Abstract 1

The common cockle (Cerastoderma edule) is an important commercial mollusk for several European countries. The diseases incurred by parasites and the disseminated neoplasic disorder have sharply affected the natural populations, with significant repercussions on the economy. The aim of this work was to develop a reliable cryopreservation protocol for larval stages to enhance the restock of wild populations and improve the hatchery production. First, toxicity tests were carried out on 48 and 72 h-old D-larvae to determine the suitable Cryoprotecting Agent (CPA) attending to harmful effects. Larvae were exposed to increasing concentrations of Ethylene-Glycol (EG), Propylene-Glycol (PG), Glycerol (GLY) and dimethyl-sulfoxide (Me 2 SO). Considering toxicity results, two CPA combinations a) 10% EG + 0.4 M Trehalose (TRE) and b) 10% PG + 0.4 M TRE (final concentrations) were considered to cryopreserve both larval stages. In addition, the effects on survival and shell size of increasing equilibrium times (15, 30 and 60 minutes) were evaluated. After equilibration, larvae were cryopreserved using a controlled freezer, as described: holding at 4°C for 2 min, then cooling at -1°C/min to -12°C (seeding), holding for 2 min, then cooling at -1/°C/min to -35°, then plunging into liquid nitrogen. Thawing was carried out by immersion of straws into a water bath at 35°C for 6 seconds. The obtained data evidenced the high tolerance of this specie to cryopreservation. Although no significant differences were found, the CPA solution with PG was better for the cryopreservation of the earlier development stage, obtaining 100% of survival with 60 minutes of equilibration. The successful cryopreservation experiment using 72 h-old D-larva provided survival rates closer to 100% regardless of the treatment selected. In both cases cryopreservation yielded a delay on larval size. Further research should focus on the long-term effects and the capacity to produce cockle spat from cryopreserved larvae.

Abstract 2

Regulatory T cells (Treg) belong to a subpopulation of CD4 + T cells with suppressor properties and play an important role in maintaining autotolerance. Currently, cord blood (CB) is considered as one of the sources of Treg for immunotherapy. This necessitates the establishing of the CB reserves using cryopreservation and lyophilization methods. The research purpose was to comparatively assess the structure and functions of Treg as well as the content of IL-10 in cryopreserved and lyophilized leukoconcentrates of human CB (HCBL). The cryopreserved HCBL (cHCBL) was obtained after freezing of HCBL according to a two- stage program (1); lyophilized HCBL (lHCBL) – as reported by A. Goltsev et al. (2). The amount of Treg, i.e. CD4 + CD25high, the average and total fluorescence intensity of the CD25 marker, and the Foxp3 protein content in cells was determined by flow cytometry (FACS Calibur, BD, USA). The concentration of IL-10 in HCBL was determined by enzyme- linked immunosorbent assay. Differences in functional parameters of Treg after cryopreservation and lyophilization of HCBL were found. The amount of Treg decreased by 2.2 times in cHCBL, while it increased in 1.6 times in lHCBL compared with their quantity in a fresh preparation, which did not affect the content of IL-10. At the same time, after cryopreservation and lyophilization, an increase in the average fluorescence intensity of the CD25 marker in Treg was noted, which may indicate a higher readiness of cells to implement their immune suppressive potential. In addition, the total fluorescence intensity for the CD25 marker and the Foxp3 protein content were maximal in Treg from lHCBL. The findings indicate the ability of cryopreservation and lyophilization to stimulate the Treg functional potential, increasing the immune regulatory properties of HCBL. Moreover, after lyophilization such an activity is manifested to a greater extent.

References: [1]Tsutsayeva AO, Grischenko VI, Kudokotseva OV , Shcheglov A.V., Tupchienko G.S., Prokopuk O.C. inventors; Institute for Problems of Cryobiology and Cryomedicine, assignee. [Method of cryopreservation of hemopoietic cells of cord blood] Ukrainian patent 31847A 1998 November 5. Ukranian. [2] Goltsev AM, Taranik GC, Grisha IG, Sokil LV, Bondarovich MO, Ostankov MV, Lutsenko OD, Goltsev KA, Ostankova LV, inventors; Institute for Problems of Cryobiology and Cryomedicine, assignee. [Method of lyophilization of leukoconcentrate of cord blood]. Ukrainian patent 113006, 2017 Jan 10. Ukranian.

