Open Pulled Straw Vitrification Method Research Articles

Thin-Film Evaporation Heat Transfer of Liquid Nitrogen and Its Application in Cell Vitrification

Abstract Cell vitrification has been an important method of cell cryopreservation. The faster the cooling rate is, the higher the cell survival rate is. However, in conventional cell vitrification methods, film boiling forms a vapor-blanket on the surface, which hinders further improvement of the cooling rate. To eliminate the problem, this article attempted to replace film boiling with thin-film evaporation (TFE) of liquid nitrogen. The experimental system was developed to investigate the TFE heat transfer characteristics of liquid nitrogen. Then, prostate cancer cells were cryopreserved by TFE vitrification method, open pulled straw vitrification method, and equilibrium freezing method. The results showed that the vitrification method of TFE obtained a higher cooling rate and better cell survival rate than the two other cell cryopreservation methods. Thus, the feasibility of this method was preliminarily proved viable when applied to the cell vitrification process. In addition, both the cooling rate and the cell survival rate are affected by the concentration of the cryoprotectant in the cell suspension. The cooling rate decreases as the concentration of the cryoprotectant increases, but the cell survival rate increases first and decrease afterward with an increase in the cryoprotectant concentration, in which an optimum value exists. This study demonstrates the practicality of the new ultrafast cell vitrification method.

47 COMPARISON BETWEEN CRYOLOOP AND OPEN PULLED STRAW VITRIFICATION METHODS FOR BOS INDICUS BLASTOCYSTS

A major obstacle of large-scale commercial application of bovine in vitro fertilization is the lack of a suitable cryopreservation method for supernumerary embryos produced. The traditional slow-freezing method has proven to be effective for embryos of a wide range of mammalian species; however, the formation of intracellular ice is still a challenge and the efficiency needs to be improved. Over the past decade, several advances have taken place in vitrification technologies, such that it can provide high efficiency with better pregnancy outcome due to its high cooling rates and the lack of crystals formed inside the cells. Most vitrification methods have been evaluated in Bos taurus cattle but more still remains to be investigated in Bos indicus races predominant in the tropics. There are several vitrification protocols and holders, including CryoLoop, open pulled straw (OPS), MS Grids, and Cryotop, among others. The CryoLoop method uses a nylon loop attached to a metal Cryovial lid were blastocysts are placed on an equilibration solution film. CryoLoop cooling rates are approximately 20.000°C min–1 and have shown very good results in humans. The OPS is a well-known support for bovine blastocysts; the embryos are taken by capillarity into the OPS and use a 1- to 2-μL drop of final equilibration solution. Cooling rates using this method are approximately 2.000°C min–1. The aim of this work was to prove CryoLoop and OPS vitrification methods in Bos indicus blastocyst and compare re-expansion and hatching rates 24 h after warming. Ovaries were collected from a local slaughterhouse and cumulus-oocyte complexes (COC) were treated for the standard IVF method. A total of 60 blastocysts were vitrified in CryoLoops and 68 blastocysts in OPS (within 4 repeats). For CryoLoops, groups of 2 blastocysts were placed in a solution of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3 min, and then were placed in a solution of 15% EG, 15% DMSO, 10 mg mL–1 of Ficoll 70, and 0.65 M sucrose for 20 s, and rapidly were put into the nylon loop and taken to the LN. For OPS, groups of 2 to 3 blastocysts were placed in a solution of 10% EG and 10% DMSO for 1 min, and then were placed in a solution of 20% EG and 20% DMSO for 20 s, and rapidly were taken by capillarity into the OPS and taken to the LN. Thawing was the same for both treatments; vitrified blastocysts were taken out from the LN and rapidly put into a solution of 0.3 M sucrose for 2 min and then put into a solution of 0.2 M sucrose for 3 min, were washed twice in TCM199 supplemented with 10% FCS, and cultured for 24 h in CR1aa media. Data were analysed using the R language. Media comparison for proportions was done using a chi-squared test. No significant difference was observed in re-expansion or hatching rates between CryoLoop and OPS supports (P = 0.01 for both); however, the CryoLoop method showed more efficiency than OPS in re-expansion rate (65 v. 44.4%, respectively) and hatching rate (30.8 v. 20%, respectively). In all cases, the CryoLoop method showed much better outcomes. The results indicate that vitrification in CryoLoops is a suitable method for cryopreservation of Bos indicus blastocysts.

Cryopreservation of Human Embryonic Stem Cells with a New Bulk Vitrification Method1

To increase the manipulation efficiency and storage capability of vitrified human embryonic stem cells, a new bulk vitrification method was established using transformed cryovials. This method vitrified a large number of cell clumps, as opposed to those cryopreserved by a slow-freezing method with conventional cryovials at one time (round). After warming, vitrified human embryonic stem cells exhibited a much higher survival rate than the slow-freezing cells. The vitrified stem cells continued to express markers of pluripotency and formed teratomas in mice with severe combined immunodeficiency, confirming the pluripotency of vitrified-warmed human embryonic stem cell clumps. The new bulk vitrification method is superior to and more practical than the open pulled straw vitrification method and the slow-freezing method for the cryopreservation of human embryonic stem cells.

