Abstract 1

Intestinal dysbiosis is one of the complications of coronavirus infection COVID-19. Factors, changing the intestinal microbiome quality and quantity in coronavirus patients are the disorders of immune system and antibiotic therapy. Probiotics, symbiotics and products of therapeutic and prophylactic nutrition are used to prevent and treat an intestinal dysbiosis. These drugs comprise living cells of microorganism probiotic strains. The most effective forms of probiotic drugs are microorganisms immobilized in gel carriers. Technologies of their long-term storage are under development. The research aim was to study the effect of disaccharides and prebiotics adding to the gel on viability of immobilized probiotics after storage at different low temperatures. Probiotic cells immobilized in gel granules (E. coli M-17, L. acidophilus, S. cerevisiae, B. bifidum, L. bulgaricus) were stored for 24 months at -20, -40, -80, -196°С. For immobilization we used 1% alginate gel and 1% alginate gel with adding 10% disaccharides (lactose, sucrose, trehalose), 10% prebiotic (inulin and fructooligosaccharides), and 5% (v / v) skim milk. The number of viable cells during storage was established to be influenced by species characteristics of structure, cell physiology, gel carrier composition, temperature and shelf life. No death of microbial cells during storage at -196°C was found. At -20, -40, -75°С microbial cells died during the entire shelf life. Number of dead cells was reduced with temperature decrease. In gel samples with adding disaccharides and prebiotics, the number of viable cells was higher versus 1% alginate gel. Thus, disaccharides or prebiotics should be added to the gel for providing a low temperature long-term storage of gel-immobilized probiotic strains of microorganisms.

Contact: embiotech@icloud.com

Abstract 2

Today, cryobiological technologies are increasingly used in modern reproductive medicine to preserve male gametes. In addition to chromosomal and gene mutations, DNA fragmentation plays a significant role in a problem of infertility. The aim of the study was to compare DNA fragmentation in cryopreserved fragments of seminiferous tubules of testes (FSTT) in various states of spermatogenesis. The study was carried out on FSTT of immature (group 1) and mature (group 2) rats (n=20). Cryopreservation was performed in fibrin gel+6% glycerol in nitrogen vapor to -70°C for 40 min, followed by transfer to liquid nitrogen (-196°C). Heating was performed in a water bath (40°C) with pre-incubation in nitrogen vapor. DNA fragmentation was evaluated by TUNEL staining (BioVision, USA). Apoptotic/necrotic processes were studied using Annexin-V-FITC (Annexin V) (BD, USA) and 7-Amino-Actinomycin D (7AAD) (BD, USA) dyes. Both tests were performed immediately before and after freezing-thawing. Statistical processing was done in Statistica 8 program (StatSoft, USA).DNA fragmentation in groups 1 and 2 after freezing-thawing was respectively 2.2- and 2.8-fold increased compared to the native samples. More cells with DNA fragmentation were observed closer to tubule lumens, and not in the basal layer of spermatogenic epithelium in all studied groups, indicating the survival of spermatogonial stem cell pool. The number of viable cells (AnnexinV-/7AAD-) was 1.7-fold decreased in cryopreserved samples of group 2 compare with group 1. The majority of cells in FSTT group 2 after cryopreservation were characterized by nuclear DNA fragmentation, which is inherent in cells at the stages of necrosis and late apoptosis (Annexin V+/7AAD++Annexin V-/7AAD+). That is, the main cell loss in FSTT of this group occurred as a result of their rapid death. Thus, the obtained data show that FSTT of prepubertal rats are more resistant to the effects of low-temperature preservation factors than samples of adult animals.

