Cryopreservation CSIR NET life sciences

Summary

This tutorial video delves into cryopreservation, the process of preserving biological tissues at extremely low temperatures, typically -195°C, using liquid nitrogen. It highlights the importance of this method for halting cellular activity to prevent cell death, and discusses the challenges of avoiding ice crystal formation which can damage cells. The concept of vitrification is introduced as a gradual temperature increase process using vitrifying agents to safely thaw preserved tissues without cell rupture, ensuring successful tissue preservation.

Takeaways

• 🧊 Cryopreservation is the process of preserving biological specimens at very low temperatures, typically around -195°C, which is the boiling point of liquid nitrogen.
• 🌡 The extreme cold is necessary to halt all cellular processes, preventing any enzymatic activity that could lead to cell death.
• 🧬 Biological tissues, including tissues and organs, are preserved to maintain their viability for future use, ensuring that they remain 'alive' during storage.
• ❄️ Liquid nitrogen is commonly used in cryopreservation due to its extremely low temperature, which is crucial for stopping all biological activity within the tissues.
• 🔬 If tissues are stored at higher temperatures, cellular activities such as enzyme functions and metabolic processes would continue, leading to cell death and failing the purpose of preservation.
• 💧 The risk of ice crystal formation during cryopreservation can damage cells; rapid freezing helps to mitigate this by preventing water from expanding and rupturing cell membranes.
• 🔬 Vitrification is a process used to slowly transition tissues from cryopreservation temperatures to normal temperatures, using agents that prevent the formation of damaging ice crystals.
• 🌡 The vitrification process involves a gradual increase in temperature, avoiding rapid changes that could cause cell membrane rupture and cell death.
• 🛠 Vitrifying agents are used to facilitate the controlled warming of tissues, ensuring stability and preventing damage during the thawing process.
• ♻️ The script emphasizes the importance of carefully managing temperature changes during both the freezing and thawing processes to maintain the integrity of biological samples.
• 📚 The tutorial aims to provide a basic understanding of cryopreservation, highlighting the significance of temperature control and the role of vitrification in preserving biological tissues.

Q & A

What is cryopreservation?

Cryopreservation is the process of preserving biological specimens or samples, such as tissues, organs, or cell lines, at extremely low temperatures, typically around -195°C, to maintain their viability for future use.

Why is liquid nitrogen commonly used in cryopreservation?

Liquid nitrogen is used because its boiling point is -195.8°C, which provides the ultra-low temperatures necessary to halt all cellular processes and prevent cell death, making it ideal for preserving the viability of biological tissues.

What is the significance of storing biological tissues at -195°C?

Storing at -195°C halts all cellular processes, including those that could lead to cell death such as apoptosis. This temperature is crucial to prevent the activation of enzymes that could degrade the tissue or lead to cell death.

What is the risk associated with rapid freezing of biological samples?

Rapid freezing can lead to the formation of ice crystals within the tissue. The expansion of water as it freezes can cause cell membranes to rupture, leading to cell death, which is why a controlled freezing process is essential.

What is vitrification in the context of cryopreservation?

Vitrification is the process of slowly warming a cryopreserved tissue from ultra-low temperatures to normal temperatures using vitrifying agents. This gradual increase in temperature helps prevent damage to the tissue caused by rapid temperature changes.

What role do vitrifying agents play in the warming process of cryopreserved tissues?

Vitrifying agents are used to prevent the formation of ice crystals during the warming process. They help to stabilize the tissue and allow for a controlled, gradual increase in temperature, reducing the risk of cell damage or death.

Why is it important to avoid rapid temperature changes when thawing cryopreserved tissues?

Rapid temperature changes can cause significant stress to the cells, potentially leading to cell membrane rupture and cell death. A controlled, gradual increase in temperature helps to minimize these risks.

What happens to cellular activities at temperatures above -195°C?

At temperatures above -195°C, cellular activities such as enzyme functions and metabolic processes can begin to occur, which could lead to cell death if the cells are not intended to be active.

How does the process of vitrification help maintain cell stability during thawing?

Vitrification involves a step-by-step increment of temperature, which allows the cells to gradually adjust to the change, thereby maintaining cell stability and reducing the risk of damage.

What is the typical temperature range for the vitrification process?

The vitrification process involves gradually increasing the temperature from -195°C, possibly through stages such as -50°C, -25°C, 0°C, and finally to 35°C, depending on the specific requirements of the tissue being thawed.

Why is the preservation of living tissues important in cryopreservation?

The preservation of living tissues is important to ensure that the biological specimens remain viable for future use, such as in transplantation or research, without the risk of cell death or degradation.

