylene glycol, are liquid and toxic at room temperature, and thus unsuitable for lyophilization. There are two possible solutions to the problem: (i) freezing using alternative cryoprotectants, which may operate as lyoprotectants as well; or (ii) removing the freezing step and resorting to alternative drying options. Freezing using alternative cryoprotectants Some sugars, trehalose included, act as lyo- as well as cryoprotectants, given that the sample is not cooled below 308C. Using such sugars may be an option, but in that case 691
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Table 4. Description of dehydration methods Dehydratation methods Freeze drying
Sublimation
Air drying Vacum drying Convective drying
Slow evaporation Fast evaporation Fast evaporation
Drum drying Spry drying
Fast evaporation Very fast evaporation
Spin drying Microwave drying
Water removal Fast evaporation
Freezing the sample and reducing the pressure to allow that the frozen water (solid phase) sublimes in vapor water (gas phase) Drying at room temperature (23–258C) – natural evaporation Drying under reduced air pressure Drying with application of heated air. Air heating reduces relative humidity which is the driving force for drying Drying on surface of heated drum Convert the sample in a fog of droplets (100 mm in diameter) and drying very fast with heated air while falling by gravity High centrifugal force leads to rapid water removal and to formation of a thin glass layer Drying induced by electromagnetic waves
all procedures for cell and gamete freezing would have to eliminate the standard permeable cryoprotectants; a task that may be complicated but not impossible. Removing the freezing step. Anhydrobiontes do not freeze before drying, suggesting that a similar process may be possible in the laboratory. The first paper showing the successful drying of LEA proteins and trehalose-expressing cells used spin-drying instead of lyophilization to remove water from the sample [36], instead of freeze-drying. In the procedure, the cells were grown onto a round cover slide, washed with a trehalose-based spin-drying solution, and loaded onto a spinning machine. Further evaporation was induced in an environment continuously purged with dry nitrogen, leaving <0.12 g water/g dry weight in the samples, but still a high concentration of water to allow long-term dry storage. Spin drying is not the only option: Table 4 lists other dry-processing techniques that may be suitable for dry storage of cells and gametes, and that await laboratory testing. A recent study provides support for the practicality of alternative drying techniques. In this study nuclei (germinal vesicles – GVs) of immature cat oocytes [42] were isolated, suspended in a drop of trehalose, and air dried. Following 32 weeks of storage at 48C, the GVs were rehydrated and transplanted by micromanipulation into enucleated cat oocytes, where they completed meiosis [42]. How long can dry cells and gametes be stored without loss of viability? A fundamental issue is the length of time dry samples can be stored. Data on dry storage of bacteria and yeast are encouraging, with a –0.018/–0.016 slope of viability per year, and a good recovery at rehydration after 20 years of storage [43]. In eukaryotes, not much information is available, but one study showed that mouse spermatozoa retained fertilization ability after 3 years of storage at 48C [43]. In addition, lyophilized cells stored for 10 years were still able to produce viable embryos once injected into enucleated oocytes (P. Loi, unpublished). Other available data on the survival of vegetal and animal species are also encouraging. The record for longevity goes to bacteria: a halotolerant bacterium has been isolated from a 250 million-year-old salt crystal [44]. Second on the longevity list are vegetal seeds, such as date palm (Palma dactifera) seeds, which were able to germinate after 2000 years [45]. 692
Aquatic invertebrates likewise show good tolerance to desiccation, primarily at the embryonic stage, with longevity ranging from 2 years (Crustacea: Notostraca Lepidurus couesii, [46]) to 332 years in Diaptomus sanguineus (Age estimated by 210Pb sediment, [47]). The longevity of species in their natural environment is largely dependent on environmental conditions [48], and this is good news, in that we can create those conditions in vitro – atmosphere, humidity, gas composition and others – to ensure the best chances for cell and gamete survival in long-term storage. Another issue that we need to address, at least theoretically, is the maximum storage time on the light of the effects exerted by cosmic rays on macromolecular stability. Importance of developing dry processing techniques We are witnessing today a worldwide surge in biobanks, for both biomedical applications and biodiversity conservation efforts. Developments in dry processing techniques could revolutionize how biobanks are created and managed. Biomedicine Biobanking resources are used in research aimed at preventing, diagnosing, and treating major pathologies, benefitting societal health and in turn, improving our quality of life. Recently, the Biobanking and Biomolecular Resources Research Infrastructure (BBMRI) was established, bringing together more than 280 organizations from over 30 countries and highlighting the newfound importance of biobanking. BBMRI, with its 10 million frozen samples, is the largest biobank in the world (http://www.bbmri.eu/). Biobanks are an essential tool for most clinical applications, from medically assisted reproduction to the emerging field of stem cell and cell replacement therapy. Gamete biobanking is predicted to increase, as more women utilize oocyte cryopreservation to protect their fertility before cancer treatment [49], or to enable future reproductive plans [50]. Cell replacement therapy is growing exponentially and promises to trigger a revolution in clinical practice. For example, newborn umbilical cord blood cells are increasingly being stored for eventual retrieving of hematopoietic stem cells for retransplant, should the need arise later in life [51]. The availability of protocols for the lyophilization of gametes and cells would simplify storage and reduce costs, potentially making biobanking safer, less prone to pathological contamination and more affordable, thus improving health outcomes.
