Freezing biological material

1. Introduction
2. Seed Lot System
3. Cryoprotection Agents
4. Preparation of Biological Materials
5. Equilibration
6. Genetically Modified Materials
7. Ampoules and Vials
8. Rate of Cooling
9. Storage
10. Thawing
11. Determination of Recovered Cells
12. Stock Inventory Control
13. Biological Materials Management
14. Safety Considerations
15. Step-by-Step for Cultured Cells

Introduction

To ensure reproducible results and continuity in research and biomedical  processes, we are faced with the task of genetically stabilizing replicable materials  such as living cells and organisms, and ensuring sub-cellular components such as nucleic acids and proteins  are preserved unchanged. Serial subculturing of replicable materials  is time consuming  and can often result in contamination or genetic drift as smaller and smaller portions of a population are selected. Improper storage and handling  of non-replicable materials  can lead to divergent  and irreproducible research results. However,  a population of living cells or a suspension of subcellular  components can be stabilized  by subjecting them to cryogenic temperatures which, for all practical  purposes, stops time.The cryopreservation technology  have led to methods  that  allow low-temperature maintenance of a variety of tissues, cell types and subcellular  materials. Techniques  are available for the preservation of microorganisms, tissues, primary  cells, established  cell lines, small multicellular organisms, complex cellular structures such as embryos, as well as nucleic acid and proteins. Cryobiological studies have led to speculation on what occurs during the freezing of living cells and how adverse phenomena can be overcome. Since water is the major component of all living cells and must be available for the chemical processes of life to occur, cellular metabolism stops when all water in the system is converted  to ice. Ice forms at different rates during the cooling process. Slow cooling leads to freezing external  to the cell before intracellular ice begins to form. As ice forms external  to the cell, water is removed from the extracellular environment and an osmotic imbalance occurs across the cell membrane  leading to water migration out of the cell. The increase in solute concentration outside the cell, as well as intracellularly as water leaves the cell, can be detrimental to cell survival. If too much water remains inside the cell, damage due to ice crystal formation and recrystallization during warming  can occur and is usually lethal.

The rate of cooling has a dramatic effect on these phenomena (Fig. 1). Rapid cooling minimizes the solute concentration effects as ice forms uniformly, but leads to formation of more intracellular ice since water has not migrated  out of the cell. Slow cooling on the other hand, results in a greater loss of water from the cell and less internal  ice being formed, but results in an increase in the solution effects. Cell permeability affects the rate of water loss;

Fast cooling rate           Slow cooling rate

More intracellular ice | Less intracellular ice

Less osmotic imbalance | More osmotic imbalance

Recommend cooling rate 1°C | Minute along with cryoprotection agent

Using cryoprotection additives, or chemicals that protect  the cells during freezing also minimizes the detrimental effects of increased solute concentration and ice crystal formation. The most commonly used cryoprotection agents are dimethylsulfoxide (DMSO) and glycerol, although many other additives have been used for specific purposes. Additionally, maintaining frozen cells at the proper storage temperature and using an appropriate warming  rate also contribute to minimizing damage to frozen cells and tissues.

A key element of a good cryopreservation program is standardization of the processes employed. Because of the complexity  of the preservation process small variations in processing and storage can lead to subtle changes in the biological materials.  By standardizing the methodologies there is greater assurance  that  research results will be consistent  and comparable. Therefore,  once a successful cryopreservation regimen is established  efforts should be made to carefully document the methodology.

Seed Lot System

To aid in maintaining the genetic stability  of cultured  cells, the frequency of subculturing beyond the original established  culture must be minimized. When freezing cells, use a system that  ensures that early passage material  is always available for producing new working  stock. One method of preserving early passage material  is to use a seed lot system.

When preparing the first frozen lot of a culture, a portion of the lot is set aside as seed material. The vials designated  as seed material are maintained separately  from the working  stocks to ensure that  they remaing unused and are not handled  during retrieval operation

When the first lot of working  stock is depleted, a vial is retrieved from the seed lot and used to prepare  a second working  stock. This continues  until all seed vials, except one have been depleted. The last seed vial is then used to prepare  a second seed lot. The second seed lot remains only one or two passages from the original material,  but may be separated by many years if the lots are adequately sized

The seed stock concept is similar to cell bankingpractices whereby a master cell bank that  is fully qualified is developed and maintained for use in preparing working  cell banks used in a manufacturing process. A similar system could also be used replicable materials, such as DNA and proteins except for a different purpose. A similar system could also be used replicable materials, such as DNA and proteins

In this case, since the material  is not renewable, the seed material  would  be avail- able solely for comparative purposes  when changes in the workingmaterial  are suspected. Sufficient material should be retained  as seed stock to allow for testing when necessary.

