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CRYOPRESERVATION

CRYOPRESERVATIO
N

Rohini s
By ,
D3Biotech
Rohini s
D3 Biotech Rohini s
Introduction

o If laboratory is using a large no. of cell lines


for various purposes , maintenance become
difficult & expensive.
o Handling a no. of cell lines increases the
chance of microbial contamination as well as
cross contamination of cell.
o therefore it is advisable to freeze cell lines in
viable
condition and thaw them whenever they are
required cryopreservation technique is
adopted.
Rationale for freezing

Reasons for freezing ;

Genetic drift due to genetic instability


senescence and resultant extinction of the cell line

phenotypic instability due to selection and

differentiation
contamination by microorganisms

cross contamination by other cell lines

incubator failure

saving time and materials

need for distribution to other users

misidenfication due to careless handling .


Acquisition of cell lines for
cryopreservaton

There are certain requirements that


should be met before cell lines are
considered for cryopreservation.

Validation ; cell lines should be shown to be


free of contamination & authentic before
cryopreservation. Proper validation should be
carried out before major stocks are frozen.
When to freeze ; based on continous or
finite cell line.
principles of cryopreservation

Theoretical background of cell


freezing
Optimal freezing of cells for maximum
viable recovery on thawing depends on minimizing
intracellular ice crystal formation and reducing
cryogenic damage from foci of high concentration
solutes formed when intracellular water freezes. This is
achieved by ;
Feezing slowly to allow water to leave the cell but
not so
slowly that ice crystal growth is encouraged.
by using a hydrophilic cryoprotectant to sequester
water
by storing the cells at lowest possible temperature
to minimize the effects of high salt concentration.
Cell concentration
cells appear to survive freezing best when frozen at
high cell concentration- improved survival.
o high concentration at freezing also allows sufficient
dilution of the cryoprotectant at reseeding after thawing
so that centrifugation is unnecessary.
o The no. of cells frozen should be sufficient to allow for 1;10
or 1;20 dilution on thawing to dilute out the cryoprotectant
keep cell concentration higher than at normal passage.
o dilutes cryoprotectant from 10% -0.5% - less toxic
Freezing medium
The cell suspension is frozen in presence of cryoprotectant
such as DMSO or glycerol.
DMSO more effective- it penetrates cell better than
glycerol.
usual concentration : 7.5% or 10% DMSO
DMSO colourless, stored in glass or polypropylene,
powerful solvent & leach out impurities .
glycerol- toxic after prolonged storage
others;
Poly vinyl pyrrolidone (PVP)
Polyethylene glycol (PEG)
Addition of cryoprotectants
A cryoprotectant is a substance that is used to protect
biological tissue from freezing damage (damage due to ice
Crystal formation).

they act as anti-freezing agent


they lower freezing temperature
they increases viscosity & prevent damage to cells.
COOLING RATE

most cultured cells survive best if they are cooled at


1c/min.

slow cooling enhances the extracellular migration of water.

when cells are transferred to the liquid nitrogen freezer, the


temperature drops rapidly to between -180 & -196c

cooling curve is obtained by,

the ambient temperature

any insulation surrounding the cell

the specific heat and volume of the ampoule content

the latent heat absorption during freezing.


FREEZING CURVE

Average cooling rate


-1c
Maximum cooling rate

use of a programmable freezer with a pbobe- senses the
temperature of the ampoule- adds liquid N2 to the freezing chambe
at the correct rate programmed cooling rate.
with the insulated container methods ,the cooling rate is proportion
to the difference in temperature between the ampoule and the
ambient air.
Itis safer to leave the ampoules at 70c overnight before
-
transferring them to liquid N2.
Neck plug cooler
cryofreezers
storage in liquid N2 freezer is currently used-

preserving cultured cells.

The frozen cells are transferred rapidly to


the cryofreezers ,when they are at or below
-70c.

cryofreezers differ in design depending on


size of the access neck ,storage system
employed and

location of liquid N2.


ECK SIZE ; canister storage system tend to have narrow neck,
Reduces the rate of evaporation of the liquid N2 but makes access
little awkward. Wide neck freezers are choosen for ease of access and
maximum capacity,usually with storage in sections within drawers.
faster freezer evaporation rate. Freezers are available with inventory
control based on square-array storage trays.