Abstract 3

This research is devoted to a comparative study of the effect of amphiphiles, differing by physicochemical properties on the sensitivity of erythrocytes under post-hypertonic shock. Post-hypertonic shock simulates the influence on cells of cryodamage factors, acting at erythrocyte thawing stage, as well as when that of cells, cryopreserved under protection of penetrating cryoprotective agent are transferred into bloodstream (1). Post-hypertonic shock (PHS) of erythrocytes was initiated by transferring cells from dehydration medium (1.65 mol/L NaCl) into rehydration one (0.15 mol/L NaCl) at 0°С. The various classes of amphiphiles (anionic sodium dodecyl sulfate, non-ionic decyl-β,D-glucopyranoside, and cationic trifluoperazine) were used. Amphiphiles were supplemented into rehydration medium prior to transferring the cell. Amphiphiles were shown to protectively affect erythrocytes in various extents. Under these conditions, decyl-β,D-glucopyranoside and trifluoperazine were more effective, for which maximum antihemolytic activity are recorded at a level of ~60%, in contrast to sodium dodecyl sulfate, which maximum antihemolytic activity was 2 times lower (~30%). A comparative study of the efficiency of amphiphiles under post- hypertonic shock of erythrocytes showed the differences in the size of concentration plateau and the values of effective concentrations. A narrowing of the plateau was revealed in a series: decyl-β,D-glucopyranoside ˃ trifluoperazine ˃ sodium dodecyl sulfate. Decyl-β,D- glucopyranoside characterize the maximum effective concentration (1,000 μmol/L) and the minimal one for sodium dodecyl sulfate (50 μmol/L). The obtained results suggest that the effectiveness of amphiphiles under PHS conditions of erythrocytes is associated with their ability to integrate into membrane, possibly, to the defect formation areas. This is accompanied by an increase in membrane surface area and, therefore, in critical hemolytic volume of cells, which allows them to swell to a larger volume in rehydration medium.

References: [1] Semionova YA, Zemlyanskikh NG, Orlova NV, Shpakova NM. Antihemolytic efficiency of chlorpromazine under posthypertonic shock and glycerol removal from erythrocytes after thawing. Problems of Cryobiology and Cryomedicine 2017, 27(1): 51-60.

Abstract 4

The piglets cryopreserved skin fragments extract is known to stimulate the wound healing after skin cryoablation, but the mechanisms of this stimulation have been poorly understood. Apoptotic (p53) and proliferative (Ki-67) cell activity was studied on days 7, 14, and 21 after skin cryoablation in hairless rats, with intraperitoneal administration of saline (group 1) or piglets cryopreserved skin fragments extract (group 2). In groups 1 and 2, at all periods of the experiment in granulation and connective tissues, p53 and Ki-67 expressed predominantly fibroblastic cells, vascular endothelial cells and immune cells. In group 1, the number of Ki-67- and p53-positive cells on days 7, 14, and 21 was 18.7 ± 2.8 and 9.5 ± 1.5, 23.4 ± 3.6 and 12.9 ± 2.0, 28.5 ± 4.6 and 15.9 ± 2.5, respectively. In group 2, the number of Ki-67- and p53-positive cells on days 7, 14, and 21 was 27.4 ± 3.9 and 14.8 ± 2.1, 32.2 ± 5.1 and 17.8 ± 2.9, 37.3 ± 5.8 and 22.5 ± 3.7, respectively. In both groups, firstly, the proliferative activity of the cells prevailed over the apoptotic one, and secondly, with a rise in the duration of the experiment, the processes of apoptosis and proliferation increased. In group 2, compared with group 1, at all periods of the experiment, the absolute number of Ki-67 and p53-positive cells was higher. Intraperitoneal administration of piglets cryopreserved skin fragments extract in a balanced manner activates the apoptotic and proliferative activity of fibroblastic cells, vascular endotheliocytes, immune cells in granulation and connective tissues, filling the wound cavity after skin cryoablation.