In addition to seed material,  a small portion of the original lot, and even portions of working  stocks, should be segregated and maintained in a location  remote from all other material. This practice assures that  the segregated materials  are not handled during stocking and retrieval activities, and remain at a constant temperature. Backup materials  should be stored in an off-site location  if possible to ensure that  preserved materials  are not lost in the event of a physical disaster at the primary  location.  Using seed lots and maintaining off-site reserve material  are of primary importance in ensuring continuity and longevity in any well-managed collection of biological materials

Cryoprotection Agents

Many compounds have been tried as cryoprotection agents, either alone or in combination, including  sugars, solvents and even serum. Although  there are no absolute  rules in cryopreservation, glycerol and DMSO have been widely used and traditionally have been demonstrated to be the most effective agents for preserving living cells and organisms. Other  cryoprotectants that  have been used occasionally,  either alone or in combination, include: poly- ethylene glycol, propylene  glycol, glycerine, polyvinylpyrolidone, sorbitol,  dextran and trehalose.

The need to preserve tissues and whole organs has led to the development of novel preservation methodologies that  are also applicable  to enhancing  the recovery of frozen cells and organisms. These include variations on the concentration of cryo-protectants, and additives that  protect  cells against  apoptosis, or programmed cell death. For many years the death of cells follow- ing freezing was assumed to be caused by events causing physical changes or damage to cells. More recently it has been discovered that  more subtle events that  contribute ultimately  to cell death may be manageable with proper  additives.

Cryoprotection agents serve several functions  during the freezing process. Freezing point depression  is observed when DMSO is used which serves to encourage  greater dehydration of the cells prior to intracellular freezing. Cryoprotection agents also seem to be most effective when they can penetrate the cell, delay intracellular freezing and minimize the solution  effects.  The choice of a cryoprotection agent is dependent upon the type of cell to be preserved. For most cells, glycerol is the agent of choice because it is usually less toxic than  DMSO. However,  DMSO is more penetrating and is usually the agent of choice for larger, more complex cells such as protists. The cryoprotection agent should be diluted to the desired concentration in fresh growth  medium prior to adding it to the cell suspension. This minimizes the potentially deleterious  effects of chemical reactions, and assures a more uniform  exposure  to the cryoprotection agent when it is added to the cell suspension,  reducing potential toxic effects. DMSO and glycerol are generally used in concentrations ranging from 5-10% (v/v), and are not usually used togetherin th The optimum concentration of the cryoprotectant varies with the cell type and for optimal  results the highest concentratione same suspension with the exception  of plant cells

The optimum concentration of the cryoprotection varies with the cell type and for optimal results the highest concentration the cells can tolerate should be used. –  Sterilization of DMSO see picture – For some material it may be be advantageous to examine the sensitivity of the cells to increasing concentrations of the cryoprotection agent to determine the optimum concentration. The lists the recommended concentrations of cryoprotection agents for each group of cells and serves as a general guide to choosing the proper agent.

You may use an DMSO free System in order to safeguard the integrity of cells e.g. Biofreezer

Glycerol and DMSO should be of reagent grade or better, sterilized prior to use, and examined  for undesirable properties. Each lot should be examined  for toxic properties by exposing sensitive cells to concentrations previously used with success. Glycerol may be sterilized by autoclaving for 15 minutes at 121°C and 15 psig. Glycerol should be protected from light during storage. DMSO must be sterilized by filtration  using a 0.2 micron nylon syringe filter or a Teflon®  PTFE syringe filter which has been pre-washed with alcohol and rinsed with DMSO (Fig. 3). Cryoprotection agents should be prepared in single-use volumes to minimize the risk of contamination and moisture introduction with repeated use from one container.

Use caution when handling DMSO as it is quickly absorbed  into the body through  the skin and may transport  harmful  substances into the body with  it.

Non-replicable materials  generally do not require additives when frozen, except when certain characteristics need to be protected. For example tissue specimens that are frozen to preserve morphology may benefit from suspension  in materials  such as optimal  cutting temperature (OCT) compound to optimize the results. Normally tissues are frozen in blocks, however combining freezing in OCT with storage in cryovials improves  the handling of small tissue specimens.