Contact: volkovana781@gmail.com

Abstract 3

Placenta, fetal membranes and umbilical cord are the promising sources of stem cells. The abundance, the absence of ethical controversy and the absence of donor injuries are among the major advantages of these tissues. The extracts, conditioned media and lysates of these tissues exhibit promising biological activity for potential application in the field of regenerative medicine. Among these forms, application of cryoextracts requires a comprehensive study of characteristic biological effects. The objective of this research was to study the effects of placental, umbilical cord and fetal membranes cryoextracts on a range of cell cultures of different origin. The impact of human placental, umbilical cord, chorionic and amniotic membranes cryoextracts on the metabolic activity of rat fibroblasts, splenocytes, hepatocytes, nerve cells and bone marrow cell cultures was assessed with the MTT assay. The extracts were added to the culture media in a ratio 1 to 9 for 24 hours. The influence of extracts on regeneration was studied with the application of scratch assay on rat fibroblasts cell culture. The anti-inflammatory effect was evaluated by assessing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation in rat leukocytes by flow cytometry. Placental cryoextracts were shown to increase the metabolic activity of the studied cell cultures. The umbilical cord cryoextract enhanced the metabolic activity of fibroblasts, nerve cells, splenocytes, and bone marrow cells. Chorionic extract stimulated the metabolic activity of fibroblasts. Amniotic cryoextract promoted the metabolic activity of fibroblasts, splenocytes, bone marrow cells, reduced the LPS-induced ROS generation in peripheral blood leukocytes. In this study, all the extracts accelerated the recovery of the fibroblast monolayer, evidenced by the scratch assay. Therefore, сryoextracts of placental derivatives improve regeneration and affect the metabolic activity of different cell cultures, opening high perspectives for application in regenerative medicine.

Contact: v.yu.prokopiuk@gmail.com

Abstract 4

This research was aimed at reducing the negative effect of cryopreservation factors due to recrystallization processes. The Research objects were the combinations of solutions: Ham's F12, 10% FBS, 1% DMSO, NDC at final concentrations of 0.02 and 1 g / L with a 2 nm particle size. The phase states of frozen samples were studied by thermoplastic deformation (TPD). Cooling was carried out at a rate of 20 deg / min, heating - at a rate of 1 deg / min. Deforming stress - 0.66 • 105 kg / m3 or 6 • 105 kg / m3, depending on the investigated temperature range. When 10% FBS and 0.02 g / L, nanocrystalline cerium dioxide (NDC) were added to Ham's F12 the TPD curve shoed melting of eutectic compositions and a less pronounced recrystallization interval before melting of the bulk of ice. With an increase in the NDC concentration in the medium with serum to 1 g / L, there was no temperature range corresponding to the recrystallization rearrangements in the sample in the TPD curve, and just melting of eutectic compositions was observed. In the specimens containing 1% DMSO + Hams F12 + 10% FBS without and with adding NDC at final concentrations of 0.02 and 1 g / L, the devitrification proceeded by 10 ° C higher than in DMSO aqueous solution (1%). In the study of recrystallization before the melting of the bulk of ice in these protective media, the already described tendency persisted. But in these cryoprotective media there was no recrystallization occurring in DMSO aqueous solutions before the DMSO eutectic melting. The findings can be used to develop protocols for cell cryopreservation.

Contact: helen.bobrova.77@gmail.com

Abstract 5

Increased lipid accumulation in oocytes is a common phenomenon in obesity. It is not known whether elevated lipid accumulation has any detrimental effects on the cryosurvival of oocytes and embryos under such conditions. The present study was designed to understand the association between maternal obesity, ER (endoplasmic reticular) stress and cryotolerance in oocytes and embryos. For the study 3 weeks old female mice were fed with high fat diet (HFD) for 8 weeks. The oocytes from HFD mice had significantly higher lipid accumulation. When these oocytes were vitrified, significant increase in the expressions of GPR78 (p<0.05), data-preserve-html-node="true" ATF4 (p<0.001) data-preserve-html-node="true" and ATF6 (p<0.001) data-preserve-html-node="true" in the frozen thawed oocytes in HFD group compared to the control group oocytes indicating ER stress response was elevated in oocytes form HFD group but the freeze-thaw process did not seem to increase the ER stress. Further, when frozen- thawed 6-8 cell stage embryos showed better cryotolerance to vitrification from HFD group compared to control group. However, expressions of GPR78 (p<0.001) data-preserve-html-node="true" and ATF4 (p<0.001) data-preserve-html-node="true" was increased in the frozen thawed oocytes in HFD group compared to the control group embryos. Further when these vitrified thawed embryos were cultured up to blastocyst stage, embryos had reduced total cell number and higher DNA damage in the HFD group compared to control group. Significant upregulation in AQP3 (P<0.05) data-preserve-html-node="true" expression was observed in the blastocysts derived from obese mice after cryopreservation which may facilitate increased membrane transportation of water and cryoprotectants helping in quick regaining of volume. Overall, the results of the present study indicate that maternal obesity does not have any significant adverse effects on the cryotolerance of oocytes and embryos. However, the quality of blastocysts derived from the obese group is poor following vitrification-thawing process.

