Cryopreservation

Cryopreservation is a technique of preserving and storing viable biological samples in a frozen state over extended periods of time. The preservation of animal cell has become dependent upon using low temperature to render the cell metabolically inert. The term ‘cryopreservation’ refers to ‘a method of cold storage.’ The optimum temperature for preserving animal germplasm is liquid nitrogen temperature of -196 degrees Celsius. Temperatures from -70 to -80 degree Celsius may be sufficient enough to maintain cell viability for a few months. Cryobiology is the study of the effects of extremely low temperatures on biological systems such as cells or organisms.

Definition

Cryopreservation is the process of cooling and storing cells, tissues and organs at very low temperatures to maintain their viability. Spermatozoa were the first mammalian cells to be cryopreserved successfully (Polge et al 1949).

Cryopreservation methods

The two most commonly used cryopreservation methods for animal germplasm are slow programmable freezing and vitrification.

Slow programmable freezing – In slow freezing, cells in a medium are slowly cooled to below freezing point. Slow cooling is needed in order to increase osmotic strength, which causes an efflux of water from the cells. Machines are used to freeze biological samples  using programmable sequences i.e. ‘freezing down’ a sample to better preserve it for eventual thawing before it is frozen or cryopreserved in liquid nitrogen. Biological samples such as oocytes, skin, blood products, embryo and stem cells are preserved by slow programmable freezing machines.

Vitrification – The term ‘vitrification’ refers to any process resulting in ‘glass formation’(‘arrested liquid state’) the transformation from a liquid to a solid in the absence of crystallization (solid-liquid).  Vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they decrease the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution becomes an amorphous ice- it vitrifies.

Stroage temperature

Cells can be stored for varying lengths of time at temperatures between -70 to -196 degrees Celsius. For short –term storage (few weeks) cells can be preserved at -70 degrees Celsius in standard mechanical freezers. For long-term storage, cells can be preserved at liquid nitrogen containers.

Liquid nitrogen containers

Liquid nitrogen containers are basically robust, heavily insulated vessels into which liquid nitrogen is poured at regular intervals in order to maintain the required temperature. Cells can be stored in either vapour phase – containers at -120 degrees Celsius or liquid phase containers at -196 degrees Celsius of liquid nitrogen. Storage at -120 degrees Celsius does not significantly reduce cell viability. But indefinite storage of cells requires liquid phase containers.

Cryoprotective agents (CPAs)

Glycerol and DMSO are the most commonly used cryoprotective agents. Fetal bovine serum (FBS) is employed in mammalian cryopreservation solutions, but it is not a cryoprotective agent. Dextrans, sorbitol, trehalose, polyethylene glycols, starches, sugars, and polyvinylpyrrolidone provide considerable cryoprotection in a variety of biologic systems (Mazur 1981).  Salts, such as magnesium chloride, have been reported to be cryoprotective agents (Karow and Carrier 1969). Cryoprotectants protect slowly frozen cells by one or more of the following mechanisms: suppressing high salt concentrations; reducing cell shrinkage at a given temperature;reducing the fraction of the solution frozen at a given temperature and minimizing intracellular ice formation. Combinations of cryoprotectants may result in additive or synergistic enhancement of cell survival (Brockbank and Smith 1993, Brockbank 1992). Intracellular cryoprotectants of low molecular weights permeate cells e.g. glycerol, dimethyl sulfoxide. Extracellular cryoprotectants with relatively high molecular weights do not penetrate cells e.g. polyvinylpyrrolidone, hydroxyethyl starch.

Advantages of cryopreservation

Germplasm cryopreservation of the sperm, eggs and embryos contributes directly to animal breeding programmes. Germplasm cryopreservation also assist the ex situ conservation for preserving the genomes of threatened and endangered species. Cryopreserved sperm, oocytes and embryos are used for artificial insemination and embryo transfer in the livestock industry. Cryopreservation also has enormous applications in the artificial propagation of widely diverse aquatic organisms. Cryopreservation of  sperm and embryonic cells has been successful in a number of teleosts and invertebrate species.  The establishment of germplasm banks using cryopreservation can contribute to conservation and extant populations in the future. Cryopreservation provides a continuous source of tissues and genetically stable living cells for a variety of purposes including research and biomedical processes. Cryopreservation reduces the risk of microbial contamination or cross contamination with other cell lines. Cryopreservation reduces the risk of morphological or genetic changes. It also reduces costs of maintenance of cells, tissues or organs.

Safety considerations

The main dangers come from explosions of ampoules or cryotubes. The technician must wear appropriate protective clothing, protective goggles, face mask, insulated gloves etc. Careless handling of cold containers can also cause burns, so it is important to use handling tongues. Nitrogen gas is colorless, odourless and tasteless. It cannot be detected by the human senses. So liquid nitrogen can be used only in well ventilated areas.

