Cryopreservation/Freezing

Cells

TC & Animal Biotechnology

• Cryobiology is to study the effects of
extremely low temperatures on biological
systems, such as cells or organisms.

• Cryopreservation is the viable freezing

of biological material and their subsequent
storage at ultra low temperatures (-196˚C).
 Why Cryopreservation?
• Genotypic drift due to genetic instability
• A senescence and extinction of cell lines
• Transformation of growth characteristics and acquiring
malignancy-associated properties
• Contamination by microorganisms
• Cross contamination with other cell lines
• Saving time and materials by maintaining only cell lines in
current use
• Distribute to other users in the same laboratory or other
laboratories
Mechanism:
• Water-bears (Tardigrada), microscopic multicellular
organisms, can survive freezing by retention of internal
water by the sugar trehalose, preventing water from
crystallization that otherwise damages cell membranes.
• Mixtures of solutes can achieve similar effects. Some
solutes, including salts, have the disadvantage that they may
be toxic at intense concentrations.
• In addition to the water-bear, wood frogs can tolerate the
freezing of their blood and other tissues. Urea is
accumulated in tissues in preparation for overwintering, and
liver glycogen is converted in large quantities to glucose in
response to internal ice formation. Both urea and glucose act
as "cryoprotectants" to limit the amount of ice that forms
and to reduce osmotic shrinkage of cells.
• The freezing process involves complex phenomena that, even
after decades of research, are not fully understood.
• 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.
Remember three things:

• Ice crystals detrimental to cells

• Higher ionic concentration detrimental to cells

and create osmotic imbalance resulting in the
loss or gain of water

• Rate of cooling during cryopreservation affects

the ice crystals
 Three types of Freezing Processes and their
effects on cells
1- Rapid Freezing
- The material is placed in vials and placed into -80˚C freezer
- The quicker the freezing, less osmotic imbalance but more the
intracellular ice crystals.
2- Slow Freezing
- Materials are slowly frozen with decrease in temperature from 0.1 to
10˚C/min
- Slow cooling permits the flow of water from the cells to the outside,
promoting extracellular ice formation instead of toxic intracellular
freezing, but more solute concentration inside hence osmotic imbalance
3- Stepwise Freezing (sloution)
• Optimum cooling at 1°C/min for most cultures
• Compromise between fast freezing minimizing ice crystal growth and
slow cooling encouraging the extracellular migration of water
• Slow freezing down to -20 to -50˚C period of approximately
30 to 50 min. and then additional rapid freezing to -196˚C
What happens to the cells when they are frozen in liquid
nitrogen= - 196°C

Everything stand still, very little molecular movement, may

be the metabolism stops hence apoptosis, differentiation,
growth etc also stop.
 Requirements before Freezing
• Status:
- Finite Cell line (Freeze at early passages)
- Continuous cell line (Clone, Select, Characterize
and amplify)

• Validation:
- Provenance (Origin, life history and properties)
- Authentication (check cell lines characteristics)
- Contamination (Microbial, Mycoplasma)
- Cross contamination and misidentification
 Process of Cryopreservation
• Cell Freezing
• Cell Concentration
• Freezing Medium
• Cooling Rate
• Cryovials or Ampoules
• Cryofreezers
• Freezing cells
• Thawing Stored Cryovials
 Cell Freezing
• Optimal freezing of cells to obtain viable recovery upon
thawing depends on minimizing crystal formation and
reducing cryogenic damage from foci of high concentration
solutes formed when intracellular water freezes.

Parameters Affecting Cell Freezing

1. Using a hydrophilic cryoprotectant to remove water
2. Storing at lowest possible temperature to minimize effect of
high salt concentration on protein denaturation
3. Thawing rapidly to minimize formation of ice crystals and
generation of solute gradients as the intracellular ice melts.
 Freezing Medium
• Cryo-protective agents (DMSO or glycerol) act like antifreeze,
they lower freezing temperature and increase viscosity.

• Dimethylsulphoxide (DMSO):
- An excellent cryprotectant, it is not toxic at low temperature,
easily permeable and having low molecular weight and is easily
washable from the cells.
- Commonly used at concentrations between 5% - 20%
- Problems: DMSO (dimethyl sulfoxide) is toxic to cells above
4°C. Toxic or induce differentiation after thawing, Ex.
Hematopoeitic cell lines (L5178Y or HL60)
Solution: change to glycerol and centrifuge to dilute conc. of
DMSO
• Other Types of Cryprotectant: Polyvinylpyrrolidone (PVP),
Polyethylene glycol (PEG) and hydroxyethyl starch (HES)
 Cooling rate
• Most cultured cells survive best if they are cooled at 1°C/min.

