Cryobiology
Theoretical background
Temperature is a measurement of internal energy in a physical system. It is this internal energy in the system that allows molecules in fluids to tumble, twist, disassociate themselves from each other, move from place to place in the fluid, and to chemically react with other molecules. As the temperature is lowered, energy becomes greatly reduced in the system, thereby failing to facilitate these types of molecular motions.
As described by Polge et al., in 1949, the first human sperm cells were successfully cry preserved. The process of crypreservation includes initial exposure to, and equilibration with the cryoprotectants, cooling to subzero temperatures, storing, thawing, and finally, dilution and removal of the cryoprotectant with return to the physiological environment that will produce the possibility of further development.
Events During Freezing
Freezing of any aqueous system involves numerous simultaneous changes.
As the temperature is reduced:
· Ice crystallizes
· Viscosity increases
· Remaining solution is reduced in volume, introducing the possibility that some solutes may reach saturation and precipitate, or if containing gases, may form bubbles
Parameters that are dependent upon more than one of these variables (e.g., pH or osmolality) may change in a complex, nonlinear fashion. When a cell is cooled at a temperature of between -5 and -15oC, ice is formed first in the extracellular medium.
Since the ice crystals are unable to cross the cell membrane, nucleation of intracellular ice is prevented. The cytoplasm itself super cools, and then has, by definition, a higher chemical potential than the water of the partly frozen external solution. Since the cell membrane is permeable to liquid water, but not to ice, water is ejected from the cell at a higher region than that of lower chemical potential, and freezes externally.
If the cell is cooled at a sufficiently slow pace, a massive amount of water will be transported out of the cell, with a reduced chemical potential difference across the cell membrane, resulting in progressive dehydration that makes formation of intracellular ice unlikely.
Basic Definitions
Cryobiology:
Study of life at below-normal temperatures. Usually cryobiology is considered as having the ability to deal with the effects of freezing and thawing. However, any temperature below normal for any given living system falls into the realm of cryobiology.
Cryopreservation – A process of preserving and storing living systems in a viable condition at low temperatures for future use. Traditionally, the meaning of cryopreservation is ‘preservation by freezing’, and the word is still used in many cases. However, the term can also be used for covering preservation by vitrification, or ice-free cryopreservation.
Cryogenics: A branch of physics that studies the causes and effects of extremely low temperatures.
Crystal: In many systems, e.g. pure water, temperature reduction below a certain point results in an abrupt reorganization of the fluid medium into an organized solid lattice known as a crystal. This is recognized as freezing.
Vitrification - Preservation at extremely low temperatures without freezing. Freezing involves ice crystal formation; however, vitrification involves the formation of a glassy or amorphous solid state which, unlike freezing, is not intrinsically damaging even to the most complicated living system.
Glass Transition Temperature: In other systems, this does not occur. Instead, temperature reduction merely causes increased slowing of molecular motions, decreased molecular mobility, and sluggish chemical reaction rates until a critical temperature is reached. Below this temperature energy is insufficient for the most mobile molecules in the fluid to move appreciably over the time scale of a typical laboratory observation. At this “glass transition temperature,” the system loses its fluidity almost completely and becomes a “solid liquid,” more formally known as a “glass,” and is said to have “vitrified.”
Vitrification prevents damage related to ice formation. This includes: mechanical disruption of extracellular structures in organized tissues and organs, cellular osmotic dehydration and shrinkage during slow freezing, intracellular ice formation and destructive intracellular ice recrystallization during rapid freezing and during thawing, and exposure to elevated intracellular and extracellular solute concentrations that can produce harmful effects or precipitate these after exceeding their solubility limits.
The physical phenomenon of vitrification takes place when the solidification of the solution occurs not by ice crystallization, but by extreme elevation in viscosity, whereby a vitreous consistency is achieved and is very similar to that of glass. Vitrification requires high cryoprotectant concentrations, and high concentrations tend to biochemically disturb living systems, producing toxic effects.
Cryoprotectants : When a cell is permeated by cryoprotectants in concentrations high enough to facilitate vitrification, all the molecular constituents of the cell become locked into the glass as it forms, and therefore over time are unable to change.Conventional cryoprotectants are glycols (alcohols containing at least two hydroxyl groups), such as ethylene glycol, propylene glycol and glycerol. The toxicity of glycerol is temperature dependent.
Dimethyl sulfoxide (DMSO) is also regarded as a conventional cryoprotectant. This is a low-molecular-weight nonelectrolyte, that with slow freezing and slow thawing, survival is better than the use of glycerol. Glycerol and DMSO have been used for decades by cryobiologists to reduce ice formation in sperm and embryos that are cold-preserved in liquid nitrogen. Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants. A mixture of formamide with DMSO, propylene glycol and a colloid was, for many years, the most effective of all artificially-created cryoprotectants.
Cryoprotectant mixtures have been used for vitrification, i.e. solidification without any crystal ice formation. Agents that reduce electrolyte concentrations in the unfrozen portion of the suspending solution protect cells from freezing injury due to solution effects. These cryoprotectants suppress the concentration of salts as a consequence of their colligative properties and the phase rule.
