Conventional Freezing Technique Research Articles

Isochoric freezing: An innovative and emerging technology for retention of food quality characteristics

Freezing is a well-established and long-term food preservation method. The most commonly used conventional freezing method follows isobaric (constant pressure) approach, which causes number of irreversible nutritional, mechanical, sensorial, and microstructural changes that make food unacceptable to consumer. The current pandemic has increased demand for food products with higher nutritional and physicochemical properties. These factors have led to the development of an isochoric freezing system. Isochoric freezing has great potential to retain the quality properties of food. Understanding the concept and application of the isochoric freezing system in the food industry is necessary to investigate its effect on various quality characteristics. Thus this review presents an outline of the working principle and impact of isochoric freezing on retention of nutrients, physicochemical and bioactive compounds of food products, and highlights current challenges and future needs by considering research published in previous years. A special section also shows the applications of isochoric freezing in medical field. Practical applications Isochoric (constant volume) freezing has emerging application in the food industry because of protects preserved food from ice damage maintains its quality properties, reduces up to 70% energy consumption, has less operating cost, and is environmentally friendly. Fewer temperature fluctuations will also experience in food products stored in the isochoric system as temperature increases continuously, resulting in ice melting inside the chamber rather than heating the food product. In conclusion, the isochoric freezing system has enormous potential in food industries in retaining nutrient and bioactive compounds compared to conventional freezing techniques.

Cryopreservation of small numbers of human spermatozoa in a Stripper tip: Report of the first live-birth worldwide

PurposeThe aim is to report the first live-birth following ICSI using spermatozoa previously vitrified in a Stripper Tip. Principal resultsA 34-year-old cryptozoospermic man was enrolled in a sperm vitrification program. After failure of conventional freezing technique, spermatozoa were vitrified using two carriers: a commercially-available, Cell Sleeper, and a “home-made” one, Stripper Tip. This man and his 30-year-old wife underwent an ICSI attempt using vitrified-warmed spermatozoa from these devices. All frozen-warmed spermatozoa were quickly recovered. Among the oocytes retrieved, six were injected with sperm from the Cell Sleeper, and seven with sperm from the Stripper tip, leading to 4 embryos in each case. Two embryos, arising from sperm frozen in the Stripper tip, were transferred, resulting in a healthy live-birth. ConclusionsThis is the first successful delivery following the use of spermatozoa frozen in an original device, the Stripper Tip, giving a promising prospect for managing severe male infertilities.

Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa.

The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O2 with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N2 gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

Short communication: The percentage of egg yolk in the freezing media affects mouflon (Ovis musimon) epididymal sperm cryosurvival

Sperm cryopreservation protocols are not well defined in many wild species such as the mouflon. The aim was to study the effect of different concentrations of EY on mouflon epididymal sperm cryosurvival. Samples were collected by the flushing method and cryopreserved by the conventional freezing technique in straws using a TEST (TES, Tris, glucose) 5% v/v glycerol medium containing either 6% v/v (n=16) or 12% v/v (n=13) clarified EY. The membrane integrity, acrosome integrity, motility, and morphological abnormalities were assessed in fresh and frozen/thawed samples. Fresh sperm quality parameters did not differ between groups except for the acrosome integrity that was lower in the TEST-6% EY than in the TEST-12% EY group (88.9 ± 2.1% vs 94.7 ± 0.8%). Membrane integrity (31.6 ± 4.6% vs 11.6 ± 4.5%), total motility (32.8 ± 4.6% vs 17.2 ± 5.6%), progressive motility (13.3 ± 2.7% vs 6.1 ± 2.9) were higher in frozen-thawed sperm with TEST-6% EY than with TEST-12% EY (p<0.05). Other motility parameters such as curvilinear velocity, straight-line velocity, average path velocity and amplitude of lateral head displacement were also higher (p<0.05) in frozen-thawed sperm with TEST-6% EY. Frozen-thawed acrosome integrity (85.1 ± 3.3% vs 91.9 ± 2.3) and morphological abnormalities (34.0 ± 3.7% vs 29.1 ± 3.6%) did not differ between extenders. In conclusion, high EY concentration had detrimental effects on post-thaw quality parameters, therefore the use of TEST based extender containing 6% EY is recommended for the cryopreservation of mouflon epididymal sperm.

