X-Message-Number: 17076
From: 
Date: Tue, 24 Jul 2001 00:01:18 EDT
Subject: Suspended Animation of Cells--Part II

Cryonet:

To follow is the second section of a reprint of a cryogenics lab manual used 
for the long-term storage of cells.  The first section appeared in 
yesterday's Cryonet edition.  An unfortunate typographical error appeared in 
yesterday's section where the word "neither" was substituted for the word 
"rather" in the following sentence regarding the efficacy of 
cryopreservatives with eukaryotic cells (e.g., mammalian cells): "These 
agents have little effect on the damage caused by fast freezing 
(intercellular ice crystal formation), but rather [not "neither" as I 
inadvertently transposed/mistyped] prevent or lessen the damage caused by 
slow freezing (dehydration and shrinkage) (8)."  I will attempt to better 
proof the following and subsequent reprint posts for the:

GENERAL GUIDE FOR CRYOGENICALLY STORING ANIMAL CELL CULTURES

by John A Ryan

[Note:  Most Cryonet readers might have little interest in the sections 
titled "STORAGE VESSELS" and "LABELING AND RECORDKEEPING" and may wish to 
skip these particular sections to follow]

QUOTE

PRACTICAL ASPECTS OF CELL FREEZING

Under the best of circumstances the process of freezing remains stressful to 
all cell cultures.  It is important that everything possible be done to 
minimize these stresses on the cultures in order to maximize their subsequent 
recovery and survival.  The following suggestions and recommendations are 
designed to augment of the protocols referred to earlier.

I. CELL SELECTION

First ensure that the cells are in their best possible condition.  Select 
cultures near the end of log phase growth (approximately 90% confluent) and 
change their medium 24 hours prior to harvesting.  Carefully examine the 
culture for signs off microbial contamination.  Facilitate this by growing 
cultures in antibiotic-free medium for several passages prior to testing.  
This allows time for any hidden, resistant contaminants (present in very low 
numbers) to reach a higher, more easily detected level. Samples or these 
cultures are then examined microscopically and tested by direct culture for 
the presence of bacteria, yeast, fungi, and mycoplasmas.

Mycoplasmas present a special problem since they can be found in cultures at 
very high concentrations (up to 10*8 organisms per milliliter of medium) 
without any visible effects or turbidity.  As a result, as many as 20% of all 
animal cell cultures are contaminated by these ubiquitous but unseen 
organisms.  Although special efforts are required to detect mycoplasmas, the 
serious consequences of their presence makes testing frozen culture stocks 
absolutely essential (9, 12).

Check for both the identity of the cultures and the presence of any expected 
special characteristics.  Monitor cell identities by karyology and isoenzyme 
analysis, ensuring that they are, at the very least, the correct species (10).

II. CELL HARVESTING

Start with the standard harvesting procedure generally recommended for the 
culture and be as gentle as possible.  Remove all dissociation agents by 
washing or inactivation (especially important when using serum-free medium).  
Centrifugation, when absolutely necessary, should only be hard enough to 
obtain a soft pellet; 100 x g for 5 to 6 minutes is usually sufficient.  To 
ensure uniformity of the final frozen stock, pool the contents or all 
harvested culture vessels.  This also makes it much easier to perform 
essential quality control testing for microbial contamination and culture 
identity.

Count and then dilute or concentrate the harvested cell suspension to twice 
the desired final concentration, which is usually 4 to 10 million viable 
cells per milliliter.  An equal volume of medium containing the 
cryoprotective agent at twice its concentration will be added later to 
achieve the desired inoculum.  Keep the cells chilled to slow their 
metabolism and prevent cell clumping.  Avoid alkaline pH shifts by gassing 
with CO2 when necessary.

III. CRYOPROTECTION

As mentioned earlier, cryoprotective agents are necessary to minimize or 
prevent the damage associated with slow freezing.  The mechanisms providing 
this protection, although not completely understood, appear to work primarily 
by altering the physical conditions of both the ice and solutions immediately 
surrounding (external to) the cells.  Permeation of the cells by 
cryoprotectants does not appear to be necessary for their proper functions 
(4).  Remember, protection against fast freezing damage (internal ice 
formation) is not provided by these agents, but rather by careful control of 
the freezing rate.  A wide variety of chemicals provide adequate 
cryoprotection, including methyl acetamide, methyl alcohol, ethylene glycol 
and polyvinyl pyrrolidone (7).  However, dimethysulfoxide (DMSO) and glycerol 
are the most convenient and widely used.  Many of these agents, although 
providing excellent cryoprotection, have toxic side effects on cultures 
making their use difficult.

