Thawing Protocol Research Articles

Effect of Different Thawing Methods on Bull's Semen Characteristics

The aim of the study was to evaluate the influence of different thawing methods on bull’s semen characteristics. The semen was collected and processed from 8 bulls (Holstein, n = 4; Czech Fleckvieh, n = 4) kept in private Sire insemination Centre. Four different thawing methods were tested: control thawing methods (temperature = 38.5 °C, time = 30 seconds), slow thawing method (temperature = 30 °C, time = 50 seconds), moderate thawing method (temperature = 50 °C, time = 15 seconds), rapid thawing rate (temperature = 70 °C, time = 3 seconds). The percentage rate of total and progressive motile spermatozoa above head as well as movement characteristics of straight‑line velocity (VSL, µm/s) and linearity (LIN, %) were recorded using CASA system immediately after thawing and after 2 hrs. of heat incubation (±38 °C). Subsequently the differences between semen characteristics measured immediately after thawing and after 2 hours of incubation were calculated. The data were analyzed with SAS software. All the evaluated semen traits reached significantly lowest values in total motility (−8 % to −10.5 %, P < 0.05), progressive motility (−5.6 % to −10 %, P < 0.05) VSL (−3.9 µm/s to −9 µm/s, P < 0.05) and LIN (−1.5 % to −4.8 %, P < 0.05) in control thawing method immediately after the thawing compared to others. However, these differences were negligible after 2 hrs. of incubation. The highest values of progressive motility and movement characteristics after thawing and after 2 hours incubation were detected using slow thawing method. Moreover, the rapid method of thawing showed the significantly the lowest results in progressive motility (−3.6 % to −6.6 %), VSL (−3.2 to −5.6 µm/s) and LIN (−3 % to −4.6 %) characteristics assessed after 2 hrs. of incubation compared the others (P < 0.05). Control method of thawing was most stable during the incubation and showed the significantly lowest decrease of total motility (−7.6 % to −11.8 %, P < 0.05), progressive spermatozoa motility above head (−7.5 % to −9.8 %, P < 0.05) and VSL characteristics (−2.4 to −9.7 µm/s, P < 0.05) during incubation. Therefore, this thawing protocol can be recommended at this study based on the lowest spermatozoa characteristics decline during incubation for using artificial insemination.

Cryopreserved human aortic root allografts arterial wall: Structural changes occurring during thawing.

BackgroundThe aim of our experimental work was to assess morphological changes of arterial wall that arise during different thawing protocols of a cryopreserved human aortic root allograft (CHARA) arterial wall.MethodsThe experiment was performed on CHARAs. Two thawing protocols were tested: 1, CHARAs were thawed at a room temperature at +23°C; 2, CHARAs were placed directly into a water bath at +37°C.Microscopic samples preparationAfter fixation, all samples were washed in distilled water for 5 min, and dehydrated in a graded ethanol series (70, 85, 95, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane for 10 minutes and air dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs, coated with gold.ResultsThawing protocol 1: All 6 (100%) samples showed loss of the endothelium and damage to the subendothelial layers with randomly dispersed circular defects and micro-fractures without smooth muscle cells contractions in the tunica media.Thawing protocol 2: All 6 (100%) samples showed loss of endothelium from the luminal surface, longitudinal corrugations in the direction of blood flow caused by smooth muscle cells contractions in the tunica media with frequent fractures in the subendothelial layerConclusionAll the samples thawed at the room temperature showed smaller structural damage to the CHARA arterial wall with no smooth muscle cell contraction in tunica media when compared to the samples thawed in a water bath.

Intersecting Worlds of Transfusion and Transplantation Medicine: An International Symposium Organized by the Canadian Blood Services Centre for Innovation.