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Repopulaon
Freeze-dried cell banking
Cloned embryo
Freeze-dried somac cells Reconstructed oocyte
Nuclear transfer
Noah’s ark: banking endangered species TRENDS in Biotechnology
Figure 1. Dry storage as an approach for repopulation of endangered species. Schematic representation of rescue of endangered species by somatic cell nuclear transfer using freeze-dried cells.
Biodiversity conservation A progressive loss of biodiversity is occurring at an unprecedented pace. Wildlife, and even domestic animals, are disappearing and being replaced by a smaller number of more productive organisms. For this reason, the Convention on Biological Diversity, ratified thus far by 193 nations, calls for the preparation of a National Biodiversity Strategy and Action Plan (NBSAP) and for the establishment, regulation, and management of a collection of biological resources from natural habitats for ex situ conservation purposes by each signatory nation. Several non-profit organizations are also collecting and storing frozen cell lines, gametes, and embryos from threatened animals, including vertebrates and invertebrates (e.g., http://www.frozenark. org and http://www.amphibianark.org). Genetic banks are naturally deep-frozen, but as discussed previously, deep-freezing has its share of problems. Dry storage would greatly simplify the establishment and management of biobanks, especially in developing countries. Stored genomes may inform future generations of the genetic makeup of the extinct animals, but may also eventually be rehydrated and used to augment animal populations through somatic cell nuclear transfer (SCNT); an endeavor we believe is especially important [52]. SCNT in its interspecific somatic cell nuclear transfer (ISCNT) variation could potentially be a ‘magic’ tool for expanding small animal populations threatened by extinction (Figure 1). An unexplored issue: rehydration Cryobiologists are well aware of the importance of warming for the viability preservation of frozen cells. Warming needs to be carefully calibrated, otherwise it can exert
cellular injuries – essentially caused by recrystallization of water – just like improper cooling [53]. Data available from freeze-dried yeast and bacteria show that rehydration dynamics affects cell viability to the same extent that freezing and drying does [54]. Membrane destabilization and loss of intracellular homeostasis following rehydration are consequent to a liquid to gel membrane transition consequent to the drying process [55]. In the few reports concerning lyophilization of mammalian nucleated cells, rehydration has been induced by the one step addition of the aliquot of water lost during desiccation [56]; yet, the consensus view gained from yeast and bacteria desiccation is that stepwise rehydration results in good survival rates. Hence, understanding the physical/chemical changes occurring in lyophilized cells during rehydration is mandatory for improving the viability. LN carbon footprint A web-based survey suggests that there are >450 000 laboratories around the world working with cryopreserved cells and gametes. If each of these laboratories uses 200 l LN per month (a realistic estimate), the annual CO2 emission resulting from the production of LN alone is 400 000 metric tons. This rough estimate does not include other associated sources of greenhouse gases, such as transportation of the LN and the high-energy requirements of the dedicated cryostorage facilities. Dry storage would minimize the environmental costs arising from the use of LN. Concluding remarks The idea of drying cells, originally met with some skepticism, is now gaining traction due to several published 693
Review proofs of concept. These reports concern not only the successful dry storage of cells [36,41], but also of genetic material from mammalian oocytes [42]; indeed, the concept of drying eggs to counteract future fertility issues is just beginning to capture the public imagination (http:// www.bionews.org.uk/page_279587.asp). In the gathering of data required for this review, it became clear that ample room for improvement exists in the dry processing of mammalian cells and gametes. Improvements appear possible in both the formulation of the drying media, and also in the procedures for drying. Lyoprotectants expressed in anhydrobiotic organisms, or possibly their improved synthetic analogs, are likely to play a major role in the future development of dry processing. Furthermore, alternative water subtraction techniques remain largely unexplored. We are confident that the development of dry biobanks of cells and gametes, which are essentially less costly and more environmentally friendly than current methods, is within our grasp. Acknowledgments The research leading to these results received funding from the European Research Council under the European Community Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement n8 210103 to GP: PRIN 2007, n8 2007MY2M92 to GP. PL acknowledges the support of the EU FP7-KBBE -2009-3 Programme, project n8 244356, NextGene, the EU FP7-KBBE -2012-6-singlestage Programme, project n8 312097, FECUND and PRIN MIUR founding (protocol n8 2009JE3CHM), project MIUR FIRB CNR ‘‘GenHome’’. Funds from the Bank Foundation Tercas (Teramo, Italy) are also acknowledged.
