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Cryopreservation is the process of freezing cell lines in viable condition for long term storage and future use. It allows for maintenance of large numbers of cell lines without risk of contamination or genetic drift over time. Key aspects of cryopreservation include slow freezing of cells with cryoprotectants like DMSO, storage in liquid nitrogen freezers, and rapid thawing for recovery of viable cells when needed. Proper design and monitoring of freezers is important for long term preservation of cell stocks.
Cryopreservation is the process of freezing cell lines in viable condition for long term storage and future use. It allows for maintenance of large numbers of cell lines without risk of contamination or genetic drift over time. Key aspects of cryopreservation include slow freezing of cells with cryoprotectants like DMSO, storage in liquid nitrogen freezers, and rapid thawing for recovery of viable cells when needed. Proper design and monitoring of freezers is important for long term preservation of cell stocks.
Cryopreservation is the process of freezing cell lines in viable condition for long term storage and future use. It allows for maintenance of large numbers of cell lines without risk of contamination or genetic drift over time. Key aspects of cryopreservation include slow freezing of cells with cryoprotectants like DMSO, storage in liquid nitrogen freezers, and rapid thawing for recovery of viable cells when needed. Proper design and monitoring of freezers is important for long term preservation of cell stocks.
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