Chilling or Cooling Injuries: The nature of these injuries is not understood, but could be related to lipid phase transitions, protein cold denaturation, or other phenomena. The factors primarily responsible for freezing injury to cells are ice formation within the cell and solution effects. The degree of cellular injury is determined by the total volume of ice within each cell, rather than by the size of individual crystals.
Thawing: two problems that occur during thawing can reduce the survival of frozen cells, i.e. recrystallization with intracellular ice formation, and osmotic shock.
Oocytes
When cells such as oocytes are included, the system becomes multicompartmental, in which the sections differ in content and are defined by the properties of the biological membranes. This matrix of inter-related phenomena should be borne in mind when evaluating experimental evidence on the efficacy of particular cryopreservation regimes.
Introduction Oocyte cryopreservation is an emerging technology with a promising future, but still requiring much developmental work to improve the survival rates and expansion potential of frozen-thawed oocytes.
Indications for Oocyte Freezing
· Improve the efficacy of IVF
· Alternative to embryo freezing
· Oocyte preservation for patients with ovarian hyperstimulation syndrome
· For oocyte donation programs
· For treatment of congenital infertility disorders
· To prevent fertility loss through surgery
· For treatment of premature ovarian failure (POF)
· In the case of patients who face infertility due to cancer therapy
During the process of cryopreservation, cells are exposed to a number of various forms of stress, which could result in lethal damage to the cell and include:
· Mechanical
· Thermal
· Chemical
Oocytes in general are more sensitive to freeze/thaw damage than later embryonic stages. Any change in the chromosomal complement (as may result from scattering or displacement from the spindle) could result in aneuploidy, with potentially severe consequences for subsequent embryonic or fetal development.
Oocyte Freezing and Storage
Human oocytes can be stored as:
· Denuded individual oocytes at metaphase-II (M-II)
· Cumulus enclosed at germinal vesicle (GV) stage immature oocytes
The protocol of freezing depends upon the stage of nuclear maturity of the oocytes. When mature MII oocytes are harvested, the gametes are commonly denuded prior to freezing to confirm their nuclear status. However, it would be physiologically far more appropriate to freeze immature oocytes with intact germinal vesicles with their cumulus cells for better in-vitro maturation after thawing.
Mature Metaphase II Oocytes
At this stage, oocytes have undergone nuclear and cytoplasmic maturation, the first polar body has been extruded and chromosomes are condensed and are arranged on the delicate MII spindle. Mature oocytes have a short fertile life, are very sensitive to chilling, and have little capacity for recuperating from the cryo-injury before fertilization. Other recent studies provided improved results with vitrification as an alternative to slow freezing protocols.
Immature Germinal Vesicle Oocytes
Germinal vesicle-stage oocytes are full-sized, but their chromatin is at the diplotene stage of first prophase, and unlike mature M-II-stage oocytes, do not have spindle apparatus. They require a period of maturation to induce the required nuclear and cytoplasmic changes before being capable of undergoing fertilization and supporting early embryo development.
Effects of Cryopreservation on Oocytes
Chromosomes and meiotic spindles comprise fragile fibres originating from the centriole at the opposing poles, and extending to the chromosomes. A loss of microtubules during freezing could separate chromosomes and cause aneuploidy. The often reduced percentage of fertilization, and the rather higher incidence of anomalies and fertilization in cryopreserved oocytes, have been hypothesized as being related to possible damage of the zona pellucidae and cortical granules that interfere with the correct interaction between them and spermatozoa. ICSI has been proposed as a solution for this problem.
General Safety Aspects
The Human Oocyte Preservation Experience (HOPE) Registry is an initiative of EMD Serono, Inc. which aims at systematically tracking the outcomes of oocyte cryopreservation cycles, and validating the efficacy and safety of techniques to freeze and thaw oocytes.
Safety Aspects – Cryopreservation of Mature Oocytes (Metaphase-II Stage)
Presence or absence of the cumulus granulosa cells during the freezing process may have a direct impact on metaphase II oocyte survival after thawing. M-II oocytes are vulnerable to cryo-injury because the meiotic spindle, to which the chromosomes have become aligned, is actually temperature sensitive. Oocyte freezing can therefore increase the incidence of aneuploidy after extrusion of the second polar body through nondisjunction of sister chromatids. This duplication of the cytoskeletal architecture may also lead to abnormal cytokinesis, retention of the second polar body, and alterations in the organization and trafficking of the molecules and organelles.
While the deleterious effects on the cytoskeleton as a result of chilling may be avoided by cryopreservation of GV, this has proved that the chromosomal integrity is not lost and there is no damage to the spindles.
Safety Aspects – Cryopreservation of Primordial Oocytes These GV-stage oocytes appear to be less vulnerable to cryo-injury than MII-stage oocytes, as they are smaller, lack zona pellucidae and cortical granules, and are relatively metabolically inactive and undifferentiated.