Stages in the cryopreservation

Selection of cell lines -à cultivation of cells------à Screening of cells -----à preparation of cells-----à freezing of cells ---à Evaluation of viability.

Preparing cells for cryopreservation

Cells are prepared by trypsinization (0.25% w/v trypsin) to detach adherent cell from flask surfaces. Cells in suspension are centrifuged at around 100xg for 5to 10 minutes and the pellet is suspended in a small volume (1-2 ml) of storage medium.

Storage medium

The cryopreservation media generally consists of a base medium, protein source and a cryoprotective agent. The cryoprotective agent protects the cells from mechanical and physical stress and reduces water content within the cells. Cryoprotective agent minimizes the formation of cell-lysing ice crystals. The formation of ice crystals may disrupt the cell membrane leading to the death of the cells. The composition of the medium used to suspend cells contains a cryoprotectant (usually glycerol or dimethyl sulphoxide, DMSO) and a high protein concentration (serum 20% v/v) cryprotectant = 7-10 % (v/v); serum – 20% (v/v).

Freezing

Since freezing is stressful, the rate of cooling from 0 to 50 degrees Celsius must be slow and controlled. A fall of about 1 degree Celsius per minute is optimal. Rapid chilling results in thermal shock and leads to cell death or injury.  Once a temperature of -50 degrees Celsius is reached, the cells must be cooled rapidly to the final holding temperature.

Thawing of frozen cells

Cells retrieved from storage must be thawed rapidly to ensure maximum survival. The ampoule should be plunged into a beaker of water at 37 degrees Celsius. The cell suspension can be transferred drop wise into a container holding about 20 ml of pre-warmed growth medium supplemented with 10% fetal calf serum.

Conclusion

Cryopreservation of gametes, embryos and embryonic cells has become of immense value in animal biotechnologies which provide an important tool for protecting the endangered species and genetic diversity. Cryopreservation protocols have been introduced as techniques for germplasm preservation of vegetatively propagated horticultural and staple food crops. The establishment of cryobanks to utilize the cryopreservation worldwide would be a significant and promising task in the future.

Ultracentrifugation

Centrifugation is the most widely used technique for understanding cellular and subcellular structures. During centrifugation, solid particles experience a centrifugal force, which pulls those outwards i.e. away from the centre. The velocity with which a given solid particle moves through a liquid medium is related to angular velocity. In other words, centrifugation a procedure involves the use of centrifugal force for the sedimentation of components of a mixture. More dense components of the mixture move away from the axis of the centrifuge, while less dense components of the mixture move towards the axis. The sedimentation process is accelerated by the centrifugal field.

Definition of centrifuge

Centrifuge is an apparatus that rotates at high speed and by centrifugal force separates substances of different densities. In other words a centrifuge is a device for separating particles or macromolecules (e.g. cells, sub-cellular components, proteins, nucleic acids) from a solution according to their size, shape, density, viscosity of the medium and rotor speed.

Definition of ultracentrifuge

Ultracentrifuge is a high-speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.

Principle of centrifugation

The principle of centrifugation is that an object moving in a circular motion at an angular velocity w is subjected to an outward force F through a radius of rotation r in cms expressed as F=w²r . F is frequently expressed in terms of gravitational force of the earth (RCF). The operating speed of the centrifuge is expressed as revolutions per minute ‘rpm’.

The velocity of the moving particle is expressed in the form of sedimentation coefficient (S) v = S (w²r). Sedimentation coefficient is characteristic constant of a molecule and is a function of size, shape and density. The rate of sedimentation can be increased by raising the revolutions per second.

General types of centrifuges

Low – speed centrifuge –(desk top or clinical centrifuges)- maximum speed 3000rpm. It is used for separating serum form blood or separation of RBC etc.

High speed centrifuge – 25,000 – 30,000rpm. It functions at low temperature of 0-4 degree Celsius. It is used for cell fractionation i.e. separation of organelles.

Ultracentrifuge – High speed at 75,000 rpm and refrigerated.  It is used for the separation of cell organelles.

Analytical centrifuge - it has a built in optical system to measure the sedimentation characteristics of macromolecules.

Components of a centrifuge

The principal components of a centrifuge are a rotor to hold sample tubes and an electric motor to spin the sample. There are 4 types of rotors: Swinging bucket rotor (horizontal orientation), fixed angle (30 degree to the axis of rotation), vertical tube (centrifuge tube orients at an angle of 90 degree).

Types of ultracentrifuges

There are two types: analytical and preparative models.

    Analytical ultracentrifuge – It consists of rotors and tubes called cells. The instrument is designed to allow the person to follow the progress of the substances in the cells, while centrifugation is in progress. By estimating sedimentation velocity during centrifugation, the molecular weight can be determined.