Preparation of Biological Materials

How materials  are processed in preparation for freezing can have an affect on the outcome  of the preservation process. For non- replicable materials  such as tissues, nucleic acids and proteins,

the preparation process consists of ensuring that  the materials  are in the proper  solution  or freezing medium in order to maximize the intended  use of the materials  when recovered. However

the stability  and recoverability of living cells and organisms  is affected by the growth  conditions and pre-freezing processing. Several factors must be considered  when preparing cells for cryopreservation. These include the type of cell, cell viability, growth  conditions, physiological  state of the cells, the number  of cells, and how the cells are handled. When preparing the initial seed stock of a new isolate or cell line, the culture  should be examined  for identity and contaminating microorganisms at a minimum. This examination should be repeated after preservation and each time a new lot of the culture  is prepared.

Microorganisms

Microbial cells, particularly bacteria  and yeast, grown under aerated  conditions demonstrate a greater resistance to the detrimental effects of cooling and freezing than  non-aerated cells. They have demonstrated that  cell permeability is greater in aerated  cultures, and that  the aerated  cells dehydrate faster during cooling than  non-aerated cells. Microbial cells harvested  from late log or early stationary cultures also demonstrate greater resis- tance to the freezing process than  younger or older cells.

Generally, the greater the number of cells present initially, the greater the recovery. For most bacteria  and yeast, approximately 10 ⁷/mL cells are required  to ensure adequate recovery. These can be conveniently harvested  from agar slants or plates, or when greater quantities are required,  grown in broth culture and harvested by centrifugation. In either case, cells are generally suspended  in fresh growth  medium containing the cryoprotection agent. Protists can also be concentrated by centrifugation, but are often suspended  in the used medium and then diluted by adding an equal volume of fresh growth  medium containing the cryoprotection agent spore-forming  fungi require  harvesting  of spores and suspension of the spores in fresh growth  medium containing the cryoprotective agent. When freezing fungal spores, care must be taken not to delay the freezing process too long to ensure that  germination does not occur prior to freezing. For fungi that  do not form spores, special procedures for harvesting  mycelia prior to freezing must be utilized. For fungi with tough mycelia, the culture is harvested  from agar growth  by cutting and removing agar plugs containing the mycelia and placing the plugs into fresh growth medium containing the cryoprotection agent. Tough mycelia that do not adhere well to agar cultures are grown in broth  culture and the mycelial mass is blended prior to freezing. The viability and an estimate of recovery should be determined both before and after freezing the culture. Viability is a measure of the culture’s ability to grow and reproduce. For some material, such as protozoan cultures, this should include several passages to ensure stability. An estimate of the number of cells recovered can be made by several means including  serial dilution, plate counts or direct cell counting.  A comparison of the counts prior to and after freezing gives an indication of the degree of recovery or the success of the preservation procedure

Mammalian Cells

When preparing mammalian cells for cryopreservation, cell populations need to be adjusted  to levels that  ensure adequate recovery without unnecessarily  growing large numbers  of cells. For most mammalian cells, a starting  population between 10 6   to10 7   cells/mL is optimum.   The cell suspension  should initially be prepared at a concentration twice that  desired for preservation so that an equal volume of cryoprotectant (2 x cryoprotection agent + medium) can be added. Alternatively,  the cell pellet can be resuspended  in the cryoprotectant (1 x cryoprotection agent + medium) to the desired cell concentration. Gentle handling  during cell harvesting  and concentration procedures will ensure healthy cells prior to subjecting them to cold stress. Vigorous pipeting and high-speed centrifugation should be avoided if possible. Where appropriate, the pH should be maintained by gassing with 5% or 10%  C02 .

Factors which can affect the recovery of cryopreserved mammalian cells include: (a) type of cell, (b) the growth  phase of the culture, (c) the stage of the cell in the cell cycle, and (d) the number and concentration of cells in the final suspension. Attempts  to improve the viability of cryopreserved cells should consider these factors, as well as the nature  of the cryoprotection agent and the freezing process.

Mammalian cell cultures are especially susceptible to contamination  by other cells, such as HeLa8, and contaminating microorganisms. The species of origin of cell lines can be verified by isoenzyme analysis, karyotyping, immunological assays, orgenomic analysis. These should be performed prior to and following preservation. Contamination by viruses and Mycoplasma is of particular concern.   A good characterization program for mammalian cell lines should include a check for contamination by bacteria, fungi, appropriate viruses, mycoplasma, and in some cases, protozoa.