DMSO is most often used at a final concentration of 5-15% (v/v).  Always use 
reagent or other high purity grades that have been tested for suitability.  
Sterilize by filtration through a 0.2 micron nylon membrane in a 
polypropylene or stainless steel housing and store in small quantities (5 
mL).  CAUTION: Take special care to avoid contract with solutions containing 
DMSO.  It is a very powerful polar solvent capable of rapidly penetrating 
intact skin and carrying in with it harmful contaminates such as carcinogens 
or toxins.  Some cell lines are adversely affected by prolonged contact with 
DMSO.  This can be reduced or eliminated by adding the DMSO to the cell 
suspension at 4C and removing it immediately upon thawing.  If this does not 
help, lower the concentration or try glycerol or another cryoprotectant.

Glycerol is generally used at a final concentration of between 5 and 20% 
(v/v).  Sterilize by autoclaving for 15 minutes in small volumes (5 mL) and 
refrigerate in the dark.  Although less toxic to cells than DMSO, glycerol 
frequently causes osmotic problems, especially after thawing.  Always add it 
at room temperature or above and remove slowly by dilution.

High serum concentrations may also help cells survive freezing.  Replacing 
standard media-cryoprotectant mixtures with 95% serum and 5% DMSO may be 
superior for some overly sensitive cell lines, especially hybridomas. Add 
cryoprotective agents to culture medium (without cells) immediately prior to 
use to obtain twice the desired final concentration (2X) to obtain the 
inoculum for freezing.  This method is less stressful for cells, especially 
when using DMSO as the cryoprotectant.

IV. STORAGE VESSELS

After the cryoprotective solution is mixed with the cell suspension, the 
resulting inoculum is added in small aliquots (usually 1 to 2 milliliters) to 
each storage vessel.  Due to the extremely low temperatures encountered 
during cryogenic storage, not all vessel materials or designs are suitable or 
safe.  Many materials become very brittle at these temperatures; vessels made 
from them may shatter or crack during storage or thawing.  Carefully check 
the vessel manufactures' recommendations on proper selection and use.

Also important is selecting the sealing system or cap design used to maintain 
the integrity of the vessel, especially for storage in liquid nitrogen.  If 
these vessels leak during storage (as many do) they will slowly fill with 
liquid nitrogen.  When they are eventually returned to room temperature, the 
liquid nitrogen quickly vaporizes causing a rapid pressure buildup.  The 
vessels may then violently blow off their caps or explode to vent the 
pressure and release their contents into the atmosphere.  This is a very 
dangerous situation, especially if the vessels contained pathogenic organisms 
or potentially toxic or harmful substances.  Storage above liquid nitrogen to 
reduce these potential hazards is strongly recommended in such situations.

Two types of vessels are commonly used for cryogenic storage; heat-sealable 
glass ampules and plastic (usually polypropylenes) screw-capped vials.  Both 
are available in a variety of sizes (1 to 5 milliliter capacity) although the 
smaller sizes are preferred for cryogenic storage (See Figure 1).

Because of sealing and labeling problems, glass ampules are no longer widely 
used in cell culture laboratories.  Invisible pinhole leaks may occur in 
vials during the sealing process; if these are later stored submerged in 
liquid nitrogen, they may explode when removed for thawing.  Pinholes can 
usually be detected before freezing by immersing sealed ampules for 30 
minutes in a chilled solution of 70% ethanol containing 1% methylene blue.  
This solution will rapidly penetrate and stain any leaky ampules; after 
rinsing with water, defective ampules are then easily detected and discarded.

Due to their greater safety and convenience, plastic vials have largely 
replaced glass ampules for cryogenic storage.  The wide variety of styles and 
special features like printed marking areas and colored caps for easier 
identification also add to their popularity.

Several cap styles are available, some with the internally-threaded stopper, 
and others with externally-threaded designs which help minimize contamination 
(See Figure 2).

