This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in thermal properties and water content. Three specific types of pollen samples based on their thermal characteristics and water content were distinguished by DSC in potato: (1) \'glassy\', with a water content lower than 0.21 g water per g dry matter; (2) \'transient\', with a water content between 0.27 and 0.34 g of water per g of dry matter; (3) \'frozen\', with a water content higher than 0.34 g of water per g of dry matter. Only the \'glassy\' pollen samples with a low water content showed suitable properties for its long-term storage using cryopreservation in potato and hops. Cryopreservation of pollen did not significantly reduce its viability, and cryopreserved pollen was successfully used to produce both potato and hop hybrids. The results indicate that cryopreservation is a feasible technique for the preservation and utilization of pollen of these crops in the breeding process.

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP\'s cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes.

BACKGROUND: Peripheral blood stem cells (PBSCs) autologous transplantation is the standard care for transplant-eligible patients with multiple myeloma (MM). This treatment option is somewhat limited due to the high consumption of economic resources and the access to Cryobank.
OBJECTIVE: Compare the results of autologous transplantation using non-cryopreserved and cryopreserved hematopoietic stem cells (HSC).
METHODS: Single center retrospective study conducted in 2018-2022.
METHODS: 78 patients with MM were included in the study. All patients got the standard immunochemotherapy programs and had remission. Patients were divided into two groups: non-CRYO (n=35) and CRYO (n=43). Cryopreservation is a standard method of storage of HSC. Native HSC are saved from +4 °C to +6 °C for 72 hours. The number of CD34+ and 7AAD- cells, colony-forming ability were made after apheresis and before transplantation.
METHODS: The results of autologous transplantation with use non-cryopreserved and cryopreserved HSC.
RESULTS: There were no differences in total number of CD34+ cells x106/kg, or in level of 7AAD- cells, or in total colony. There was a significant difference in the percentage of loss of CD34+ cells from the moment of apheresis to the moment of reinfusion. We suppose it was caused by adverse effects of temperature changes in CRYO group. Neutrophil recovery was achieved at 11th day (range 9-14) and platelets at 12th day (range 8-19) in non-CRYO group, 10th day (range 8-14) and 12th day (range 8-20) in CRYO group, respectively. The partial response before autoHSCT was achieved in 37%, very good partial response-40%, complete response - 23% in the non-CRYO group and 72%, 14%, 14% in the CRYO group, respectively. Partial response after autoHSCT was achieved in 23% patients, very good partial response in 40%, complete response in 37% in the non-CRYO group compared to the CRYO group (47%, 21%, 32%, respectively). The frequency of MRD-negative response before autoHSCT was 37.5%, after transplantation - 54.5% in the non-CRYO group and 16% and 33% in the CRYO group.
CONCLUSIONS: The method of storage of PBSCs without cryopreservation is equal to traditional method controlled freezing and can be used in hospitals which have no Cryobank.

OBJECTIVE: To study the presence of certain proteins - EGF (epidermal growth factor), KGF (keratinocyte growth factor), IL-10 (interleukin 10), HGF (hepatocyte growth factor), Alpha2-macroglobulin and IL-1RA (interleukin 1 receptor antagonist) in cryopreserved amniotic membranes at 1 and 18 months and, as a secondary objective, to detect mRNA corresponding to KGF, IL-1Ra, Alpha2-macroglobulin, Fas Ligand, TGF beta (transforming growth factor beta) and Lumican by RT-PCR in membranes preserved at 1 and 18 months.
METHODS: Four samples of amniotic membrane were divided into 2 groups: the first group (N=2) cryopreserved for 1 month and the second group (N=2) cryopreserved for 18 months, in order to be studied by RT-PCR and ELISA.
RESULTS: RT-PCR detected KGF, IL-1Ra, Alpha2-macroglobulin, Fas Ligand, and Lumican. Of these, FAS Ligand mRNA was found in samples preserved for 1and 18 months. KGF, Lumican, and alpha2-microglobulin mRNA were found only at 1 month, and IL-1Ra mRNA was absent in both sample groups. RT-PCR for TGF-beta was inconclusive. ELISA was performed for detection and quantification of 6 proteins (EGF, KGF, IL-10, HGF, Alpha2-macroglobulin and IL-1Ra) in both amniotic membrane groups. All 6 proteins were found in all samples, with a lower concentration at 18 months compared to 1 month of preservation.
CONCLUSIONS: This study shows that membranes cryopreserved in 50% glycerol for 18 months do retain the proteins necessary for regeneration of the corneal surface, giving these membranes their biochemical properties.