Preparative ultracentrifuge – It is used for the purification of the components of macromolecules and all determinations are made at the end of centrifugation. The instrument does not have a monitoring device. Centrifugation is accomplished by rotors which are either swinging bucket type or fixed angle rotors. Swinging bucket rotors in which the buckets become horizontal, while in motion. In fixed angle rotors, the tubes of the centrifuge are set at fixed angles and the rotor moves in a specified plane at all times.

Description of ultracentrifuge

The ultracentrifuge consists of a fixed angle rotor of aluminium or titanium revolving at high speed about an axis in an evacuated chamber. The solution containing macromolecules is taken in the cell having quartz windows.  The cell is almost filled with the solution and sealed to withstand the pressure developed in the intense centrifugal field. A beam of light is allowed to pass through the solution, which then falls on a detector – normally a photographic plate.  Since light passing through an area of the sample is proportional to the molecules present in that region, the darkening produced in the photographic plate indicates the concentrations at various depths of the centrifuge tube.

The ultracentrifuge can be useful in two different methods: sedimentation equilibrium and sedimentation velocity methods.

In sedimentation equilibrium method, equilibrium is attained between the rate of settling down of the molecules and at the rate at which they diffuse back because of the thermal motion and Brownian movement under action of gravity. This  method takes several days to complete because of the low centrifugal forces (10,000-100,000g).

In sedimentation velocity method, higher centrifugal forces (up to 500,000 times the gravity)  are applied to accelerate sedimentation. This method starts with a well defined boundary or layer of solution near the center of the rotation and follows the movement of this layer toward the outside of the cell as a function of time. When a solution containing uniformly distributed solutes is centrifuged at high speed (55,000rpm), the particles migrate outwards from the centre of rotation, forming a well defined boundary between the solvent portions with or without particles.

Analytical Ultracentrifugation (AUC)

All analytical techniques require the use of an ultracentrifuge and can be classified into differential centrifugation and density gradient centrifugation. The density gradient centrifugation is further subdivided as zonal and isopycnic centrifugation.

Differential centrifugation is a technique commonly used by biochemists. A tissue sample such as liver is homogenized at 32 degrees in a sucrose solution that contains a buffer. The homogenate is then placed in a centrifuge and spun at constant centrifugal force and at constant temperature. After sometime a sediment forms at the bottom of the centrifuge tube called pellet and the overlying solution is called supernatant. The overlying solution is then placed in another tube, which is then spun at higher speeds. 

Applications – Differential centrifugation is used to determine the number of components and number of species; detection of impurities, molar mass of species; kinds and stoichiometry of chemical reactions present in solution including association with ligands, self- association etc. Materials analyzed include macromolecules such as proteins, polysaccharides, nucleic acids; small molecules such as drugs, ligands, gases and large aggregates such as viruses and organelles.

Density gradient centrifugation

The separation is  done in a medium having different density gradients. The selection of gradient medium is an important prerequisite. The gradient medium should not affect the cell sample. The medium should be easily sterilizable, recoverable and non-corrosive. Most common media includes sucrose, glycerol, sorbitol etc. There are discontinuous and continuous density gradients. In discontinuous density gradient medium, the density increases one layer to another. This medium is useful in the separation of whole cells, sub-cellular organelles or lipoproteins. In continuous density gradient medium, the density decreases from the bottom of the solution to the meniscus. This medium is useful in the separation of ribosomes, viruses, proteins and enzymes.  Density gradient centrifugation can be of two types such as rate zonal centrifugation and isopycnic centrifugation.

Rate zonal centrifugation – centrifugation is carried out at a very low speed for a short time so that the particles settle down. However centrifugation should be stopped before the particles of any zone settle at the bottom e.g. separation of nucleic acids, ribosomal subunits.

Isopycnic centrifugation – Isopycnic means “of the same density.” Isopycnic = equal density and separation is on the basis of different densities of the particles. Molecules are separated on equilibrium position, not by rates of sedimentation. The particles of solution move according to their buoyant densities and become static at a place, where the density is greater than their own. This requires a very long time centrifugation and high speed. As an example, the pellet obtained by centrifugation of the tissue homogenate at 10,000g is suspended in increasing densities of sucrose solution and centrifuged for several hours at 40,000rpm. Now the individual organelles move to the region of their own equilibrium density and remain at the specific regions. This method is useful in the separation of proteins, intracellular organelles and nucleic acid fraction.

Applications of density gradients

Density gradients are widely used to separate and purify, on a preparative scale, a variety of cells, organelles and macromolecules such as nucleic acids or proteins. Gradients are required for analytical experiments for example to measure the apparent buoyant densities or sedimentation coefficients of particles; to estimate the size, conformation or turnover rates of proteins and nucleic acids; and to investigate the  effects of chemical, physical or biochemical treatment of the sample material.