Abstract 5

Fish spermatozoa cryopreservation protocols are characterized by high species specificity. An unique method of their low-temperature storage should be created for each species [1]. Taking into account that research is often carried out in the field, there is a need to use simple and universal tools to determine the functional characteristics of spermatozoa. In order to solve this problem we designed a method that allows studying osmotic response of spermatozoa using photoelectric colorimeter, based on changes in the optical density of the suspension [2]. We described the osmotic response of spermatozoa using such parameters as membrane permeability and activation energy of water transfer through the membrane. In addition, we used cells osmotic resistance as cells lysis time in osmotic shock. To determine the membranes permeability of fish spermatozoa to water or cryoprotectant molecules, spermatozoa were added to the cuvette, filled either with NaCl aqueous solution or NaCl based solutions of ethyleneglycol, 1,2-propanediol, methanol, glycerol or other cryoprotectants. Permeability coefficients of spermatozoa plasma membranes for either water (L p ) or cryoprotectant (K p ) molecules were determined by fitting the experimental dependences of relative cell volumes versus time and the solutions of theoretical model equations. The activation energy (Е а ) of substance transfer through cell membranes was calculated from the lnL p or ln K p dependencies versus temperature, the slope of those according to the Arrhenius equation was Е а /R, where R was the universal gas constant.

Thus we investigated the spermatozoa osmotic response of such freshwater fish as carp, silver carp, crucian carp, tench, grass carp, pike perch, pike, sterlet, Russian sturgeon and some others. There is a tendency for membrane permeability to correlate with sperm cryoresistance with some restrictions and conditions. This is consistent with previously obtained data [3]. Also determining the value of this parameter allows the selection of suitable cryoprotective media.

References: 1 Kopeika E. F., Kopeika J. E. Variability of sperm quality after cryopreservation in fish // Fish spermatology / Editors: S. H. M. Alavi, J. Cosson, K. Coward, G. Rafiee. Alpha Science International Ltd. Oxford, 2007. P. 347–396. 2 Puhovkin A. Y., Kopeika E. F. Investigation of water and cryoprotectants molecules transfer through common carp (Cyprinus carpio, L.) spermatozoa membranes (CRYO conference of the Society for Cryobiology, Ostrava, Czech Republic, 2015) // Cryobiology. 2015. Vol. 71, № 3. P. 567. 3 Hypotonic treatment prior to freezing improves cryoresistance of common carp (Cyprinus carpio L.) spermatozoa / B. Dzyuba, J. Cosson, G. Yamaner, O. Bondarenko, M. Rodina, D. Gela, V. Bondarenko, A. Shaliutina, O. Linhart // Cryobiology. 2013. Vol. 66. P. 192–194.

Abstract 6

In vitro embryo production and embryo transfers in goats are a developing industry in Ukraine. Introducing effective technique for in vitro fertilization would lead to better results in goat reproduction. The objective of this study was to compare embryo development after oocyte fertilization with fresh versus frozen sperm in Saanen goats. 3 mature Saanen goats (84 ± 8 kg) were used as donors. Estrus synchronization was performed using intravaginal sponges with 45mg flugestone acetate for 14 days. On day 7 after sponge insertion, ovarian stimulation was induced by injecting intramuscularly 5 doses of follicular stimulating hormone (4D – 500IU, 1D - 250IU) with intervals of 24 h and administrating a single intramuscular injection of 1000IU of a pregnant mare's serum gonadotropin 36 h before sponge removal and oocyte aspiration. Laparoscopic ovum pick-up was performed using a 23G Aspic needle connected to 20mL syringe. Following in vitro maturation, 29 recovered oocytes (9.7 ± 2.1 per goat) were randomly divided into groups (20 and 19 oocytes) and in vitro fertilized using washed fresh and frozen-thawed semen derived from Saanen buck. After 7 days of in vitro culture 11 blastocysts (55%) and 9 blastocysts (47,4%) were developed in groups with fresh vs. frozen sperm consequently. To conclude fertilization with fresh sperm leads to better embryo development rate than with frozen-thawed sperm in goats.