• A compromise between fast freezing minimizing ice crystal

growth and slow cooling encouraging the extracellular migration
of water.

The shape of the cooling curve is governed by:

(a) the ambient temperature,
(b) any insulation surrounding the cells, including the ampoule,
(c) the specific heat and volume of the ampoule contents, and
(d) the latent heat absorption during freezing.
When cells are frozen in an insulated container placed in an ultra
deep freeze, results in a curve with a rapid cooling rate at the
start, when the temperature differential is greatest, down to a
minimum at the eutectic point, a slight rise as freezing
commences, followed by a plateau as the latent heat of freezing is
absorbed, and then a more rapid fall as freezing is completed,
gradually slowing down as the temperature of the freezing
chamber is reached
 Cryovials
• Plastic vials made of polypropylene
• Label with: cell strain, date, user’s initial and #
of cells or estimated confluency
• Labels are printed and barcoded
• Different colored caps helps in the
identification
• Cell culture stored in liquid nitrogen may
outlive you, use proper labeling so it can be
used by others
 Freezing Cells
• Harvest cells by:
- Trypsinization with 0.25% Trypsin-EDTA solution after
washing the cells 2x with PBS
- Resuspend the trypsinized cells with growth medium and
collect the cells by centrifugation
- Resuspend the cells in freezing medium:
- 80 % Growth Medium
- 20 % DMSO
• Transfer 1ml-aliquots in cryo-vials labeled with name of the
cell line, passage number, date, cell density and your initial.
• Freeze samples in a -80°C freezer over night; thereafter the
vials are transferred to the gaseous phase of a liquid nitrogen
storage vessel.
Nalgene Nunc Cooler. Plastic holder with fluid-filled base.
The specific heat of the coolant in the base insulates the
container and gives a cooling rate of ∼1°C/min in the
ampoules.
 Cryofreezers
• Frozen cells are transferred to a cryofreezers when they are at -70°C, - 80°C freezers are used
for short term storage (1-3 years) . Storage in liquid nitrogen the most suitable method, it can
stay for centuries.
Cryofreezers differ depending on:
• Neck size: 1. canister storage systems have narrow necks which reduces evaporation but access
is awkward. 2. Wide neck freezers: Easy access, larger capacity but faster evaporation rate.
• Storage System: 1. Cane system uses vials clipped to an aluminum cane inserted into a
cardboard. Tubes are placed within a cylindrical canister
2. Storage in rectangular drawers.
• Phase of Nitrogen:
- Liquid phase, storage will last longer but risk of
contamination from surrounding.
- Vapor phase, preferred. There is a temp gradient
form the surface of the liquid to the neck of about
80°C (-190°C to -110°C)
• Monitoring & Replenishing liquid Nitrogen regularly.
 Thawing Cells
• Cells are thawed and seeded, it should be thawed rapidly
to eliminate intracellular ice crystal growth during the
warming process
• It is done by putting the vial containing the cells in a
warm water bath (37˚C)
• The immersed vial is swirled just to the point of ice
disappearance
• Cell suspension should be diluted slowly, sudden dilution
can cause severe osmotic damage and reduce cell
survival by half
• Transfer the sample immediately into growth medium,
mix by pipetting up and down
• Most cells do not require centrifugation, but medium
should be changed the next day to dilute out the
cryoprotectant.
• Suspension cells are more sensitive to DMSO and may
require centrifugation and resuspending with fresh growth medium
 Freezing Flasks

• Grow the cells to late log phase

• Add 5%-10% DMSO to the smallest volume
that would cover the monolayer
• Place in a polystyrene container with 15mm
wall thickness in -80°C freezer, it will freeze at
1°C/min.
The summary of cell response to
cryopreservation as discussed early.
The following is the summary of the
processes and also their solutions
 Cell Response to Cryo-preservation
– Reduced Metabolic Activity
– Ionic balance and Osmotic balance are disrupted
– Formation of large ice crystals inside the cell
– Intracellular concentration of solutes increase toxic levels before
or during freezing as a result of dehydration

Solutions:
– Tissue culture media
– Serum (10-90%)
– Cryo-protective agents act like antifreeze, they lower freezing
temperature and increase viscosity
END

Tissue Culture & Hybridoma Technology

(1450361) / Ban Al-Joubori