Stem Cells

Stem cells are cryopreserved in a manner  similar to other mammalian cells, with some exceptions  to enhance recovery and clonogenic activity. Cryoprotection is normally  afforded  by using DMSO, sometimes in combination with serum, and freezing slowly is preferred. Trehalose  can be used to reduce the potential toxicity associated  with other cryoprotectants. Rapid warming is also preferred,  and viability may vary depending  on the cell type.

Vitrification  can also be used to preserve stem cells. The protocol involves the cellsuspension in a concentrated mixture  typically composed  of more than  one cryoprotectant. For vitrification,  the freezing and re-warming processes are rapid  to avoid ice crystal formation, and in some studies the vitrification  process resulted in greater viability. DMSO, glycerol and propylene  glycol have all been used successfully to cryopreserve stem cells (or Biofreezer)

Plant Cells

Plant cells respond  to cryopreservation in a manner  similar to other cells.    The stage in the growth  cycle from which they are harvested  can affect their recovery, most optimum being late log phase. Also, cell density may play a role in recovery, the optimum cell density depending  on the species being preserved.

Combinations of cryoprotection agents are sometimes more effective than  agents used singly. The cooling rate is important, and in many cases a two-step  cooling process where the cells are held at -30°C to -40°C for a period of time before cooling to liquid nitrogen  temperatures, is beneficial. This process enhances the dehydration of the cytoplasm  prior to freezing. Rapid thawing

is preferred,  but there is evidence that  slow warming  is just as effective in some cases. Vitrification  can also be used to preserve plant cells by using concentrated cell suspensions  and rapid  rates of cooling.

Hardening of plants leads to greater tolerance  of stressful conditions, such as experienced  during the freezing process. Plants produce  increased  quantities of some compounds such as sugars and even glycerol which contribute to protecting the cells from osmotic stress during freezing. Undifferentiated callus tissue is often preserved in an effort to stabilize characteristics that  can be affected by continued cultivation.

Preservation  of seeds is also an acceptable  method  of stabilizing plant germ plasma,  and the most common  method  is storage at low humidity  and cool temperatures. However  some seeds are tolerant of the increased  desiccation  associated  with freezing and cryogenic storage, and can be stored at liquid nitrogen  temperatures.

Viruses

Most viruses can be frozen as cell-free preparations without difficulty and do not require  controlled cooling.   The exceptions are those viruses cultured  in viable infected cells which require controlled cooling. For cell-adapted viruses the preservation process should be applicable  to survival of the host cell. When viruses are harvested  from eggs, the high protein  content  of the allantoic fluid or yolk sac provides protection during the freezing process.

Plant viruses can be preserved either in infected plant tissue or as purified virus preparations. The virus preparations are suspended in DMSO or another cryoprotectant prior to freezing. Recovery is generally best when the cooling rate is controlled, although most plant viruses will tolerate  a rapid  freezing procedure. Recovery of plant viruses simply involves thawing  in a warm bath, followed by inoculation into the appropriate plant host

Embryos

Embryos have been preserved both by controlled cooling and vitrification.  Recovery depends on the stage of embryonic development, and is measured  by successful implantation leading to fetal development.

Genetically  Modified  Materials

Non-Replicable Materials

Non-replicable materials  such as whole blood, serum, tissues, nucleic acids and proteins  do not usually have any special requirements for successful preservation. The materials  are generally frozen without a cryoprotectant, and the freezing process can be rapid. However,  the process used depends on the end-use of the material.  Successful recovery of the properties of whole blood requires  cryoprotection and controlled cooling, and the quality of frozen tissues can be improved  by using a suspension  such as optimal  cutting temperature (OCT) compound.

Equilibration

The period of time between mixing the cryoprotectant with the cell suspension  and beginning the cooling process is called the equilibration period.  For most cells, equilibration should occur for at least 15 minutes,  but no longer than 45-60 minutes.  The cryoprotection agent may be toxic to the cells if the equilibration time is too long. For tissues frozen in OCT,  not technically a cryoprotectant,  the period of equilibration is generally not significant since OCT does not usually penetrate the tissue but simply provides support during freezing and subsequent sectioning of the tissue. Equilibration, which should take place at ambient temperature, allows time for the cryoprotection agent to penetrate the cells, with larger and less permeable cells as well as embryos requiring a longer equilibration period. During this period of equilibration the cell suspension may be dispensed into vials and otherwise manipulated in preparation for freezing. An optimal equilibration time should be determined empirically for the cells being cryopreserved to maximize later recovery.

