Cryopreservation

Cryo is Greek word. (krayos – frost)

It literally means preservation in “frozen state.”

The principle - to bring plant or animal cells or tissue to a zero metabolism and non dividing
state by reducing the temperature in the presence of cryoprotectant.

• Cryopreservation is the method of keeping the live cells, tissues and other biological samples in a
deep freeze at subzero temperatures for the storage or preservation.
 There are various methods of storage :

1. Cryopreservation : Generally involves storage in liquid

nitrogen.

2. Cold storage: It involves storage in low and non freezing temperature.

3. Low pressure : It involves partially reducing the atmospheric pressure of

surrounding.

4. Low oxygen storage: It involves reducing the oxygen level but

maintaining the pressure.
Cryopreservation Steps

The complete procedure steps involved in preserving the obtained biological samples
are as
follows:

1. Harvesting or Selection of material– Few important criteria should be

followed while selecting the biological materials such as – volume, density, pH,
morphology, and without any damage.

2. Addition of cryo-protectant– Cryoprotective agents such as glycerol, FBS (fetal

bovine serum), salts, sugars, glycols are added to the samples as it reduces the freezing
point of the medium and also allow slower cooling rate, which reduces the risk of
crystallization.

3.Freezing– Different methods of freezing are applied in this method of

cryopreservation to protect cells from damage and cell death by their exposure to the
warm solutions of cryoprotective agents.
It can be done :

Low temperature deep freezer (at -80 degree )

In vapor phase nitrogen (at -150 degree)

In liquid nitrogen (at -195.8 degree Celsius)

 Why preservation is important ?

• Until two decades ago the genetic resources were getting depleted owing to the
continuous depredation by man.

• It was imperative therefore that many of the elite, economically important

• Endangered species are preserved to make them available when needed.

• The conventional methods of storage failed to prevent losses caused due to various
reasons.

• A new methodology had to be devised for long term preservation of material.

Applications of Cryopreservation
• Cryopreservation is a long-term storage technique, which is mainly used for preserving and maintaining
viability of the biological samples for a longer duration.
• This method of preservation is widely used in different sectors including cryosurgery, molecular biology,
ecology, food science, plant physiology, and in many medical applications.
• Other applications of cryopreservation process are:
1. Seed Bank.
2. Gene Bank.
3. Blood transfusion.
4. In vitro fertilization.
5. Organ transplantation.
6. Artificial insemination.
7. Storage of rare germplasm.
8. Freezing of cell cultures.
9. Conservation of endangered plant species.
10. Biodiversity conservation
Batch and Continuous culture
 Microorganisms such as bacteria and fungi are very beneficial to various types of
industries. For industrial usage, microorganisms should be grown in large scale during
the fermentation or for other process in order to extract the necessary products resulting
from the microbial metabolism.
 A special apparatus called industrial fermenter is used to cultivate and maintain microbial
biomass.
 There are two types of industrial fermentation cultures commonly adopted in industries

1) Batch culture
2) Continuous culture
Batch Culture:- Batch culture is a technique used to grow microorganisms under limited
nutrient availability in a closed system.

• In batch culture technique, nutrients are provided at the beginning and the particular

microorganism is inoculated into the fermenter.

• The fermenter is closed and the temperature and pH are maintained for the growth of

microorganisms. Microorganism grows inside and utilizes the provided nutrients and other

conditions.
• Microorganisms are cultured or grown in a closed system viz. flasks, culture tubes.

• Nutrients are not renewed in the culture system.

• Population growth remains exponential for only a few generations and then enters a stationary

phase due to nutrient limitation and waste accumulation.

Batch Culture
 Continuous culture system

• Continuous culture is a technique used to grow microorganisms under optimum and

continual supply of nutrients in an open system in industries.

• Continuous culture is another technique which grows useful microorganisms.

• It aims to maintain a continuously growing microbial culture at exponential phase.

• It can be achieved by supplying fresh nutrients continually, removing accumulated waste and products

at the same rate and keeping other conditions at the optimum values.
• Open culture system

• Continuous nutrient supply

• Continuous waste removal

• Exponential phase can be maintained for longer periods

• This culture system maintains a constant volume to which fresh medium is added at

a constant rate, while an equal volume of spent culture medium with cells is removed

at the same rate.

• Once such a system is in equilibrium – chemostat volume, cell number and nutrient

status remains constant.

Continuous culture system
Differences between batch and continuous culture