References: Bellver J, et al. Obesity and assisted reproductive technology outcomes. Reprod Biomed Online, 2006;12:562–568; Ma W, et al. Maternal obesity in mice not only affects fresh embryo quality but also aggravates injury due to vitrification. J Assist Reprod Genet, 2016; 33:1515–1523; Ma W, et al. Obesity does not aggravate vitrification injury in mouse embryos: a prospective study. Reprod Biol Endocrinol, 2012;10:68; Rao A, et al. High-fat diet leads to elevated lipid accumulation and endoplasmic reticulum stress in oocytes, causing poor embryo development. Reprod Fertil Develop, 2020;32:1169–1179.

Contact: sandhya.patil@manipal.edu

Abstract 6

The unicellular alga Dunaliella salina is unique in its ability to adapt to extreme environmental conditions and β-carotene synthesis. Although D. salina can accumulate large amounts of intracellular glycerol, we did not observe viable samples after cryopreservation with no use of cryoprotectants. Vitrification are based on the cell dehydration by highly concentrated cryoprotective solutions followed by their immersion into liquid nitrogen. The use of plant vitrification solutions (PVS) for cryopreservation of microalgae has been insufficiently studied. There are only a few publications on this issue.Therefore, the aim of the work was to study the effect of the 50% modified PVS1 (22% glycerol, 13% 1,2-propylene glycol, 13% ethylene glycol, 6% Me2SO4, 0,4M sucrose), PVS2, PVSN (15% glycerol, 15% ethylene glycol, 34% sucrose) and 44% PVS3 on halophile unicellular microalgae D. salina. At the first stage of the work, we evaluated the effect of individual components that made up the PVS on the motility, integrity and osmotic reactions of the microalgae D. salina with depending on concentration (1-40%) and exposure time (5-30 min). It was shown that D. salina cells were osmotollerants to the action of the test cryoprotectants during 30 minutes of exposure. The cells also retained their motility and integrity throughout the experiment. Exposure of D. salina cells in PVS showed that the cells did not lose their viability and motility. We noticed no significant changes in the relative cell surface area after 30-minute exposure. Based on the obtained data on high osmotolerance of D. salina cells, the investigated cryoprotectant solutions can be used for cryopreservation of D. salina cells by vitrification. As a result, the probability of intracellular ice formation and damage may decrease.

Contact: nadiiachernobai@gmail.com

Abstract 7

Gametes, embryos and larvae of marine invertebrates have been cryopreserved with different levels of success through the years. Although oocyte cryopreservation is still under research as no successful protocol has been developed yet (Campos et al. 2021). Nowadays, vitrification coupled with ultra-rapid laser warming is another alternative approach to cryopreserve different type of animal cells. However, vitrification initially needs the addition of high concentrations of cryoprotecting agents (CPAs), but in the case of ultra-fast laser warming dehydration of the cells may allow to reduce the concentration of CPAs needed but a heat conductive substance, carbon black (India Ink) need to be into the solution (Jin and Mazur 2015), which dissolves poorly on sea water and forms lumps. The aim of this work is to try and solve these two problems by using sea water with low salinity in combination with different CPAs. By using low salinity water: the cells dehydrate so lower concentration of CPAs is needed (less toxicity) and also, India Ink dissolution may improve. Sea urchin Paracentrotus lividus eggs were used to see the effects of salinity in combination with different CPAs on their viability. Eggs were kept in sea water (SW) with different salinities (45%, 60%, and 100%) for 2 and 5 minutes. We also add two different CPAs (Dimethyl sulfoxide (Me2SO) and Ethylene glycol (EG)) using concentrations of 0.5M, 1M, 1.5M and 2M. Results have shown that exposure to concentrations of CPAs higher than 1M with 45%, 60% and 100% salinity during 2 and 5 minutes had a negative effect on eggs viability, as barely any development was achieved. This study showed that salinity can be lowered until 45% and add 1M of CPA without affecting viability, thus maybe solving the problems associated with vitrification coupled with ultra-rapid laser warming.