TORAGE SYSTEM ; two main steps of storage


-The cane system, based on the storage of sperm in straws, uses ampo
clipped on to an aluminium cane,Inserted into a cardboard tube and
placed within cylindrical canisters in the freezer.
-advantages; multiple ampoules can be handled at a time,. Easier
freezing & quicker.
OULES

astic ampoules are preferred for the average experimental laboratory


r and more convenient.
ome cell banks prefer glass ampoules for seed stocks- long term storage.
astic ampoules are usully polypropylene & 1.2 mL. (popular size). They may be l
a fine-tipped marker ( alcohol resistent and able to withstand low temperatue o
freezer(cryomarkers and cryolabels). Different coloured caps also help identificat
LOCATION OF LIQUID N2

Storage in liquid phase- container filled with liquid N2


Introduction of improved insulation and reduced evaporation,
vapour-phase
storage is preferable.
Gradient in the temperature range- design and composition of
the racking
ststem may help to eliminate this gradient.
Some freezers have the liquid nitrogen located within the the wall of
the freezer and not in the storage compartment.
-automatic feed with high and low level controls
-evaporated N2 is released via a relief valve
advantages ; gas storage system, lower consumption of liquid N2,
elimination of the temperature gradient

MONOTORING AND REPLENISHING


LIQUID N2

Strict monitoring unit


electronic liquid level alarms
monitoring at least once per
week,recording
two independent temperature recorders
NITROGEN FREEZER DESIGN
Resuspend at 2x10 6c
ell/Ml.
Dilute one of the cryoprotectants (DMSO 5-10%) in growth
medium to make freezing medium.
3.Dilute the cell suspension 1:1 with freezing medium,
4.Dispense the cell suspension into prelabledd ampoules.
5.Place the ampoules on canes for canister storage.
6.Freeze the ampoules at 1c/min
7.When the ampoules have reached -70c,
for insulated container methods, it takes 4- hours.
8. Trasfer the ampoules into liquid N2 freezer.
THAWING STORED
AMPOULES

When required , cells are thawed and reseeded at a


relatively high concentration to
optimize recovery.
The ampoules should be thawed as rapidly as possible, to
minimize intracellular ice
crystal growth during the warming process.
This can be done in warm water, in a bucket or waterbath.
The cell suspension should be diluted slowly after thawing
as rapid dilution reduces
viability DMSO- sudden dilution can cause severe osmotic
damage and reduces
cell survival by half.
Most cells do not require centrifugation.
THAWING FROZEN
CELLS
1) Check the index for the location of the ampoule to be thawed
PROTOCOL
2) .Collect all materials,prepare the medium,and label the cuture flask.
3) Retrieve the ampoule from the freezer place it in sterile water at
37c
in a beaker,plastic bucket,or waterbath.
4) When the ampoule has thawed,double check the label to conform
the
identity of the cell; then swab the ampoule thoroughly with 70%
ethanol.
5) Transfer the contents of the ampoule to a culture flask with a 1mL
pippette.
6) Add medium slowly to the cell suspension, gradully diluting the cells
and the cryoprotectant.
7) The dregs in the ampoule may be stained with naphthalein black to
Freezing Vs THAWING
DESIGN AND CONTROL OF FREEZER STOCKS

1. FREEZER INVENTORY CONTROL

The seed stock should be protected and not be made available

for general issue.

when an ampoule is thawed to check the viabity of the seed


stock freezing, and if usage of the cell line is anticipated in the
future; ampoules from this stock are issued to individuals as
required.

Individuals users requiring stocks over a prolonged period

should then freeze should be discarded when work is


finished.

User stocks should never be passed on to another user as they

will not have been fully validated.


SERIAL REPLACEMENT OF CULTURES

Stock culture should be replaced from the freezer at regular intervals-


minimize the effects of genetic drift and phenotypic variation.
After a cell line has been culture for 2 months, thaw out another
vial- make sure that it is free from contamination.
Discard the existing stocks when they have been out of freezer for 3
months,move on to the new stock.
Repeat this process every 3 months with cells that have a population-
doubling time(PDT) of approximately 24h.
Cell lines with shorter or longer PDT- shorter or longer replacement
intervels respectively.
CELL BANKS

several cell banks exist for the secure storage and

distribution of validated cell lines.

Necessary characterization and quality control

have been done.

Most cell banks also make their catalog information


available on-line major source of information.

Provides a vast increase in the amount of material

that is potentially available.