22. Avoiding the problems of ice in tissues via vitrification

Cryopreservation involving freezing in the presence of protective agents such as dimethyl sulfoxide is a well-established method for storing cells and small tissues. While successful techniques for cryopreservation have been developed over the past seven decades, they are generally related to small specimens, in the scale-range of cell suspensions and clusters to small-organized tissues ( μ m to mm range), with blood cells, stem cells, embryos and pancreatic islets as examples. Cryopreservation of larger-size specimens (cm and above) has been accomplished only in cases where the mechanical functionality has a higher priority need than the recovery of biological functionality, as with heart valves. There is evidence that ice formation within the extracellular matrix of multicellular tissues is the principal event that limits the survival of cryopreserved tissues using conventional freezing techniques. This mode of injury can be circumvented by using an ice-free cryopreservation approach, known as vitreous cryopreservation. Vitrification, the amorphous solidification of a supercooled liquid, can be achieved by adjusting the solute composition and thermal processing rates such that nucleation and growth of ice crystals are essentially prevented. Avoidance of ice by vitrification can be generally achieved with one of two approaches or a combination of both. The first approach employs cooling highly concentrated solutions (typically > 50% w/w) that become sufficiently viscous at low temperatures to suppress crystallization rates. Typically, a vitrified material is considered solid when the viscosity reaches 10 15 poise. Vitrification can also be achieved by selecting sufficiently high cooling rates to prevent ice crystallization in relatively dilute solutions ( 50% w/w). This second approach generally produces a metastable state that is at risk of devitrification and/or recrystallization during warming. Ice formation during warming is just as potentially injurious as during cooling. Applications of the vitrification approach to the cryopreservation of viable tissues such as blood vessels and cartilage has been demonstrated to result in a dramatic improvement in post-rewarm functionality from a meagre 20% after classical freezing to a respectable > 80% after ice-free cryopreservation. These achievements provide confidence and enthusiasm that this approach holds great promise for future cryobanking of complex bulky tissues and organs, with an isolated case of kidney cryopreservation having been reported. Nevertheless there are well recognized challenges that remain to be investigated and resolved. While vitrification is a well-understood phenomenon, its application to biological systems comes with potentially harmful effects of toxicity of the cryoprotectants and structural damage due to thermo-mechanical stresses. In fact, these effects represent competing needs important for selecting cryoprotectants and their concentrations, and represent a significant barrier to the development of cryopreservation technology. New approaches to alleviate this couple molecules that modulate ice nucleation and growth with the cryoprotectant cocktail. Both natural and synthetic compounds are being explored as a new class of additives that are anticipated to be essential for ice-free cryopreservation of bulky tissues and organs. In addition, new innovative technologies are conceived to address the challenges associated with the needed for uniform heating of vitrified tissues to avoid the problems of devitrification and recrystallization during warming.

Feline spermatozoa from fresh and cryopreserved testicular tissues have comparable ability to fertilize matured oocytes and sustain the embryo development after intracytoplasmic sperm injection

Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%–1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing technique with glycerol as a principle cryoprotectant is simplified and applicable for cat testicular tissue cryopreservation. We also demonstrate for the first time that feline spermatozoa derived from frozen-thawed testicular tissues retain their fertilizing ability and can be used to produce ICSI-derived embryos.