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16 Watanabe, H. et al. (2009) Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes. J. Assist. Reprod. Genet. 26, 531–536 17 Hochi, S. et al. (2008) Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year. Mol. Reprod. Dev. 75, 890–894 18 Martins, C.F. et al. (2007) Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm. Theriogenology 67, 1307–1315 19 Kwon, I.K. et al. (2004) Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa. Biol. Reprod. 71, 1430–1436 20 Liu, J.L. et al. (2004) Freeze-dried sperm fertilization leads to full-term development in rabbits. Biol. Reprod. 70, 1776–1781 21 Czarny, N.A. et al. (2009) Acrosomal integrity, viability, and DNA damage of sperm from dasyurid marsupials after freezing or freeze drying. Theriogenology 1, 72 22 Gianaroli, L. et al. (2012) DNA integrity is maintained after freezedrying of human spermatozoa. Fertil. Steril. 97, 1067–10730 23 Loi, P. et al. (2002) Nuclei of nonviable ovine somatic cells develop into lambs after nuclear transplantation. Biol. Reprod. 67, 126–132 24 Loi, P. et al. (2008) Freeze-dried somatic cells direct embryonic development after nuclear transfer. PLoS ONE 3, 2978 25 Ono, T. et al. (2008) Nuclear transfer preserves the nuclear genome of freeze-dried mouse cells. J. Reprod. Dev. 54, 486–491 26 Das, Z.C. et al. (2010) Lyophilized somatic cells direct embryonic development after whole cell intracytoplasmic injection into pig oocytes. Cryobiology 61, 220–224 27 Natan, D. et al. (2009) Freeze-drying of mononuclear cells derived from umbilical cord blood followed by colony formation. PLoS ONE 4, 5240 28 Buchanan, S.S. et al. (2010) Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage. PLoS ONE 5, 12518 29 Huang, Z. and Tunnacliffe, A. (2004) Response of human cells to desiccation: comparison with hyperosmotic stress response. J. Physiol. 558, 181–191 30 Crowe, J.H. et al. (2005) Stabilization of dry mammalian cells: lessons from nature. Integr. Comp. Biol. 45, 810–820 31 Eroglu, A. et al. (2000) Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat. Biotechnol. 18, 163–167 32 Guo, N. et al. (2000) Trehalose expression confers desiccation tolerance on human cells. Nat. Biotechnol. 18, 168–171 33 Eroglu, A. (2010) Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates. Cryobiology 60 (3 Suppl.), S54–S59 34 Cihan, E. et al. (2011) Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation. Curr. Biol. 21, 1331–1336 35 Blackman, S.A. et al. (1991) Maturation proteins associated with desiccation tolerance in soybean. Plant Physiol. 96, 868–874 36 Li, S. et al. (2012) Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation. PNAS 109, 20859– 20864 37 Hand, S.C. et al. (2011) LEA proteins during water stress: not just for plants anymore. Annu. Rev. Physiol. 73, 115–134 38 Roberts, J.K. et al. (1993) Cellular concentrations and uniformity of cell-type accumulation of two lea proteins in cotton embryos. Plant Cell 5, 769–778 39 Wu, X. et al. (2013) Functional assignment to maize group 1 LEA protein EMB564 within the cell nucleus using computational analysis. Bioinformation 9, 276–280 40 Tolleter, D. et al. (2010) A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state. Biochim. Biophys. Acta 1798, 1926–1933 41 Marunde, M.R. et al. (2013) Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein. J. Insect Physiol. 59, 377–386 42 Graves-Herring, J.E. et al. (2013) Retention of structure and function of the cat germinal vesicle after air-drying and storage at suprazero temperature. Biol. Reprod. 88, 139 43 Kaneko, T. and Serikawa, T. (2012) Long-term preservation of freezedried mouse spermatozoa. Cryobiology 64, 211–214
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54 Leach, R.H. and Scott, W.J. (1959) The influence of rehydration on the viability of dried micro-organisms. J. Gen. Microbiol. 21, 295–307 55 Crowe, J.H. and Crowe, L.M. (1982) Induction of anhydrobiosis: membrane changes during drying. Cryobiology 19, 317–328 56 Garcı´a de Castro, A. and Tunnacliffe, A. (2000) Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells. FEBS Lett. 487, 199–202 57 Crowe, J.H. et al. (2010) Stabilization of cells during freeze-drying: the trehalose myth. In Life in the Frozen State (Fuller, B.J. et al., eds), pp. 581–601, CRC Press 58 Thorat, L.J. et al. (2012) Trehalose as an indicator of desiccation stress in Drosophila melanogaster larvae: a potential marker of anhydrobiosis. Biochem. Biophys. Res. Commun. 419, 638–642 59 Menze, M.A. et al. (2009) Occurrence of mitochondria-targeted ate embryogenesis abundant (LEA) gene in animals increases organelle resistance to water stress. J. Biol. Chem. 284, 10714–10719 60 Mizrahi, T. et al. (2012) Heat Shock proteins and survival strategies in congeneric land snails (Sphincterochila) from different habits. Cell Stress Chaperones 17, 523–527 61 Chakrabortee, S. et al. (2012) Catalytic and chaperone-like functions in an intrinsically disordered protein associated with desiccation tolerance. Proc. Natl. Acad. Sci. U.S.A. 109, 20859–220864 62 Gasulla, F. et al. (2013) The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach. Plant J. 75, 726–741 http://dx.doi.org/10.1111/tpj.12241 63 Watanabe, M. (2006) Anhydrobiosis in invertebrates. Appl. Entomol. Zool. 41, 15–31
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Towards storage of cells and gametes in dry form P. Loi, D. Iuso, M. Czernik, F. Zacchini, and G. Ptak Department of Comparative Biomedical Sciences, University of Teramo, Piazza Aldo Moro 45, 64100, Teramo, Italy