The principal theme of the symposium was centered on how the world of regenerative medicine intersects with that of transfusion medicine, with a particular focus on hematopoietic stem cells (HSCs) and stem cell therapies. The symposium highlighted several exciting developments and identified areas where additional research is needed. A revised map of human hematopoietic hierarchy was presented based on the functional and phenotypic analysis of thousands of single stem and progenitor cells from adult bone marrow and fetal liver. These analyses revealed that multipotency is largely restricted to the HSC and multipotent progenitor compartments in adult bone marrow where most progenitors are unipotent, whereas fetal liver contains a large number of distinct oligopotent progenitors. Furthermore, unlike adult bone marrow, multipotency is extended in the downstream progenitors in the hierarchy in the fetal liver stage. Production of platelets ex vivo from HSCs is emerging as a potentially viable option because of advances in culture techniques that combine cytokine mixtures, small molecules, and shear stress. However, limited HSC expansion and low platelet yield from culture-derived megakaryocytes remain problematic. Evidence was presented to support stricter guidelines for transfusion of platelets and red blood cells practices in allogeneic HSC transplant patients, although evidence is often extrapolated from general indications. Basic principles of human leukocyte antigen testing in HSC transplant were described, emphasizing the need for a national (and global) stem cell donor registry. Ongoing research is aimed at improving cellular cryopreservation including the establishment of a new thawing protocol that improves viability of umbilical cord blood CD34+ cells. Umbilical cord blood transplantation practices have also been improved; recent studies suggest noninferior outcomes when patients are transplanted with umbilical cord blood vs a matched adult donor. Finally, mesenchymal stem cell infusion is an example of a cellular therapy useful for immunomodulation. Preclinical trials suggest that mesenchymal stem cells may be effective in managing sepsis. In conclusion, practices and research surrounding HSCs are continuing to evolve rapidly as new information is obtained.

Clinically applied procedures for human ovarian tissue cryopreservation result in different levels of efficacy and efficiency

PurposeDifferent protocols are being used worldwide for the cryopreservation of human ovarian tissue for fertility preservation purposes. The efficiency and efficacy of the majority of these protocols has not been extensively evaluated, possibly resulting in sub-optimally cryopreserved ovarian tissue. To address the impact of this issue, we assessed the effects of two clinically successful human ovarian tissue slow-freezing cryopreservation procedures on the quality of the cryopreserved tissue.MethodsTo differentiate between cryopreservation (C) versus thawing (T) related effects, four combinations of these two (A and B) very different cryopreservation/thawing protocols (ACAT, ACBT, BCAT, BCBT) were studied. Before and after cryopreservation and thawing, the percentage of living and morphologically normal follicles, as well as the overall tissue viability, was assessed.ResultsOur experiments revealed that the choice of the cryopreservation protocol noticeably affected the overall tissue viability and percentage of living follicles, with a higher viability after protocol BC when compared to AC. No statistically significant differences in tissue viability were observed between the two thawing protocols, but thawing protocol BT required considerably more human effort and materials than thawing protocol AT. Tissue morphology was best retained using the BCAT combination.ConclusionOur results indicate that extensive and systematical evaluation of clinically used protocols is warranted.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm

ABSTRACTIn addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Cryopreservation of whole bovine ovaries: comparisons of different thawing protocols

ObjectiveThe aim of this study was to perform a comparative investigation of several different thawing protocols and to determine an appropriate protocol for thawing whole bovine frozen ovaries. Study designBovine ovaries were slowly frozen and then thawed by applying different protocols. Ultrastructural change, follicle viability, and the hormone levels of culture supernatant were measured. ResultsThe percentage of morphologically normal primordial follicles and the hormone levels of culture supernatant in group D (two-step, thawing in water at 39°C) were significantly higher than those in any other group. Moreover, the ultrastructural alteration of oocyte in group D (two-step, thawing in water at 39°C) was slighter than those in any other group. ConclusionsThe two-step protocol involving short-term exposure to water at a moderately high temperature (39°C) proved to be a suitable for thawing bovine whole ovaries.

An Effective and Reliable Xeno-free Cryopreservation Protocol for Single Human Pluripotent Stem Cells.

Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined, xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free, single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10μM Rho-associated kinase inhibitor Y-27632 into two types of xeno-free, defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (~90 %) and cell expansion (~70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells.

Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta

AbstractDiverse progenitor cell populations, including mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells can be isolated from the amniotic fluid and the placenta. At least some of the amniotic and placental cells share a common origin, namely the inner cell mass of the morula. Indeed, most types of progenitor cells that can be isolated from these two sources share many characteristics. This unit will focus solely on the mesenchymal stem cells, the most abundant progenitor cell population found therein and, unlike some of the other stem cell types, present all through gestation. Protocols for isolation, expansion, freezing, and thawing of these cells are presented. Preference is given to the simplest methods available for any given procedure. © 2015 by John Wiley & Sons, Inc.

Donor eggs: on ice, still nice?