References: Campos S, et al. Major challenges in cryopreservation of sea urchin eggs. Cryobiology, 2021;98:1-4; Jin B & Mazur P. High survival of mouse oocytes/embryos after vitrification without permeating cryoprotectants followed by ultra-rapid warming with an IR laser pulse. Sci Rep, 2015;5:1-6.

Contact: sara.campos.rosende@uvigo.es

Abstract 8

Prolongation of liver simple cold storage (SCS) term is actual trend of cryobiology and transplantology. Enrichment of preservation solution with growth factor and cytokine cocktail is promising way to strengthen organ resistance under hypoxia/re-oxygenation. Earlier we showed that animal pretreatment or supplementation of preservation solution with trophic factors of fetal origin strongly reduced liver damage after SCS and warm reperfusion (WR). The aim of this work – to study the effects of mesenchymal stem cell conditioned media (MSC-CM) added to preservation solution on liver state after CS and WR.Conditioned media were obtained after one day of serum-free human dermal MSC culturing (5-6 passages), concentrated and standardized by protein content. Rat livers were stored in sucrose-based solution (SBS) for 24 h at 4°С without or with bioregulators, and then reperfused during 60 min at 37°С. Screening by ELISA showed presence in conditioned media of HGF, EGF, VEGF, PDGF-BB, NGF-β, SCF, FGF-β, and trace quantity of TNF-α. After storage and following WR, oxidative stress rise, uncoupling of oxidative phosphorylation, ATP level and bile production fall, massive liver cell death, organ architecture disarrangement, and increase of transaminase perfusate activity were observed in SBS-group. Addition of MSC-CM led to decrease of liver TBARS level after WR 2.2 times as compared with SBS-group. Bioregulators prevented mitochondria dysfunction resulting in respiratory control index maintenance almost at level of freshly isolated organs and promoting partial ATP content recovery, which was higher 1.7 times than in SBS-group. Bile flow was 40% faster; activity of perfusate ALT and AST was lower 3 and 2 times respectively. Liver morphological structure was similar to intact organs and a large number of binuclear hepatocytes was found. The pronounced protective effect of MSC-CM on livers during SCS indicates the prospects of their application as compounds of preservation solutions.

Contact: daria_cherkashina@ukr.net

Abstract 9

The aim of research was to study the effect of three-dimensional microenvironment on the survival of rat fetuses neural stem/progenitor cells (NSPCs) after cryopreservation.Neural cells were isolated from brain tissues of rats fetuses (15-16 days of gestation). Cell viability was 15-70%. The cells were cultured in DMEM/F12 medium (1-4×106 cells/ml) in the presence of serum. The cells samples were frozen under the protection of 10% DMSO in the presence of serum at the rate of 1 °C/min to -80 °C, and then the samples were transferred into liquid nitrogen. DMSO was removed from the thawed samples by washing using centrifugation. During cultivation the viable NSPCs formed multicellular aggregates. After the attachment of aggregates, the cells migrated, spread out and formed a monolayer. With further cultivation, β-tubulin 3-positive cells with neuroblast morphology and colonies of undifferentiated nestin/vimentin-positive cells appeared on the monolayer. Loose aggregates up to 30 µm in size did not attach and disintegrated during further cultivation. Under conditions preventing attachment, the aggregates to form spheroids that increased in size during further cultivation, which indicates NSPCs proliferation. In contrast to cryopreservation of a cell suspension, cells aggregates didn’t significantly depend on the initial cell viability. Cryopreservation of aggregates both with high (50-70%) and low (15-30%) viability was the same in its effectiveness. The viability of NSPCs in aggregates after cryopreservation didn’t depend on the presence of serum. In turn, serum was an important component for effective cryopreservation of a cell suspension. Cryopreserved aggregates under cultivation conditions behaved similarly to the initial ones. An essential condition for the survival of NSPCs in cryopreserved aggregates was the retention of their morphological integrity. The findings show the formation in the aggregates of conditions conducive to the recovery, survival and effective functioning of rat fetuses NSPCs after their isolation and cryopreservation.