Abstract 7

Treatment by means of donor lymphocyte infusions (DLI) after allogeneic haematopoietic progenitor cell (HPC) transplantation represents, now the main indication for cryopreservation of PBMC. Usually a small part (20-30 ml), surplus of the collected allogeneic HPC concentrate is frozen in several units for administration in increasing CD3+ cell doses (standard scheme once or twice: 0.5x10 5 /kg, 1x 10 6 /kg, 5x 10 6 /kg,1x 10 7 /kg of the patient s weight) to achieve guided GvD. If the sufficient number of cell doses cannot be achieved, additional collection of non- mobilized PBMC must be performed in an original HPC donor. Results of cryopreservation of concentrates collected in 16 donors (originated from Czech Republic in 9 cases, from Germany in 5 cases, from Poland in 2 cases) and sent to the EU TE Code CZ000426 are presented. The standard operating processing and cryopreservation procedure using clean room facility, 10%DMSO, and controlled rate freezing with storage in liquid nitrogen vapour phase was used. The WBC count including detailed differential, CD3+ count, cell viability and sterility tests were performed from the collected suspension and the thawed control samples. The median values of collected suspensions were: volume 152 ml, NC concentration 75.2 x 10 9 /l, TNC 144.2 x 10 8 , MC percentage 96.4. The MC post- thaw viability was 95 %, post-thaw recoveries: viable TNC 88.25%, viable TMNC 87,93%, viable CD3+ cells 88.62%. Annual DLI application rate has shown a slowly increasing trend between 20 to 79 infusions in the last 5 years. The authors plan to use the described experience in a near future in cryopreservation of non-mobilized autologous PBMC as starting material for manufacturing of genetically modified advanced therapy medicinal product Kymriah (tisagenlecleucel) CTL 019, Novartis that should expand the spectrum of therapeutical tools in treatment of malignant lymphomas, multiple myeloma and certain types of leukaemia.

Abstract 8

Retinal degenerative diseases are one of the main clinical causes of severe, progressive vision impairment. Due to limited availability of retinal tissue the ideal solution would be the formation of retinal cryobanks. Stored retinas could be used for research purposes, epidemiological studies, validation of newly developed drugs etc. Retinal cryopreservation is underresearched, with only two studies published 1,2 , both studying immature murine retinal tissue. We focused on mature mouse retina and investigated the impact of a slow-cooling to -10°C using different freezing media (FM). Retinas from adult C57BL/6 mice were preconditioned on ice using mouse artificial cerebrospinal fluid (maCSF) with addition of 10% DMSO for 4 hours with a constant supply of carbogen. After preconditioning, samples were transferred to different FM: (1) 90% retinal organ culture media (50% DMEM-GlutaMAX, 25% HBSS, 25% FBS, 5.75 g/l glucose, and 0.1M sucrose) with 10% DMSO (2) 90% FBS and 10% DMSO; (3) Cryostore; (4) Nutrifreeze; and (5) Bambanker. Samples were cooled down at 1°C per minute to -10°C using a VIA Freeze TM Research controlled rate freezer (Asymptote, UK) and kept cool for 24 hours. Afterwards, the samples were warmed to 37°C, washed and transferred for recovery to maCSF with a constant supply of carbogen for 24 hours at room temperature. After recovery, the tissue was fixed and processed for immunofluorescent and hematoxylin & eosin staining. Taking into consideration macroscopic and microscopic damage, we found that cooling with Nutrifreeze best preserved retinal layer organisation and tissue morphology, including all retinal cell types. Our findings indicate that murine retinas can be effectively stored at -10°C for at least 24 hours without significant impact on the tissue organization and cell morphologies using Nutrifreeze, or DMSO in combination with either FBS or retinal organ culture media. Further experiments will confirm functionality of these tissues.

References

1. Aramant, R. and M. Seiler, Cryopreservation and transplantation of immature rat retina into adult rat retina. Brain Res Dev Brain Res, 1991. 61(2): p. 151-9.

2. Jensen, S., T. Sorensen, and J. Zimmer, Cryopreservation of fetal rat brain tissue later used for intracerebral transplantation. Cryobiology, 1987. 24(2): p. 120-34.

Abstract 9

The aim of the study was a comparative assessment of the thawing regimes on the morphofunctional characteristics of the immature rat seminiferous tubules after vitrification. The samples of immature rat testicles of 2-3 mm 3 size were exposured in cryomedia (Me 2 SO+glycerol+sucrose) of increasing concentrations based on a fibrin gel for 5 min at 4°C and then were vitrified by rapid immersion in liquid nitrogen. The following regimes were used for thawing: I - at 22°C; II - at 50°C; III - at 70°C by successive transfer of samples to a sucrose solution of decreasing concentration (1M, 0.5M, 0.25 M, 0M) for 5 min. Intact control was used as a comparison group. Histomorphological data, MTT-test, total antioxidant defense system (TAS) activity were studied. The results were processed with Kruskal-Wallis ANOVA test with multiple comparisons. The indicates of MTT-test and TAS activity in the samples warmed at 50°C were higher by 1.9 and 1.7 times respectively relative to regime I. The use of regime II and III did not significantly differ from each other in the investigated parameters. However, these indicates in all experimental groups remained significantly lower than in intact control. The warming at regime I negatively affected histological structure seminiferous tubules: there were extensive destruction zones, and spermatogenic epithelium lost its organization. The samples warmed at regime II retained their architecture (slight retraction and vacuolization of the nuclei of individual cells, which were located throughout the epithelial layer). After thawing of samples at regime III, the spermatogenic epithelium was characterized by a sharp retraction and desquamation cells, which resulted in defects of the spermatogenic layer. The thawing at 50°C allows effective preserving of the morphofunctional properties of vitrified seminiferous tubules. These findings relate to reproductive medicine and can be used for development of effective thawing regimes for vitrified seminiferous tubules.