The vitrification and thawing of mature human eggs are among the newest of assisted reproductive technologies (ART), and present the opportunity for creating donor egg banks. Given some of the advantages to the recipient patient of using vitrified eggs (VE), e.g., greater selection of donors, lower cost, and more convenience with no need for cycle synchronization with the donor, ART laboratories may encounter VE cycles more frequently, requiring development of expertise in using VE. To learn how VE compare with fresh eggs (FE) and identify the technical differences in using each egg type, we compared clinical outcomes for recipients of VE and FE from July 1, 2012 through December 31, 2014. VE were purchased from a single egg bank in this study. Retrospective data analysis of IVF cycles of recipients of FE or VE from a single VE bank. Statistical analyses were conducted by Student's t-test and Mann-Whitney U test. Recipient cycles of FE or VE were compared for average number of eggs per cycle, fertilization rate, #embryo transfers, average # embryos transferred, pregnancy rate,and chance for the recipient to have supernumerary embryos for cryopreservation. FE were inseminated conventionally or by ICSI; VE received ICSI exclusively. Assisted hatching was used electively on cleavage stage embryos (CSE)from FE but on all VE CSE. Embryologists were trained in the specific thawing protocol of VE obtained from a single egg bank. 80 FE and 168 VE cycles were analyzed, with 1595 and 1171 eggs, respectively, available for the recipients (averages of 19.9 FE vs 7 VE per recipient). Fertilization rates of 76.±18.5% and 84.±18.0%)of inseminated FE and VE, respectively, were achieved(significant, p=0.0011) leading to averages of 1.2 (FE) and 1.3 (VE) embryos per transfer (no statistical difference)and 49/75 FE and 91/158 VE clinical pregnancies per ET (not significant, chi-squared=1.2699, p=0260).72.5% of FE recipients had frozen embryos after ET compared to 51.2% of VE. Despite the almost 3-fold higher number of eggs available per treatment cycle for FE vs. VE, comparable fertilization rates, application of elective single embryo transfer, and clinical pregnancy rates per ET were obtained. FE patients had an advantage in more frequently having supernumerary embryos for freezing from their cycles. VE can provide more cost-effective and convenient treatment to patients but require specific training in the thawing protocol prescribed by the egg bank to realize this advantage.

Safe and effective cryopreservation methods for long-term storage of human-amniotic-fluid-derived stem cells.

Stem cells (SCs) can be isolated from amniotic fluid (AF) for a variety of perinatal applications. In view of this, we compared different cryopreservation protocols for these AFSCs. We screened seven freezing and thawing protocols using two well-established human AFSC lines: freezing protocol 1 (FP1), 10% dimethyl sulfoxide (DMSO); FP2, 2.5% DMSO, caspase inhibitor, and catalase; FP3, 5% glycerol, caspase inhibitor, and catalase; FP4, sperm freezing medium; FP5, slow-freezing solution; FP6, ethylene glycol, sucrose, and Ficoll 70; and FP7, vitrification solution. Outcome measures were post-thawing cell viability, recovery, doubling time and mesenchymal SC markers. The three best performing protocols were subsequently tested on cells isolated from clinical consecutive freshly harvested AF samples from two fetal medicine units. Protocols 1, 5, and 6 performed significantly better on well-characterized cell lines. They performed equally well on cell pellets from freshly harvested AF (n = 28). We identified three suitable cryopreservation protocols because of high cell recovery and unchanged SC characteristics. Given one of these, the slow-freezing solution, is compatible with current good manufacturing practice legislation, it may be ultimately clinically used.

Cryopreservation of Sambar deer semen in Thailand.

Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer in developing countries.

Osmolality values of previously frozen human milk from Guatemalan mothers: Effects of temperature range for thawing and of storage duration.