We review published data on cell/gamete lyophilization. Most studies have utilized the same established protocols for cryopreservation (storage in liquid nitrogen) as for cell lyophilization (dehydration of frozen samples by water sublimation). Surveying natural lyoprotectants, we suggest trehalose and late embryogenesis abundant (LEA) proteins as ideal candidates for the reversible desiccation of mammalian cells/gametes. We find that despite the numerous water subtraction techniques, scientists have relied almost exclusively on lyophilization. There is thus room for improvement in both medium formulation and water subtraction strategies for dry cell/gamete storage. We believe the development of dry processing protocols for use in biobanks of cells/ gametes, at reduced cost and with minimal carbon footprint, is within our grasp. Introduction The demand for long-term eukaryotic cell storage methods emerged in the first half of the 20th century once experimentalists began successfully exploiting spermatozoa and cells for medicine, research, and animal breeding. Initially, the options for storage were freezing, vitrification, and freeze drying (i.e., lyophilization) [1–3]. For the difficulties to maintain structural integrity in dry cells, lyophilization was partially dropped, whereas freezing was developed further; helped also by technological advances in mechanical freezer development, coupled with the availability of liquid nitrogen (LN). LN was first liquefied by two Polish physicists, Wroblewski and Olszewski in 1883 [4], and was widely used after World War II, becoming a key element in early cryobiology. The route to successful cryopreservation was far from straight and owes a great deal to serendipity. The first successful cryopreservation of spermatozoa [5] was accomplished with a bottle of sucrose – the common cryoprotectant in vogue at that time – contaminated with glycerol. The accidental discovery of glycerol and its analogs as the most powerful cryoprotectants paved the way for deep-freezing cells in LN. The science of cryobiology started in 1940 and laid down all physical/chemical Corresponding authors: Loi, P. ([email protected]); Ptak, G. ([email protected]). Keywords: lyophilization; liquid nitrogen; anydrobiosis; gametes; long-term storage. 0167-7799/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2013.09.004
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requirements enabling a cell to withstand reversibly the long-term exposure at subzero temperatures. A common feature of all cryopreservation protocols is the addition of a suitable permeable cryoprotectants, for example, glycerol or ethylene glycol, to the freezing medium. The cryoprotectant binds to intracellular water, thus reducing intracellular damage. Then the sample undergoes controlled cooling in order to induce progressive dehydration prior to being plunged into LN. The dehydration is essential, because it avoids/reduces intracellular ice injury. The search for avoiding intracellular ice formation resulted in the evolution of the freezing paradigms, which is now named vitrification. Vitrification relies on the ability of highly concentrated solutions of cryoprotectants to supercool to low temperatures, forming a glassy matrix without the formation of ice [6]. Since the first report, there has been massive investment in vitrification research, which has now become the first cryostorage choice for human oocytes and embryos [7,8]. Overall, the current freezing protocols are straightforward and efficient, with a good recovery rate at thawing [9], but they are not devoid of problems. Liquid nitrogen storage is expensive, requiring continuous monitoring of its levels and supply [9]; it is inconvenient, necessitating dedicated facilities and equipment, especially for many developing countries; it is potentially dangerous for the operator and can become contaminated by viruses or other pathogens [10]; and finally, it makes the shipping of samples difficult (Table 1). Aside from these technical and practical inconveniences, LN storage also poses an environmental concern, given that its industrial production and the maintenance of its storage centers have a high carbon footprint. Given the increasing demand of biobanks for various purposes, including medicine, research, pharmaceutical endeavors, and biodiversity preservation, there is a pressing need to develop alternative storage options. In this review we critically analyze the data published on dry preservation of mammalian cells and gametes; surveying all organisms undergoing desiccation in their life cycle; listing the naturally available lyoprotectant molecules; and finally, we speculate about their potential use for dry storage of cells and gametes. Freeze-dried spermatozoa Following the successful lyophilization of spermatozoa [5], in the 1950s, some attempts were made to lyophilize red blood cells and platelets [11,12]. Although drying red blood cells proved unsuccessful, the lyophilized platelets nevertheless preserved the morphology and function of the
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Table 1. Pros and cons of cryoconservation versus lyophilization storage Method Ease/practicality Specialized equipment Cost for long-term storage Hazard Pathogen contamination
Cryoconservation + ++++ +++++ +++++ +++++
Lyophilization +++++ +++++ + + +
cells [13]. Why were nucleated cells or cell particles such as platelets not the first targets for lyophilization trials? We speculate that there were at least two reasons; one practical and one biological. In practical terms, freeze-dried blood would simplify the storage and transport of blood banks enormously. Biologically, red blood cells and platelets are devoid of nuclei, and it is likely that early ‘lyobiologists’ feared that the nuclear structure would be incompatible with dry storage. At the end of 1998, the cell and gamete storage paradigm shifted, when Wakayama and Yanagimachi demonstrated that lyophilized mouse spermatozoa, stored at room temperature for 3 months, were able to generate normal offspring [14]. The spermatozoa were nonviable and motionless at rehydration, but the handicap of the lost motility was easily overcome by their direct injection into the oocytes. These findings demonstrated for the first time that nuclear and cellular viability are not equivalent. The lyophilized spermatozoa were essentially dead, yet were still able to interact with the programming machinery of the oocytes, giving rise to normal pups. This discovery was an important milestone; the fertilizing ability of lyophilized spermatozoa has since been confirmed in other species including horses [15], dogs [16], rats [17], cattle [18], pigs [19], rabbits [20], marsupials [21], and humans [22]. Other experiments, performed after the publication of Wakayama and Yanagimachi’s data, demonstrated that somatic cells subjected to thermal stress (558C and 758C) for 30 min, and obviously killed by the treatment, were capable of generating normal lambs upon transfer into enucleated sheep oocytes [23]. Hence, it was established that