95 THE EFFECT OF FREEZING TECHNIQUES ON QUALITY OF CAT TESTICULAR SPERM

Cryopreservation of testicular tissue is beneficial for valuable animals that die unexpectedly or when elective castration is required. Until recently, knowledge regarding cryopreservation of testicular tissue/sperm in the domestic cat has been limited. The objective of this study was to determine the effects of freezing techniques and cryoprotectants on quality of testicular sperm. In Experiment 1, each testis was cut into 10 equal small pieces (∼2 × 3 × 5 mm) and cryopreserved in freezing medium containing with 5% (v/v) glycerol using conventional (10 min in liquid nitrogen vapors) or controlled-rate freezing techniques. In Experiment 2, testicular tissues were conventionally frozen with different types of 5% (v/v) cryoprotectants [glycerol (Gly), dimethylsulphoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG)]. Non-cryopreserved testicular sperm was used as a control. After thawing, testicular sperm were extracted and examined for viability and DNA integrity using non-membrane-permeable DNA staining (Ethidium homodimer-1) and TUNEL assay, respectively. Viability of testicular sperm cryopreserved by controlled-rate cryopreservation (45.9 ± 3.7) was significantly lower than non-frozen control (60.3 ± 0.9) and conventional freezing technique (55.0 ± 2.7). Gly (58.2 ± 2.6) and EG (53.3 ± 2.3) yielded a similar viability compared with non-frozen control (P &gt; 0.05), whereas DMSO and PrOH demonstrated an inferior cryoprotectant for feline testicular sperm (% viability for DMSO and PrOH: 46.3 ± 3.3 and 44.3 ± 2.9, respectively). In both experiments, DNA integrity of frozen–thawed testicular sperm did not significantly differ from the control group. In conclusion, cat testicular tissue can be frozen as small pieces using both conventional technique and controlled rate freezing. However, freezing technique and type of cryoprotectant markedly affect the post-thawed quality of testicular sperm. Further study requires examination of the optimal cooling rate during cryopreservation and also the fertilizability of frozen–thawed testicular sperm. This study was financially supported by CHE-TRF Senior Research Scholars RTA-5080010.

Directional freezing as an alternative method for cryopreserving rhesus macaque ( Macaca mulatta) sperm

The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.3, 61.1, and 50.3%, respectively). Furthermore, sperm cryopreserved with 5% glycerol and a DF technique had similar frozen-thawed sperm motility to those cryopreserved by a CF technique (63.7 vs. 53.9%, P > 0.05). The function of sperm cryopreserved at the optimized cooling rate using a DF technique was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated rhesus macaques. Of the 38 mature oocytes collected, 78.9% were fertilized and 71.1, 47.4, and 42.1% of the oocytes developed to the 2-cell, morulae, and blastocyst stages, respectively. In conclusion, rhesus macaque sperm was effectively cryopreserved using a DF technique, providing a new and effective method for genetic preservation in this important species.

Protocol Development for Vitrification of Tissue-Engineered Cartilage.

The success of tissue-engineered cartilage constructs (TECCs) as treatment options for healing cartilage defects can only be achieved if suitable preservation methods are found that can maintain their viability and function. Simply lowering the temperature of cells and tissues to below their freezing point invariably destroys them due to ice crystals that form in the water-laden cells and tissues. In addition, high salt concentrations that result from removal of water due to ice formation create a toxic imbalance. If the formation of ice crystals can be minimized while still halting metabolic activity of cells at low temperatures, then the viability and functionality of the preserved tissue may be maintained. Several important variables have been studied that affect cryopreservation of cells and tissues to maximize cell survival and tissue functionality: cryoprotectant agents, cryoprotectant concentrations, methods of introduction and elution, cooling and warming rates, and storage temperatures.[1–5] In 1949, Polge et al.[6] discovered a significant breakthrough in the role of glycerol as an intracellular cryoprotectant, which permeates cells to minimize intracellular ice formation. Later on, other intracellular cryoprotectants were discovered such as dimethylsulfoxide (Me2SO)[7] and extracellular cryoprotectants such as starches and sucrose, which protect cell membranes or act as osmotic buffers.[2,8] Formamide, acetamide and other such chemicals reduce the toxicity of cryoprotectants used in high concentration. Amides, in general, are weak cryoprotectants but improve cell viability when combined with cryoprotectants.[9] Combinations of intracellular and extracellular cryoprotectants can have additive or synergistic effects on cell viability upon rewarming of cryopreserved samples.[1] There are two main approaches to cryopreservation for cells, tissues, and organs. The first is the conventional freezing technique where up to 30% of cell water is substituted by a cryoprotective compound, usually Me2SO, permitting the storage of many types of cells in vitrified channels within ice.[1] Conventional freezing relies on slow cooling and warming rates. In contrast, vitrification uses high concentrations of cryoprotectants resulting in greater than 50% replacement of water in the cell or tissue. When combined with fast cooling and rewarming rates, both intracellular and extracellular ice formation is avoided. This method minimizes or prevents formation of ice, creating a glass-like material, while still halting metabolic activity and maintaining an osmotic balance between cells and their environment. The major limitation of vitrification, however, is the potential cytotoxicity of the high cryoprotectant concentrations employed.[1] Vitrification has been shown to provide effective preservation for a number of tissues including oocytes, early embryos, cartilage, skin, blood vessels (natural and engineered), and heart valves.[10–19] Furthermore, vitrified arterial blood vessels and tissue-engineered vascular grafts have been shown to maintain viscoelastic properties similar to fresh vascular tissues while demonstrating superior biomechanical performance when compared with frozen cryopreserved specimens.[15–17] Song et al. achieved significantly better viability by employing vitrification (80%) with a 55% (w/v) vitrification solution (VS), known as VS55, than with conventional freezing (12.8%) employing 0.6 mm thick rabbit articular cartilage.[20] Guan et al. demonstrated that 51% viability occurred when bovine cartilage was vitrified, and only 5% viability when bovine cartilage was preserved without cryoprotectants.[21] Subsequently, we found that VS55 was inadequate for preservation of thicker, large mammal cartilage and that better preservation was obtained using a more concentrated 83% vitrification formulation.[22, 23] Kuleshova et al. demonstrated the need to consider different approaches in the preservation of engineered tissues due to potential interactions between the scaffold material and tissue matrix.[24] There are three main variables affecting cell viability during cryopreservation by vitrification (cooling/warming rates, effective tissue permeation, and cytotoxicity) and protocols that work for one tissue type do not necessarily apply for other tissues or, in the case of cartilage, even the same tissue type from different species. The investigation of long-term storage is an important field in tissue engineering. The main reason for this study was to determine whether vitrification protocols developed for native articular cartilage were effective for tissue-engineered cartilage constructs. TECCs were harvested from a bioreactor, vitrified employing different protocols, and cell viability was assessed after rewarming and cryoprotectant removal. The end goal of this study was to employ a bioreactor, the perfusion concentric cylinder bioreactor, to aid in the cryopreservation of tissue-engineered cartilage.