Contact: alexsukach587@gmail.com

Abstract 10

The elucidation of mechanisms of cell injuries caused by a cold stress (CS) resulting from a drop in the ambient temperature from 0°С to +2°С followed by a return to 37°C (normothermia), is an urgent objective in current cryobiology. The search for compounds that can prevent CS-attributed apoptosis and necrosis is of immediate interest. In this regard, the use of neuropeptides, in particular synthetic leu-enkephalin (dalargin), as protectors is promising. The in vitro study was performed on L929 mouse fibroblast cell line. The impact of CS and dalargin was evaluated by morphological parameters, distortion of cell membrane asymmetry and release of cytochrome C into cytoplasm. To assess the proliferative potential of fibroblasts, mechanical damage to the monolayer was modeled as a scratch wound. In vivo study was performed in rats under CS and subsequent return to normothermia. The effect of CS and dalargin on potassium ions content in the serum (ionometry method) were evaluated.The study showed that CS induced the apoptosis in L929 fibroblasts and reduced proliferation in the fibroblast monolayers after mechanical damage. Dalargin was demonstrated to exert a protective effect on proliferation and against apoptosis during CS. Using opioid receptor antagonist naloxone, we revealed that the protective mechanism of dalargin appeared to be due to activation of δ-opioid receptors of L929 fibroblasts, which affected the development of apoptosis. In vivo showed that CS significantly increased in the potassium ions content in the serum and its recovery after return of animals to normothermia. The injections of dalargin to rats before CS led to a decrease in the content of potassium ions in the serum. The protective effect of dalargin can be mediated by regulating the concentration of stress hormones in the blood and reducing the intensity of free radical oxidation which ensures the body's adaptation to the CS.

Contact: profgulevskyy@gmail.com

Abstract 11

The heparin-binding epidermal growth factor (HB-EGF) reaches its expression peak 5-8 days after the ovulation in uterus epithelial cells and plays an important role in regulating blastocyst implantation. Due to the antiapoptotic effect of pituitary adenylate cyclase activating polypeptide (PACAP) and its widespread presence in the organ system, PACAP is considered as a general cytoprotective peptide. The peptide was found in the gonads in high levels, that is what drew attention to the peptide might play a central role in reproduction. The aim of our study is to assess the application possibilities of PACAP treatment during embryo vitrification to the development and the HB-EGF gene expression. BDF1 female mice were superovulated (7.5 IU eCG i.p., followed by 7.5 IU hCG i.p. 48 hours later) and paired with males for a night. The zygotes were collected on the subsequent morning, then they were cultured in G1 medium for 96 hours. After that we examined the developmental stage and vitrificated the blastocyst stage embryos. We treated the embryos with PACAP in two different doses during the vitrification (group 1 and group 2) and after thawing, in the culture medium (group 3 and group 4). After 24 hours culturing, we recorded survival rate and determined the HB-EGF gene expression by qPCR. Our results showed a higher rate of survival and higher level of HB-EGF gene expression in the group of higher concentration of PACAP-treatment during vitrification compared to both the vitrified and non-vitrified control groups, indicating that PACAP treatment during vitrification has a beneficial effect on embryo survival and HB-EGF gene expression, thus on probability of implantation in dose-dependent manner.

Acknowledgements: Project no. 134887 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the FK_20 funding scheme.