cell viability and nuclear viability are likewise not equivalent in somatic cells. Soon after, sheep somatic cells – granulosa cells and lymphocytes – lyophilized with the addition of trehalose in the freezing medium in Israel, were shipped by ordinary mail to Italy. The lyophilized cells were kept on a shelf in a cardboard box and used 5 years later for nuclear transfer. Surprisingly, the reconstructed embryos developed to the blastocyst stage [24]. Subsequent work reproduced these results in mice [25] and pigs [26]. The current list of successful nuclear transfers of lyophilized cells includes sheep, pigs, mice, and cattle (Japanese Brown Cattle; Matzukawa, personal communication). Thus, multiple research teams have shown that lyophilized cells can be successfully reprogrammed after nuclear transfer and can develop until the blastocyst stage. The next step would be to demonstrate that these cloned embryos can develop into normal offspring, and experiments to that end are currently ongoing. The above results prompted scientists to look beyond LN for the storage of cells. Two independent research
groups reported the successful dry storage of human mesenchymal stem cells and cord blood cells. In both cases, the cells maintained the capacity to form colonies under appropriated conditions in vitro [27,28]. The findings are significant in showing that dry storage of cells and gametes is a viable option. We are clearly in the early stages of dry storage of cells and gametes, and there is a large margin for improvement. For one, if we exclude the addition of trehalose, the freezing protocol we used in our original study [24], but also in those leading to successful dry storage of spermatozoa [14], is even more primitive that those used for drying yeast. The cells and spermatozoa were suspended in small aliquots (200 ml) of standard buffered medium, snap-frozen in LN, and water-extracted with a conventional lyophilizer. Yeast cells are instead lyophilized efficiently using a combination of sugars and yeast extracts, which probably exert a bulk effect analogous to the egg yolk in sperm freezing medium. None of these ingredients have ever been used for cell lyophilization. It appears likely that improvements in the composition of lyophilization media will improve the dry storage of cells and gametes overall, but there is also room for improvement in the drying procedures. Is lyophilization the only way, or are alternative options also worthy of exploration? Selection of ideal lyoprotectants The discovery of the protective action of glycerol ensured the success of deep-freezing gametes and cells [4]. Since then, other cryoprotectants have been identified and optimal cell type–cryoprotectant combinations have been established. We must do the same for dry cell and gamete storage, starting with the individuation and selection of the best lyoprotectants. This is particularly important for eukaryotic cells and gametes. Mammals are sensitive to water loss, and dehydration can be life-threatening; therefore, a coordinated physiological pathway keeps water loss to a minimum. Eukaryotic cells sense desiccation and respond by increasing stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein kinases (MAPKs [29]); a general protective pathway activated by a wide range of stimuli. However, even if the cells sense water loss, the cellular response protects cells from an increase in ionic concentration and osmotic stress, and not water loss [29]. The sugar trehalose was the first lyoprotectant; its effectiveness was shown by Crowe et al. in platelet-drying studies, following the observation that many anhydrobiotes accumulate the sugar when drying [13]. The protective effects of trehalose and the underlying mechanisms have been described in many authoritative reviews [30]. The induced expression of trehalose in cultured cells confers a partial desiccation tolerance in fibroblasts [31,32]. However, whether the cells can resume growing following rehydration is unclear. Trehalose has also been used as a cryoprotectant, for oocyte freezing [33], but it is during drying that trehalose is most helpful. In a recent study, Caenorhabditis elegans was engineered to express the sugar. Only trehalose-expressing dauer larvae survived dehydration, whereas controls did not [34]. Even in our 689
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Table 2. Anhydrobiotic organisms; adapted from [63] Anhydrobiotic organism Rotifers
Tardigrades
Nematodes
Bacteria
Fungi Seeds of several plants
Eggs of certain crustaecans
Larvae of certain insects
Large animal
Species Philodina roseola Mniobia sp., Mniobia magna, Mniobia russeola, Macrotrachela musculosa Macrobiotus hufelandi Macrobiotus dispar Ramazzottius oberhaeuseri Scottnema lindsayae Aphelenchus avenea Caenorhabditis elegans, Steinernema feltiae Tylenchus polyhypnus Anguina tritici Heterodera avenae Clostridium sporogenes Deinococcus radiodurans Nostoc commune Chroococcidiopsis Escherichia coli Pseudomonas putida Thermoplasma acidophilum Schizophyllum commune Saccharomyces cerevisiae Matricaria chamomilla Sinapis arvensis Nelumbo nucifera Gossypium hirsutum Artemia salina Artemia francescana Branchinecta packardi Streptocephalus sealii Branchinecta mackini Thamnocephalus platyurus Streptocephalus proboscideus Macrobiotus sp. Branchinecta lindahli Polypedilum vanderplanki Margarodes vitium Eburia quadrigeminata, Buprestis aurulenta Drosophila melanogaster Placobdella parasitica Margarodes vitium Eburia quodrigeminata Buprestis aurulenta Allolobophora chlorotica
first paper on somatic cell lyophilization, trehalose was a crucial element for maintaining nuclear viability [24]. However, trehalose alone does not allow somatic cells or oocytes to undergo lyophilization followed by rehydration and remain viable [24]; hence, other lyoprotectants are needed to fulfill the task. Fortunately, we can look to nature for some clues: a plethora of model organisms (Table 2) have developed elegant mechanisms for coping with water loss, and their physiologies may provide us with worthwhile lyoprotectant ideas. Table 3 lists the major lyoprotectants expressed by anhydrobiots, classified on the basis of their molecular structures and functions. Most of these exert bulk effects, establishing hydrogen bonds with structural and functional proteins and lipids, or increasing the viscosity of the cytosol, thus facilitating its transition to an amorphous, biologically inertial ‘glassy’ state. 690
