Cryobiology

Cryobiology

Definition

Cryobiology is the branch of biology that studies the effects of low and ultra-low temperatures on living cells, tissues, organs, and biological systems. It mainly deals with understanding cellular responses to freezing and thawing and developing methods for long-term preservation of biological material at sub-zero temperatures, especially using liquid nitrogen (−196°C).

Basic Principle of Cryobiology

At extremely low temperatures, metabolic activities, enzymatic reactions, and biochemical processes slow down or stop completely. This state allows cells to remain viable for long periods without genetic or physiological changes, provided freezing and thawing are properly controlled.

Fig: -  Cryopreservation steps

Cellular Events During Freezing

Freezing causes both physical and biochemical stress in cells:

1.     Ice Crystal Formation

o    Extracellular ice forms first, increasing solute concentration outside the cell.

o    This creates an osmotic gradient, causing water to move out of the cell.

o    Intracellular ice formation is lethal as it damages membranes and organelles.

2.     Osmotic Stress

o    Excessive dehydration or rapid rehydration during thawing can rupture membranes.

3.     Solution Effects Injury

o    High solute concentration during freezing can denature proteins and disrupt membranes.

Cryoprotectants

Cryoprotectants reduce freezing damage by preventing ice crystal formation.

Types:

• Penetrating cryoprotectants
• Glycerol
• Dimethyl sulfoxide (DMSO)
• Ethylene glycol
• Non-penetrating cryoprotectants
• Sucrose
• Trehalose
• Polyvinylpyrrolidone (PVP)

Functions:

• Reduce ice nucleation
• Stabilize proteins and membranes
• Control osmotic balance

Cryopreservation Techniques

1. Slow Freezing

• Cooling rate: ~1°C/min
• Allows controlled dehydration
• Commonly used for plant tissues, microorganisms, sperm, embryos

2. Rapid Freezing

• Faster cooling, limited dehydration
• Risk of intracellular ice formation

3. Vitrification (High-Yield Topic ⭐)

• Ultra-rapid cooling with high cryoprotectant concentration
• Water solidifies into a glass-like amorphous state without ice crystals
• Widely used in embryo and oocyte preservation

Storage

• Biological samples are stored in:
• Liquid nitrogen (−196°C)
• Nitrogen vapor phase (−150°C to −170°C)

Thawing Process

• Rapid thawing is preferred to prevent recrystallization.
• Cryoprotectants must be removed gradually to avoid osmotic shock.

Comparison: Slow Freezing vs Vitrification

Feature	Slow Freezing	Vitrification
Definition	Controlled cooling method where cells are frozen gradually	Ultra-rapid cooling method converting solution into a glass-like state
Cooling rate	Slow (≈ 0.5–1°C per minute)	Extremely fast (thousands of °C per minute)
Ice crystal formation	Small extracellular ice crystals may form	No ice crystals formed
Mechanism	Gradual dehydration of cells before freezing	Solidification without crystallization
Cryoprotectant concentration	Low to moderate	Very high
Type of cryoprotectants	Glycerol, DMSO (low concentration)	DMSO, ethylene glycol + sugars (high concentration)
Cell dehydration	Occurs gradually	Minimal dehydration
Risk of damage	Osmotic injury, ice crystal damage	Cryoprotectant toxicity
Storage temperature	−196°C (liquid nitrogen)	−196°C (liquid nitrogen)
Equipment required	Programmable freezer required	Simple equipment (no freezer needed)
Technical complexity	Technically simpler	Requires high skill and precision
Cell survival rate	Moderate	Very high
Common applications	Microorganisms, plant tissues, sperm	Embryos, oocytes, stem cells
Cost	Relatively low	Relatively high
Suitability	Large sample volumes	Small sample volumes
Recrystallization during thawing	Possible	Minimal

Applications of Cryobiology

1.     Medical & Clinical

o    Cryopreservation of sperm, ova, embryos

o    Stem cell banking

o    Blood and bone marrow preservation

2.     Plant Biotechnology

o    Conservation of germplasm

o    Preservation of rare and endangered species

o    Storage of somatic embryos and meristems

3.     Microbiology

o    Long-term storage of bacteria, fungi, and viruses

4.     Animal Biotechnology

o    Preservation of genetic material

o    Conservation breeding programs

Advantages

• Long-term preservation without genetic alteration
• Maintains cell viability and metabolic integrity
• Reduces cost and labor of continuous culture maintenance

Limitations

• Cryoprotectant toxicity
• Ice crystal damage
• Requires precise temperature control and expertise

CSIR-NET Important Points (Quick Revision)

• Storage temperature: −196°C
• Most common cryoprotectants: DMSO, glycerol
• Most lethal damage: intracellular ice formation
• Best method to avoid ice crystals: Vitrification
• Fast thawing > slow thawing

Conclusion

Cryobiology plays a crucial role in biotechnology, medicine, conservation biology, and reproductive science. Understanding cellular responses to freezing, cryoprotectant action, and preservation techniques is essential for successful cryopreservation, making cryobiology a high-scoring topic in CSIR-NET and other competitive life-science examinations.

 

Cryopreservation MCQ Test

1. Cryopreservation mainly involves storage at:

−20°C
−80°C
−196°C
0°C

2. Most lethal injury during freezing is:

Protein denaturation
Intracellular ice formation
DNA mutation
pH change

3. Which is a penetrating cryoprotectant?

Sucrose
Trehalose
DMSO
Starch

4. Vitrification prevents damage by:

Slow cooling
Ice crystal formation
Glass-like solidification
Dehydration

5. Cooling rate in slow freezing is about:

100°C/min
10°C/min
1°C/min
50°C/min