Ampoules and Vials

A variety of small containers such as flame-sealed glass ampoules and screw-cap plastic vials can be used for storing cells at ultra- low temperatures. The most commonly  used sizes are 1.2- to 2.0 mL cryovials, the size and configuration of which maximize storage capacity while retaining  ease of handling  during stocking and retrieval activities. Generally, 0.5-1.0 mL of the cell suspension is placed into each container. Several factors must be considered (Maximum storage volume of about 70%/Vial)

Internal thread (manly with silicon sealing O-ring) External thread

when selecting a container, including  its cryotolerance, storage conditions, type of cells to be stored, and safety considerations.

Quick guide

Recommendation for using Biofreezer for Mammalian cells

Biofreezer – Cryoprotection new freezing medium. It creates an environment for cell viability comparable with conventional freezing media, but has key advantages: DMSO-free: The function of dimethyl sulphoxide (DMSO), which is highly toxic to cells, is replaced by a less toxic anti-freezing agent. Biofreezer is demonstrably free of substances of animal origin and is also free of genetically modified organisms. Biofreezer is particularly suitable for all mammalian cells that should remain free of animal products: for the manufacture of medicines made from cell cultures or to enable tissue re- construction.

2   Recommended cell count               

Aliquot in 1 ml freezing media per cryovial and place on ice.

3   Thawing cells

1. Remove the frozen culture from the nitrogen tank
2. Quick thawing in a water bath at 37 °C
3. Transfer the cell suspension into a tissue culture tube with 10 ml medium and 10-20 % FBS

4. Centrifugation (300 x g)
5. Discard the supernatant and resuspend the cells in the designated medium
6. Transfer the cell suspension in a tissue flask
7. Change the medium the next day

For temperatures above -100°C where low-temperature mechanical  stresses are less severe, a variety of containers may be used. However,  when storing material  at liquid nitrogen temperatures, containers specifically designed to withstand cryogenic temperatures must be used. A variety of containers specifically designed for cryogenic use are available. Plastic vials have screw-on closures with external  or internal  threads  . The rate of warming  may be affected by the type of container used since plastic vials usually require  a longer warming  period for complete thawing  than  glass ampoules. This difference in warming  rate may be significant for some fastidious  cells, but for most cells does not contribute to a loss of viability.

Other  containers can be used to store cryopreserved materials including  straws traditionally used for embryos, and microtiter plates commonly  used for freezing cell arrays or clones. The container of choice should be one that  maximizes the ability to maintain viable material  during storage, retrieval and handling. Glass ampoules  may be flame sealed, however care must betaken that  sealing is performed properly,  since improperly sealed glass ampoules  may have microchannels  that  lead to liquid nitrogen  penetration over time. When these are retrieved from liquid nitrogen  to ambient  temperature, rapid  conversion  of the liquid nitrogen  to vapor inside the ampoule  can result in explosion of the ampoule.  Plastic vials with screw-top  closures are also susceptible to liquid nitrogen  penetration  and while the explosion potential is minimized, liquid can spray from the cap/vial interface with potential dissemination of the vial contents  during warming  and handling. For 100% seal proof storage use straws or other heat sealing plastic tubing systems.

Rate of Cooling

Once the cells and the cryoprotectant have been combined  and dispensed into vials, the next step is to cool the suspension. The rate of cooling is important since it affects the rate of formation and size of ice crystals, as well as the solution  effects that  occur during freezing. Different types of cells may require  different cooling rates, however a uniform  cooling rate of 1°C per minute from ambient  temperature is effective for a wide variety of cells and organisms.

Generally, the larger the cells, the more critical slow cooling becomes. Most bacteria  and spore-forming fungi will tolerate less-than-ideal  cooling rates and can be frozen by placing the material  at -80°C for a period of time. More fastidious  bacteria and non-sporulating fungi require  more uniform  rates of cooling. Protists, mammalian cells and plant cells often require even greater control  of the cooling rate including  special manipulation to minimize the detrimental effects of under-cooling and the heat liberated  during the phase change from water to ice.

Despite the control  applied to the cooling of cells, most of the water present will freeze at approximately -2°C to -5°C. The change in state from liquid to crystalline form results in the release of energy in the form of heat; this is known  as the latent heat of fusion. Warming  of the sample occurs until the equilibrium freezing point is reached,  at which temperature ice continues  to form. To minimize the detrimental effects of this phenomenon, under-cooling must be minimized by artificially inducing the formation of ice. This can be accomplished by seeding the suspension  with ice or some other nucleating  agent, or by rapidly dropping the temperature of the external  environment to encourage ice crystal formation

To achieve uniform,  controlled cooling rates, use a programmable- rate cell freezing apparatus. Simple units allow only the selection

of a single cooling rate for the entire temperature range. More sophisticated units, however, allow a selection of variable rates for different portions of the cooling curve. Less costly and easier-to-use systems are available for simulating a controlled-rate cooling process by placing the vials in a mechanical  freezer at -60°C to -80°C. In order to accomplish a uniform  rate of cooling, the vials must be placed in specially designed containers.