Family Philodinidae Philodinidae Philodinidae Philodinidae Philodinidae Macrobiotidae Macrobiotidae Hypsibiidae Cephalobidae Aphelenchoididae Rhabditidae Steinernematidae Tylenchinae Anguinidae Heteroderidae Clostridiaceae Deinococcaleae Nostocaceae Xenococcaceae Enterobacteriaceae Pseudomonadaceae Thermoplasmataceae Schizophyllaceae Saccharomycetaceae Asteraceae Brassicaceae Nelumbonaceae Malvaceae Artemiidae Artemiidae Branchinectidae Streptocephalidae Branchinectidae Thamnocephalidae Streptocephalidea Macrobiotidae Branchinectidae Chironomidae Margarodidae Cerambycidea Buprestidae Drosophilidae Glossiphoniidae Margarodidae Cerambycidae Buprestidae Lumbricidae
Anhydrobiotic longivity n/a 9 years (eggs) 2.5 months (adult) 2.5 months 2–3 months (adult)
9 years (eggs) n/a 2.2 years n/a n/a 39 years (adult) 32 years (adult) 5.5 years 35 years till 100 years
15 yeras 35 years n/a from 50 to about 1000 years
n/a n/a 15 years 16 years 15 years 14 years 13 years 6 years 7 years 4 years 17 years n/a n/a n/a n/a n/a 17 years 40 years (larvae) 26 years n/a
The most interesting lyoprotectant candidates are the LEA proteins, normally expressed during the final maturation of seeds [35]. First, LEA proteins are expressed not only in seeds,but also in other organisms (i.e., Drosophila melanogaster and Artemisia franciscana) and surprisingly in eukaryotes, suggesting that mammalian cells may be protected by the same mechanisms, although the path to lyophilization of larger organisms is not so straightforward [36,37]. Second, LEA proteins are unfolded and randomly coiled in the presence of water but acquire functional structures in the form of a helices and b sheets, upon dehydration [38]. Third, LEA proteins target subcellular organelles such as mitochondria [37], nucleus [39], cytosol [37], and membranes [40], including the endoplasmic reticulum. The protective action of LEA proteins varies, depending on membrane binding, DNA protection, and
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Table 3. List of lyoprotectant molecules and their action Lyoprotectant molecules SUGAR
PROTEIN
Name Trehalose Sucrose Raffinose Fructans LEA
Heat shock protein (e.g., p26, p27, and p70) Anhydrin
OTHER
Extracellular polysaccharides (EPSs)
Lipids Hydroxyethyl starch (HES) Proline
Yeast extract Skimmed milk
Action Sugars confer protection of cellular and macromolecular structure by replacing water and formation of a glassy matrix in the cytoplasm. In particular, sugars can reduce lipid membranes melting temperature (Tm), inhibit fusion between membranes and stabilize them Highly hydrophilic and unstructured proteins that acquire secondary structures during desiccation. They protect cells from desiccation by - reducing aggregation of denatured proteins, - stabilization of target proteins via chaperon-like activity, - protection of cell membranes - stabilization of vitrified sugar glasses by increasing glass transition temperature (Tg) - sequestration of divalent ions - synergic interaction with other lyoprotectants, as trehalose Chaperon activity by preventing protein aggregation
Refs [57,58]
Highly hydrophilic and unstructured proteins that acquire secondary structures during desiccation. They act as - chaperon-like molecular shields by creating electrostatic and/or steric obstacle that avoid aggregation of denatured proteins in nucleus - endonuclease that might be involved in repair process of desiccation-induced DNA damage incurred during anhydrobiosis EPS can - help to maintain the integrity, due to their ability to retain water layer around the cells, - stabilize cellular membranes when in combination with nonreducing sugars, such as trehalose and sucrose Lipids, as phosphatidylinositol, contribute to protein and membrane stabilization thanks to hydroxyl groups HES inhibits fusion between membranes. It has been observed that a combination of glucose and HES can increase desiccation tolerance Proline can act as osmoprotectant amino acid. It has been suggested that proline can be involved in membrane and protein stabilization, in reduction of DNA Tm and response to oxidative stress Bulk action Bulk action
[61]
ion chelation (Table 3). Finally, a synergistic effect between trehalose and LEA proteins has been described [37]. Several recent studies have considered that LEA proteins confer desiccation tolerance in somatic cells. In one study, a cell line (human HepG2) stably expressing two LEA proteins – one localized to the cytoplasm and nucleus (AfrLEA2), and one targeted to mitochondria (AfrLEA3m) – plus a trehalose transporter, was deprived of water with using spin drying [36]. The results were encouraging, with 98% of the cells expressing LEA plus trehalose alive after rehydration. The resistance to desiccation was primarily attributable to the LEA proteins. The same group repeated the work on cells of another model organism, D. melanogaster, with similar findings [41]. The preliminary conclusion is that LEA proteins, by themselves but preferably in association with a sugar like trehalose, can contribute to a successful lyophilization medium. These papers [36,41] provide a proof of principle that LEA proteins can confer desiccation tolerance in somatic cells. However, the approach taken in these studies, involving genetic transformation of the cells, is not broadly applicable. Hence, simpler and more practical solutions are urgently needed. Further molecular studies exploring which part of the LEA proteins confers the desiccation
[41–59]
[60]
[62] [57]
tolerance, may help the development of simpler protocols. An ideal approach may involve short peptides that can be easily introduced into cells using electroporation or another membrane permeabilization technique. Alternatives to lyophilization Lyophilization is an established industrial process for foodstuff, pharmaceutical, and biological specimens preservation, involving two steps: freezing and vacuum drying. When cells or gametes are frozen, the experimentalist typically defaults to using LN. LN freezing requires a high concentration of permeable cryoprotectants. However, the most common cryoprotectants, such as glycerol or ethylene glycol, are liquid and toxic at room temperature, and thus unsuitable for lyophilization. There are two possible solutions to the problem: (i) freezing using alternative cryoprotectants, which may operate as lyoprotectants as well; or (ii) removing the freezing step and resorting to alternative drying options. Freezing using alternative cryoprotectants Some sugars, trehalose included, act as lyo- as well as cryoprotectants, given that the sample is not cooled below 308C. Using such sugars may be an option, but in that case 691