BackgroundFreezing and thawing of milk alters a number of assay values and a practical perspective for osmolality is needed.ObjectivesTo determine the consistency of osmolality values in human milk (Mosm) after varying frozen storage‐time and thawing protocols.Methods14 lactating women gave a 1st full‐breast expression after 90 min of nursing abstinence and 11 returned within a wk for a 2nd extraction. 3 aliquots were stored at ‐20oC: 1st brought to 21°C and measured within 1 wk; 2nd with same procedure ~10‐11 wks later; and 3rd warmed to 37oC in a water bath and then cooled to 21oC also at this later time. Single milk collections from 16 additional women were analyzed at the final time‐point by both thawing routines. Mosm was determined on a Vogel Löser 815 osmometer and expressed as mOsm/kg.ResultsFor the subgroup of 25 samples measured twice after standard thawing (early and late), the respective Mosm medians were 288 mOsm/kg and 309 mOsm/kg (p=<0.001, Wilcoxon test). The Pearson r‐value was 0.772 (p=<0.001). This is consistent with the typical osmolality of ~300 mOsm/kg. The overall descriptive Mosm median for the 46 milk specimens measured after simple thawing was 308 mOsm/kg compared to 304 mOsm/kg; this 1.3% difference was significant (p=0.011). The Pearson correlation coefficient was r=0.847 (p=<0.001). 85% of values were within a band of ±5% of the median.ConclusionMinor variations in storage and handling produce significant, but not practically meaningful, differences for osmolality research with human milk.

Effect of freeze/thaw cycles on several biomarkers in urine from patients with kidney disease.

Urine samples were collected from eleven randomly selected patients with kidney disease, including diabetic nephropathy, chronic nephritis, and nephritic syndrome. Urine samples were treated with one of four protocols for freezing and thawing: freeze directly and thaw directly; freeze directly and thaw by temperature gradient; freeze by temperature gradient and thaw directly; and freeze by temperature gradient and thaw by temperature gradient. After one to six freeze/thaw cycles at -20°C or -80°C, different biomarkers showed differential stabilities. The concentrations of total protein, calcium, and potassium did not change significantly after five freeze/thaw cycles at either -20°C or -80°C. Albumin could only sustain three freeze/thaw cycles at -20°C before it started to degrade. We recommend that urine be stored at -80°C as albumin and the organic ions could sustain five and six freeze/thaw cycles, respectively, using the simple "direct freeze and direct thaw" protocol. Furthermore, in most cases, gradient freeze/thaw cycles are not necessary for urine sample storage.

68 DIFFERENCE IN THAWING CURVE OF STALLION FROZEN SEMEN DILUTED IN 2 EXTENDERS

Commercial freeze extenders have different composition and ratio of cryoprotectors; freezing and thawing protocols are different for each extender. The aim of this experiment was to observe the effect of thawing curve in stallion frozen semen with 2 commercial extenders. Two ejaculates from each of 9 stallions of different breeds (Quarter Horses and Mangalarga Marchador) were used. Semen was collected using an artificial vagina, and the ejaculate was divided into 2 groups following the manufacture's protocol: group 1 (INRA), in which the semen was diluted 1 : 1 with the extender INRA 96TM (IMV, Paillette Crista, France) and group 2 (BC), in which the semen was diluted (1 : 1) with the extender Botu-SemenTM (Botupharma, Brazil). The samples of the 2 groups were centrifuged at 600 × g for 10 min, the supernatant was discarded, and the pellet was resuspended with INRA FreezeTM (group INRA, IMV) and with BotucrioTM (group BC, Botupharma) at the concentration of sperm 100 × 106 sperm mL–1. After this, the semen was packaged in 0.5-mL straws. For each group the freezing process was carried out according to the manufacturer's instructions. The straws were thawed in a water bath with 3 different thawing curves: 37°C for 30 s (37/30), 46°C for 20 s (46/20), and 75°C for 7 s (75/7) before analysis. The aim of these rates is to keep the semen in 37°C post-thaw. The sperm kinetic analysis was performed by computerized method (CASA, HTM-IVOS, IMV, USA) and the analysis of plasma membrane integrity by flow cytometer (BD LSR Fortessa, Becton Dickinson, Mountain View, CA, USA). Data of sperm kinetic and of plasma membrane integrity were compared among the 3 thawing curves for one extender using analysis of variance. Differences were considered significant at a probability level of 5%. No differences were observed in total motility (%, BC 37/30 = 72.8 ± 14.4; BC 46/20 = 70.0 ± 14.2; BC 75/7 = 70.3 ± 12.0 v. INRA 37/30 = 57.2 ± 19.1; INRA 46/20 = 50.0 ± 21.9; BC 75/7 = 58.8 ± 20.8), progressive motility (%, BC 37/30 = 36.9 ± 8.2; BC 46/20 = 34.4 ± 10.5; BC 75/7 = 33.6 ± 7.8 v. INRA 37/30 = 25.3 ± 12.7; INRA 46/20 = 21.9 ± 13.9; BC 75/7 = 28.9 ± 14.8), rapid sperm (%, BC 37/30 = 59.7 ± 16.4; BC 46/20 = 56.8 ± 17.1; BC 75/7 = 58.1 ± 14.9 v. INRA 37/30 = 38.3 ± 20.9; INRA 46/20 = 35.3 ± 22.9; BC 75/7 = 44.4 ± 23.8), and plasma membrane integrity (%, BC 37/30 = 49.1 ± 14.8; BC 46/20 = 43.1 ± 13.1; BC 75/7 = 46.7 ± 11.8 v. INRA 37/30 = 32.2 ± 10.7; INRA 46/20 = 29.6 ± 10.1; BC 75/7 = 37.4 ± 9.1) among the 3 thawing curves for INRA and BC groups. In this study, we can conclude there is no influence of the 3 tested thawing curves in sperm quality for stallion frozen semen with INRA Freeze and Botucrio extenders.

