cryopreservation

1. CRYO-
PRESERVATION

2. CRYOPRESERVATION
 Cryopreservation is use of very low temperature to preserve the cells and tissues that are structurally intact.
 Cryopreservation is the method of keeping the live cells, tissues and other biological sample in a deep freeze
at temp. for the storage or preservation the sample is commonly kept at -196◦C.
 At such low temperature all the biological activities of the cells stop and the cell dies, cryopreservation helps
the cells to survive freezing and thowing.
 The ice formation inside the cells can break the cell membrane this can preventend by regulating the freezing
rate and carefully choosing the freezing medicine .
CRYOPRESERVATION PROCESS:
 In thus process, biological materials including cells, oocytes , spermatozoa, tissue, ovarian tissues, pre –
implantation embryos, organs etc. are kept in extremely cold temperature without affecting the cell visbility.
 Dry ice and liquid nitrogen are generally used in this method.

3. CRYOPRESERVATION STEPS :
 The complete procedure steps involved in preserving the obtained biological samples are as follows:
HARVESTING OR SELECTION OF MATERIAL/SAMPLES
 Few important criteria should be followed while selecting the biological materials such as volume, density ,
morphology and without any damage .
ADDITION OF CRYOPRESERVATION
 Cryoprotectants agents such as Glycrol , FBS ,Salts , Sugars ,glycols , DMSO (Dimethyl sulfoxide)are added to
the samples as it reduces the freezing point of the medium and also allow slower cooling rate , which
the risk of crystallization.
 The 2 major sources of cryoinjury excessive mechanical damage due to the excessive concentration of
intracellular solute resulting from ice formation . Water loss during freezing may reduce the cell volume
a critical level necessary for survival.
 The general order of cryoprotection seems to be PROLINE ≥ DMSO + GLYCEROL=DMSO + PROLINE ≥

4. FREEZING
 Different methods of freezing cryopreservation to protect cell from damage & death by their by their
to the warm solutions of cryoprotective agents.
 Different tissues have different sensitivities for cooling rate these are 3 strategies for freezing:
 A) slow cooling
 B) rapid cooling
 C) freezing following dehydration
a) Slow freezing –the material is cooled at the rate of 0.5 to 4◦C per minute upto 40◦c to 100◦c temperature
for 20 – 45 minutes before it is introduced into liquid nitrogen. It reduces the amount of intracellular water.
b) Rapid cooling – some tissues cannot survive at slow cooling rate and required rapid cooling of 200 -
1000◦c/minute.
c) Freezing following dehydration – excised single node segment from 6 to 8 week old plant lets were pre
cultured for 2 days on 0.7 sucrose & them dehydrated with silica gel before immersing them in liquid
nitrogen.

5. STORAGE IN LIQUID NITROGEN
 The cryopreserved samples are stored in extreme cold or 80◦c in a freezer for at least 5 to 24 hrs this before
Transfering it to storage vessels .
THAWING
 The process of warming the biological samples in order to control the rate of cooling and prevent the cell
damage cause by crystallisation or thawing is the bringing of cryopreserved material back to the normal

6. Thankyou..