116 IMPROVED SURVIVAL BY CRYOPRESERVING RHESUS MACAQUE (MACACA MULATTA) SPERMATOZOA WITH DIRECTIONAL FREEZING TECHNIQUE

A significant increase in nonhuman primate models of human diseases will be expected in the near future since the successes in production of genetically engineered rhesus monkey models of human diseases. Sperm banking can provide an effective way to preserve valuable genetic resources. Our objective was to (1) develop a protocol using directional freezing technique (DFT) for rhesus monkey spermatozoa cryopreservation, which allows precise control of the velocity and the morphology of the ice-front propagation by transferring the tubes loaded with 2 mL sperm samples at a controllable velocity through two separate chambers with controllable temperature settings, and (2) achieve survival rate that was higher than that achieved with conventional freezing technique (CFT), by which sperm samples were cryopreserved in 0.25 mL straws with liquid nitrogen vapor in a styrofoam box. Sperm motility, acrosomal integrity, and in vitro fertilization (IVF) assay were used to assess the function of frozen-thawed spermatozoa. Data were analyzed by ANOVA and Fisher protected LSD test. Experiment 1 was aimed at optimizing the cooling rate using DFT. Tubes were frozen using the multi-thermal gradient freezing device (MTG 516, Harmony CryoCareTM, IMT Ltd.) at fast (16°C/min), medium (12°C/min), and slow (7°C/min) cooling rates, which corresponded to the transferring velocities (2.5, 1.5, and 0.5 mm s-1, respectively). The results showed that spermatozoa frozen at fast and medium cooling rates showed significantly higher frozen-thawed motility than those frozen at slow cooling rate (61% and 59% v. 50%, P &lt; 0.05). However, no difference was observed on sperm acrosomal integrity among the experimental groups (84, 80, and 78%, respectively, P &gt; 0.05). The purposes of Experiment 2 were determined to examine if using DFT at the optimized cooling rate (12°C/min) can improve the cryo-survival of rhesus monkey spermatozoa compared with CFT. Our results showed that spermatozoa cryopreserved by using DFT achieved significantly higher frozen-thawed sperm motility that those cryopreserved by using CFT (64 v. 54%, P &lt; 0.05). However, no difference was observed on acrosomal integrity between spermatozoa cryopreserved by DFT and CFT (84 and 83%, respectively; P &gt; 0.05). The function of spermatozoa cryopreserved by using DFT was further evaluated by IVF. Females were treated with rhFSH twice-daily for 8 days after the onset of menses and following a treatment of hCG injection on Day 9. Cumulus-oocyte complexes were collected by laparoscopic follicular aspiration 32 h later. Of the inseminated oocytes, 79% were fertilized and 90 and 53% of the resulting zygotes developed into 2-cell and blastocysts, respectively. The fertilization rate was lower and the blastocyst rate was slightly higher than our previous report when fresh spermatozoa were used for IVF (94 and 52%, respectively). Our results indicate that spermatozoa of rhesus monkeys can be effectively cryopreserved using DFT in large volume. This finding provided a new and effective way for genetics preservation purposes in this important species.