43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions. Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry

Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze–thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze–thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.

Impact of using a fast-freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n=25) were frozen by conventional or a fast-freezing technique. Frozen semen was thawed by two thawing protocols (37°C 30s(-1) or 75°C 7s(-1) ). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n=25) were inseminated with 300 (n=11) or 150 (n=14) million spermatozoa. A greater (P<0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20min post-thawing in the fast-freezing technique than in the conventional one. Plasma membrane integrity was also greater (P<0.05) in semen frozen with the fast-freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast-freezing technique was 76% (19/25), and did not differ (P>0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep-horn insemination maximises the use of equine semen. The fast-freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.

111 Cryopreservation of stem cells inside alginate 3D constructs after high-voltage encapsulation

Application of stem cells in regenerative medicine require improving cryopreservation protocols for long term storage, especially for tissue-imitating 3D constructs containing cells. Encapsulation of stem cells into alginate-based 3D constructs that repeat an extracellular matrix may provide a mild environment during cryopreservation as well as the possibility for large-scale expansion. Furthermore, smaller alginate micro-beads ⩽3 μm offer additional advantages over larger ones due to higher specific surface area, less water content and improved heat and mass transfer. The gel-like structure and mild environment inside alginate micro-beads may affect the metabolic activity of encapsulated cells post-cryopreservation. The effects of high voltages, alginate encapsulation and pre-incubation time on morphology, viability and proliferation of MSCs post-cryopreservation were evaluated. Mesenchymal Stem Cells derived from the Common marmoset monkey Callithrix jacchus (MSC) were encapsulated into 1.5% (w/v) sterile medium viscosity unmodified alginate (Sigma Aldrich, filter-sterilized using 0.8–0.45–0.22 mm filter set) with a concentration 1*106 cells per ml of alginate using electro-spraying method. Final alginate micro-beads were obtained with less then 300 μm in diameter under the next process parameters: applied voltages 15, 20 and 25 kV, spraying distance 10 cm, and alginate flow rate 10 ml/h. As a negative control to high voltages, the air flow encapsulation was run in parallel. Micro-beads (0.5 ml) containing MSCs (0.5*106 cells) were either immediately frozen after encapsulation (0 h) or cultured overnight prior to cryopreservation (24 h). Cryopreservation was conducted with 1 °C/min cooling rate down to −80 °C with 10% Me2SO (v/v) as a cryoprotective agent. After 24 h of storage at low temperatures micro-beads were thawed at 37 °C with further removing of alginate using 55 mM sodium citrate for 2 min with gentle shaking. Then MSCs were either seeded for MTT test at 104 cells/well immediately after thawing (protocol from manufacturer) or cultured for 5 days followed by MTT assay. Membrane integrity of encapsulated cells before cryopreservation and after thawing was evaluated by Trypan Blue exclusion method. Metabolic activity and proliferation efficiency of MSCs were evaluated using MTT Nonradioactive Proliferation Assay (CellTiter 96, Promega, USA). The MTT data were normalized on respective control groups. The one-way ANOVA was run for statistical analysis of obtained results. MSCs can be encapsulated into alginate micro-beads with desired concentration and without significant changes in viability and metabolic activity post-encapsulation. MSCs showed normal morphology, attached and proliferated well after thawing. The incubation of MSCs inside alginate micro-beads prior to cryopreservation resulted in lower proliferation rate as compared to immediately frozen (18 ± 6% and 45 ± 8% in respect to native control) and at the same rate as frozen control. The same behavior was observed for re-passage efficiency of encapsulated and frozen MSCs. However, immediately frozen cells after encapsulation recovered at the same rate as native control, indicating an effectiveness of cell cryopreservation inside alginate micro-beads. This study shows an importance of cell cryopreservation inside unmodified alginate directly after encapsulation. Due to toxic effect of Me2SO, cryopreservation of MSCs inside alginate 3D structures using other less toxic CPAs will be conducted. Source of funding: This work is supported by funding from the Cluster of Excellence REBIRTH (DFG EXC 62/1). Conflict of interest: None declared. gryshkov@imp.uni-hannover.de