The freezing container 1°C  provides a simple-to-use  system designed to achieve a rate of cooling very close to 1°C per minute

Typical cooling rates for home-made freezing systems lead to uncontrolled cooling that averages 1°C per minute but the cells actually experience more rapid  rates of cooling during some parts of the cooling curve. Home-made freezing systems are also non-repeatable. The freezing container eliminates the need for direct immersion in an alcohol bath. This feature eliminates the potential for contamination due to wicking of the alcohol, as well as the presence of residual alcohol on the exterior of the vials. During handling at colder temperatures, the presence of alcohol on the vials makes the vials colder to the touch and extremely slippery

Cryopreservation of embryos requires  even greater control  of the process because of their multicellular structure. In addition to the controlled rate freezing commonly used for single cells, a vitrification process is also used for preserving embryos. This requires suspending  the embryos in a highly viscous solution,  and rapidly cooling the suspension  to eliminate  the formation of ice crystals. The resulting frozen mass is a vitreous glass that  requires  storage at liquid nitrogen  temperatures. If the storage temperature rises above -130˚C ice crystals will form resulting in damage to the embryo.

Storage

When the sample has been frozen for 48 hours,  a vial should be thawed  to determine  whether  the cells are viable and able to establish a cell population, (i.e. if they survived the freezing procedure).

Determination of Recovered  Cells, below.

The temperature at which frozen preparations are stored affects the length of time after which the material  can be recovered.

The lower the storage temperature, the longer the viable storage period. Ultimate stability  of frozen cells cannot  be assured unless the material  is maintained below -130˚C.18   Some bacteria  and spore-forming fungi may tolerate  storage temperatures of -60˚C to -80˚C for long periods of time. However,  more fastidious  cells, such as mammalian tissue cultures, hybridomas and stem cells must be maintained below -130˚C to assure long-term  stability. It has been demonstrated that  some cells survive for less than  one year when stored at -80˚C. For ultimate  security and maximum stability, living cells and embryos should be stored in liquid nitrogen  freezers. However,  there are risks associated  with immersing vials directly into liquid nitrogen, as discussed previously. Liquid nitrogen units that provide all-vapor  storage are ideal as long as the working  temperature at the opening  of the unit remains below -130˚C.

To assure that  a liquid nitrogen  freezer maintains the proper working  temperature, the volume of liquid nitrogen  in the unit should be adjusted  to a level that  results in a temperature of

-150°C just above the stored material  when the lid of the unit is removed19 (Fig. 7). An adequate working  temperature can be attained in most liquid nitrogen  freezers; however, the design of some models requires  that  the amount of liquid nitrogen necessary to attain  the proper  working  temperature will reduce the amount of usable storage space.

Cryotank (Liquid nitrogen LN2 – Tank)

If vials are to be immersed in the liquid phase of liquid nitrogen, they must be correctly sealed.  Improper use may cause entrapment of liquid nitrogen inside the vial and lead to pressure build up, resulting in possible explosion  or biohazard release. Liquid phase LN penetration can also be a source of contamination for submerged  samples not properly. In most cases, vapor phase storage at –130˚C is adequate and avoids the hazards  of liquid phase storage. Mechanical freezers that  cool to -150˚C are also available.

Improper handling  of material  maintained at cryogenic temperatures can have a detrimental effect on the viability of frozen cells. Each time a frozen vial is exposed to a warmer  environment, even briefly, it experiences a dramatic change in temperature. Storage systems should be designed to avoid exposure  of stored material to warmer  temperatures, as well as minimizing prolonged exposure  of personnel  during specimen retrieval. Box stacking systems (i.e. stainless steel racks) necessitate exposure  of boxes at the top to warmer  temperatures when retrieving boxes at lower temperatures. When box stacking systems are used, maintain a small number  of vials of each preparation in the top box of the rack and store the remaining  vials of each preparation in lower boxes. By doing this, a vial of one preparation can be retrieved without exposing all vials of any particular culture or lot.