Contact: torok.dora@univet.hu

Abstract 12

In the field of reproductive science, there has been an increased interest in the application of ovarian preantral follicles as an alternative option for improved reproductive efficiency of domestic animals with poor superovulatory responses. In human medicine, improving follicle culture in vitro of the cryopreserved preantral follicles could be of high relevance to fertility preservation in cancer patients undergoing chemotherapy. The conceptual method for the preantral follicles is to be isolated from the ovaries and vitrified/thawed, then cultured in vitro, and fertilized the ovulated oocyte in vitro. The aim of our research was to examine the viability of preantral follicles after the vitrification process while using different cryoprotectants and holders. The preantral follicles with the diameter of 80-120 μm were mechanically dissected from ovaries of 8 to 12-week-old BDF1 mice. Experiment 1: A selection of the acquired preantral follicles was vitrified, with mouse embryo cryopreservation solution. Before the vitrification procedure, preantral follicles were incubated for 30 minutes in vitrification medium (1-control) or vitrification medium supplemented with (2) retinol or (3) cytochalasin B. Experiment 2: Preantral follicles were vitrified in OPS applying embryo freezing protocol. Live/dead fluorescence viability assay based on metabolic processes, was used to determine the survival rate of the follicles. Retinol has shown a beneficial effect during vitrification process, since significant increase in the survival rate of the follicles in the retinol-treated group was found compared with cytochalasin B. OPS vitrification supports high viability of vitrified/thawed preantral follicles and can be used appropriately for preantral follicle freezing.

Acknowledgements: Project no. 134887 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the FK_20 funding scheme.

Contact: bordas.lilla@univet.hu

Abstract 13

The low effectiveness of existing antiviral drugs against the SARS-CoV2 virus necessitates the search for a new strategy to prevent the development and treatment of this infection, using cryotechnologies. The research aim was to study the immunobiological activity of cryopreserved human cord blood leukoconcentrate (cHCBL) and its components during preventive intranasal administration. Human cord blood leukoconcentrate was obtained from healthy women and cryopreserved in autologous plasma according to a two-stage program. The samples were heated at a temperature of 40°C. Plasma and nucleated cells (NCs) were isolated from cHCBL after thawing and centrifugation. Balb / C mice were divided into 6 groups. Six months before infection with influenza virus strain A / Victoria mice of groups 1-3 were intranasally administered with cHCBL, plasma and NCs respectively, 0.05 ml; group 4 comprised the animals with influenza virus at a dose of LD25; group 5 did Laferobion (14 ME 5 times a day for 3 days); group 6 was 0.9% NaCl (0.05 ml). Indices of intact animals served as controls. At 6 months, mice of all groups were infected with influenza virus at a dose of LD100 and the survival of animals was assessed for 14 days. Phagocytic activity was determined in alveolar macrophages; the number of CD11b+ cells was examined by flow cytometry. Intranasal administration of cHCBL to healthy animals was shown to significantly increase the phagocytic activity of alveolar macrophages. This effect was kept for six months, ensuring 86% survival of mice with introduced cHCBL after infection with virus and compared with 72% in those introduced plasma and 43% when using NCs, emphasizing the amplifying effect of each of cHCBL components. Successful experience of prevention of respiratory viral infection with cryopreserved cord blood product allows to predict its possible use in COVID-19.

