Vitrification Solutions for Plant Cryopreservation: Modification and Properties

Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and must not be toxic for plants. This review introduces the compounds used for vitrification solutions (VSs), their properties indicating a modification of different plant vitrification solutions, their modifications in the compounds, and/or their concentration. An experimental comparison is listed based on the survival or regeneration rate of one particular species after using more than three different VSs or their modifications. A brief overview of various cryopreservation methods using the Plant Vitrification Solution (PVS) is also included. This review can help in alert researchers to newly introduced PVSs for plant vitrification cryoprotocols, their properties, and the choice of their modifications in the compounds and/or their concentration.

Short-term Storage of Seeds and Cryopreservation of Embryonic Axes of Lepisanthes fruticosa

Aims: This work highlights short-term storage of recalcitrant Lepisanthes fruticosa seeds and long-term conservation attempts of its embryonic axes (EAs) through cryopreservation. Study design: This study adopted the Completely Randomized Design (CRD). Ten samples were used for each experiment and replicated for 3 – 5 times. Place and Duration of Study: Cryopreservation Laboratory, Agrobiodiversity and Environment Research Centre, MARDI Headquarters, Malaysia, in 2017 and 2018. Methodology: Short-term storage was carried out using fresh seeds at 54% moisture content and stored at 8±1 °C and 25±2 °C for 7 weeks. Three variations to sterilization were attempted to optimize survival while keeping contamination low. Cryopreservation using two different methods were tested, namely vitrification and the encapsulation vitrification method. Vitrification technique involved the pre-culturing of EAs overnight in different sucrose pre-culture concentrations (0, 0.2, 0.4 and 0.6 M) prior to, loading, dehydration with vitrification solution (PVS2), rapid immersion into liquid nitrogen (-196°C), rapid warming, unloading and recovery. While, encapsulation vitrification involved encapsulation of the EAs using 3% sodium alginate followed by exposure to different duration (0, 10, 20, 30, 40 and 50 minutes) of vitrification solution (PVS2) prior to cryopreservation. Results: L. fruticosa seeds can be safely stored for short-term up to 7 weeks of storage either at 8±1 °C or 25±2 °C with no loss in germination. This study also showed that EA was amenable to cryopreservation and 13.33 – 66.67% of viability was obtained when the EAs were cryopreserved using the vitrification technique. The best result was obtained with 66.67% viability, when the EAs were pre-cultured with 0.4M sucrose prior to exposure to PVS2 and liquid nitrogen. Cryopreservation of EAs using the encapsulation-vitrification method was unsuccessful. Conclusion: Seeds of L. fruticosa can be stored for short-term (up to 7 weeks) using hydrated/non-dried seeds where they can be successfully stored at 8±1 °C and 25±2 °C for up to 7 weeks. For long-term (cryopreservation), EAs can be cryopreserved upon pre-culture with 0.4M sucrose prior to exposure to PVS2 and liquid nitrogen through vitrification technique.

Effects of sucrose and glycerol on vitrification of buffalo oocytes

Background: Vitrification, ultra-rapid cooling can be used to cryopreserve oocytes for embryo technology. The objective of this study was to evaluate the effects of sucrose and glycerol on vitrification of buffalo oocytes.Methods: Cumulus-oocyte complexes (COCs) were aspirated from slaughtered buffalo ovaries. In experiment 1, the vitrification solution was supplemented with either 0, 0.25 or 0.5 M sucrose. In experiment 2, the vitrification solution was supplemented with either 0, 5 or 10 M glycerol together with 0.5 M sucrose. COCs were exposed into equilibration solution and vitrification solution for 5 min and 1 min, respectively. Then the oocytes were submerged into liquid nitrogen for 10 min using cryotops. The oocytes were thawed, diluted and washed in washing solution. Vitrified oocytes were cultured for maturation at 38.5°C for 24 hrs at 5% CO2. Then oocytes were fixed in acetic acid and ethanol and stained with aceto-orcein to examine the meiotic stages.Results: In experiment 1, a significantly higher number of morphologically normal oocytes and cumulus cell expansion were found in 0.5 M sucrose group than others. In addition, a proportion of oocytes resumed meiosis but none of those developed to the metaphase II (MII) stage. In experiment 2, a significantly higher number of oocytes showed cumulus cell expansion as well as higher morphologically normal oocytes in 5 M and 10 M glycerol than in 0 M (control) group. In addition, 18% oocytes matured to MII stage in 5 M glycerol group.Conclusions: Buffalo oocytes can be vitrified with a combination of sucrose and glycerol to maintain its developmental potential.