Survival of frozen or vitrified bovine blastocysts produced in vitro in synthetic oviduct fluid

Simplification of in vitro culture conditions offers the advantage of limiting problems due to variation in composition of biological fluids and co-culture with epithelial cells. The aim of this work was to study cryosurvival of bovine embryos produced under partially defined conditions. A conventional freezing technique and a vitrification method that allow for direct transfer of embryos after thawing were compared. Following IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 h post insemination, or, in the absence of FCS, in a humidified atmosphere of 5%O 2, 5%CO 2, 90%N 2. Morphologically normal Day 8 and 9 blaslocysts were classified into 3 groups and were cryopreserved. Group A were blastocysts (< 150 μm); Group B consisted of expanded blastocysts (> 150 μm); while Group C were hatching or hatched blastocysts. The freezing solution consisted of 1.36 M glycerol and 0.25 M sucrose in PBS. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured on granulosa cells and survival was defined as the re-expansion and maintenance of the blastocoel over 24 and 72 h, respectively. Survival rates following vitrification were similar or better than after freezing (Day 8 embryo survival rate at 72 h was 72 vs 57%). More advanced embryos of the same group seemed to survive better. Embryos cultured without serum survived cryopreservation significantly better than those produced with addition of serum at 48 h post insemination (86 vs 61% at 72 h; P < 0.01). Hatched blastocysts survived freezing or vitrification well (80 to 100%). One calf was born following the transfer of 6 vitrified embryos. The results indicate that both freezing and vitrification methods are suitable for cryopreservation of bovine embryos produced in SOF.

Realization of the Indium Freezing Point

The freezing and melting behaviour of indium samples with nominal purities of 99,9999%, 99,999% and 99,99% has been studied to find the influence of conventional freezing techniques on the freezing temperature and to assess the effect of trace impurities on the quality of a fixed-point sample. Plateaus of constant temperature (± 0,1 mK) are readily obtained from both the induced and outside-chilled induced freezes, while freezing ranges of about 2 mK are observed in both the normal and outside-chilled freezes for all the samples. The maximum freezing temperatures of the conventional freezes are found to coincide very closely for each sample and to within ± 0,1 mK for the purest sample. The purest sample showed a narrow melting range on the melting curve after very fast freezing, in contrast to the other samples which showed the melting ranges expected of alloys. The average melting temperature of the purest sample closely agreed with the maximum freezing temperature. The difference between the average melting temperature after the very fast freeze and the maximum freezing temperature appears to be a useful criterion for assessing the quality of indium as a fixed-point sample.

Cryosectioning plant roots prepared by high-pressure freezing

Obtaining frozen-hydrated sections of plant tissues for electron microscopy and microanalysis has been considered difficult, if not impossible, due primarily to the considerable depth of effective freezing in the tissues which would be required. The greatest depth of vitreous freezing is generally considered to be only 15-20 μm in animal specimens. Plant cells are often much larger in diameter and, if several cells are required to be intact, ice crystal damage can be expected to be so severe as to prevent successful cryoultramicrotomy. The very nature of cell walls, intercellular air spaces, irregular topography, and large vacuoles often make it impractical to use immersion, metal-mirror, or jet freezing techniques for botanical material.However, it has been proposed that high-pressure freezing (HPF) may offer an alternative to the more conventional freezing techniques, inasmuch as non-cryoprotected specimens may be frozen in a vitreous, or near-vitreous state, to a radial depth of at least 0.5 mm.