37. Cell Encapsulation into Alginate Micro-Capsules Provides Living Cells with a Mild Environment During Cryopreservation

Cell encapsulation technology has been proposed to treat a variety of chronic disorders. However, the availability of some cell types such as human stem cells, in turn, promotes the need for such cells to be preserved for longer periods of time. For this, cryopreservation procedures are commonly used, which can cause injuries to the cells. A semi-permeable membrane might protect the cells during cryopreservation, serving as reservoir for cryoprotective agents (CPAs) and further additives [1] . Furthermore, smaller alginate-based micro-capsules (diameter 250μm) offer additional advantages over larger ones for transplantation and cryopreservation owing to higher specific surface area, less water content as well as improved heat and mass transfer [2] . In this work we indicate on the possibility to encapsulate living cells into semi-permeable alginate micro-capsules via electrospraying in order to improve viability of living cells post-cryopreservation as well as to support their normal proliferation rate after thawing. NIH 3T3 fibroblasts (5∗106 cells/ml) were encapsulated into 1,5% or 2,0% (w/v) alginate micro-capsules (diameter 250μm) under sterile conditions using high-voltage processes based on previously optimized parameters. Cryopreservation was conducted under 2 K/min to − 30°C and 5 K/min from − 30°C to − 80°C freezing protocol using DMEM, 20% FCS, 10% DMSO as freezing medium. Thawing was performed at 20°C using standardized equipment. Proliferation and viability of encapsulated cells before cryopreservation and after thawing was measured using MTT and Calcein AM/Ethd assays respectively. The change in morphology of alginate micro-capsules was observed under Carl Zeiss Axiovert 200 M microscope using 5x or 10x magnifications and AxioVision V 4.8.2.0 built-in software. Alginate micro-capsules have been entrapped with NIH 3T3 fibroblasts cells at a density of 130 ± 24 cells per capsule. Preliminary results on cell survival after encapsulation showed that electrospraying is a suitable technique for living cells entrapment. Microscopic observations revealed that low temperature treatment and further thawing did not significantly affect the morphology of alginate capsules as they appeared to be stable and round in shape. The results of cell viability assays indicated an increase in viability of encapsulated cells post-cryopreservation as compared to non-encapsulated by 10%. The MTT proliferation assay showed that cells proliferate well after thawing. In order to further improve the viability of cells, the most suitable freezing and thawing protocol as well as the finding of alternative and less-harmful for the cells CPAs are of necessary and will supplement this study.

Effects of five cryoprotective agents on quality of sheep epididymal spermatozoa during pre-freezing

The objective of this study was to test five cryoprotective agents during ram epidydimal spermatozoa incubation (at 4°C), of up to 3h, typical equilibration time before the freezing step begins, in order to establish a starting point for future freezing and thawing protocols. The parameters analyzed were: progressive motility (PM), vitality, and the plasma membrane functional integrity by the hypoosmotic swelling (HOS) test. Testes and epididymides were collected immediately after death. The tail of both epididymides were isolated and spermatozoa were recovered constituting one sample (n=20). A Tes–Tris–Yolk extender was employed. The extender contained five alternative CPAs: Dimethylacetamide (DMA), Dimethyl sulfoxide (DMSO), Ethylene glycol (EG), Glycerol (GLY) and Propylene glycol (PG) at three final concentrations: 2.5%, 5.0% and 10.0%. Control groups consisted of samples mixed only with the extender, without any CPA. All sample groups were exposed to the CPAs for 1h or 3h at 4°C. EG exposure yielded better responses in both PM and HOS test parameters compared to extender only and also the other CPAs. There was no difference among all the treatments regarding vitality. EG (with best results at 2.5%) is thus proposed as a good CPA (followed by DMA as an explorable alternative) for the implementation of forthcoming ram epididymal spermatozoa freezing protocols.