Outlines

00:00

🧊 Cryopreservation Basics and Temperature Significance

This paragraph introduces the concept of cryopreservation, which is the preservation of biological samples at extremely low temperatures. The primary focus is on the significance of using temperatures as low as -195°C, which is the boiling point of liquid nitrogen. The purpose of such low temperatures is to halt all cellular processes, preventing cell death due to apoptosis or other enzymatic activities that could occur at higher temperatures. The paragraph also touches on the importance of rapid freezing to prevent the formation of water crystals that could damage cells.

05:02

🔄 The Vitrification Process in Cryopreservation

The second paragraph delves into the process of vitrification, which is essential for safely thawing cryopreserved biological samples. It explains the need for a gradual temperature increase to avoid cell damage that could occur from rapid temperature changes. Vitrification agents are used to facilitate this slow and controlled temperature rise, ensuring the stability of the cells. The paragraph outlines the step-by-step process of incrementally raising the temperature from -195°C to physiological levels, emphasizing the avoidance of a direct and rapid transition that could be detrimental to cell integrity.

Keywords

💡Cryopreservation

Cryopreservation refers to the process of preserving biological specimens or samples at extremely low temperatures, typically around -195°C or 77 Kelvin. This is crucial for maintaining the viability of tissues, organs, or cell lines for future use. In the video, cryopreservation is the central theme, as it discusses the importance of using such low temperatures to halt all cellular processes and prevent cell death.

💡Liquid Nitrogen

Liquid nitrogen is highlighted as the medium used for cryopreservation due to its extremely cold temperature of -195°C. It is pivotal in the process as it provides the necessary cold environment to preserve biological tissues effectively. The script mentions that liquid nitrogen is used because of its boiling point, which aligns with the required temperature for cryopreservation.

💡Cellular Activities

Cellular activities encompass the various biochemical processes that occur within cells, such as enzyme activity, replication, transcription, and metabolism. The script explains that these activities must be halted to preserve the cells, which is why cryopreservation at such low temperatures is necessary, as it inhibits the enzymes that would otherwise lead to cell death.

💡Apoptosis

Apoptosis, also known as programmed cell death, is a process where cells are systematically dismantled and removed. The script mentions that storing cells at temperatures that allow certain enzymes to activate can lead to apoptosis, which would defeat the purpose of preservation. Cryopreservation at -195°C prevents this by halting all such deadly pathways.

💡Vitrification

Vitrification is a process mentioned in the script that involves the slow and controlled increase of temperature to bring a cryopreserved sample back to normal conditions. This is important to prevent the formation of water crystals that can damage cells. The script describes vitrification as the careful step-by-step increment of temperature, using vitrifying agents to avoid rapid changes that could rupture cell membranes.

💡Vitrifying Agents

Vitrifying agents are substances used in the vitrification process to prevent the formation of ice crystals and to stabilize the sample as it is warmed. The script explains that these agents are essential for the slow and gradual increase in temperature, ensuring the cell membrane can adapt to the changes without rupturing.

💡Temperature Control

Temperature control is a critical aspect of cryopreservation and vitrification. The script emphasizes the need for rapid freezing at extremely low temperatures to prevent cell damage and the gradual increase in temperature during the thawing process to avoid cell rupture. Proper temperature control ensures the preservation and revival of biological tissues.

💡Enzymes

Enzymes are biological catalysts that facilitate various chemical reactions within cells. The script discusses how enzymes, particularly those involved in apoptosis, can be inhibited at cryopreservation temperatures, preventing cell death. It also mentions the need to avoid conditions that would activate these enzymes during the preservation process.

💡Cell Membrane

The cell membrane is a selectively permeable barrier that surrounds and protects the cell. In the context of the script, it is highlighted that rapid temperature changes can cause the cell membrane to rupture, leading to cell death. Vitrification helps to protect the cell membrane by controlling the rate of temperature increase.

💡Biological Tissues

Biological tissues are collections of cells that perform a specific function within an organism. The script discusses the importance of cryopreserving these tissues to maintain their viability for future use. The preservation of these tissues at -195°C halts all cellular processes, ensuring they remain in a state that can be revived.

Highlights

Cryopreservation is the process of preserving biological specimens at extremely low temperatures.

The term 'cryopreservation' originates from 'cryo' indicating cold and 'preservation' for maintaining the state of the samples.

Biological tissues, organs, and cell lines can be preserved through cryopreservation.

The ideal temperature for cryopreservation is approximately -195°C, which is the boiling point of liquid nitrogen.

Liquid nitrogen is commonly used in cryopreservation due to its extremely low temperature.

At -195°C, cellular activities and enzymatic reactions are halted, preventing cell death.

Cryopreservation inhibits apoptosis or programmed cell death, thus preserving the viability of the tissue.

Vitrification is a process in cryopreservation that prevents the formation of water crystals which can damage cells.

Rapid freezing is crucial to prevent water from expanding and bursting the cell membranes.

The thawing process must be gradual to avoid cell membrane rupture due to rapid temperature changes.