Short-term storage of seeds and cryopreservation of embryonic axes of Lepisanthes fruticosa

This work highlights short-term storage of recalcitrant Lepisanthes fruticosa seeds and long-term conservation attempts of its embryonic axes (EAs) through cryopreservation. Short-term storage was carried out using fresh seeds at 54 % moisture content and stored at 8 ±1 °C and 25 ±2 °C for 7 weeks. Three variations to sterilization were attempted to optimize survival while keeping contamination low for cryopreservation. Cryopreservation using two different methods were tested, namely vitrification and the encapsulation vitrification method. Vitrification technique involved the pre-culturing of EAs overnight in different sucrose pre-culture concentrations (0, 0.2, 0.4 and 0.6 M) prior to, loading, dehydration with plant vitrification solution (PVS2), rapid immersion into liquid nitrogen (-196 °C), rapid warming, unloading and recovery. While, encapsulation vitrification involved encapsulation of the EAs using 3 % sodium alginate followed by exposure to different duration (0, 10, 20, 30, 40 and 50 minutes) of PVS2 prior to cryopreservation. L. fruticosa seeds can be safely stored for short-term with no loss in germination up to 7 weeks of storage either at 8 ±1 °C or 25 ±2 °C. This study also showed that EA of L. fruticosa was amenable to cryopreservation, 13.0 – 66.67% of viability was obtained when the EAs were cryopreserved using the vitrification technique while the best result was obtained (66.67 % viability) when the EAs were pre-cultured with 0.4 M sucrose prior to exposure to PVS2 and liquid nitrogen. Cryopreservation of EAs using the encapsulation-vitrification method was unsuccessful.

Vitrification of Intact Porcine Femoral Condyle Allografts Using an Optimized Approach

Objective Successful preservation of articular cartilage will increase the availability of osteochondral allografts to treat articular cartilage defects. We compared the effects of 2 methods for storing cartilage tissues using 10-mm diameter osteochondral dowels or femoral condyles at −196°C: (a) storage with a surrounding vitrification solution versus (b) storage without a surrounding vitrification solution. We investigated the effects of 2 additives (chondroitin sulfate and ascorbic acid) for vitrification of articular cartilage. Design Healthy porcine stifle joints ( n = 11) from sexually mature pigs were collected from a slaughterhouse within 6 hours after slaughtering. Dimethyl sulfoxide, ethylene glycol, and propylene glycol were permeated into porcine articular cartilage using an optimized 7-hour 3-step cryoprotectant permeation protocol. Chondrocyte viability was assessed by a cell membrane integrity stain and chondrocyte metabolic function was assessed by alamarBlue assay. Femoral condyles after vitrification were assessed by gross morphology for cartilage fractures. Results There were no differences in the chondrocyte viability (~70%) of 10-mm osteochondral dowels after vitrification with or without the surrounding vitrification solution. Chondrocyte viability in porcine femoral condyles was significantly higher after vitrification without the surrounding vitrification solution (~70%) compared to those with the surrounding vitrification solution (8% to 36%). Moreover, articular cartilage fractures were not seen in femoral condyles vitrified without surrounding vitrification solution compared to fractures seen in condyles with surrounding vitrification solution. Conclusions Vitrification of femoral condyle allografts can be achieved by our optimized approach. Removing the surrounding vitrification solution is advantageous for vitrification outcomes of large size osteochondral allografts.

28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).