Contact: cryo.ua@gmail.com

Abstract 14

The ability of spermatozoa to fertilize an oocyte and, consequently, form a normal embryo depends on its motility, viability, and DNA integrity. Cryopreserved spermatozoa are effectively used in assisted reproductive technologies of goats on farms. However, many factors during cryopreservation such as centrifugation, equilibration with cryoprotectant, freezing and thawing may negatively affect sperm functions. This might result in reduced motility, viability, and DNA integrity. The aim of this study was to evaluate motility, viability, and DNA fragmentation of goat spermatozoa after cryopreservation in breeding and non-breeding season. Ejaculates from 5 sexually matured goats were obtained during breeding (September-December) and non-breading season (January-March). Immediately after collection total concentration of spermatozoa, motility, viability, and DNA fragmentation were calculated. Following seminal plasma removal each ejaculate was diluted with HEPES based media supplemented with 10% glycerol and 20% egg yolk. Extended semen was equilibrated 15 min at 25℃ and loaded into 0.25 mL straws (Minitube, Germany). Then samples were equilibrated 2.5 h at 5℃, placed horizontally 4 cm above liquid nitrogen for 15 min and plunged into the liquid nitrogen. Thawing was performed on a water bath at 37℃ for 30 sec. Sperm motility, viability and DNA fragmentation were evaluated after thawing and removing cryoprotectant. Investigated sperm parameters were significantly (p≤0.05) higher in breeding season than in non-breeding season. Cryopreservation caused a decrease of the sperm motility and viability and an increase of DNA fragmentation rate both in breeding and non-breeding season. However, the difference in motility and viability of cryopreserved sperm in breeding season was not significant. DNA fragmentation of cryopreserved spermatozoa in non-breeding season was 2,5 times higher than in breeding season. In conclusion, motility and viability of goat spermatozoa might be better cryopreserved in breeding season, however, DNA integrity is negatively affected by cryopreservation both in breeding and non-breeding season.

Contact: baa1995ua@gmail.com

Abstract 15

There is an increasing interest in extending the germplasm storage capabilities to a wider and more diverse cohort of plants, both wild and cultivated, with agricultural, biotechnological or diversity preservation aims. Among the candidates for preservation are some seeds characterized by both having a significant oil content and suffering unexpected storage aging. When seeds are stored at water contents and temperature conditions under the glass transition temperature of their aqueous solutions, the lack of mobility of water molecules is guaranteeing an absolute halt to any diffusion-driven chemical reaction, while at the same time, ice crystals (if under the freezing point) are not developed. These factors usually ensure indefinite preservation. Some fat-rich seeds, however, show unexplained aging and viability and germination decreases when stored in those conditions. To gain some information on the causes of this storage-aging, oil-rich seeds (peanut: Arachis hypogeae and papaya: Carica papaya) were stored at a range of sub-cero temperatures, after equilibration at different relative humidities (RH). Their temporal evolution was studied by cryo-SEM (low-temperature scanning electron microscopy), a technique that is able to differentiate between cells able to form ice crystals and vitrified ones. Seeds were transferred from storage conditions to the microscope stage quenched in liquid nitrogen, where they were fractured in situ and observed, after etching and metal-coating. The micrographs show volume and tissue structural changes (lipid phase reorganization, alterations of cell wall unions) that can be possible related to lipid changes during storage. This lipid evolution could be related to the aging behaviour observed, either through mechanical effects derived from crystallinity changes or via the release of fat bound water that could be an unexpected ice crystals source.

Contact: antoniom@ictan.csic.es

Abstract 16

Cell culture is a critical platform for research and industrial processes. However, methods for transporting cells are largely limited to cryopreservation, which is logistically challenging, expensive, and can result in poor cell recovery. Transporting cells at ambient temperatures would alleviate these issues. We have developed CellShip®, a novel, xeno free transportation medium for mammalian cells. Commercially relevant cell lines, (HEK293, CHO, HepG2, K562, Jurkat, A549 and HeLa) were successfully shipped/stored for 72h-120h at ambient temperature, after which, cells were recovered into standard culture conditions. Viability (%) and cell numbers, were examined, before, following the transport/storage period and following the recovery period. In all experiments, cell numbers had exceeded pre-transport/storage concentrations within 48h of recovery. When compared to cells revived from cryopreservation, HepG2 and Jurkat cells that had been transported/stored for 72h at ambient, were analysed. Following transport/storage in CellShip® cells recovered more quickly than cells recovered from cryopreservation. Within 48h, HepG2 transported at ambient had become fully adherent and displayed typical growth morphology which was not seen in the cryopreserved samples. Jurkat cells recovered from cryopreservation took >72h to return to pre-cryopreservation cell numbers, whereas cells recovered from transport at ambient demonstrated a 2 fold increase within 48h of recovery, indicating that cells re-entered the cell-cycle with minimal delay.

Contact: EmmaBuick@lifesciencegroup.co.uk