Modern biobanks maintain valuable living materials for medical diagnostics, reproduction medicine, and conservation purposes. To guarantee high quality during long-term storage and to avoid metabolic activities, cryostorage is often conducted in the N2 vapour phase or in liquid nitrogen (LN) at temperatures below - 150 °C. One potential risk of cryostorage is microbial cross contamination in the LN storage tanks. The current review summarises data on the occurrence of microorganisms that may compromise the safety and quality of biological materials during long-term storage. We assess the potential for the microbial contamination of LN in storage tanks holding different biological materials based on the detection by culture-based and molecular approaches. The samples themselves, the LN, the human microbiome, and the surrounding environment are possible routes of contamination and can cause cross contaminations via the LN phase. In general, the results showed that LN is typically not the source of major contaminations and only a few studies provided evidence for a risk of microbial cross contamination. So far, culture-based and culture-independent techniques detected only low amounts of microbial cells, indicating that cross contamination may occur at a very low frequency. To further minimise the potential risk of microbial cross contaminations, we recommend reducing the formation of ice crystals in cryotanks that can entrap environmental microorganisms and using sealed or second sample packing. A short survey demonstrated the awareness for microbial contaminations of storage containers among different culture collections. Although most participants consider the risk of cross contaminations in LN storage tanks as low, they prevent potential contaminations by using sealed devices and - 150 °C freezers. It is concluded that the overall risk for cross contaminations in biobanks is relatively low when following standard operating procedures (SOPs). We evaluated the potential sources in detail and summarised our results in a risk assessment spreadsheet which can be used for the quality management of biobanks. KEY POINTS: • Identification of potential contaminants and their sources in LN storage tanks. • Recommendations to reduce this risk of LN storage tank contamination. • Development of a risk assessment spreadsheet to support quality management.

Improvements in ex situ storage of genetic and reproductive materials offer an alternative for endangered livestock breed conservation. This paper presents a dataset for current ex situ collections and in situ population for 179 Spanish livestock breeds of seven species, cattle, sheep, pig, chicken, goat, horse and donkey. Ex situ data was obtained via survey administered to 18 functioning gene banks in Spain and relates to the reproductive genetic materials (semen doses) of 210 livestock breeds distributed across the gene banks. In situ data combines CENSUS information with linear regression techniques and relates to the geographic distribution of 179 Spanish autochthonous livestock breeds (2009-2018), and in situ population projections and extinction probabilities (2019-2060). We use a decision variable defining an \"acceptable level of risk\" that allows decision makers to specify tolerable levels of in situ breed endangerment when taking ex situ collection and storage decisions.