Abstract 10

The aim of this work was to verify the possibility of using the cryopreservation method in the breeding process of potatoes and hops in the form of explant cultures and pollen. Two methods of dehydration of potato shoot tips were tested: 1) dry air over silica gel, 2) PVS3 cryoprotective solution and the only the first mentioned method for hop shoot tips. Thermal characteristics were determined using a Q2000 differential scanning calorimeter (TA Instruments) at isolated shoot tips of potatoes or hops before or after dehydration specified above. In the case of potato explants, dehydration of the growth peaks with a cryoprotective solution was found to be more effective compared to dry air dehydration. In the case of hop explants, it was found that the optimal dehydration of the growing peaks occurs in the range of 1.5 to 2 hours, when a decrease in the proportion of frozen water to 6 to 4% was found. When comparing the results of dehydration of potato and hop explants, the presence of two endotherms was found when the proportion of frozen water decreased by 18%, regardless of the method of dehydration of plant material and plant species. While an endotherm that has been detected at a higher temperature appears to be related to the presence of free water, the significance of the second endotherm is unclear and will be the subject of further research. Thermal analysis of the pollen did not detect the presence of frozen water in the potatoes or hops. The presence of a wide glass transition in the range of -55 ° C to 0 ° C with a mean value of approximately -25 ° C was detected in all tested materials. This finding suggests the possibility of widespread use of the cryopreservation method for preserving potato and hop pollen for its use in the breeding process. Acknowledgement: This study was supported by research projects of the Ministry of Agriculture of the Czech Republic QK1910277 and MZE RO0418.

Abstract 11

The current state of nanotechnologies development allows the obtaining of multifunctional nanomaterials, applicable in cryobiology, such as nanocrystalline cerium dioxide (CeO 2 NPs), which can be used as an auxiliary component of cryoprotective media to reduce the Me 2 SO concentration, commonly applied to freeze cells, but being toxic. However, CeO 2 NPs can also exhibit toxic effects, and therefore determination of CeO 2 NPs non-toxic concentrations is important. Two series of experiments were performed: detection of toxic CeO 2 NPs concentration for L929 mouse fibroblast cells by MTT test and evaluation of apoptosis / necrosis in L929 mouse fibroblast cells cryopreserved in media containing CeO 2 NPs using AnexinV and 7AAD dyes by flow cytometry (FACS Calibur, USA). In the first series of experiments, the cells were cultured in the nutrient medium with the addition of CeO 2 NPs at final concentrations of 1.0; 0.2 and 0.02 g/l, followed by their evaluation using the MTT test on the fourth cultivation day. In the next series of experiments, the fibroblasts were cryopreserved in a protective media containing 1% and 5% Me 2 SO. As an additional component 1.0 g/l and 0.02 g/l of CeO 2 NPs were added. Control were the cells cryopreserved in a medium containing 1% Me 2 SO without the addition of CeO 2 NPs. According to the MTT test, it was found that only 1 g/l was toxic of all three studied CeO 2 NPs concentrations added to the medium during cell culture. However, according to flow cytometry, at this CeO 2 NPs concentration there is a decrease in the number of AnnexinV+/7AAD+ cells after cryopreservation in a medium containing 1% and 5% Me 2 SO. CeO 2 NPs at a concentration of 0.02 g/l and 0.2 g/l have no any toxic effect during the cultivation of fibroblasts but also do not have a cryoprotective effect. Thus, further research may be aimed at finding a nontoxic concentration of CeO 2 NPs having a cryoprotective effect.