Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification

A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after de- and rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0°C during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure.     

Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.     

Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM) and glycerol at ?80 °C and at ?196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at ?196 and ?80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10⁶; 1.59, after 6 weeks in DMSO:FBS at ?196 °C 3.0 × 10⁶; 2.38 and at ?80 °C 2.1 × 10⁶; 1.60, in DMEM:Glycerol at ?196 °C 3.6 × 10⁶; 2.33 at ?80 °C 23 × 10⁶; 1.66 and at 4 °C 3.3 × 10⁶; 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at ?196 °C showed morphology similar to fresh AM in both preservation media and AM stored at ?80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at ?196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.     

Low-temperature preincubation enhances survival and regeneration of cryopreserved Saussurea involucrata callus

Saussurea involucrata Kar. et Kir. is one of the most well-known Chinese medicinal plants, and it is utilized for a variety of medical conditions. Due to the overexploitation of this endangered species, it is crucial to develop methods for both conservation and propagation. To address this issue, we have developed and optimized a simple and effective vitrification process for the cryopreservation of S. involucrata callus tissue. The optimized method consisted of a 3-d incubation period on medium containing 0.3 M sucrose, transfer to a plant vitrification solution (PVS2) containing 30% (v/v) glycerol, 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, and 0.4 M sucrose first at 60% PVS2 for 40 min, then at 100% PVS2 for 60 min, followed by immediate immersion and storage in liquid nitrogen. To thaw the tissue, tissues were rewarmed at 40°C for 2 min. This method resulted in a survival rate of approximately 56% and a regrowth rate of approximately 40%. Survival and regrowth were significantly improved by the addition of a low-temperature preincubation step. Incubating the calli at 4°C for 12 d prior to initiating the optimized cryopreservation protocol increased the survival rate of the tissue to 75%, increased the regrowth rate to 60%, and more than doubled the number of regenerated shoots per explant. Following cryopreservation, greater than 90% of the regenerated shoots formed complete plantlets, and 81% of the regenerated plantlets survived and grew vigorously under greenhouse conditions.     

Cryopreservation,early seedling development,and genetic stability of <Emphasis Type="Italic">Oncidium flexuosum</Emphasis> Sims

An efficient cryopreservation protocol was developed for mature seeds of Oncidium flexuosum Sims. Seed morphology, protocorm formation, and early seedling development were also assessed. The effects of phloroglucinol and Supercool X-1000^® as cryoprotectant additives in the vitrification solution were investigated. Dehydration using the plant vitrification solution 2 (PVS2) for 60 and 120 min prior to immersion in liquid nitrogen promoted the highest frequency of in vitro seed germination 6 weeks following culture on half-strength Murashige and Skoog (½ MS) medium. Mature seeds submitted to vitrification for 120 min in PVS2 and 1 % phloroglucinol at 0 °C enhanced germination by 68 %, whereas in PVS2 and 1 % Supercool X-1000^® germination was just moderately enhanced (26 %). In vitro-germinating seedlings developed healthy shoots and roots without the use of plant growth regulators. After 6 months of growth, there were no differences between in vitro- and ex vitro-grown seedlings for various phenotypic characteristics, including shoot length, number of leaves, number and length of roots, and fresh and dry weight. Seedlings were transferred to greenhouse conditions and successfully acclimatized, further developing into normal plants with over 90 % survival. Comparative analysis of seedlings from control and vitrified seeds using flow cytometry indicated that no change in ploidy levels occurred as a result of cryopreservation, therefore maintaining seedlings genetic stability. In this study, vitrification with PVS2 for 120 min with the addition of 1 % phloroglucinol offers a simple, safe, and feasible protocol for cryopreservation of O. flexuosum mature seeds.     

Cryopreservation of ex-vitro-grown Rosa chinensis ‘Old Blush’ buds using droplet-vitrification and encapsulation-dehydration

Axillary buds from greenhouse-grown plants of Rosa chinensis ‘Old Blush’ were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20–80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 °C gave the best regrowth rates (82–86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.     

The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g^?1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.     

Safety and efficacy of human amniotic membrane in primary pterygium surgery

Grafts made from human amniotic membrane are used to prevent recurrence of pterygium after excision. The success of the procedure can be affected by the quality of preparation and preservation of the grafts. We prospectively evaluated the safety and efficacy of cryopreserved amniotic membrane prepared at the research tissue bank of the Biotechnology Research Center in Tripoli, Libya, and used as adjunct therapy in primary pterygium excision. Twenty-six patients (15 males and 11 females) aged 21–78 years and indicated for primary pterygium excision were transplanted at the Tripoli Eye Hospital with the amniotic membrane grafts. Sixteen patients (62 %) were available for all three follow-up visits scheduled at 1, 3 and 6 months post-surgery. By the third visit, two patients (12.5 %) developed granuloma and three (18.8 %) had pterygium recurrence. The grafts were used after cryopreservation for ≤180 days or >180 days, but statistical analysis showed that the complications were not associated with the length of storage. Moreover, the high rate of complications in this study was not caused by use of cryopreserved AM. In conclusion, locally produced cryopreserved AM is safe as an adjunct therapy for treatment of primary pterygium excision.     