V. LABELING AND RECORDKEEPING

Providing for long-term location and identification of frozen cultures is the 
most frequently overlooked area of cryogenic storage.  A cryogenic cell 
repository is expected to outlast the laboratory workers who contribute to 
it, but poorly maintained or missing inventory records, and improperly or 
illegibly labeled vials and ampules may prevent this, especially after the 
people responsible have gone.

Labels must contain enough information to locate the appropriate records; 
usually the culture's identity, date frozen, and initials of the person 
responsible are sufficient.  Most plastic vials have printed marking spots or 
areas for easy labeling.  On vials and ampules without marking spots use 
cloth labels with special adhesives formulated for cryogenic conditions.  
Special ceramic-based inks are available for labeling glass ampules.  These 
are applied prior to filling and then baked onto the glass, usually during 
dry heat sterilization.  Permanent marking spots can be applied on glass 
ampules with white nail polish.  A laboratory marking pen is then used to 
write on the spot once it has dried.

No matter which labeling method is chosen, use special care to check its 
permanency under cryogenic conditions.  Some marking spots, inks, and labels 
may flake off or fade during long-term storage; a trial run of at least 
several weeks is recommended.

Fully detail in the records the culture's storage conditions, including all 
of the following information: culture identity, passage or population 
doubling level, date frozen, freezing medium and method used, number of cells 
per vial, total number of vials initially frozen and the number remaining, 
their locations, their expected viability and results of all quality control 
tests performed (sterility, mycoplasma, species, karyotype, etc.).  
Additional culture information, especially their origin, history, growth 
parameters, special characteristics, and applications, is also helpful and 
should be included whenever possible.

Make special efforts to keep all records up to date and ensure everyone in 
the facility is properly using them.  Use pre-printed forms to make the 
information recording process easier and more likely to be completed.  Keep 
updated, duplicate copies of all critical records in a safe place removed 
from the laboratory area to guard against their accidental loss or 
destruction.  This is  especially important if a computer-based recordkeeping 
system is used; a current back up copy should always be maintained in 
addition to the information stored in the computer.

VI. COOLING RATE

The cooling rate used to freeze cultures must be just slow enough to allow 
the cells time to dehydrate, but fast enough to prevent excessive dehydration 
damage.  A cooling rate of -1C to -3C per minute is satisfactory for most 
animal cell cultures.  Larger cells, or cells having less permeable membranes 
may require a slower freezing rate since their dehydration will take longer.

The best way to control cooling rates is using electronic programmable 
freezing units.  Although expensive, they allow precise control of the 
freezing process, give very uniform and reproducible results, and can freeze 
large numbers of vials or ampules.  Most units are available with chart 
recorders for a permanent record of the cooling process.

There are a variety of mechanical freezing units that provide adequate 
control of the cooling rate and are relatively inexpensive.  Some units use 
racks designed to hold vials at predetermined depths in the neck of a liquid 
nitrogen freezer.  The cooling rate is dependent on the total umber of vials 
and the depth at which the rack is placed.  Another design uses an alcohol 
filled metal canister containing a rack with a capacity of 24 vials.  The 
filled canister is placed in an ultracold mechanical freezer where the 
alcohol acts as a bath to achieve more uniform heat transfer and cooling.  
After freezing 4 to 5 hours, the vials are removed from the canister and 
transferred to their final storage locations.

Insulated cardboard or polystyrene foam boxes are commonly used as freezing 
chambers in ultracold freezers.  These homemade devices work well with many 
cell lines but do not always give controlled, reproducible or uniform 
cooling.  As a result, their may be serious differences in viability among 
the vials upon thawing.  This homemade approach is not recommended for 
valuable or irreplaceable cultures.

No matter which cooling method is used, transfer from the cooling chamber or 
device to the final storage location must be done quickly to avoid warming of 
the vials.  Use an insulated container filled with dry ice or liquid nitrogen 
as a transfer vessel to ensure that the cells remain below -70C.  UNQUOTE

[More tomorrow.  I note that the last sentence above appears to specify -70C 
as presumably being a "lab tested" maximum temperature limit for keeping 
cells viable for future rewarming.  For this reason, it may be worth 
considering the addition of some amount of dry ice to liquid nitrogen storage 
dewars for "insurance" as it might "buys time" in the event of unit failure 
(e.g., lost vacuum in hard vacuum LN2-based systems).  I do not know if this 
is common practice or not]

David C. Johnson, Raleigh, NC

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