Effect of Different Combinations of Cryoprotectants onIn VitroMaturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open-Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open-pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 +/- 1.9% in EG, 47.5 +/- 3.4% in EG + DMSO, 36.8 +/- 1.2% in EG + glycerol and 29.9 +/- 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 +/- 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase-I or metaphase-II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 +/- 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 +/- 0.6% for EG + glycerol and 17.0 +/- 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.

101 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

In the production of cloned pigs, a large number of nuclear transfer (NT) embryos generally need to be transferred into a single surrogate. Thus, attempts to conduct embryo transfer can be frustrating when either a synchronized surrogate is not available, or enough NT embryos are not produced. This problem would be solved if one could cryopreserve the porcine nuclear transfer embryos. Cryopreservation of porcine embryos has been successful only for in vivo-derived embryos. In vitro-derived porcine embryos are sensitive to chilling, and this sensitivity has been attributed to the lipid droplets in the cytoplasm. In previous reports, the viability of cyropreserved embryos was improved by removal of lipid drops from the cytoplasm. Therefore we designed a procedure to cryopreserve cloned blastocysts by a combination of the open pulled straw (OPS) vitrification method with removal of lipid drops from the oocyte. In vitro-matured MII oocytes were enucleated, and centrifuged (10 000 rpm, 5 min) to polarize the lipid droplets. This was followed by removal of the polarized lipid droplets and transfer of a donor fetal-derived fibroblast cell into the perivitelline space by micromanipulation. After electrical activation and fusion, the NT embryos were cultured in PZM-3 medium with 4 mg/mL BSA. Day 5 and Day 6 blastocysts (manipulation day was Day 0) were vitrified by equilibration with 25 mM HEPES-buffered TCM-199 containing 10% ethylene glycol, 10% DMSO, and 20% fetal calf serum for 2 min, followed by exposure to 20% ethylene glycol and 20% DMSO. Embryos were loaded into an OPS straw and immediately plunged into liquid nitrogen. The process from exposure of embryos to vitrification solution to plunging was 25–30 s. Embryos were thawed by immersing the end of the OPS straw in 0.3 M sucrose in which embryos were kept for 5 min, and then in 0.2 M sucrose for 5 min. Some embryos were cultured in PZM-3 for 12 h to determine the percentage and cell number of re-expanded blastocysts. The others were transferred to the uterus of a surrogate gilt within 3 h of thawing. Lipid removal appeared to have no harmful effect on embryo development and cell number of the blastocysts. Interestingly, a higher blastocyst percentage (28.8%, 178/619) was obtained with NT embryos from which the lipid had been removed as compared to normal NT (19.6%, 44/225; P &lt; 0.01). The cell number (31.2 ± 7.7) of re-expanded blastocysts in the delipation group was comparable with normal NT blastocysts (33.6 ± 14.1, P = 0.33). The survival rate of blastocysts after freezing and thawing was enhanced after delipation (delipation group: 66.7%, 14/21; normal NT group: 21.9%, 9/42; P &lt; 0.01). Two hundred and fourteen delipatized NT blastocysts were transferred to four surrogates after freezing and thawing. Three of the surrogates showed a delayed estrus cycle and one is still pregnant as confirmed by ultrasound scanning. We show that the combination of the OPS vitrification method with removal of lipid drops of oocyte cytoplasm might be an efficient way to cryopreserve porcine NT blastocysts. Funding for this project was from the NIH HL51670 and RR018877 and Food for the 21st Century.

Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts

The aims of this study were: (1) to evaluate the effect of the number of previous estrus of recipient gilts on effectiveness of intrauterine insertion of a flexible catheter designed for non-surgical deep intrauterine catheterization during diestrus in pigs; and (2) to determine the farrowing rate and the litter size after non-surgical deep intrauterine embryo transfer (ET) of porcine blastocysts vitrified by the open pulled straw (OPS) method. In experiment 1, 27 large white hyperprolific gilts (LWh) with 2–6 previous estrus were used. Intrauterine insertions of the flexible catheter were carried out at day 5.5–6 of the estrous cycle (D0 = onset of estrus). During insertions, no or only moderate reactions were observed in 88.9% of gilts and was not related ( P > 0.05) to the number of estrus prior to the insertion periods. The number of the estrus had a significant effect ( P < 0.05) on the difficulties found during the procedure. In the 100% of gilts with two estrus ( N = 6) it was not possible to insert the flexible catheter through the cervix. In gilts with three or more estrus, it was possible to pass the cervix and to progress along a uterine horn in 80.9% of the cases. In 86.7% of the gilts, the tip of the flexible catheter achieved the second or third quarter of the uterine horn. In experiment 2, following non-surgical deep intrauterine transfer of 20 vitrified/warmed blastocysts, 9 Meishan recipients (42.9%) farrowed an average of 5.4 ± 0.8 piglets (range 3–9) of which 0.6 ± 0.3 piglets (range 0–2) were born dead. In conclusion, this study shows that it is possible to obtain birth of piglets following non-surgical deep intrauterine embryo transfer (ET) of vitrified/warmed blastocysts. Non-surgical deep intrauterine ET and OPS vitrification methods are promising procedures to be used together for the introduction of new genetic material in a farm.

Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.

Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.