Vitrification of one-cell mouse embryos in cryotubes

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-μl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

Conservation,ex vitro direct regeneration,and genetic uniformity assessment of alginate-encapsulated nodal cuttings of <Emphasis Type="Italic">Sphagneticola calendulacea</Emphasis> (L.) Pruski

A well-organized procedure was established for the conservation and distribution of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] for the first time, using alginate-encapsulated nodal segments (NSs) as synthetic seeds. The ideal beads were obtained through a combination of 2.5% sodium alginate and 75 mM calcium chloride with 84.40 ± 2.20% rate of shoot emergence. The maximum regeneration (88.84 ± 2.24%) from synthetic seeds was achieved on liquid 1/2Murashige and Skoog (MS) medium in comparison to its other formulations. Furthermore, superior frequency (91.09 ± 2.24%) of complete plantlet (having both shoots and roots) formation was achieved when synthetic seeds were cultured on liquid 1/2MS (1.5% sucrose) fortified with 1.0 mg L^?1 N₆-benzyladenine plus 0.25 mg L^?1 α-naphthalene acetic acid. Synthetic seeds could be effectively stored at low temperature (8 °C) up to 90 days with a survival rate of 52.38 ± 3.06%, whereas higher temperature (25 °C) did not support regeneration after 75 days of storage. The plantlets were successfully acclimatized to natural conditions with ~ 89% survival frequency. To by-pass the time-consuming in vitro culture step after encapsulation, synthetic seeds were directly regrown into complete plantlets ex vitro on sand, soil, and vermicompost (1:1:1; w/w). Regeneration rate of 42.22 ± 2.22% was attained when NSs were pretreated on 1/2MS medium containing 4.0 mg L^?1 indole-3-acetic acid for 24 h in dark, prior to encapsulation. The random amplified polymorphic DNA and intersimple sequence repeat fingerprinting profiles demonstrated genetic uniformity amongst the regenerated plantlets, in vitro mother plant, as well as in vivo wild plant.     

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

In vitro conservation of chestnut (Castanea sativa) by slow growth

Slow growth storage has been achieved for Castanea sativa (cv. ‘Montemarano’) shoot cultures over a duration of 48 mo at a temperature of 8°C, where 82% of explants survived and were able to resume normal growth after transfer to standard culture conditions at 23°C. The evaluation of the chlorophyll content of leaves also showed no differences between material stored for 48 mo and control material subcultured at 23°C. With a storage temperature of 4°C, the survival of shoots was significantly lower at approximately 56% after 12 mo, and no plants recovered after 24-mo storage. The presence of 6-benzyladenine 0.44 μM in the culture medium proved to be necessary for the recovery of healthy shoots, while pre-treatments with different concentrations of abscisic acid did not significantly influence the survival of shoots following storage conditions. A low level of light during slow growth storage resulted in positive effects on the rate of shoot survival over the longest preservation periods.     

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.     

Selenium Modifies the Effect of Short-Term Chilling Stress on Cucumber Plants

The objective of this study was to investigate the effect of selenium (Se) supply (0, control; 2.5, 5, 10, or 20 μM) on cucumber (Cucumis sativus L.) cv. Polan F1 plants grown under short-term low temperature stress. About 14–16 day-old seedlings, grown at an optimal temperature (25/20°C; day/night), were exposed to short-term chilling stress with a day/night temperature of 10°C/5°C for 24 h, for a further 24 h at 20°C/15°C, and then transferred to 25/20°C (re-warming) for 7 days. Se did not affect the fresh weight (FW) of plants at a concentration of 2.5–10 μM, but in the presence of 20 μM Se, the biomass of shoots significantly decreased. The contents of chlorophylls and carotenoids witnessed no significant change after Se supplementation. Compared with the control, the Se-treated plants showed an increase of proline content in leaves, once after chilling and again after 7 days of re-warming. However, proline levels were much higher immediately after chilling than after re-warming. The malondialdehyde (MDA) content in the root of plants treated with 2.5–10 μM Se decreased directly after stress. This was in comparison with the plants grown without Se, whereas it increased in roots and leaves of plants exposed to 20 μM Se. Seven days later, the MDA level in the root of plants grown in the presence of Se was still lower than those of plants not treated with Se and generally witnessed no significant change in leaves. Although Se at concentrations of 2.5–10 μM modified the physiological response of cucumber to short-term chilling stress, causing an increase in proline content in leaves and diminishing lipid peroxidation in roots, the resistance of plants to low temperature was not clearly enhanced, as concluded on the basis of FW and photosynthetic pigments accumulation.     

Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro

Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.     

Cryopreservation of pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method

Our present study constitutes the successful and efficient protocol for cryopreservation of Dendrobium chrysanthum. D. chrysanthum Wall. ex Lindl. is a pharmaceutically valuable, ornamental epiphytic orchid of temperate and subtropical regions. On account of excellent herbal medicinal value and horticultural importance, D. chrysanthum is becoming rare due to over exploitation. For long-term conservation of this orchid, protocorm-like bodies of D. chrysanthum were excised and used for cryopreservation by encapsulation–vitrification. In this cryogenic procedure, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix (comprising of 3 % (w/v) sodium alginate and 0.1 M CaCl₂). Encapsulated protocorm-like bodies (PLBs) were then precultured on MS liquid medium supplemented with different concentrations of sucrose (0.06, 0.3, 0.5, 0.7 M), and loaded in a loading solution (comprised of 2 M glycerol and 0.4 M sucrose) for different duration to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the PLBs were subjected to PVS2 (Sakai et al. 1990) treatment at different time of exposure (minutes) and temperatures (0 °C and 25 °C). Encapsulated–vitrified PLBs were plunged directly into liquid nitrogen and stored for 1 h. Optimum result (survival 63.2 % and regrowth 59.9 %) was obtained when the beads treated with loading solution for 80 min followed by PVS2 treatment for 100 min. Regenerated plants showed normal morphology as that of control plants.     

The use of autologous fibrin as a scaffold for cultivating autologous conjunctiva in the treatment of conjunctival defect

The purpose of this study was to compare the use of autologous fibrin to human amniotic membrane (HAM) as a scaffold in cultivating autologous conjunctiva for transplantation in treatment of conjunctival defect. An experimental study was performed using 18 adult New Zealand white strain rabbits which were divided into 3 groups. Each group consists of 6 rabbits. The conjunctiva on the temporal site was excised to create a conjunctival epithelial defect. The excised area in the Group 1 was transplanted with autologous conjunctiva cultivated on autologous fibrin; Group 2 was transplanted with autologous conjunctiva cultivated on HAM and Group 3 was left bare. The rabbits were followed up at regular intervals until 6 weeks. The mean period of complete conjunctival epithelization was 11.50 ± 8.22 days for the autologous fibrin group, 15.33 ± 11.80 days for the HAM group and 25.33 ± 5.32 days in the bare sclera group. The epithelization rate for the autologous fibrin group was faster compared to the other two groups. However all the results were not statistically significant (p value >0.05). There were no postoperative complications noted during the follow up. Autologous fibrin is comparable to HAM as a scaffold for cultivation of conjunctiva in the treatment of conjunctival defect.     

Impact of prolonged oocyte incubation time before vitrification on oocyte survival,embryo formation,and embryo quality in mice

Oocyte incubation time before freezing is one of the factors affecting oocyte vitrification. In the assisted reproductive technology (ART) clinics, it is sometimes decided to perform oocyte vitrification after a long period of incubation time due to various conditions, such as inability to collect semen samples, unsuccessful urological interventions (PESA, TESE, etc.), or unexpected conditions. A time factor of up to 6 h has been studied in the available reports. Therefore, this study was designed to evaluate oocyte incubation time before freezing at 0, 6, 12, 18, and 24 h after retrieval. Metaphase II (MII) oocytes were obtained from NMRI female mice after being randomly divided into the five groups of 0, 6, 12, 18, and 24 h of freezing via hormonal stimulation following retrieval and entered into the vitrification-warming process. The thawed oocytes were evaluated according to the survival criteria and then inseminated with the sperms of male mice for in vitro fertilization. The next day, the embryo formation rate and embryo quality were assessed. Our results demonstrated that even after 24 h of incubation, the survival rate of oocytes was 51.35% with the embryo formation rate of 73.21%. However, the survival and embryo formation rates significantly decreased within 12, 18, and 24 h after retrieval compared to the groups vitrified at 0 h. The embryo quality was significantly reduced by vitrification at 0 to 24 h after retrieval. According to our data, although a prolonged incubation time before freezing reduced the survival rate, there was still a chance for oocytes to stay alive with acceptable embryo formation and quality rates after vitrification warming of oocytes.