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Table 4. Description of dehydration methods Dehydratation methods Freeze drying
Sublimation
Air drying Vacum drying Convective drying
Slow evaporation Fast evaporation Fast evaporation
Drum drying Spry drying
Fast evaporation Very fast evaporation
Spin drying Microwave drying
Water removal Fast evaporation
Freezing the sample and reducing the pressure to allow that the frozen water (solid phase) sublimes in vapor water (gas phase) Drying at room temperature (23–258C) – natural evaporation Drying under reduced air pressure Drying with application of heated air. Air heating reduces relative humidity which is the driving force for drying Drying on surface of heated drum Convert the sample in a fog of droplets (100 mm in diameter) and drying very fast with heated air while falling by gravity High centrifugal force leads to rapid water removal and to formation of a thin glass layer Drying induced by electromagnetic waves
all procedures for cell and gamete freezing would have to eliminate the standard permeable cryoprotectants; a task that may be complicated but not impossible. Removing the freezing step. Anhydrobiontes do not freeze before drying, suggesting that a similar process may be possible in the laboratory. The first paper showing the successful drying of LEA proteins and trehalose-expressing cells used spin-drying instead of lyophilization to remove water from the sample [36], instead of freeze-drying. In the procedure, the cells were grown onto a round cover slide, washed with a trehalose-based spin-drying solution, and loaded onto a spinning machine. Further evaporation was induced in an environment continuously purged with dry nitrogen, leaving <0.12 g water/g dry weight in the samples, but still a high concentration of water to allow long-term dry storage. Spin drying is not the only option: Table 4 lists other dry-processing techniques that may be suitable for dry storage of cells and gametes, and that await laboratory testing. A recent study provides support for the practicality of alternative drying techniques. In this study nuclei (germinal vesicles – GVs) of immature cat oocytes [42] were isolated, suspended in a drop of trehalose, and air dried. Following 32 weeks of storage at 48C, the GVs were rehydrated and transplanted by micromanipulation into enucleated cat oocytes, where they completed meiosis [42]. How long can dry cells and gametes be stored without loss of viability? A fundamental issue is the length of time dry samples can be stored. Data on dry storage of bacteria and yeast are encouraging, with a –0.018/–0.016 slope of viability per year, and a good recovery at rehydration after 20 years of storage [43]. In eukaryotes, not much information is available, but one study showed that mouse spermatozoa retained fertilization ability after 3 years of storage at 48C [43]. In addition, lyophilized cells stored for 10 years were still able to produce viable embryos once injected into enucleated oocytes (P. Loi, unpublished). Other available data on the survival of vegetal and animal species are also encouraging. The record for longevity goes to bacteria: a halotolerant bacterium has been isolated from a 250 million-year-old salt crystal [44]. Second on the longevity list are vegetal seeds, such as date palm (Palma dactifera) seeds, which were able to germinate after 2000 years [45]. 692
Aquatic invertebrates likewise show good tolerance to desiccation, primarily at the embryonic stage, with longevity ranging from 2 years (Crustacea: Notostraca Lepidurus couesii, [46]) to 332 years in Diaptomus sanguineus (Age estimated by 210Pb sediment, [47]). The longevity of species in their natural environment is largely dependent on environmental conditions [48], and this is good news, in that we can create those conditions in vitro – atmosphere, humidity, gas composition and others – to ensure the best chances for cell and gamete survival in long-term storage. Another issue that we need to address, at least theoretically, is the maximum storage time on the light of the effects exerted by cosmic rays on macromolecular stability. Importance of developing dry processing techniques We are witnessing today a worldwide surge in biobanks, for both biomedical applications and biodiversity conservation efforts. Developments in dry processing techniques could revolutionize how biobanks are created and managed. Biomedicine Biobanking resources are used in research aimed at preventing, diagnosing, and treating major pathologies, benefitting societal health and in turn, improving our quality of life. Recently, the Biobanking and Biomolecular Resources Research Infrastructure (BBMRI) was established, bringing together more than 280 organizations from over 30 countries and highlighting the newfound importance of biobanking. BBMRI, with its 10 million frozen samples, is the largest biobank in the world (http://www.bbmri.eu/). Biobanks are an essential tool for most clinical applications, from medically assisted reproduction to the emerging field of stem cell and cell replacement therapy. Gamete biobanking is predicted to increase, as more women utilize oocyte cryopreservation to protect their fertility before cancer treatment [49], or to enable future reproductive plans [50]. Cell replacement therapy is growing exponentially and promises to trigger a revolution in clinical practice. For example, newborn umbilical cord blood cells are increasingly being stored for eventual retrieving of hematopoietic stem cells for retransplant, should the need arise later in life [51]. The availability of protocols for the lyophilization of gametes and cells would simplify storage and reduce costs, potentially making biobanking safer, less prone to pathological contamination and more affordable, thus improving health outcomes.