To maximize the available space in liquid nitrogen freezers and minimize exposure  of material  during retrieval, use small storage boxes or aluminium canes. Press the vials onto the canes, putting no more than  one lot of one culture on each cane. Canes provide a flat surface for coding their position  and easy identification during retrieval. Place the canes into cardboard or plastic sleeves to eliminate  the potential for vials to fall from the canes. When retrieving vials from canes, the cane should be lifted only to a level that  exposes the first available vial, without removing the remaining  vials from the working  temperature of the freezer.

Cryostick (several sticks can be loaded into the LN2 tank – mainly smaller tanks)

Reconstitution (Thawing)

For most cells, warming  from the frozen state should occur as rapidly  as possible until complete thawing  is achieved. To achieve rapid  warming, place the frozen vial into a 37°C water bath. Remember, material  frozen in plastic vials will take longer to thaw than  that  in glass ampoules,  and sometimes gentle agitation of the vial during warming  will accelerate the thawing  process. Care must be taken, however, not to vigorously agitate vials containing fragile cells such as protists and mammalian cells. As soon as the contents  of the vial have been thawed,  remove the vial from the water bath. To minimize the risk of contamination during reconstitution, disinfect the external  surface of the vial by wiping with alcohol-soaked gauze prior to opening.

Immediately  transfer  the contents  of the vial to fresh growth medium following thawing  to minimize exposure  to the cryoprotection agent. For most cultures, the entire contents  of the vial may be placed into fresh media, however further  dilution  may be necessary for cell lines. It is recommended that the cell suspension be centrifuged  at 100 x g for 10 minutes after initial dilution, the supernatant removed, and the cells resuspended  into fresh growth medium to remove residual cryoprotection chemicals.

Some materials  that  are not sensitive to the cryopreservation process may tolerate  thawing  and re-freezing. Most replicable cells will not tolerate refreezing unless they are in a resistant  form such as a spore. However,  for non-replicable materials  such as serum, nucleic acids, and proteins,  thawing  and refreezing may be acceptable. Keep in mind that each time an aliquot  is thawed  and re-frozen subtle changes may occur in the character of the mate- rial that  could impact future use. An alternative to thawing  and refreezing is to store material  in smaller aliquots  for single use.

Determination of Recovered Cells

Methods used to estimate the number  of viable cells recovered following freezing depend on the type of material  preserved. Visual inspection  alone can be deceptive, and although staining and dye exclusion are effective in determining the presence of viable cells for most mammalian cells, they do not indicate an ability to establish the cell population. For microbial  cells, serial dilution  and plate counts are effective in quantifying the population of cells recovered.  Although  there may be some vial-to-vial variation within a given lot, with constant storage conditions the number  of recovered cells will generally be the same in all vials. Vial-to-vial variation may be an indication of problems  occurring  during storage and handling.

For stem cells the recovery is determined in the same manner  as for other mammalian cells by estimating  the number  of viable cells. In addition measuring  the differentiation capacity and clone forming capability  are also important in assuring complete stem cell recovery.  Embryo recovery can be determined via morphological  examination, and verified by implantation and fetal development. Recovery of non-replicable materials  is determined by acceptable  results following intended  use.

Inventory Control

Appropriate record keeping is important in any laboratory and there are a number  of methods  available for keeping records on cryopreserved materials.20 When establishing  your own method, keep in mind that  there is key information which will be important  for future use: (a) the preservation methodology used; (b) the location  and identification of the stored material;  (c) preservation date; and (d) number  of passages for replicable material. The item number  should be linked to associated  data for that  material,  and for some purposes  each container may require  a unique identifying code linked to specific information for that  particular aliquot.

Identification begins with proper labeling of the storage container. The label information should include a name or identification

code for the frozen material,  as well as a lot number. The information  on the label should be kept with the inventory  records that  include the location  code for each vial. These records can be maintained as paper documents, or preferably  as electronic files. Duplicate  inventory  records should be maintained in a location separate  from working  records. Locator  codes should be specific enough to allow rapid  and easy retrieval for a specific lot and should include freezer unit number, a code for a freezer section or inventory  rack, a box or canister number, and possibly even a grid spot within the box or a cane number  when canes are used.

Detailed locator  codes minimize hunting  for material  which risks warming  the freezer unit, exposure  of other materials  to warmer temperatures, and prolonged exposure  of laboratory personnel  to extremely cold temperatures.

