Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum     

Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Structures in Sieve Elements Cut with a Cryostat Following Different Rates of Freezing

Vascular bundles of the internodes of squash (Cucurbita pepo)were frozen while still attached to the stem by their ends.Four different methods of freezing were employed. With slowercooling rates the sieve tube contents showed distinct evidenceof damage due to ice-crystal formation while tissues were wellpreserved when rapidly frozen. The sieve tubes contained longitudinallyorientated structures which, in rapidly frozen tissues, apparentlyconsisted of discrete strands which appeared to pass into thesieve plate pores. It is concluded that the method of freezing permits the cuttingof sections which represent the in vivo structure of phloemand the results support the concept of translocation by meansof transcellular strands in sieve tubes.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

Long-term skin preservation by combined use of tissue culture and freezing techniques.

Simple but effective methods for shipping newly excised rabbit skin to a distant central laboratory for in vitro culture on a pigskin base, followed by freezing in a cryoprotective agent (DMSO) for frozen storage and subsequent reshipment to the originating laboratory while still frozen are described. At a suitable time the frozen tissue was rapidly thawed and transplanted to the autologous recipient rabbit. Of 12 cultures, seven indicated good to excellent cell growth and maturation on the host. The successful method combined the use of in vitro tissue culture and freezing to permit a central laboratory to grow the skin and ship it to the originating center for autografting at a convenient time.     

A chamber for the simulation of radiation freezing of plants

Frost injury to plants can occur following episodic radiation frosts. In the UK this is particularly important to spring sown crops such as potatoes. Most laboratory based frost studies simulate freezing using either conductive or convective freezing chambers. Such frost tests do not simulate overnight freezing events adequately. A freezing chamber based on radiative cooling is described which mimics overnight radiative freezing. The chamber is rectangular in design (1 m × lm × 2 m high) with a radiative cooling plate at the top of the chamber cooled to -40°C to -45°C using HFC coolants, which acts as a cold black body. The sides of the chamber are also cooled to variable temperatures down to -5°C in order to prevent the chamber walls radiating to the plant material during testing. Using thermocouples to measure air temperature and plant temperature the chamber has been characterised to simulate the radiative cooling conditions found in the UK during autumn and spring. Exotherm detection upon plant freezing is simplified by virtue of the reduction in temperature fluctuation normally experienced at the plant surface during natural freezing. Radiation frosts and subsequent frost damage to potatoes have been recorded in the temperature range -4°C to –5°C. The equipment is recommended for studies of frost damage to plants normally caused by episodic radiation frost events.     

Simple freezing apparatus for resolving rapid metabolic events associated with smooth muscle activation.

A method is described for freezing thin strips of smooth muscle by replacing physiological saline in the muscle chamber with cold organic solvent in <100 ms. Calculations suggest that, with a perfectly stirred boundary at the tissue surface, freezing could occur within approximately 15 ms at the center of a 200-microm-thick piece of tissue by use of acetone coolant at -78.5 degrees C and in approximately half the time with either isopentane at its freezing point (-160 degrees C) or aluminum chilled with liquid nitrogen. Myosin light chain phosphorylation in muscles frozen with cold acetone began to rise approximately 200 ms earlier than force and increased at a much more rapid rate. The difference in onsets of the two processes reflects the delay in arresting phosphorylation plus two lags associated with force generation, attachment of phosphorylated bridges followed by force generating movements of the attached bridges. The much more rapid rise of phosphorylation, once it began, suggests that most of this delay is due to physiological lags and not to slow arrest of metabolism.     

A mathematical model for the freezing process in biological tissue

A mathematical model has been developed to study the process of freezing in biological organs. The model consists of a repetitive unit structure comprising a cylinder of tissue with an axial blood vessel (Krogh cylinder) and it is analysed by the methods of irreversible thermodynamics. The mathematical simulation of the freezing process in liver tissue compares remarkably well with experimental data on the structure of tissue frozen under controlled thermal conditions and the response of liver cells to changes in cooling rate. The study also supports the proposal that the damage mechanism responsible for the lack of success in attempts to preserve tissue in a frozen state, under conditions in which cells in suspension survive freezing, is direct mechanical damage caused by the formation of ice in the vascular system.     

The influence of high pressure freezing on mammalian nerve tissue

Summary Vitrification of biological specimens in liquid nitrogen can be achieved under high pressure (2,100 bars). This procedure obviates the use of aldehyde fixation and cryoprotection (glycerol). The present work demonstrates its applicability to the freeze-etching of mammalian brain tissue. Freeze-fracture replicas from rat cerebellar cortex and subfornical organ prepared by this method are compared to conventionally processed material using aldehyde fixation, glycerination and freezing with Freon. The formation of large ice crystals is prevented in tissue blocks up to 0.5 mm thick; deep etching is markedly enhanced. Cytoplasmic microstructures such as mitochondrial cristae, microtubules and microfilaments, are readily observable against a finely granulated cytosol matrix. An additional advantage is the combined application with freeze-substitution.     

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.     