The aim of the study was to evaluate of the effects of glycerol and DMSO, belonging to the endocellular type of cryoprotective agents (CPAs), as well as polyethylene glycol, dextran, sucrose, and mannitol, related to exocellular CPAs, on proteins of the membrane-cytoskeleton complex (MCC) of human erythrocytes at the stage preceding freezing. The assessment of protein modifications was performed by SDS-PAGE using different approaches when preparing samples for analysis. The use of β-mercaptoethanol in the solubilizing buffer showed no changes in the MCC polypeptide profile of erythrocytes preincubated with CPAs thus suggesting good biocompatibility of the studied substances. The use of the cross-linking reagent diamide for assessment of protein modifications did not reveal structural abnormalities that would result in significant changes in the localization of -SH groups and an increase in the production of high-molecular-weight polypeptide complexes identified by SDS-PAGE without β-mercaptoethanol. However, the recognized changes in the electrophoretic mobility of proteins in the area of band 5 in erythrocytes incubated with CPA in the presence of diamide suggest a reorganization of the structural state of actin protofilaments, which can be caused by alterations of actin monomers themselves or initiated by modifications of actin-binding proteins in the presence of CPAs. In addition, an increase in the amount of the protein fraction located between bands 5 and 6 in the MCC profiles of erythrocytes incubated with CPA and diamide was revealed. Despite the similarity of the reaction of erythrocyte proteins to different CPAs, the properties of cells depending on MCC, may differ due to modifications in the macromolecule structures, which are not associated with changes in the localization of the -SH-groups of proteins. The results obtained indicate that CPAs may have a significant impact on the erythrocyte MCC, and this requires further research.
Tsel\' issledovaniia zakliuchalas\' v opredelenii vliianiia glitserola i dimetilsul\'foksida (DMSO), prinadlezhashchikh k éndotselliuliarnomu tipu krioprotektornykh agentov (KPA), a takzhe poliétilenglikolia, dekstrana, sakharozy i mannitola, otnosiashchikhsia k ékzotselliuliarnym KPA, na belki membrano-tsitoskeletnogo kompleksa (MTsK) éritrotsitov cheloveka na étape, predshestvuiushchem zamorazhivaniiu. Otsenka modifikatsiĭ belkov vypolnena metodom élektroforeza v poliakrilamidnom gele v prisutstvii dodetsilsul\'fata natriia (SDS-PAAG-élektroforeza) s primeneniem raznykh podkhodov pri podgotovke obraztsov dlia analiza. Ispol\'zovanie β-merkaptoétanola v sostave soliubiliziruiushchego bufera prodemonstrirovalo otsutstvie izmeneniĭ polipeptidnogo profilia MTsK éritrotsitov, predvaritel\'no inkubirovannykh s KPA, chto kharakterizuet khoroshuiu biosovmestimost\' issledovannykh veshchestv. Primenenie “sshivaiushchego” reagenta diamida dlia otsenki modifikatsiĭ belkov ne vyiavilo strukturnykh narusheniĭ, kotorye privodili by k znachitel\'nym izmeneniiam lokalizatsii −SH-grupp i uvelicheniiu obrazovaniia vysokomolekuliarnykh polipeptidnykh kompleksov, identifitsiruemykh s pomoshch\'iu SDS-PAAG-élektroforeza bez primeneniia β-merkaptoétanola. Vmeste s tem, obnaruzheny izmeneniia élektroforeticheskoĭ podvizhnosti belkov v zone polosy 5 v éritrotsitakh, inkubirovannykh s KPA v prisutstvii diamida, chto ukazyvaet na reorganizatsiiu strukturnogo sostoianiia aktinovykh protofilamentov, kotorye mogut byt\' obuslovleny izmeneniiami samikh aktinovykh monomerov ili initsiirovany modifikatsiiami aktin-sviazyvaiushchikh belkov v prisutstvii KPA. Krome togo, vyiavleno uvelichenie soderzhaniia belkovoĭ fraktsii, raspolozhennoĭ mezhdu polosami 5 i 6 v profiliakh MTsK éritrotsitov, inkubirovannykh v prisutstvii KPA s diamidom. Nesmotria na vidimost\' odinakovoĭ reaktsii belkov éritrotsitov na raznye KPA, svoĭstva kletok, zavisiashchie ot MTsK, mogut razlichat\'sia blagodaria modifikatsiiam v strukture makromolekul, ne sviazannym s izmeneniiami lokalizatsii −SH-grupp belkov. Poluchennye rezul\'taty svidetel\'stvuiut o tom, chto pod vliianiem KPA v MTsK éritrotsitov mogut proiskhodit\' opredelennye perestroĭki, detal\'nye aspekty kotorykh trebuiut dal\'neĭshikh issledovaniĭ.