Review
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Repopulaon
Freeze-dried cell banking
Cloned embryo
Freeze-dried somac cells Reconstructed oocyte
Nuclear transfer
Noah’s ark: banking endangered species TRENDS in Biotechnology
Figure 1. Dry storage as an approach for repopulation of endangered species. Schematic representation of rescue of endangered species by somatic cell nuclear transfer using freeze-dried cells.
Biodiversity conservation A progressive loss of biodiversity is occurring at an unprecedented pace. Wildlife, and even domestic animals, are disappearing and being replaced by a smaller number of more productive organisms. For this reason, the Convention on Biological Diversity, ratified thus far by 193 nations, calls for the preparation of a National Biodiversity Strategy and Action Plan (NBSAP) and for the establishment, regulation, and management of a collection of biological resources from natural habitats for ex situ conservation purposes by each signatory nation. Several non-profit organizations are also collecting and storing frozen cell lines, gametes, and embryos from threatened animals, including vertebrates and invertebrates (e.g., http://www.frozenark. org and http://www.amphibianark.org). Genetic banks are naturally deep-frozen, but as discussed previously, deep-freezing has its share of problems. Dry storage would greatly simplify the establishment and management of biobanks, especially in developing countries. Stored genomes may inform future generations of the genetic makeup of the extinct animals, but may also eventually be rehydrated and used to augment animal populations through somatic cell nuclear transfer (SCNT); an endeavor we believe is especially important [52]. SCNT in its interspecific somatic cell nuclear transfer (ISCNT) variation could potentially be a ‘magic’ tool for expanding small animal populations threatened by extinction (Figure 1). An unexplored issue: rehydration Cryobiologists are well aware of the importance of warming for the viability preservation of frozen cells. Warming needs to be carefully calibrated, otherwise it can exert
cellular injuries – essentially caused by recrystallization of water – just like improper cooling [53]. Data available from freeze-dried yeast and bacteria show that rehydration dynamics affects cell viability to the same extent that freezing and drying does [54]. Membrane destabilization and loss of intracellular homeostasis following rehydration are consequent to a liquid to gel membrane transition consequent to the drying process [55]. In the few reports concerning lyophilization of mammalian nucleated cells, rehydration has been induced by the one step addition of the aliquot of water lost during desiccation [56]; yet, the consensus view gained from yeast and bacteria desiccation is that stepwise rehydration results in good survival rates. Hence, understanding the physical/chemical changes occurring in lyophilized cells during rehydration is mandatory for improving the viability. LN carbon footprint A web-based survey suggests that there are >450 000 laboratories around the world working with cryopreserved cells and gametes. If each of these laboratories uses 200 l LN per month (a realistic estimate), the annual CO2 emission resulting from the production of LN alone is 400 000 metric tons. This rough estimate does not include other associated sources of greenhouse gases, such as transportation of the LN and the high-energy requirements of the dedicated cryostorage facilities. Dry storage would minimize the environmental costs arising from the use of LN. Concluding remarks The idea of drying cells, originally met with some skepticism, is now gaining traction due to several published 693
Review proofs of concept. These reports concern not only the successful dry storage of cells [36,41], but also of genetic material from mammalian oocytes [42]; indeed, the concept of drying eggs to counteract future fertility issues is just beginning to capture the public imagination (http:// www.bionews.org.uk/page_279587.asp). In the gathering of data required for this review, it became clear that ample room for improvement exists in the dry processing of mammalian cells and gametes. Improvements appear possible in both the formulation of the drying media, and also in the procedures for drying. Lyoprotectants expressed in anhydrobiotic organisms, or possibly their improved synthetic analogs, are likely to play a major role in the future development of dry processing. Furthermore, alternative water subtraction techniques remain largely unexplored. We are confident that the development of dry biobanks of cells and gametes, which are essentially less costly and more environmentally friendly than current methods, is within our grasp. Acknowledgments The research leading to these results received funding from the European Research Council under the European Community Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement n8 210103 to GP: PRIN 2007, n8 2007MY2M92 to GP. PL acknowledges the support of the EU FP7-KBBE -2009-3 Programme, project n8 244356, NextGene, the EU FP7-KBBE -2012-6-singlestage Programme, project n8 312097, FECUND and PRIN MIUR founding (protocol n8 2009JE3CHM), project MIUR FIRB CNR ‘‘GenHome’’. Funds from the Bank Foundation Tercas (Teramo, Italy) are also acknowledged.
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