Biological Materials Management

Despite its use in stabilizing living material, low-temperature preservation can stress cells. Care must be taken to ensure that preserved material remains unchanged following preservation. A good preservation program should include effective characterization and cataloguing programs, both of which combined, provide optimum biological materials  collection

practices.  There is little use in preserving material  that is of little value, inadequately characterized, contaminated or misidentified. The first step in maintaining a collection of biological materials

is to assess the material  to be preserved to ensure that it is of use and is worth  keeping. This practice should continue  throughout the collection process to minimize the accumulation of preserved material  to unmanageable levels. To avoid duplication of collection materials,  a system of identification of each item should be established. This can be done by devising unique numbering systems or number/letter combinations. Each identification number should be cross-referenced to other information about

the biological material.  Preservation of living cells ensures stability but does not correct any problems  already present in the material. All material  to be preserved should be examined thoroughly for contamination, proper  identification, and other key characteristics unique to the cells, prior to preservation. Since freezing can stress cells and handling  exposes cells to the risks of contamination, characterization must continue  after successful preservation is accomplished. Each time a new lot of frozen material  is prepared, complete characterization of the material should be carried out. Cataloguing and data record keeping are important aspects of all biological material  collection programs. Cataloguing ensures that duplication of material  does not occur and is especially useful when collection material  is to be made available to others.  Maintaining records on data generated  during the characterization and preservation of collection materials ensures that any future problems  can be adequately addressed.

An important aspect of good biological materials  management is constant assessment of the usefulness of the material,  and removal of materials that are no longer needed.

Avoid any contact with thread or inner cap

Aseptic – minimize contamination risk

Safety Considerations

1) Safety precautions must be observed throughout the preservation and maintenance process. All work with hazardous cultures should be performed under proper  containment, and U.S. Public Health  Service Biosafety Level guidelines should be adhered  to at all times.

2) Human and other primate  cells may contain  adventitious viral agents that  require  special handling,  and all primate  cells that have not been thoroughly characterized should be handled  at Biosafety Level II. At this level, laboratory staff must have training in handling  pathogenic agents and work under the direction  of a competent scientist. Access to the laboratory must be limited and biological safety cabinets must be used for large-volume  work or when aerosols are generated.

3) Low-temperature storage of cells presents unique hazards  that  necessitate safety precautions. Cryogenic temperatures can result in exposure  of personnel  to extremely cold conditions, and precautions must be taken to protect  personnel  during operations in liquid nitrogen  freezers. Insulated  gloves and long-sleeved laboratory coats or other garb protect  the skin from exposure. It is extremely important to wear a full face and neck shield when working  in the liquid portion of a liquid nitrogen  freezer. As noted previously, improperly sealed glass ampoules  may explode when retrieved from liquid nitrogen. To minimize the risk of potential explosions, leave vials retrieved from the liquid phase of the freezer in the vapor phase of the same freezer for a minimum  of 24 hours. A face shield that  provides neck protection should be mandatory when retrieving vials from liquid nitrogen.

4) Special precautions must be taken when working  with hazardous biological materials  at liquid nitrogen  temperatures. Always thaw and open vials containing hazardous material  inside a biological safety cabinet. Be prepared for exploding  and leaking ampoules/ vials. Broken ampoules  in a liquid nitrogen  freezer are a potential source of contamination and contaminants may survive, despite the extremely cold temperatures.

5) When a liquid nitrogen freezer becomes contaminated, the entire unit should be decontaminated after warming  to room temperature. When closing down a liquid nitrogen  freezer that  is not obviously contaminated, remove all material  to be retained, warm the unit to room temperature and disinfect it prior to further handling.

Step-by-Step for Cultured Cells

(Not Stem Cells)

1. Harvest cells from late log or early stationary growth.
   Scrape cells from the growth  surface if they are anchorage dependent. Centrifuge broth  or anchorage independent cultures to obtain  a cell pellet, if desired.

2. Prepare pre-sterilized DMSO or glycerol in the concentration desired in fresh growth  medium. When mixing with a suspension of cells, prepare the cryoprotection agents in twice the desired final concentration.
3. Add the cryoprotectant solution to the cell pellet or mix the solution  with the cell suspension.  Begin timing the equilibration period.
4. Gently dispense the cell suspension into vials.
5. Begin cooling the cells after the appropriate equilibration time.
   1. Uncontrolled cooling—place the vials on the bottom of a -60˚C freezer for 90 minutes.
   2. Semi-controlled cooling—use Dr. Frost freezing container to freeze the vials in a -70˚C freezer.
   3. Controlled cooling—use a programmable cooling unit to cool the cells at 1°C per minute to -40˚C.
6. Remove the cells from the cooling unit and place them at the appropriate storage temperature.
7. To reconstitute, remove a vial from storage and place into a water bath at 37˚C. When completely thawed, gently transfer the entire contents  to fresh growth medium.