Genetic modifications such as mutation and transformation are powerful tools to study the function of genes and proteins in the model liverwort Marchantia polymorpha, but maintaining the resulting germplasm requires a practical, reliable method. Cryopreservation methods allow researchers to maintain mutant and transgenic lines of M. polymorpha. To date, two methods have been developed for cryopreservation of M. polymorpha gemmae: in the first method, unencapsulated gemmae are stored in liquid nitrogen at --196 °C, and in the second method, encapsulated gemmae are stored in liquid nitrogen at --196 °C or a deep freezer at -80 °C. In the present study, we developed a simple method named CRUNC (cr yopreservation of un en c apsulated gemmae), which can be used to store unencapsulated, dried gemmae of wild-type and transgenic M. polymorpha lines in liquid nitrogen and in freezers at -80 °C and -20 °C. Using the CRUNC method, we observed a high recovery rate (as high as 100%) and successful long-term (5 months) storage of the gemmae. Therefore, the CRUNC method is practical for maintaining valuable M. polymorpha germplasm.

Fecal Microbiota Transplantation (FMT) is suggested as an efficacious therapeutic strategy for restoring intestinal microbial balance, and thus for treating disease associated with alteration of gut microbiota. FMT consists of the administration of fresh or frozen fecal microorganisms from a healthy donor into the intestinal tract of diseased patients. At this time, in according to healthcare authorities, FMT is mainly used to treat recurrent Clostridium difficile. Despite the existence of a few existing stool banks worldwide and many studies of the FMT, there is no standard method for producing material for FMT, and there are a multitude of factors that can vary between the institutions. The main constraints for the therapeutic uses of FMT are safety concerns and acceptability. Technical and logistical issues arise when establishing such a non-standardized treatment into clinical practice with safety and proper governance. In this context, our manuscript describes a process of donor safety screening for FMT compiling clinical and biological examinations, questionnaires and interviews of donors. The potential risk of transmission of SARS-CoV-2 virus by the use of fecal microbiota for transplantation must be taken urgently into consideration. We discuss a standardized procedure of collection, preparation and cryopreservation of fecal samples through to the administration of material to patients, and explore the risks and limits of this method of FMT. The future success of medicine employing microbiota transplantation will be tightly related to its modulation and manipulation to combat dysbiosis. To achieve this goal, standard and strict methods need to be established before performing any type of FMT.

Canine semen cryoconservation was used since 1969, and this process is still nowadays in progress. This review aims to have an overview of two factors leading to a successful freezing-thawing semen. The success and efficiency of freezing process can be measured by the post-thawing sperm mobility. The first factor is the best extender used as a cryoprotectant to have a similar osmolarity and pH compared to the seminal plasma to enable sperm survival. Historically, chicken egg yolk was used since 1940, but due to microbial risks and to the presence of granules (which interfere with counting dead spermatozoa and inhibits a spermatozoal respiration), despite these disadvantages, egg yolk is considered an excellent cryoprotectant for sperm of different animal species. The low-density lipoproteins (LDL), contained in EY, when used at a concentration of 6% in a freezing medium associated with 20 mM of glutamine, show a mobility up to 54.5%, which is the best combination found. However, the sperm protection mechanism by LDL during freezing-thawing process only begins to be decrypted. But extraction protocols of LDL are not efficient for an industrial use. Therefore, egg yolk plasma is used within liquid or lyophilized state, and offering similar efficiency as the 6% LDL middle. The equilibration step, in which the diluted sperm is placed for a variable period of time at a temperature of +4°C, before freezing it. The studies show that 6 hr is the optimal duration for the canine sperm equilibration. The future of canine sperm cryopreservation is expected in liposome use and synthetic substances, which mimics LDL role.