Vitrifying agents are used to facilitate the controlled increase in temperature during the thawing process.

Incremental temperature increase helps maintain cell stability and prevent cell death post-thawing.

Cryopreservation is vital for the long-term storage of biological samples without loss of viability.

The process of cryopreservation halts all cellular processes, ensuring the preservation of the sample's integrity.

Cryopreservation has significant practical applications in medical and biological research.

Understanding the principles of cryopreservation is essential for successful preservation and revival of biological samples.

The tutorial provides a basic understanding of cryopreservation techniques and their importance in preserving biological tissues.

Transcripts

[Music]

welcome back friends in this video

tutorial we'll be talking about

cryopreservation it's just basic small

video about

cryopreservation

cryo

preservation okay so what do we mean by

cryopreservation as the term suggest

cryo preservation means preserving

biological specimens or samples like

tissues or different organs or different

cell lines in cold temperature because

when cryo is associated we always talk

about cold climate and cold temperature

so

preservation so

preservation

of tissue which is living

obviously in cold temperature now the

question comes how much cold right and

the answer is here it is almost let's

say - 195° c or let's say 77 Kelvin or

something so - 197 which is

below

below

Subzero temperature minus 195° cus now

why this particularly minus 195° C

because this is the boiling point

of of liquid

nitrogen boiling point of liquid

nitrogen okay so usually in cry

preservation of tissue grafts and C

preservation of biological tissue

specimens we usually use this liquid

nitrogen for that okay now liquid

nitrogen is very cold because you can

see minus 195° C very very very cold uh

in temperature now why we require that

much very small amount of uh temperature

to store them we can store it freezing

temperature like 0 -4 -5 - 10 but is -

195° c and the answer for that the key

for the cry preservation is that we need

to store the biological tissues which

are living tissues so that they still

leave right because if you store them in

higher temperature the activity of there

all the cellular activities like

activity of enzymes like polymerous

enzyme for the replication transcription

different enzymes for metabolism they

will start to work because for all the

biological enzymes to work it requires a

particular temperature more than 0°

celus usually it acts better at

physiological temperature like 35 to 37°

CSUS many of them works well in the home

temperature like 25 to 30° C but very

few of them can also Act on that 5 10

12° cus temperature so if we store them

in low temperature like 510 or close to

zero it will still act on it and the

cell will finally go and die right

because for the cell death it requires

some enzymes some deadly Pathways called

apoptosis or programmed cell death right

so if we put if those enzymes

responsible for program cell death are

activated at a particular temperature

and if you store this cell in that

temperature if those enzymes are active

then the cell will die and the purpose

for preserving tissue will be failed so

for that reason we have seen that it

atus 195° C temperature none of those

enzymes are active to kill the cell or

to guide the cell through apoptosis so

all of the processes I repeat all of the

cellular processes that are usually

going on inside the cell are kind of

halted are kind of in inhibited at that

low temperature that you have talked

that's why we used to store our uh

samples in that much cold temperature

okay that's the reason and second thing

another question CSI net people can ask

you is that what is vitrification now

let me explain this term

vitrification now one important thing

about this C preservation in any kind of

cold preservation is once you put it

into that high uh very low temperature

it there's a chance of water crystal

formation in your tissue now if water

crystal form we know that at Crystal

form at at the solid form at the icy

form water expands more so it can burst

your cell but usually for that reason we

need to rapid rapid freeze them in this

temperature that's a very important

point now let's say you've made the

sample you've taken the sample prepare

the graph and you put it rapidly into

the freezing Machinery everything is

okay everything is fine it is stored it

is preserved now you need to take that

particular sample out from that cold

temperature and you need to PL place it

in the normal temperature right so what

you need to do we need to increase the

temperature now remind you in any

condition if you increase the

temperature rapidly just like freezing

it rapidly it will kill the cell why

because increasing temperature rapidly

will cause certain changes very fast

into your cell and the cell membrane

will not be able to hold on to all those

changes at very fast so the cell

membrane will rupture and the cell will

die to prevent that what we need to do

we need to increase the temperature

little by little with time right and

using certain amount of different agents

different watering agents different uh

water supplying agents and different

enzymatic agents now those agents are

called vitrifying agents and the process

of slowly taking out of the tissue graph

from the liquid nitrogen to the normal

temperature is called the vitrification

process in this process what we do we do

stepbystep increment of

temperature so we take let's say it's

previously 195° cius temperature we add

some vitrification agent or vitrifying

solution one for example and the

temperature comes like let's say minus

50 then slightly let's 25 then it's kind

of zero then from it slightly we're

increasing towards 35° C and this is the

process not directly we're not going

this this path because this pathway is

very very dangerous this pathway is

dangerous so it is not taken either we

take the small stages because it helps

uh to maintain the St cell stable okay

so that is the vitrification process

okay so that's kind of it I hope that's

helpful thank you thank

you