Improved and reproducible cell viability in the superflash freezing method using an automatic thawing apparatus

Cell cryopreservation stops the biological activity of cells by placing them in the frozen state, and can be used to preserve cells without subculturing, which can cause contamination and genetic drift. However, the freezing process used in cryopreservation can injure or damage the cells due to the cytotoxicity of cryoprotecting agents (CPAs). We have previously reported a CPA-free cryopreservation method based on inkjet technology. In this method, the vitrified cells were exposed to the room temperature atmosphere during the transport of the cells using tweezers, which caused devitrification due to the increased temperature and often lowered the cell viability. In the present study, we developed an automatic thawing apparatus that transports the vitrified cells rapidly into a prewarmed medium using a spring hinge. Observations with a high-speed camera revealed that the spring hinge drops the cells into the prewarmed medium within 20 ms. All heat-transfer simulations for the apparatuses with different designs and rotation speeds showed that the cells remained below the glass-transition temperature during the transport. Finally, the apparatus was evaluated using mouse fibroblast 3T3 cells. The cell viability was improved and its reproducibility was enhanced using this apparatus. The results indicate that the combination of superflash freezing with the rapid thawing process represents a promising approach to circumvent the problems typically associated with the addition of CPAs.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation

The aim of our investigations was to compare the effectiveness of two methods for cryopreservation of sheep ovarian tissue, slow freezing and vitrification. The quality of cryopreserved tissues was evaluated after 5 days of thawing and chorioallantoic membrane (CAM) transplantation. Follicular structure, stromal integrity and neovascularization were assessed. The areas of fibrosis and necrosis were measured using MICROVISIBLE software, and proliferation was assessed with Ki-67 immunostaning. After 5 days of culture, the proportion of primordial follicles decreased, whereas the primary and intermediary follicles increased insignificantly (p > .05). Only necrosis in the vitrified culture group increased significantly (p < .05). It was established also that 5 days CAM culture was not suitable methodology for detection of folliculogenesis. Follicular quality decreased after culture, but was better in fresh and slow frozen tissues than after vitrification (p < .05). Cellular proliferative activity fell, but it preserved to some extent in all groups. In conclusion, follicles was preserved better in grafted tissue after slow freezing than vitrification and stroma was more susceptible to ischemia in vitrified rather than conventional freezing in this view. Vitrification may not be a suitable alternative to the slow freezing.     

A novel simplified ultra-rapid freezing technique for cryopreservation of tissue slices

Cryopreservation offers the potential to maximize the use and availability of biological materials that have a limited supply. This study demonstrates an enhanced technique for the parallel cryopreservation of a series of liver tissue slices using a tray modeled from aluminium foil and low concentrations of a cryoprotectant. Cooling and warming rates of approximately 2000 and 3900 degrees C min(-1), respectively, were achieved as the thermal capacity of the foil-tray was significantly reduced compared to the aluminium sandwich device introduced by Day et al. [S.H. Day, D.A. Nicoll-Griffith, J.M. Silva, Cryopreservation of rat and human liver slices by rapid freezing, Cryobiology 38 (1999) 154-159]. Additionally, the two critical steps involved in the sandwich approach, i.e., clamping the plates and complete filling of the entire space between the plates with liquid, can be omitted using the foil tray. The viability of the slices was verified by measuring tetrazolium salt reduction capacity, cytosolic enzyme lactate dehydrogenase leakage, and ethoxycoumarin metabolism.     

Cryopreservation of human ovarian tissue: Comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Large-scale, high-density freezing of hybridomas and its application to high-density culture

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 x 10(8) cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 x 10(8)/mL. The number of cells stored in a bag (3.0 x 10(9) cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 x 10(6) cells/mL) than normal inoculation, and the cells proliferated to 10(7) cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.     

Preservation of gastrointestinal bacteria and their microenvironmental associations in rats by freezing.

The use of frozen rat gastrointestinal tissue samples for both the recovery of viable bacteria and for observation of microbial communities associated with the tissue was investigated. A decrease of 1 log in lactobacilli, bifidobacteria, and anaerobes was observed when the numbers of bacteria recoverable from frozen tissue (stored 7 to 9 days) were compared to those recoverable from fresh nonfrozen tissue (zero time control). However, freezing did not appear to decrease the numbers of recoverable coliforms. Tissues, cleaved with razor blades after being frozen and stored for 7 to 9 days, showed bacterial communities situated on the mucosa and in the lumen of gastrointestinal specimens. This freezing technique preserved structures not previously observed in the gastrointestinal tract. This indicates that freezing is a good method to use to study such fragile microenvironments.     

Escherichia coli viability in an isochoric system at subfreezing temperatures

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between −5 °C and −20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to −10 °C and −15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at −20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at −15 °C and −20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.     

Effect of varying freezing and thawing rates in experimental cryosurgery

Six different freezing/thawing programs, which varied freezing rate, duration of freezing, and thawing rates, were used to investigate the effect of these factors on cell destruction in dog skin. The range of tissue temperatures produced was from -15 to -50 degrees C. The extent of destruction was evaluated by skin biopsies 3 days after cold injury. In single, short freezing/thawing cycles, the temperature reached in the tissue was the prime factor in cell death. Longer freezing time and slow thawing were also important lethal factors which increased destruction of cells. Cooling rate, whether slow or fast, made little difference in the outcome. The experiments suggested that present-day, commonly employed cryosurgical techniques, which feature fast cooling, slow thawing, and repetition of the freeze/thaw cycle, should be modified by the use of maintenance of the tissue in the frozen state for several minutes and slow thawing. Thawing should be complete before freezing is repeated. These modifications in technique will maximize tissue destruction, an important consideration in cancer cryosurgery.